Low temperature stress in crop plants: the role of the membrane: proceedings of an international...

LOW TEMPERATURE STRESS IN CROP PLANTS

THE ROLE OF THE MEMBRANE

ACADEMIC PRESS RAPID MANUSCRIPT REPRODUCTION

Proceedings of an International Seminar on Low Temperature Stress in Crop Plants Held at the East-West Center, Honolulu,

Hawaii, March 26-30, 1979

Sponsored by The United States National Science Foundation

The Australian Department of Science The College of Agricultural and Environmental Sciences,

university of California at Davis


T H E R O L E O F T H E M E M B R A N E

Edited by

JAMES M. LYONS

Plant Physiology Unit CSIRO Division of Food Research and School of Biological Sciences

Macquarie University North Hyde, N.S.W., Australia

ACADEMIC PRESS A Subsidiary of Harcourt Brace Jouanouich, Publishers

New York London Sydney Toronto San Francisco 1979

Department of Vegetable Crops University of California at Davis

Davis, California

DOUGLAS GRAHAM JOHN K. RAISON

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CONTENTS

Participants ix Preface xi/i Photograph of Participants xu

I N T R O D U C T I O N

The Plant Membrane in Response to Low Temperature: An Overview 1

7. M. Lyons, J. K. Raison, and P. L. Steponkus Adaption to Chilling: Survival, Germination, Respiration, and Protoplasmic Dynamics 25

B. D. Patterson, D. Graham, and R. Paull Seed Germination at Low Temperatures 37

E. W. Simon Drought Resistance as Related to Low Temperature Stress 47

J. M. Wilson Low Temperature Responses of Three Sorghum Species 67

D. J. Bagnall Physiology of Cool-Storage Disorders of Fruit and Vegetables 81

N. L. Wade

P R O P A G A T I O N O F T H E T E M P E R A T U R E R E S P O N S E

A. Chilling Injury

Sequence of Ultrastructural Changes in Tomato Cotyledons during Short Periods of Chilling 97

R. Ilker, R. W. Breidenbach, and J. M. Lyons Movement and Loss of Organic Substances from Roots at Low Temperature 115

Μ. N. Christiansen

ν

vi Contents

Cold-Shock Injury and Its Relation to Ion Transport by Roots 123 F. Zsoldos and B. Karvaly

Ion Leakage in Chilled Plant Tissues 141 T. Murata and Y. Tatsumi

Effects of Chilling on Membrane Potentials of Maize and Oat Leaf Cells 153

P. H. Jennings and T. A. Tat tar Temperature Sensitivity of Ion-Stimulated ATPases Associated with Some Plant Membranes 163

E. J. McMurchie Membrane Lipid Transitions: Their Correlation with the Climatic Distribution of Plants 177

J. K. Raison, E. A. Chapman, L. C. Wright, and S. W. L. Jacobs

The Useful Chloroplast: A New Approach for Investigating Chilling Stress in Plants 187

R. M. Smillie Low Temperature Response of Chloroplast Thylakoids 203

M. P. Garber The Influence of Changes in the Physical Phase of Thylakoid Membrane Lipids on Photosynthetic Activity 215

D. C. Fork

B. Freezing Injury

Freeze-Thaw-Induced Lesions in the Plasma Membrane 231 P. L. Steponkus and S. C. Wiest

Membrane Structural Transitions: Probable Relation to Frost Damage in Hardy Herbaceous Species 255

C. Rajashekar, L. V. Gusta, and M. J. Burke Possible Involvement of the Tonoplast Lesion in Chilling Injury of Cultured Plant Cells 275

S. Yoshida, T. Niki, and A. Sakai Freezing Stress in Pota to 291

P. H. Li, J. P. Paha, and Η. H. Chen

T H E P R I M A R Y T E M P E R A T U R E SENSOR

A. Characterization of Fluidity Parameters

Fluorescence Polarization Studies of Membrane Phospholipid Phase Separations in Warm and Cool Climate Plants 305

C S. Pike, J. A. Berry, and J. K. Raison Differential Thermal Analysis of Tomato Mitochondrial Lipids 319

A. W. Dalziel and R. W. Breidenbach

Contents vii

Some Physical Properties of Membranes in the Phase Separation Region and Their Relation to Chilling Damage in Plants 327

J. Wolfe Lipid Phase of Membrane and Chilling Injury in the Blue-Green Alga, Anacystis nidulans 337

N. Murata, T. Ono, and N. Sato

B. Control of Membrane Fluidity

Molecular Control of Membrane Fluidity 347 G. A. Thompson, Jr.

In Vitro Membrane Lipid Reconstitution and Enzyme Function 365 A. Waring and P. Glatz

The Influence of Fatty Acid Unsaturation on Fluidity and Molecular Packing of Chloroplast Membrane Lipids 375

D. G. Bishop, J. R. Kenrick, J. H. Bayston, A. S. Macpherson, S. R. Johns, and R. I. Willing

Temperature Regulation of Plant Fatty Acyl Desaturases 391 P. Mazliak

Chemical Modification of Lipids in Chilling Sensitive Species 405 J. B. St. John

Chemical Modification of Lipids during Frost Hardening of Herbaceous Species 411

C Willemot Chill-Induced Changes in Organelle Membrane Fatty Acids 431

E. J. Bartkowksi, T. R. Peoples, and F. R. H. Katterman

C. Membrane-Cytoplasmic Interactions

Diffusion Relationships between Cellular Plasma Membrane and Cytoplasm 437

A. D. Keith, Α. Μ astro, and W. Snipes

D . Direct Effects of Temperature on Proteins

Temperature Effects on Phosphoenol Pyruvate Carboxylase from Chilling-Sensitive and Chilling-Resistant Plants 453

D. Graham, D. G. Hockley, and B. D. Patterson

GENETIC POTENTIAL F O R C O L D RESISTANCE

Cell Culture Manipulations as a Potential Breeding Tool 463 P. J. Dix

Genetic Diversity of Plants for Response to Low Temperatures and Its Potential Use in Crop Plants 473

C. E. Vallejos

viii Contents

Adaptation to Chilling Stress in Sorghum 491 J. R. Mc William, W. Manokaran, and T. Kipnis

Chilling Injury Assays for Plant Breeding 507 R. E. Paull, B. D. Patterson, and D. Graham

SPECIAL T O P I C S R E L A T E D T O T H E USE O F A R R H E N I U S PLOTS

Breaks or Curves? A Visual Aid to the Interpretation of Data 523 Μ. E. Willcox and B. D. Patterson

Statistical Tests to Decide between Straight Line Segments and Curves as Suitable Fits to Arrhenius Plots or Other Data 527

J. Wolfe and D. J. Bagnall Maximum Likelihood Estimation of Breakpoints and the Comparison of the Goodness of Fit with that of Conventional Curves 535

/. F. Potter and G. J. 5. Ross Epilogue 543 Appendix 549

Table of Temperature Coefficients of Various Plant Responses

index 559

PARTICIPANTS

David J. Bagnall Division of P lan t Indust ry , C S I R O , Canbe r ra City, ACT 2600, Australia

David G. Bishop, Plant Physiology Unit, C S I R O , Division of Food Research and School of Biological Sciences, Macquarie University, Nor th Ryde, N.S.W. 2113, Australia

R. William Breidenbach, Plant Growth Laboratory, University of California, Davis, California 95616

Michael J. Burke, Department of Horticulture, Colorado State University, Ft. Collins, Colorado 80521

Meryl N. Christiansen, Plant Stress Laboratory, Plant Physiology Institute, U S D A - R S , Beltsville, Maryland 20705

Adam W. Dalziel, Plant Growth Laboratory, University of California, Davis, California 95616

Philip J. Dix, Department of Genetics, Ridley Building, Claremont Place, University of Newcastle Upon Tyne, Newcastle Upon Tyne, N E I 7 R U , Great Britain

David C. Fork, Depa r tmen t of Plant Biology, Carnegie Inst i tute of Washington, 290 Panama Street, Stanford, California 94305

Melvin P. Garber, Tree Propagation Scientist, Weyerhaeuser Company, Southern Forestry Research Center, P. O. Box 1060, Hot Springs, Arkansas 71901

Douglas Graham, Plant Physiology Unit, C S I R O Division of Food Research and School of Biological Sciences, P. O. Box 52, North Ryde, N.S.W. 2113, Australia

Larry V. Gusta, Crop Development Center, University of Saskatchewan, Saskatoon, Canada S7N O W O

Reinfriede Ilker, General Foods Corporat ion, Technical Center, Tarrytown, New York

Paul H. Jennings, Department of Plant and Soil Sciences, Bowditch Hall, University of Massachusetts, Amherst, Massachusetts 01002

Frank Katterman, Department of Plant Sciences, University of Arizona, Tucson, Arizona 85721

ix

χ Participants

Alec D. Keith, Department of Biochemistry & Biophysics, The Pennsylvania State University, University Park, Pennsylvania 16802

Paul H. Li, Laboratory of Plant Hardiness, Department of Horticultural Science, 286 Alderman Hall, University of Minnesota, 1970 Folwell Avenue, St. Paul, Minnesota 55108

James M. Lyons, Department of Vegetable Crops, University of California, Davis, California 95616

James R. McWilliam, Department of Agronomy & Soil Science, University of New England, Armidale, N.S.W. Australia

E. J. McMurchie, Plant Physiology Unit, C S I R O Division of Food Research and School of Biological Sciences, Macquarie University, Nor th Ryde, N.S.W. 2113 Australia

Paul Mazliak, Laboratorie de Physiologie Cellulaire, Universite Pierre et Marie Curie, 4, Place Jussieu, Tour 53, 75005 Paris, France

Norio Murata, Department of Biology, University of Tokyo, College of General Education, Komaba, Meguro-Ku, Tokyo 153, Japan

Takao Murata, Laboratory of Postharvest Physiology and Preservation of Fruits and Vegetables, Faculty of Agriculture, Shizuoka University, Oya, Shizuoka 422, Japan

Τ Nikki, Inst i tute of Low Tempera tu re Science, H o k k a i d o Universi ty, Sapporo, Japan 060

Brian D. Patterson, Plant Physiology Unit, C S I R O Division of Food Research and School of Biological Sciences, P . O. Box 52, North Ryde, N.S.W. 2113, Australia

Robert Paull, Department of Botany, University of Hawaii at Manoa, 3190 Maile Way, Honolulu, Hawaii 96822

Tim Peoples, Department of Plant Sciences, University of Arizona, Tuscon, Arizona 85721

Carl Pike, Department of Plant Biology, Carnegie Institute of Washington, 290 Panama Street, Stanford, California 94305

John K. Raison, Plant Physiology Unit, C S I R O Division of Food Research and School of Biological Sciences, Macquarie University, Nor th Ryde, N.S.W. 2113, Australia

Eric W. Simon, Botany Department, The Queen's University of Belfast, Belfast BT7 INN, North Ireland

Robert M. Smillie, Plant Physiology Unit, C S I R O Division of Food Reserach and School of Biological Sciences, P. O. Box 52, North Ryde, N.S.W. 2113, Australia

Peter L. Steponkus, Department of Agronomy, Cornell University, Ithaca, New York 14853

Judith B. St. John, Plant Physiologist, USDA, RS , AEQU, Weed Science Laboratory, Beltsville, Maryland 20705

F. Tagawa, Institute of Low Temperature Science, Hokkaido University, Sapporo, Japan 060

Participants xi

Yasuo Tatsumi, Laboratory of Postharvest Physiology and Preservation of Fruits and Vegetables, Faculty of Agriculture, Shizuoka University, Oya, Shizuoka 422, Japan

Guy A. Thompson, Jr. Department of Botany, The University of Texas, Austin, Texas 78712

C. Eduardo Vallejos, Depa r tmen t of Vegetable Crops , University of California, Davis, California 95616

Neil L. Wade, N.S.W. Department of Agriculture, C S I R O Division of Food Research, P. O. Box 52, North Ryde, N.S.W. 2113, Australia

Alan J. Waring, Johnson Research Foundat ion G4 and Physical Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19174

Mary E. Willcox, C S I R O Division of Mathematics and Statistics, located at C S I R O Division of Food Research, P. O. Box 52, North Ryde, N.S.W. 2113, Australia

Claude Willemot, Station de Recherches, Agriculture Canada, 2560, Boul. Hochelaga, Ste-Foy, Quebeck, Canada GIV 2J6

John M. Wilson, School of Plant Biology, Memorial Building, University College of Nor th Wales, Bangor LL572UW, Great Britain

Joe A. Wolfe, Department of Applied Mathematics, Research School of Physical Science, Australian National University, Canberra City, ACT 2600, Australia

Shizuo Yoshida, Institute of Low Temperature Science, Hokkaido University, Sapporo, Japan 060

Ferenc Zsoldos, Department of Plant Physiology, Attila Jozsef University, H-6701 Seged, P. O. Box 428, Hungary

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PREFACE

This volume is based on the proceedings of an international seminar on "Low Temperature Stress in Crop Plants: The Role of the Membrane," which was held at the East-West Center, Honolulu, Hawaii, March 26-30, 1979. It contains a series of articles which focused on exploration of the fundamental mechanisms involved in the temperature response of crop plants.

The seminar was sponsored by the United States Nat iona l Science Foundation and the Australian Department of Science under the auspices of the United States-Australian Cooperative Science Program, as well as the College of Agricultural and Environmental Sciences, University of California at Davis. The primary focus of the seminar was to examine the hypotheses related to the primary temperature sensor in crop plants and to evaluate evidence in support of current concepts in the mechanisms of low temperature injury. As food reserves approximate the amount of annual production, the impact of fluctuating weather and climate become increasingly important. Selection of plant materials and breeding of improved varieties has historically been done against a background of climatic fluctuations and environmental variables. Intolerable low temperature is one of the climatic variables that imposes stress on crop production. Avoiding low temperature damage by growing shorter-season varieties on the edges of more suitable production areas is one approach to the solution of variable low temperatures. However, the necessity to incorporate increased genetic potential for resistance to these low temperatures remains as a high priority for improving food production.

It is hoped that the discussions that are included in this volume will make a significant contribution to the researchers involved in understanding and attenuating crop losses by low temperature and that specific research questions have been identified which should provide the best approaches to planning research strategies and finding solutions that will aid in expanding the limits of crop production.

We would like to express our appreciation to Mr. J im McMahon and his staff at the East-West Center for their superb management of the conference facilities during the week's program. Because of the fine facilities and ease in presentation, a very full week was easily handled.

xiii

xiv Preface

We would also like to acknowledge Ms. Betty Perry, Department of Vegetable Crops, University of California at Davis, for her impeccable secretarial work and preparation of the camera-ready manuscript.

Symposium participants and contributors (left to right). First row: David C. Fork, Eric Simon, Yasuo Tatsumi, T. Nikki, Robert M. Smillie, Paul H. Li, Tim Peoples, Eduardo Vallejos, Philip Dix, Alec Keith, F. Zsoldos, Guy A. Thompson, Shizuo Yoshida, F. Tagawa. Second row: Paul Mazliak, Norio Murata, Takao Murata, David G. Bishop, Carl Pike, A. Dalziel, Reinfriede Ilker, T. McMurchie, John Raison, Douglas Graham, Jim Lyons, M. Garber, Judith St. John, Bill Breidenbach, Claude Willemot. Third row: Alan Waring, Joe Wolfe, Robert Paul, F. Katterman, L. Gusta, J. McWilliam, Michael Burke, Meryl Christiansen, Brian Patterson, David Bagnall, John Wilson, Peter Steponkus, Paul Jennings.


L O W T E M P E R A T U R E S T R E S S I N C R O P P L A N T S

THE PLANT MEMBRANE IN RESPONSE TO LOW TEMPERATURE: AN OVERVIEW

J. M. Lyons

D e p a r t m e n t of Vege tab le Crops Univers i ty of Cal i forn ia

Davis , Ca l i forn ia

J. K. Raison

CSIRO Plan t Physiology Unit School of Biological Sciences

Macqua r i e Univers i ty Nor th Ryde , N.S.W. Aus t r a l i a

Peter L. Steponkus

D e p a r t m e n t of Agronomy Cornel l Univers i ty I t haca , New York

I. INTRODUCTION

The world 's n e e d for food i nc r ea se s eve ry y e a r . C u r r e n t l y , food for s o m e 4 bill ion inhab i t an t s is r equ i r ed . In the yea r Z000 food for a t l eas t 6 billion will be needed . As food r e s e r v e s a p p r o x i m a t e t he amoun t of annual p roduc t ion , the i m p a c t of f l uc tua t ing w e a t h e r and c l i m a t e b e c o m e increas ing ly i m p o r t a n t . Eff ic iency and p roduc t ion of food have improved s t ead i ly to m e e t man 's n e e d s . Se lec t ion of p lan t m a t e r i a l s and b reed ing of improved v a r i e t i e s has h i s to r ica l ly been done aga ins t a background of c l ima t i c f luc tua t ions and e n v i r o n m e n t a l va r i ab le s . Al though t h e r e is t he gene ra l consensus t ha t env i ronmen ta l s t r e s se s l imit c rop d i s t r ibu t ion and p roduc t ion , it is usual ly a c c o m p a n i e d by an a t t i t u d e of a c c e p t i n g r a t h e r than address ing t h e d e v a s t a t i n g e f fec t of c l i m a t i c e x t r e m e s . Within the p lan t kingdom t h e r e is an e x t r e m e r a n g e of g e n e t i c d ivers i ty in t h e c a p a c i t y to w i th s t and low t e m p e r a t u r e s . While many spec ies of t rop ica l and sub t rop ica l reg ions a r e injured by low t e m p e r a t u r e s above 0 C, numerous t e m p e r a t e zone spec ies can

Copyright * 197Θ by Academic Press, inc. 1 All rights of reproduction in any form reserved

I S B N a i 2 46056O 5

2 J. Μ. Lyons et al

wi ths t and - 1 9 6 ° C . Explo i ta t ion of t h e g e n e t i c d ivers i ty for low t e m p e r a t u r e t o l e r a n c e is however h a m p e r e d by a universa l o b s t a c l e . A c o m p l e t e unders tand ing of what c o n s t i t u t e s low t e m p e r a t u r e r e s i s t a n c e or even how low t e m p e r a t u r e s r esu l t in injury is l ack ing . In o rder to u l t i m a t e l y improve p lan t r e s i s t a n c e to low t e m p e r a t u r e one mus t 1) c h a r a c t e r i z e the s t r e s s imposed on the p l an t , Z) d e t e r m i n e t he r epe rcuss ion of t he s t r e s s on t he ce l lu lar env i ronmen t and a r c h i t e c t u r e , and 3) d e t e r m i n e what c o n s t i t u t e s injury a t the ce l lu lar and molecu la r l eve l .

When such in fo rmat ion is ava i lab le , t h e physical and b iochemica l a s p e c t s of a d a p t a t i o n , e i the r cons t i t u i t i ve or f a c u l t a t i v e , will be b e t t e r unders tood . With such an approach , p rocedu re s for t he a s se s smen t and i m p r o v e m e n t of low t e m p e r a t u r e r e s i s t a n c e will e m e r g e for use in eva lua t ion of ge rmp la sm, in b reed ing p r o g r a m s , and in fo rmula t ion of a p p r o p r i a t e cu l tu ra l and m a n a g e m e n t p r a c t i c e s .

This "Overview" s u m m a r i z e s some a s p e c t s of wha t is known about the mechanism(s) of how low t e m p e r a t u r e con t ro l s bo th chil l ing and f reez ing p rocesses leading to injury and ident i f ies ques t ions and a r e a s whe re i n a d e q u a t e in format ion or under s t and ing ex i s t s . Hopefully, t hese ques t ions will aid in focusing r e s e a r c h s t r a t e g i e s to develop a b e t t e r unders tand ing and c o n t r i b u t e to finding solut ions to low t e m p e r a t u r e l imi t a t ions of world food p roduc t ion .

A. Chilling Injury

Many i m p o r t a n t crop spec ies of t rop ica l or sub t rop ica l origin (r ice, corn , t o m a t o , c o t t o n , soybean, e tc . ) a r e sens i t ive to low t e m p e r a t u r e s in t h e r ange of ZO down to about 0 C. Below some c r i t i c a l t e m p e r a t u r e , they a r e sharply r e s t r i c t e d in ge rmina t ion , g rowth , r ep roduc t ion , and pos tha rves t longevi ty . This physiological d a m a g e to p lan t t i ssues no t involving f reez ing is commonly r e f e r r e d to as "chill ing injury." Such chill ing sens i t ive spec ies a r e d r a m a t i c c o n t r a s t s to spec ies of t e m p e r a t e origin which do not mani fes t this sens i t iv i ty to low t e m p e r a t u r e , and in some cases have t e m p e r a t u r e op t ima in this r a n g e .

The physiological dysfunct ions t h a t occur upon exposure of chi l l ing-sens i t ive spec ies to low t e m p e r a t u r e s lead to a v a r i e t y of visible s y m p t o m s . The e x t e n t of this visible injury is a funct ion of t he t e m p e r a t u r e e x t r e m e , dura t ion of exposure to cold condi t ions , p lan t spec ies , and morphological and physiological condi t ion of the p lan t m a t e r i a l a t t i m e of exposure . The obse rva t ions include e f f ec t s on p lan t m a t e r i a l s a t all s t ages of deve lopmen t . To offer a few example s : Low t e m p e r a t u r e s have been shown to be harmful in the ge rmina t ion of sens i t ive spec ies such as c o t t o n (10), muskmelons and peppe r s (Z7) t o m a t o e s (99, 113), wild t o m a t o spec ies (79), and many o the r spec ies (11Z). If exposure to cold o c c u r r e d a f t e r t he p lan t has b e c o m e es tab l i shed , the most s t r ik ing symptom in a number of h e r b a c e o u s p l an t s such as the cucumber and bean is w a t e r loss and wil t ing (67, 1Z7, 1Z8).

The Plant Membrane in Response to Low Temperature 3

Leaf s t o m a t e s in some in s t ances a r e unable to c lose a t chill ing t e m p e r a t u r e s and the abi l i ty of l eaves to t r an spo r t w a t e r is impa i r ed . In sorghum, night t e m p e r a t u r e s of 13 C or less dur ing meiosis induces ma le s t e r i l i t y (8). The prob lem of low t e m p e r a t u r e lesions has been s tud ied in tens ive ly by those i n t e r e s t e d in t he p o s t h a r v e s t s t o r a g e and handl ing of t he se chil l ing sens i t ive c o m m o d i t i e s , s ince low t e m p e r a t u r e is the most e f f ec t ive m e a n s of ex tend ing s t o r a g e l i fe . A n u m b e r of physiological p rob lems develop when ch i l l ing-sens i t ive spec ies expe r i ence e i the r a p r e - or p o s t h a r v e s t exposure to low t e m p e r a t u r e s . For example , t he fa i lure of t o m a t o e s to r ipen norma l ly a t low t e m p e r a t u r e (90), the su r f a c e p i t t i ng and a c c e l e r a t e d d e c a y of c u c u m b e r frui ts (21), d i sco lora t ion of l a t e x vessels and sub -ep ide rma l d i sco lora t ion in bananas (38), and many s imi lar a b e r a t i o n s a r e all c o m m o n o c c u r r e n c e s following a chil l ing t r e a t m e n t . In addi t ion many of the t e m p e r a t e f rui ts display undes i rab le s y m p t o m s when held a t low t e m p e r a t u r e s . Apple scald (superficial sca ld) , a brown d i sco lora t ion giving the skin a cooked a p p e a r a n c e occur s in some v a r i e t i e s upon l ong - t e rm s t o r a g e a t 0-4 C (123). Low t e m p e r a t u r e b reakdown of apples , also caused by s t o r a g e a t such t e m p e r a t u r e s , occur s in t he c o r t e x and in c e r t a i n v a r i e t i e s involves most of t h e flesh (125).

Ano the r f e a t u r e of t he chil l ing p h e n o m e n a is i t s r eve r s ib i l i ty following s h o r t - t e r m exposure to a chil l ing t r e a t m e n t . The physiological dysfunct ion resu l t ing from t h e molecu la r changes -induced a t low t e m p e r a t u r e can be r e v e r s e d (or repa i red) if t he t i s sue is r e t u r n e d to non-chil l ing t e m p e r a t u r e s be fo re a c t u a l injury occu r s (12, 50). This r eve r s ib i l i t y can be accompl i shed e x p e r i m e n t a l l y by a l t e r n a t i n g low/high t e m p e r a t u r e s over some shor t t i m e cyc le (116) but it also r e f l e c t s field condi t ions whe re , in the highland reg ions of the t rop ics for e x a m p l e , e ach cold night is fol lowed by a warm day (see Vallejos, th is vo lume) . Thus, while physiological dysfunct ion is i n i t i a t e d below some c r i t i c a l t e m p e r a t u r e , it does no t l ead to visible m a n i f e s t a t i o n of injury or e f f ec t s on p a r a m e t e r s such as g rowth r a t e and deve lopmen t b e c a u s e t he dysfunct ion has been r e v e r s e d be fo re it could b e c o m e p e r s i s t e n t .

Condi t ioning or harden ing chil l ing sens i t ive t i ssues by exposure to t e m p e r a t u r e s jus t s l ight ly above the chil l ing r a n g e for some per iod of t i m e be fo re p lac ing them a t an injurious t e m p e r a t u r e can p r e p a r e them to w i th s t and low t e m p e r a t u r e s for a somewha t longer per iod before showing visible s y m p t o m s (3). This condi t ioning also induces changes in compos i t ion of m e m b r a n e lipids (116, 118).

This brief r ev iew p r e s e n t s some of t h e phenomenology a s soc i a t ed wi th the chil l ing response and inc ludes a number of physiological even t s which have been s tud ied in an endeavor to unde r s t and the even t s leading to t he obse rva t ions . For the sake of c l a r i ty , t he following def in i t ions will be used:


chilling - t h e exposure of p lan t m a t e r i a l to some low t e m p e r a t u r e (above f reez ing) .

chilling injury - a d a m a g e to p lan t t i s sues , ce l l s , or organs which r e su l t s from the imposi t ion of low t e m p e r a t u r e (i .e. , chil l ing, or a chi l l ing t r e a t m e n t ) for a per iod of t i m e suff ic ient to cause p e r m a n e n t or i r r eve r s ib le d a m a g e .

dysfunction - a d i sordered or impa i red funct ion in response to t e m p e r a t u r e which, if a l lowed to occur for sufficient time, will cause an injury.

primary response to temperature - t h e p r i m a r y even t in t h e cel l t h a t causes some dysfunct ion which, in t i m e , leads to injury.

The purpose of this overview is to discuss m e c h a n i s m s and ev idence which focus on the primary response to temperature in chill ing sens i t ive p lan t spec ies . As the r e a d e r will d i scover , t h e r e is a bias in this overview toward reduc ing the poss ibi l i t ies ava i lab le to a physical e f fec t e i t he r on a p ro te in , on the ce l lu lar m e m b r a n e s , or on the i n t e r a c t i o n of t h e two . The causes of chilling injury a r e t he many sequen t ia l even t s which resu l t from a physiological or b iochemica l dysfunction occur r ing as a resu l t of chilling over some t i m e per iod .

B. Freezing Injury

While a d i s t inc t ion b e t w e e n chill ing sens i t ive and r e s i s t a n t spec ies can be made , chil l ing r e s i s t a n t spec ies can be fur ther d is t inguished on the basis of the i r r e s i s t a n c e to f reez ing t e m p e r a t u r e s . F r e e z i n g injury may be incur red over a b road spec t rum of sub-ze ro t e m p e r a t u r e s depending on the spec ies in ques t ion and t h e s t a g e of a c c l i m a t i o n . In a non-a c c l i m a t e d condi t ion, most p l an t s will be kil led by f reez ing to -1 to -3 C. In t he a c c l i m a t e d condi t ion, some spec ies will only w i th s t and f reez ing to t e m p e r a t u r e s s l ightly below the f reez ing point while o t h e r s will w i th s t and f reez ing to -196 C. Within this s p e c t r u m , examples of i n t e r m e d i a t e deg rees of f reez ing r e s i s t a n c e a r e read i ly d o c u m e n t e d . Al though t h e r e is e x t r e m e d ivers i ty in t h e r ange of f reez ing t e m p e r a t u r e s which can be t o l e r a t e d , t h e r e is however one common f e a t u r e of f reezing r e s i s t a n c e .

M e m b r a n e d a m a g e is a universa l man i f e s t a t i on of f reez ing in biological sy s t ems and is commonly in fe r red to be the p r i m a r y cause of injury. The f laccid, w a t e r - s o a k e d a p p e a r a n c e of var ious p lan t t i ssues and organs following thawing s t rongly sugges t s t ha t exposure to l e tha l low t e m p e r a t u r e s r e su l t s in m e m b r a n e dis rupt ion. The rap id i ty with which injury is man i fe s t ed fur ther sugges ts .that injury is no t p r imar i ly due to me tabo l i c dysfunct ion (as in chill ing injury). Addi t ional ly , nea r ly all of the me thods used to e v a l u a t e t i ssue viabi l i ty following a f r e e z e - t h a w cycle (plasmolysis and deplasmolys is , v i ta l s ta in ing , and so lu te leakage) a r e based on the r e t e n t i o n of i n t a c t ce l lu lar m e m b r a n e s (102) even the m e t a b o l i c r educ t ion of t r iphenyl t e t r a z o l i u m chlor ide (104). Desp i t e the overwhelming c i r c u m s t a n t i a l ev idence to w a r r a n t the conclusion tha t


m e m b r a n e d a m a g e is t h e p r i m a r y cause of injury, L e v i t t and D e a r (46) have emphas i zed t h a t . . c a t e g o r i c a l proof of d i r ec t injury to t he p l a s m a m e m b r a n e during e x t r a c e l l u l a r f reez ing is st i l l lacking."

An unders tand ing of wha t c o n s t i t u t e s f reez ing injury a t t he ce l lu lar and molecu la r levels is i m p o r t a n t for the fo rmula t ion of m e c h a n i s m s of f reez ing injury. Most hypo theses r ega rd ing t h e mechan i sm of f reez ing injury h a v e evolved from the analys is of the phys i co -chemica l even t s occur r ing during f reez ing and only p ro jec t ions of p o t e n t i a l m e m b r a n e d a m a g e have been provided [see Mazur (57, 58, 59, 69); Meryman (65); Meryman et al. (66) for r e v i e w s ] . Only r e c e n t l y has a t t e n t i o n been focused on wha t c o n s t i t u t e s f reez ing d a m a g e to ce l lu lar m e m b r a n e s [for r ev i ews see Steponkus et al. (107), Steponkus (103, 104, 105), Steponkus and Weist (106)] . Of u t m o s t conce rn is t he d e m o n s t r a t i o n of a m e m b r a n e lesion r e su l t ing from a f r e e z e - t h a w cyc le which q u a n t i t a t i v e l y a c c o u n t s for t he observed ce l lu la r injury.

As ce l lu la r m e m b r a n e s a r e in fe r red to be t h e p r i m a r y s i t e of f reez ing injury, cold a c c l i m a t i o n must involve ce l lu lar a l t e r a t i o n s which allow t h e m e m b r a n e s to survive subzero t e m p e r a t u r e s . Such a l t e r a t i o n s may be in t he ce l lu lar env i ronmen t or in the m e m b r a n e s per se. A l t e r a t ions in t h e ce l lu la r env i ronmen t may a l t e r t h e s t r e s se s t h a t occur dur ing f reez ing or may resu l t in d i r ec t p r o t e c t i o n of the m e m b r a n e . Both poss ibi l i t ies w e r e iden t i f ied by ea r ly i nves t i ga to r s [see Chand le r (9)] and a r e sti l l v iable today . For i n s t a n c e , Heber and Santa r ius (31) consider t h a t one componen t of cold a c c l i m a t i o n involves the fo rmat ion of p r o t e c t i v e compounds which can resu l t in p r o t e c t i o n through nonspeci f ic co l l iga t ive di lut ion of tox ic compounds . A l t e rna t i ve ly , c r y o p r o t e c t i v e p ro t e in s m a y confer p r o t e c t i o n in a non-co l l iga t ive manner (29). Olien (72, 73) has sugges ted t h a t the d e g r e e of hard iness depends on a rabo-xy lan po lymers which modify t h e f reez ing s t r e s s e s . A l t e r a t i o n s in the m e m b r a n e may inf luence the s t r e s s e s r e su l t ing from f reez ing or may r ende r t h e m e m b r a n e m o r e t o l e r a n t of t he f reez ing s t r e s s e s . As ea r ly as 1936, Sca r th and Lev i t t (93) proposed t ha t cold a c c l i m a t i o n could resu l t in an a l t e r a t i o n in m e m b r a n e w a t e r p e r m e a b i l i t y to p e r m i t the rap id r e m o v a l of w a t e r to ex t r ace l l u l a r s i t e s of i ce nuc l ea t i on . This p rov ided an exp lana t ion for t h e obse rva t ion t h a t i n t r ace l lu l a r ice fo rmat ion o c c u r r e d a t s lower f reez ing r a t e s in n o n a c c l i m a t e d t i ssues than in a c c l i m a t e d t i s sues . However , subsequent work by Stout et al. (110) i nd i ca t e s t h a t t h e r e may not be a d i r ec t cause and e f fec t r e l a t ionsh ip b e t w e e n the two obse rva t ions . Evidence t h a t cold a c c l i m a t i o n r e su l t s in changes in t h e t o l e r a n c e of t h e p l a sma m e m b r a n e has been provided by Sca r th et al. (94) and Siminovi tch and Lev i t t (98). C lea r ly , many a l t e r a t i o n s a r e involved in t he p roces s of cold a c c l i m a t i o n , and e luc ida t ion of t h e speci f ic physiological and b iochemica l a s p e c t s r equ i r e s an unde r s t and ing of the mechan i sm of f reez ing injury.

As ea r ly as 1912, Maximov (56) conc luded t h a t f reez ing d a m a g e was t h e resu l t of f r eeze - induced r e m o v a l of w a t e r from t h e su r face of t h e p l a sma m e m b r a n e . In 1940, Scar th et al. (94) addressed changes in

6 J. Μ. Lyons et ai

p la sma m e m b r a n e s t r u c t u r e in r e l a t i on to cold a c c l i m a t i o n and f reez ing injury. On the basis of severa l previous pub l ica t ions (47, 48 , 94), they i nd i ca t ed t ha t bo th i n t r ace l lu l a r and e x t r a c e l l u l a r i c e fo rma t ion r e su l t in d a m a g e speci f ica l ly to the p l a sma m e m b r a n e , a lbe i t for d i f fe ren t r ea sons . Moreover , while ex t r ace l l u l a r i ce fo rma t ion r e su l t s in ce l lu la r dehydra t ion and plasmolysis followed by deplasmolys is upon thawing , p lasmolysis per se was no t t he injurious even t . R a t h e r , r u p t u r e of t h e p l a s m a m e m b r a n e o c c u r r e d upon deplasmolys is . The s ign i f icance of t hese observa t ions w e r e , however , overshadowed by numerous incurs ions in to t h e cy top lasm to explain the m e c h a n i s m s of f reez ing injury and cold a c c l i m a t i o n . In 1970, Lev i t t and Dea r (46) ) i nd ica t ed t ha t a t t e n t i o n was again being d i r e c t e d to t he p l a sma m e m b r a n e , however , i n t e r e s t in m e m b r a n e s was s t i m u l a t e d by s tud ies of a l t e r a t i o n s in mi tochondr i a l (41, 42) and chloroplas t m e m b r a n e s (28, 30), r a t h e r than s tud ies of t h e p l a s m a m e m b r a n e d i r ec t ly . Numerous pape r s by Heber and coworke r s [see Heber and Santa r ius , (31)] provided a wea l th of in fo rmat ion on the e f f e c t s of f reez ing on the function of ch loroplas t thy lakoids . Subsequent ly , G a r b e r and Steponkus (23, 24) and coworke r s (105, 107) ex t ended this work providing in format ion on the r epe rcuss ions of f reez ing and cold a c c l i m a t i o n on thylakoid s t r u c t u r e and funct ion a t t h e molecu la r l eve l . To d a t e in format ion a t a c o m p a r a b l e level for the p l a sma m e m b r a n e awa i t s e luc ida t ion .

Π PROPAGATION OF THE TEMPERATURE RESPONSE

As desc r ibed above , a number of phys ica l , physiological and b iochemica l p h e n o m e n a have been closely c o r r e l a t e d wi th low t e m p e r a t u r e s t r e s s in p lan t spec ies . Each of t he se p h e n o m e n a will be examined wi th r e s p e c t to i n t e r p r e t a t i o n of the d a t a as it might r e l a t e to an unders tand ing and ass ignment of even t s a t t h e molecu la r l eve l .

Λ. Chilling Injury

1. Cytological Responses. An a r r ay of pa tho log ica l responses have been observed a t the e l e c t r o n mic roscope level when sens i t ive p lan t t i ssues h a v e been exposed to a chill ing t r e a t m e n t . For example , a p p a r e n t loss of cell tu rgor , vacuol iza t ion , r educ t ion in t h e a p p a r e n t volume of bo th t h e cy top lasm and the vacuole or p ro t e in bodies , a p p a r e n t deposi t ion of new m a t e r i a l in t he cel l wal ls , gene ra l d i sorgan iza t ion of o rgane l les , and a genera l loss of cy top l a smic s t r u c t u r e have been r e p o r t e d (36). G iaqu in ta and Ge ige r (25) showed t ha t an inc reased ves icu la t ion of t he s ieve tube cy top lasm, d isrupt ion of t h e s ieve tube r e t i c u l u m , and the a p p e a r a n c e of e x t r a n e o u s masses inside vacuoles o c c u r r e d in bean pe t io l e s a f t e r only 30 min a t 0 C. A r e c e n t s tudy on the t i m e - c o u r s e of t h e e f f ec t s of low t e m p e r a t u r e on ce l lu la r u l t r a s t r u c t u r e in t h e chil l ing sens i t ive t o m a t o (see I lker , e t a l , this volume)


d e m o n s t r a t e d t h a t m e m b r a n e a l t e r a t i o n s p r e c e d e d o t h e r ce l lu lar changes , and d i f fe ren t l eng ths of exposure w e r e r equ i r ed to d a m a g e d i f fe ren t types of o rgane l l e s . Surprisingly, t h e p l a s m a l e m m a appea red r e l a t i v e l y more r e s i s t a n t to chil l ing than the o the r m e m b r a n e s in these s tud ies . As l i t t l e as 2 hours of chil l ing a t 5 C induced observab le changes in u l t r a s t r u c t u r e in some but no t all ce l l s .

P a t t e r s o n , et al (this volume) also observed ves icu la t ion a t t h e l ight mic roscope level in living t r i c h o m e s of seve ra l chil l ing sens i t ive spec ies . These obse rva t ions c o r r e l a t e d wi th d i f fe rences observed in p ro top l a smic s t r e a m i n g . They showed t ha t t he r a t e of s t r e a m i n g a t 5 C was g r e a t e s t from e c o t y p e s of t he wild t o m a t o L. hirsutwn co l l e c t ed from the highest a l t i t u d e s , sugges t ing t h a t t h e r a t e s c o r r e l a t e d wi th gene t i c a d a p t a t i o n to the low t e m p e r a t u r e s expe r i enced a t h igher a l t i t udes (76). These wild t o m a t o spec ies n a t i v e to Equador and n o r t h e r n Pe ru have a d a p t e d to a wide r a n g e of t e m p e r a t u r e e x t r e m e s (88). These obse rva t ions p r e s e n t a fair ly c l ea r p i c t u r e of t he changes in ce l lu lar o rgan iza t ion in response to low t e m p e r a t u r e but do not afford the oppor tun i ty to d i f f e r e n t i a t e t he p r i m a r y t e m p e r a t u r e response and how it is p r o p a g a t e d in to t he obvious d i so rgan iza t ion t ha t occu r s .

2. The Plasma Membrane. Ano the r physiological even t observed as t h e resu l t of a chil l ing e x p e r i e n c e t ha t has focused a t t e n t i o n on m e m b r a n e i n t e g r i t y has been the loss of e l e c t r o l y t e s from t i ssue injured by low t e m p e r a t u r e . An enhanced loss of po tass ium from chil led s l ices of s w e e t p o t a t o r o o t s (50)^ c a r b o h y d r a t e and amino ac ids from the roo t s of c o t t o n seedl ings a t 5 C (11) of o t h e r e l e c t r o l y t e s from chi l led leaf t i ssues (12, 129) and seedl ings (114) has been observed . This loss of e l e c t r o l y t e from low t e m p e r a t u r e d a m a g e d t i ssues has been used in a t t e m p t s to q u a n t i t a t i v e l y m e a s u r e the e x t e n t of chil l ing injury. For example , in t h e a s se s smen t of chil l ing of c u c u m b e r l eaves (128) and as a m e a s u r e of d i f f e rences b e t w e e n Passiflora spec ies t o l e r a n t to a r a n g e of c l i m a c t i c env i ronmen t s (78). Each of these obse rva t ions can be c o r r e l a t e d to the phys ica l e f f ec t s of chil l ing t e m p e r a t u r e s on changes in m e m b r a n e p e r m e a b i l i t y and /o r m e m b r a n e i n t e g r i t y .

The p l a sma m e m b r a n e s t r u c t u r e and funct ion in response to low t e m p e r a t u r e is a key issue as it con t ro l s the u p t a k e or loss of var ious e l e c t r o l y t e s as desc r ibed above . For example , t h e efflux of rubidium has been observed for roo t s of ch i l l ing-sens i t ive spec ies below 10 C (131) and d i f f e rences in t he compos i t ion of bo th t he cel l wal ls and m e m b r a n e s of roo t cel ls of t he rmophy l i c p l an t s d i f fe red from non- the rmophy l i c p l an t s in the i r i on -exchange p r o p e r t i e s , espec ia l ly in the apica l reg ion of the roo t t ip (132). The p l a sma m e m b r a n e has also b e e n shown to play a key ro le in f reez ing injury of t e m p e r a t e spec ies (see Steponkus and Wiest , this vo lume, for r ev iew) . M e m b r a n e p e r m e a b i l i t y to w a t e r and non-e l e c t r o l y t e s can also be modif ied by C a (101). This e f fec t is sugges ted to be t h e resu l t of a binding of Ca to t h e polar head groups of t h e m e m b r a n e phospholipids which widen the d i s t a n c e b e t w e e n the phospholipid molecu les and al lows m o r e w a t e r molecu les to be l o c a t e d


b e t w e e n t h e hydrocarbon ends . It would be i n t e r e s t i n g to examine t he se Ca e f f ec t s on m e m b r a n e phospholipids in sens i t ive spec ies wi th esr and o the r t echn iques descr ibed in this vo lume .

3. Mitochondrial Membranes. The response of mi tochondr i a l m e m b r a n e s in chil l ing sens i t ive p lan t t i ssues during a low t e m p e r a t u r e t r e a t m e n t has been r e c e n t l y r ev i ewed by Raison (81). P l an t s t h a t a r e t o l e r a n t to low t e m p e r a t u r e s exhibi t a d e c r e a s e in r e s p i r a t o r y a c t i v i t y as a resu l t of t he genera l loss in k ine t i c energy and this r educ t ion is s imi lar to the d e c r e a s e in a c t i v i t y of o the r me tabo l i c r e a c t i o n s . Thus for most p l an t s , t he r e s p i r a t o r y ac t i v i t y a t low t e m p e r a t u r e s r e m a i n s in b a l a n c e wi th glycolysis and o the r closely a s soc i a t ed r e a c t i o n s and t h e r e is l i t t l e change in t he r e s p i r a t o r y quo t i en t . In c o n t r a s t , t he r e s p i r a t o r y a c t i v i t y of the chil l ing sens i t ive p lant spec ies d e c r e a s e s d i sp ropor t iona te ly when t h e t e m p e r a t u r e is lowered below about 10 C (120, 121). This d i spropor t iona l i ty has also been in fe r red from non- l inear Arrhenius p lo t s of r e s p i r a t o r y r a t e as a funct ion of t e m p e r a t u r e for bo th i n t a c t p lan t p a r t s and i so la ted mi tochondr i a (83). It has been sugges ted tha t a change in molecu la r order ing of t he m e m b r a n e lipids is t h e p r i m a r y even t in t he low t e m p e r a t u r e response of these chil l ing sens i t ive spec ies and t ha t this change in physical s t a t e a l t e r s t h e confo rma t ion and h e n c e a c t i v i t y of the r e s p i r a t o r y enzymes of the mi tochondr i a which leads to a d i sp ropor t iona te d e c r e a s e in t he r a t e of mi tochondr ia l r e sp i r a t i on r e l a t i v e to t he r a t e of glycolysis [cf., Raison (80), Lyons and Raison (54), Raison and C h a p m a n (83)] . The me tabo l i c imba l ances , and e f f ec t s on g rowth and deve lopmen t which occur subsequent to t h e a l t e r a t i o n in m e m b r a n e s t r u c t u r e and the even tua l i r r evers ib le loss of ce l lu la r i n t eg r i t y of the t i ssues ma in ta ined a t chil l ing t e m p e r a t u r e s , a r e v iewed by the se workers as secondary even t s dependen t on t he p r i m a r y s t r u c t u r a l change in t he m e m b r a n e .

In r e l a t e d e x p e r i m e n t s Dix and S t r ee t (19) showed t ha t t he Arrhenius plot for oxygen u p t a k e by mi tochondr i a e x t r a c t e d from a chil l ing t o l e r a n t l ine of Capsicum annuumexhibited a cont inuous l inear funct ion wi th t e m p e r a t u r e s b e t w e e n 0 and 30 C, unlike t h a t for u n s e l e c t e d l ines which showed a d iscont inui ty around 10 C typ ica l of ch i l l ing-sens i t ive m a t e r i a l s . While it r e m a i n s ques t ionab le as to the abi l i ty to r e g e n e r a t e such cell l ines, it appea r s as though the p o t e n t i a l is suff ic ient to w a r r a n t fur ther explora t ion wi th these t echn iques . (For addi t iona l discussion see Dix, this volume) .

4. Chloroplast Membranes. One of t he ea r ly responses observed in chi l l ing-sens i t ive p lan t s growing under field condi t ions when exposed to low t e m p e r a t u r e is a d e c r e a s e in p h o t o s y n t h e t i c a c t i v i t y (14, 111). A number of s tud ies have been u n d e r t a k e n to examine the i m p a c t of low t e m p e r a t u r e on chloroplas t funct ion and pa r t i cu l a r l y m e m b r a n e r e l a t e d r e sponses . For example , an Arrhenius plot of the pho to reduc t ion of NADP from w a t e r by ch lorop las t s i so la ted from t o m a t o and bean shows a m a r k e d d iscont inu i ty a t about 12 C whe rea s a s imi lar plot for chill ing


r e s i s t a n t l e t t u c e and p e a shows a cons t an t s lope over t he t e m p e r a t u r e r a n g e from n e a r 0 to 25 C (97). A dev ia t ion from this c o r r e l a t i o n was observed by Nolan and Smill ie (70). They observed t h a t t h e uncoupler s t i m u l a t e d pho to r educ t i on of 2 ,6-dichlorophenolendophenol by ch lorop las t s shows an i n c r e a s e in Ea below about 12 C with both chil l ing sens i t ive and chil l ing t o l e r a n t p l a n t s . These d a t a ques t ion t h e n e c e s s i t y of pos tu l a t ing t h e e x i s t e n c e of m e m b r a n e changes below 10 C - 1 2 C t o explain inhibi t ion of g rowth a t chil l ing t e m p e r a t u r e s (Smillie, this volume) . Studies on t h e molecu la r a r c h i t e c t u r e of thylakoid m e m b r a n e s of a n u m b e r of p l a n t s in r esponse to t e m p e r a t u r e a l t e r a t i o n s has been r e c e n t l y r e v i e w e d (82). These d a t a i nd i ca t e t h e r e is a co r r e l a t i on b e t w e e n a phys ica l change in t h e m e m b r a n e sys tem in response to low t e m p e r a t u r e and changes in t he physiological r e sponses of i n t a c t t i s sues .

B. Freezing Injury

1. The Freezing Process. The phys i co -chemica l e v e n t s which occur dur ing the f reez ing of a cel l suspension and the r ed i s t r ibu t ion of w a t e r wi th r e s p e c t to bo th i t s physica l s t a t e and loca t ion have been d e t a i l e d by Mazur (57, 58, 59, 60). Briefly when cooled below 0 C, t h e cel ls and the surrounding medium ini t ia l ly r e m a i n unfrozen due to depress ion of t h e f reez ing point by t h e so lu tes p r e s e n t and, to some e x t e n t , due to supercool ing . Ice nuc l ea t i on will in i t ia l ly occur in the e x t r a c e l l u l a r solut ion b e t w e e n -2 and - 1 5 C, depending on the so lu te c o n c e n t r a t i o n and e x t e n t of supercool ing . The in t r ace l lu l a r solut ion r e m a i n s unf rozen and supercooled , p re sumab ly b e c a u s e t he p l a sma m e m b r a n e p r e v e n t s the g rowth of i ce c rys t a l s in to the ce l l . This r e su l t s in a disequi l ibr ium in t he chemica l p o t e n t i a l of w a t e r in t he in t r ace l lu l a r solut ion r e l a t i v e to the chemica l p o t e n t i a l of the pa r t i a l l y f rozen e x t r a c e l l u l a r solut ion. The lower chemica l p o t e n t i a l of w a t e r molecu les in i c e is a d i r ec t funct ion of t e m p e r a t u r e .

Equil ibrium may be ach ieved e i t he r by ce l lu lar dehydra t ion and con t inued ex t r ace l l u l a r ice fo rma t ion or by in t r ace l lu l a r ice fo rma t ion . The manner of equi l ibra t ion is inf luenced by t h e r a t e a t which the cel l is cooled and the min imum t e m p e r a t u r e ach ieved r e l a t i v e to the c a p a c i t y for w a t e r flux out of t he ce l l . The amount of w a t e r which must be r e m o v e d to ach i eve equi l ibr ium by ce l lu la r dehydra t ion will depend on t h e in i t ia l osmola l i ty of t h e i n t r ace l l u l a r solut ion and the minimum t e m p e r a t u r e to which t h e cell suspension is cooled . Whether th is amoun t of w a t e r will be r e m o v e d depends p r imar i ly on the p e r m e a b i l i t y of the p l a sma m e m b r a n e and the su r f ace a r e a ava i lab le for efflux r e l a t i v e to t h e cel l vo lume in r e l a t i o n to t h e cooling r a t e and t e m p e r a t u r e . These f a c t o r s i n t e r a c t to d e t e r m i n e the magn i tude of disequi l ibr ium t h a t will r esu l t a t any s t a t e in t h e disequil ibr ium con t inuum. This disequi l ibr ium is m a n i f e s t e d as super -cool ing of t h e i n t r ace l l u l a r solut ion, and t h e p robab i l i ty of i n t r ace l l u l a r i c e fo rma t ion b e c o m e s g r e a t e r as t he e x t e n t of supercool ing i n c r e a s e s . In o the r words ,

ΙΟ J. Μ. Lyons et al.

if w a t e r flux is a d e q u a t e r e l a t i v e to t he cooling r a t e , excess ive supercool ing of the in t r ace l lu l a r solut ion will be p rec luded and the cel l will d e h y d r a t e and i ce fo rmat ion will only occur ex t r ace l l u l a r l y . A l t e rna t i ve ly , if w a t e r flux is not a d e q u a t e , h e a t t r ans fe r m e c h a n i s m s will d o m i n a t e over mass t r ans fe r mechan i sms , and excess ive supercool ing of the in t r ace l lu l a r solut ion will r e su l t in i n t r ace l l u l a r i ce fo rma t ion .

The manne r of equi l ibra t ion is of cons iderab le conce rn b e c a u s e in t r ace l lu l a r i ce fo rmat ion is genera l ly cons idered to be l e tha l , a l though t h e r e a r e some r e p o r t e d excep t ions [see Mazur (59)] . Ex t r ace l l u l a r i ce fo rma t ion and a t t e n d a n t cel l dehydra t ion may or may not be l e tha l , depending on o the r f a c t o r s . The manne r in which e x t r a c e l l u l a r i ce fo rma t ion causes ce l lu lar injury, pa r t i cu l a r l y to the p l a s m a m e m b r a n e , is of fo remos t conce rn to t h e field of cryobiology.

2. Repercussions of Freezing on the Cellular Environment. The repercuss ions of t h e f reez ing p rocess on the ce l lu lar env i ronmen t of the cel l and i t s componen t s a r e n u m e r o u s . These changes inc lude the obvious d e c r e a s e in t e m p e r a t u r e , t he p r e s e n c e of i ce c rys t a l s , and dehydra t ion of the ce l l . There is a gene ra l concensus t h a t , under condi t ions of e x t r a c e l l u l a r i c e fo rma t ion , d e c r e a s e s in t e m p e r a t u r e or t h e p r e s e n c e of i ce c rys t a l s per se a r e not t h e p r i m a r y causes of f reez ing injury (31) and t ha t t he p rocess of ce l lu lar dehydra t ion is t he most d i s rupt ive and injurious repercuss ion of t he f reez ing p rocess (58). There a r e , however , severa l consequences of dehydra t ion which inc lude: a r educ t ion in cell volume and su r f ace a r e a , an i n c r e a s e in c o n c e n t r a t i o n of so lu tes , p r ec ip i t a t i on of some buffer ing sa l t s r e s u l t ing in pH changes , and r emova l of w a t e r of hydra t ion of m a c r o m o l e -cu les . Mazur (57, 58) r e f e r s to these as 'solution e f f ec t s ' and no t e s t ha t all occur as a monoton ic funct ion of t e m p e r a t u r e .

It is within this a r r a y t ha t t h e r e is a g r e a t d ive rgence in hypo theses on the mechanism of f reez ing d a m a g e . All of the r epe rcuss ions , e i the r s ingularly or in var ious combina t ions , have se rved as t he basis for mechan i sms of f reez ing d a m a g e [see Mazur (57, 58, 59. 60), Heber and Santa r ius (31), Meryman (65), Meryman et ol. (66) for rev iews] . Some of t hese hypo theses have fai led to p rov ide an a d e q u a t e exp lana t ion : for i n s t ance , t h e sulfhydryl hypothes is of L e v i t t (45) has been gradual ly diminished [see Mazur (57, 58) and Heber and San ta r ius (31) for cr i t ic isms] O t h e r s r ema in pa r t i a l ly a t t r a c t i v e (52, 64), but fail to explain all of t h e obse rva t ions [see Mazur (60)] . The very a t t e m p t to explain all of t h e man i f e s t a t i ons of f reez ing injury may be the major shor t coming of e ach .

Since f reez ing r e su l t s in a m u l t i t u d e of s t r e s s e s , it is r easonab le to assume tha t the overal l mechanism of f reez ing injury is a compos i t e of t he many hypo theses put for th and they should not be cons idered as mutua l ly exc lus ive . It might be more app rop r i a t e to view the f reez ing p rocess as a sequen t ia l se r ies of po t en t i a l l y l e tha l s t r e s s b a r r i e r s . Thus, any one single f reez ing s t r e s s is a po ten t i a l l y injurious fac tor—depending on the i m m e d i a t e condi t ions and the successful su rmount ing of previously e n c o u n t e r e d s t r e s s b a r r i e r s .

T h e P l a n t M e m b r a n e in R e s p o n s e to L o w T e m p e r a t u r e

In summary,the phenomenology involved in chill ing injury and f reez ing s t r e s s has been br ief ly desc r ibed . It is now app rop r i a t e to e x a m i n e ev idence which might e l u c i d a t e t he p r i m a r y t e m p e r a t u r e sensor . In addi t ion , we will a t t e m p t to def ine a r e a s where fu ture e x p e r i m e n t a t i o n should focus, and to def ine c e r t a i n t e rmino logy which should be used in descr ib ing p h e n o m e n a a s soc i a t ed wi th low t e m p e r a t u r e s and m e m b r a n e lipid o rde r .

ΠΙ. PRIMARY TEMPERATURE SENSOR

A. Membrane Fluidity and Chilling Injury

Over t h e pas t t en y e a r s or so t h e r e have been a n u m b e r of r e p o r t s t ha t have r e l a t e d physiological even t s in low t e m p e r a t u r e s t r e s s in p l an t s to m e m b r a n e "phenomonology." A p a r t of this s t imulus was der ived from the obse rva t ion tha t Arrhenius p lo t s of oxygen u p t a k e by mi tochondr i a i so la ted from chil l ing sens i t ive p lan t t i ssues d e m o n s t r a t e d a non- l inear t e m p e r a t u r e dependency or "break" a t the t e m p e r a t u r e c r i t i c a l for chil l ing injury in t he i n t a c t p lan t (54). F u r t h e r m o r e , app l ica t ion of esr t echn iques provided a physica l me thod which showed t ha t the i n t a c t m e m b r a n e s and t h e lipid c o m p o n e n t s of t h e m e m b r a n e s of t hese mi tochondr i a unde rwen t a change in molecu la r order a t the s ame t e m p e r a t u r e as t h a t of t h e "break" in t h e Arrhen ius p lo t for oxygen u p t a k e (85). These r e su l t s sugges ted t ha t the t e m p e r a t u r e - i n d u c e d change in t e m p e r a t u r e - s e n s i t i v e m e m b r a n e s is an in t r ins ic p r o p e r t y of the m e m b r a n e lipid and a d i r e c t c o r r e l a t i o n b e t w e e n the phys ica l s t a t e of m e m b r a n e componen t and e n z y m e a c t i v i t y was in fe r red . These obse rva t ions es tab l i shed physiological dependency on m e m b r a n e s t r u c t u r e and led to a r t i c u l a t i o n of a hypothes i s to explain t he mechan i sm of the p r i m a r y t e m p e r a t u r e sensor leading to chil l ing injury in sens i t ive p l a n t s — t h i s model was based on t h e assumpt ion t h a t "cel lular m e m b r a n e s in sens i t ive p l an t s undergo a phys ica l -phase t r ans i t i on from a no rma l f lexible l iqu id-c rys ta l l ine to a solid gel s t r u c t u r e a t t he t e m p e r a t u r e c r i t i c a l for chil l ing injury" (53, 85). The change in molecu la r order ing of m e m b r a n e lipids a t about 10-1Ζ C in sens i t ive spec ies was v iewed as the p r i m a r y response to low t e m p e r a t u r e s and u l t i m a t e dysfunct ion and injury was t h e n e t resu l t of a se r ies of subsequent sequen t ia l e v e n t s .

This model , based on the molecu la r s t r u c t u r e of m e m b r a n e s y s t e m s , was a r e f i n e m e n t of the h i s to r i ca l belief t ha t "sol idif icat ion of the p ro top l a smic lipids" could accoun t for t h e observed d e a t h or injury of sens i t ive p l an t s a t low t e m p e r a t u r e s (110). R e f e r e n c e to a phase t r ans i t i on from "a l iqu id-c rys ta l l ine s t r u c t u r e to a solid gel" in t he model was der ived from the s tud ies of L u z z a t i and Husson (55) employing model lipid s y s t e m s . Appl ica t ion of the i r t e rmino logy to complex biological m e m b r a n e s from higher p l an t s was , in r e t r o s p e c t , an overs impl i f ica t ion t h a t has led to con t rove r sy , but it p rov ided a c e r t a i n u t i l i ty a t t he t i m e .

1 1

1 2 J. Μ. Lyons et ai

1. Characterization of Fluidity Parameters. A n u m b e r of p o w e r ful t echn iques - - esr , nmr , f luo rescence , X-ray s c a t t e r i n g , dsc —are ava i lab le to d iscern physical p h e n o m e n a in biological m e m b r a n e s (2). The task be fo re us is to a t t e m p t to c o r r e l a t e t hese d i f fe ren t t echn iques and d i f fe ren t r e su l t s in to a common p i c t u r e of molecu la r e v e n t s in m e m b r a n e s y s t e m s of p l an t s sens i t ive to low t e m p e r a t u r e s and to develop a common language t ha t will r e d u c e a c e r t a i n amoun t of confusion t ha t ex is t s in t he l i t e r a t u r e .

For example , t h e esr d a t a from t h e f irs t s tud ies on m e m b r a n e s der ived from chil l ing sens i t ive p lan t s we re used to infer "mel t s" in the hydrocarbon zones of phospholipids (85). The abrup t , t e m p e r a t u r e -induced a l t e r a t i o n s in the molecu la r o rder ing of m e m b r a n e lipids d i sce rned by esr w e r e r e f e r r e d to as m e m b r a n e "phase changes ." Appl ica t ion of these t echn iques to the chil l ing sens i t ive mung bean showed t ha t two changes in molecu la r order ing of t h e m e m b r a n e linids of mi tochondr ia and ch loroplas t s could be d e t e c t e d , one a t about 28 C and one a t about 15 C (83). The t e rmino logy deve loped from s tud ies of b inary mix tu res of phospholipids and m e m b r a n e s of E. coli was a d o p t ed, des igna t ing the change in physical s t a t e of t he m e m b r a n e lipids observed a t the lower t e m p e r a t u r e as Τ and a t t he h igher t e m p e r a t u r e T^ (95). Hence , t h e changes occur r ing in t he m e m b r a n e s of mung bean t i ssue we re v iewed as a change from a fluid to a m i x t u r e of fluid + gel below 28 C (T f) and from a fluid + gel to a p r edominan t ly gel phase below 15

WC (T ) and Τ and T f , we re cons idered t h e t e m p e r a t u r e for t he

in i t ia t ion and t e r m i n a t i o n of a t h e r m a l phase t r ans i t ion in the m e m b r a n e l ipids.

F u r t h e r s tud ies on mi tochondr i a e x t r a c t e d from whea t also ind ica t ed two phase changes , one a t about 0 C and a t about 30 C (84). Again, these changes w e r e i n t e r p r e t e d t o show tha t the m e m b r a n e lipids undergo a mel t ing t r ans i t ion , and t ha t within t he t e m p e r a t u r e r ange of growth for w h e a t , the m e m b r a n e lipids we re in a mixed l iquid-c rys ta l l ine , gel form. Much of our knowledge about t he physical s t a t e of biological m e m b r a n e s is der ived from ex t r apo la t i on of r e su l t s ob ta ined wi th mix tu re s of syn the t i c or n a t u r a l phospholipids d ispersed in w a t e r and from the observa t ion t ha t phospholipid b i layers undergo a r eve r s ib l e , t h e r m o t r o p i c ge l - l iqu id-c rys ta l l ine phase t r ans i t ion which is der ived from a coope ra t i ve mel t ing of the hydrocarbon chains in the in t e r io r of t he b i layer . These physical t r a n s f o r m a t i o n s in phospholipid b i layers have been c o r r e l a t e d to g rowth of Acholeplasma laidlawii whe re it was r e p o r t e d t ha t both the abso lu te r a t e s and the t e m p e r a t u r e coef f ien t s of cell g rowth w e r e s imi lar in cel ls whose m e m b r a n e lipids ex i s ted en t i r e ly in t he l iqu id-crys ta l l ine s t a t e , but the i r abso lu te g rowth r a t e s dec l ined rapidly and t e m p e r a t u r e coef f i c ien t s i nc r ea sed when most of t h e m e m b r a n e lipids b e c a m e solidified (61). A subsequent s tudy using d i f fe ren t ia l t h e r m a l analysis did not suppor t t h e obse rva t ions wi th e l e c t r o n spin r e s o n a n c e spec t roscopy t h a t t he minimum and max imum growth t e m p e r a t u r e s of t he organism might be d i r ec t ly d e t e r m i n e d by the solid/f luid phase t r ans i t ion boundar ies of t h e m e m b r a n e lipids (62).

The Plant Membrane in Response to Low Temperature 1 3

Their new ev idence sugges t ed t h a t t h e e l e c t r o n spin r e s o n a n c e t echn iques did no t d e t e c t the gel to l iqu id-c rys ta l l ine phase t r ans i t ion of t h e bulk m e m b r a n e lipids as could be d i sce rned by d i f fe ren t i a l t h e r m a l ana lys is . Severa l s tud ies h a v e shown t h a t a t l eas t half the lipids in the m e m b r a n e phospholipids of E. coli and A. laidlawii mus t be in t h e d i sordered s t a t e to allow for no rma l g rowth but E. coli is able to grow with a lmos t all of i t s lipid in t he d i so rdered s t a t e which sugges t s t ha t t he b r e a d t h of t h e lipid phase t r ans i t i on is due to t h e d ive rs i ty of f a t t y acyl chains normal ly found (13). A r e c e n t r ev iew by Cronin (13) has shown t h a t a number of s tudies using f r e e z e - f r a c t u r e e l e c t r o n mic roscopy have been i n t e r p r e t e d in t e r m s of t h e s eg rega t i on of m e m b r a n e p ro te in in p a t c h e s of fluid or d i so rdered lipid. The smooth a r e a s d i sce rned a r e seen as p a t c h e s of o r d e r e d lipid and t h e a g g r e g a t e s as m e m b r a n e p ro t e in s t ha t have been exc luded from t h e o rde red lipid p a t c h e s in to p a t c h e s of fluid l ipids. Phys ica l i so la t ion and analysis of t h e d i f fe ren t reg ions has been ach ieved and shown t h a t t hese smoo th a r e a s have a s o m e w h a t higher p ropor t ion of s a t u r a t e d f a t t y ac ids than do t h e p a r t i c l e r i ch a r e a s [see Cronin (13) for r e v i e w s ] .

While i t has been t e m p t i n g to i n t e r p r e t e t h e desc r ip t i ve a s p e c t s of the physica l e v e n t s d e t e r m i n e d using m e m b r a n e p robes , cau t ion must be exe rc i sed in ex tend ing t h e r e su l t s ob ta ined with b i layers composed of b inary m i x t u r e s of phospholipids or wi th b a c t e r i a l m e m b r a n e s to the c o m p l e t e lipids in p lan t m e m b r a n e s .

As will be r e p o r t e d in subsequent c h a p t e r s in this vo lume (see Dalz ie l and Bre idenbach , Waring and G l a t z , Bishop, et al), i t is c l ea r t h a t t h e even t s observed wi th esr p robes do not r e f l e c t a p h a s e - t r a n s i t i o n from "a l iqu id-c rys ta l l ine to gel" t r a n s f o r m a t i o n or "mel t s" of t h e bulk lipids; but r a t h e r some change in molecu la r o rder ing of d i s c r e t e domains within t he m e m b r a n e .

F u r t h e r e luc ida t ion of t h e molecu la r a r c h i t e c t u r e of t h e m e m b r a n e and /o r m e m b r a n e componen t s in response to t e m p e r a t u r e is one of t h e key a r e a s t h a t should a t t r a c t r e s e a r c h emphas i s in t h e field of low t e m p e r a t u r e s t r e s s in p l a n t s .

2. Control of Molecular Ordering of Membrane Lipids. If t h e mo lecu la r o rder ing of m e m b r a n e l ipids in r esponse to t e m p e r a t u r e is c r i t i c a l in d e t e r m i n i n g the chil l ing r e sponse , then any of the f ac to r s a l t e r ing p r o p e r t i e s of t h e m e m b r a n e and the t r ans i t ion t e m p e r a t u r e must be under s tood . Because of the p rob lems a s soc i a t ed wi th loca l iz ing d i s c r e t e pools or a r e a s of speci f ic lipid compos i t ion within m e m b r a n e s , cons ide ra t ions based on analys is of bulk lipids e x t r a c t e d from p lan t t i ssues or e x t r a p o l a t i o n s from t h e o r e t i c a l cons ide ra t ion of p u r e compounds or model s y s t e m s (see Bishop et al. and Wil lemot , this volume) as an exp lana t ion of the con t ro l of m e m b r a n e f luidi ty by t e m p e r a t u r e mus t be t r e a t e d wi th cau t ion .

a. Acyl chain composition. Much of our knowledge on t h e ro le of f a t t y ac id compos i t ion in d e t e r m i n i n g t h e r m o t r o p h i c phase t r ans i t ions in m e m b r a n e s c o m e s from s tud ies wi th mic roo rgan i sms . For

1 4 J. Μ. Lyons et al.

example , McElhaney (61) showed t h a t g rowth of A. laidlawii in t h e p r e s e n c e of specif ic f a t t y acid supp lemen t s shif ted t h e m e m b r a n e phase t r ans i t ion to a t e m p e r a t u r e above t h e abso lu te min imum growth t e m p e r a t u r e and this shift was c lea r ly con t ro l l ed by the f a t t y ac id compos i t ion of t h e cell m e m b r a n e . F u r t h e r m o r e , p e r m e a t i o n r a t e s of n o n - e l e c t r o l y t e s pass ively diffusing in to s y n t h e t i c l iposomes , A. laidlawii ce l l s , or l iposomes der ived from A. laidlawii m e m b r a n e l ipids, w e r e s t rongly dependen t on t h e chemica l s t r u c t u r e and chain l eng th of the i r f a t t y ac ids (16, 63 , 89). Incorpora t ion of b r anched -cha in , m o r e u n s a t u r a t e d , or sho r t e r - cha in f a t t y ac ids , which i nc r ea se m e m b r a n e f luidi ty , all i nc reased n o n - e l e c t r o l y t e p e r m e a b i l i t y to a s imi lar e x t e n t in bo th cel ls and l iposomes . A c t i v e t r anspo r t of g lucose by A. laidlawii (86) and t h e β -ga lac tos ide and β-glucoside t r anspo r t s y s t e m s of E. coli (51, 71) have been c lea r ly shown to depend on t h e acyl chain compos i t ion of t h e m e m b r a n e l ipids. Ba ldassa re et al (4) have shown t h a t t h e p a r t i c u l a r f a t t y ac id spec ies p r e s e n t in t h e m e m b r a n e lipids of E. coli d e t e r m i n e t h e funct ion of severa l m e m b r a n e a s soc i a t ed e n z y m e s .

A l t e r a t i o n of t h e f a t t y acid compos i t ion of t h e m e m b r a n e s of a number of o rgan isms in o rder to ma in ta in m e m b r a n e f luidi ty a t d i f fe ren t g rowth t e m p e r a t u r e s has r e l e v a n c e to t h e problem of cold sens i t iv i ty . For example , P a t o n et al (75) have r e c e n t l y r e p o r t e d t h a t a d e c r e a s e in g rowth t e m p e r a t u r e of Bacillus amyloliquefaciens was a c c o m p a n i e d by an i nc rease in t h e r a t i o of b r a n c h e d - t o s t r a i g h t - c h a i n f a t t y ac ids and m a r k e d inc rease in t he level of unsa tu r a t i on of t h e b r a n c h e d - c h a i n f a t t y acids and they sugges ted a d i r ec t co r r e l a t i on b e t w e e n m e m b r a n e f luidity and the suscep t ib i l i ty to cold shock. Ano the r example of t h e in f luence of f a t t y ac id compos i t ion on m e m b r a n e f luidi ty is found in t h e se r ies of r e p o r t s on m e m b r a n e p rope r t i e s of Tetrahymena pyriformis during t e m p e r a t u r e a c c l i m a t i o n (40, 43). These s tud ies i nd i ca t ed t h a t t h e a c t i v i t y of m e m b r a n e a s soc i a t ed f a t t y ac id d e s a t u r a s e s w e r e con t ro l l ed by m e m b r a n e f luidi ty in response to low t e m p e r a t u r e r a t h e r than t e m p e r a t u r e per se. In addi t ion to t he r e l a t i v e amoun t of s a t u r a t i o n / u n s a t u r a t i o n and the b ranch ing of the acyl cha ins , t he chain length of t he f a t t y acyl mo ie t i e s of t h e phospholipids is also a major d e t e r m i n a n t in t he f luidity and phase t r ans i t ion of lipids [see rev iew by Cronin , (13)] .

M e m b r a n e phospholipid acyl chain compos i t ion can r e f l e c t in p a r t t h e composi t ion of t h e f a t t y acids ava i lab le to t h e o rgan i sm. Animals fed corn oil (68), mic roorgan i sms cu l tu r ed in t he p r e s e n c e of f a t t y ac ids (34), yeas t grown in medium conta in ing f a t t y ac ids (1), and p o t a t o tuber t i ssue s l ices i ncuba t ed in medium conta in ing f a t t y ac ids and f a t t y acid de r iva t ives (117) all show changes in t he f a t t y acyl chain compos i t ion of t h e o rgan isms phospholipids to a p p r o x i m a t e t h a t of t h e supp lemen t . F a t t y ac id compos i t ion of t he m e m b r a n e lipids can be a l t e r e d by means o the r than d i rec t supp l emen ta t i on . Pyr idaz inone de r iva t ives (108, 1Z4) have been r e p o r t e d to i n t e r f e r e wi th f a t t y ac id b iosynthes is and a l t e r t he acyl chain composi t ion of p lan t m e m b r a n e l ipids. Similar ly , t r e a t m e n t of t o m a t o seedl ings wi th e t h a n o l a m i n e - t w e e n - o l e a t e induced a l t e r e d


phospholipid compos i t ion and s o m e w h a t d iminished t h e s y m p t o m s of chilling (118).

Can the f a t t y acyl chain compos i t ion have any inf luence over m e m b r a n e f luidi ty in h igher p l an t s ? It is c lea r t ha t molecu la r o rder ing of m e m b r a n e lipids in some s y s t e m s can be a l t e r e d by va r i a t ions in f a t t y acyl compos i t ion . It r e m a i n s to be shown if a s imi lar s i tua t ion ex is t s in higher p l an t s , p a r t i c u l a r l y those sens i t ive to chil l ing t e m p e r a t u r e s .

b. Polar head group composition. The ro le of phospholipid polar head groups in r e g u l a t i n g t he f luidi ty of m e m b r a n e s of h igher p l an t s has not been a s c e r t a i n e d . It has been shown t h a t t h e r e is m o r e than a 30 C d i f f e rence in t h e t e m p e r a t u r e of t he t r ans i t ion from the gel to l iqu id-c rys ta l l ine s t a t e for aqueous dispers ions of pure phosphat idy lchol ine and phospha t idy l e thano lamine having the s a m e s a t u r a t e d acyl chain compos i t ion (13). That the polar head group of phospholipids can e x e r t an inf luence on t h e a c t i v i t y of a s soc i a t ed e n z y m e s is c l ea r (69, 115) but the r e l a t i v e i m p o r t a n c e in r e l a t i o n to o the r f ac to r s is ye t to be d e t e r m i n e d in r e l a t i o n to t h e chil l ing r e sponse .

C. Sterol composition. In model m e m b r a n e s y s t e m s , adding cho les t e ro l to t h e phospholipids a l t e r s the i r molecu la r pack ing caus ing them to be m o r e condensed above and m o r e fluid below the i r t r ans i t ion t e m p e r a t u r e [ s e e rev iew by D e m e l and De Kruyff, (18)] . Because of those unique p r o p e r t i e s it has been proposed t h a t cho le s t e ro l and o the r s t e ro l s such as those common in p l an t s may funct ion as r e g u l a t o r s of m e m b r a n e f luidi ty in e u c a r y o t e s and in p r o c a r y o t e s t h a t have s t e ro l s in the i r m e m b r a n e s (61). Cho les t e ro l c o n t e n t has been shown to in f luence t h e p h a s e - t r a n s i t i o n t e m p e r a t u r e for m e m b r a n e - a s s o c i a t e d e n z y m e s (17, 63 , 91). These r e p o r t s i nd i ca t e t h a t f a c t o r s o the r than phospholipids and the i r acyl chain compos i t ion can be involved in m e m b r a n e phase t r ans i t ions and by i n f e r ence could be involved in chil l ing sens i t iv i ty .

It is of i n t e r e s t to n o t e , however , t h e a lmos t p rec i se co r r e l a t i on t h a t ex is t s in the t e m p e r a t u r e a t which d i scon t inu i t i e s in Arrhenius p lo t s of oxygen u p t a k e , in t he t e m p e r a t u r e coef f ic ien t of spin label mot ions wi th i n t a c t m e m b r a n e s , and wi th ves ic les of phospholipids e x t r a c t e d from those m e m b r a n e s f ree of s t e ro l s (and p ro t e in s for t ha t m a t t e r ) . F u r t h e r m o r e , s ince the phospholipid ves ic les a r e also f ree of p r o t e i n s , t h e r e is s t rong ind ica t ion t h a t t h e lipids a r e t he c o m p o n e n t s sensing t h e t e m p e r a t u r e change .

d. Membrane protein. The in t r ins ic p ro t e in s of m e m b r a n e s do not appear to inf luence the t e m p e r a t u r e a t which the m e m b r a n e lipids undergo a change in molecu la r o rder ing which would sugges t only a smal l p ropor t ion of the m e m b r a n e lipids a r e involved in hydrophobic i n t e r a c t i o n s with p ro te in (80). This view is suppor ted by t h e fac t t ha t t he t e m p e r a t u r e of the phase change in mi tochondr i a l m e m b r a n e s is t he s a m e r ega rd l e s s of w h e t h e r t h e p ro t e in is h e a t d e n a t u r e d or in a n a t i v e s t a t e and coincides wi th the t e m p e r a t u r e of the phase change in e x t r a c t e d phospholipids . F u r t h e r m o r e , a compar i son of t he h e a t of


t r ans i t ion for t he lipids of M. laidlawii m e m b r a n e s wi th t h a t for t he e x t r a c t e d lipids ind ica t e s t ha t 90% of the m e m b r a n e lipids a r e in a b i layer conf igura t ion and only 10% a r e involved in hydrophobic i n t e r a c t i o n s , p resumably wi th p ro t e in (87). Sharp b reaks in the t e m p e r a t u r e prof i les of a c t i v i t i e s of many e n z y m e s coincides qu i t e closely wi th the phase t rans i t ion d e t e c t e d in the i so la ted l ipids, implying t h a t t he m e m b r a n e phase t r ans i t ion is u n a f f e c t e d by the p r e s e n c e of t h e p ro t e in (71). Studies wi th ATPase a c t i v i t y however have shown t h a t the annulus (a layer of about 30 lipid molecu les i m m e d i a t e l y surrounding t h e e n z y m e prote ins) e x e r t s the p r edominan t e f fec t and sugges t s t ha t the f luidity g rad ien t and lipid chains in t h e i m m e d i a t e v ic in i ty of t h e p ro t e in may be subs tan t i a l ly less than the g rad ien t observed in the bulk m e m b r a n e (119).

e. Chemical perturbers. In addi t ion to chemica l t r e a t m e n t s d i r e c t e d toward modifying m e m b r a n e phospholipids and acyl chain compos i t ion per se, some chemica l s h a v e been shown to d i r ec t l y p e r t u r b the m e m b r a n e and main ta in f luidity wi thout chemica l modi f ica t ion of m e m b r a n e componen t s . For example , bu ty l a t edhyd roxy to luene (BHT), because of i t s high lipid solubil i ty and e x t r e m e l y low aqueous solubi l i ty r e su l t s in a s t rong i n t e r a c t i o n wi th t h e hydrocarbon zones of m e m b r a n e s and p e r t u r b s m e m b r a n e - a s s o c i a t e d even t s (22, 100). Similar ly, a d a m a n t a n e , ano the r l ipophylic molecu le shif ts t he m e m b r a n e phase change in yeas t cel ls and as a consequence a f f e c t s biological funct ion (22). In addi t ion to these compounds which have been shown to d i r ec t l y p e r t u r b the m e m b r a n e , c e r t a i n an t iox idan t s have been used to r e d u c e the s y m p t o m s of chil l ing injury. For example , t h e use of d ipheny lamine , e i t he r as a coa t ing on the apple skin or in wrappe r s a round the frui t p r e v e n t e d superf ic ia l scald in apples a f t e r pro longed s t o r a g e a t 0-4 C (35). Jones et al (38) t e s t e d a number of chemica l a g e n t s for the i r abi l i ty to p r even t chil l ing injury of bananas and showed t ha t p o s t h a r v e s t t r e a t m e n t with d imethy lpo lycy loxane , saff lower oil, and minera l oil w e r e e f f ec t ive in p r even t ing unde r -pee l d i sco lora t ion of bananas from a chill ing t r e a t m e n t . Similar ly, Wang and Baker (116) could p r e v e n t some of the symptoms of chil l ing injury using sodium b e n z o a t e and e thoxyquin , compounds which scavange f r ee - r ad i c l e s . Whether t hese compounds a c t by p reven t ing lipid perox ida t ion to main ta in m e m b r a n e f luidi ty, or a c t addi t ional ly as m e m b r a n e p e r t u r b e r s is not c l ea r a t this t i m e .

In summary,it is both t e m p t i n g and diff icult to e x t r a p o l a t e from t h e r e su l t s wi th model b inary mix tu re s of phospholipids and mic roo rgan i sms , having r e l a t i ve ly s imple phospholipids in the i r m e m b r a n e s , to t h e complex m e m b r a n e of h igher p l a n t s . However , in fo rmat ion about the f ac to r s cont ro l l ing m e m b r a n e f luidi ty is i m p o r t a n t in unders tand ing t h e co r r e l a t i on b e t w e e n chil l ing sens i t iv i ty and m e m b r a n e f luidi ty . R e c e n t r e p o r t s t ha t show a co r r e l a t i on b e t w e e n chil l ing sens i t iv i ty in avocado fruit and lipid composi t ion as a funct ion of r ipening (44), and the re la t ionsh ip b e t w e e n a l t e r e d mi tochondr ia l a c t i v i t y and changes in t he f a t t y ac id composi t ion of mi tochondr ia l lipids in mango fruit s t o red a t low t e m p e r a t u r e (39) a r e in c o n t r a s t to t he r e su l t s ob ta ined with lipids


e x t r a c t e d from Pa$$ifl(pra spec ies vary ing in the i r r e s i s t a n c e to chil l ing where t h e r e was no co r r e l a t i on wi th t he d e g r e e of u n s a t u r a t i o n of t h e lipids (77).

A b e t t e r unders tand ing of t h e f a c t o r s cont ro l l ing m e m b r a n e fluidity is e s sen t i a l in o rder t o assign observed physiological p h e n o m e n a to t he c o r r e c t mo lecu l a r e v e n t s .

B. Membrane-Cytoplasmic Interactions

While changes in t h e physical n a t u r e of t h e m e m b r a n e can inf luence d i r ec t l y those funct ions t ha t a r e an i n t eg ra l p a r t of t h a t s t r u c t u r e , eg . , a m e m b r a n e - b o u n d e n z y m e , cons ide ra t ion must be given to t h e i n t e r a c t i o n b e t w e e n the m e m b r a n e b i layer and the cy top lasm and c y t o s k e l e t a l e l e m e n t s of t h e ce l l . For example cessa t ion or i m p a i r m e n t of p ro top l a smic s t r e a m i n g in ch i l l ing-sens i t ive spec ies is one of the more i m m e d i a t e e f f ec t s t h a t has been r e p o r t e d as t h e resu l t of exposure to chill ing t e m p e r a t u r e s . As ea r ly as 1864, Sachs (92) r e p o r t e d tha t p ro top la smic s t r e a m i n g c e a s e d a t about 10 to 1Z C in roo t ha i r s of cucumber and t o m a t o p l an t s , while those of chil l ing t o l e r a n t spec ies con t inued s t r e a m i n g down to or n e a r 0 C . Lewis (49) found t h a t s t r e a m i n g c e a s e d or was jus t p e r c e p t i b l e a f t e r one or two minu t e s a t 10 C in p e t i o l e t r i c h o m e of chil l ing sens i t ive p l an t s and cea sed p rompt ly a t 5 C or 0 C . These obse rva t ions w e r e conf i rmed by Wheaton (1ZZ). In

ο c o n t r a s t to t hese r e su l t s w h e r e 1 0 C has been shown to be t he lower l imit of s t r e a m i n g for the p l an t s s tud ied , P a t t e r s o n and G r a h a m (76) have r e p o r t e d t h a t s t r e a m i n g did no t a c tua l l y c ea se a t some c r i t i c a l t e m p e r a t u r e but t ha t the t e m p e r a t u r e coef f ic ien t for r a t e of s t r e a m i n g marked ly d i f fe red b e t w e e n sens i t ive and t o l e r a n t spec ie s . Their r e su l t s w e r e in a g r e e m e n t wi th s imi lar work on the chil l ing sens i t ive t o b a c c o (15).

In euka ryo t i c cel ls , cy top l a smic mic ro tubu le s con ta in ing tubulin, cy top l a smic f i l amen t s con ta in ing ac t i n and a s soc i a t ed myosin, a r e cons idered to be t h e pr inc ipa l c o m p o n e n t s of t h e c y t o s k e l e t o n . Cy top l a smic mic ro tubu le s a r e involved in main ta in ing cel l shape , in the i n t r ace l l u l a r s t r a t i f i c a t i o n of o rgane l l es , in t r a n s p o r t and s e c r e t i v e p roces se s , and cel l su r f ace topography . The ge la t ion of a c t i n s , myosins , and ac t in -b ind ing p ro t e in s is t e m p e r a t u r e and p r e s s u r e dependen t , and t h e gel is de s t royed by low t e m p e r a t u r e s above 0 C (74). The m o v e m e n t and mechan i ca l work funct ions of many kinds of ce l ls appear to depend on t h e p r e s e n c e of a ge l - l ike cy top la sm, b e c a u s e low t e m p e r a t u r e s or e x t r e m e p re s su re des t roy the gel and i n t e r f e r e wi th cyclosis and cytokines is (74). The i n t e r f e r e n c e of low t e m p e r a t u r e wi th cyclosis in p l an t s has been i n v e s t i g a t e d ex tens ive ly [for r e c e n t rev iew see P a t t e r s o n and G r a h a m , (76)] . The d i s t r ibu t ion of tubu l in -con ta in ing s t r u c t u r e s has been s tud ied in cu l t u r ed an imal ce l ls and in a lgae , but only to a very l im i t ed e x t e n t in h igher p l an t s (7). Micro tubu le r br idges to the p l a sma m e m b r a n e have been desc r ibed in u l t r a s t r u c t u r a l s tud ies of

1 8 J. Μ. Lyons et al

p lan t s [see Heppler and Pa l iv i t zh (33) for r e v i e w ] . Henry , et al (32) employed w a t e r soluble spin labels to d e m o n s t r a t e t h a t a l t e r a t i o n s in m e m b r a n e compos i t ion in mic roorgan i sms inf luence cy top la smic v iscos i ty . Ke i th (unpublished data) has found t h a t t h e v iscos i ty of w a t e r in t h e cy top lasm of chil l ing sens i t ive t i ssues as d i sce rned by w a t e r -soluble spin labels changes marked ly below about 10 C and t h e r e is a loss of diffusion ba r r i e r s in the cy top lasm of the sens i t ive t i ssues no t obse rved in chil l ing r e s i s t a n t t i ssues . The exac t mechan i sm cont ro l l ing cy top la smic viscosi ty and the low t e m p e r a t u r e response is no t c l ea r . However , i t s e e m s s ignif icant t h a t t he obse rva t ion of t h e e f f ec t s of low t e m p e r a t u r e on p ro top la smic s t r e a m i n g and chil l ing sens i t ive spec ies t a k e n t o g e t h e r wi th t h e known inf luence of t e m p e r a t u r e on r eve r s ib l e d issassoc ia t ion of the c y t o s k e l e t a l po lymers point to t he i n t e r a c t i n g and i n t e g r a t e d response of t h e m e m b r a n e cy to ske l e t a l s y s t e m s .

C. Direct Effect of Low Temperature on Proteins

The discussion to this point has focused on the molecu la r o rder ing of the m e m b r a n e lipids as the cont ro l l ing e l e m e n t in the low t e m p e r a t u r e r e sponse . It is now a p p r o p r i a t e to address t h e ques t ion of t h e ro le of p ro te ins in the t e m p e r a t u r e r e sponse . Apparen t d i scon t inu i t i es in Arrhenius p lo ts as t he resu l t of a conformat iona l change or d isassoc ia t ion of p ro t e in s have been r e p o r t e d for a number of sy s t ems (5, 26). While some enzyme p ro te ins may undergo these confo rmat iona l changes , it is ve ry i m p o r t a n t to r e a l i z e tha t t h e r e is n e i t h e r any t h e o r e t i c a l r eason to expec t nor e x p e r i m e n t a l ev idence to i nd i ca t e , t h a t many enzymes undergo confo rma t iona l changes over the na r row r a n g e of t e m p e r a t u r e observed for t he chill ing r e sponse . F u r t h e r , wi th but few excep t ions as n o t e d below, the c r i t i c a l t e m p e r a t u r e for physiological dysfunct ion has not been c o r r e l a t e d with any p ro te in confo rmat iona l change as has been done wi th m e m b r a n e f luidity changes . If p ro t e in confo rma t iona l changes a r e cons idered as a p r imary d e t e r m i n a n t of t h e c r i t i ca l t e m p e r a t u r e for chil l ing, then the broad r a n g e of consequences to ce l lu lar funct ion would seem to r equ i r e t ha t t he p ro te in be p r e s e n t in major quan t i t i e s in all of the sens i t ive spec ies , or , have a means of rapidly amplifying and p ropaga t ing i t s confo rmat iona l change to a f fec t many ce l lu lar a s p e c t s . In addi t ion some ev idence should be p r e s e n t e d to show tha t me tabo l i c dysfunct ion leading to a physiological even t occurs a t t he s t ep c a t a l y z e d by t h e e n z y m e .

T h e r e is l i t t l e ev idence of any m a s t e r p ro t e in or m a s t e r r e a c t i o n t ha t can explain the commona l i t y of the physiological dysfunct ions in chil l ing sens i t ive spec ies when t e m p e r a t u r e s a r e lowered below about ΙΟΙ 2 C. In his rev iew of g e n e t i c r egu la t ion of t e m p e r a t u r e responses in mic roo rgan i sms , Ingraham (37) ind ica ted tha t while the c h e m i c a l basis for loss of funct ion of a p ro te in a t high t e m p e r a t u r e s is r e l a t i ve ly well unders tood , (i .e. , those chemica l bonds which ma in ta in the p roper secondary and t e r t i a r y s t r u c t u r e of p ro t e in s b e c o m e weakened a t


e l e v a t e d t e m p e r a t u r e s , r e su l t ing in d e n a t u r a t i o n and loss of funct ion of the p ro te in ) , t he loss of funct ion a t low t e m p e r a t u r e is not r ead i ly a p p a r e n t , (al though weaken ing of hydrophobic bonds is involved) . His r e s e a r c h ind ica t ed t ha t co ld -sens i t ive m u t a n t s of E. coli owed the i r cold sens i t iv i ty to a slight a l t e r a t i o n in t he s t r u c t u r e of e n z y m e s involved in r ibosome synthes i s a t low t e m p e r a t u r e . The e n z y m e s in these m u t a n t s w e r e more sens i t ive to f eed -back inhibi t ion a t all t e m p e r a t u r e s .

With higher p l an t s t h a t a r e chil l ing sens i t ive , Yamak i and Ur i t an i (129, 130) cons idered t ha t the p r i m a r y response was in the abi l i ty of m e m b r a n e p ro t e in s to bind phospholipids as a r e su l t of chill ing t r e a t m e n t s . F u r t h e r m o r e , Shirahashi et al (96) have shown tha t p y r u v a t e , o r t hophospha t e d ik inase from m a i z e is cold labi le and tha t this cold labi l i ty c o r r e l a t e s wi th a sharp change in a c t i v a t i o n energy of t h e dikinase - c a t a l y z e d r e a c t i o n obse rved nea r 12 C . Studies h a v e shown tha t some e n z y m e s a s soc i a t ed wi th m e m b r a n e s y s t e m s in chil l ing sens i t ive p l an t s undergo an anomalous change in r a t e a t about t h e s a m e t e m p e r a t u r e as t ha t shown for the phys ica l change in m e m b r a n e c h a r a c t e r i s t i c s ; in c o n t r a s t , n o n - m e m b r a n e a s s o c i a t e d e n z y m e s main ta in a r e l a t i v e l y l inear r e l a t ionsh ip wi th d ec r ea s in g t e m p e r a t u r e (6, 85). Downton and Hawker (20) have shown t h a t t h e soluble enzymes respons ib le for s t a r c h synthes is in chil l ing sens i t ive m a i z e , avocado and swee t p o t a t o exhibi t a d i scon t inu i ty in Arrhen ius p lo t s a t about 12 C which c o r r e l a t e wi th physiological dysfunct ion in those t i s sues . In c o n t r a s t s imi lar enzymes from t h e chil l ing r e s i s t a n t p o t a t o exhibi t a l inear p lo t in response to t e m p e r a t u r e over t h e r ange from 23 C down to 0 C . Their r e su l t s i nd i ca t ed t h a t t h e r e was an assoc ia t ion b e t w e e n s t a r c h s y n t h e t a s e and the lipid, lyso le th ic in , and they sugges ted t ha t the d i scon t inu i t i es observed in t h e p lo t s from chil l ing sens i t ive p l an t s r e f l e c t e d a t r ans i t ion in lipid a t t he c r i t i c a l t e m p e r a t u r e s imi lar to t h a t observed wi th t he m e m b r a n e - b o u n d e n z y m e s r e p o r t e d previously for those spec ie s . These s tud ies of Downton and Hawker (20) focus a t t e n t i o n on t h e lipid r a t h e r than expec t ing t h e p ro t e in enzymes from the d i f fe ren t spec ies to respond d i f fe ren t ly to t e m p e r a t u r e .

Again, a s s ignment of t h e p r i m a r y t e m p e r a t u r e response to a d i r ec t e f fec t on a p ro t e in would have to provide a mechan i sm to explain how t h a t response could be p r o p a g a t e d th roughout a n u m b e r of c rop spec ies a t a r e l a t i ve ly nar row t e m p e r a t u r e r a n g e focusing around 10-12 C.

In summary, th is vo lume is i n t ended to p r e s e n t wha t is or might be known about the p r i m a r y molecu la r even t s involved in the response of p l an t s to low t e m p e r a t u r e and, as such, will hopefully e n h a n c e our abi l i ty to plan a p p r o p r i a t e r e s e a r c h to fill knowledge gaps w h e r e they might ex is t .


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124. Wil lemot , C. Plant Physiol. 60, 1-4 (1977). 125. Wills, R. Β. H., Sco t t , K. J . Phytochem. 10, 1783-1785 (1971). 126. Wilson, J . M. New Phytol. 76, 257-270 (1976). 127. Wright , M. Planta 120, 63-69 (1974). 128. Wright , M., and Simon, E. J. Expt. Bot. 24, 400-418 (1973). 129. Yamak i , S., and Ur i t an i , I. Agr. Biol. Chem. 36, 47-55 (1972). 130. Yamak i , S., and Ur i t an i , I. Plant Cell Physiol. 15, 385-393 (1974). 131 . Zsoldos, F . Plant and Soil 37, 469-478 (1972). 132. Zsoldos, F . and Karva ly , B. Physiol . P l an t 43 , 326-330 (1978).


ADAPTATION TO CHILLING: SURVIVAL, GERMINATION, RESPIRATION AND PROTOPLASMIC DYNAMICS

Brian D. Patterson and Douglas Graham

Plan t Physiology Unit CSIRO Division of Food R e s e a r c h

School of Biological Sciences Macquar i e Univers i ty

Nor th Ryde , N.S.W. 2113, Aus t r a l i a

Robert Paull

D e p a r t m e n t of Botany Univers i ty of Hawai i a t Manoa

3190 Maile Way Honolulu, Hawai i

I. INTRODUCTION

In this pape r we desc r ibe e x p e r i m e n t s wi th two so r t s of p l an t s wi th c o n t r a s t i n g ch i l l i ng - re s i s t ance . The f irst a r e v a r i e t i e s of t h e t rop ica l spec ies Lycopersicon hirsutum, a wild t o m a t o , which is indigenous to t h e Andes of Ecuador and Pe ru . Var ie t ies a t t h e h igher a l t i t ude s of i t s n a t i v e h a b i t a t e x p e r i e n c e t e m p e r a t u r e s below 10 C th roughout t h e yea r (minimum night t e m p e r a t u r e s ) , while va r i e t i e s growing n e a r s ea level r a r e l y or neve r e x p e r i e n c e t e m p e r a t u r e s below 18 C (12). Var ie t ies from the lower a l t i t ud es a r e a t l eas t as chi l l ing-sens i t ive as t h e d o m e s t i c t o m a t o Lycopersicon esculentum (9). The second group of p l a n t s c o m e e i t h e r from " t e m p e r a t e " c l i m a t e s or from t h e t rop ica l lowlands . Most of t h e " t e m p e r a t e " group w e r e d o m e s t i c a t e d in t he w in t e r ra infa l l a r e a s of t h e M e d i t e r r a n e a n . Such p l an t s a r e b e t t e r a d a p t e d to survive pro longed chil l ing than high a l t i t u d e t rop ica l p l a n t s . However , t hey have t h e d i sadvan tage t h a t they a r e not c losely r e l a t e d to t h e sens i t ive t rop ica l p l an t s wi th which it is useful to c o m p a r e t h e m . It is t h e r e f o r e o f ten diff icult to dec ide which physiological d i f fe rences b e t w e e n , say, t he onion and the w a t e r melon a r e speci f ica l ly a d a p t a t i o n s to chil l ing. For i n s t a n c e , t h e g r e a t e r frost r e s i s t a n c e of t h e onion might

Copyright · 1979 by Academic Press, inc. 2 5 All rights of reproduction in any form reserved

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2 6 Β. D. Patterson et al.

be r e f l e c t e d in a d i f fe ren t m e m b r a n e lipid compos i t ion . Hence t h e usefulness of compar ing the physiology of c lo se ly - r e l a t ed spec ies such as t h e t o m a t o e s which vary in chill ing sens i t iv i ty , a l though none is c o m p l e t e l y chil l ing r e s i s t a n t . In these we have examined lipid p r o p e r t i e s , r e sp i r a t ion , ge rmina t ion and t h e d iurnal f l uc tua t ion of chil l ing r e s i s t a n c e . We have then c o m p a r e d the i m m e d i a t e e f f ec t of cold on t h e s t r u c t u r e of t h e cy top lasm of t h e very chil l ing r e s i s t a n t onion (Allium cepa) and t h e very chill ing sens i t ive w a t e r melon (Citrullus la-natus).

Π. RESULTS AND DISCUSSION

A. Lipid Properties of Lycopersicon Varieties

In prev ious s tudies (7, 8) Passiflora spec ies w e r e shown to differ in chil l ing r e s i s t a n c e . However , t h e compos i t ion of the i r polar lipids did no t vary apprec iab ly . D i f f e rences in lipid u n s a t u r a t i o n which would accoun t for d i f fe rences in chill ing r e s i s t a n c e w e r e no t found. D i f f e r ences in t h e physica l p r o p e r t i e s of t he lipids was , however , sugges ted by e l e c t r o n spin r e s o n a n c e s tud ies (ESR). T h e r e w e r e a p p a r e n t changes in t h e t e m p e r a t u r e coef f ic ien t of spin label mot ion a t two t e m p e r a t u r e s , i .e . t h e r e was an upper b reak t e m p e r a t u r e and a lower b reak t e m p e r a t u r e . The r e su l t s sugges ted a re la t ionsh ip b e t w e e n the t e m p e r a t u r e of t h e lower b reak and t h e chill ing sens i t iv i ty of t h e p a r t i c u l a r Passiflora s p e cies or hybrid. We t h e r e f o r e p r e d i c t e d t h a t t he m e m b r a n e lipids of L. hirsutum and those of t h e d o m e s t i c t o m a t o L. esculentum would show cor responding b reak t e m p e r a t u r e s which would also r e l a t e to the i r chil l ing sens i t iv i ty . F igure 1 shows a p lot of t h e log of t h e spin label mot ion p a r a m e t e r τ aga ins t t h e r ec i i p roca l of abso lu te t e m p e r a t u r e (Arrhenius plot) for ves ic les of polar lipids from t h e domes t i c t o m a t o L. esculentum. It is of ten difficult to dec ide when to r e j ec t models for such d a t a which p o s t u l a t e s t r a igh t l ines s e p a r a t e d by b reaks , in favour of a smooth cu rve . The re fo re in this c a se , be fo re f i t t ing s t r a igh t l ines , fo re sho r t ened p lo ts which e x a g g e r a t e curves but p r e s e r v e l inea r i ty w e r e m a d e by the m e t h o d of Wilcox and P a t t e r s o n (13). F igure Ζ shows t h e r e su l t : a smooth curve was a t l eas t as convincing a model for t h e d a t a as one based on t h r e e s t r a igh t l ines wi th b r eaks . Very s imi lar r e su l t s w e r e ob ta ined for a l t i tud ina l v a r i e t i e s of L. hirsutum. A b reak in t he t e m p e r a t u r e coef f ic ien t of spin label mot ion in lipids from the t o m a t o has been r e p o r t e d a t 13 C (11). The changes in slope w e r e s imi lar to those r e p o r t e d h e r e for L. esculentum and L. hirsutum. Our r e su l t s sugges t t ha t ob jec t ive t e s t s which p e r m i t the opt ion of smooth curves should be appl ied to ESR d a t a . This is p a r t i c u l a r l y i m p o r t a n t in view of t h e p r o m i n e n c e of b reaks in ESR for t he lipid theory of chill ing sens i t iv i ty .

Adaptation to Chilling 2 7

FIGURE 1. Spin label (12NSMe) motion in vesicles of polar lipids prepared from leaves of tomato (L. esculentum cv. Rutgers). Lipid preparation and ESR techniques were as described previously (7).

FIGURE 2. Visual demonstration that a curve model fits the ESR data shown in Figure 1. The plot was foreshortened by five times in order to magnify any changes in slope, as described elsewhere (13). The increased angle between the original axes is shown. No breaks are indicated.


B. Temperature Effects on Respiration

The t e m p e r a t u r e dependence of r e sp i r a t i on has been r e p o r t e d to show abrup t changes a t specif ic t e m p e r a t u r e s ("breaks"), espec ia l ly a round 10 C in t h e case of chi l l ing-sens i t ive p l an t s such as c u c u m b e r (3). When mac roscop ic t i ssues a r e used ( leaves, r o o t s , frui ts) , it is qu i t e diff icul t to m a k e a c c u r a t e m e a s u r e m e n t s of t h e e f fec t of t e m p e r a t u r e on p lan t r e sp i r a t ion , espec ia l ly a t low t e m p e r a t u r e s , because smal l changes in oxygen c o n c e n t r a t i o n a r e diff icult to m e a s u r e in gas s t r e a m s . However , pol len has a high r a t e of r e sp i r a t i on which can be m e a s u r e d in an oxygen e l e c t r o d e . T o m a t o pollen gra ins have t he d imensions of mic roorgan i sms , the i r n u t r i e n t r e q u i r e m e n t s a r e s imple , and they can be h a r v e s t e d as needed . Even close to 0 C, t h e reproduc ib i l i ty of r e sp i r a t i on m e a s u r e m e n t is as high as a t Z0 C . F igure 3 shows t h e e f fec t of t e m p e r a t u r e on the r a t e of r e sp i r a t i on of pol len from two v a r i e t i e s of L. hirsutum; a r e l a t i v e l y chil l ing r e s i s t a n t one from high a l t i t u d e (3200m) and a chi l l ing-sens i t ive one from low a l t i t u d e (30m). N e i t h e r shows a s ignif icant change in t h e t e m p e r a t u r e coef f ic ien t of r e sp i r a t i on b e t w e e n 0 and 20 C . This resu l t might be e x p e c t e d for t he high a l t i t u d e form, for in i t s n a t i v e h a b i t a t it sha res t he t e m p e r a t u r e env i ronmen t of wild and d o m e s t i c a t e d p o t a t o e s , which a r e genera l ly cons idered to be ch i l l ing- res i s tan t (2). However , t he form from low a l t i t u d e is m o r e ch i l l ing-sens i t ive , as would be e x p e c t e d for a p lan t from a lowland t rop ica l env i ronmen t . While t hese r e su l t s r e f e r qu i t e speci f ica l ly to pol len and do not necessa r i ly r e f l ec t t he r e sp i r a t ion of o t h e r t i ssues , they show t h a t abrupt changes in t he t e m p e r a t u r e coef f ic ien t of r e sp i r a t i on a r e no t invar iably a s soc i a t ed wi th chill ing sens i t iv i ty of t h e p a r e n t p l an t .

C. Genetic Association of Chilling Adaptations

Chill ing r e s i s t a n c e could be a s imple p r o p e r t y , r e su l t ing say from the physical p r o p e r t i e s of m e m b r a n e lipid, or i t could be t h e man i f e s t a t i on of a whole se r ies of u n c o n n e c t e d a d a p t a t i o n s to t e m p e r a t u r e as has been desc r ibed for mic roorgan i sms (1). If it w e r e a single p r o p e r t y , i t would be e x p e c t e d to be i nhe r i t ed in a un i t a ry m a n n e r . The re fo re t he e x t e n t to which d i f fe ren t a d a p t a t i o n s to low t e m p e r a t u r e in Lycopersicon w e r e a s soc i a t ed was i nves t i ga t ed . These w e r e low t e m p e r a t u r e pol len ge rmina t ion and low t e m p e r a t u r e seed ge rmina t i on . F igure 4 shows t h e e x t e n t to which pol len from two a l t i tud ina l v a r i e t i e s of L. hirsutum g e r m i n a t e d when held a t d i f fe ren t t e m p e r a t u r e s for 18 h. Apprec iab le d i f fe rences in the minimum t e m p e r a t u r e of ge rmina t ion a r e shown. The t e m p e r a t u r e a t which 50% ge rmina t i on was ach ieved was then p l o t t e d agains t a m e a s u r e of low t e m p e r a t u r e seed ge rmina t ion for a number of a l t i t ud ina l v a r i e t i e s of L. hirsutum. Table I shows t h a t t h e r e is no a p p a r e n t r e l a t i on b e t w e e n these c h a r a c t e r s .


1

α

ο" φ

0.1

0 10 20 Temperature C

FIGURE 3. Pollen respiration. Pollen (1 mg.ml was suspended in 10% sucrose, 0.01% H^BO^ cmd the rate of oxygen uptake plotted as a proportion of the rate at zO C (log scale). Each point represents the initial rate of uptake determined from a time course. Oxygen concentration in the electrode cell was maintained at 250 ± 20 μ mol-es.ml . (%), pollen from low altitude L. hirsutum; (0), pollen from high altitude L. hirsutum. Both plots show a constant Q 1f)of between 2 and 3.

TABLE I. Ability of Seed to Germinate Quickly at Low Temperature and Ability of Pollen to Germinate at Low Temperature

Altitude of Temperature at Temperature at origin which 50% seed which 50% pollen

(metres) germination occurs germination occurs after 10 days after 15 h

(°C) (°C)

400 15.4 8.8 1000 15.2 7.0 1500 13.3 6.8 2100 11.0 7.5 2650 13.0 7.7 3200 10.8 7.2

Seed germination (radical extrusion) at different temperatures, and the temperature at which 50% germination occurred after 10 days was found. The temperature at which 50% germination of pollen occurred was found as described in Fig. 4.


ι 1 1 1 1 1 r — r

I I I I 1 1 1 1 1—I 4 6 8 10

Temperature °C

FIGURE 4. Minimum temperatures of germination of pollen for different varieties of L. hirsutum. Pollen was germinated on 10% sucrose, 0.01% boric acid, solidified with 1% agar. After 15 h, the pollen was fixed with formaldehyde vapour and photomicrographs made, from which percentage germination was assessed. For these and other altitudinal varieties of L. hirsutum, the temperature at which 50% germination occurred was used as a standard for comparison.

D. Effect of Chilling on the Structure of Cytoplasm

Our r e su l t s sugges t t h a t a d a p t a t i o n to chil l ing t e m p e r a t u r e s may r e q u i r e a d a p t a t i o n a t many s i t e s wi thin a ce l l . We have t h e r e f o r e looked for e f f ec t s of chil l ing which do no t necessa r i ly r e l a t e to t he loss of tu rgor which even tua l ly occur s and which is i r r evers ib le (see Sect ion E). One such e f fec t is t he change in t h e s t r u c t u r e of cy top lasm which occu r s when chil l ing sens i t ive p l an t s a r e chi l led . We found tha t wi thin seconds of cooling living t r i c h o m e s of w a t e r melon, t o m a t o or o t h e r chil l ing sens i t ive p lan t s a d i s t inc t ive change could be seen using phase c o n t r a s t mic roscopy . F igu re 5 shows the a p p e a r a n c e of a w a t e r melon cell which has been cooled to 4 C. The no rma l thin s t r ands of cy top lasm which span the vacuo le have d i sappeared and have been r e p l a c e d by spher ica l ves ic les . Also shown is t h e s a m e cell a f t e r it was w a r m e d to 15 C. B e t w e e n 1Z and 15 C t h e ves ic les d i sappea red and t h e thin s t r ands of cy top lasm w e r e r e f o r m e d . Qui te u n r e l a t e d p l an t s show the s a m e e f fec t as long as they a r e chil l ing sens i t ive , for i n s t a n c e corol la ha i r s of Datura (Reid, P a t t e r s o n and Ferguson , unpublished fi lm, 1978). In c o n t r a s t , p l an t s which can t o l e r a t e pro longed chil l ing, such as t he onion, ma in t a in n o r m a l cel l s t r u c t u r e s down to 0 C (Fig. 5). The r ap id i ty of t h e response of p ro top l a smic s t r u c t u r e to cold sugges t s t h a t we a r e observing someth ing close to a p r imary e f fec t on t h e ce l l . However , we have observed t h a t t h e r e p l a c e m e n t of t he fine s t r ands of cy top lasm by ves ic les is also a genera l r esponse to s t r e s s . In bo th chil l ing sens i t ive and r e s i s t a n t ce l l s , tox ic agen t s , m e c h a n i c a l d a m a g e or b a c t e r i a l in fec t ion


produce a s imi lar loss of l inear s t r u c t u r e and t h e s imul t aneous a p p e a r a n c e of spher ica l ves i c l e s . It has been r e p o r t e d t ha t a s imi lar e f fec t occur s when f ixa t ives a r e used in e l e c t r o n mic roscopy (4). The mic ro tubu le e l e m e n t s t h a t p a r t l y m a k e up t h e cy to ske l e ton of euka ryo t i c cel ls a r e known to be d i s rup ted by chil l ing t e m p e r a t u r e s in vitro and in vivo (5) and this d issoc ia t ion of t h e mic ro tubu la r e l e m e n t s is r eve r s ib l e on warming . The re fo re , our r e su l t s a r e cons i s t en t with a model in which a cy toske l e ton which suppor t s l inear s t r ands of cy top lasm is d i s soc ia ted a t chil l ing t e m p e r a t u r e s in to a pool of tubulin subuni ts . On r e w a r m i n g , po lymer i sa t ion is favoured, and t h e cy toske l e ton is r e f o r m e d , so t h a t t he cy top la smic s t r ands a r e s t ab i l i zed . If this model is c o r r e c t , it should be possible to d e m o n s t r a t e d i f fe rences in t he t e m p e r a t u r e d e p e n d e n c e of mic ro tubu le fo rma t ion for ch i l l ing- res i s tan t and chi l l ing-sens i t ive p l a n t s .

E. Diurnal Variation in Chilling Sensitivity

When physiologis ts classify spec ies accord ing to the i r chill ing sens i t iv i ty , they of ten use c r i t e r i a which vary depending on w h e t h e r they a r e r e f e r r ing to whole p l an t s or the i r d e t a c h e d organs , such as t ube r s or f ru i t . In o rder t o c o m p a r e d i f fe ren t kinds of wild and d o m e s t i c t o m a t o , t h e c r i t e r ion of chill ing r e s i s t a n c e we have mainly used is t h e abi l i ty to survive exposure to 0 C (9). In o rde r to s t a n d a r d i z e t h e physiological s t a t e of the p l an t s , they w e r e grown wi th a day leng th of 1Z h and sampled for chil l ing half way through t h e light per iod . However , it was found t h a t when p lan t s w e r e chi l led in the dark , but beginning a t d i f fe ren t t i m e s in t h e day /n igh t cyc l e , the i r c a p a c i t y t o survive chil l ing va r i ed s t r ik ingly . F igure 6 shows the diurnal va r i a t ion in the loss of tu rgor induced by chil l ing t h e seedl ings a t 0 C for 6 days . The env i ronmen t of t h e seedl ing was kept s a t u r a t e d wi th mo i s tu re so t h a t the loss of tu rgor was no t a resu l t of w a t e r loss. If p l an t s w e r e chi l led from 1 h a f t e r t h e onset of t h e l ight per iod (7.00 h) t h e r e was a max imum sens i t iv i ty to chil l ing m e a s u r e d as loss of t u rgor . The p l a n t s w e r e t hen w a r m e d t o Z0 C in a humidif ied a t m o s p h e r e , t r a n s f e r r e d to cont inuous l ight a t ZZ C and a f t e r 1 week the p ropor t ion of survivors assessed . F igure 6 shows t h a t , using t h e c r i t e r ion of survival , t h e p l an t s a r e l eas t sens i t ive to chil l ing when they a r e p l aced a t 0 C 4 h a f t e r t h e onse t of t h e dark per iod (ZZ.00 h). The t i m e a t 0 C r equ i r ed to kill half t h e seedl ings was 6 days for t h e leas t sens i t ive t i m e of t h e day , bu t only 3 days for t he mos t sens i t ive p a r t of t h e day. We assume t h a t t h e d i f f e rences in chil l ing s y m p t o m s for d i f fe ren t t i m e s of the day r e f l e c t d i f f e rences in m e t a b o l i s m . The r e su l t s a r e cons i s t en t wi th t h e hypothes i s t h a t chil l ing causes a m e t a b o l i c i m b a l a n c e (Z, 10); in addi t ion , a way to t e s t t he hypothes i s is sugges ted . If a p a r t i c u l a r m e t a b o l i t e is p r imar i ly respons ib le for t h e chil l ing d a m a g e , i t is r e a s o n a b l e t o a s sume i t mus t be p roduced to a g r e a t e r e x t e n t when chil l ing is s t a r t e d a t 7.00 h than when it is s t a r t e d a t ZZ.00 h under our condi t ions . We a r e t h e r e f o r e i nves t iga t ing which m e t a b o l i t e s do vary in t h e e x p e c t e d manne r during chi l l ing.

< 3

2

33

FIGURE 5. Structural changes in cytoplasm during chilling. A. Water melon (chilling sensitive) trichomes were chilleQ on a microscope slide with an attached thermocouple (6). The photomicrograph at 4.1 C shows large and small cytoplasmic vesicles. The thinnest cytoplasmic strands have disappeared. The photomicrograph taken after warming to 15.5 C shows that the vesicles have disappeared, and thin cytoplasmic strands bridging the vacuole have reappeared. B. Onion (chilling resistant) epidermal cells were subjected to cold also. The cell at the chilling and non-chilling temperatures does not differ in its content of thin cytoplasmic strands or vesicles.

3 4 Β. D. P a t t e r s o n et al

FIGURE 6. Diurnal variation in chilling sensitivity of tomato seedlings. Seeds (L. esculentum cv. Grosse Lisse) were sown at intervals of 2h through a growing cycle of 12 h day/night and 22 C/18 C. After exactly 9 days, each group of seedlings was chilled to 0°C at 100% R.H. After 6 days exactly, each group was examined for turgor loss (curve A). After warniing to 20 C, plants were returned to conditions of constant light at 22 C, and assessed for % killed after 7 days (curve B). The shaded bars show the dark period.

II. CONCLUSIONS

Using t h e t o m a t o and i t s wild r e l a t i v e L. hirsutum, no ev idence was found wi th ESR techn iques for an abrup t change in lipid p r o p e r t i e s nea r 13 C. Ne i t he r was t he t e m p e r a t u r e d e p e n d e n c e of pol len r e sp i r a t i on found to change nea r this t e m p e r a t u r e ^ so t ha t t h e Q , Q for oxygen u p t a k e was cons t an t b e t w e e n 0 and 20 C . Two d i f fe ren t a d a p t a t i o n s to chil l ing t e m p e r a t u r e s (low t e m p e r a t u r e g e r m i n a t i o n of pol len and seed) w e r e not necessa r i ly a s soc i a t ed in t h e s a m e p l a n t . Abi l i ty to survive chil l ing in t o m a t o seedl ings was not c o n s t a n t , but va r ied on a diurnal r h y t h m . In a v a r i e t y of chil l ing sens i t ive p l an t s , chil l ing has a very rap id e f fec t on cy top la smic s t r u c t u r e ; th is may be the r e su l t of a d i r ec t e f fec t on t he mic ro tubu le s of t h e c y t o s k e l e t o n .


ACKNOWLEDGMENTS

We thank Mrs . L. P a y n e for t echn ica l a s s i s t an ce , Mrs. J . Kenr ick for help wi th lipid p r e p a r a t i o n , and Mr. N. F . Tobin for ESR d e t e r m i n a t i o n s . One of us (R.P.) was suppor t ed by a g ran t from the Rura l Cred i t Deve lopmen t Fund of the R e s e r v e Bank of Aus t r a l i a . We thank Professor C. M. Rick and Mr. E. Vallejos for t h e gift of a l t i tud ina l v a r i e t i e s of L. hirsutum.


1. Ing raham, J . L. In " T e m p e r a t u r e and Life" (H. P r e c h t , J . Chr i s tophe r sen , H. Hensel and W. Larcher , eds . ) , pp . 60-86 . Springer Verlag, Berlin (da te) .

2. Lyons, J . M. Ann. Rev. Plant Physiol. 24, 445-466 (1973). 3 . Lyons, J . M. and Raison , J . K. Plant Physiol. 45, 386-389 (1970). 4 . Mersey , B. and McCul ley , Μ. E. J. Microsc. 114, 49-76 (1978). 5. O lms t ed , J . B. and Borisy, G. G. Ann. Rev. Biochem. 42, 507-539

(1973). 6. P a t t e r s o n , B. D . and G r a h a m , D. J. Exptl. Bot. 28, 736-743 (1977). 7. P a t t e r s o n , B. D. Kenr ick , J . and Raison , J . K. Phytochem. 17, 1089

(1978). 8. P a t t e r s o n , B. D. , Mura t a , T., and G r a h a m , D. Aust. J. PI. Physiol.

3, 435-442 (1977). 9. P a t t e r s o n , B. D. , Paul l , R. and Smill ie , R. M. Aust. J. PI. Physiol.

5, 609-617 (1978). 10. Raison, J . K. Symp. Soc. Exp. Biol. 27, 485-512 (1973). 11 . Raison , J . K. In "Mechanisms of Regu la t ion of P lan t Growth" (R. L.

Bieleski , A. R. Ferguson and Μ. M. Cresswel l , eds.) , pp . 487-497 . Bull. 12, Royal Soc. New Zea land , Well ington. (1974).

12. Schwerd t f ege r , W. "World Survey of C l imato logy" , Vol. 12 (Elsevier, New York) (1976).

13. Wilcox, Μ. E. and P a t t e r s o n , B. D. This vo lume (1979).



SEED GERMINATION AT LOW TEMPERATURES

E. W. Simon

D e p a r t m e n t of Botany The Queen 's Univers i ty of Belfas t

N o r t h e r n I re land

The seeds of some spec ies a r e able to g e r m i n a t e , a lbe i t slowly, a t t e m p e r a t u r e s n e a r f reez ing , or even on i c e . O t h e r s fail to g e r m i n a t e when held cont inuously a t low t e m p e r a t u r e s , a l though they will g e r m i n a t e no rma l ly if t r a n s f e r r e d subsequen t ly to w a r m e r condi t ions . A th i rd type of r e sponse is seen in those seeds said to exhibi t chil l ing injury; if they a r e p l aced in cold w a t e r a t the s t a r t of imbibi t ion the seedl ings fo rmed during subsequent g rowth in t he warm show s y m p t o m s of injury (3, 9).

It is t h e purpose of this pape r to survey br ief ly wha t is known about this form of chil l ing injury and the fa i lure of ge rmina t i on in the cold, and to assess t he ro le in e a c h response of a change in m e m b r a n e f luidi ty .

I. CHILLING INJURY DUE TO IMBIBITION IN COLD WATER

When t h e seeds or embryos of c e r t a i n spec ies a r e exposed to low t e m p e r a t u r e s for a per iod a t the s t a r t of imbibi t ion they suffer chil l ing injury. Thus c o t t o n (which m a k e s no g rowth if held cont inuously a t 5 C) will n e v e r t h e l e s s g e r m i n a t e r a t h e r slowly if f irst exposed to w a t e r a t 5 C and then t r a n s f e r r e d to 31 C - a l though under these condi t ions t h e r e is a t e n d e n c y for t he p r i m a r y roo t to abo r t , l a t e r a l r o o t s growing out ins tead (3, 5). As l i t t l e as 30 minu t e s a t 5 C is enough to induce roo t abnorma l i t i e s and r e d u c e the speed of g e r m i n a t i o n , but longer per iods of chil l ing have a m o r e p ronounced e f f e c t . Likewise if soybean embryos a r e imbibed for 30 minu t e s a t 1Ζ C or less , t h e p e r c e n t a g e of embryos able to g e r m i n a t e and the subsequent g rowth of t h e axis a r e bo th r e d u c e d (1).

The consequences of a few days of chil l ing a t 5 or 10 C a r e sti l l very ev iden t in t he p l an t s long a f t e r w a r d s . C o t t o n p l an t s , for i n s t a n c e , st i l l grew m o r e slowly than con t ro l s 4 weeks a f t e r a per iod of chil l ing a t 10 C, dry weight and he ight being r e d u c e d in p ropor t ion to t he number of days of chil l ing (4). Similar ly soybean p l a n t s m a d e less dry weight in Ζ weeks and w e r e less tal l if t hey had been imbibed for 1Ζ hours a t 5 C r a t h e r than

Copyright © 1979 by Academic Press, inc. 3 7 All rights of reproduction In any form reserved

ISB N σ ΐ 2 4 β 0 5 6 Ο 5

3 8 Ε. w. Simon

Z5°C (Zl). Even more r e m a r k a b l e a r e t he d a t a of Highkin and Lang (11) who g e r m i n a t e d pea p lan t s a t a r a n g e of t e m p e r a t u r e s and then grew them on in a g reenhouse . Seeds g e r m i n a t e d a t 3 C p roduced p l an t s t h a t w e r e subs tan t i a l ly smal le r than those from seeds g e r m i n a t e d a t Z7 or 31 C, wi th s lower g rowth and fewer f lowers , pods and seeds .

Exposure to chil l ing t e m p e r a t u r e s does no t inev i tab ly resu l t in t h e deve lopmen t of injury s y m p t o m s even in sens i t ive p l a n t s . Thus c o t t o n seeds and l ima bean axes a r e r e n d e r e d i m m u n e to cold w a t e r if t hey a r e f irs t a l lowed to imbibe for a few hours a t Z5 or 31 C (6, 24); soybean embryos do not suffer chil l ing injury if they first t a k e up enough warm w a t e r to r e a c h a w a t e r c o n t e n t of 3 5 % (1). Ano the r way to p r e v e n t chil l ing injury is to expose seeds to mois t air unt i l the w a t e r c o n t e n t in c o t t o n and soybean seed r i ses to a t l eas t 13% (7, 1Z), t h a t of corn to 13-16% (Z), and the axes of l ima bean seed to Z0% (Z3).

As chil l ing injury can be avoided by an ini t ia l pe r iod of imbib i t ion wi th warm w a t e r or mois t a ir , i t follows t h a t only when dry seeds imbibe cold w a t e r do they sus ta in injury. Evidence has been a s sembled e l s ewhe re (Z8, Z9) t h a t in dry seed t i ssues , t he w a t e r c o n t e n t is l ikely to be so low tha t the m e m b r a n e phospholipids a r e forced in to t he hexagona l conf igura t ion and only come to adopt t he fami l ia r l ame l l a r a r r a n g e m e n t when the w a t e r c o n t e n t r i ses above about Z0% (or possibly m o r e r e l e v a n t , when w a t e r p o t e n t i a l r i ses above -80 ba r s (31). Dur ing t h e few seconds or minu te s when the phospholipid molecu les a r e r e o r i e n t i n g t hemse lve s from hexagonal to l ame l l a r con fo rma t ion in e a c h cell as i t s w a t e r c o n t e n t r i ses above the c r i t i c a l level , t h e r e is a shor t per iod when cel l c o n t e n t s a r e becoming hyd ra t ed , but t h e m e m b r a n e is no t y e t fully r e es tab l i shed as a s e m i - p e r m e a b l e ba r r i e r a round the cel l and so lu tes can leak ou t . This l eakage is especia l ly rap id in t he f irs t few m i n u t e s of imbibi t ion , then waning in in tens i ty a l though it con t inues slowly for many hours . Removing t h e seed coa t al lows fas t e r imbibi t ion of w a t e r and inc reases t he in i t ia l r a t e of l eakage .

T h e r e a r e severa l r easons for l inking chil l ing injury wi th an e n h a n c e m e n t of the l eakage of so lu tes from seeds . F i r s t , chil l ing injury is induced by exposing seeds to cold w a t e r a t t he s t a r t of imbibi t ion - two minu te s in cold w a t e r is enough to injure soybean co ty ledons (1); this ear ly per iod of imbibi t ion is jus t t h e t i m e when m e m b r a n e r e s t i t u t i o n is under way and l eakage is mos t profuse . Second, l e akage from imbibing seeds and embryos is i tself in tens i f ied a t low t e m p e r a t u r e s (14, ZZ, Z7). Low t e m p e r a t u r e thus has a dual e f f ec t , enhanc ing l eakage but r e s t r i c t i n g growth (1 , Z3).

The s eve r i t y of chil l ing injury and l eakage a r e bo th a b a t e d if t h e t i ssues a r e f irst a l lowed to imbibe in t h e warm (Z8). Final ly i t mus t be po in ted out t ha t rap id imbibi t ion of warm w a t e r is i tself enough to r e d u c e seedl ing vigour. If i so la ted embryos a r e a l lowed to imbibe w a t e r and a r e then p l a n t e d out in sand they grow poorly by compar i son wi th i n t a c t seeds (which imbibe m o r e slowly); p e a embryos grew in to p l an t s which w e r e only 70% as high as t he cont ro ls a f t e r 18 days (15, Ζ5).

In s u m m a r y , chill ing injury and l eakage respond al ike to changes of

Seed Germination at Low Temperatures 3 9

t e m p e r a t u r e and seed hydra t ion ; bo th r e d u c e seedl ing vigour and both a r e man i f e s t a t i ons of e v e n t s in ea r ly imbib i t ion . F u r t h e r m o r e chil l ing is known to i n c r e a s e l e a k a g e . These pa ra l l e l s b e t w e e n chil l ing and l e akage lead to the hypothes is t ha t chil l ing injury is a consequence of enhanced l eakage (25), r e su l t ing from a s lower r e s t i t u t i o n of m e m b r a n e i n t e g r i t y in the cold. Al though the loss of so lu tes from seeds may be qu i t e ex tens ive (29), l e akage should pe rhaps be r e g a r d e d as an ex t r ace l l u l a r ind ica t ion of wha t is also happening within cel ls - t ha t is to say, a t e m p o r a r y loss of c o m p a r t m e n t a t i o n which may have ser ious repercuss ions for the subsequent ope ra t ion of t he ce l l .

Is it also possible t h a t chil l ing injury could be due to a m e m b r a n e phase shift caus ing mi tochondr i a l dysfunct ion? This possibi l i ty mus t be v iewed with some r e s e r v e in view of t h e ev idence (29) t h a t mi tochondr ia a r e " i m m a t u r e " and i nac t i ve in the f irst few hours of imbibi t ion , the t i m e when seeds a r e mos t suscep t ib le to chil l ing injury. Cohn and Obendorf (8) h a v e examined the s i tua t ion a t a l a t e r s t a g e when the in t eg ra l m e m b r a n e p ro t e in s would h a v e r e t u r n e d to the i r r ightful p l aces a longside t he phospholipids so r e - e s t ab l i sh ing mi tochondr i a l m e m b r a n e i n t e g r i t y . The a c t i v i t y of mi tochondr i a from corn wi th e i t he r 5 or 13% ini t ia l mo i s tu r e

ο c o n t e n t was examined a f t e r 12, 24 or 48 hours of imbibi t ion a t 5 C. The low mo i s tu r e grains grew m o r e slowly than the o t h e r s in subsequent g rowth t e s t s , showing t h a t they had suf fe red chil l ing d a m a g e . However , t he r e sp i r a t i on r a t e , ATP c o n t e n t and energy cha rge w e r e the s a m e in high and low m o i s t u r e gra ins a f t e r imbibi t ion in the cold. Mi tochondr ia l oxidase a c t i v i t y , A D P / O r a t i o and r e s p i r a t o r y con t ro l r a t i o w e r e also subs tan t i a l ly independen t of m o i s t u r e leve l . Cohn and Obendorf c a m e to the conclusion t ha t "a d is rupt ion of ene rgy me tabo l i sm is no t a p r i m a r y cause of imbibi t ional chil l ing injury."

Π. THE LOW TEMPERATURE LIMIT FOR GERMINATION

The e f fec t of t e m p e r a t u r e on t h e ge rmina t i on of seeds has been under inves t iga t ion for m o r e than a c e n t u r y . Al ready in 1874 Haber l and t r e c ogn i z e d t h a t ba r l ey , w h e a t and o a t s would sti l l g e r m i n a t e when t h e t e m p e r a t u r e was as low as 2 or 3 C, but t h a t a number of o t h e r crop p l an t s such as corn , r i c e , c o t t o n and t o b a c c o fai led to g e r m i n a t e below about 10-12 C. Taken in conjunct ion wi th t he work on chil l ing injury discussed e l s ewhe re in this Volume these ear ly obse rva t ions seem to imply t h a t p l an t s can be divided in to two groups ( the ch i l l - r e s i s t an t and the chi l l -sensi t ive) bo th as r e g a r d s the i r behaviour as m a t u r e p l an t s and also in r e s p e c t of the i r power to g e r m i n a t e . Below about 1 0 C t h e p l an t s a r e injured and the i r seeds fail to g e r m i n a t e . It would be a shor t s t e p from this to suppose t h a t c o m p a r a b l e m e c h a n i s m s under l ie chil l ing in t he two s i t ua t ions .

4 0 Ε. w. Simon

R e c e n t work ca s t s doubt on this v iew. F i r s t it should be said t ha t spec ies l ike Agrostemma githago (Fig. 1), l e t t u c e (33) and m u s t a r d (30) a r e able to g e r m i n a t e read i ly down to t e m p e r a t u r e s nea r f r eez ing . On an Arrhenius plot such d a t a yield a single l ine (Fig. Z) cor responding to an a c t i v a t i o n energy of Ζ5.6 kcals (Agrostemma) and 17.1 kcals (mus ta rd) .

However , many spec ies p roduce curves of a d i f fe ren t kind, 50% of t h e seeds only ge rmina t ing over a l imi t ed r ange of t e m p e r a t u r e . For Ajuga reptans (Fig. 1) this ex tends from 35 C down to about 16 C w h e r e t he curve appea r s to b e c o m e ve r t i c a l ind ica t ing a very sharp cut-off , sugges t ing t h a t 50% ge rmina t ion would no t be a t t a i n e d even a f t e r pro longed per iods a t 15 C or less . C u c u m b e r seeds h a v e a sharp cut -off a t 11.5 C, and seeds ma in t a ined a t 10 C ach ieved no m o r e than 14% ge rmina t ion in 100 days (30). The t r ans i t ion from t e m p e r t u r e s a t which seeds g e r m i n a t e read i ly , to t e m p e r a t u r e s a t which few if any seeds will eve r g e r m i n a t e , is surpris ingly abrupt (3Z).

The t e m p e r a t u r e a t which this cut -off occu r s shows a wide va r i a t ion from spec ies to spec ies . Mason (17) has r e c o r d e d d a t a for 115 n a t i v e Bri t ish spec ies of which 74 had a sharp cut-off , a t a t e m p e r a t u r e ranging from 6°C for Trifolium pratense up to 30°C for Polygonum persicaria (Fig. 1). It mus t be emphas ized t h a t this las t is no r a r e excep t ion . Seeds jus t emerg ing from do rmancy under t he in f luence of a f t e r - r i pen ing (dry s t o r a g e a t room t e m p e r a t u r e ) or s t r a t i f i c a t i o n (s torage a t 5 C in damp sand or soil) can a t f irst only g e r m i n a t e over a nar row t e m p e r a t u r e r ange ; for some seeds this ini t ia l r ange is pos i t ioned a t a surpris ingly high poin t on t he t e m p e r a t u r e sca l e . Thus Vegis (34) has a d iagram for the ge rmina t ion of five weed spec ies be fo re a f t e r - r i pen ing from which it can be seen tha t t he low t e m p e r a t u r e l imit for t h r e e spec ies was 33 , and for two 3 6 ° C .

The low t e m p e r a t u r e l imi t may also vary within a single spec ies , b e t w e e n d i f fe ren t cu l t iva r s or d i f fe ren t seed lo t s . The min ima for a se r ies of cu l t iva r s of c a r r o t , leek and Brassicaeach vary over a r ange of 2 -3°C , while t he minimum for Petrorhagia proliferain G e r m a n y was 5 C, and in Hungary 9 C (33). A survey of 1Z spec ies examined by Thompson ind ica te s tha t on a v e r a g e the low t e m p e r a t u r e l imit may shift in this way by about 3-4 C.

Much m o r e r e m a r k a b l e is the shift t h a t may occur as seeds lose thei r do rmancy . Thompson (3Z) found t h a t seeds of Silene conoidea would g e r m i n a t e down to 8 C i m m e d i a t e l y a f t e r ha rves t but the minimum then fell to 3 C a f t e r one year ' s s t o r a g e a t room t e m p e r a t u r e . Hemerocal-lis s eed would only give 50% ge rmina t ion b e t w e e n Z0 and Z5 C a f t e r one week of s t r a t i f i c a t i o n , but t he lower l imi t fell in 8 weeks to less than ο 10 C (10K Accord ing to Vegis (34) fresh b i rch seeds only g e r m i n a t e a t about 30 C but acquire t he abi l i ty to g e r m i n a t e down to t e m p e r a t u r e s l i t t l e m o r e than 0 C a f t e r severa l mon ths of s t r a t i f i c a t i o n . These cons iderab le shifts in the low t e m p e r a t u r e l imit during e m e r g e n c e from dormancy , and the oppos i te m o v e m e n t said tc occur as seeds e n t e r d o r m a n c y (34, 35), must c lear ly be cons idered in any discussion of t he mechan i sm t h a t s e t s the low t e m p e r a t u r e l imit for ge rmina t i on (19).

S e e d G e r m i n a t i o n a t L o w T e m p e r a t u r e s 4 1

FIGURE 1. The effect of temperature on seed germination. Following the practice introduced (32) the time required for 50% germination is shown as a function of temperature. The lines representing the high temperature limits for the germination of each species are interrupted to draw attention to the position of the low temperature limits. A, Agrostemma githago; B, Ajuga reptans; C, Silene nutans; D, Carex nigra; E, Polygonum persicaria. A, B, C are from (32); D and Ε from (17).

4 2 Ε. W. S i m o n

FIGURE 2. Arrhenius plots of germination for three species shown in Figure 1.

The r a n g e of t e m p e r a t u r e s over which the lower l imit for ge rmina t ion may occur t h e r e f o r e ex tends from n e a r f reez ing r ight up to about 35 C, qu i t e d i f fe ren t from the r ange up to 10 or 1Z C t h a t is s o m e t i m e s r e g a r d e d as typ ica l for chil l ing injury. The r a n g e for the low t e m p e r a t u r e l imi t is also wider than t h a t of t he d i scont inu i ty in Arrhenius p lo t s for membrane -bound e n z y m e s in ch i l l - sens i t ive p l an t s , which s e e m s to run from about 4 C in apple (18) up to 15 C in mung bean (26) and Z0 C in p o s t c l i m a c t e r i c avocado (13). Moreover t h e r e does no t seem to be any c o u n t e r p a r t in t he mi tochondr ia l work to t h e shift t h a t occur s as seeds e m e r g e from do rmancy during dry s t o r a g e .

When t h e ge rmina t ion d a t a a r e expressed as Arrhenius p lo t s (Fig. Z) it b e c o m e s appa ren t t ha t they differ from mi tochondr ia l oxidase p lo t s in two addi t ional r e s p e c t s . In Ajugathere is a qu i t e ex t en d ed r ange of


TABLE I. Activation Energies (kcal) for Germination Above and Below the Break in the Arrhenius Plot.

Species Above break Below break Reference

Cucumber 12 95 (30) Mung bean 12 77 (30) Fragaria vesca 19 61 (32) Ajuga reptans 12 92 (32) Primula farinosa 14 170 (32) Silene nutans 23 89 (32) Cistus creticus 25 210 (32) Silene otites from Germany 32 170 (32)

t e m p e r a t u r e s over which ge rmina t i on p r o c e e d s a t a r a t e l i t t l e inf luenced by t e m p e r a t u r e (presumably b e c a u s e the physica l p roces s of imbibi t ion is then r a t e - l i m i t i n g ) . This is fol lowed by a r a n g e of t e m p e r a t u r e encompass ing the b r e a k in the plot and finally the curve b e c o m e s v e r t i c a l a t t he sharp cut-off t e m p e r a t u r e . The a c t i v a t i o n energy for ge rmina t i on a t t e m p e r a t u r e s below the d i scon t inu i ty (Table 1) is much higher than the f igure of Z3.0 kcal quo ted by Simon et al., (30) as an a v e r a g e from 35 Arrhen ius p lo t s for t h e a c t i v i t y of sub-ce l lu la r f r ac t ions from chi l l -sens i t ive p l an t s . F u r t h e r m o r e t h e r e is only a smal l t e m p e r a t u r e span b e t w e e n t h e d i scont inu i ty and t h e cut -off point for ge rmina t ion , amoun t ing to 1.5 C in Ajuga and Silene (Fig. Z) as c o m p a r e d to a m e a n of 7.5 C in Z0 published Arrhenius p lo t s for m e m b r a n e - b o u n d e n z y m e s y s t e m s from p l a n t s .

These d i f f e rences b e t w e e n t h e Arrhen ius p lo t s for ge rmina t i on and for e n z y m e s y s t e m s r a i s e ser ious doubts as to w h e t h e r t h e s a m e under ly ing mechan i sm is respons ib le for each . It would be i n t e r e s t i n g to know w h e t h e r Arrhen ius p lo t s for t h e a c t i v i t y of mi tochondr i a i so la t ed from e a c h of t h e seeds shown in F igs . 1 and Ζ would be s imilar in shape to t h e ge rmina t ion d a t a . However , i n fo rma t ion of this sor t is no t y e t ava i l ab le , but Ar rhen ius p lo ts for the r e sp i r a t ion of 16 hour imbibed seeds of cucumber and mung bean show no d i scon t inu i ty , a l though t h e seeds fail to g e r m i n a t e t h e cold (30), an obse rva t ion which s e e m s c o n t r a r y to t h e thes is of a common under lying m e c h a n i s m .

Moreover if t h e block to ge rmina t i on w e r e due to mi tochondr ia l dysfunct ion t h e r e should be an a c c u m u l a t i o n of toxic p roduc t s such as e thano l . Subsequent t r ans fe r of t h e seeds in to warm condi t ions would allow them to g e r m i n a t e a f t e r a lag per iod while such p roduc t s w e r e m e t a b o l i z e d away . No such de lay is in fac t observed , t h e seeds g e r m i n a t i n g a l i t t l e sooner a f t e r such a t e m p e r a t u r e s t ep -up than if they had been a t Z0 C from the s t a r t (30).

4 4 Ε. W. Simon

The ev idence p r e s e n t e d above does l i t t l e to susta in t he view t h a t t he inabi l i ty of seeds to g e r m i n a t e in the cold is m e d i a t e d by a dysfunct ion of mi tochondr i a or o the r membrane -bound enzyme s y s t e m s . T h e r e s e e m s on the c o n t r a r y to be a d i f f e rence b e t w e e n the r e sponse to cold by seeds and by m a t u r e p l a n t s . Thus a l though the r e sp i r a t i on of imbibed c u c u m b e r seeds yields an Arrhenius plot wi thou t d i scon t inu i ty (30), t h e r e a r e c lea r b reaks a t 1Z C in p lo t s of cucumber leaf r e sp i r a t ion (Z0) and cucumber fruit succinoxidase (16). Again p l an t s such as Carex n ig raand Polygonum persicaria which can g e r m i n a t e when exposed to t e m p e r a t u r e s of ZZ and Z9 C r e s p e c t i v e l y (Fig. 1), could hard ly survive for long in a Bri t ish s u m m e r if t he adult p l an t s b e c a m e chi l led as soon as t h e t e m p e r a t u r e dropped any lower . T e m p e r a t u r e s below the lower l imit for ge rmina t ion ev ident ly do not injure t he seeds or p l an t s of t he se spec ies ; they mere ly p r e v e n t ge rmina t i on . In brief, ge rmina t i on is p r e v e n t e d in these n a t i v e spec ies a t t e m p e r a t u r e s far above those respons ib le for chi l l ing injury. Why then , do these seeds fail to g e r m i n a t e a t such t e m p e r a t u r e s ?

Fa i lu re of cell division or a r e q u i r e m e n t for some s imple ho rmone or m e t a b o l i t e do not seem to be respons ib le . Nor do e n h a n c e m e n t of m e m b r a n e p e r m e a b i l i t y and loss of c o m p a r t m e n t a t i o n seem to be i m p o r t a n t consequences of imbibi t ion in the cold, because c u c u m b e r seeds can sti l l g e r m i n a t e read i ly a f t e r 350 hours i m b i b i ^ o n a t 5°C (30). Inhibi tor e x p e r i m e n t s and work on the incorpora t ion of C- leuc ine in to imbibing cucumber seed provide no suppor t for the idea t ha t t h e r e is a c o m p l e t e b reakdown of p ro te in synthes is in the cold, a l though inabi l i ty to syn thes ize c e r t a i n p a r t i c u l a r p ro t e in s v i ta l to the p rocess of ge rmina t ion r e m a i n s a possibi l i ty (19).

It is also possible t ha t p ro t e in d e n a t u r a t i o n under l ies t h e fa i lure of ge rmina t i on in the cold. This hypothes i s is in a cco rd wi th the wide r a n g e of t e m p e r a t u r e s a t which the lower l imi t for ge rmina t ion may occur , wi th the very high a c t i v a t i o n energy observed in the cold, wi th the sharp cut-off jus t below the t e m p e r a t u r e of t h e b reak , and also with the t e m p e r a t u r e s t ep -up d a t a . In addi t ion it has i n t e r e s t i n g consequences for our unders tand ing of do rmancy , which I hope to develop e l s e w h e r e .

III. R E F E R E N C E S

1. Bramlage , W. J . , Leopold, A. C. and Par i sh , D. J . Plant Physiol 61, 5Z5-5Z9 (1978).

Z. Cal , J . P . and Obendorf, R. L. Crop Science, 12, 369-373 (197Z). 3 . Chr i s t i ansen , Μ. N. Plant Physiol. 38, 5Z0-5ZZ (1963). 4 . Chr i s t i ansen , Μ. N. Crop Science, 4, 584-586 (1964). 5. Chr i s t i ansen , Μ. N. Plant Physiol. 42, 431-433 (1967). 6. Chr i s t i ansen , Μ. N. Plant Physiol. 43, 743-746 (1968). 7. Chr i s t i ansen , Μ. N. Bel twide C o t t o n Produc t ion R e s e a r c h

Confe r ence , 50-51 (1969).


8. Cohn, M. A. and Obendorf , R. L. Crop Science, 16, 449-452 (1976).

9. Cohn, M. A. and Obendorf , R. L. Amer. J. Bot. 65, 50-56 (1978). 10. Gr i e sbach , R. A. and Voth, P . D. Bot. Gaz. 118, 223-237 (1958). 11 . Highkin, H. R. and Lang, A. Planta (Berl), 68, 94-98 (1966). 12. Hobbs, P . R. and Obendorf, R. L. Crop Science, 12, 664-667

(1972). 13. Kosiyachinda , S. and Young, R. E. Plant Physiol. 60, 470-474

(1977). 14. Kra f t , J . M. and Erwin, D. C. Phytopath. 57, 866-868 (1967). 15. Larson , L. A. Plant Physiol. 43, 255-259 (1968). 16. Lyons, J . M. and Raison , J . K. Plant Physiol. 45, 386-389 (1970). 17. Mason, G. Ef fec t s of T e m p e r a t u r e on t h e G e r m i n a t i o n and Growth

of N a t i v e Species , Using T e m p e r a t u r e - G r a d i e n t Techniques . Ph .D . Thes is . Univers i ty of Sheffield (1976).

18. McGlasson, W. B. and Raison, J . K. Plant Physiol. 52 390-392 (1973).

19. McMenamin , Μ. M. T e m p e r a t u r e L imi t s for Seed G e r m i n a t i o n . Ph .D. Thesis . Queen 's Univers i ty , Belfas t (1978).

20. Minchin, A. and Simon, E. W. J. Exp. Bot. 24, 1231-1235(1973) . 2 1 . Obendorf , R. L. and Hobbs, P . R . Crop Science, 10, 563-566

(1970). 22. P e r r y , D. A. and Harr i son , J . G. J. Exp. Bot. 21, 504-512(1970) . 23 . Pol lock, Β. M. Plant Physiol. 44, 907-911 (1969). 24. Pol lock, Β. M. and Toole , V. K. Plant Physiol. 41, 221-229 (1966) . 25 . Powel l , A. A. and M a t t h e w s , S. J. Exp. Bot. 29, 1215-1229 (1978). 26. Raison , J . K. and Chapman , E. A. Aust. J. Plant Physiol. 3, 2 9 1 -

299 (1976). 27. Shor t , G. E. and Lacy , M. L. Phytopath. 66, 182-187 (1976). 28. Simon, E. W. New Phytol. 73, 377-420 (1974). 29. Simon, E. W. In "Dry Biological Sys tems" (J. H. Crowe and J . S.

Clegg , eds.) , pp . 205-224. A c a d e m i c P res s , New York (1978) . 30. Simon, E. W., Minchin, Α., McMenamin , Μ. M. and Smith , J . M.

New Phytol. 77, 301-311 (1976). 3 1 . Simon, E. W. and Wiebe, Η. H. New Phytol. 74, 407-411 (1975). 32 . Thompson, P . A. Nature (Lond.) 225, 827-831 ( 1 9 7 0 ) . 3 3 . Thompson, P . A. In "Seed Ecology" (W. Heydecke r , ed.) , pp . 31-58 .

B u t t e r w o r t h s , London (1973). 34. Vegis, A. In "Envi ronmenta l Con t ro l of P l an t Growth" (L. T.

Evans , ed.) , pp . 265-287. A c a d e m i c P res s , New York (1963). 35 . Vegis, A. Ann. Rev. Plant Physiol. 15, 185-224 (1964).



DROUGHT RESISTANCE AS RELATED TO LOW TEMPERATURE STRESS

J. M. Wilson

School of P lan t Biology Univers i ty Col lege of Nor th Wales

Bangor, Gwynedd LL57 ZUW

I. INTRODUCTION

The f irst s y m p t o m s of chi l l ing-injury to t he l eaves of many agr icu l tu ra l ly i m p o r t a n t t rop ica l and sub- t rop ica l spec ies a r e rapid leaf wi l t ing and t h e deve lopmen t of sunken, n e c r o t i c p a t c h e s within a few hours of t h e s t a r t of chil l ing (e.g. Phaseolus vulgaris and Gossypium hirsutum). On r e t u r n of t h e p l an t s to t h e w a r m t h , t h e leaf marg ins and n e c r o t i c p a t c h e s usual ly dry out giving the leaf a m o t t l e d and b r i t t l e a p p e a r a n c e . In t h e e x t r e m e l y ch i l l - sens i t ive o r n a m e n t a l spec ies Epis-cia reptans a chill ing t r e a t m e n t of only Ζ hrs a t 5 C r e su l t s in t he loss of leaf tu rgor and the deve lopmen t of w a t e r soaked p a t c h e s on the leaf su r f ace which b e c o m e n e c r o t i c if chil l ing is pro longed or the p l an t s a r e r e t u r n e d to the w a r m t h . These obse rva t ions have led to inves t iga t ions on w h e t h e r t he p e r m e a b i l i t y of t h e p l a s m a l e m m a to w a t e r and ions i nc rea se s as a resu l t of a phase change in t he m e m b r a n e l ipids.

In many crop p l an t s , such as Phaseolus vulgaris, leaf wi l t ing and injury a t 5 C can be p r e v e n t e d for up to 9 days by ch i l l -hardening the p lan t s a t 1Z C, 8 5 % R.H. ( re la t ive humidi ty) for 4 days be fo re chil l ing (Z3, Z4). Injury to these spec ies can also be p r e v e n t e d by d rough t -harden ing t h e p lan t s a t Z5 C, 4 0 % R.H. , by withholding w a t e r from t h e roo t s so t ha t t he l eaves wilt over a 4 -day per iod . Drough t -ha rden ing has been shown to be as e f f ec t i ve as ch i l l -hardening in p r even t ing chi l l ing-injury (Z5). Chil l ing-injury a t 5 C can also be p r e v e n t e d in many crop spec ies s imply by ma in ta in ing a s a t u r a t e d (100% R.H.) a t m o s p h e r e around the leaf by enclosing t h e p lan t inside a po ly thene bag before t r ans fe r from Z5 to 5 C (Z5).

Broadly speaking, ch i l l - sens i t ive p l an t s can be divided in to two c a t e g o r i e s based on A) t h e sens i t iv i ty of t h e spec ies to chil l ing-injury, B) t he abi l i ty to ha rden aga ins t chil l ing-injury and C) w h e t h e r chi l l ing-injury can be p r e v e n t e d on d i r ec t t r ans fe r from Z5 to 5 C by main ta in ing

Copyright · 1 9 7 9 by Academic Press. Inc. 4-7 All rights of reproduction in any form reserved

ISBN σ 1 2 4 6 0 5 6 0 -5

4 8 J. Μ. Wilson

TABLE I. The Division of Chill-Sensitive Species into Two Categories Based on their Sensitivity to Chilling, their Ability to Chill and Drought-Harden Against Chilling Injury at 5 C, 85% R.H., and on Whether Chilling Injury can be Delayed on Direct Transfer from 25 to 5°C by Maintaining a Saturated(100% R.H.) Atmosphere.

Category 1. e.g. Episcia reptans, Episcia cupreata, Nautilocalyx lynchii (a) Extremely chill-sensitive species which show injured spots

after only 2 h at 5°C. (b) These plants cannot be chill-hardened at 12 C, 85% R.H. or

drought-hardened at 25 C, 40% R.H. to withstand chilling injury at 5 C. Even prolonged periods of acclimatization at 15°C result in little increase in chill-tolerance.

(c) Maintaining a saturated atmosphere at 5 C does not delay the onset of injury.

Category 2. e.g. Phaseolus vulgaris, Cucumis sativus, Gossypium hirsutum (a) Less chill-sensitive species usually incurring severe leaf injury

after 24 h at 5°C, 85% R.H. (b) Chill-hardening and drought-hardening can protect the leaves

against chilling injury at 5 C, 85% R.H. for up to 9 days in P. vulgaris.

(c) Maintaining a saturated atmosphere at 5 C can prevent chilling-injury for up to 9 dayôn direct transfer of Phaseolus vulgaris leaves from 25 to 5 C. Chilling-injury can also be prevented for up to 3 days in Cucumis sativus leaves and 2 days in Gossypium hirsutum leaves by maintaining 100% R.H. at 5 C.

a s a t u r a t e d a t m o s p h e r e around the leaf. Table I shows t ha t spec ies such as Phaseolus vulgaris, Gossypium hirsutum and Cucumis sativus a r e p l aced in c a t e g o r y Ζ as t hese p lan t s a r e less ch i l l - sens i t ive and usually only incur 50% leaf injury a f t e r Z4 hrs a t 5 C, 8 5 % R.H. In addi t ion t h e s e p lan t s can be chill and d rough t -ha rdened agains t chil l ing-injury and injury can be p r e v e n t e d for up to 9 days in Phaseolus vulgaris by m a i n ta in ing a s a t u r a t e d a t m o s p h e r e around the leaf on d i r ec t t r ans fe r from Z5 to 5 C. The re fo re , chil l ing-injury to these spec ies is p r imar i ly due to w a t e r loss . However , when chil l ing is prolonged for seve ra l days by main ta in ing 100% R.H. , me tabo l i c changes must even tua l ly lead to cel l d e a t h .

In t he e x t r e m e l y ch i l l - sens i t ive c a t e g o r y 1 spec ies (Episcia reptans), w a t e r loss is less i m p o r t a n t in t he deve lopmen t of chil l ing-injury as t h e r a t e of injury cannot be s ignif icant ly r educed by main ta in ing a s a t u r a t e d a t m o s p h e r e around the l eaves . In addi t ion it is no t possible to chill or d rough t -ha rden the l eaves of this spec ies to wi ths tand chi l l ing-

Drought Resistance as Related to Low Temperature Stress 4 9

injury. Even a pro longed per iod of a c c l i m a t i z a t i o n in a cool, well v e n t i l a t e d g reenhouse a t 15 C r e s u l t e d in l i t t l e i nc rea se in chi l l -t o l e r a n c e . The re fo re , chi l l ing-injury to the l eaves of c a t e g o r y 1 spec ies is p r imar i ly m e t a b o l i c . Tropica l f ru i ts also possess l i t t l e abi l i ty to ha rden aga ins t chi l l ing-injury. A t t e m p t s a t ha rden ing s w e e t p o t a t o e s have not b e e n successful in r educ ing chil l ing-injury (Zl) and harden ing c u c u m b e r s is only e f f ec t i ve aga ins t sl ight chil l ing (1).

II. FATTY-ACID CHANGES DURING CHILL-HARDENING

A ro le for lipids and f a t t y - a c i d s in t he p reven t ion of chil l ing-injury is sugges ted by inc reases in the d e g r e e of u n s a t u r a t i o n and, of ten , weight of lipid during the a c c l i m a t i z a t i o n of p l an t s as well as po ik i lo the rmic and h o m o e o t h e r m i c an imals to low t e m p e r a t u r e s (Z4). Inc reases in the d e g r e e of unsa tu r a t i on of t h e m e m b r a n e f a t t y - a c i d s of 5 to 1Z% may p r e v e n t chi l l ing-injury by lower ing the phase t r ans i t ion t e m p e r a t u r e to below 5 C. P lan t cell m e m b r a n e s usual ly con ta in a t l eas t 70% of the i r f a t t y - a c i d s in the u n s a t u r a t e d form and Lyons and Asmundson (14) showed t h a t , in a r t i f i c ia l m ix tu re s of u n s a t u r a t e d and s a t u r a t e d f a t t y -ac ids a t this c o n c e n t r a t i o n , a 10% inc rea se in u n s a t u r a t i o n could lower t he sol id i f ica t ion t e m p e r a t u r e by as much as Z0 C . In a g r e e m e n t wi th this hypothes i s Wilson and Crawford (Z4) r e p o r t e d i nc r ea se s of 5 to 1Z% in t h e d e g r e e of unsa tu r a t i on of t h e f a t t y - a c i d s a s soc i a t ed wi th t he phospholipids of P. vulgaris and G. hirsutum l e aves during ch i l l -ha rdening a t 1Z C (Table Π). These i n c r e a s e s in t o t a l p e r c e n t a g e u n s a t u r a t e d f a t t y - a c i d w e r e mainly due to an i n c r e a s e in t he p e r c e n t a g e of l inoleic acid (Z4). No i n c r e a s e in the d e g r e e of unsa tu r a t i o n of t h e glycolipids was d e t e c t e d . Table Ζ also shows t h a t no i nc r ea se in t h e d e g r e e of u n s a t u r a t i o n of t h e phospholipids o c c u r r e d during t h e ine f f ec t ive ha rden ing of Episcia reptans a t 15 C excep t for a slight i nc r ea se in phospha t idy lchol ine .

Phase changes in t h e l eaves of ch i l l - sens i t ive p l an t s a t 12 C and below may resu l t in chil l ing-injury by inc reas ing the a c t i v a t i o n energy of m e m b r a n e bound enzymes thus caus ing m e t a b o l i c imba lances wi th non-m e m b r a n e bound s y s t e m s (13). In addi t ion phase t r ans i t ions may also i n c r e a s e t h e p e r m e a b i l i t y of m e m b r a n e s to w a t e r and e l e c t r o l y t e s leading to a loss of cel l c o m p a r t m e n t a t i o n and even tua l ly d e a t h of the l e aves . The following sec t ions e x a m i n e t h e ev idence for me tabo l i c imba l ances and inc reased m e m b r a n e p e r m e a b i l i t y in t rop ica l l eaves a t chil l ing t e m p e r a t u r e s .

ΠΙ. INCREASED ACTIVATION ENERGIES

The most f requent ly quo ted e f fec t of a r educ t ion in t he r a t e of t r i carboxyl ic acid cyc le a c t i v i t y below 12 C due to a lipid phase t r ans i t ion ,

5 0 J. Μ. Wilson

TABLE II. Changes in Total Percent Unsaturated Fatty-Acid Associated with the Phospholipids During the Chill-Hardening of Phaseolus vulgaris and Gossypium hirsutum Leaves at 12 C, 85% R.H. Changes in Unsaturation guring the Ineffective Hardening of Episcia reptans at 15 C are Included for Comparison.

Total percent unsaturated fatty-acid

Lipid* Phaseolus Gossypium Episcia vulgaris hirsutum reptans

25°C 12°C 25°C 12°C 25°C 12°C

PC 69.3 80.8 70.6 81.3 73.8 74.8 PE 63.2 66.8 60.9 67.7 72.8 71.1 PI 69.4 64.7 52.6 60.6 58.3 57.6 PA 66.9 80.8 62.6 72.4 60.7 52.0 PG 54.4 59.7 45.2 55.3 66.8 61.8

PC - Phosphatidylcholine: PE - Phosphatidyl-ethanolamine; PI -Phosphatidylinositol; PA - Phosphatidic acid; PA - Phosphatidyl-glycerol.

without a s imi lar r educ t ion in t h e r a t e of glycolysis , is t h e accumula t i on of the end p roduc t s of glycolysis (e.g. e thano l and ace ta ldehyde) to toxic leve ls , r e su l t ing in cell d e a t h . However , in t rop ica l p lan t l eaves and frui ts ev idence for an i nc rease in e thanol or a c e t a l d e h y d e c o n c e n t r a t i o n as a resu l t of chil l ing is s c a r c e . In E. reptans l eaves Wilson (Z2) d e t e c t e d an i n c r e a s e in the e thano l c o n t e n t a f t e r 6 hrs chil l ing a t 5 C. In ο banana pulp chi l led a t 6 C for 15 days M u r a t a (15) has shown an i nc r ea se in the levels of e thano l , a c e t a l d e h y d e , p y r u v a t e and a - k e t o g l u t a r a t e . Whether t h e levels of t hese m e t a b o l i t e s a r e suff ic ient ly high to cause injury to t he cel ls has not been d e t e r m i n e d .

Inc reases in t he a c t i v a t i o n energy of NAD reduc t ion by the ch loroplas t s of P. vulgaris ' e a v e s (18) and impa i red phosphoryla t ion in G. hirsutum l eaves (19) have also been a t t r i b u t e d to lipid phase t r ans i t ions . It has been sugges ted t h a t t hese changes lead to t he degene ra t i on of t h e chloroplas t s t r u c t u r e and a level of ATP which would be insuff ic ient to ma in ta in the me tabo l i c i n t eg r i t y of t h e cy top la sm. The s igni f icance of ATP changes during chill ing is d iscussed in a l a t e r sec t ion of this c h a p t e r .


IV. INCREASED MEMBRANE PERMEABILITY

Physica l changes in m e m b r a n e s t r u c t u r e such as a phase t r ans i t ion in the m e m b r a n e lipids can be e x p e c t e d to a l t e r m e m b r a n e p e r m e a b i l i t y . Trauble and Haynes (20) sugges t ed t h a t an i nc rease in m e m b r a n e p e r m e a b i l i t y would be e x p e c t e d to a c c o m p a n y the phase t r ans i t ion due to a) a d e c r e a s e in m e m b r a n e th ickness , b) changes in t h e s t r u c t u r e of t h e hydrocarbon chains i m p o r t a n t for diffusion ac ross the m e m b r a n e , or, c) changes in t h e a r r a n g e m e n t of t h e polar head groups i m p o r t a n t for t h e e n t r y of p e r m e a n t s in to the m e m b r a n e . In addi t ion it has been specu l a t ed t h a t ' c r acks ' or ' channels ' m a y appea r in t h e m e m b r a n e a t low t e m p e r a t u r e s due to the sol id i f ica t ion of the lipid, t he r eby inc reas ing m e m b r a n e p e r m e a b i l i t y . In a g r e e m e n t wi th t he se hypo theses t he major i ty of s tud ies on m e m b r a n e p e r m e a b i l i t y a t chil l ing t e m p e r a t u r e s h a v e shown an i n c r e a s e in t h e r a t e of e l e c t r o l y t e l e akage from chi l l -sens i t ive t i s sues . L i e b e r m a n n et al. (12) w e r e t h e f irs t to show t h a t t h e r a t e of l e akage of ions, mainly po tas s ium, from s w e e t p o t a t o discs was i n c r e a s e d a t 7.5 C. In addi t ion Chr i s t i ansen et al. (4) and Guinn (19) d e t e c t e d an a c c e l e r a t e d r a t e of l e akage of e l e c t r o l y t e s , p ro t e in s and c a r b o h y d r a t e s from chi l led c o t t o n r o o t s and co ty ledons . Enhanced l e akage of e l e c t r o l y t e s from chi l led leaf t i s sue has been r e p o r t e d by Wright and Simon (27); C r e e n c i a and B r a m ' a g e (5) and P a t t e r s o n et al. (16). However , in all t he se s tud ies t h e r a t e of l eakage only b e c a m e rap id

a f t e r many hours or days a t t he chil l ing t e m p e r a t u r e and this a rgues aga ins t any rap id r i se in p e r m e a b i l i t y which can be a t t r i b u t e d to lipid phase t r ans i t i ons .

15 0

0 6 1 2 1 8

Hour s afte r immersio n i n wate r

2 4

FIGURE 1. Leakage of electrolytes from leaves of Phaseolus vulgaris chilled at 5 C, 85% R.H. for 24h(k) in comparison to unchilled leaves (O), placed either in water at 25°C ( ) , or 5°C (—).

5 2 J. Μ. Wilson

Wilson (25) has c o m p a r e d the r a t e s of ion l e akage from P. vulgaris l eaves a f t e r chill ing in air a t 5 C, 8 5 % R.H. , wi th t h e r a t e of l e a k a g e on d i r ec t t r ans fe r from air a t 25 C to w a t e r a t 5 C. F ig . 1 shows t h a t l eaves

ο of P. vulgaris

T~ak e l e c t r o l y t e s if they a r e chi l led in air for 24 h r s a t 5 C,

8 5 % R.H. , so tha t the l eaves a r e badly iniured and wi l t ed before they a r e t r a n s f e r r e d to dis t i l led w a t e r a t e i t he r 25 or 5 C. The r a t e of l e a k a g e

ο from t h e air chi l led l eaves in w a t e r a t 25 C is a lmos t t w i c e as fast as t he l eaves p l aced in w a t e r a t 5 C. This may be due to a h e a t shock on t r ans fe r from 5 to 25 C. In c o n t r a s t , t he con t ro l l eaves t r a n s f e r r e d d i r ec t ly from 25 C to w a t e r a t e i the r 25 or 5 C show a very slow r a t e of e l e c t r o l y t e l e a k a g e . The behaviour of the con t ro l l eaves appea r s t o be c o n t r a r y to t h e phase t rans i t ion hypo thes i s . If t h e phase t r ans i t ion in t h e m e m b r a n e lipids causes an i nc rease in m e m b r a n e p e r m e a b i l i t y then we would expec t t he l eaves to leak e l e c t r o l y t e s when t r a n s f e r r e d d i r ec t l y from 25 C to w a t e r a t 5 C (i.e. wi thou t chil l ing in a i r ) . However , F ig . 1 shows t ha t t h e l eakage of e l e c t r o l y t e s from unchi l led l eaves of P. vulgaris t r a n s f e r r e d d i r ec t ly to w a t e r a t 5 C is very s ^ w and t h a t l e akage is f a s te r from t h e cont ro l l eaves t r a n s f e r r e d d i r ec t ly to w a t e r a t 25 C. This resu l t sugges t s tha t ini t ia l ly a phase t r ans i t ion in the m e m b r a n e lipids of t h e p l a s m a l e m m a leads to a d e c r e a s e in i t s p e r m e a b i l i t y to ions, such as Κ , a t low t e m p e r a t u r e . In support of this a r g u m e n t Blok et al. (2) have shown t h a t t h e w a t e r p e r m e a b i l i t y of l iposomes p r e p a r e d from s y n t h e t i c lec i th in d e c r e a s e s d ras t i ca l ly below t h e t r ans i t ion

FIGURE 2. Leakage of electrolytes from leaves of Emscia reptans transferred directly from 25 C, 85% R.H. to water at 25 C ( ) , or 5 C (— ). Total quantity of electrolytes in leaves - 175 \iS.


t e m p e r a t u r e . F u r t h e r m o r e , ves ic les of Escherichia coli p r e loaded wi th label led pro l ine did no t lose the i r r a d i o a c t i v i t y when incuba t ed a t t e m p e r a t u r e s below t h e t r ans i t ion (7). I nc reased r a t e s of ion l eakage from leaf t i ssues may t h e r e f o r e only occur a f t e r p ro longed chil l ing which has r e s u l t e d in the d a m a g e or d e a t h of the cel l m e m b r a n e s . F ig . 1 shows t h a t , in l eaves , cons iderab le w a t e r loss and ce.ll d a m a g e must occur during chill ing in air a t 5 C, 8 5 % R.H. , for t h e l eaves to lose the i r e l e c t r o l y t e s rapidly when t r a n s f e r r e d to dis t i l led w a t e r .

The leaves of some ch i l l - sens i t ive spec ies leak e l e c t r o l y t e s m o r e rapid ly than P. vulgaris l eaves on t r ans fe r from air a t Ζ5 C to w a t e r a t 5 C . For e x a m p l e , F ig . 2 shows t h a t l eaves of t h e e x t r e m e l y chi l l -sens i t ive c a t e g o r y 1 spec ies , Episcia reptans, lose a p p r o x i m a t e l y 25% of the i r t o t a l e l e c t r o l y t e s within 5 hrs of t r ans fe r from 25 C to w a t e r a t ο 5 C In this spec ies visible signs of cell d e a t h a c c o m p a n y the r i se in t h e conduc t iv i ty of the w a t e r so t ha t it is not possible to d i f f e r e n t i a t e b e t w e e n w h e t h e r l eakage is an i m m e d i a t e e f fec t due to a phase t r ans i t ion in the m e m b r a n e l ipids, or , w h e t h e r l eakage is a secondary e f fec t due to cel l d e a t h . At t h e p r e s e n t t i m e efflux e x p e r i m e n t s using r a d i o a c t i v e t r a c e r s a r e in p rogress to t r y to r e so lve w h e t h e r t h e r e is any rapid change in t h e p e r m e a b i l i t y of t he p l a s m a l e m m a of t he above spec ies a t low t e m p e r a t u r e which can be a t t r i b u t e d to a lipid phase t r ans i t i on . Changes in t he p e r m e a b i l i t y of leaf cel ls of Phaseolus vulgaris dur ing pro longed chill ing a t 5 C, 100% R.H. , a r e also being i n v e s t i g a t e d .

V. THE CAUSE OF LEAF WILTING IN PHASEOLUS VULGARIS LEAVES

To i n v e s t i g a t e t h e s ign i f icance of t h e i n c r e a s e in t h e d e g r e e of u n s a t u r a t i o n of t he f a t t y - a c i d s during ch i l l -hardening of P. vulgaris l eaves (Table Π), t he changes in t h e f a t t y - a c i d compos i t ion of t h e phospholipids dur ing d rough t -ha rden ing w e r e fol lowed. Al though d rough t -ha rden ing was as e f f ec t i ve as ch i l l -hardening in p r even t ing chil l ing-injury a t 5 C, 8 5 % R.H. , no i n c r e a s e in t h e d e g r e e of u n s a t u r a t i o n of t h e phospholipids or glycolipids was d e t e c t e d . In genera l the d e g r e e of unsa tu ra t i on of the f a t t y - a c i d s a s soc i a t ed wi th the phospholipids d e c r e a s e d during d rough t -ha rden ing . For e x a m p l e , Table ΠΙ shows t ha t t h e r e is a d e c r e a s e in the t o t a l p ropor t ion of u n s a t u r a t e d f a t t y - a c i d a s soc i a t ed wi th phospha t idy lchol ine during d rough t -ha rden ing so t h a t , acco rd ing to the phase t r ans i t ion hypothes i s , we would expec t t h e d r o u g h t - h a r d e n e d p lan t s to be m o r e ch i l l - sens i t ive and not chi l l -r e s i s t a n t . Chi l l -hardening of P. vulgaris l eaves a t 12 C is no t e f f ec t i ve if t he p l an t s a r e ma in t a ined a t 100% R.H. (25). Al though the d e g r e e of u n s a t u r a t i o n of t h e phospholipids i nc r ea sed during an ine f fec t ive ha rden ing t r e a t m e n t of 4 days a t 12 C, 100% R.H. , t h e r e was no i n c r e a s e in ch i l l - t o l e r ance , ind ica t ing t h a t ch i l l -hardening is not dependen t on a highly u n s a t u r a t e d f a t t y - a c i d compos i t ion . The phase t r ans i t ion

http://ce.ll

5 4 J. Μ. Wilson

TABLE III. Changes in % Fatty Acid Composition of Phosphatidyl Choline from Leaves of Phaseolus vulgaris during Chill-Hardening at 12°C, 85% R.H. and Drought-Hardening at 25°C, 40% R.H.

Fatty acid composition of phosphadidylcholine (%)

Fattv Control Chill hardened at Drought-hardened at acid

a 12°C 85% R.H. 2 5 ° C , 40% R.H.

for 4 days for 4 days

14:0 3. .8 2. 1 5. .3 16:0 20. .4 12. 8 24. .3 16:1 0. .9 1. .0 0. .7 16:2 0. .9 1. 1 0. .4 18:0 6. .5 4. 3 6. .2 18:1 4. .0 3. .5 2. .0 18:2 27. .5 40. .0 24. .1 18:3 36. .0 35. 2 37. .0

Total % unsaturated 69. .3 80. .8 64. .2 fatty acid

The numbers shown are the ratios of the number of carbon atoms to the number of double bonds in the molecule.

hypothes is is also unable to accoun t for t he p reven t ion of chil l ing-injury to l eaves of P. vulgaris by enclosing t h e p lan t inside a po ly thene bag be fo re t r ans fe r to 5 C. P l an t s ma in t a ined in a s a t u r a t e d a t m o s p h e r e for 7 days do not wilt a t 5 C. If lipid phase t r ans i t ions r e s u l t e d in an i nc r ea se in t he p e r m e a b i l i t y of t h e p l a s m a l e m m a of t h e leaf cel ls a t 5 C then we would expec t t he l eaves to wilt on t r ans fe r to 5 C, 100% R.H. , as t h e turgor p res su re of the cel l would f a c i l i t a t e the loss of w a t e r and e l e c t r o l y t e s .

It has been d e m o n s t r a t e d t ha t t he p r imary cause of chil l ing-injury to P. vulgaris l eaves a t 5 °C , 8 5 % R.H. , is w a t e r loss due to t he opening of the s t o m a t a a t a t i m e when the p e r m e a b i l i t y of the r o o t s to w a t e r is low (25). The opening of the s t o m a t a a f t e r 2 hrs a t 5 C, 8 5 % R.H. , (Fig. 3), is surpr is ing as the leaf is wi l t ed and in most p l an t s the s t o m a t a c lose in the ear ly s t ages of w a t e r s t r e s s be fo re visible wi l t ing occu r s . The r e p l a c e m e n t of the w a t e r lost by evapo t r ansp i r a t i on from the leaf is p r e v e n t e d by t h e low p e r m e a b i l i t y of t h e roo t s to w a t e r a t 5 C (Fig. 4),


Ι 2 Γ

2 μ

ι 1 I i I ι ι • 10.00 12.00 14.00 16.00 18.00 20.00 22.00

Tim e o f day

FIGURE 3. Changes in stomatal aperture on transferring entire plants of Phaseolus vulgaris directly from 25 C, 85% R.H. to (a) 5 C, 85% R.H. (h), (b) 12%, 85% R.H. (Φ), compared to the controls maintained at 25 C, 85% R.H. (O). Arrow shows start of night period.

Τ ' — ι — ' 1 " 1 1 34 35 36 34 35 36

I04/Root temperature (K-

1)

FIGURE 4. Arrhenius plots of the effect of root temperature on the rate of water absorption by Phaseolus vulgaris plants grown at 25°C, 85% R.H. ( Δ λ chill-hardened at 12°Ct 85% R.H. (Φ), and drought-hardened at 25 C, 40% R.H. (O). (a) Shows the rate of water uptake plus exudation under 50 cm Hg vacuum and (b) the rate of exudation alone. Each point represents the average value from at least five plants.

5 6 J. Μ. Wilson

1 loo ο ο *S α>

S 7 5 c Β α> Q. 2 5 0 ο

β» I » «Λ β>

"θ «j ,

0 6 1 2 1 8 2 4

Hour s chille d

FIGURE 5. Changes in leaf fresh weight on chilling either the leaves alone (O), roots alone (Φ), or the whole plant of Phaseolus vulgaris (Δ), for 24 h at 5 C, 85% R.H.M, Denotes plants grown at 12 C, 100% R.H. for 4 days before whole plant chilled at 5 C, 85% R.H. Figures beside the points show the percentage of the leaf which became necrotic after 24 h chilling and 2 days recovery at 25 C, 85% R.H.

r e su l t ing in rap id leaf dehydra t ion and injury. H e n c e t he s eve r i t y of chil l ing-injury depends on a synerg i s t i c e f f ec t b e t w e e n s t o m a t a l opening and r e d u c e d p e r m e a b i l i t y of t h e r o o t s to w a t e r a t 5 C. F ig . 5 shows t ha t when only t h e l eaves of P. vulgaris w e r e chi l led a t 5 C, 8 5 % R.H. , t h e r e was no d e t e c t a b l e fresh weight loss a f t e r 24 h rs , excep t for a sl ight wilt a f t e r 3 to 4 hrs chi l l ing. Chil l ing t h e l eaves a lone for 24 hours r e s u l t e d in only 6% injury to t h e leaf a f t e r 2 days r e c o v e r y a t 25 C, 8 5 % R .H . (Fig. 5). In the r e v e r s e e x p e r i m e n t , when roo t s a lone we re chi l led and the l eaves held a t 25 C, 8 5 % R.H. , t h e r e was a 30% d e c r e a s e in fresh weight a f t e r 24 hrs but this r e s u l t e d in only 10% injury. Chil l ing the e n t i r e p lan t of P. vulgaris a t 5°C , 8 5 % R.H. , p roduced a m o r e rapid fresh weight loss than chil l ing e i t he r t he r o o t s or l eaves a lone . F ig . 5 shows t h a t chil l ing t h e whole p lan t for 24 hrs r e su l t ed in a 50% d e c r e a s e in fresh weight and 4 2 % injury on r e t u r n to 25 C. The cause of s t o m a t a l opening in chi l l -sens i t ive spec ies a t 5 C is unknown. Pe rhaps a phase t r ans i t ion in t h e m e m b r a n e s of the guard cel ls a l t e r s the i r p e r m e a b i l i t y p r o p e r t i e s or , a l t e r n a t i v e l y , Κ ,H - A T P a s e pumps may be i n a c t i v a t e d a t low t e m p e r a t u r e s .


i 2 r

2h

I I _J I I I I I 10.00 12.00 14.00 16.00 18.00 20.00 22.00

Time o f da y

Figure 6. Changes in stomatal aperture of Phaseolus vulgaris plants on chilling at 5 C, 85 % R.H. after being hardened at 12°C, 85% R.H. (Φ), or ineffectively hardenedat 12°C, 100% R.H. (by enclosure in a polythene bag) ( 9 ) , compared to the controls maintained at 25 C, 85% R.H. (O). Arrow shows start of night period.

VI. THE MECHANISM OF CHILL- AND DROUGHT-HARDENING

Chi l l -harden ing a t 1Z°C, 8 5 % R.H. , p r e v e n t s leaf dehydra t ion by condi t ioning t h e s t o m a t a so t ha t they close on t r ans fe r to 5 C, 8 5 % R.H. (Fig. 6). Similar ly, d rough t -ha rden ing causes s t o m a t a l c losure and the s t o m a t a r e m a i n c losed on t r ans fe r to 5 C, 8 5 % R.H. Al though chi l l -ha rden ing r e s u l t e d in an i n c r e a s e in the p e r m e a b i l i t y of the r o o t s to w a t e r a t low t e m p e r a t u r e , d rough t -ha rden ing p roduced a l a rge d e c r e a s e in roo t p e r m e a b i l i t y (Fig. 4) and y e t d rough t -ha rden ing was as e f f ec t i ve as ch i l l -hardening in p r e v e n t i n g leaf injury. The re fo re , t h e mos t i m p o r t a n t f a c t o r in t h e p r e v e n t i o n of chi l l ing-injury to P. vulgaris during chi l l - and d rough t -ha rden ing is t he c losure of t h e s t o m a t a . This can be d e m o n s t r a t e d by spraying t h e l eaves of p l an t s grown a t 25 C, 8 5 % R.H. , wi th 100 μ m abscis ic ac id (ABA) which causes s t o m a t a l c losure wi thin 24 h r s . On t r ans fe r to 5 C, 8 5 % R.H. , t h e sp rayed l eaves do not wil t as t he s t o m a t a r e m a i n closed and injury is p r e v e n t e d for a p p r o x i m a t e l y 2 days by which t i m e t h e e f f ec t i venes s of t h e ABA has d e c r e a s e d . This d e c r e a s e in the e f f ec t iveness of appl ied ABA is surpr is ing as one might expec t t he p lan t to syn thes i ze ABA during this 2-day per iod . P e r h a p s 5 C is too low a t e m p e r a t u r e for t he synthes is of ABA in ch i l l - sens i t ive p l a n t s .

5 8 J. Μ. Wilson

During chi l l -hardening a t 1Z°C, 8 5 % R.H. , t h e p lan t e x p e r i e n c e s a w a t e r s t r e s s (as shown by the t e m p o r a r y wi l t ing of the leaves) due to t he opening of t he s t o m a t a (Fig. 3) and the d e c r e a s e in t he p e r m e a b i l i t y of t he r o o t s t o w a t e r (Fig. 4) . However , a t t h e i n t e r m e d i a t e t e m p e r a t u r e of 1Z C t h e s t r e s s is not s eve re enough to resu l t in d a m a g e and t h e wi l t ing vanishes a f t e r 1Z h r s . Similar ly, during d rough t -ha rden ing the w a t e r s t r e s s is imposed simply by withholding w a t e r from t h e r o o t s under condi t ions of high evapo t r ansp i r a t i on so t ha t the l eaves wi l t . P l an t s m a i n t a i n e d a t 5 or 1Z C, 100% R.H. , do no t ha rden b e c a u s e they e x p e r i e n c e no w a t e r s t r e s s . Even though the s t o m a t a a r e open under t he se condi t ions no w a t e r can be lost from t h e leaf so t h a t t h e s t o m a t a r e m a i n fully open on t r ans fe r to 5 C, 8 5 % R.H. , (Fig. 6). P l a n t s held a t 1Z°C, 100% R.H. , for 4 days and then chi l led for Z4 hrs a t 5 °C , 8 5 % R.H. , suffer a 55% d e c r e a s e in fresh weight and a p p r o x i m a t e l y 50% leaf injury, a f t e r Ζ days r e c o v e r y a t Z5 C (Fig. 5). The re fo r e , enc losure in a po ly thene bag is no t a me thod which can be used to lower the ha rden ing t e m p e r a t u r e below 1Z C. Al though the above e x p e r i m e n t s i nd i ca t e t h a t ABA is not syn thes ized in P. vulgaris l eaves a t 1Z C, 100% R.H. , it is no t known whe the r low t e m p e r a t u r e a lone , in the absence of w a t e r s t r e s s , can induce ABA syn thes i s . However , t h e co r r e l a t i on b e t w e e n chill and d rough t -ha rden ing has shown t h a t an i n t e r m e d i a t e t e m p e r a t u r e of 1Z C is not essen t ia l for ha rden ing . The re fo re , w a t e r s t r e s s and not low t e m p e r a t u r e per se is t he p r i m a r y f ac to r inducing hardening agains t chil l ing-injury in P. vulgaris l e aves .

Vn. CAUSES OF CHILLING-INJURY TO PHASEOLUS VULGARIS LEAVES AT 5°C , 100% R.H.

Al though changes in s t o m a t a l a p e r t u r e and roo t p e r m e a b i l i t y to w a t e r a r e able to explain the rap id wi l t ing , dehydra t ion and u l t i m a t e l y injury to t h e l eaves during chill ing a t 5 C, 8 5 % R.H. , an a l t e r n a t i v e explana t ion must be sought for the d e a t h of the l eaves a f t e r 9 days a t 5 C, 100% R.H. The loss of tu rgor , b leach ing and nec ros i s of P. vulgaris l eaves a f t e r 9 days a t 5 C, 100% R.H. , sugges t s t h a t injury is probably due to a combina t ion of f ac to r s which may develop from a phase t r ans i t ion even t in t he m e m b r a n e l ipids.

A. Lipid and Fatty-Acid Changes

Pre l imina ry e x p e r i m e n t s on t h e changes in m e m b r a n e lipid composi t ion during prolonged chil l ing have r e v e a l e d no s ignif icant d e c r e a s e in t h e weigh ts of phospholipids or glycolipids during t h e f irst 4 days a t 5 C, 100% R.H. This i nd ica t e s t h a t up to this t ime t h e r e is no i nc r ea se in phosphol ipase a c t i v i t y . However , during this per iod t h e r e was a d e c r e a s e in t h e p e r c e n t a g e of l inoleic ac id a s soc i a t ed wi th t he


phospholipids but no change in t h e f a t t y - a c i d compos i t ion of t h e glycol ipids . Changes in the f a t t y - a c i d and lipid compos i t ion during pro longed chil l ing may l ead to changes in m e m b r a n e p e r m e a b i l i t y and funct ion and seve re ly a f f ec t the r eve r s ib i l i t y of the phase change on r e t u r n of t h e p l an t s to t h e w a r m t h .

B. Photo-oxidation of Plant Pigments

The b leach ing of some P. vulgaris l e aves a f t e r 7 days chil l ing a t 100% R.H. i nd i ca t e s pho to -ox ida t i ve deg rada t i on of the leaf p i g m e n t s . Hasse l t and S t r ikwerda (10) have shown tha t s e v e r e pho toox ida t ion of t h e leaf p i g m e n t s and m e m b r a n e lipids occurs in c u c u m b e r l eaves a f t e r Ζ to 3 days chil l ing a t 1 C. At t e m p e r a t u r e s h igher than 1 C this type of d a m a g e develops m o r e slowly.

C. Photosynthesis and Translocation

It is well known t h a t pho tosyn thes i s is m o r e sens i t ive to low t e m p e r a t u r e than r e sp i r a t i on so t h a t s t a r v a t i o n of p lan t t i s sue may occur dur ing pro longed chi l l ing. T rans loca t ion is inhib i ted in ch i l l - sens i t ive spec ies a t 5 C which may lead to t he s t a r v a t i o n of n o n - p h o t o s y n t h e t i c p a r t s of t he p l an t , and the a c c u m u l a t i o n of s t a r c h in the ch loroplas t may fu r ther inhibit pho tosyn thes i s . G iaqu in ta and Geiger (8) sugges ted t h a t t h e ces sa t ion of t r ans loca t ion in ch i l l - sens i t ive spec ies a t 5 C is due to a phase change in the m e m b r a n e lipids of the p l a s m a l e m m a of the s ieve tube r e su l t i ng in t he col lapse of t h e m a t e r i a l l ining the cell and t h e b lockage of the s ieve p l a t e by the flow of cy top l a sm, o rgane l l e s , P -p ro t e in and m e m b r a n e s in to i t .

D. ATP Supply

Fig . 7a shows t h a t t he ATP and ADP levels in P. vulgaris l eaves chi l led a t 5 C, 100% R.H. , i n c r e a s e d over t he f irst Z4 hrs and r e m a i n e d high dur ing the following seven days . Even a f t e r 8 or 9 days chil l ing in a s a t u r a t e d a t m o s p h e r e t h e r e was only a sl ight fall in t he ATP and A D P levels and this coincided wi th t he deve lopmen t of visible signs of chi l l ing-injury to t h e leaf. A d e c r e a s e in ATP supply below t h a t n e c e s s a r y to ma in ta in the m e t a b o l i c i n t e g r i t y of the cy top lasm canno t t h e r e f o r e be cons idered to be t h e cause of chi l l ing-injury to l eaves a t 5 C, 100% R .H . In a g r e e m e n t with this r e su l t Jones (11) has also r e p o r t e d an i n c r e a s e in t h e ATP level of P. vulgaris l eaves a t low t e m p e r a t u r e s . The i nc rea se s in ATP level of P. vulgaris l eaves a t 5°C may be due to t h e cold sens i t iv i ty of ATPase which is r ead i ly i n a c t i v a t e d a t low t e m p e r a t u r e s (17).

6 0 J. Μ. Wilson

ο IZOOr - (o ) I 2 0 0 r (b )

100 0

0 1 2 3 4 5 6 7 8 9

FIGURE 7. Changes in the levels of ATP (Φ), ADP (0), and AMP (A) in the leaves of Phaseolus vulgaris, (a) during the prevention of chilling-injury and water loss by maintaining a saturated (100% R.H J atmosphere around the leaves on transfer from 25°C to 5°C and (b) during the development of chilling-injury and water loss on direct transfer from 25 C to 5 C, 85% R.H. Each point is the mean of three replicates.

«δ 6 0 0 p

α 4 8 1 2 1 6 2 0 2 4 Hour s chille d

FIGURE 8. Changes in the levels of ATP (Φ), ADP (O) and AMP (Δ ) in the leaves of Episcia reptans during chilling in the dark at 5 C. Each point is the mean of three replicates.


In c o n t r a s t , chil l ing l eaves of P. vulgaris a t 5 °C , 8 5 % R.H. , r e s u l t e d in rap id leaf wi l t ing and injury and the ATP and A D P levels d e c r e a s e d a f t e r 1Z hours chil l ing (Fig. 7b). Al though t h e l eaves chi l led a t 5 C, 8 5 % R.H. , w e r e app rox ima te ly 50% injured a f t e r 24 hrs , t h e level of ATP had d e c r e a s e d by less than 3 3 % , ind ica t ing t ha t leaf dehydra t ion and not a fall in ATP supply is t h e cause of cell ' d e a t h . The impa i r ed phosphory la t ion of c o t t o n l eaves a t 5 C r e p o r t e d by S t e w a r t and Guinn (19) can be a t t r i b u t e d to the e f f e c t s of w a t e r s t r e s s and not low t e m p e r a t u r e per se.

Vm. CAUSES OF CHILLING-INJURY TO EPISCIA REPTANS LEAVES

A. ATP Supply

Although chil l ing l eaves of Episcia reptans for 5 hr a t 5°C produced s e v e r e leaf injury t h e r e was no rap id d e c r e a s e in ATP level dur ing the f irs t 5 hr of chi l l ing. F ig . 8 shows t h a t t h e ATP and ADP levels fell by only 2 5 % a f t e r 5 hr of chi l l ing. Pro longed chil l ing b e t w e e n 5 and 24 hr r e s u l t e d in a fu r the r g radua l dec l ine in ATP level and a s t ab i l i za t ion of ADP and AMP leve l s . A r e d u c e d A T P supply is t h e r e f o r e unl ikely to be t h e cause of chil l ing-injury to E. reptans (26).

B. Leaf Respiration

Although changes in r e s p i r a t o r y behaviour h a v e been widely i nves t i ga t ed in r e l a t i on to chi l l ing-injury in f rui ts t h e r e h a v e been c o m p a r a t i v e l y few s tud ies of changes in t h e r e sp i r a t i on r a t e of chi l l -sens i t ive l eaves a t 5 C, espec ia l ly in very sens i t ive l eaves such as Episcia reptans. In cucumber f rui ts Eaks and Morris (6) d e t e c t e d a doubling of t he r e sp i r a t i on r a t e a f t e r 8 days a t 5 C and this i nc r ea se coincided wi th t he onse t and deve lopmen t of chi l l ing-injury. This was followed by a dec l ine in t h e r e sp i r a t i on r a t e a t t h e t i m e of t h e genera l d e a t h of t he t i s sue . Al though Eaks and Morris w e r e unab le to explain t he cause of t he r e s p i r a t o r y i n c r e a s e C r e e n c i a and Bramlage (5) have shown, using t h e uncoupl ing agen t 2, 4 -d in i t rophenol (DNP), t ha t the r e s p i r a t o r y i n c r e a s e in Zea mays l eaves a f t e r p ro longed chil l ing for 24 to 48 hrs a t 0.3 C is p a r t l y due to t h e uncoupling of ox ida t ive phosphory la t ion a t chil l ing t e m p e r a t u r e s .

In E. reptans l eaves F ig . 9a shows t h a t a f t e r only 80 min a t 5 C t h e oxygen u p t a k e r a t e is t h r e e t i m e s higher than tha t of the con t ro l s m a i n t a i n e d a t 25 C. The r a t e of ca rbon dioxide p roduc t ion also i nc reased to a max imum a f t e r 80 min a t 5 C but only to a max imum value of one - th i rd of the r a t e of oxygen u p t a k e (Fig. 9b). The r e s p i r a t o r y peak a f t e r 80 min a t 5 C co inc ided wi th t h e d e v e l o p m e n t of dark w a t e r soaked p a t c h e d on the leaf and the loss of leaf tu rgor . P l an t s chi l led for 80 min

6 2 J. Μ. Wilson

3 · 0 Γ (α) 3 · 0 Γ (b)

4 / \

Minute s chillin g

FIGURE 9. (a) Oxygen uptake and (b) carbon dioxide evolution in the dark by Zegf discs of Episcia reptans chilled ( ) at 5°C (Δ), 10°C (Φ) and 11 C (m)in comparison to unchilled ( ) leaf discs at 12.5 C (b)and25°C (O).

a t 5°C showed few signs of injury on r e t u r n to 2 5 ° C . However , m o r e prolonged chil l ing r e s u l t e d in a gradual dec l ine in r e sp i r a t ion r a t e and an i nc rease in leaf nec ros i s unt i l a f t e r 5 hrs a t 5 C none of t h e l eaves r e c o v e r e d on subsequent t r ans fe r to 25 C.

Chill ing a t t e m p e r a t u r e s h igher than 5 C de layed the onset of t h e r e s p i r a t o r y r i se and r e d u c e d i t s he ight (Fig. 9a) . In l eaves chi l led a t 10 C the r e sp i r a t ion r a t e was max imal a f t e r 100 min and peak height was r e d u c e d by one-f i f th in compar i son to l eaves chi l led a t 5 C. The f irst visible signs of chil l ing-injury a t 10 C also o c c u r r e d a t app rox ima te ly t he s a m e t i m e as max imum resp i r a t ion r a t e . The e x t r e m e ch i l l - sens i t iv i ty of E. reptans is d e m o n s t r a t e d by t h e 90% injury incur red a f t e r 5 hrs a t t h e r e l a t ive ly high chill ing t e m p e r a t u r e of 10 C followed by 2 days a t 25 C (26). At 11 C t h e r e was an ini t ia l d e c r e a s e in r e sp i r a t i on r a t e followed by a ^gradual i nc r ea se to the level of the con t ro l l eaves ma in t a ined a t 25 C a f t e r 160 min (Fig. 9a). The deve lopmen t of chi l l ing-injury was also slow a t 11 C, t h e l eaves incurr ing only 20% injury a f t e r 5 h r s chil l ing and 2 days r e c o v e r y a t 25 C, which i nd i ca t e s t h a t 11 C is nea r the upper t e m p e r a t u r e l imi t for chil l ing-injury in this spec ie s . An upper t e m p e r a t u r e l imi t of 11 to 12 C for chil l ing-injury in E. reptans is suppor ted by t h e absence of any i n c r e a s e in t h e r e sp i r a t ion r a t e of t h e l eaves held for 5 hrs a t 12.5 C (Fig. 9a) and t h e deve lopmen t of only 5% injury on t r ans fe r to 25 C (26). At 12.5 C the r e sp i r a t ion r a t e d e c r e a s e d to app rox ima te ly one - th i rd of t h e r a t e of t h e con t ro l l eaves a t 25 C and r e m a i n e d a t this level over t he 5 hr per iod . However , prolonged chil l ing


over severa l days a t 12 to 15 C can cause chil l ing-injury to E. Reptans (24). In c o n t r a s t to E. reptans t h e r e was no i n c r e a s e in t h e r e sp i r a t i on r a t e of Phaseolus vulgaris l e aves dur ing t h e f irs t 12 hrs of chil l ing a t 5°C (26).

E x p e r i m e n t s on t h e cause of this r e s p i r a t o r y i n c r e a s e in E. reptans l e aves a t 5 C, have been m a d e using 2 ,4-din i t rophenol (DNP) and po tass ium cyan ide (KCN). F ig . 10 shows t h a t t r e a t i n g leaf discs of E. reptans wi th 1 mM D N P doubled t h e oxygen u n t a k e r a t e a t 25 C. However , t r ans fe r r ing t h e D N P - t r e a t e d discs to 5 C did no t lessen t h e e f fec t of D N P , as would be e x p e c t e d if chil l ing caused the uncoupl ing of ox ida t ive phosphory la t ion . F ig . 10 shows t h a t chil l ing t h e D N P - t r e a t e d leaf discs r e s u l t e d in an e x t r e m e l y rap id th ree - fo ld i n c r e a s e in oxygen u p t a k e within 40 min of t h e s t a r t of chi l l ing, whilst in t h e u n t r e a t e d con t ro l s a t 5 C, t h e oxygen u p t a k e did not a t t a i n i t s max imum va lue unt i l 80 min a f t e r the s t a r t of chi l l ing. The rap id a c c e l e r a t i o n of r e sp i r a t i on by t h e addi t ion of D N P showed t h a t this m e t h o d could no t be used to d e t e r m i n e w h e t h e r any p a r t of t he r e s p i r a t o r y i n c r e a s e was due to the uncoupl ing of ox ida t ive phosphory la t ion . F ig . 10 also shows t h a t t he r e sp i r a t i on r a t e of leaf discs t r e a t e d wi th 10 mM KCN a t 5 C was a c c e l e r a t e d in the s a m e manne r as the D N P - t r e a t e d d iscs . A seven-fold

4 · 5 Γ

4 0 8 0 12 0 16 0 2 0 0 2 4 0 2 8 0

Minute s

FIGURE 10. The effects of 1 mM DNP (Φ) and 10 mM KCN (O) on the oxygen uptake ( ) and carbon dioxide evolution ( ) rates of Episcia reptans leaf discs at5C in comparison to untreated, control discs at 5 C

6 4 J. Μ. Wilson

i n c r e a s e in t he oxygen u p t a k e r a t e of KCN t r e a t e d l eaves o c c u r r e d within 40 min of the s t a r t of chi l l ing. The rap id i n c r e a s e in the r a t e of oxygen u p t a k e by t h e D N P - and K C N - t r e a t e d leaf discs a t 5 C is no t a c c o m p a n i e d by a higher r a t e of carbon dioxide p roduc t ion (Fig. 10). The addi t ion of D N P and KCN to E. reptans l e aves on chil l ing is thought to i nc r ea se the r a t e of oxygen u p t a k e by dec reas ing m e m b r a n e s t ab i l i t y a t low t e m p e r a t u r e s .

It is sugges ted t ha t the cause of this rapid wound- induced r e sp i r a t i on in E. reptans l eaves a t 5 C is an i n c r e a s e in t he p e r m e a b i l i t y of t h e cel l m e m b r a n e s a t low t e m p e r a t u r e due to e i t he r lipid phase t r ans i t ions (13), p ro te in d e n a t u r a t i o n (3), changes in l ip id-pro te in i n t e r a c t i o n (28), or a combina t ion of these e v e n t s . The m a g n i t u d e of t he phase change in the m e m b r a n e s of E. reptans l eaves may be far g r e a t e r than in P. vulgaris and this may accoun t for t he more rapid deve lopmen t of chil l ing-injury in E. reptans. A rapid change in t he p e r m e a b i l i t y of t h e p l a s m a l e m m a and tonoplas t would accoun t for t h e speed of injury to E. reptans and t h e rapid r ise in r e sp i r a t ion r a t e due to t he loss of cell c o m p a r t m e n t a t i o n which would allow e n z y m e s inc reased a c c e s s to s u b s t r a t e s . It is s p e c u l a t e d t h a t t h e oxidat ion of phenols r e l e a s e d from the vacuole as a r e su l t of a change in tonoplas t p e r m e a b i l i t y may a c c o u n t for the rap id r i se in wound- induced r e sp i r a t ion in this spec ies .

Expe r imen t s a r e in p rogress on t h e changes in m e m b r a n e p e r m e a b i l i t y and lipid compos i t ion of E. reptans l eaves during chil l ing. An e l ec t ron mic roscope s tudy of t h e deve lopmen t of chil l ing-injury in Episcia reptans l eaves has r e v e a l e d unusual changes in cy top la smic s t r u c t u r e .

IX. R E F E R E N C E S

1. Apeland, J . Bull. Int. Inst. Refrig. 46, 325 (1966). 2. Blok, M. C , van Deenen , L. L. M. and De Gier , Biophys. Acta.

433, 1 (1976). 3 . Brand t s , J . F . "Heat Ef fec t s on P ro t e in s and Enzymes . " In Thermo-

biology (Ed. by A. H. Rose) . A c a d e m i c P res s , London (1967). 4 . Chr i s t i ansen , Μ. N. , C a m s , H. R. and Slyter , D. J . PI. Physiol. 46,

53 (1970). 5. C r e e n c i a , R. P . and Bramlage , W. J . PI. Physiol. 47, 389 (1971). 6. Eaks , I. L. and Morris , L. L. PI. Physiol. 31, 308 (1956). 7. Esfahan, M., Limbr ick , A. R., Knu t ton , S., Oka, T. and Wakil, S. J .

Piur. Nat. Acad. Sci. U.S.A. 68, 3180 (1971). 8. Giaqu in ta , R. T. and Geiger , D. R. PI. Physiol. 51, 372 (1973). 9. Guinn, G. Crop Sci. 11, 101 (1971). 10. Hasse l t , Ph .R . van and S t r ikwerda , J . T. Physiol. Plant. 37, 253

(1976). 11 . Jones , P . C. T. J. Exp. Bot. 21, 58 (1970). 12. L iebe rmann , M., Cra f t , C . C , Audia, W. V. and Wilcox, M. S. PI.

Physiol. 33, 307 (1958).


13. Lyons, J . M. Ann. Rev. Plant Physiol. 24, 445 (1973). 14. Lyons, J . M. and Asmundson, C. M. J. Am. Oil Chem. Soc. 42,

1056 (1965). 15. M ura t a , T. Physiol. Plantarum, 22, 401 (1969). 16. P a t t e r s o n , B. D. , Mura t a , T. and G r a h a m , D. Aust. J. Plant Physiol.

3, 435 (1976). 17. Penefsky , H. S. and Warner , R. C. J. Biol. Chem. 250, 4694 (1965). 18. Shneyour, Α., Raison, J . K. and Smill ie , R. M. Biochem. Biophys.

Acta. 292, 152 (1973). 19. S t e w a r t , J . McD. and Guinn, G. PI. Physiol. 44,605 (1969). 20. Trauble , H. and Haynes , D. H. Chem. Phys. Lipids, 7, 324 (1971). 2 1 . Wheaton , T. A. and Morr is , L. L. Proc. Am. Soc. Hort. Sci. 91,

529 (1967). 22. Wilson, J . M. Ph .D . Thesis , Univers i ty of St. Andrews (1974). 23 . Wilson, J . M. and Crawford , R. Μ. M. J. Exp. Bot. 25, 121 (1974a). 24. Wilson, J . M. and Crawford , R. Μ. M. New Phytol. 73,805 1974b). 25. Wilson, J . M. New Phytol. 76, 257 (1976). 26. Wilson, J . M. New Phytol. 80, 325 (1978). 27. Wright , M. and Simon, E. W. J. Exp. Bot. 24, 400 (1973). 28. Y a m a k i , S. and Ur i t an i , I. PI. Cell Physiol. 15, 385 (1974).



LOW TEMPERATURE RESPONSES OF THREE SORGHUM SPECIES

David Bagnall

CSIRO Division of P l an t Indust ry C a n b e r r a C i ty , 2601 , Aus t r a l i a

I. INTRODUCTION

P l a n t s possess ing t h e C^ p a t h w a y of pho tosyn thes i s (13) a r e of ten suscep t ib le to injury a t t e m p e r a t u r e s above 0 C (32), wi th g rowth and d e v e l o p m e n t usual ly very slow within t h e chil l ing r a n g e of 0 to 15 C (6, 15, 33). The grain c e r e a l Sorghum bicolor L. (Moench) is a C p lan t t h a t is p a r t i c u l a r l y chil l ing sens i t ive (8, 10, 20, 29)> requ i r ing nof only frost f r ee per iods for g rowth but t e m p e r a t u r e s above 10 C for ge rmina t ion and e m e r g e n c e (25). Specif ic s t a g e s of deve lopmen t can be p a r t i c u l a r l y t e m p e r a t u r e sens i t ive ; for example night t e m p e r a t u r e s of 13 C or less a t t h e t i m e of pol len meiosis m a y induce m a l e s t e r i l i t y (3, 10).

O t h e r spec ies of sorghum a r e less suscep t ib le to chil l ing injury than S. bicolor (9, 30). Johnson grass (S. halepense) and an Aus t ra l i an wild sorghum (S. leiocladum) a r e two spec ies able to w i th s t and t h e s e v e r e c l i m a t e of C a n b e r r a , Aus t r a l i a , having in one yea r survived 40 s e v e r e f ros ts and m e a n daily t e m p e r a t u r e s of 6 C in t h e mon ths of July and Augus t . S. leiocladum is a n a t i v e grass of e a s t e r n Aus t r a l i a and is t he only n a t i v e sorghum found in t h e co lder sou the rn t ab le lands reg ion of Aus t r a l i a (4). Johnson grass , a n a t i v e of t h e M e d i t e r r a n e a n reg ion , is a w e e d in Aus t ra l i an p a s t u r e s . It has a s igni f icant ly h igher g rowth r a t e than S. bicolor a t low day /n igh t t e m p e r a t u r e s - 15 / 1 0 C (30). In t h e e x p e r i m e n t s of T a y l o r e t al.(30) l eaves ofS. bicoZorwere ch lo ro t i c while those of Johnson grass and i t s hybrids w e r e g r e e n e r than o the r chil l ing r e s i s t a n t spec ies and l ines . The abi l i ty of Johnson grass to survive f rost ing t e m p e r a t u r e s may however be due to t he e x i s t e n c e of ex t ens ive c reep ing r h i z o m e s which allow t h e p lan t to g e n e r a t e new shoots in spring (9), and not to some c h a r a c t e r i s t i c of t h e p lan t ' s leaf t i s sue which mainly dies back in w in t e r .

In this pape r t h e g rowth responses of v e g e t a t i v e p lan t s of t he se t h r e e spec ies a r e examined a t d i f fe ren t t e m p e r a t u r e s to d e t e r m i n e t he r e l a t i v e i m p o r t a n c e of night t e m p e r a t u r e , pho tosyn thes i s , chlorophyll f o rma t ion and deg rada t i on , and w a t e r s t r e s s to the i r g rowth and survival

Copyright · ΙΘ7Θ by Academic Press. Inc. 6 7 All rights of reproduction in any form reserved

ISBN 0 1 2 46056O5

6 8 D. Bagnall

a t low t e m p e r a t u r e s . Within this s tudy t h e c r i t i ca l t e m p e r a t u r e s is def ined as t ha t t e m p e r a t u r e a t which a p lan t fails to a c c u m u l a t e dry m a t t e r over one week (27). This t e m p e r a t u r e cor responds c losely to t he t e m p e r a t u r e a t which the p lan t does not survive in the long t e r m and a t which chill ing lesions appea r as desc r ibed by Taylor and Rowley (32).

A. Experimental

1. Relative Growth Rates at Different Constant Temperatures. Plan t s of t he spec ies S. bicolor L. (Moench) cv Texas 610, S. leiocladum (Hack) G. E. Hubbard and S. halepense (L.) P e r s . w e r e g e r m i n a t e d and grown in 13 cm po t s conta in ing equal p ropor t ions of p e r l i t e and v e r m i c u l i t e in a 21 /16 C glasshouse of t h e C a n b e r r a p h y t o t r o n (23). The p lan t s w e r e t r a n s f e r r e d to na tu ra l ly lit g lasshouse C c a b i n e t s (23) held cont inuously a t 5, 10, 15, 20 and 25 C for 10 days and r e l a t i v e g rowth r a t e s c o m p u t e d . The ages of t h e p l an t s a t t he t i m e of t r ans fe r w e r e 72 days for S. leiocladum, 36 days for S. halepense and 21 days for S. bicolor. These d i f fe rences in age w e r e used to obta in p lan t s of s imi lar s i ze . Maximum rad i a t i on in t h e cab ine t s r e a c h e d ' 1 , 5 5 0 μ Ε m s a t midday .

2. Relative Growth Rates at Various Night Temperatures. S. bicolor p l an t s t h a t had been grown in a 21 / 16 C glasshouse w e r e t r a n s f e r r e d to a r ange of night t e m p e r a t u r e s 18 days from p lan t ing . Con t ro l p l an t s con t inued to r e c e i v e 8 h (day) a t 21 C and 16 h a t 16 C in t h e n a t u r a l l y li t g lasshouse . B e t w e e n 0800 h and 1600 h ^ o t h e x p e r i m e n t a l and con t ro l p l an t s w e r e held a t 21 C in t he g lasshouse . At 1600 h t he e x p e r i m e n t a l p l an t s we re t r a n s f e r r e d to a r t i f i c ia l ly lit L.B. c a b i n e t s . B e t w e e n 1600 and 1800 h and 0600 and 0800 h t h e e x p e r i m e n t a l p lan t s w e r e held a t 13 C in t h e dark , while b e t w e e n 1800 and 0600 h they w e r e sub jec ted to t e m p e r a t u r e s of 1.5 , 4 , 7 , 1 0 or 13 C in t he dark .

3. Photosynthetic Response to Low Temperature. Carbon dioxide exchange r a t e was measu red in a w a t e r - c o o l e d ass imi la t ion c h a m b e r (14) under no rma l L.B. cab ine t l ight ing (23) supp l emen ted wi th a 1000W Phillips^rL^LR Mercury Vapour L a m p . The i r r a d i a n c e a t t h e leaf ^was 950 μ Ε m s . Air flow r a t e s va r i ed from 1.5 to 2.0 l i t r e s min and t h e cab ine t t e m p e r a t u r e was held a t 20 C . The t e m p e r a t u r e of t h e leaf zone within the j a c k e t was lowered from 20 to 5 C over a per iod of about 2 h, t he leaf zone was then kep t below 5 C for t i m e s ranging from 10 min to 3 h (average about 75 min) and then subsequent ly r e h e a t i n g from 5 to 20 C over about 2 h. Var ia t ion of t i m e a t low t e m p e r a t u r e was used to assess w h e t h e r t he t i m e f ac to r was i m p o r t a n t in t h e p a t t e r n of r e c o v e r y . The t e m p e r a t u r e of t h e leaf zone in t h e c h a m b e r was m e a s u r e d wi th a c o p p e r - c o n s t a n t a n t he rmocoup le p l aced aga ins t t h e unders ide of t he leaf.

Low Temperature Responses of Three Sorghum Species 6 9

4. Relative Growth Rates and Light Intensity. P l an t s of t h e t h r e e spec ies t h a t had been grown in t he Zl / 1 6 C glasshouse w e r e t r a n s f e r r e d to na tu r a l l y l i t g lasshouse Β c a b i n e t s (Z3) held a t c o n s t a n t t e m p e r a t u r e s of 8.5 , 1 1 . 0 and 13.0 C . In e a c h cab ine t one-half of t h e seedl ings w e r e kept under shade c lo th t h a t r e d u c e d r ad i a t i on to 4 0 % of t h a t within t he c a b i n e t . The seedl ings w e r e 35 day old for S. bicolor, 40 day old for S. halepense and 115 day old for S. leiocladum a t t he t i m e of t r ans fe r and w e r e held a t t h e chil l ing t e m p e r a t u r e s for 40 days . Rad i a t i on levels within t h e cab ine t s w e r e m o n i t o r e d wi th a Li -cor quan tum m e t e r and R i m c o i n t e g r a t i n g p y r a n o m e t e r s under shaded and unshaded condi t ions .

5. Low Temperature, Water Stress and Chlorophyll Destruction. S. bicolor p l a n t s t h a t had been grown to t h e 10th leaf s t a g e a t 30° /Z5°C w e r e t r a n s f e r r e d to a glasshouse Β cab ine t held a t 7 C c o n s t a n t . Chlorophyll c o n t e n t and r e l a t i v e w a t e r c o n t e n t (RWC) w e r e mon i to red over t h e subsequent 10 days using the me thods of Arnon (Z) and Wea the r l ey (35) r e s p e c t i v e l y . Chlorophyl l was sampled from the las t fully expanded leaf from t h e top (usually t h e th i rd leaf) while RWC was d e t e r m i n e d in t h e 5th leaf from t h e t op . Wate r p o t e n t i a l was m e a s u r e d using a model 1000 P.M.S. p r e s su re c h a m b e r (16). A con t ro l p lan t was ο ο kept in t h e 30 /Z5 C g lasshouse .


A. Relative Growth Rates at Constant Temperatures

The response of p l an t s to low t e m p e r a t u r e can be m e a s u r e d in severa l ways but r e l a t i v e g rowth r a t e is t he fundamen ta l m e a s u r e of o rgan ic g rowth and it is a very sens i t ive ya rds t i ck (36). Over t h e 10 day e x p e r i m e n t a l pe r iod (Figure 1) Johnson grass and S. bicolor had s imi lar g rowth r a t e s th roughout t h e t e m p e r a t u r e r ange ,whi l e S. leiocladum had s igni f icant ly s lower g rowth a t t h e h igher t e m p e r a t u r e s . At a cons t an t 5 C, however , all t h r e e spec ies w e r e suffer ing from s e v e r e chil l ing les ions . The inabi l i ty of t he se t h r e e spec ies to grow a t 5 C appea r s to be a t v a r i a n c e with field d a t a t h a t show t h a t t hese spec ies can w i t h s t a n d mild f ros t ing t e m p e r a t u r e s [S. bicolor (Z0)] or s e v e r e f ros ts [Johnson grass - (9)] . The fa i lure of t h e t h r e e spec ies to grow a t a cons t an t 5 C while being able to survive f ros ts a t night r a i ses t h e possibi l i ty t h a t a d i f fe ren t c r i t i c a l t e m p e r a t u r e ex is t s a t night from t h a t dur ing t h e day .

B. Effect of night temperature on relative growth rate

The growth response of S. bicolor t o d i f fe ren t night t e m p e r a t u r e s is shown in F igure Z. S. bicolor is t h e mos t chil l ing sens i t ive of t he t h r e e spec ies in this se r ies of e x p e r i m e n t s . All of t he p l an t s w e r e grown

7 0 D. Bagnall

0-20

0-15

ο "Ό

Ο

Ο

ο

ο

ΟΊΟ

0-05

0-0

-0Ό51

• S. bicolor

• S. halepense

A S. leiocladum

20 25 5 10 15 Temperature (°C)

FIGURE 1. Relative growth rates of three Sorghum species over 10 days at 5 constant temperatures. Each point is the mean value of 15 plants + 1 s.e.

a t 21 C in t he day while night t e m p e r a t u r e was va r i ed b e t w e e n 16 C (control) and 1.5 C. Al though growth t ended to d e c r e a s e wi th dec reas ing night t e m p e r a t u r e , this was only a smal l drop and it con t inued a t a r a t e about equal to g rowth a t a cons t an t 15 C . All of t h e e x p e r i m e n t a l p l an t s showed r e d u c e d growth and deve lopmen t , but none showed any sign of chill ing d a m a g e . Tissue fo rmed during t h e seven days when night t e m p e r a t u r e fell to 1.5 C had s imi lar chlorophyll c o n t e n t (1.6 m g / g fresh wt) to con t ro l p l an t s (night t e m p e r a t u r e equal to 16 C) . The C O ^ exchange r a t e s of l eaves from the ^rontrolând 1.5 C night t r e a t m e n t s w e r e also s imilar (27.3 mg CO^dm hour

The absence of a c r i t i ca l night t e m p e r a t u r e for S. bicolor cv 61^0 above 1.5 C was conf i rmed by field t r ia l d a t a . Begg and Turner (unpublished data) found tha t night t e m p e r a t u r e s b e t w e e n 0° and 10°C did not s top crop growth and tha t only f ros ts caused d a m a g e to m a t u r e sorghum p l a n t s .

Low Temperature Responses of Three Sorghum Species 71

•10

•0 9

^ Ό8 |

ο 2 Ό 7

ο

•0 6

•0 5

ο -0U

- 0 31

σ CD

^•0 2

•01h

I—ι 1 ι • 0 5 1 0 1 5

nigh t temperatur e (°C )

FIGURE 2. Relative growth rates of S. bicolor over 7 days at different night temperatures. Day temperature was 21°C in all cases. Each point is the mean value of 25 plants + 1 s.e.

C. Photosynthetic Response to Low Temperature

The d i f f e rence b e t w e e n c r i t i c a l n ight t e m p e r a t u r e as found in t h e field and p h y t o t r o n and the c r i t i c a l t e m p e r a t u r e when the day as well as t h e night t e m p e r a t u r e is low (Fig. Ζ vs F ig . 1) might be a t t r i b u t a b l e to t he p h o t o s y n t h e t i c r e sponse of the p l an t s to low t e m p e r a t u r e . F igure 3 shows t h e C O ? Exchange R a t e (CER) in a leaf zone (5 cm length) of S. bicolor and S. leiocladum as t e m p e r a t u r e was lowered from Z0 to 3 C, and then subsequent ly r e t u r n e d to Z0 C . The leaf zone was kept below 5°C for va r iab le a m o u n t s of t i m e from 10 min to 3 h a l though on an a v e r a g e this t i m e was 75 min .

7 2 D. Bagnall

0 5 1 0 1 5 temperatur e (°C )

FIGURE 3. CO2 exchange rate of S. leiocladum and S. bicolor under decreasing (solid symbols) and then increasim (hollow symbols) temperature. Both species were grown at 21°/16 C in a glasshouse (S. bicolor; square symbols, S. leiocladum: circular symbols). Each loop is a mean of 5 plants + 1 s.e.

Plo ts of t h e CER as a funct ion of t e m p e r a t u r e t a k e t he form of hys t e re s i s loops. During t h e cooling phase CER drops rapid ly wi th t e m p e r a t u r e down to 3 C, but during t h e warming phase a lag occu r s . This lag was ev iden t in bo th spec ies i r r e s p e c t i v e of how long the p l an t s w e r e kep t below 5 C (from 10 min to 3 h). The lag was , however , g r e a t e r in S. bicolor than in wild sorghum and this may be an adap t i ve mechan i sm tha t al lows S. leiocladum to respond to w a r m e r t e m p e r a t u r e s a f t e r cold n igh ts f a s t e r than S. bicolor.

Afte r t h e leaf zone had been r e w a r m e d to 20 C, c o m p l e t e r e c o v e r y of CER did no t occur even a f t e r 12 h. This p h o t o s y n t h e t i c r educ t ion could be e i t he r a s t o m a t a l r esponse (24) or photo inhib i t ion (26), but this a spec t was not pursued in these e x p e r i m e n t s .


Ο

0) Ο

cr

Ο

C D

Φ >

~Ό Φ

cr

0-04

0-02

0-0

oo4

0-02

0-0

0·02 ί

0-0 4

0-02

0-0

S.bicob r

-J 1 1 ι

δ. halepens e (Johnso n Grass )

• 0

S. leiocladu m

I. ' _J L

8 1 0 1 2 Temperatur e (° C )

FIGURE 4. Relative growth rates of three Sorghum species at 3 constant temperatures and two irradiance levels over 40 days. Hollow symbols represent unshaded plants while solid symbols represent the shaded plants. Each point represents the mean value of 10 plants +ls.e.

o Wild sorghum had a higher p h o t o s y n t h e t i c r a t e th roughout t he r ange 3 to Z0 C than S. bicolor (Fig. 3) a l though genera l ly having a lower g rowth r a t e over most of this t e m p e r a t u r e r ange (Fig. 1). The higher CER of wild sorghum a t low t e m p e r a t u r e s may provide a r eason for i t s

7 4 D. Bagnall

abi l i ty to grow at low t e m p e r a t u r e s than S. bicolor but i t does no t r e so lve why both spec ies suffer s eve re chill ing d a m a g e a t a cons t an t 5 C, when both have pos i t ive C E R s .

D. Effect of Light Intensity on Relative Growth Rate

Severe lesions occur in S. hicolor p l an t s sub jec ted to high light i n t ens i ty and I O C c o n s t a n t , wi thin 2 days a f t e r t r ans fe r to t h e cold (32). Chloroghyll levels dropped rapidly within these les ions . Under low light and I O C t h e chil l ing e f f ec t s w e r e much less s e v e r e . Light in t ens i ty is t h e r e f o r e i m p o r t a n t to chill ing injury. F igure 4 shows t h e r e l a t i v e g rowth r a t e s of t h e t h r e e sorghum spec ies a t t h r e e t e m p e r a t u r e s and under two l ight i n t ens i t i e s during 40 days a t low t e m p e r a t u r e . The h ighes t i r rad iance^ r e a c h e d in the cab ine t s on cleîr junny days a t midday we re 1480 ]JE m s (unshaded) and 605 μΕ m s (under shade_gloth) a l though on a v e r a g e t he plants^ only r e c e i v e d 470 g cal cm day (unshaded) and 165 g cal cm (shaded, over t he 40 days a t low t e m p e r a t u r e .

All of t h e p lan t s from the t h r e e spec ies survived and nut on dry weight in bo th shaded and unshaded t r e a t m e n t s a t 11 and 13 C, but a t 8.5 C Johnson grass and Sorghum bicolor lost dry weight under shaded and unshaded condi t ions while in S. leiocladum 2 of t h e 10 p l an t s ο survived a t 8.5 C in t h e open and all grew successful ly under t h e shaded condi t ions . The re fo re , under l ight i n t ens i t i e s typ ica l of win te r t he perennia l S. leiocladum can st i l l grow a t a cons t an t 8.5 C, a l though only very slowly.

TABLE I. Effect of Low Temperature and Shade on Chlorophyll Content of Three Sorghum Species

Species

Temperature Leaf age S. bicolor S. leiocladum S. halepense

8.5°C old 15.3 + 1.9 61.8 + 5.2 12.6 + 3.2 (shaded) new * 39.5 *

21/16°C old+new 49.6 + 4.0 74.0 + 6.0 19.7 + 6.4

-2 Chlorophyll was measured in \ig cm or leaf area. Each value is

mean of 6 plants + s.e. Old leaves were formed before transfer to 8.5°C, new leaves were formed during 40 d. at low temperature.

*No new leaf tissue was formed at 8.5 C in these species.


TABLE II. Effect of Temperature on Chlorophyll Content of Old and New Leaves of Three Sorghum Species

Species

Temperature Leaf age S. bicolor S. leiocladum S. halepense

11°C old new

22. 0.

.9

.8 + +

5. 0.

.8

.0 60. 56.

.4

.0 + +

1.5 14.5

20. 0.

.8

.2 + +

2. 0.

.5

.0

13° C old new

31. 6.

.6

.5 + +

1. 3.

.8

.4 63. 50.

.2

.6 + +

1.7 4.5

25. 7.

.1

.4 + +

6. 5.

0 .8

21/16°C old+new 49. .6 + 4. .0 74. .0 + 6.0 19. .7 + 6. .4

Chlorophyll was measured in \ig cm of leaf area. There were not significant differences in the chlorophyll contents of shaded and unshaded plants. Each value is the mean of 12 plants + s.e.

TABLE III. Effect of Temperature on the Chlorophyll Content of Week Old Seedlings of Three Sorghum Species

Temperature

Species

Temperature S. bicolor S. leiocladum S. halepense

15.5°C 0.04 + 0.00 0.76 + 0.04 0.87 + 0.04 16.5°C 0.37 + 0.04 - -17.5°C 1.00 + 0.02 - -

Chlorophyll was measured in mg g fresh weight. Each value is the mean of 3 samples of 3-6 seedlings that were germinated and grown on moist blotting paper in glasshouse cabinets.

7 6 D. Bagnall

The a p p e a r a n c e of t h e p lan t s undergoing t h e t h r e e t e m p e r a t u r e t r e a t m e n t s va r ied g r e a t l y . None of t h e unshaded p l an t s a t 8.5 C had s igni f icant chlorophyll levels in the i r l eaves a t t h e end of 40 days . At 8 .5°C, under t he shade , t he Johnson grass and S. bicolor p l an t s survived but bo th spec ies had suffered chill ing injury of t h e type desc r ibed by Taylor and Rowley (3Z) wi th s ignif icant ly r e d u c e d chlorophyll c o n t e n t (Table I). Wild sorghum, however , p roduced addi t ional dry m a t t e r (Fig. 4), r e t a i n e d chlorophyll in i t s old t i ssue and fo rmed chlorophyll in i t s new l e a v e s . At 11 C and 13 C, t he S. bicolor and Johnson grass p l an t s r e t a i n e d chlorophyll in t he t i ssue fo rmed be fo re t r ans fe r to t h e cold, but t h e new leaves fo rmed in t he cold l acked chlorophyll (Table Π). This o c c u r r e d under both shaded and unshaded t r e a t m e n t s for t hese two spec ie s . Wild sorghum was able to m a n u f a c t u r e chlorophyll a t bo th of t he se t e m p e r a t u r e s as well as under shade a t 8.5 C. Rhykerd et aZ.(Z9) found t ha t chlorophyll fo rmat ion l imi t ed g rowth below 16 C in S. bicolor while Alberda (1) found t h a t m a i z e seedl ings s ca r ce ly grew a t 15 C b e c a u s e of thei r fa i lure to "green up" bu t , de sp i t e t h e fa i lure to p roduce chlorophyll in new leaf t i ssue of older p l an t s , t h e g rowth r a t e of t hese was la rge ly ma in t a ined by t h e p re -ex i s t i ng g reen t i s sue .

Seedlings of S. bicolor w e r e g e r m i n a t e d and grown a t 15.5 C, 16.5 C and 17.5 C for 1 week and chlorophyll c o n t e n t s c o m p a r e d wi th Johnson grass and wild sorghum grown a t 15.5 C (Table III). The r educed chlorophyll fo rmat ion in S. bicolor obse rved below 16 C mus t l imit g rowth , pa r t i cu l a r ly in t he seedl ing s t a g e . Wild sorghum and Johnson grass formed s ignif icant levels of chlorophyll a t 15.5 C and a r e l ikely to survive ea r l i e r in spring than crop sorghum b e c a u s e of th i s .

E. Water Relations

It has been sugges ted t h a t p lan t w a t e r r e l a t i ons play a p a r t in chill ing injury (5, 7, 37). When shoots and roo t s w e r e chi l led in p l an t s , bo th pho tosyn thes i s and s t o m a t a l opening subsequent ly d e c r e a s e d due to t e m p o r a r y condi t ions of w a t e r s t r e s s (7). Wright (37) also working wi th a C^, p lan t concluded t ha t a pa r t i a l w a t e r def ic i t was a p r e r equ i s i t e for chil l ing injury and t h a t w a t e r de f ic i t s in the shoots a r i se when the roo t s a lone a r e chil led, probably due to r e d u c e d w a t e r u p t a k e .

Changes in r e l a t i v e w a t e r c o n t e n t (RWC) and chlorophyll over 10 days of chill ing a t 7 C in S. bicolor p l an t s a r e shown in F igure 5. The fall-off in RWC was p r e c e d e d by chlorophyll d e s t r u c t i o n in this spec ies .

T h e J e a f w a t e r p o t e n t i a l up to t he morning of day 5 var ied b e t w e e n -1 .1 χ 10 Pa and -1 .5 χ 10 Pa (1 bar = 10 Pascal) for all of t h e low t e m p e r a t u r e p l a n t s . At day 9 when RWC had dropped to 60%, t h e m e a n leaf w a t e r p o t e n t i a l for all t i s sue was -5 .1 ^ 10 Pa while t he g reen , hea l t hy t i ssue had a m e a n value of -3 .3 χ 10 |?a and the b l eached and d a m a g e d l eaves gave a m e a n va lue of -6 .1 χ 10 P a .

P lan t w a t e r p o t e n t i a l s we re in f ac t qu i t e high when c o m p a r e d wi th those of p lan t s of a s imi lar age and v a r i e t y undergoing w a t e r s t r e s s .


days

FIGURE 5. Changes over a 10 day period in relative water content (upper graph) αιψ chlorophyll content (lower graph) of S. Qicolor plants transferred to 7 C. Plants were transferred from a 30 /25 C glasshouse at 0900 h on day 1. Hollow symbols represent control plant held in a 30 /25 C glasshouse, solid symbols are experimental plants at a constant 7°C. Each experimental point is a mean of 10 plants.

Wate r s t r e s sed S. bicolor cv 610 p l an t s wi th RWCs of 60% have leaf w a t e r p o t e n t i a l s lower than -20 χ 10 (Turner and Long, unpublished) . Wate r s t r e s sed p l an t s and chil l ing injured p l an t s w e r e t o t a l l y d i f fe ren t in a p p e a r a n c e . W a t e r - s t r e s s e d p l an t s had s e n e s c e n t lower l eaves wi th genera l ly he a l t hy upper l eaves showing some browning around t h e edges of t he younger l e a v e s . Chil l ing injured p l an t s had lost all chlorophyll from leaf t i s sue exposed pe rpend icu la r ly to sunl ight . Upper l eaves w e r e mos t a f f e c t e d while t i ssue t h a t was shaded or c lose to t he leaf shea th was less a f f e c t e d .

7 8 D. Bagnall

To assess t h e i m p o r t a n c e of roo t w a t e r u p t a k e in chill ing injury in this spec ies the response of d e t a c h e d leaves in flasks of w a t e r was c o m p a r e d wi th whole p lan t s in well w a t e r e d po t s a t 7 C c o n s t a n t . The l eaves and the whole p lan t s looked qua l i t a t ive ly s imi lar th roughout : chill ing lesions a p p e a r e d on the second day of low t e m p e r a t u r e , while wi l t ing did not occur in e i the r se t of p l a n t s .

These r e su l t s suggest t ha t w a t e r s t r e s s was not t he in i t ia l cause of low t e m p e r a t u r e s t r e s s in S. bicolor. The loss in w a t e r c o n t e n t then is s y m p t o m a t i c of chil l ing injury r a t h e r than a causa l f a c to r . It p robably r e f l e c t s b reakdown of m e m b r a n e s , l e akage of cell c o n t e n t s and drying of t he leaf. It appea r s l ikely t h a t r educed w a t e r u p t a k e may be a f ac to r in chil l ing s t r e s s in p l an t s but not in p l an t s (7).

F. Formation and Destruction of Chlorophyll and its Importance to Low Temperature Injury

Chlorophyll fo rmat ion and d e s t r u c t i o n a r e major f a c t o r s in chill ing injury in p l a n t s . Reduced chlorophyll fo rmat ion has been found to l imi t t h e g rowth of m a i z e a t low t e m p e r a t u r e (1) and chlorophyll deve lopmen t was shown to be t e m p e r a t u r e and light in t ens i ty dependen t in this spec ies (Zl). It appea r s t h a t a pho tosens i t i zed ox ida t ive de s t ruc t i on of chlorophyll can occur under high l ight condi t ions a t low t e m p e r a t u r e (1Z, 17, 3Z). It has been sugges ted t h a t t he r a t e s of C O ^ f ixat ion a r e insuff icient to u t i l i ze the phosphory la t ive and r educ ing c a p a c i t y of the ch loroplas t under low t e m p e r a t u r e condt ions (31), an e f fec t possibly r e l a t e d to s t o m a t a l c losure a t low t e m p e r a t u r e (Z6, Z8).

Millerd and McWilliam (ZZ) found t ha t t he minimum t e m p e r a t u r e for chlorophyll fo rmat ion could be lowered using f lashing l ight , and sugges ted t ha t t he i n t e r m i t t e n t dark per iod avoided pho tooxida t ion of p igmen t s following the ini t ia l pho to reduc t i on . R e c e n t e x p e r i m e n t s have shown tha t the an t iox idan t tocophero l r e d u c e s t he chil l ing breakdown in ch lorop las t s and r e su l t s in p r o t e c t i o n of u n s a t u r a t e d lipids (18).

G. Plant Critical Temperatures

Raison and C h a p m a n (Z7) have p o s t u l a t e d t h a t a single c r i t i ca l t e m p e r a t u r e may exist for p lan t g rowth , but an examina t ion of the l i t e r a t u r e sugges ts t h a t this is an overs impl i f ied v iew. Mar t in (ZO) for example found d i f ferent t e m p e r a t u r e s l imi t ing growth a t d i f fe ren t s t ages of t h e life cyc le of sorghum. Ivory and Whi teman (15), when measur ing the e f fec t of t e m p e r a t u r e on the g rowth of severa l C^ g rasses , found t ha t "c r i t i ca l" night t e m p e r a t u r e a p p e a r e d to vary depending on the accompany ing day t e m p e r a t u r e and t h a t c r i t i c a l day t e m p e r a t u r e also var ied with accompany ing night t e m p e r a t u r e . These changes can be expla ined by changes in n e t pho tosyn thes i s and may be due to s t o m a t a l r esponse , s t a r c h accumula t i on or dark r e sp i r a t ion (19, Z4, 34).


The r e s u l t s r e p o r t e d h e r e do not suppor t t h e concep t of a single c r i t i c a l t e m p e r a t u r e for p lan t g rowth s ince the e f f ec t depends on the s t a g e of g rowth and t h e level of i r r a d i a n c e .

ACKNOWLEDGMENT

I g ra te fu l ly acknowledge adv ice from Ian Wardlaw, Ross Downes and Neil Turner and superb a s s i s t ance from the s taff of the p h y t o t r o n and Mike Moncur .

ΠΙ. R E F E R E N C E S

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Tablelands of N.S.W. Angus and Robe r t son , Sydney (1966). 5. Chu, Τ. M., Ju sa i t i s , M., Aspinal l , D. , and Pa l eg , L. G. Physiol.

Plant. 43, 254-260 (1978). 6. Cooper , J . P . and Tain ton , Ν. M. Herb. Ab. 38, 167-176 (1968). 7. Crooks ton , R. K., O'Toole, J . , Lee , R., Ozbun, J . L., and Wal lace , D.

H. Crop Sci. 14, 457-464 (1974). 8. D o g g e t t , H. "Sorghum" Longmans , London (1970). 9. Downes , R. W. In "Sorghum in Sevent ies" (N.G.P. R a o , and L. R.

House , eds.) , pp . 265-274. Oxford and IBH, New Delhi (1972). 10. Downes , R. W. and Marshal l , D. R. Aust. J. Exp. Agric. Anim.

Husb. 11, 352-356 (1971). 11 . Hasse l t , P . R. van Acta Bot. Neerl. 21, 539-543 (1972). 12. Hasse l t , P . R. van Acta Bot. Neerl. 23, 159-169 (1974). 13. H a t c h , M. D. , Slack, C . R. , and Johnson, H. S. Biochem. J. 102,

417-422 (1967). 14. He lms , K., and Wardlaw, I. F . Phytopathology 67, 344-350 (1977). 15. Ivory, D . Α., and Whi teman , P . C. Aust. J. Plant Physiol. 5, 138-

148 (1978). 16. Jones , Μ. M. and Turner , N . C . PI. Physiol. 61, 122-126 (1978). 17. Kandle r , O. and Sironval, C . Biochim. Biophys. Acta 33, 207-214

(1959). 18. Kok, L. J . de , van Hasse l t , P . R. and Kuiper , P . J . C . Physiol.

Plant. 43, 7-12 (1978). 19. Ludlow, Μ. M. and Wilson, G. L. Aust. J. Biol. Sc. 24, 1065-1075

(1971). 20. Mar t in , J . H. U.S. Dept. of Ag. Yearbook, 343-347 (1941). 2 1 . McWill iam, J . R . and Naylor , A. W. PL Physiol. 42, 1711-1715

(1967).

8 0 D. Bagnall

22. Millerd, A. and McWill iam, J . R . PL Physiol. 43, 1967-1972 (1969). 23 . Morse , R. N. and Evans , L. T. J. Agric. Eng. Res. 7, 128-140 (1962). 24. P a s t e r n a k , D . and Wilson, G. L. New Pytol. 71, 682-689 (1972). 25. P in thus , M. J . and Rosenblum, J . Crop Sci. 1, 293-296 (1961). 26. Powles , S. B. and Osmond, C. B. Aust. J. Plant Physiol. 5, 619-629

(1978). 27. Raison, J . K. and Chapman , E. A. Aust. J. Plant Physiol. 3, 2 9 1 -

299 (1976). 28. R a s c h k e , K. Planta 91, 336-363 (1970). 29. Rhykerd , C . L., Cross , C . F . and Sullivan, E. F . Crops and Soils

12/9, 24 (1960). 30. Taylor , A. O. , Hal l igan, G. and Rowley , J . A. Aust. J. Plant Phy

siol. 2, 247-257 (1975). 3 1 . Taylor , A. O., Slack, C. R. and McPherson , H. G. In "Mechanisms

of Regu la t ion of P lan t Growth" . (R. L. Bieleski , A. R. Ferguson and Μ. M. Cresswel l , eds.) Bull. 12, Royal Soc. N.Z., 519-524 (1974).

32 . Taylor , A. O. and Rowley , J . A. PI. Physiol. 47, 713-778 (1971). 3 3 . Tee r i , J . A. and Stowe, L. G. Oecologia 23, 1-12 (1976). 34. West , S. H. Proc. XI Int. Grassl. Congr. 514-517 (1970). 35 . Wea the r l ey , P . E. New Phytol. 49, 81-87 (1950). 36. Will iams, R. F . "The Shoot Apex and Leaf Growth" . Cambr idge

Univers i ty P re s s , London (1974). 37. Wright , M. Planta 120, 63-69 (1974).


PHYSIOLOGY OF COOL-STORAGE DISORDERS OF F R U I T AND VEGETABLES

N. L. Wade

N.S.W. D e p a r t m e n t of Agr i cu l tu re l o c a t e d a t CSIRO Division of Food R e s e a r c h

P .O . Box 52 Nor th Ryde , N.S.W. 2113, Aus t r a l i a

I. INTRODUCTION

C o o l - s t o r a g e is an i m p o r t a n t t e chn ique for p rese rv ing fresh p lan t p r o d u c e . D e t e r i o r a t i o n due to r ipening , s e n e s c e n c e and d i sease can be r e t a r d e d by s t o r a g e a t r e d u c e d t e m p e r a t u r e , wi th t he op t imum t e m p e r a t u r e of ten being s l ight ly above the f reez ing point of the p r o d u c e . Many c o m m o d i t i e s , however , suffer injury when coo l - s to red a t t e m p e r a t u r e s above the i r f reez ing po in t . By def in i t ion , such c o m m o d i t i e s might be class i f ied as "chill ing sens i t ive" (19), but this des igna t ion m a y s o m e t i m e s be ambiguous .

Low t e m p e r a t u r e has been asc r ibed as t he i n c i t a n t of many s t o r a g e d isorders wi thou t c r i t i c a l appra i sa l . When p roduce is coo l - s to red i t s l ife is usual ly i nc reased , p e r m i t t i n g s e n e s c e n t d isorders to develop which a r e neve r observed a t h igher t e m p e r a t u r e s due to rap id t i ssue d i s in t eg ra t ion or invasion by d i sease . Low t e m p e r a t u r e can also a g g r a v a t e an injury inc i t ed by some o the r cause , such as h e a t or mechan i ca l injury.

The c r i t i c a l th reshold t e m p e r a t u r e below which injury develops r anges from less than 5 C for apple and o range fruit to 10-13 C for mango and 12-13°C for b a n a n a fruit (18). C u c u m b e r , eggp lan t , p a p a y a and p e a c h fruit suffer injury below about 7 C, while t h e l ime , muskmelon and p ineapple have thresholds of 7-10 C. T e m p e r a t u r e and t i m e i n t e r a c t in the deve lopmen t of coo l - s t o r age injury. As the t e m p e r a t u r e a t which p roduce is s t o r e d d e c r e a s e s below t h e c r i t i c a l th reshold t e m p e r a t u r e , t he s eve r i t y of u l t i m a t e injury i nc r ea se s , a l though the r a t e of deve lopmen t of injury during coo l - s to r age d e c r e a s e s (36).

In t rop ica l fruit such as t he banana , s e v e r e injury is expressed a f t e r exposures to chil l ing t e m p e r a t u r e s of only a few hours , and each fruit in a p a r t i c u l a r s ample suffers injury (9). In o the r co mmo d i t i e s such as apple and g rapef ru i t , injury can t a k e weeks or mon ths of s t o r a g e to deve lop ,

8 1 Copyright ® 1979 by Academic Press. Inc.

All rights of reproduction in any form reserved ISBN 012-4605605

8 2 Ν. L. Wade

and only a p ropor t ion of t he fruit in a p a r t i c u l a r sample may mani fes t injury. Biological va r ia t ion in the c r i t i c a l threshold t e m p e r a t u r e for chill ing within samples of s imi lar m a t e r i a l was p o s t u l a t e d by van der Plank and Davies (36). These au thors observed tha t only a p ropor t ion of g rapef ru i t in a sample suf fered chilling injury and t ha t this p ropor t ion r e a c h e d a p l a t e a u which did no t change fur ther wi th t i m e . They proposed t ha t when a sample of fruit is coo l - s to red , a por t ion of t h e s ample is "out of equi l ibr ium" (i .e. suffers a me tabo l i c imbalance) from the "very in s t an t of cooling, and is p r edes t i ned to subsequent injury". T ime does no t cause this i m b a l a n c e in the suscep t ib le por t ion but mere ly enables t h e deve lopmen t of visible les ions. The r e s t of t h e sample does not suffer injury because these fruit have a c r i t i c a l threshold t e m p e r a t u r e which is below the s t o r age t e m p e r a t u r e .

This pape r discusses ev idence which has a c c r u e d about t h e mechan i sms by which coo l - s to rage t e m p e r a t u r e s can injure fresh p roduce . The p lant organs which we ha rves t for food usually conta in no dividing cel ls and have of ten ceased expansion g rowth . These d e t a c h e d organs a r e no longer dependen t on t he p a r e n t p lan t for w a t e r , minera l s and p h o t o s y n t h a t e , and their me tabo l i sm is d i r e c t e d towards m a i n t e n a n c e of exis t ing t i ssues . In t he coo l - s to rage env i ronment p roduce is subject to a cons t an t t e m p e r a t u r e , unl ike p lan t s growing in the field, where diurnal t e m p e r a t u r e f luc tua t ions may r e l i eve chill ing condi t ions for p a r t of eve ry day.

II. ROLE OF THE MEMBRANE IN COOL-STORAGE DISORDERS

The injury response observed when t rop ica l commodi t i e s a r e s to red below the threshold t e m p e r a t u r e for chil l ing is cons i s ten t wi th the hypothes is of Lyons and Raison (ZO) t h a t an i n s t an t aneous t e m p e r a t u r e -induced change in m e m b r a n e s t r u c t u r e causes the injury. Where , however , t he s y m p t o m s of injury develop a f t e r months of coo l - s to rage and suscept ib i l i ty to injury var ies amongs t organs from the s a m e clone and the same t r e e s , a s imple cause-and-e f f ec t r e l a t i on b e t w e e n a change in m e m b r a n e s t r u c t u r e and injury is obscured . The re is very l i t t l e ev idence which enables us to r e l a t e d isorders of this type with the c r i t i ca l t e m p e r a t u r e , Τ (Z4) below which m e m b r a n e s t r u c t u r e and function b e c o m e abnorma l . Few s t o r a g e e x p e r i m e n t s have been conduc ted in which Τ of cell m e m b r a n e s was mon i to red throughout the s t o r a g e per iod . Studies wi th coo l - s to red a r t i c h o k e tuber t i ssue showed t h a t Τ was not cons t an t but changed with t i m e (Z). The re is both d i rec t and p r e s u m p t i v e ev idence t ha t the va lue of Τ changes as a fruit r ipens (see sec t ion IV A). It is not suff ic ient to d e t e r m i n e the value of Τ on a s few samples of a p a r t i c u l a r commodi ty and assume tha t this va lue appl ies to o the r samples from d i f fe ren t h a r v e s t s be fo re and during s t o r a g e .

Physiology of Cool-Storage Disorders of Fruit and Vegetables 8 3

An a t t e m p t has been m a d e to c o r r e l a t e t he low t e m p e r a t u r e b reakdown disorder of apple fruit wi th t h e Τ of mi tochondr ia l m e m b r a n e s from pulp t i ssue sampled be fo re s t o r a g e (21). The appa ren t Τ ^ e s t i m a t e d using both succ inoxidase a c t i v i t y and spin- labels , was 4 -ΐ β C, depending on v a r i e t y and loca l i ty . T h e r e was no co r r e l a t i on b e t w e e n a p p a r e n t T § and suscep t ib i l i ty to b reakdown, s ince some of t h e v a r i e t i e s t e s t e d w e r e c o m p l e t e l y r e s i s t a n t to the d isorder whilst o t h e r s showed s y m p t o m s when s t o r e d below 5 C. Some r e s e r v a t i o n s mus t , however , be held about t he re l i ab i l i ty of t h e Τ e s t i m a t e s b e c a u s e of t h e subsequent work of Raison et al. (25), who d e m o n s t r a t e d how er roneous high e s t i m a t e s of T g can be der ived .

Unt i l r e l i ab le e s t i m a t e s of Τ a r e ava i l ab le , c r i t i ca l appra isa l of t h e ro l e of m e m b r a n e s t r u c t u r e in many coo l - s to r age d isorders of fresh p roduce will no t be poss ib le . The i n h e r e n t va r iab i l i ty of h o r t i c u l t u r a l p roduce and t h e e f f ec t s of r ipening and s t o r a g e h i s to ry on Τ d i c t a t e t h a t Τ should be mon i to red th roughout each s t o r a g e e x p e r i m e n t . A s imple me thod is n e e d e d to ach ieve this a i m . Assays of succ inoxidase a c t i v i t y or of spin- label mot ion a r e n e i t h e r s imple nor r o u t i n e .

ΠΙ. CHILLING INJURY AND THE ACCUMULATION OF METABOLITES DURING COOL STORAGE

A. Glycolytic Products

Inhibi t ion or d isrupt ion of mi tochondr ia l funct ion may lead to an a c c u m u l a t i o n of p y r u v a t e , which may in turn be me tab o l i z ed to such compounds as a c e t a l d e h y d e , e thano l and a c e t a t e . Lyons (19) observes t ha t such a me tabo l i c i m b a l a n c e would resu l t from a t e m p e r a t u r e -induced change in m e m b r a n e s t r u c t u r e which inhibi ts m e m b r a n e - b o u n d e n z y m e s , such as those of the t r i ca rboxy l i c ac id cyc l e , whilst not a f f ec t ing t h e g lycoly t ic enzymes of t h e cy top l a sm.

P y r u v a t e , a c e t a l d e h y d e , e thano l and α-ke to ac ids a c c u m u l a t e in chi l led banana fruit t i ssues (22). A c e t a t e (43) and a c e t a l d e h y d e (31) a c c u m u l a t e in coo l - s to red apple f ru i t s .

The o c c u r r e n c e of abnormal c o n c e n t r a t i o n s of var ious m e t a b o l i t e s in co ld - s t r e s sed t i ssues is unequivoca l . It is d i f f icul t , however , to i n t e r p r e t t he s igni f icance of such obse rva t ions . Changes in m e t a b o l i t e c o n c e n t r a t i o n s may occur as a d i r ec t r e su l t of a p r i m a r y e f fec t of chill ing s t r e s s , as p roposed by Lyons and Raison (20), or as a t e rmina l even t in t i ssue injury. The p r e s e n c e in visibly injured t i ssues of high c o n c e n t r a t i o n s of subs tances such as those ar is ing from the i n c o m p l e t e oxidat ion of p y r u v a t e may be c o m m o n p l a c e in morbid t i s sues . Changes in m e t a b o l i t e c o n c e n t r a t i o n s which p r e c e d e visible injury a r e m o r e i n fo rma t ive , s ince confusion wi th i m m e d i a t e p r e - and p o s t - m o r t e m even t s is avoided.

8 4 Ν. L. Wade

A fur the r di f f icul ty a r i ses in deciding if a m e t a b o l i t e which a c c u m u l a t e s in response to chil l ing s t r e s s e x e r t s a tox ic e f f ec t on the t i s sue . In a review of t h e ro le of a c e t a l d e h y d e in fruit d i sorders , Smagula and Bramlage (31) concluded tha t it was no t possible from exis t ing in fo rmat ion to dist inguish if a c e t a l d e h y d e a c c u m u l a t i o n is a cause or an e f fec t of t i ssue d i so rgan iza t ion . A s imi lar conclusion can probably be drawn for r e l a t e d m e t a b o l i t e s such as e thano l . Proof of tox ic i ty a t physiological c o n c e n t r a t i o n s is no t a lways easy to ob ta in . When banana fruit s l ices w e r e t r e a t e d with e thanol solut ions and then induced to r ipen wi th e t h y l e n e , the s l ices showed a n o r m a l r e s p i r a t o r y r i se and r ipened normal ly , as judged by pee l colour , a r o m a , sof tening and soluble solids a c c u m u l a t i o n (Table I). Assuming c o m p l e t e di lut ion of the applied e thano l wi th t i s sue w a t e r , in f i l t ra t ion wi th 1.0 Μ e thano l gives a t i ssue c o n c e n t r a t i o n of 100 mM e thano l . The h ighes t endogenous e thano l c o n c e n t r a t i o n s measu red in the pulp of chi l led, g reen banana fruit was about 15 mM, and in chil led, yellow fruit was about 50 mM (22). Rega rd le s s of the r a t e a t which applied e thano l vo la t i l i zes or is de toxi f ied , injury sus ta ined by t h e t i ssue a t t he t i m e of app l ica t ion should p r e v e n t a normal response to appl ied e t h y l e n e .

B. Oxaloacetate versus Acetate

Hulme et al. (14) observed a s t rong pos i t ive co r r e l a t i on b e t w e e n the accumula t i on of o x a l o a c e t a t e in coo l - s to red apples and the subsequent deve lopmen t of low t e m p e r a t u r e b reakdown. A shor t in t e r im per iod of s t o r a g e a t higher t e m p e r a t u r e r e d u c e d both t he accumula t i on of o x a l o a c e t a t e and t h e seve r i ty of b reakdown. O x a l o a c e t a t e did not a c c u m u l a t e in some v a r i e t i e s of apple which do not suffer from low t e m p e r a t u r e b reakdown. This promis ing co r r e l a t i on has not been sus ta ined . In ano the r s tudy no d i f fe rences w e r e found in the levels of t r i ca rboxyl ic acid cyc le i n t e r m e d i a t e s which could be a s soc i a t ed with d i f fe rences in b reakdown (39)·

The inc idence of low t e m p e r a t u r e b reakdown in apples is c o r r e l a t e d wi th the c o n c e n t r a t i o n of a c e t a t e in the fruit (40, 43) . The seve r i ty of t h e d isorder can be e x a c e r b a t e d by in jec t ing a c e t a t e in to t he fruit and a l l ev ia t ed by t r e a t m e n t s which d e c r e a s e the a c e t a t e c o n c e n t r a t i o n in t h e t issues by increas ing t h e r a t e of evolut ion of a c e t a t e e s t e r s . The long t i m e lapse observed b e t w e e n inject ion of a c e t a t e and a p p e a r a n c e of the disorder suggest t h a t a m e t a b o l i t e of a c e t a t e is t he toxin (44). Indeed, seve ra l compounds including the a c e t a t e d e r i v a t i v e , m e v a l o n a t e , a r e p o t e n t inducers of b reakdown (41).

TABLE I. Effect of Ethanol on the Response of Banana Fruit Slices to Applied Ethylene

No Ethylene

Ethylene Applied

Untreated control

Untreated control

Water 0.10 treated

0.25 0.50 Μ e t h a n o l

1.00

Soluble solids content

(%)* 2.1+0.2 17.7+0.7 16.0+0.4 15.3+0.6 16.0+1.2 15.3+1.3 15.7+2.7

Transverse slices of preclimacteric banana fruit (6mm thick) were vacuum-infiltrated with water or ethanol solutions and incubated in air at 20 C for 7 days. Ethylene (10\i %/%] was then applied and the soluble solids content of juice expressed from the pulp was determined 14 days after ethanol application.

* g sucrose equivalents/100 g fresh weight.

8 6 Ν. L. Wade

C. a-farnesene

F a r n e s e n e has been i m p l i c a t e d in t h e super f ic ia l sca ld d isorder of coo l - s to red apples (11), and the skin injury sus t a ined by chi l led banana f ru i t s (44). Al though t h e c o n c e n t r a t i o n of th i s compound can i n c r e a s e s o m e w h a t in r esponse to chil l ing s t r e s s p r io r to t h e onse t of t i s sue morb id i ty , a b e t t e r co r r e l a t i on ex is t s b e t w e e n the c o n c e n t r a t i o n of f a r n e s e n e in t he pee l t i ssues of apples and bananas a t h a r v e s t and the subsequen t s e ve r i t y of injury a f t e r c o o l - s t o r a g e . F a r n e s e n e can be r e g a r d e d as a p r e - ex i s t i ng p o t e n t i a l toxin , which b e c o m e s ha rmfu l only a f t e r a chil l ing s t r e s s has been appl ied. F a r n e s e n e i tself does no t appea r t o be tox ic to pee l t i s sues , but i t p roduces tox ic hydroperoxides when it oxidizes dur ing coo l - s to rage (12). Chil l ing m a y p r o m o t e oxidat ion of f a r n e s e n e by damaging ce l lu la r c o m p a r t m e n t a t i o n .

D. Sorbitol

Sorbitol can a c c u m u l a t e in apple fruit during coo l - s to r age , and this a c c u m u l a t i o n is a ccompan ied by low t e m p e r a t u r e b reakdown (9). A causal connec t ion was no t es tab l i shed in this work. Inf i l t ra t ion of apple fruit wi th sorbi to l solut ions can also i n c r e a s e the inc idence of b reakdown (1). The invo lvement of sorbi tol in coo l - s to rage injury is discussed in sec t ion IV.E.5.

IV. STORAGE TREATMENTS WHICH ALLEVIATE COOL-STORAGE INJURY

A number of empi r i ca l s t o r a g e me thods h a v e been d i scovered which r e d u c e the o c c u r r e n c e or s eve r i t y of injury during c o o l - s t o r a g e . Some of t he se me thods a r e of cons iderab le p r a c t i c a l c o m m e r c i a l i m p o r t a n c e , and they a r e also a useful tool in the s tudy of coo l - s t o r age d i so rders . Specula t ion about the mode of ac t ion of a p a r t i c u l a r s t o r a g e m e t h o d can lead to useful hypo theses , and the eva lua t ion of t hese hypo theses is a ided by t h e abi l i ty to induce or con t ro l t h e d isorder a t will .

A. Delayed Cool-Storage

A r e m a r k a b l e r educ t ion in t h e subsequent deve lopmen t of injury has been observed in c e r t a i n in s t ances where fruit a r e i ncuba ted a t room t e m p e r a t u r e for a shor t t ime be fo re coo l - s t o r age . Davies et al. (3, 4) obse rved t h a t p e a c h fruit bene f i t ed if s t o r a g e was de layed for about two days . All r e p o r t e d in s t ances of a benef ic ia l r e sponse to de layed s t o r a g e appea r to involve fruit which w e r e r ipening a t ha rves t or began to r ipen i m m e d i a t e l y a f t e r h a r v e s t . The c ruc ia l e f fec t of delaying s t o r a g e is ,

Physiology of Cool Storage Disorders of Fruit and Vegetables 8 7

t h e r e f o r e , to a l t e r t h e s t a g e of r ipening a t which the fruit e n t e r s t o r a g e . The max imum benef i t s of de layed s t o r a g e have been ob ta ined when rap id , uniform r ipening was ensured by t r e a t m e n t wi th a hydrocarbon gas (5).

One consequence of t h e s t a g e of r ipeness a t which a fruit e n t e r s s t o r a g e is obvious. The r ipening mechan ism is a c u t e l y sens i t ive to chil l ing s t r e s s and an inev i t ab le symptom of chil l ing in unr ipe frui ts is t h e subsequent fa i lure of the fruit to r ipen (8). Avocado fruit a r e most sens i t ive to chil l ing s t r e s s during the c l i m a c t e r i c r i se and a t t he c l i m a c t e r i c peak (16). The fruit is marked ly less sens i t ive when p r e - or p o s t - c l i m a c t e r i c , wi th p o s t - c l i m a c t e r i c fruit being l eas t sens i t ive . The possible invo lvement of m e m b r a n e s t r u c t u r e in this phenomenon has been s tud ied , by obta in ing Arrhenius p lo t s for succ inoxidase a c t i v i t y of m i tochond r i a i so la t ed from avocado fruit a t d i f fe ren t s t a g e s of r ipeness (17). These au thor s conc luded t h a t t h e a p p a r e n t Τ was about 9°C in both the p r e c l i m a c t e r i c s t a g e and the c l i m a c t e r i c r i s e . At the c l i m a c t e r i c peak , a p p a r e n t Τ rose to 11-12 C, whils t in t h e p o s t - c l i m a c t e r i c phase Τ fell to 2-5 C. These r e su l t s a g r e e qua l i t a t i ve ly wi th the r e l a t i v e suscep t ib i l i t i e s of fruit to chil l ing injury a t e a c h r ipeness s t a g e , but they do not explain why, for example , p r e c l i m a c t e r i c (but no t c l i m a c t e r i c rise) fruit could be s t o r e d a t 2 C for about 30 days be fo re inc ip ient chil l ing injury appea red . Kosiyachinda and Young (17) proposed t ha t a change in m e m b r a n e lipid compos i t ion during r ipening could accoun t for t h e changes in a p p a r e n t Τ .

Studies wi th t he r ipening banana frui t h a v e shown t h a t app rec i ab le changes in m e m b r a n e lipid compos i t ion o c c u r in this fruit dur ing r ipening (37). The change is g r e a t e s t in t h e phospholipid f rac t ion and en ta i l s an i n c r e a s e in t he p ropor t ion of e s t e r i f i ed l inolenic acid and in t he t o t a l u n s a t u r a t i o n of t h e phospholipids (Table Π). The f luidi ty of l iposomes p r e p a r e d from the e x t r a c t e d phospholipids i nc r ea sed as the p ropor t ion of l inolenic ac id i n c r e a s e d (Table ΙΠ) and i t c a n be p r e d i c t e d t h a t Τ would d e c r e a s e c o n c o m i t a n t l y . This does no t imply , however , t h a t t he lbanana fruit should b e c o m e less sens i t i ve t o chil l ing as it r ipens . The threshold t e m p e r a t u r e for chil l ing m a y c h a n g e during r ipening of the banana and avocado , but if e i t h e r f rui t is s t o r e d well below t h e threshold , t hen t h e observed sens i t iv i ty t o chi l l ing wil l depend on the m e t a b o l i c s t a t e of t h e fruit a t t h e t i m e . The d i s rup t ive e f fec t of chil l ing a r ipening fruit m a y be p a r t i c u l a r l y s e v e r e due t o the i n t ense m e t a b o l i c a c t i v i t y which c h a r a c t e r i z e s r ipen ing .

β . Intermittent Warming

In t e r rup t i on of cool s t o r a g e by per iods of exposure t o w a r m t e m p e r a t u r e s pos tpones the deve lopmen t of injury. The benef ic ia l e f f e c t of warming a f t e r a per iod of cool s t o r a g e appea r s to r e s ide in r e c o v e r y from the harmful e f f e c t s of the s t r e s s , a l though the r e c o v e r y e f f e c t c a n be confounded wi th t h e r ipening which also occu r s a t t h e h ighe r t e m p e r a t u r e .

8 8 Ν. L. Wade

TABLE II. Changes in the Fatty Acid Composition of Banana Pulp Phospholipids during Ripening (37).

Fatty acid Days after ethylene applied 0 3 6

Palmitic 42.0 31.2 35.1 Oleic 6.5 7.3 6.2 Linoleic 31.6 34.7 26.4 Linolenic 6.6 13.5 25.5

TOTAL UNSATURATED (includes 16:1, 16:2)

53.3 65.2 63.2

Banana fruit were induced to ripen with 10μ 1/fL ethylene and sampled at the times shown for extraction, isolation and analysis of fatty acids esterified to phospholipids. Results for the four major compoents only are shown.

TABLE III. Changes in the Physical Properties of Banana Pulp Phospholipids during Ripening (37)

Temperature Days after ethylene applied of

measurement 0 3 6 (0°C)

V 10 0.765 0.764 0.742 20 0.699 0.690 0.671 30 0.628 0.624 0.608 40 0.579 0.573 0.561

* Sn is the order parameter (13). Liposomes were prepared from the phospholipid samples analyzed in

Table II and the motion of infused 5-nitroxide stearic acid spin label monitored. The order parameter, S , decreased both as the measurement temperature increased and as the time of ripening increased, showing that the liposomal membranes from the lipids of ripe tissue were more flexible or fluid than those from unripe tissue.


I n t e r m i t t e n t warming has r e c e i v e d p a r t i c u l a r a t t e n t i o n in t he s t o r a g e of s tone frui t , such as the Vic tor ia plum (33), a l though apples also respond to this t r e a t m e n t (34). It has been a rgued t h a t t r ans fe r to a warm t e m p e r a t u r e p e r m i t s t he fruit to m e t a b o l i z e a tox ic m e t a b o l i t e which has a c c u m u l a t e d in cool s t o r a g e (32). Unless spec ia l p r e c a u t i o n s a r e t aken , i nc reased w a t e r loss will occur when p roduce is t r a n s f e r r e d to a h igher t e m p e r a t u r e . The consequence of this is d iscussed in I V . C

C. Humidity Control

Chil l ing injury can be r e d u c e d if t h e s t o r a g e humid i ty is e i t he r high or low, depending upon the p a r t i c u l a r c o m m o d i t y . High humid i ty may simply suppress t h e express ion of s y m p t o m s , by reduc ing des i cca t ion of n e c r o t i c t i ssues (19). Low humid i ty enhances the loss of vo la t i l e e s t e r s of a c e t a t e , a s u s p e c t e d toxin (ΙΠ.Β) in coo l - s to red apples (38). Simon (29) has sugges ted t ha t an ea r ly s t a g e in the e t io logy of low t e m p e r a t u r e b reakdown of apples is w a t e r - s o a k i n g of t h e i n t e r ce l l u l a r spaces , fol lowed by h y d r o s t a t i c r u p t u r e of the p r o t o p l a s t s . Evapora t ion of i n t e r ce l l u l a r w a t e r would r a i se t he ton ic i ty of t h e r ema in ing solut ion and r e d u c e this p o s t u l a t e d r u p t u r e of p r o t o p l a s t s .

D. Controlled Atmospheres

Cont ro l l ed a t m o s p h e r e s which a r e d e p l e t e d in oxygen and /or en r i ched in ca rbon dioxide r e l a t i v e to air a r e used to delay r ipening and s e n e s c e n c e in fresh p roduce , usually in conjunct ion wi th cool s t o r a g e . In many in s t ances con t ro l l ed a t m o s p h e r e s h a v e e x a c e r b a t e d coo l - s to rage d isorders (7), a l though t h e r e a r e examples w h e r e con t ro l l ed a t m o s p h e r e s h a v e p roven benef i c i a l . A harmful e f f ec t of con t ro l l ed a t m o s p h e r e s can be read i ly expla ined in p roduce which is suffer ing a s e v e r e m e t a b o l i c i m b a l a n c e due to chil l ing. The inhibi t ion of v i ta l m e t a b o l i c p a t h w a y s by coo l - s t o r a ge will probably be e x a c e r b a t e d by r e d u c e d oxygen and i n c r e a s e d ca rbon dioxide tens ions . For example , inhibi t ion of succ inoxidase a c t i v i t y by high c o n c e n t r a t i o n s of ca rbon dioxide may be highly injurious in a t i s sue w h e r e mi tochondr ia l funct ion is also being inhib i ted by low t e m p e r a t u r e .

The benef ic ia l e f f ec t s which a r e s o m e t i m e s observed during con t ro l l ed a t m o s p h e r e t r e a t m e n t a r e less a m e n a b l e to exp lana t ion . An example of such a response is given by t h e p e a c h f ru i t . Addi t ion of ca rbon dioxide to t h e s t o r a g e a t m o s p h e r e r e d u c e s injury in coo l - s to red p e a c h e s (Fig. 1). In an a t m o s p h e r e of 20% v/v ca rbon dioxide (balance a i r ) , no injury (mealy t e x t u r e or flesh browning) was d e t e c t e d a f t e r s t o r a g e for 30 days a t 1 C, fol lowed by 7 days a t 20 C, w h e r e a s t he s t o r a g e life in air was only 14-21 days .

9 0 Ν. L. W a d e

5 -I

Ο Ό

3 ·

I2

'

\ \ \ \

Us

\ \ \ \

\ \ \ 11

Ο 5 10 /5 2 0 %00ζ

FIGURE 1. Peach fruit (cv. J. Η. Hale) were stored at 1 C for 30 days in air containing the amounts of adged carbon dioxide indicated. After a further 7 day ripening period at 20 C, the fruit were subjectively assessed for the presence of flesh browning (d) and mealy texturetioz). A score of 1 denotes no flesh browning or mealy texture and 5 denotes a severe level of each attribute.

Coo l - s to r age d is rupts normal r ipening in t he g e a c h f ru i t . When p e a c h e s w e r e s t o r e d a t 1°C, fruit t r a n s f e r r e d to 20 C a f t e r 14 days or less evolved e t h y l e n e a t rap id r a t e s a f t e r t r an s f e r (Fig. 2) and unde rwen t a r e s p i r a t o r y r i se (Fig. 3). Af te r 21 days a t 1.0 C e t h y l e n e evolut ion was g r ea t l y r educed upon r e m o v a l and a f t e r 28 days was very low (Fig. 2). The r e sp i r a t ion r a t e s of fruit t r a n s f e r r e d a t 21 to 28 days we re ini t ia l ly abnormal ly high and then dec l ined , showing no ev idence of a no rma l r e s p i r a t o r y r i se . Inc ip ient s y m p t o m s of flesh mea l iness and r e t a r d e d ca ro teno id p igm e n ta t i on w e r e observed in fruit t r a n s f e r r e d a t 21 days , and s e v e r e s y m p t o m s of flesh mea l iness and browning w e r e p r e sen t a t 28 days .

When 20% v/v carbon dioxide was added to t h e s t o r a g e a t m o s p h e r e and fruit w e r e r e m o v e d to air a t 20 C a f t e r 30 days , a l a rge i nc rease in e thy lene evolut ion p r e c e d e d by a lag phase of six days was observed (Fig. 4). These p o s t - s t o r a g e changes in gas exchange r e s e m b l e d those of a i r -s t o r e d fruit r e m o v e d within the f irst fou r t een days of s t o r a g e . P e a c h e s coo l - s to red in air lose t h e abi l i ty to r ipen normal ly upon r emova l from s t o r a g e , whilst peaches s t o r ed in 20% v/v carbon dioxide r e t a i n the abi l i ty to r ipen .

The mechan i sm by which carbon dioxide e x e r t s this p r o t e c t i v e e f f ec t on peach fruit is unknown. Cont ro l e x p e r i m e n t s using low oxygen a t m o s p h e r e s verify t ha t a t r u e e f fec t of carbon dioxide is being observed . The possible s i t e s of carbon dioxide ac t ion in the cel l a r e n u m e r o u s . In t h e c o n t e x t of t h e ro le which m e m b r a n e s p lay in chil l ing s t r e s s , i t is i n t e r e s t i n g tha t a d i r ec t e f fec t of carbon dioxide on the hydra t ion and p e r m e a b i l i t y of m e m b r a n e s has been proposed (26).

Physiology of Cool-Storage Disorders of Fruit and Vegetables Θ1

0 10 10 30

FIGURE 2. Peach fruit (cv. J. H. Hale) were stored in air at 1 or 20QC. Ethylene evolution was measured during continuous storage at 20 C ( A — i )fwhile ethylene production at 1 C was barely detectable. At the times indicated by the arrows, samples of frmt were removed from 1 to 20 C and their ethylene production at 20 C (O 0) was measured for several days.

FIGURE 3. Peach fruit (cv. J. H. Hale) were stored in air at 1 or 20°C. Carbon dioxide evolution was measured during continuous storage at 20°C ( Δ — A) and 1°C (Δ Δ λ At the times indicated by the arrows, samples of fruit were removed from 1 to 20 C and their respiration rates at 20°C (O O) measured for several days.

9 2 Ν. L. W a d e

FIGURE 4. Peach fruit (cv. J. H. Hale) were stored for 30 days at 1°C in air containing 0% (Δ — Δ ) , 15% (Ο Ο) or 20% (Φ · ; of added carbori dioxide. The ethylene evolution of the fruit upon transfer to air at 20 C was measured.

E. Chemical Treatments

1. Antioxidants. D ipheny lamine , e thoxyquin and b u t y l a t e d hydroxy to luene (10, 11) r e d u c e t he s eve r i t y of superf ic ia l scald in apple f rui t . These t h r e e compounds a r e an t iox idan t s and may a c t by reduc ing t h e oxidat ion of f a rnesene (EI.C). Dipheny lamine , however , is also known to inhibit c a ro t eno id b iosynthes is (6) and b u t y l a t e d hydroxy to luene can p e r t u r b m e m b r a n e s t r u c t u r e (35).

2. Oils. A t r e a t m e n t for t h e con t ro l of superf ic ia l scald in apple fruit which p r e c e d e d the use of an t iox idan t s en ta i l ed wrapping each fruit in pape r i m p r e g n a t e d with minera l or v e g e t a b l e oils . The minera l oil wraps absorbed f a rnesene from the pee l of the fruit (11), but oil which c o n t a m i n a t e d t h e pee l may have also a f f e c t e d t he oxidat ion of t he f a rnesene and i t s de r iva t ives which r e m a i n e d in the pee l (III.C). R e c e n t l y , Jones et al. (15) have found t h a t t r e a t m e n t of b a n a n a f rui ts wi th d imethylpo lys i loxane , saff lower oil and mine ra l oil p r e v e n t s p e e l -d i sco lora t ion a f t e r a brief chill ing t r e a t m e n t . It was sugges t ed t ha t these m a t e r i a l s may p e r t u r b m e m b r a n e s t r u c t u r e , but ano the r valid hypothes is is t h a t t h e oxidat ion of f a rnesene or i t s de r iva t ives is a f f e c t e d .


3. Gibberellic acid and phorone (2,6-dimethyl-2,5-heptadien-4-one). G ibbere l l i c ac id (41) and phorone (42) r e d u c e subsequent w a s t a g e from low t e m p e r a t u r e b reakdown when appl ied to apple f ru i t . The mode of a c t i o n of t hese compounds is unknown.

4. Benzimidazole derivatives. The fungicide th i abendazo le (2-(4-thiazolyl) benz imidazole) r e d u c e s su r f ace p i t t i ng in coo l - s to red g rapef ru i t (26). Benz imidazo le and i t s de r i va t i ve s display weak cytokinin a c t i v i t y (30). Th iabendazo le may inhibit s e n e s c e n t changes which lead to su r f a c e p i t t i ng .

5. Calcium. Ca lc ium a c c u m u l a t e s in growing frui ts wi th di f f icul ty , and m a t u r e frui ts o f ten suffer from c a l c i u m - r e l a t e d d isorders (29). Good co r r e l a t i ons h a v e been found b e t w e e n t i ssue ca lc ium c o n t e n t and the suscep t ib i l i ty of the p roduce to co o l - s t o r ag e d isorders (23). P r o d u c e wi th r e l a t i ve ly high ca lc ium c o n t e n t is less l ikely to suffer from coo l - s t o r a ge d isorders than p roduce wi th low c a l c i u m . The appl ica t ion of ca lc ium to frui t a f t e r ha rves t r educes t he inc idence of d i sorders such as low t e m p e r a t u r e b reakdown of apples (1, 27). Var ia t ions in t i ssue ca lc ium may explain why c o m m o d i t i e s such as apple fruit do no t respond uniformly to chi l l ing, so t h a t only a p ropor t ion of frui t in a p a r t i c u l a r s amp le mani fes t injury.

Sorbitol has been i m p l i c a t e d in t h e b reakdown of apples in cool -s t o r a g e (III.D). Banger th et al. (1) found t h a t in f i l t r a t ion of fruit wi th sorbi to l i nc reased b reakdown, whilst addi t ion of ca lc ium to the in f i l t r a t ion solut ion p r e v e n t e d this adve r se e f f e c t . It was sugges ted t h a t ca lc ium enhances the u p t a k e and c o m p a r t m e n t a t i o n of s u b s t r a t e s such as sorb i to l . Simon (29) sugges t s t ha t apples a r e p red isposed to b reakdown by p e r m e a t i o n of the i n t e r ce l lu l a r spaces wi th phloem sap . The a f f e c t e d t i ssues b e c o m e inc iden ta l ly high in sorbi to l b e c a u s e phloem sap con ta ins so rb i to l . O s m o t i c u p t a k e of this i n t e r ce l lu l a r w a t e r causes individual pulp cel ls to swel l . In ca l c ium-de f i c i en t t i ssues t he cell walls a r e unable to w i th s t and the turgor p re s su re and the cel ls r u p t u r e , causing the n e c r o t i c lesions c h a r a c t e r i s t i c of b reakdown . The ro le of t e m p e r a t u r e in this hypothes i s is unc l ea r , s ince the sorbi to l is ex t e rna l l y -de r ived and not syn thes i zed by t h e chi l led t i s sues . P e r h a p s apple f ru i t s a r e a speci f ic example of the s i t ua t ion r e f e r r e d to in Sect ion I, whe re t e m p e r a t u r e is no t a p r i m a r y i n c i t a n t of injury but m e r e l y f a c i l i t a t e s t h e express ion of s y m p t o m s . C o n t r a r y to this propos i t ion is the ev idence t ha t apples a r e normal ly coo l - s to red below t h e c r i t i ca l t e m p e r a t u r e T g (Section Π).

V. CONCLUSION

The e t io logies of t h e coo l - s to r age d i sorders of fresh p roduce a r e a t bes t poorly unders tood . A unifying concep t has been provided by the hypothes i s t h a t a t e m p e r a t u r e - i n d u c e d change in m e m b r a n e s t r u c t u r e

9 4 Ν. L. W a d e

causes changes in cell c o m p a r t m e n t a t i o n and imba lances in m e t a b o l i s m . The re is , howeve r , a d e a r t h of e x p e r i m e n t a l ev idence which e i t h e r suppor t s or r e fu t e s this hypothes i s for many s t o r a g e d i sorders , s ince in many in s t ances it is no t known if p roduce is being s t o r e d above or below the c r i t i ca l t e m p e r a t u r e a t which t h e p o s t u l a t e d change in m e m b r a n e s t r u c t u r e begins . A problem also exis t s in l inking the p o s t u l a t e d change in m e m b r a n e s t r u c t u r e wi th t h e var ious abno rma l i t i e s in me tabo l i sm which h a v e been found to follow chil l ing s t r e s s . In no case does it s eem possible to se t out in de ta i l a s equence of even t s s t a r t i n g wi th a change in m e m b r a n e s t r u c t u r e and ending in cel l d e a t h . Rega rd l e s s of wha t p rogress is m a d e in our unders tand ing of t he e f fec t of t e m p e r a t u r e on m e m b r a n e a r c h i t e c t u r e , the conclusion appea r s inescapab le t ha t t he e f f ec t s which t e m p e r a t u r e has on t h e cel ls and t i ssues of s t o r ed p roduce will r e m a i n e n i g m a t i c .

VI. R E F E R E N C E S

1. Banger th , F . , Dil ley, D. R., and Dewey , D. H. J. Amer. Soc. Hort. Sci. 97, 679-682 (1972).

2. Chapman , Ε. Α., Wright , L., and Raison, J . K. Plant Physiol, 63, 363-366 (1979).

3 . Dav ies , R., Boyes, W. W., and de Vill iers, D . J . R . Union of South Africa Department of Agriculture and Forestry Low Temperature Research Laboratory, Capetown. Annual Report. 1936-1937, pp . 53-67 (1938).

4 . Dav ies , R., Boyes, W. W., and de Vill iers, D . J . R . Union of South Africa Department of Agriculture and Forestry Low Temperature Research Laboratory, Capetown. Annual Report 1937-1938, pp . 51-53 (1939).

5. Dav ies , R., and Boyes, W. W. Union of South Africa Department of Agriculture and Forestry Low Temperature Research Laboratory, Capetown. Annual Report. 1938-1939, p p . 41-43 (1940).

6. Dav ies , D. D. , Giovanel l i , J . , and Ap. R e e s , T. "Plant Biochemis-try"p. 340. Blackwell Oxford (1964).

7. Eaks , I. L. Proc. Amer. Soc. Hort. Sci. 67, 473-478 (1956). 8. F id ler , J . C , and Coursey , D. G. In "P roceed ings of t h e C o n f e r e n c e

on Tropical and Subtropica l F ru i t s , 1969" p p . 103-110. Tropical P roduc t s I n s t i t u t e , London (1970).

9. F id ler , J . C , and Nor th , C. J . J. Hort. Sci. 45, 197-204 (1970). 10. Gough, R. E., Shutak, V. G., Olney, C . E., and Day , H. J. Amer.

Soc. Hort. Sci. 98, 14-15 (1973). 11 . Huelin, F . E., and Coggiola , I. M. J. Sci. Food Agric. 19, 397-301

(1968). 12. Huelin, F . E., and Coggiola , I. M. J. Sci. Food Agric. 21, 44-48

(1970).

Physiology of Cool Storage Disorders of Fruit and Vegetables 9 5

13. Hues t i s , W. H., and McConnel l , Η. M. Biochem. Biophys. Res. Commun. 57, 726-732 (1974).

14. Hu lme , A. C , Smith , W. H., and Wool tor ton , L. S. C . J. Sci. Food Agric. 15, 303-307 (1964).

15. Jones , R. L. , F r e e b a i r n , Η. T., and McDonnel l , J . F . J. Amer. Soc. Hort. Sci. 103, 219-221 (1978).

16. Kosiyachinda , S., and Young, R. E. J. Amer. Soc. Hort. Sci. 101, 665-667 (1976).

17. Kosiyachinda , S., and Young, R. E. Plant Physiol. 60, 470-474 (1977).

18. L u t z , J . M., and Hardenburg , R. E. "The C o m m e r c i a l S to rage of F r u i t s , Vege tab les , and F lor i s t and Nurse ry Stocks ." Agr i cu l tu re Handbook No. 66 pp . 19 and 34. Uni t ed S t a t e s D e p a r t m e n t of Agr i cu l tu re , Washington D . C . (1968).

19. Lyons, J . M. Anna. Rev. Plant Physiol. 24, 445-466 (1973). 20. Lyons, J . M., and Raison , J . K. Plant Physiol. 45, 386-389 (1970). 2 1 . McGlasson, W. B., and Raison , J . K. Plant Physiol. 52, 390-392

(1973). 22. Mura t a , T. Physiol. Plant 22, 401-411 (1969). 23 . Pe r r ing , M. A. J. Sci. Food Agric. 19, 640-645 (1968). 24. Raison , J . K. and C h a p m a n , E. A. Aust. J. Plant Physiol. 3, 2 9 1 -

299 (1976). 25 . Raison , J . K., C h a p m a n , Ε. Α., and Whi te , P . Y. Plant Physiol. 59,

623-627 (1977). 26. Sch i f fmann-Nadel , M., C h a l u t z , E., Waks, J . , and L a t t a r , F . S.

HortScience 7, 394-395 (1972). 27. Sco t t , K. J . , and Wills, R. Β. H. HortScience 10, 75-76 (1975). 28. Sears , D . F . , and Eisenberg , R. M. J. Gen. Physiol. 44, 869-887

(1961). 29. Simon, E. W. New Phytol. 80, 1-15 (1978). 30. Skene, K. G. M. J. Hort. Sci. 47, 179-182 (1972). 3 1 . Smagula , J . M., and Bramlage , W. J . HortScience 12, 200-203

(1977). 32 . Smith , W. H. Nature (London) 159, 541-542 (1947a). 3 3 . Smith , W. H. J . Pomol . H o r t . Sci. 23, 92-98 (1947b). 34. Smith , W. H. Nature (London) 181, 275-276 (1958). 35 . Snipes, W., Person , S., Ke i th , Α., and Cupp, J . Science 188, 64 -66

(1975). 36. Van der Plank, J . E., and Dav ies , R. J. Pomol. Hort. Sci. 15, 226-

247 (1937). 37. Wade, N . L., and Bishop, D. G. Biochim. Biophys. Acta 529, 454-

464 (1978). 38 . Wills, R. Β. H. J. Sci. Food Agric. 19, 354-356 (1968). 39 . Wills, R. Β. H., and McGlasson, W. B. Phytochemistry 7, 7 3 3 - 739

(1968). 40 . Wills, R . Β. H., and McGlasson, W. B. J. Hort. Sci. 46, 115-120

(1971). 4 1 . Wills, R . Β. H., and P a t t e r s o n , B. D. Phytochemistry 10, 2983-2986

(1971).

9 6 Ν. L. Wade

42 . Wills, R. Β. H., and Sco t t , K. J . J. Hort. Sci. 49, 199-202 (1974). 4 3 . Wills, R. Β. H., Sco t t , K. J . , and McGlasson, W. B. J. Sci. Food

Agric. 21, 42-44 (1970). 44 . Wills, R. Β. H., Bai ley, W. M c C , and Sco t t , K. J . Plant Physiol.

56, 550-551 (1975).


SEQUENCE O F ULTRASTRUCTURAL CHANGES IN TOMATO COTYLEDONS DURING SHORT

PERIODS O F CHILLING

Reinfriede Rker

Gene ra l Foods Corpora t ion Techn ica l C e n t e r

Ta r ry town , New York

R. W. Breidenbach

Plan t Growth L a b o r a t o r y D e p a r t m e n t of Agronomy & R a n g e Sc ience

Univers i ty of Cal i forn ia Davis , Ca l i forn ia

J. M. Lyons

D e p a r t m e n t of Vege tab le Crops Univers i ty of Cal i forn ia


I. INTRODUCTION

When m a t u r e g rape f ru i t s (9) or t o m a t o f rui ts (8) w e r e exposed to chil l ing t e m p e r a t u r e s for ex t ended per iods , subcel lu lar fine s t r u c t u r e was ex tens ive ly modif ied . Chlorop las t convers ion to ch romop la s t s was inhibi ted , m i tochondr i a we re swollen and more opaque , and the nuc leop lasm of ten a p p e a r e d m o r e t r a n s p a r e n t , wi th a c c u m u l a t i o n s of condensed h e t e r o c h r o m a t i n . E lec t ron mic rog raphs of co ty ledons of t o m a t o seedl ings exposed to 5 C for 3 days also showed s e v e r e d a m a g e a t eve ry level of ce l lu lar o r g a n i z a t i o n . The cy top la smic vo lume appea red diminished, t h e m e m b r a n e s y s t e m s w e r e genera l ly d isorganized , t h e r e was a loss of cy top l a smic u l t r a s t r u c t u r e . Cel l d e a t h o c c u r r e d in a r a t i o of one in t en cel ls (4).

9 7 Copyright © 1979 by Academic Press, Inc.

All rights of reproduction in any form reserved ISBN 0 1 2 4 6 0 5 6 0 5

9 8 R. Ilker et al.

Evidence also sugges ts t h a t d i f fe ren t m e m b r a n e sy s t ems within t he cell may be d i f fe ren t ia l ly sens i t ive to chi l l ing. Minchin and Simon (7) r e p o r t e d t h a t ae rob ic r e sp i ra t ion was impa i r ed a t 1Z C in expanding cucumber l eaves , but t ha t w a t e r and so lu tes unde rwen t l eakage a t 8 C, implying tha t the tonoplas t and p l a s m a l e m m a a r e less sens i t ive than the mi tochondr ia l m e m b r a n e s . So-cal led "lipid c lus te r ing" (16) seen as d i s c r e t e a r e a s wi th smooth f r a c t u r e faces and o the r a r e a s of a g g r e g a t e d p a r t i c l e s in f r e e z e - f r a c t u r e e l e c t r o n - m i c r o s c o p e s tudies of m a m m a l i a n lymphocy tes and Tetrahymena pyriformis, also sugges ted t h a t d i f fe r en t m e m b r a n e s y s t e m s in t he cell undergo t h e r m o t r o p i c t r ans i t ions a t d i f fe ren t t e m p e r a t u r e s (5).

The in t ens i ty of chil l ing injury depends on the t e m p e r a t u r e and on the dura t ion of exposure . This r e p o r t on t o m a t o co ty ledons used t i m e i n c r e m e n t s to assess when and whe re t he f irst e f f ec t s on ce l lu lar u l t r a s t r u c t u r e we re seen and how they p rogressed during exposure for per iods up to 24 h.

F igures 1, Z, 6, 7, 8, 1Z, and 18 i l l u s t r a t e t h e u l t r a s t r u c t u r e of mesophyll cel ls from 8-day-old unchi l led t o m a t o co ty l edons . Organe l l e envelopes we re d i s t inc t , well def ined boundar ies . C i s t e r n a e of t he endoplasmic r e t i cu lum and d i c tyosomes were r e l a t i v e l y in f requen t . Micro tubules with i n t e r c o n n e c t i n g br idges and connec t ions to t he p l a sma m e m b r a n e were n u m e r o u s . Vacuolar s t o r a g e p ro t e in was abundant and t h e gene ra l ce l lu lar u l t r a s t r u c t u r e was typica l for p l an t ce l l s , espec ia l ly those of s t o r age o rgans .

The Ζ h of chil l ing a t 5 C may have induced smal l changes in u l t r a s t r u c t u r e in some ce l l s , but no t in o t h e r s . These changes may be seen as a sl ight d e c r e a s e in t he def ini t ion of m e m b r a n e prof i les , or as d i scon t inu i t i e s in the thylakoids and envelopes of p las t ids and the ou t e r m e m b r a n e of mi tochondr i a . At 4 h (Figs. 3 , 9, 13, 19), de f in i te changes due to chil l ing exposure we re observed , including vacuo l iza t ion of the cy top lasm (Fig. Z), a loss of def ini t ion of t h e thylakoids and p r o l a m m e l a r bodies (Fig. 8), and the deve lopmen t of d i scon t inu i t i e s in the enve lopes of mi tochondr i a (Figs. Ζ, 1Z), pe rox i somes (Fig. 1Z), and nucle i (Fig. 18). The p l a s m a l e m m a and ground p lasm (Fig. Z), including the c o r t i c a l mic ro tubu les , a p p e a r e d unchanged . At 8 h, t h e ground plasm a p p e a r e d genera l ly less s t r u c t u r e d wi th a loss of o rder and def in i t ion of r i bosomes . The p l a s m a l e m m a sti l l a p p e a r e d i n t a c t (Fig. 13). The p las t id enve lopes , p ro lame l l a r bodies , and thylakoids (Fig. 9), as well as the mi tochondr ia l (Fig. 13) and t h e nuc l ea r m e m b r a n e s (Fig. 19), b e c a m e less de f in i t e . An inc reased e l ec t ron opac i t y of t h e o rgane l l e m a t r i c e s was c o m m o n . Micro tubules w e r e now r a r e l y s e e n . Swelling of t h e endoplasmic r e t i cu lum and d i c tyosome c i s t e r n a e and loss of bo th the pe rox i somal envelope and m a t r i x had a l r e a d y o c c u r r e d a t 4 h (Fig. 1Z). In many in s t ances , the p las t id m e m b r a n e s w e r e ba re ly reso lved a t 1Z h (Fig. 4), while s ta in ing of t h e p las t id m a t r i x was i n t ense . Changes in t h e cel l wal ls cons i s ted of changes in o p a c i t y and occas iona l swel l ing of t he middle l ame l l a . At 16 h (Fig. 10, 14) all t h e chil l ing s y m p t o m s a p p e a r e d in tens i f ied . In addi t ion , t h e p ro t e in p r e c i p i t a t e s wi thin t h e vacuo le s

S e q u e n c e of Ul t ras t ruc tura l C h a n g e s in T o m a t o C o t y l e d o n s 9 9

FIGURE 1. Tomato (Lycopersicon esculentum c v . VF145) mesophyll cells from cotyledons of non-chilled seedlings. Seedlings used for all treatmentsQwere germinated and grown in vermiculite in the dark for 6 days at 20 C. The seedlings were then transferred to slant boards as described by Waring et al. (1976) after carefully washing them free of vermiculite. The seedlings were established on the slant Jgoards for 1 day at 20 C. Exposure to the chilling temperature of 5 was initiated beginning with those seedlings receiving the longest exposure so that all seedlings were the same age when killed and fixed. Ultrathin sections were prepared as described by Rker et al (4) and observed with a JEOL 100S electron microscope. Plastid, PL; mitochondria, M; storage protein, P; vacuole, V; nucleus, N; spherosome, G; microbody, M.B. dictysome, D; middle lamella, L.

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FIGURE 2. Part of a mesophyll cell after 4 h of chilling.

ΙΟΙ

FIGURE 3. View of mesophyll cells after 12 h of chilling. One cell is slightly plasmolyzed (arrow) cell walls are swollen at the region of middle lamella.

FIGURE 4. Appearance of mesophyll cells after 24 h of chilling. Remnant of what could be microtubules are seen at the arrow. Double arrows indicate the nuclear envelope.

Sequence of Ultrastructural Changes in Tomato Cotyledons 1 0 3

FIGURE 5. A view of an unchilled mesophyll cell to illustrate the distribution of microtubules (arrows). Note the sharpness of all cellular details.

FIGURE 6. An enlargement of Figure 5 to show that microtubules are bridged between themselves and to the plasmalemma.

1 0 4 R. Ilker et al

FIGURE 7. Appearance of prolammelar body (PB) from a non-chilled cell. A fairly regular hexagonal pattern is seen (x 58,000).

FIGURE 8. After 4 h of chilling, the prolammelar body appears collapsed. Thylakoids are intact.

FIGURE 9. After 12 h of chilling, most membraneous parts of plastids are indistinct.

FIGURE 10. After 16 h of chilling, the membrane changes are still progressing; the entire plastid stains darkly.

Sequence of Ultrastructural Changes in Tomato Cotyledons 1 0 5

FIGURE 11. The appearance of mitochondria from wnchilled tissue.

FIGURE 12. Mitochondrion (M) from tissue chilled for 4 h. Envelope and cristae are indistinct. Peroxisome (MB) and plastid membrane are indistinct (x 42,000).

1 0 6 R. Ilker et al.

FIGURE 13. Mitochondria after 8 h of chilling. Envelopes appear faint or disassembled. Matrix is darkened (x 42,000). Arrow points to microtubular remnants.

FIGURE 14. Mitochondrion after 16 h of chilling. Mitochondrial structure appears to have stabilized because injury appears about the same as at 8 h (x 42,000).

S e q u e n c e of Ul l ras t ruc tura l C h a n g e s in T o m a t o C o t y l e d o n s 1 0 7

FIGURE 15. Mitochondrion from a vascular cell after 20 hours. Cristae are distended, envelope is missing. Fat body(G) and cytoplasmic details, including plasma membrane (double arrows) are highly damaged (x 42,000).

FIGURE 16. Mitochondria after 24 h of chilling. Most membranes are indistinct (x 42,000).

1 0 8 R. Ilker et al.

FIGURE 17. Nucleus (N) from an unchilled mesophyll cell. Both nuclear envelopes appear distinct. The nucleoplasm is evenly dispersed.

FIGURE 18. The nucleus after 4 h of chilling envelope has small discontinuities.

FIGURE 19. The nucleus after 12 h of chilling, large parts of the envelope may be missing.

FIGURE 20. Nuclear envelope after 20 h of chilling seems "smeared-out". A small amount of heterochromatin has accumulated.

FIGURE 21. Nuclear envelope after 24 h of chilling. In this instance a filamentous "beard' is associated with the nuclear surface.

S e q u e n c e of Ul t ras t ruc tura l C h a n g e s in T o m a t o C o t y l e d o n s 1 0 9

a p p e a r e d to have diminished. T w e n t y and 24 h of chil l ing of ten r e su l t ed in an a lmos t c o m p l e t e loss of m e m b r a n e s , as i l l u s t r a t e d in F ig . 5. P las t id , endoplasmic r e t i c u l u m , and nuc l ea r and mi tochondr ia l m e m b r a n e s we re absent or nea r ly absen t . The tonoplas t and p l a s m a l e m m a (see F ig . 15), as well as any r e m n a n t s of m ic ro tubu l e s , w e r e poorly reso lved . Both nuc l ea r m e m b r a n e s of ten a p p e a r e d to be c o m p l e t e l y d i sassembled (Fig. 20), wi th f i l amentous pe r inuc l ea r m e m b r a n e assoc ia t ions (Fig. 21). However , de sp i t e the a p p a r e n t absence of m e m b r a n e s , ce l lu lar o rgan iza t ion r e m a i n e d (its loss was obse rved only r a r e l y in th is s tudy) . F u r t h e r m o r e , cy top l a smic dehydra t ion and ce l l -vo lume changes , which might be e x p e c t e d from the a p p a r e n t loss of s t r u c t u r e and funct ion of t h e p l a s m a l e m m a , we re u n c o m m o n . At this t i m e , the cel l wall exh ib i ted m a r k e d i r r e g u l a r i t i e s . It a p p e a r e d th inner in some a r e a s , while in o t h e r s i t was expanded , wi th i n c r e a s e d m a t e r i a l in t h e middle l ame l l a .

Π. DISCUSSION

E lec t ron mic rog raphs from t o m a t o co ty ledon cel ls show t h a t changes from chil l ing exposures occur very quickly a t the m e m b r a n e l eve l . Slight d i scon t inu i t i es in some of t h e m e m b r a n e prof i les of the p las t ids and mi tochondr i a a f t e r very brief exposure to chil l ing t e m p e r a t u r e s give ev idence t ha t t h e t i s sue has been sub jec ted to a s t r e s s . D a m a g e in m e m b r a n e s is seen seve ra l hours be fore the a p p e a r a n c e of any p l a smoly t i c p h e n o m e n a or changes in s ta in ing .

Af te r pro longed chil l ing (4, 9), t h e mi tochondr i a genera l ly a p p e a r e d less seve re ly a l t e r e d than the o t h e r o rgane l l e s . However , the r e l a t i v e l y shor t exposures of 2, 4, and 8 h used in t h e p r e s e n t s tudy r e s u l t e d in d i scon t inu i t i e s in the mi tochondr i a l enve lope , fol lowed by swel l ing of the c r i s t a e and i n c r e a s e d opac i ty of t h e m a t r i x . It appea r s t ha t the s t r u c t u r e of the mi tochondr i a may somehow s t ab i l i ze during the longer chil l ing exposures , whe rea s t h a t of t he o t h e r o rgane l les con t inues to change .

The p las t ids of t o m a t o mesophyl l a r e highly suscep t ib le to chil l ing, and visible d i so rgan iza t ion p rogres ses m o r e quickly and c o m p l e t e l y in the p las t ids than in t he mi tochondr i a or o the r o rgane l l e s . An i m p o r t a n t cause of this d i f fer ing behavior could be t he unique lipid compos i t ion of p las t ids , wi th a l a rge a m o u n t of g a l a c t o and sulfo lipids and r e l a t i ve ly smal l a m o u n t s of phosphol ipids . This may r e l a t e to the r e p o r t of Wilson and Crawfo rd (13), who found t h a t t h e d e g r e e of s a t u r a t i o n could be a l t e r e d in the phospholipids , but no t in the glycolipids during the ha rden ing of cucumber seedl ings . Shneyour et al. (11) found a d iscont inu i ty in t h e p lo t s of 1/T vs t h e log of t h e r a t e of pho to reduc t i on of NADP by the ch lorop las t s a t a round 12 C, about t he s a m e t e m p e r a t u r e w h e r e d i scon t inu i t i es a r e obse rved for m e m b r a n e - b o u n d mi tochondr ia l enzymes(6) .

The n u c l e a r enve lope wen t th rough a p rogress ive loss of def ini t ion, cu lmina t ing in what of ten a p p e a r e d to be f i l amen tous pe r inuc l ea r a s soc ia t ions . F r a n k e (2) has desc r ibed such pe r inuc l ea r "beards" in HeLa

1 ΙΟ R. Ilker et al

and o t h e r ce l l s . He be l ieves t h a t they occur a t t i m e s of g e n e r a l m e m b r a n e d isassembly , as for example , jus t be fo re mi tos i s .

Desp i t e the a p p a r e n t loss of t h e nuc l ea r envelope (Fig. 5), t he nuc leoplasm did no t undergo the ex tens ive changes observed wi th 3 days of chil l ing (4). Only occas ional ly we re t h e r e smal l a c c u m u l a t i o n s of h e t e r o c h r o m a t i n . Most of the t i m e the g ranu la r i t y of the nuc l ea r m a t r i x r e m a i n e d unchanged .

Co r t i c a l mic ro tubu les a r e be l ieved to se rve as a cy to ske l e ton in expanding p lan t ce l l s . In an imal ce l l s , t h e r e is ev idence t ha t i n t e r a c t i o n s b e t w e e n mic ro tubu les , m ic ro f i l amen t s , and the p l a s m a l e m m a aid p ro t e in mobi l i ty a t the cel l su r face (1 , 10). Our e l e c t r o n mic rog raphs i nd i ca t e t h a t mic ro tubu les may d i sassemble or col lapse a f t e r shor t per iods of chi l l ing. Since these s t r u c t u r e s a r e known to depo lymer i ze a t low t e m p e r a t u r e in vitro, the i r a lmos t c o m p l e t e d i s a p p e a r a n c e by 8 h of exposure is no t surpr is ing.

The swell ing and d iscolora t ion of t h e cell walls s t a r t i n g a t 12 h may i nd i ca t e a l eakage and /or a c c u m u l a t i o n of so lu tes from the cel ls a t t ha t t i m e . The p r e m a t u r e loss of s t o r a g e p ro te in may no t be a s soc i a t ed wi th the no rma l me tabo l i sm of ac t ive ly growing seedl ings . The s t o r a g e p ro te in could be dissolved and d ispersed or hydro lyzed wi thout fu r ther me tabo l i sm during t h e chill ing per iod .

U l t r a s t r u c t u r a l changes in t h e p l a s m a l e m m a o c c u r r e d a t 20 and 24 h, much l a t e r than in the o the r m e m b r a n e s . We be l ieve t h a t the absence of visible cy top lasmic dehydra t ion in this s tudy is due p r imar i ly to t he higher r e s i s t a n c e of this m e m b r a n e to chi l l ing. In an imal ce l l s , a loss of ce l l -vo lume regu la t ion i n i t i a t e s a s equence of u l t r a s t r u c t u r a l changes tha t can lead to d e a t h (12). In p l an t s , Guinn (3) has po in ted out t ha t dehydra t ion is a p r e r e q u i s i t e to mac roscop ic chill ing injury because c o t t o n leaf -d iscs f loa ted on 0.2 Μ manni to l r e m a i n e d f ree of s y m p t o m s during chil l ing, whe reas exposure of whole p l an t s injured or kil led the l eaves . Wright and Simon (14) found t ha t phospholipids dec l ine during chill ing only a f t e r w a t e r loss is cons ide rab le . Accord ing to Minchin and Simon (7), so lu tes leak from cucumber leaves a t t e m p e r a t u r e s below 12 C, the t e m p e r a t u r e where d i scont inu i t i es occur in t h e plot for t h e u p t a k e of oxygen.

In lymph cel ls (15, 16) and in Tetrahymena (5), t h e p l a sma m e m b rane is highly r e s i s t a n t to chi l l ing- induced f r e e z e - f r a c t u r e p a t t e r n s , a p r o p e r t y a s soc ia t ed , a t l eas t in p a r t , to the s t rong damping e f fec t of s t e ro l s (ch lores te ro l and t e t r a h y m e n o l , r espec t ive ly ) upon lipid f luidity of these m e m b r a n e s . Even though l i t t l e is known about the lipid compos i t ion in p lan t p l a sma m e m b r a n e s , s imi lar pr inc ip les may ac t to m a k e them m o r e r e s i s t a n t to chil l ing.

Each m e m b r a n e type in f r e e z e - f r a c t u r e d Tetrahymena i n i t i a t e s smooth m e m b r a n e reg ions or "lipid c lus ter ing" a t speci f ic t e m p e r a t u r e s . While e l e c t r o n mic rographs of thin sec t ions canno t r e v e a l such p rec i se re la t ionsh ips , they did i l l u s t r a t e a progress ion or cont inuum of chil l ing injury, and the d i f fe ren t ia l sens i t iv i ty to t e m p e r a t u r e s of individual m e m b r a n e types .

TABLE I. Dependency ofnChilling Symptoms on Time in Various Compartments of Tomato Cotyledon Chilled at 5° C.

Duration of Plasma- Tono- Mito Plastids Nuclear Peroxi Micro

Chilling lemma plast chondria envelope somes tubules (h)

2 + + 4 + + + + + — 8 -f ++ + + absent

12 + ++ +++ + + absent 16 + + +++ ++++ + ++ absent 20 + ++ +++ ++++ +++ ++ absent 24 ++ ++ +++ ++++ ++++ ++++ absent

aAbout six samples of each treatment were observed. — = no injury

+ = slight ++ - moderate

+++ - severe ++++ - extreme

R. Ilker et al

In s u m m a r y , t h e u l t r a s t r u c t u r a l chil l ing s y m p t o m s of t o m a t o -seedl ing co ty ledons (held a t 5 C from 2 to 24 h) m a n i f e s t e d t h e m s e l v e s p r imar i ly as a progress ion of m e m b r a n e d e t e r i o r a t i o n s (see Table I). We have found t h a t : 1) m e m b r a n e a l t e r a t i o n s and t h e a p p e a r a n c e of smal l vacuoles p r e c e d e d o the r ce l lu lar changes ( these w e r e followed by m o r e s e v e r e a l t e r a t i o n s of t he m e m b r a n e s of p las t ids , mi tochondr i a , endoplasmic r e t i c u l u m , pe rox i somes , and nuc le i , of ten p roceed ing to c o m p l e t e loss of u l t r a s t r u c t u r e ) ; 2) s e v e r e cy top la smic dehydra t ion , new ce l l -wal l depos i t s , and accumula t i on of osmiophi l ic m a t e r i a l , previous ly observed a f t e r 3 days of chil l ing a t 5 C (4), was uncommon with the shor t e r exposures used h e r e ; 3) d i f fe ren t lengths of exposure we re r equ i r ed to d a m a g e d i f fe ren t types of o rgane l l es ; and 4) t he p l a s m a l e m m a appea red r e l a t i ve ly more r e s i s t a n t to chil l ing than the o the r m e m b r a n e s .

ACKNOWLEDGMENTS

We thank Dr . Ε. M. Gifford, D e p a r t m e n t of Botany, Univers i ty of Cal i forn ia , Davis , for kindly al lowing us to use his l a b o r a t o r y . We thank Dr . C . Rick, D e p a r t m e n t of Vege tab le Crops , Univers i ty of Cal i forn ia , Davis , for providing us wi th t o m a t o seeds .


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2. F r a n k e , W. W. Protoplasma 73, 263-292 (1971). 3 . Guinn, G. Crop Sci. 11, 11 -12(1971) . 4 . I lker , R., Waring, A. J . , Lyons, J . M., Bre idenbach , R. W. Proto

plasma 90, 229-252 (1976). 5. Ki ta j ima , Y., Thompson, G. A. J. Cell Biol. 72, 744-755 (1977). 6. Lyons, J . M., Raison, J . K. Plant Physiol. 45, 386-389 (1970). 7. Minchin, Α., Simon, E. W. J. Exp. Bot. 24, 1231-1235 (1974). 8. Moline, Η. E. Phytopathology 66, 617-624(1976) . 9. P l a t t - A l o i a , Κ. Α., Thomson, W. W. Cryobiology 13, 95-106

(1976). 10. P o s t e , G., Papahadjopoulos , D. Proc. Natl. Acad. Sci. USA 73,

1603-1607 (1976). 11 . Shneyour, Α., Raison, J . K., Smill ie , R. M. Biochem. Biophys.

Acta 292, 152-161 (1975). 12. Trump, B. F . , Croke r , B. P . , J r . , Mergher , W. J . In Cel l M e m b r a n e s

(Rich te r , G. W., Scaprel l i , D . G., eds.) The Will iams and Wilkins Co . , Ba l t imore (1971).

13. Wilson, J . M., Crawford , R. Μ. M. New Phytol. 73, 805-820 (1974).

1 12

S e q u e n c e of Ul t ras t ruc tura l C h a n g e s in T o m a t o C o t y l e d o n s

14. Wright , J . , Simon, E. W. J. Exp. Bot. 23, 400-411 (1973). 15. Wunderl ich, F . , Hoelz l Wal lach, D. F . , Speth , V., F i sher , H. Bio

chim. Biophys. Acta 373, 34-43 (1974). 16. Wunder l ich , F . , Rona i , Α., Speth , V., Seelig, J . , Blume, A. Bio-

chem. 14, 3730-3735 (1975).

1 13



MOVEMENT AND LOSS O F ORGANIC SUBSTANCES FROM ROOTS AT LOW TEMPERATURE

Μ. N. Christiansen

U.S. D e p a r t m e n t of Agr i cu l tu re Science and Educa t ion Admin i s t r a t i on

Agr icu l tu ra l R e s e a r c h P lan t Physiology I n s t i t u t e

P lan t S t ress L a b o r a t o r y Bel tsvi l le , Maryland

Subs tances r e l e a s e d from r o o t s a r e commonly ca l led e x u d a t e s , be they pass ively or ac t ive ly t r a n s p o r t e d to the roo t r h i zosphe re . Root e x u d a t e s inc lude sugars , amino ac ids , p ro t e in , and most o t h e r en t i t i e s common to p lan t roo t ce l l s . Severa l env i ronmen ta l s t r e s s e s including w a t e r def ic iency , anaerobios i s , low pH, r e d u c e d l ight i n t ens i ty , and t e m p e r a t u r e e x t r e m e s , a r e known to i nc rease loss of o rganic and inorganic subs t ances from r o o t s . A l t e r a t i o n of t he chemica l qua l i ty of e x u d a t e s is also r e p o r t e d as a consequence of s t r e s s . The loss of subs t ances to t he rh i zosphe re has been r e l a t e d to subsequent i nc reased roo t d i sease inc idence and has , t h e r e f o r e , been of cons iderab le i n t e r e s t to p l an t pa tho log i s t s . Al le lopa th ic e f f ec t s or t he c r e a t i o n of unfavorab le condi t ions for ne ighbor ing p l an t s or subsequent p lan t ings may be due to exuda t e s (13). The ea r l i e s t r e p o r t s of t h e n a t u r e of r oo t exuda t ion we re by Knudson (10). R o v e r i a (14, 15) has p r e s e n t e d seve ra l r ev i ews of the sub jec t . Our i n t e r e s t in t h e subject s t e m s from a convic t ion t h a t s t r e s s -induced exuda t ion r e su l t s from m e m b r a n e mal func t ion , and tha t exuda t ion is an easi ly m e a s u r e d and re l i ab le in vivo i nd ica to r t h a t ad ve r se t r e a t m e n t s h a v e induced injury.

The d o c u m e n t a t i o n t h a t s t r e s se s induce g r e a t e r exuda t ion genera l ly suppor t s the concep t t ha t t he mechan i sm r e l a t e s d i r ec t l y to t he physical n a t u r e of m e m b r a n e s or to m e t a b o l i c even t s t h a t suppor t m e m b r a n e funct ion and i n t e g r i t y . F rom this bas is , one can e x t r a p o l a t e to t he thes is t h a t t e m p e r a t u r e e x t r e m e s a r e a f fec t ing t h e con t ro l sy s t ems t h a t enc lose , c o m p a r t m e n t a l i z e and t r an spo r t o rganic and inorganic subs t ances .

Copyright « 1979 by Academic Press. Inc. All rights of reproduction in any form reserved

ISBN Ο12 46056O5 1 15

1 1 6 Μ. Ν. C h r i s t i a n s e n

It is well e s tab l i shed t h a t mos t o rganic subs t ances found in p lan t r oo t cel ls also occur in exuda te s [see r ev iews by Rove r i a (14; Schroth and Hi ldebrand (16) ] . O t h e r ev idence has been p r e s e n t e d t h a t top ica l ly appl ied non-endogenous chemica l s can also be t r a n s p o r t e d within t he p lan t and e xuda t ed from roo t s (9). As one might expec t , e x u d a t e s from v a c u o l a t e cel ls may vary from those of n o n - v a c u o l a t e cel ls (1). G a r r a d and Humphreys (6) showed var ia t ion in qua l i ty of s accha r ides from corn s cu t e l l a r t i ssue in varying condi t ions .

The re is much ev idence t h a t m e m b r a n e s under cold s t r e s s fail in the i r ro le as m e t a b o l i c b a r r i e r s . Banana (Musa sp.) is an exce l l en t example ; only brief chil l ing is n e c e s s a r y to induce marked fruit d i sco lora t ion (ZO). The color change is a consequence of oxida t ion of phenols by r e l e a s e of membrane -bound polyphenol ox idase . O t h e r examples of leaf (8) and roo t exuda t ion (Z) show a rapid a l t e r a t i o n of m e m b r a n e s and r educed con t ro l of cel l c o n t e n t s . Exudat ion , t h e r e f o r e , provides in vivo ev idence t ha t t e m p e r a t u r e e x t r e m e s a l t e r m e m b r a n e s .

One of t h e ques t ions p r e s e n t e d by chi l l ing- induced exuda t ion is whe the r it is a consequence of cold inhibi t ion of me tabo l i c s y s t e m s tha t a r e essen t ia l for m a i n t e n a n c e of m e m b r a n e i n t e g r i t y or if it is due to a s imple biophysical d isorder ing of the lipid b i layer . The ques t ion can pe rhaps in p a r t be answered by obse rva t ion of response t i m e . A rap id chil l ing induct ion of l eakage might well i nd i ca t e physical m e m b r a n e "channel iza t ion" a l t e r a t i o n , while a response t ha t r equ i r e s hours of even days may be a consequence of me tabo l i c b lockage , energy unba lance or toxin accumula t i on .

Resp i r a t ion inhibi t ion by cold can be dup l i ca ted by chemica l inhib i tors or O- de f ic iency and can induce exuda t ion r a t h e r rapidly which somewha t null if ies an a r g u m e n t for c a t e g o r i z i n g s t r e s s e f f ec t s wi thin a shor t or long t i m e f rame as r e l a t e d to physical or m e t a b o l i c e f f ec t s on m e m b r a n e s . Rapid low t e m p e r a t u r e induced exuda t ive response of l eaves or roo t s (Fig. 1) as c o n t r a s t e d to fleshy organs such as f ru i ts or tubers may be only a consequence of t i ssue mass and h e a t loss . Sweet p o t a t o (Ipomoea batatas, (L.) Lam.), for example , only develops l eakage and t i ssue browning a f t e r a pro longed exposure of Z-4 weeks a t 10 (1Z). The s a m e s i tua t ion occurs in in t e rna l browning of app le .

The i n t e rven t ion of d iva lent ca t ions such as ca lc ium or magnes ium in cold- induced exuda t ion provides ev idence t h a t t h e p rocess is physical in n a t u r e . Ca lc ium is known to rapidly s t ab i l i ze roo t cell m e m b r a n e funct ion (3). Much of t he published in fo rmat ion concern ing Ca and m e m b r a n e s does not r e l a t e to cold s t r e s s but to t h e ro le of C a in med ia t ing m e m b r a n e ion d i sc r imina t ion or u p t a k e (5, 18). The need for Ca for normal roo t fo rmat ion and funct ion was n o t e d by Sorokin and So m m e r (17). The p r e s e n c e of Ca can lead to a genera l e n h a n c e m e n t of ion absorpt ion commonly ca l led t h e "Viets" e f fec t (19) which is a t t r i b u t e d to t he e f fec t of Ca on m e m b r a n e p e r m e a b i l i t y . The manne r in which Ca funct ions in m e m b r a n e s is a point of cons iderab le d e b a t e . Dodds and Ellis (4) hold tha t membrane -bound ATPase is a c t i v a t e d by Ca . R e m o v a l of Ca wi th a che la t ing agen t (EDTA) r e d u c e s r e sp i r a t i on and r e su l t s in loss of nuc leo t ides from roo t s (7). This again i m p l i c a t e s

M o v e m e n t & L o s s of O r g a n i c S u b s t a n c e s from R o o t s a t L o w T e m p e r a t u r e 1 1 7

r e sp i r a t ion as an i m p o r t a n t f ac to r in m a i n t e n a n c e of m e m b r a n e i n t eg r i t y . It has also been es tab l i shed th^ t Ca m e d i a t e s m e m b r a n e con t ro l of cel l o rganic c o n t e n t s . G a r r a r d and Humphreys (6) have shown tha t Ca or Mg p r e v e n t s suc rose l eakage from corn scu te l l a r t i s sue , espec ia l ly a t low t e m p e r a t u r e s . They t h e o r i z e t h a t C a binds anionic groups of the m e m b r a n e s t r u c t u r e to form cross br idges b e t w e e n m e m b r a n e s t r u c t u r a l componen t s , t h e r e b y main ta in ing a selecting p e r m e a b i l i t y by po re rad ius or su r f ace c h a r g e . O t h e r s tudies wi th C a i nd i ca t e t ha t Ca loca l i zes on the su r face of roo t ce l ls (11), t he r eby sugges t ing a p l a s m a l e m m a m e m b r a n e s t r u c t u r a l m a i n t e n a n c e ro le in r o o t s .

Chr i s t i ansen et al. (2) n o t e d t h a t C a e x e r t s a m a r k e d e f fec t on co ld - s t r e s s - induced exuda t ion (Table I). Cold or O^ def ic iency induced exuda t ion is b locked by Ca or Mg; low pH (3.0) induced exudat ion could no t be con t ro l l ed by added C a . The inhibi t ion of exuda t ion was e f f ec t ive as a p r e - t r e a t m e n t ( T r e a t m e n t F , Table I), or by addi t ion a f t e r induct ion of exuda t ion ( T r e a t m e n t E). The b lockage of ongoing exuda t ion by C a is qu i te rap id (within minutes) and s t rongly sugges t s a biophysical binding of m e m b r a n e s t r u c t u r e r a t h e r than a s t imu la t i on of ATPase funct ion or an e f fec t on o the r me tabo l i c e v e n t s . This also sugges t s t ha t co ld- induced exuda t ion is due to a d i r ec t phys ica l a l t e r a t i o n of m e m b r a n e s r a t h e r than an i m p a c t on m e m b r a n e - a s s o c i a t e d m e t a b o l i s m .

We have also p e r f o r m e d e x p e r i m e n t s c o n c e r n e d wi th l a t e n t e f f ec t s of chi l l ing. The aim of t hese s tud ies was to d e t e r m i n e the chil l ing dosages r equ i r ed to induce exudat ion ; to develop chemica l ame l io r a t i on t echn iques ; and, to d e t e r m i n e by chemica l inhib i tors if me tabo l i sm was involved in r e c o v e r y or "mending t h e ba r r i e r s . " In gene ra l , four days a t 1 0 C was t he point of no r e t u r n for r e c o v e r y of c o t t o n r o o t s . Ca lc ium applied a f t e r prolonged cold did l i t t l e to h a s t e n r e c o v e r y of m e m b r a n e con t ro l . We found no chemica l me thods of ame l io r i za t i on , but did find many "me tabo l i c inhibi tors" t ha t slow r e c o v e r y as well as induce roo t exuda t ion . In gene ra l , r e sp i r a t i on inhib i tors cause i nc rea se s in exuda t ion ; p ro te in synthes is inhib i tors have l i t t l e e f f ec t , and the uncouple rs such as D N P induce exuda t ion as well as p r e v e n t r e c o v e r y a f t e r chil l ing (Tables Π and HI).

In using "specif ic" m e t a b o l i c inh ib i tors , one l ea rns t h a t t hey a r e more of ten dull r e s e a r c h tools , f i rs t because they a r e not specif ic in m e t a b o l i c ac t ion s i t e , secondly b e c a u s e l i t t l e can be d e t e r m i n e d about r a t e of influx to ac t i on s i t e s . The most i n t e r e s t i n g point in these d a t a is t h a t EDTA (Table I) e x e r t e d a much g r e a t e r induc t ive e f fec t than i o d o a c e t a t e , f luor ide , or f l u o r o c i t r a t e a l though EDTA is r e p u t e d to move in to t i s sue much m o r e slowly (21). One might surmise t h a t EDTA a c t s a t the m e m b r a n e su r f ace by r emov ing C a or o t h e r s tab i l i z ing ca t ions r a t h e r than by exe r t ing an e f fec t upon m e t a b o l i s m , w h e r e a s t h e r e sp i r a t ion inhib i tors must p e n e t r a t e t h e cel l m i tochondr i a to a f f ec t r e sp i r a t i on .

1 18 Μ. Ν. Christiansen

FIGURE 1. Cumulative radicle loss of carbohydrate at 31° and 5° by seedlings germinated 1 day at 31 .

TABLE Ι. Cotton Radicle Exudation at Low Temperature in or at Low pH

Exudation μ g/seedling/hour

Treatment carbohydrate

A. Control 31° 4 5 B. Control 5°

Control + CaSO. (JO M) 5° + CaSO. (fo'

bM) R

5° for 2 hours - 5° CaSO4(10~°M) 31° + CaS04 (10~

bM) •> 5° Water

14 4 C . D . E. F.

Control 5° Control + CaSO. (JO M) 5° + CaSO. (fo'

bM) R

5° for 2 hours - 5° CaSO4(10~°M) 31° + CaS04 (10~

bM) •> 5° Water

2 4 2 4

0 4 1 4

G. N2 , -6 22 0

Η. N9 + CaSO. (10 °M) 1 7 I. ρή 3-31°

4

pH 3 + CaS04 £10 M) 121 0

J. ρή 3-31°

4

pH 3 + CaS04 £10 M) 106 0 K. EDTA (2 χ 10

dM) 109 0

M o v e m e n t & L o s s of O r g a n i c S u b s t a n c e s from R o o t s a t L o w T e m p e r a t u r e 1 1 9

TABLE II. Induction of Radicle Exudation by Chemical Agents

Treatment Carbohydratee Exudation

\ig/seedlingAiour Function inhibited0

50 Respiration 48 Respiration glycolysis 24 Respiration enolase 20 Krebs cycle 66 Oxidative phosphorylation

5 Respiration 20 Protein synthesis 4.5

Sodium Azide 3^5 χ Iodoacetate 10 Μ _ς

Sodium Fluoride 10 b

M Fluorocitratz 10 Μ 2,4DNP 10 Μ _5

Sodium Arsenate 10

10~5M

~ Λ t Chloroamphenicol 10 Μ Control

Davenports Law: "The specificity of an inhibitor is inversely proportional to how much is known about it" (21).

CO Ο 525 Μ h J Q W W CO CO

ο

CO CO

ο

600 I

400

200

1 2 3 4

DAYS AT 5°

FIGURE 2. Loss of C M Glycine at 30° after chilling at 5°.

1 2 0 Μ. Ν. Christiansen

TABLE III. Carbohydrate Exudation after Cold as Affected by Metabolic Inhibitors, Calcium and ATP.

Treatment

Control H9Q 3 days 5KJ

CaS04 (10~όΜ)

Sodium Azide (3.5 χ 10 Μ) DNP (10

όΜ)

Chloroamphenicol (1Q~ M) Cyclohexamide (10 M) DNP (10

όΜ) + ATP (10~

4M)

Sodium Azide (10~qM) + ATP

Treatment

Hours of treatment 31 ~~T 2 3

μ g/hour/seedling

Control 3Γ HJO CaS04 (io'3m Sodium Azide (3.5 χ DNP (1θ'

όΜ)

10'4M)

- 4 , Chloroamphenicol (10 M) Cyclohexamide (10~ Μ) A

DNP (10~6M) + ATP (10'

qM)

Sodium Azide (10" M) + ATP

73 27 20 14 65 31 22 14 67 77 91 73 53 126 143 110 38 18 10 9 53 37 27 27 60 133 131 117

(10 '4Μ) 38 29 24 37

Hours at 31° after 4 days cold 1 2 3 4

81 37 36 26 71 34 23 16 65 70 59 48

100 124 141 105 63 25 23 20 60 35 32 32

. 4 50 98 138 114 (10 M) 32 19 29 30

20 12 9 6 i 38 49 49 52

Control-No Cold r4> Sodium Azide (10 ^M)-No Cold

INTERNAL SOLUTE MOVEMENT AND R O O T EXUDATION

In an effor t to d e t e r m i n e organic m o v e m e n t within seedl ing t i ssue and the e f fec t of ̂ h i l l i n g on these p rocesses , a m e t h o d involving topica l app l ica t ions of C - l abe led glycine was used. The l abe led s u b s t a n c e was appl ied quan t i t a t i ve ly in 0 .5% agar to cut su r faces of co ty ledons of seedl ings which had been g e r m i n a t e d 30 hours a t 31 (2 cm uniform rad i c l e length) . The seedl ings w e r e p l a c e d upr ight in ho lders wi th 1 cm of t h e rad ic le t ip e m e r s e d in dis t i l led w a t e r . Roo t loss of topica l ly appl ied g lycine i nc reased a t a cons t an t r a t e over 4 days ' t r e a t m e n t a t 5 C (Fig.

M o v e m e n t & L o s s of O r g a n i c S u b s t a n c e s f rom R o o t s a t L o w T e m p e r a t u r e 1 2 1

TABLE IV. Calcium Effect on Loss of Topically Applied C14 Glycine

from Radicles of Chilled and Nonchilled Cotton Seedlings

Glycine lossfiir from 10 seedlings as counts per second

6 hr reading

Temperature Regime

30 Control 5 after 8 hours at 30° 30° after 8 hours at 5° 5 continuous

3 hr reading Ca

10 47

144 102

No Ca Ca No Ca

8 11 10 35 63 50

350 48 108 166 44 102

2). The response is c o m p a r a b l e to previously r e p o r t e d exuda t ion of sugars and amino ac ids from r o o t s (2). The r e s u l t s i nd i ca t e t ha t chil l ing inf luences m e m b r a n e s of t h e e n t i r e seedl ing m o r e or less equal ly with t h a t of the roo t cel l plasmalemma. P r e sumab ly the m o v e m e n t of co ty l edona ry appl ied C glycine would be th rough cel ls of t h e co ty l edona ry mesophyl , t he ph loem cel ls of the axis and r o o t , th rough c o r t e x cel ls of t h e roo t to t h e rh i zosphe re , or in o the r words , t h e p a t h w a y of o rgan ic n u t r i e n t flow from co ty ledons to t h e r o o t .

We also a t t e m p t e d to fu r the r e l u c i d a t e t he ro le of ca lc ium ^ incorpora t ing C a S O . in to t h e top ica l ly appl ied aga r -g lyc ine C m i x t u r e . It had no e f fec t on m o v e m e n t a t 30 C. In seedl ings t r a n s f e r r e d from 30 to 5 C ? l i t t l e e f fec t was n o t e d unt i l a f t e r 6 hours a t 5 C, glycine m o v e m e n t through the seedl ing axis and loss from the roo t was r e d u c e d 1/3 to 1/2 (Table IV). The s i t e of t h e ca lc ium e f fec t in t h e seedl ing has no t been d e t e r m i n e d . Possibly it loca l ized nea r the co ty ledon s i t e of app l ica t ion and e x e r t e d a major l imi t ing inf luence a t t ha t po in t . A l t e rna t i ve ly , it could diffuse throughout the seedl ing . Ca lc ium is normal ly r a t h e r immobi le i n | j s s u e due to i t s r e a c t i v i t y within t h e p l a n t . The answer l ies in use of C a t r ac ing which we have no t y e t done .

The p r e s e n t in fo rma t ion ind ica t e s t h a t ch i l l ing-sens i t ive c o t t o n seedl ings suffer m e m b r a n e a l t e r a t i o n throughout the p lan t which can a f fec t so lu te as well as w a t e r m o v e m e n t . The rap id i ty of induct ion of roo t exuda t ion sugges t s physica l a l t e r a t i o n of m e m b r a n e s ; the reve r s ib i l i ty by ca lc ium and t h e ac t i on of EDTA l ikewise suppor t a l t e r a t i o n of s t r u c t u r e r a t h e r t han m e t a b o l i c e v e n t s .

1 2 2 Μ. Ν. C h r i s t i a n s e n

R E F E R E N C E S

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46, 53-56 (1971). 3 . Chr i s t i ansen , Μ. N., and Foy, C. D. Comm. in Soil Sci. and Pit.

Analysis 10, 427-442 (1979). 4 . Dodd, J . Α. Α., and Ellis, R. J . Biochem. J. 101, 31 (1966). 5. Eps te in , E. Plant Physiol. 36, 437-444 (1961). 6. G a r r a r d , L. Α., and Humphreys , Τ. E. Phytochem. 6, 1085-1095

(1967). 7. Hanson, J . B. Plant Physiol. 35, 372-379 (I960). 8. Guinn, G. Crop Science 11, 101-102 (1971). 9. H u r t t , W., and Foy, C. L. Plant Physiol. (Sup.) 40, 58 (1965). 10. Knudson, L. Am. J. Bot. 7, 371-379 (1920). 11 . L e g g e t t , J . E., and Gi lbe r t , W. A. Plant Physiol. 42, 1658-1664

(1967). 12. L i ebe rman , M., C ra f t , C. C , Audia, W. V., and Wilcox, M. S.

Plant Physiol. 33, 307-311 (1958). 13. Risser , P . G. Bot. Rev. 35, 251-284 (1969). 14. Rover i a , A. D. In "Ecology of Soil-Borne Pa thogens . " K. Baker

and W. Snyder (eds.). pp . 170-186. Univers i ty of Cal i forn ia P res s , Los Angeles (1965).

15. Rover i a , A. D. Bot. Rev. 35, 35-57 (1969). 16. Schroth , Μ. N. , and Hi ldebrand, D. C. Ann. Rev. Plant Phyto-

pathol. 2, 101-132 (1964). 17. Sorokin, H., and Sommer , A. L. Am. J. Bot. 27, 308-318(1940) . 18. True , R. H. Am. J. Bot. 1, 255-273 (1914). 19. Viets , F . G., J r . Plant Physiol. 19, 466 -480(1944) . 20. Wardlaw, C. W. In "Banana Diseases Including P lan ta ins and

Abaca ." Pub . Longmans , London (1961). 2 1 . Webb, J . Leyden . In "Enzyme and Metabo l ic Inhibi tors ," Vol. 1,

A c a d e m i c P re s s , N.Y. and London.


COLD-SHOCK INJURY AND ITS RELATION TO ION T R A N S P O R T BY ROOTS

F. Zsoldos

D e p a r t m e n t of P lan t Physiology A t t i l a Jozsef Univers i ty

Szeged, Hungary

B. Karvaly^

I n s t i t u t e of Biophysics Biological R e s e a r c h C e n t e r

Hungar ian A c a d e m y of Sciences Szeged, Hungary

I. INTRODUCTION

The overwhe lming major i ty of e x p e r i m e n t a l s tud ies on t he e f f ec t s of l o w - t e m p e r a t u r e s t r e s s on var ious life p rocesses of p l an t s have ind ica t ed t h a t m e m b r a n e - l i n k e d e v e n t s , including ion t r a n s p o r t , a r e p r imar i ly d a m a g e d when p l an t s a r e sub jec ted to a sudden fall in t e m p e r a t u r e . Owing to t h e t e m p e r a t u r e - b a l a n c i n g p r o p e r t i e s of soil , r oo t s a r e usually s o m e w h a t less exposed to sudden va r i a t ions in t e m p e r a t u r e . In sp i te of th is , the i r s c a n t y adap t iveness to e x t r e m e t e m p e r a t u r e s and the i r higher sens i t iveness to rapid t e m p e r a t u r e f luc tua t ions m a k e i t unde r s t andab l e why t h e r o o t t e m p e r a t u r e is c r i t i c a l as r ega rds t h e surviving of e i t h e r h igh- or l o w - t e m p e r a t u r e s t r e s s and shock (21).

The key- ro le of po tass ium in ma in ta in ing s t r u c t u r e s and physiological funct ions a t a ce l lu lar level is genera l ly acknowledged (6). This, as well as a l o w - t e m p e r a t u r e anomaly observed solely for Κ (and Rb ), i n i t i a t ed c o m p a r a t i v e inves t iga t ions on t h e l o w - t e m p e r a t u r e Κ -t r anspo r t of r o o t s of co ld - r e s i s t an t and co ld-sens i t ive ( thermophil ic)

Present address: Chemistry Department, University of California, Los Angeles, 405 Hilgard Avenue, Los Angeles, California 90024, U.S.A.

1 2 3 Copyright * 1979 by Academic Press, inc.

All rights of reproduction in any form reserved ISBN 0-12 4 6 0 5 6 0 5

1 2 4 F. Z s o l d o s a n d B. K a r v a l y

p lan t s (30, 3 1 , 32, 34, 35, 37, 38, 39). In t he following an acco u n t will be given for the most i m p o r t a n t r e su l t s ob ta ined wi th win te r whea t (Tri-ticum aestivum L. cv. Mironowskaya 808), as a model spec ies of non-the rmophi l i c c h a r a c t e r , and r i c e (Oryza sativa L. cv . Dunghan Shali), r e p r e s e n t i n g the rmophi l i c spec ies . Most e x p e r i m e n t a l f indings, to be s u m m a r i z e d below will p e r t a i n to t h e s e r e l a t e d spec ies (cereals) ; t hey a r e , however , typ ica l of o t h e r non- the rmoph i l i c (e.g. win te r ba r l ey , e tc . ) and the rmophi l i c (e.g. cucumber , melon, sorghum, e tc . ) seedl ings , r e s p e c t i v e l y . Mostly l o w - t e m p e r a t u r e e f f ec t s will be discussed.

Condi t ions of g rowth and handl ing of seedl ings , t h e e x p e r i m e n t a l p r o c e d u r e used, t h e reproduc ib i l i ty , reliability and accuracy of data have been desc r ibed previously (34, 38). Rb label l ing was employed for Κ , t h e su i tab i l i ty of this be^ng c a r e f ^ l y checked and s u b s t a n t i a t e d in e x p e r i m e n t s wi th double ( Rb and K) label l ing.


Α. Κ Uptake by Excised Roots Following Cold-Shock Treatment

When 6-7 cm long, exc ised roo t s a r e suddenly i m m e r s e d in to a C a ^+-

f ree up t ake solut ion of a given t e m p e r a t u r e , t he in i t ia l (60 min) Κ u p t a k e exhibi ts a very c h a r a c t e r i s t i c t e m p e r a t u r e p a t t e r n , depending upon both va r i e ty and spec ies (Fig. 1). R o o t s of non- the rmoph i l i c p lan t s display a monotonously dec reas ing Κ u p t a k e when t he t e m p e r a t u r e is lowered , as usual ly e x p e c t e d . For the rmophi l i c spec ies an anomalous Κ u p t a k e occurs , wi th n e g a t i v e t e m p e r a t u r e coef f ic ien t , which may exceed t ha t in the physiological ly op t imum t e m p e r a t u r e r a n g e . The t rough- l ike t e m p e r a t u r e p a t t e r n is typ ica l only of seedl ings of the rmophi l i c spec i e s . As r ega rds the posi t ions of t he min ima of t he t rough- l ike curves , they l ie a t higher t e m p e r a t u r e s t he m o r e thermophi l i c t h e spec ies and /o r v a r i e t y (muskmelon > cucumber > r i c e > sorghum).

Anomalous Κ u p t a k e ar i ses solely when r o o t s a r e suddenly exposed to and not gradual ly cooled down to l o w - t e m p e r a t u r e (Fig. 2). Ano the r kind of anomaly occurs in t he in i t ia l Κ uptake when e x p e r i m e n t s a r e p e r f o r m e d on suddenly p recoo led roo t s in Ca - f r e e absorp t ion medium and a t 25°C (Fig. 3). The ini t ia l Κ u p t a k e m e a s u r e d a t 25°C exceeds t he no rma l physiological va lue , " r emember ing" t he precool ing t e m p e r a t u r e much m o r e than recogniz ing t he a c t u a l , more favourable u p t a k e t e m p e r a t u r e . As a funct ion of t h e precool ing t e m p e r a t u r e , t he 25 C Κ -u p t a k e r a t e is very r e m i n i s c e n t of t he curve given in F ig . 1. No such "memory -e f f ec t " can be found, however , if Ca is p r e s e n t . These observa t ions i nd ica t e t ha t anomalous in i t ia l Κ u p t a k e is a dynamic p r o p e r t y with a fairly l a rge i ne r t i a and a p o t e n t i a l i t y for i r r eve rs ib i l i ty . Moreover , t hese f e a t u r e s cannot be asc r ibed to individual molecu la r p rocesses , but r a t h e r to changes in molecu la r o rgan iza t ion of t h e t r anspor t ba r r i e r , p r imar i ly t he p lasma m e m b r a n e . The s t r ik ing d i f fe rences b e t w e e n responses of ini t ia l Κ up takes of non- the rmoph i l i c

C o l d - S h o c k Injury a n d Its Re la t ion to i on T r a n s p o r t b y R o o t s 1 2 5

• musk-melo n ο winte r whea t

n 1 1 • 1 1 ι 0 5 1 0 1 5 2 0 2 5

Uptak e temperatur e (°C )

8G FIGURE 1. Temperature-dependence of initial Κ ( Rb) uptake by

excised roots of various plants, after sudden cooling to the uptake temperature. Absorption solution: 0.5 mM KCl; uptake time: 50 min.

and the rmophi l i c p l an t s to co ld-shock suggest t h a t s ignif icant d i ss imi la r i t i es must exist in the c h e m i c a l compos i t ions and o rgan iza t ion pr inc ip les of t h e p l a sma m e m b r a n e s in t h e r o o t s of the rmophi l i c p l a n t s .

1. Êffect of pH. As shown in F ig . 4, e x t e r n a l pH inf luences t he in i t ia l Κ u p t a k e only a t t e m p e r a t u r e s out of t h e r ange of anomaly . Nea r 0 C profound changes occur , however , when t h e ex t e rna l pH is lowered : a sharp r e d u c t i o n of t h e anomalous Κ u p t a k e a c c o m p a n i e d dec reas ing pH from 6.5 to 5.5, and t h e anomaly c o m p l e t e l y d i sappea red below 5.5 (33). The abrup t change in t h e anomalous Κ u p t a k e canno t be asc r ibed simply e i t he r to a pH induced change in m e m b r a n e s t r u c t u r e or to t he r e d u c t i o n of a s e l e c t i v e Η -Κ exchange diffusion due to a ba lanc ing pH g rad i en t . Consequen t ly , t h e observed t i t r a t ion^ l ike behaviour must be c o n n e c t e d wi th t h e p H - d e p e n d e n t af f in i ty of Κ for t h e roo t m a t e r i a l .

2+ 2 +2. Effect of Ca . I n t h e physiological r a n g e of t e m p e r a t u r e ,

Ca usual ly e x e r t s a s t imu la t ion on Κ absorp t ion (Fig. 5) (1, 4, 25, 36, 38) in bo th groups of p l a n t s . An inhibi t ion of Κ u p t a k e , however , occu r s a f t e r cold-shock t r e a t m e n t . The anomaly (at e i t he r 0 C or 25 C) is

126 F. z s o l d o s a n d B. Ka rva ly

• sudde n coolin g

Ogradua l coolin g (0.1 5 °C|min )

10 1 5 Uptak e temperatur e (°C )

20

~h 86 FIGURE 2. Temperature-dependence of initial Κ ( Rb) uptake by

excised roots of rice, after gradual cooling to the uptake temperature. Absorption solution: 0.5 mM KCl; uptake time: 50 min.

gradual ly r e d u c e d when^_the Ca is i nc reased and is comple t e ly e l imina t ed when the Ca_^ is p r e s e n t well ^ excess of a threshold c o n c e n t r a t i o n of about 10 M. In our case , Ca appa ren t l y c o u n t e r a c t s t h e s p e c t a c u l a r e f f ec t s of cold-shock on Κ u p t a k e , comple t e ly abolishing the anomaly , but it does no t p r e v e n t +t h e g rowth d i s t u r b a n c e s . F igure 6 shows t h a t t h e in i t ia l anomalous^^K u p t a k e can be no t only e l imina ted , but also r e v e r s e d by adding CJa , which is r e m i n i s c e n t of a c a t i o n - e x c h a n g e r as well (4).g^This Ca -p roduced r e v e r s a l could be a t t r i b u t e d to t h e loss of Κ ( Rb) absorbed, due to pass ive exchange gaining ascendancy over u p t a k e . Such a mechanism a lone , however , canno t accoun t q u a n t i t a t i v e l y for t he obse rva t ions . The re fo re , it is compulsory to r eason t h a t the anomalous Κ u p t a k e may be closely r e l a t e d to t he pecu l ia r c a t i o n - e x c h a n g e p r o p e r t i e s of the roo t m a t e r i a l p roduced by cold-shock.

3. Interactions of Κ with Other Electrolytes. Ca t i ons , bo th m o n o - and d iva len t , g r ea t l y a f fec t the anomaly , de fend ing upon va lency and c o n c e n t r a t i o n . Monoposi t ive ions (Li , Na , Cs , Rb , NH J only r e d u c e ,

C o l d - S h o c k injury a n d Its Re la t ion to Ion T r a n s p o r t b y R o o t s 127

rice winte r whea t

· - 0°C V / F Δ - 1 0

Β0 λ / /

- a - 2 0 ° C / f i t

· - o ° c o - 6°C

_ a - 1 0 ° C *

1 1 1

• - 2 5 ° C

1 1 1 1 1 1 1 1 1 2 0 4 0 6 0 2 0

Uptak e t ime(min ) AO 6 0

FIGURE 3. Time-dependence of 25 C Κ ( Rb) uptake by excised rice and winter wheat roots after precooling. Absorption solution: 0.5 mM KCl; precooling time: 50 min.

but do no t e l iminate t h e anomaly , wi th varying e f f ec t ivenes s . As a t e n t a t i v e o rder , Li < Na < Cs < Rb < NH^) can be given, which coincides qu i t e well wi th t he s equence of t h e su r f ace cha rge dens i ty on t h e r e s p e c t i v e ca t ion in hydra t ion fo rm. This finding is also in l ine wi th t h e assumpt ion of a c o m p e t i t i v e t r ans loca t i on of monopos i t ive ions th rough n e g a t i v e l y - c h a r g e d s i t e s , e ^ . in caption-exchange r e g i m e s .

Diva len t ca t ions such as Ca , Mg , Sr and Mn wi thou t excep t ion abolish t h e anomaly , wi th practical ly t h e s a m e ef f ic iency . The p r e s e n c e of d i f fe ren t anions (NO^, CI , SO , H^PO^) does no t e x e r t any s ignif icant inf luence on the anomalous u j âke p roces s . The observed i m p a c t s of d i f f e ren t ions on the Κ u p t a k e anomaly do no t pa ra l l e l the i r o rde r ing /d i so rde r ing e f f ec t s on lipids (8, 13). H e n c e , the i r i n t e r a c t i o n s with Κ a r e not d i r ec t ly r e l a t e d to m e m b r a n e s .

4. Efflux and Exchange. The efflux of ions from p lan t r o o t s is. usually r e g a r d e d as a pass ive p rocess wi th very m o d e r a t e t e m p e r a t u r e -dependence , occur r ing via e i the r s imple or exchange diffusion along the e l e c t r o c h e m i c a l g rad ien t (17, 18, 19). F igure 7 shows t h a t t he non-the rmophi l i c whea t r o o t s obey this qu i t e genera l r u l e , even when they

128 F. Z s o l d o s a n d B. Ka rva ly

~h 86 FIGURE 4. pH-dependence of initial Κ ( Rb) uptake by excised

rice roots at different temperatures. Absorption solution: 0.5 mM KCl: uptake time: 60 min (33).

w e r e exposed suddenly | o cold, r ega rd l e s s of w h e t h e r Ca^ is p r e sen t or n o t ^ I n c o n t r a s t , t h e Κ efflux r a t e for r i c e , which exhibi t s a m o d e r a t e Ca - d e p e n d e n c e a t ZO C, responds d r a m a t i c a l l y to cold shogk, increas ing marked ly to an e x t e n t independent of t he p r e s e n c e of C a No s ignif icant change was found in t he t i m e - c o u r s e of Κ efflux when the pH was lowered from 6.5 to 4.5 where the u p t a k e anomaly is abol ished (Fig. 4). Under^ exchange condi t ions t he cor responding r a t e s a r e inf luenced by C a s imi lar ly as for t h e efflux in bo th e x p e r i m e n t a l s y s t e m s .

A compar i son of t he c h a r a c t e r s of t h e Κ - u p t a k e , -eff lux and -exchange p rocesses sugges ts t ha t c o l d - t r e a t m e n t of the rmophi l i c p lan t r o o t s opens new, s e p a r a t e p a t h w a y s for t he pass ive inward and ou tward m o v e m e n t s , r e spec t i ve ly , of Κ , whichâre very d i f fe ren t in n a t u r e , and tha t it t r ans fo rms the overa l l pass ive Κ t r anspo r t of the rmophi l i c r o o t s in an i r r evers ib le way (see also t h e "memory e f fec t " in F ig . 3).

C o l d - S h o c k Injury a n d Its Re la t ion to Ion T r a n s p o r t b y R o o t s 129

0 * Ϊ 0 ' 20 1 3 0 ' UO 1 50 Uptak e temperature(°C )

FIGyi^g 5. Effect of Ca upon the temperature-dependence of initial Κ ( Rb) uptake by excised roots of rice and wheat. Absorption solution: 1 mM KCl with or without 1 mM CaCl9; uptake time: 60 min.

B. Relations of Cold-Shock Effect to Root Zones

1. K+ uptake distribution along roots. F igure 8 p r e s e n t s t h e

zonal d i s t r ibu t ions of the^^K u p t a k e in r i c e and win te r whea t r o o t s and the i r r e sponses to Ca , a t 0 C and Ζ 5 C. Indisputably , t h e ex t r ao rd ina r i ly l a rge Κ influx does no t ex t end over t he whole roo t ; i t is confirmed to t h e ap ica l r oo t po r t ions , which a r e also t h e most respons ive to Ca . As r e g a r d s t he non - the rmoph i l i c win te r w h e a t , t he r e s p e c t i v e u p t a k e p a t t e r n s a r e s imi lar to those publ ished by o t h e r s (Z, 5, 16) and bea r wi tness to c o m p l e t e l y d i f fe ren t behaviour , as d iscussed in de t a i l r e c e n t l y (38).

1 3 0 F. Z s o l d o s a n d B. K a r v a l y

TO 2 0 3 0 4 0 5 0 6 0 Uptak e time(min )

+ gÎGURE 6. Reversal of low-temperature agpmalous initial Κ ( Rb) uptake of rice roots by addition of 1 mM Ca during uptake. Absorption solution: 1 mM KCl.

2. Potassium content distribution along roots. The overa l l p o t a s sium dis t r ibut ion p a t t e r n s (Fig. 9) for u n t r e a t e d r o o t s exhibi t qua l i t a t ive ly r a t h e r s imi lar a p p e a r a n c e s ; a high po tass ium c o n t e n t was found in the f irst 1 cm sec t ion , gradual ly dec reas ing (39). The absence of Ca from the u p t a k e solut ion exe rc i s e s , in gene ra l , only a m o d e r a t e inf luence on t h e u p t a k e p a t t e r n s , wi th t he excep t ion of r i c e a t 0 C. In this l a t t e r case a marked loss of Κ occur s especia l ly in t he first two roo t zones , which is i r r e v e r s i b l y and canno t be p r e v e n t e d by Ca . In addi t ion, the p r e s e n c e of Ca fur ther i nc r ea se s (and does no t a r res t ) t h e loss of Κ . Similar obse rva t ions on o the r the rmophi l i c p lan t r oo t s (39) enable us to conc lude t ha t such a behaviour of t he ap ica l m e r i s t e m a t i c reg ion of r o o t s is pecu l i a r to the rmophi l i c p l a n t s . It is i m p o r t a n t t o emphas i ze t h a t t he h ighes t p o t a s s i u m c o n t e n t ex is t s in t he ap ica l m e r i s t e m a t i c region, whe re Κ is t aken up anomalous ly .

3. Calcium content d i s t r i b u t i o n along roots. F igu re 10 dep ic t s t h e r e su l t s ob ta ined for Ca d i s t r ibu t ion . A compar i son of d a t a £or con t ro l , 25 C and 0 C e x p e r i m e n t s , r e spec t i ve ly , in t he absence of C a ,


-J I I 1 1 L. 10 2 0 3 0 4 0 5 0 6 0

E f f lux tim e (min )

FIGURE 7. Effects of temperature and Ca upon the time-course of efflux from excised roots of rice and wheat. Corresponding data for wheat fell within the ruled area, i.e. they coiricgged within experimental uncertainties. Labelling solution: 1 mM Κ ( Rb)Cl; efflux medium: distilled water with or without 1 mM CaCl9; pre-incubation time: 50 min.

sugges t s th^J t C aL T

ex is t s in roo^s in a t l ea s t t h r e e d i f fe ren t pools : (1) soluble C a r e m o v a b l e in a C a f ree absorp t ion solut ion a t 25 C; (2) s t rongly-bound £_Ca r ema in ing inQ t h e roo t tis^sûe even a f t e r an incuba t ion in Ca f ree solut ion a t 25 C; and (3) Ca r e t a i n e d in excess t o t he s t rpngly-bound C a when cold-shock t r e a t m e n t is appl ied. Mobile Ca , + is c e r t a in ly s i t u a t e d in t h e w a t e r - f r e e space (4); s t rong ly -bound Ca is thought to be adsorbed on t h e cell wall and t h e ex t e rna l su r face of t hg jp l a smolemma, and l o c a t e d in var ious cel l c o m p a r t m e n t s (4), while <£a r e t a i n e d a f t e r cold-shock is a p ropor t ion of t h e soluble mobi le Ca access ib le for and adsorbed on co ld-shock-produced , new

132 F. Z s o l d o s a n d B. K a r v a l y

Distanc e fro m roo t apex(cm )

FIGURE 8. Effects of Ca and temperature upon Κ ( Rb) uptake distribution patterns along primary roots of rice and winter wheat. Absorption solution: 1 mM KCl with or without 1 mM CaCl9; uptake time: 60 min (38).

binding s i t e s . If t h e ex t e rna l solut ion con ta ins Ca , t hen this ion is a c c u m u l a t e d in a cons iderab le excess , in the apical region for r i c e and in t h e m o r e m a t u r e d s e g m e n t s for win te r whea t following cold-shock t r e a t m e n t . 2 +

It should be s t r e s sed t h a t a t 0 C the en t ry of Ca in to t he r e s p e c t i v e r i c e roo t segments t akes p l a c e a t t he expense of Κ (Figs. 8, 9 and 10). In o the r words , Ca is p r e f e r en t i a l l y bound by roo t zones where t he Κ u p t a k e o p e r a t e s anomalous ly . This poin ts again to t he fac t t h a t , in the case of the rmophi l i c p l an t s , co ld-shock t r e a t m e n t creates new c a t i o n - b i n d i n g s i t e s within apical roo t t i ssue , for which Ca and Κ c o m p e t e , Ca having t h e g r e a t e r af f in i ty .

It has been d e m o n s t r a t e d in t he foregoing t h a t the anomalous Κ up t ake by the rmophi l i c p lan t roo t s is r e s t r i c t e d to t he apical m e r i s t e m a t i c region, where in t ense loss of Κ and p r e f e r e n t i a l Ca


FIGURE 9. Effects of Ca and temperature upon the distribution of potassium content along primary roots of rice and winter wheat. Otherwise as Figure 9 (39).

a c c u m u l a t i o n also occur following cold-shock. All t h e s e d a t a under l ine t h a t t h e observed anomaly in t h e Κ u p t a k e is r e l a t e d to , and may t h e r e f o r e be an ind ica to r for co ld - s t r e s s sens i t iv i ty ( thermophi ly) . This finding r e c e i v e s s t rong suppor t from t h e wel l -vis ible chronic growth d i s tu rbances of r o o t s : t he a r r e s t a t i o n of p r i m a r y - r o o t e longat ion and the in t ense s ide - roo t fo rma t ion behind t h e roo t t ip a f t e r chil l ing t r e a t m e n t (39). P re l imina ry morphologica l s tud ies show tha t a cold-shock injury p roceeds towards c o m p l e t e d i sorgan iza t ion in t h e ap ica l roo t sec t ion (40).

C. Uptake of Other Ions by Excised Roots Following Cold-Shock Treatment

As men t ioned be fo re , only po tass ium and i t s r ep lac ing ion, rubid ium, exhib i ted t h e anomalous inward m o v e m e n t a t 0 C and in t h e absence of Ca . This is now d e m o n s t r a t e d in F ig . 11 for t h e absorp t ions of N H . ,


- 7 5 σι

50 en

c 2 5

ο

0°C

rice

25 eC Contro l

)-C a winte r whea t i*C a

O'C • 25° C Contro l

1 ' > ι 1 1—— 1 1 1—*-H 1 Γ

0 4 0 4 0 4 0 4 0 4 0 4 Distanc e fro m roo t apex(cm )

FIGURE 10. Distribution of calcium content along primary roots of rice and winter wheat. Otherwise as Figure 8.

NO^, H ^ P O ^ a n d l , r e spec t i ve ly , by r i c e r o o t s . The NH^ and NO^ up t akes inc rease monotonously wi th r is ing t e m p e r a t u r e , e i the r in the p r e s e n c e or in t he absence of Ca . A higher absorp t ion can be expe r i enced for NH^, however , t h e most e f f ec t ive c o m p e t i t o r of Κ a n c ^ R b a t low t e m p e r a t u r e s and in t he absence of Ca_ . In c o n t r a s t , Ca cons iderably p r o m o t e s t he accumula t i ons of H ^ P O ^ and I ions a t e ach t e m p e r a t u r e . In gene ra l , s imi lar u p t a k e p a t t e r n s w e r e found for the non- the rmoph i l i c p l an t s (e.g. w in te r whea t ) . These r e su l t s c lea r ly d o c u m e n t t h a t t h e observed anomaly p roduced by cold-shock is a p r o p e r t y of the rmophi l i c spec ies and pecu l ia r to monova len t ca t ions such as Κ and Rb . Thus, of all t h e above e f f ec t s , t he Κ u p t a k e anomaly can provide t he key to a b e t t e r unders tand ing of the mechan i sm of cold-shock injury.

ΠΙ. PROPOSED MECHANISM FOR CHILLING-INJURY OF THERMOPHILIC ROOTS

The indispensabi l i ty of po tass ium in main ta in ing s t r u c t u r a l and funct ional o rgan iza t ion in p l an t s , in addi t ion to very p r o m p t r e a c t i o n of

C o l d - S h o c k Injury a n d Its Re la t ion to Ion T r a n s p o r t b y R o o t s 1 3 5

NH£ NO J H 2PO^ I "

ο

10 0

50 h

0 1 0 2 5 0 1 0 2 5 0 1 0 2 5 0 1 0 2 5 Uptak e temperature(°C )

FIGURE 11. Temperature-dependence of initial uptake of different ions by excised rice roots after sudden cooling to the uptake temperature. The labelled absorption solution contained 0.5 mM of NH.Cl, NaNOr NaJ each and 0.1 mM KH9P04, respectively. Uptake time: 60 minutes ( or -Ca, m+Ca).

potass ium m o v e m e n t and r e t e n t i o n to , and the pro longed e f f ec t s of cold-shock, suggest t ha t the potass ium economy and r e l a t e d even t s form key-e l e m e n t s of t he cold-injury of the rmophi l i c p lan t r o o t s .

The above obse rva t ions enable us to out l ine a t e n t a t i v e proposal for t he possible mechan i sm of some p r i m a r y p rocesses involved in the cold-shock injury of r o o t s of the rmophi l i c p l a n t s . The d i s t inc t ive f e a t u r e s of the rmophi l i c p l an t s can be br ief ly s u m m a r i z e d as follows:

1) only a sudden +drop in t e m p e r a t u r e br ings about t he anomalous inward m o v e m e n t of Κ wi th n e g a t i v e t e m p e r a t u r e coef f ic ien t (Figs. 1 and Z);

Z) p recoo l ing r e su l t s in an anomaly of s imi la r c h a r a c t e r in t h e Z5 C u p t a k e of Κ (a f te r - or " m e m o r y - e f f e c t " , F ig . 3);

3) t h e p H - d e p e n d e n c e of anomalous Κ u p t a k e is ve ry r e m i n i s c e n t of a t i t r a t ion^curve (Fig. 4); +

4) C a no t only e l im ina t e s t h e Κ u p t a k e anomaly (Fig. 5) but even r e v e r s e s t he anomalous Κ u p t a k e (Fig. 6), which is typ ica l of a c a t i o n - e x c h a n g e r e g i m e ;

5) e l e v a t e d l o w - t e m p e r a t u r e efflux e l i c i t ed by cold-shock is p r a c t i c a l l y insens i t ive to C a + (Fig. 7);

6) t h e anomalous Κ u p t a k e is r e s t r i c t e d to t he apica l


m e r i s t e m a t i c zone (Fig. 8) whe re ^ m a r k e d C a ^ - d e p e n d e n t po tass ium loss occurs (Fig. 9), and w h e r e Ca a c c u m u l a t e s p r e f e r e n t i a l l y as well (Fig. 10).

Bear ing all th is in mind, one has to a s sume t h a t , as a f irst s t e p , rap id co ld-exposure very probably leads to the fo rmat ion of r andomly d i s t r i bu ted pores and a p r o m p t ex t rus ion of w a t e r and so lu tes (preferen t ia l ly Κ ), espec ia l ly from t h e cel ls in t h e apica l reg ion (3, 10, 11,12,20,39). Then, the cy top la sm, because of i t s abrupt ly d ec r ea s in g vo lume, tends to ca r ry and thus , to t e a r away t h e ou t e r p l a sma m e m b r a n e adher ing to the suppor t ing cel l wal l , the r e s t r i c t e d p l a s t i c i t y of m e m b r a n e in t he m o r e o rde red s t a t e not al lowing it to follow the volume shr inkage smoo th ly . The d r a s t i c r e m o v a l of the p l a s m a l e m m a from the p r i m a r y cel l wall (which p redominan t ly ex i s t s in t h e ap ica l zone) (22), ce r t a in ly a c c o m p a n i e s the b reak ing-up of ionic , p ro t e in - ce l l wall and l ipid-cel l wall l inkages and t h e f ree ing of d issociable groups shielded original ly from w a t e r by m e m b r a n e c o n s t i t u e n t s . Consequen t ly , new, p o t e n t ca t ion-b inding s i t e s a r e c r e a t e d on and in t h e cel l wal l , which s e rve as c a t i o n - e x c h a n g e p a t h w a y s for Κ . Such an i n t e r p r e t a t i o n is in l ine wi th the e x p e r i m e n t a l d a t a p r e s e n t e d in F igs . 1-5. Since t h e ^ o w -t e m p e r a t u r e efflux of Κ proved to be p r a c t i c a l l y ind i f fe ren t to Ca , it is compel l ing to deduce t h a t t h e r e evolve s e p a r a t e p a t h w a y s for Κ u p t a k e and r e l e a s e in the (apical reg ion of) r o o t s of the rmophi l i c p l an t s during cold-shock t r e a t m e n t . It is easy to see t h a t t he n e w l y - c r e a t e d ca t ion-b inding s i t e s , which a r e p r e r equ i s i t e s of anomalous Κ u p t a k e , o p e r a t e as pass ive p a t h w a y s pecu l ia r to a Κ (monovalent cat ion) p ro ton an t ipo r t sy s t em, t h e Κ m o v e m e n t being d i r e c t e d towards and p ro tons leaving t h e roo t t i s sue .

As r ega rds t h e origins of excess p ro tons within t h e cel l , they a r e ce r t a in ly p roduced by the e l e v a t e d r e sp i r a t i on and the a r r e s t a t i o n of the "pro ton-consuming" p rocesses , including ATP syn thes i s . In t h e proposed model , t he highly mobi le K-ions accompl i sh the i r uphill m o v e m e n t a t t he expense of t he e l e c t r o c h e m i c a l p o t e n t i a l of p ro tons p roduced inside t he roo t ce l l s , and Κ and Η a r e se l ec t ive ly exchanged due to t he p r e f e r e n c e of t he c a t i o n - e x c h a n g e r e g i m e for Κ . This l a t t e r mus t be c o n n e c t e d wi th the pecul ia r ca t ion-b inding p r o p e r t i e s of the c a r b o x y l a t e ch romophores of p e c t i c acid, cons idered as pr inc ipa l c o n s t i t u e n t of p r i m a r y cell wall (9). It is t h e r e f o r e sugges ted t h a t this pass ive Κ / Η an t ipo r t man i fe s t s i tself in t he un id i rec t iona l , anomalous Κ m o v e m e n t . Moreover , t h e pa ra l l e l working of th | s pass ive Κ / Η an t ipo r t sys tem with probably pa r t i a l l y impa i red Κ u p t a k e leads to t he a f t e r - or " m e m o r y - e f f e c t " observed a t 25 C (Fig. 3). Accordingly , cold-shock on r o o t s of the rmophi l i c p l an t s br ings about in i t ia l e v e n t s leading to s t r u c t u r a l and funct ional injury a t t he levels of bo th p l a sma m e m b r a n e and in t r ace l lu l a r (e.g. mi tochondr ia l ) m e m b r a n e s . This is v isual ized in a t e n t a t i v e n e t w o r k d iagram in Fig . 12, which is an ex tens ion of Lyons' proposal (15), and appl ies to the ap ica l reg ion of r o o t s of the rmophi l i c p l an t s .

MICROFIBRILLAR CELL-WALL

PLASMALEMMA (LIQUID-

'CRYSTALLINE)

? MICROFIBRILLAR " CELL-WALL

WATER GAP

PLASMALEMMA (SOLID GEL)

INJURY ΔΝ Ο DEATH OF APICAL MERISTEMATIC ZONES/GROWTH DISTURBANCES

INCREASED ACTIVATION ENERGY OF ENZYMES

COLD-SHOC K

CESSATION OF PROTOPLASMIC

I STREAMING

REDUCED ATP

SUPPLY

FIGURE 12. Suggested schematic pathway of membrane-linked events involved in cold-shock and chilling injuries of the apical meristematic zones os thermophilic plant roots. The model is an extension of that proposed originally by Lyons (15).


Final ly , men t ion should be m a d e about the possible r e l a t ion b e t w e e n lipid and f a t t y ac id composi t ions of m e m b r a n e s and cold-shock t o l e r a n c e . This is espec ia l ly i m p o r t a n t , because the t h e r m o t r o p i c p rope r t i e s of m e m b r a n e lipids and l o w - t e m p e r a t u r e responses of p l an t s have appea red to c o r r e l a t e in a l a rge number of s tud ies (14, 26, 28, 29), a l though the lack of any co r r e l a t i on has also been r e p o r t e d (27). Very r e c e n t r e su l t s ob ta ined with shoots of a co ld-sens i t ive whea t (Penjamo 62), when i ts f r o s t - r e s i s t a n c e was gradual ly improved by r ad i a t i on t r e a t m e n t , fail t o suppor t a def in i te para l le l i sm b e t w e e n unsa tu r a t i on and f r o s t - r e s i s t a n c e (24). It s e e m s very l ikely, however , t ha t the " s to ich iomet ry" of t he C^g family and p ro top la smic p ro te ins jo in t ly d e t e r m i n e the c o l d - t o l e r a n c e , bo th being dec i s ive as r e g a r d s w a t e r economy (permeabi l i ty and r e t en t ion ) (7). Similar conclusions can be drawn from pre l imina ry f a t t y acid ana lyses of roo t s e g m e n t s as well (Toth and Karva ly , unpublished) . For these r easons , we v e n t u r e to a s sume tha t the rap id , c rys ta l l ine - to - so l id gel t r ans i t ion of m e m b r a n e lipids, caused by cold-shock, may resu l t in non-equi l ibr ium m e m b r a n e s t r u c t u r e s : pores differ ing in e i the r popula t ion dens i ty or s ize d is t r ibu t ion , or bo th . (Beyond doubt , this is t igh t ly and a lmos t exclusively bound up wi th the f a t t y ac id composi t ion.) This can explain conclusively not only t he r e su l t s p r e s e n t e d in this pape r , but even the bases of d i ss imi la r i t i es in the behaviours of co ld - r e s i s t an t and cold-t o l e r an t p l an t s upon cold-shock.


1. Bowling, D. J . F . "Uptake of Ions by P l an t Roo t s . " C h a p m a n and Hall , London (1976).

2. Canning , R. E. and K r a m e r , P . J . Amer. J. Bot. 45, 378-382. (1958).

3 . Chr i s t i ansen , Μ. N. , C a m s , H. R. and Slyter , D. J . Plant Physiol. 46, 53-56. (1970).

4 . Eps te in , E. "Mineral Nu t r i t i on of P l an t s : Pr inc ip les and P e r s p e c t i v e s . " Wiley and Sons, New York (1972).

5. Eshel , A. and Waisel , Y. Plant Physiol. 49, 585-589 (1972). 6. In t e rna t iona l Po tash I n s t i t u t e . "Potass ium in Biochemis t ry and

Physiology." Proc. 8th Coll. Intern. Potash Inst., Uppsala 1971. Publ . IPI Berne , Swi tzer land (1971).

7. K a c p e r s k a - P a l a c z , Α., Dlugokecka , E., Bre i t enwald , J . and Wcislinska, B. Biol. Plant. (Praha) 19, 10-17 (1977).

8. Karva ly , B. and Loshchi lova, E. Biochim. Biophys. Acta 514, 274-285 (1977).

9. Kohn, R. and S t iczay , T. Collection Czechoslov. Chem. Commun. 42, 2372-2378 (1977).

10. L e v i t t , J . "Responses of P l an t s to Env i ronmenta l S t resses . " Acad . P res s , New York (1972).


11 . L e v i t t , J . In "Regula t ion of Cel l M e m b r a n e Ac t iv i t i e s in P lan t s" (E. M a r r e and D. Ci f fer i , eds), pp . 103-119. A m s t e r d a m : N o r t h -Holland Publishing C o m p a n y (1977).

12. L i ebe rmann , M., C r a f t , C . C , Audia, W. V. and Wilcox, M. S. Plant Physiol. 23, 307-311 (1958).

13. Loshchi lova, E. and Karva ly , B. Biochim. Biophys. Acta 470, 492-493 (1978).

14. Lyons, J . M., Wheaton , T. A. and P r a t t , Η. K. Plant Physiol. 39, 262-268 (1964).

15. Lyons, J . M. Ann. Rev. Plant Physiol. 24, 445 -466 (1973) . 16. Marschner , H. and R i c h t e r , Ch. Z. Pflanzenernahr. Bodenkd. 135,

1-5 (1973). 17. Mengel , Κ. Z. Pflanzenernahr. Dung. Bodenkd. 103, 193-206

(1964). 18. Mengel , K. and Herwig , Κ. Z. Pflanzenphysiol. 60, 147-155

(1969). 19. Mengel , K. and Pf luger , R. Plant Physiol. 49, 16-19 (1972). 20. Minchin, A. and Simon, E. W. J. Exp. Bot. 24, 1231-1235(1973) . 2 1 . Nielsen, K. F . In "The P lan t Roo t and I ts Envi ronment" (Ε. V.

Carson , ed.) , p p . 293-333 . Univ. P re s s of Virginia (1974). 22. Nobel , P . S. " In t roduct ion to Biophysical P lan t Physiology."

F r e e m a n and Company , San F ranc i s co (1974). 23 . Smolenska, G. and Kuiper , P . J . Physiol. Plant. 41, 29-35 (1977). 24. To th , Ε. T., Vigh, L. , Karva ly , B. and F a r k a s , T. Physiol. Plant.

(In press) (1979). 25. Viets , F . G. Plant Physiol. 19, 446-486 (1944). 26. Wil lemot , C . Plant Physiol. 60, 1-4 (1977). 27. Wilson, J . M. and Crawford , R. Μ. M. New Phytol. 73, 805-820

(1974). 28. Wilson, J . M. and Rinne , R. W. Plant Physiol. 57, 270-273 (1976). 29. Yoshida, S. and Sakai , A. Plant Cell Physiol. 14, 353-359 (1973). 30. Zsoldos, F . Z. Pflanzenernahr. Bodenkd. 119, 169-173 (1968a). 3 1 . Zsoldos, F . Z. Pflanzenphysiol. 60, 1-4 (1968b). 32 . Zsoldos, F . Acta Agr. Acad. Sci. Hung. 18, 121-126 (1969). 3 3 . Zsoldos, F . Z. Pflanzenernahr. Bodenkd. 126, 210-217 (1970). 34. Zsoldos, F . Plant and Soil 37, 469-478 (1972a). 35 . Zsoldos, F . Acta Biol. Szeged. 18, 121-129 (1972b). 36. Zsoldos, F . Proc. 10th Congr. Intern. Potash Inst., Budapest 1974,

pp . 197-204. Publ . IPI Berne , Swi tze r l and (1975). 37. Zsoldos, F . and Karva ly , B. Experientia 31, 75-76 (1975). 38 . Zsoldos, F . and Karva ly , B. Physiol. Plant. 43, 326-330 (1978a). 39. Zsoldos, F . and Karva ly , B. Physiol. Plant. 43, 331-336 (1978b). 40 . Zsoldos, F . and Gulyas , S. Acta Biol. Szeged. (In press) (1979).



ION LEAKAGE IN CHILLED PLANT TISSUES

Takao Murata and Yasuo Tatsumi

F a c u l t y of Agr i cu l tu r e Shizuoka Univers i ty

Oya, Shizuoka 4ZZ, J a p a n

The re a r e a number of s y m p t o m s of chil l ing injury of fruit and v e g e t a b l e s which may be a t t r i b u t e d to the d e n a t u r a t i o n of b io -m e m b r a n e s . Browning, p i t t i ng , scald , w a t e r y b reakdown, shr ivel ing may be class i f ied in this c a t e g o r y of s y m p t o m s . There may be changes in m e m b r a n e p e r m e a b i l i t y of phenol subs t ances be fo re or during the o c c u r r e n c e of browning. Changes in p e r m e a b i l i t y of w a t e r must t a k e p l a c e in t h e course of shr ivel ing of chil l ing injured t i s sues . Since L i e b e r m a n et al (3) i nd i ca t ed a cont inuous i nc r ea se in m e m b r a n e p e r m e a b i l i t y of po tass ium ion of t h e t i ssues of s w e e t p o t a t o roo t s t o r e d a t chil l ing t e m p e r a t u r e severa l inves t iga t ions concern ing m e m b r a n e p e r m e a b i l i t y and chil l ing injury have been publ ished (1, Z, 4, 6, 7, 9, 10. 11, 1Z). However , t h e re la t ionsh ip b e t w e e n t h e changes in m e m b r a n e p e r m e a b i l i t y and the o c c u r r e n c e of chil l ing injury of fruit and v e g e t a b l e s sti l l r e m a i n s to be c lar i f ied .

In this sec t ion , t h e e f fec t of t e m p e r a t u r e s on ion l eakage from t i ssue s l ices of chil l ing sens i t ive and insens i t ive p l an t s will be r e p o r t e d in t h e form of Arrhen ius p lo t s of t he r a t e of po tass ium ion l eakage and changes in ion l eakage during s t o r a g e a t chil l ing t e m p e r a t u r e s .

I. MATERIALS AND METHODS

Most p lan t m a t e r i a l s used in th is s tudy we re pu rchased in local m a r k e t s . Cucu rb i t s , bell pepper and egg-p lan t f rui ts w e r e freshly p icked a t t h e t ab l e r ipe s t a g e of m a t u r i t y from p l an t s grown in soil in a g reenhouse or on a farm of t h e F a c u l t y of Agr i cu l tu re , Shizuoka Univers i ty .

To d e t e r m i n e t h e e f fec t of d e t e r g e n t s on t he o c c u r r e n c e of chill ing injury, cucumber fruit was s t o r ed a t 1 C a f t e r dipping in to aqueous solut ion of 0 . 1 % Tr i ton X-100 (HLB 13.5) for 3 hours .

Copyright · 1979 by Academic Press. Inc. 141 AH rights of reproduction in any form reserved

ISBNOI2 4€056O5

142 Τ. M u r a t a a n d Y. T a t s u m i

Discs of 4mm d i a m e t e r punched from t h e t i ssues of fruit and v e g e t a b l e s we re cut in to s l ices of 5 mm th ickness unless o the rwi se men t ioned . In t he cases of bell pepper and t o m a t o f ru i t s , th ickness of the discs was 2-3mm and 3-5mm, depending on the th ickness of the o u t e r wall of the f ru i t s , r e s p e c t i v e l y . Samples of discs w e r e i m m e r s e d in de ionized w a t e r or in 0.4 Μ manni to l solut ion a t d i f fe ren t t e m p e r a t u r e s in t h e r anges of 0-30 C. Af te r incuba t ion for 2 hours , the c o n t e n t of l eaked ions of po tas s ium, sodium and magnes ium in the incuba t ion medium was m e a s u r e d using a Hi tach i 207 type a t o m i c absorp t ion s p e c t r o p h o t o m e t e r .

E l e c t r o l y t e c o n t e n t of t h e incuba t ion medium was m e a s u r e d wi th a TOA conduc t iv i ty m e t e r a t a cons t an t t e m p e r a t u r e of 20 C during the course of incuba t ion . Af te r incubat ion for 4 hours , the medium conta in ing t h e discs was boi led for 15 minu te s to l each all of t he ions from the discs of t i ssues in to the med ium. The r a t e of ion l eakage was t h e amoun t of l eaked ions expressed as a p e r c e n t a g e of t he t o t a l amoun t of ions of t h e t i s sues .


A. Arrhenius Plots of Rate of Potassium Ion Leakage

L Arrhenius Plots of Ion Leakage from Cucurbitaceae Fruit Tissues. F ru i t of cucurb i t s a r e usually chil l ing sens i t ive and they have to be s t o r e d a t 7-10 C. F igure 1 shows the Arrhenius p lo t s of po tass ium ion l eakage from the discs of t i ssues of cucumber (Cucumis sativus L.), o r i en t a l pickl ing melon (Cucumis melo L. common MAKI -NO), pumpkin (Cucurbita moschata DUCH) , s u m m e r squash (Cucurbita pepo L.) and c h a v o t e (choko; Sechium edule SWARTZ) for t h e t e m p e r a t u r e r ange of 0 to 30 C. In all ca ses , t h e r e w e r e b reak po in t s a t 5-10 C t h a t cor responded closely wi th t h e c r i t i c a l t e m p e r a t u r e s for chil l ing injury of t he se frui ts during s t o r a g e . Higher r a t e s of po tass ium ion l eakage w e r e observed a t t he lower t e m p e r a t u r e s below t h e c r i t i ca l po in t s .

F igure 2 shows the Arrhenius p lo t s of r a t e of po tass ium ion l eakage from t h e discs of cucumber fruit s t o red a t 5 C and 1 0 C for 1 to 9 days . Break poin ts w e r e observed in Arrhenius p lo t s from t h e t issues of fruit s t o r ed a t 10 C t h a t was safe from chill ing injury. Break po in ts b e c a m e obscure during s t o r a g e and the poin ts b e c a m e ha rd to dist inguish a f t e r s t o r a g e for 9 days a t 5 C . However , c l ea r b reak po in ts in t h e Arrhenius p lo t s w e r e ob ta ined from t h e fruit which had been s to r ed a t t e m p e r a t u r e s above t h e c r i t i ca l point (10 C) .

Arrhenius p lo t s of r a t e of po tass ium ion l eakage from t h e t i ssues of bell pepper fruit and s w e e t p o t a t o roo t (chilling sensi t ive) also exhib i ted a s imilar t endency which involved typica l b reak po in t s .

i on L e a k a g e in Chi l led P l a n t T i s s u e s 143

2.5

1.2.0

3 Φ

cn

id - 1.5 cn ο

1.0 30 33

• cucumbe r

Ο orienta l picklin g melo n

A pumpki n

Δ pep o

• chayot e

20

34 1 / Τ x 10

10 τ—' 1 35 36

u

o c

37

FIGURE 1. Arrhenius plots of rate of potassium ion leakage from the discs of Cucurbitaceae fruits ( · · ; cucumber, Ο Ο ; oriental pickling melon, A 4 ; pumpkin,Δ Δ; summer squash (pepo), 1 Β ; chayotej .

2. Arrhenius Pio is of /on Leakage from Chilling Insensitive Plant Tissues. F igure 3 shows t h e Arrhenius p lo t s of r a t e of po tass ium ion l eakage from t h e discs of p o t a t o tuber t i ssues (Solanum tubersum L.) which is chil l ing insens i t ive . A b reak point was not observed in t h e r a n g e b e t w e e n 0 to 25 C, so t h a t t he Arrhenius p lo t s showed a l inear l ine . However , in t he ases of onion bulb (Allium cepa L.) and c a r r o t roo t (Daucus carrota L. sativa DC) which a r e also chil l ing insens i t ive , t he shapes of t he l ines in t h e Arrhenius p lo t s w e r e d i f fe ren t from those observed wi th p o t a t o t u b e r s . For onion bulb and c a r r o t r o o t , b r eakpo in t s w e r e obse rved which s e e m e d no t to co r respond wi th t h e t e m p e r a t u r e s of any kind of physiological c h a r a c t e r s of t hese p lan t t i s sues .

3. Effect of Detergent on Arrhenius Plots. If d e n a t u r a t i o n of b i o - m e m b r a n e s a t low t e m p e r a t u r e s is t h e p r i m a r y cause of chil l ing injury, i t is possible t h a t t r e a t m e n t wi th some kinds of d e t e r g e n t s may enhance t he o c c u r r e n c e of chil l ing injury of fruit and v e g e t a b l e s a t low t e m p e r a t u r e s . Arrhenius p lo t s of t h e r a t e of l e akage of po tass ium ion from t h e discs of cucumber fruit which w e r e p r e t r e a t e d wi th 0.1%


5 2 . 5 h

I cn cn 3 cn OJ J* αί •S 2.0 cn ο

1.5H-

storage at 10°C

20 10 Ore • In JL

9 days -O = Q_

5°C

10 —τ— 35

- O -

0°C

3A 35 36 34 1/T χίΟ

36

FIGURE 2. Arrhenius plots of rate of potassium ion leakage from the discs of cucumber fruit (cv. Natsuakihushinari) stored at 5 C or 10°C for 1-9 days.

Tri ton X-100 a r e shown in F igure 4 . O c c u r r e n c e of chil l ing injury of cucumber fruit during s t o r a g e a t low t e m p e r a t u r e was i nc reased sl ight ly by t h e t r e a t m e n t with t he d e t e r g e n t , but t h e r e was no s ignif icant e f fec t of d e t e r g e n t on the shapes of Arrhenius p lo t s shown in F igure 4; b reakpo in t s r e m a i n e d in t he Arrhenius p lo t s of r a t e of l eakage of Dotassium ion from the discs of t r e a t e d t i ssues during s t o r a g e a t 1 C.

It is r easonab le to consider t ha t t he phase t r ans i t ion (and/or phase separa t ion) in the m e m b r a n e s of t i ssues of chil l ing sens i t ive p l an t s occurs a t a c r i t i c a l t e m p e r a t u r e . However , fu r ther c la r i f i ca t ion is r equ i r ed because b reak poin ts a r e also found to occur in chil l ing insens i t ive spec ies .

Ion L e a k a g e in Chi l led P l an t T i s s u e s 145

2.5 h

FIGURE 3. Arrhenius plots of rate of potassium ion leakage from chilling insensitive plant tissues (Ο Ο ; potato tuber, Ο Ο ; onion bulb, Δ Δ; carrot root).

B. Changes in the Rate of Ion Leakage

1. Changes in Ion Leakage from Chilling Sensitive Plant Tissues during Storage. The r a t e of ion l e akage in to de ion ized w a t e r by discs of cucumber fruit did no t change during s t o r a g e a t 10 C and Z0 C (i.e. above the c r i t i c a l t e m p e r a t u r e ) . However , t h e r a t e of ion l eakage from t h e discs of fruit s t o r e d a t 0 C and 5 C s t a r t e d to i nc r ea se abrup t ly on t h e seven th day of s t o r a g e and r e a c h e d app rox ima te ly tw ice t he r a t e of those a f t e r 12 days s t o r a g e a t 10 C or 20 C (Fig. 5 le f t ) . R a t e of ion l eakage in to aqueous manni to l from the discs of fruit s t o r e d a t 5 C or 20 C showed bas ica l ly t h e s a m e t endency as t h a t in to de ion ized w a t e r (Fig. 5 r igh t ) . Also, a s imi lar i nc reased r a t e of ion l eakage was observed in chi l led t i ssues of snap bean pod which is a chill ing sens i t ive p l an t .

The r a t e of l eakage of po t a s s ium, sodium and magnes ium ions in to w a t e r from discs of s w e e t p o t a t o roo t s t o r ed a t 20 C showed a r e l a t i ve ly cons t an t level th roughout t h e s t o r a g e per iod . However , t he r a t e a t 5 C s t a r t e d to r i se suddenly a f t e r s t o r a g e for 2 weeks (Fig. 6).


3.0

cn

cn

2.5

σ ι ο

2.0

1.5

o Control • T r i t o n X-10 0

15 day s

2,0

33 34 35 1/ T x 1 0 A

36 37

FIGURE 4. Arrhenius plots of rate of potassium ion leakage from discs of cucumber fruit (cv. Horai) stored at 1 C for 15 days with and without treatment with 0.1% Triton X-100.

The r a t e of l e akage of po tass ium, sodium and magnes ium ions from s w e e t p o t a t o roo t s t o r ed a t 5 C was about 5 t i m e s t h a t from r o o t s s t o r e d a t 20 C a f t e r 6 weeks .

Changes in t h e r a t e of ion l eakage from the discs of bell pepper (Capsicum annum L.)Qand t o m a t o ^Lycopersicon esculentum Mill) f rui ts during s t o r a g e a t 2 C and 12.5 (i.e. below and above t h e c r i t i ca l t e m p e r a t u r e ) exhib i ted d i f fe ren t cu rves c o m p a r e d wi th those of cucumber and s w e e t p o t a t o (Fig. 7 le f t ) . A d r a m a t i c r i se in t he r a t e of ion l eakage from t h e discs of bell pepper fruit was no t observed during s t o r a g e a t 2 C or 12.5 C. Changes in the ion l eakage from t h e t i ssues of egg p lan t fruit exh ib i ted a s imi lar t endency to t h a t of bell peppe r . These r e su l t s sugges t t h a t some chill ing sens i t ive p lan t t i ssues show l i t t l e change in m e m b r a n e p e r m e a b i l i t y during s t o r a g e a t chil l ing t e m p e r a t u r e s . In t h e case of t o m a t o fruit (ma tu re g r e e n - b r e a k e r s t ages ) , t h e r a t e of ion l eakage from t h e discs of fruit s t o r ed a t 2 C and 12.5 C cont inued to i nc rease during s t o r a g e . The s a m e t endency was observed in t h e changes of t h e l e akage from b a n a n a fruit t i ssues (half g r e e n - g r e e n t ip s t ages ) .

Ion L e a k a g e in Chi l led P l a n t T i s s u e s 147

FIGURE 5. Changes in the rate of ion leakage into deionized water (Left; cv. Natsuakihushinari) and 0.4 Μ mannitol solution (Right ; cv. Horai) from discs of cucumber fruit stored at 0-20 C. (% of leaked ion to total ion after incubation for 4 hours).

2. Changes in Ion Leakage from Chilling Insensitive Plant Tissues during Storage. Changes in t h e r a t e of ion l eakage from discs of p o t a t o tube r s s t o r e d a t Ζ C and 1Z.5 C a r e shown in F igure 7-r ight . R a t e s w e r e very low c o m p a r e d wi th those of o t h e r p lan t t i ssues . T h e r e was no sudden r i se of p e r m e a b i l i t y during s t o r a g e a t Ζ C and 1Z.5 C. No s ignif icant d i f f e rence was observed in t h e r a t e s of l eakage from the discs of p o t a t o tuber s t o r e d a t Ζ C and 1Z.5 C a t t h e end of s t o r a g e .

T h e r e a r e l a rge f luc tua t ions of chil l ing sens i t iv i ty of seeds in pod of Leguminosae such as soybean, p e a and snap bean . In t h e case of pod t i ssues of pea , which is a chil l ing insens i t ive p l an t , no i n c r e a s e of ion l eakage was obse rved during s t o r a g e a t Ζ C . However , a cont inuous i nc r ea se of ion l eakage was obse rved in t h e t i ssues of pods of young


50 0 κ 5°C /

30 0 Ι Α 20"C

10 0

0 1 ι ,

10 0

0

5 0

? Ω. Q. Φ cn 3 0

Na

A 5 ° c /

? Ω. Q. Φ cn 3 0

OJ φ

10 (

1 • •

20°C

-O I

0 1 5

Mg 5°C /

10 •

5

1 1 1_

20°C

1 L_ I ' I I I L·

0 1 2 3 4 5 6 Week s afte r storag e

FIGURE 6. Changes in the rate of leakage of potassium, sodium and magnesium ions from discs of sweet potato root stored at 5 C or 20°C.

soybean (chilling insensi t ive) and of snap bean (chilling sensi t ive) during s t o r a g e a t 2 C.

Inc reased r a t e s of ion l eakage of chi l led t i ssues have been found in the t i ssues of fruit and v e g e t a b l e s (2, 3 , 10) and l eaves (1, 4, 7, 9, 11 , 12). Regard ing leaf t i ssues , it is known t h a t r e l a t i v e humid i ty during growth of p lan t s a f f e c t e d the r a t e of ion l eakage from the t i ssues (11, 12). Wright (11) has ind ica t ed t h a t chill ing a lone , or w a t e r def ic i t

FIGURE 7. Changes in the rate of ion leakage from discs of bell pepper and tomato fruits (Left) and potato tuber (Right) stored at 2 C and 12.5°C.

(Ο/0)Θ

6Β>|ΡΘ

Ι UO

I


alone , did not l ead to i nc reased l eakage of l eaves of seedl ings of Phaseolus vulgaris L. Simon (8) has discussed the e f fec t of chil l ing t r e a t m e n t a t d i f fe ren t humid i t i es on m e m b r a n e p e r m e a b i l i t y of leaf t i s sues . Morris and P la ten ius (5) have shown tha t t h e s eve r i t y of chil l ing injury of cucumber fruit was con t ro l l ed by ra is ing t h e r e l a t i v e humid i ty during s t o r a g e . It is well known t h a t low humid i ty enhances t h e o c c u r r e n c e of visual s y m p t o m s of chill ing injury of c i t rus fruit and bell pepper f rui t . However , l i t t l e in fo rmat ion is ava i lab le concern ing the e f fec t of w a t e r def ic i t s on t h e changes in e l e c t r o l y t e l eakage of chi l led t issues of fruit and v e g e t a b l e s .

Our r e su l t s ob ta ined for t he changes in e l e c t r o l y t e l eakage of t i ssues of cucumber frui t , o r i en t a l pickl ing melon frui t , snap bean pod and swee t p o t a t o roo t coincide wi th t h e r e su l t s of prev ious r e p o r t s . However , no inc reased r a t e of ion l e akage is observed in chi l led t i ssues of bell pepper and egg p lan t f rui ts which a r e chil l ing sens i t ive p l an t s . In the cases of t o m a t o and banana f ru i ts , a high r a t e of ion l eakage from the t i ssues is observed even a t t e m p e r a t u r e s above t h e c r i t i ca l point during s t o r a g e , probably due to thei r r ipening .

N e v e r t h e l e s s , all of t h e published inves t iga t ions on e l e c t r o l y t e l eakage have sugges ted inc reased r a t e s of l eakage from chill ing sens i t ive p lan t t i ssues occur a t low t e m p e r a t u r e s . Our d a t a suggest t he ex i s t ence of chil l ing sens i t ive p lan t t i s sues , such as bell pepper and egg p lan t f ru i t s , which exhibi t no i nc rease of e l e c t r o l y t e l eakage a t t e m p e r a t u r e s below the c r i t i c a l po in t . The re fo r e , it is r ea sonab le to infer t h a t high r a t e s of e l e c t r o l y t e loss a t chil l ing t e m p e r a t u r e s is not necessa r i ly a gene ra l p r o p e r t y of chil l ing sens i t ive p lan t t i s sues . It might be cons idered t h a t d i f fe ren t types of mechan i sms rega rd ing e l e c t r o l y t e loss from t issues may exis t in the chil l ing injury of fruit and v e g e t a b l e s .

ΙΠ. R E F E R E N C E S

1. C r e e n c i a , R. P . , and Bramlage , W. J . Plant Physiol. 47, 389-392 (1971).

2. Lewis , T. L., and Workman, M. Aust. J. Biol. Sci. 17, 147-152 (1964).

3 . L i ebe rman , M., C ra f t , C. C , Audia, W. V. and Wilcox, M. S. Plant Physiol. 33, 307-311 (1958).

4 . Minchin, Α., and Simon, E. W. Jour. Expt. Bot. 24, 1231-1235 (1973).

5. Morr is , L. L. and P la t en ius , H. Proc. Amer. Soc. Hort. Sci. 36, 609--613 (1938).

6. Nobel , P . S. Planta 115, 369-372 (1974). 7. P a t t e r s o n , B. D. , Mura t a , T., and G r a h a m , D. Aust. J. Plant Phys

iol. 3, 435-442 (1976). 8. Simon, E. W. New Phytol. 73, 377-420 (1974). 9. Tanczos , O. G. Physiol. Planta. 41, 289-292 (1977).

Ion L e a k a g e in Chi l led P lan t T i s s u e s 151

10. T a t s u m i , Y. and Mura t a , T. J. Japan. Soc. Hort. Sci. 47, 105-110 (1978).

11 . Wright , M. Planta. 120, 63-69 (1974). 12. Wright , M., and Simon, E. W. Jour. Expt. Bot. 24, 400-411 (1973).



E F F E C T S OF CHILLING ON MEMBRANE POTENTIALS OF MAIZE AND OAT LEAF CELLS

Paul H. Jennings

D e p a r t m e n t of P lan t and Soil Sc iences

and

Terry A. Tattar

D e p a r t m e n t of P l an t Pa tho logy Univers i ty of M a s s a c h u s e t t s

A m h e r s t , M a s s a c h u s e t t s

I. INTRODUCTION

The in t roduc t ion to this symposium rev i ews a l a rge body of in fo rmat ion encompass ing the var ious r e sponses of p l a n t s , o rgans , t i ssues and biological m e c h a n i s m s a f f e c t e d by l o w - t e m p e r a t u r e chil l ing injury. We will not r e p e a t or a t t e m p t to e l a b o r a t e on tha t exce l l en t overview but will point out an approach which we consider to have p o t e n t i a l in providing an addi t iona l t e chn ique in s tudying chil l ing injury in p l a n t s .

Various app roaches have been used in s tudying l o w - t e m p e r a t u r e e f f ec t s on p l an t s , ranging from obse rva t ions of visible injury, such as wi l t ing, chlorosis and nec ros i s , to t echn iques which m e a s u r e e l e c t r o l y t e l eakage from var ious p lan t p a r t s in r e sponse to chi l l ing, and cu r r en t l y to soph i s t i ca t ed s tud ies employing spin- label ing of t h e m e m b r a n e (9, 13, 19)· An approach which has not r e c e i v e d as much a t t e n t i o n in low-t e m p e r a t u r e s tud ies is e f f e c t s on m e m b r a n e p o t e n t i a l .

Maize was chosen for s tudy p a r t l y as a resu l t of o t h e r c u r r e n t low-t e m p e r a t u r e s tud ies being conduc ted wi th it but also b e c a u s e it is genera l ly r e c o g n i z e d as an i m p o r t a n t ch i l l ing-sens i t ive c rop p l a n t . O a t s , as a non-chi l l ing sens i t ive grass , was chosen for compar i son in these

1 Paper No. 2287, Massachusetts Agricultural Experiment Station,

University of Massachusetts at Amherst. This research supported from Experiment Station Projects No. Hatch 364 and Hatch 426.

153 Copyright © 1Θ7Θ by Academic Press, inc.

All rights of reproduction in any form reserved 1SBN012 4 6 0 5 6 0 5

154 P. Η. J e n n i n g s a n d Τ. A. T a t t a r

s tud ies . Maize exhibi ts visible l o w - t e m p e r a t u r e injury in t h e form of ch lo ro t i c and n e c r o t i c lesions which form bands ac ross the leaf (15). This banding p a t t e r n , s o m e t i m e s r e f e r r e d to as Fa r i s bands (3), is a p p a r e n t l y a response of leaf cel ls a t a c r i t i ca l s t a g e in deve lopmen t to low-t e m p e r a t u r e s t r e s s (16). The resu l t is fa i lure of var ious cel ls and ce l lu lar componen t s to develop resu l t ing in the very c h a r a c t e r i s t i c ch lo ro t i c band which even tua l ly b e c o m e s n e c r o t i c . O t h e r dysfunct ions in m a i z e h a v e been observed including inc reased e l e c t r o l y t e l eakage from s t r e s sed leaf t i ssue as well as var ious m e t a b o l i c changes (1). The dura t ion of s t r e s s in these l a t t e r examples d e t e r m i n e d the e x t e n t and r eve r s ib i l i t y of the r e sponse . With these r e su l t s and o t h e r s wi th d i f fe ren t p lan t m a t e r i a l it s e e m e d tha t s tud ies of the e f f ec t s of chil l ing injury on m e m b r a n e p o t e n t i a l might provide addi t ional useful in fo rmat ion in a t t e m p t s to e luc ida t e mechan i sms of l o w - t e m p e r a t u r e injury.

An e l e c t r i c a l p o t e n t i a l (E) ex is t s b e t w e e n t h e inside of t h e p lan t cel l and the outs ide med ium. This p o t e n t i a l is ma in t a ined p r imar i ly ac ross t h e p lasma m e m b r a n e and is usual ly t e r m e d a m e m b r a n e p o t e n t i a l (Em) (6). Cons iderab le ev idence suppor t s the theory t ha t much of the Em in p l an t s is ma in t a ined a t t h e expense of m e t a b o l i c energy supplied by r e sp i r a t ion and /o r pho tosyn thes i s (6, 12). It has also been d e m o n s t r a t e d t h a t the e l e c t r i c a l energy g rad ien t in t he Em is used to pe r fo rm t r anspor t work ac ross the p l a sma m e m b r a n e (12, 17). More r e c e n t l y changes in Em have been r e l a t e d to u p t a k e of amino acids and c a r b o h y d r a t e s (2, 11).

L i t t l e in fo rmat ion is ava i lab le on t h e e f f ec t s of chil l ing on Em. Some inves t i ga to r s , however , have used low t e m p e r a t u r e (less than I O C ) incubat ion to inhibit me tabo l i sm and to d e m o n s t r a t e i t s depolar iz ing e f fec t on Em, much t h e s ame way as po tass ium cyan ide and sodium az ide have been used to depo la r i ze Em chemica l ly (4, 5, 8). We r e p o r t on the e f f ec t s of chill ing on the Em of m a i z e and o a t s as well as t he i n t e r a c t i v e e f f ec t s of var ious me tabo l i c inhibi tors and na tu r a l l y occur r ing organ ic compounds .

Π. MATERIALS AND METHODS

A. Tissue Preparation

The p lant m a t e r i a l used in t he se s tud ies cons i s ted of c o m m e r c i a l va r i e t i e s of m a i z e (Zea mays cv Seneca Chief) ob ta ined from t h e Joseph Harr i s Seed Company and oa t s (Avena sativa cv Garry) ob t a ined from Agway (a f a rmers ' coope ra t i ve ) . Seed w e r e soaked overnight in w a t e r and g e r m i n a t e d in v e r m i c u l i t e in a g rowth chamber under t he following condi t ions : 15/9 hr day /n igh t l ight cyc le ; l ight in tens i ty of 110 Wm ^provided by cool wh i t e f luorescen t and incandescen t bulbs; 24 / 2 4 C day /n igh t t e m p e r a t u r e cyc le . P l an t s w e r e w a t e r e d wi th a c o m p l e t e Hoagland solut ion (7) modif ied to con ta in 3X i ron. In e x p e r i m e n t s se t up to d e t e r m i n e low growth t e m p e r a t u r e e f f e c t s on p o s t s t r e s s m e m b r a n e po t en t i a l a g rowth t e m p e r a t u r e cyc le of 24 / 8 C

Effects of Chil l ing o n M e m b r a n e P o t e n t i a l s 155

was used. P lan t t i s sue for m e m b r a n e p o t e n t i a l m e a s u r e m e n t s was ob ta ined using the f i rs t leaf when the p l an t s w e r e b e t w e e n 12 and 16 days old. Tissue was p r e p a r e d by r emov ing t h e leaf from t h e p lan t wi th a r a z o r b lade and in the ca se of m a i z e c u t t i n g the leaf in half longi tudinal ly a long t h e mid-ve in . The lower ep ide rmis was then r e m o v e d using a pair of e l e c t r o n mic roscopy fo rceps . Sect ions of s t r ipped leaf m a t e r i a l (approx 2 x 8 mm) w e r e then f loa ted s t r ipped su r f ace down in p e t r i dishes con ta in ing 1 mM potass ium phospha t e buffer pH 6.5 wi th 1 mM ca lc ium chlor ide . This s a m e buffer was used to per fuse the t i ssue in t he perfusion c h a m b e r desc r ibed below. The leaf sec t ions w e r e i ncuba t ed 20-24 hr under t he g rowth c h a m b e r condi t ions desc r ibed above .

B. Measurement of Em

Tissue sec t ions w e r e per fused wi th buffer in a L u c i t e c h a m b e r t h a t was a t t a c h e d to a hor izon ta l ly m o u n t e d mic roscope (2). Glass cap i l la ry m i c r o e l e c t r o d e s , t ip diam less t han 1 p m , w e r e p r e p a r e d from f iber -fi l led glass cap i l l a r ies wi th an e l e c t r o d e pul ler (Industrial Sc ience) . M i c r o e l e c t r o d e s w e r e fi l led wi th 3 Μ po tass ium chlor ide and had a t ip r e s i s t a n c e of 10-20 Meg ohms . The Em was m e a s u r e d b e t w e e n an e l e c t r o d e fi l led wi th 3 Μ po tass ium chlor ide in 2% agar , in t h e ba th ing solut ion, and t h e m i c r o e l e c t r o d e in t h e ce l l . Both e l e c t r o d e s w e r e c o n n e c t e d to an ampl i f ie r (WPI I n s t r u m e n t s , Model 725) and t h e Em r e c o r d e d on a s t r ip c h a r t r e c o r d e r .

C. Effects of Light, Inhibitors, Glycine and Sucrose

All in i t ia l e x p e r i m e n t s w e r e conduc t ed wi th buffer a t 21°C and s t ab l e Em m e a s u r e m e n t s w e r e ach ieved for 5 m i n u t e s in the dark be fo re any t r e a t m e n t s w e r e imposed . In light, e x p e r i m e n t s t i s sue was i l l umina ted wi th a l ight in t ens i ty of 670 Wm for 5 minu t e s and then t h e l ight was tu rned off for 5 m i n u t e s . In inhibi tor e x p e r i m e n t s , t he s t a n d a r d 1 mM po tass ium phospha t e buffer was m a d e 1 mM wi th r e s p e c t to e i t he r po tass ium cyan ide or sodium az ide , and the t i s sue exposed to inhibi tor by swi tch ing from perfusion wi th buffer a lone to buffer plus inhibi tor wi thout i n t e r rup t ion of liquid f low. In some e x p e r i m e n t s wi th po tass ium cyan ide t h e t i s sue was exposed to l ight a f t e r max imum depo la r i za t ion had o c c u r r e d . Tissue was per fused wi th suc rose (50 mM) or g lycine (50 mM) in buffer in a s imi lar manne r for 5 minu te s and was followed by 5 minu te s of buffer a lone .

D. Effects of Cold Buffer

Buffer a t 0 -2°C was passed th rough t h e perfusion c h a m b e r conta in ing t h e m a i z e or oa t leaf s ec t ions in o rder to m e a s u r e d i r ec t e f f ec t s of chil l ing t e m p e r a t u r e on Em. The t e m p e r a t u r e in t h e c h a m b e r


was cont inuously mon i to red wi th a t h e r m o p r o b e (YSI T e l e -t h e r m o m e t e r ) . Ef fec t s of l ight vs dark on Em and e f f ec t s of 50 mM glycine or 50 mM sucrose w e r e d e t e r m i n e d in cold buffer (8 C) as previously desc r ibed for 21 C buffer .

III. RESULTS

A. Effects of Light

No d i f fe rences w e r e d e t e c t e d in t he Em of con t ro l or chi l led m a i z e or o a t s , grown a t e i t he r 24 / 2 4 C or 24 / 8 C, when Em m e a s u r e m e n t s w e r e m a d e wi th 21 C buffer (Table I). T h e r e w e r e also no d i f fe rences in response of Em to light with e i t he r con t ro l or chi l led leaf sec t ions of m a i z e wi th 21 C buffer (data not shown). The re fo re , all subsequent e x p e r i m e n t s w e r e conduc ted wi th p lan t m a t e r i a l grown a t 24 / 2 4 C, excep t whe re no t ed . Incubat ion wi th 8 C buffer in t he dark caused a 30% depo la r i za t ion of Em in both m a i z e and oa t leaf ce l l s , c o m p a r e d with 21 C buffer (Fig. 1). The Em of oa t leaf cel ls a t 8°C in t he dark, however , would of ten slowly hyperpo la r i ze to levels s imi lar to Em a t 21 C in t he dark (data not shown). When oa t and m a i z e t i ssues we re i l lumina ted in 8 C buffer , oa t leaf cel ls again hyperpo la r i zed to an Em c o m p a r a b l e to cel ls of t ha t spec ies in 21 C buffer , but t he Em of m a i z e leaf cel ls exposed to light a t 8 C depo la r i zed and did no t r e c o v e r even to the i r in i t ia l Em in t he dark .

TABLE I. Effects of Inhibitors and Growth Temperatures on Em of Corn and Oat Leaf Cells

Em in mV Maize Oats

Treatment 24°/24° 24°/8° 24°/24° 24°/8°

Control 120 + 2 125 + 7 147 t 4 142 + 6

CN 68 + 13 58 + 13 -N3 57 ±9 63 + 6 69 t

6 73 + 6

Em measurements were made with buffer at 21 C in the dark. Plants were grown with a 15/9 hr day/night light cycle.


I 1 2 m i n

FIGURE 1. Effect of light and temperature on leaf cell Em of maize and oats.

B. Effects of Metabolic Inhibitors

Addit ion of 1 mM potass ium cyan ide or 1 mM sodium az ide a t 21°C caused an i m m e d i a t e co l lapse of Em to a p p r o x i m a t e l y 50% of the in i t ia l levels in bo th m a i z e and o a t s (Table 1). A s imi lar Em response followed addi t ion of sodium az ide in o a t s . In KCN e x p e r i m e n t s whe re m a i z e was i l lumina ted a f t e r max imum Em depo la r i za t ion had been ach ieved , t he Em of bo th chi l led and con t ro l sec t ions r e c o v e r e d to app rox ima te ly the in i t ia l da rk levels in less than 10 minu te s (data not shown). No d i f fe rences in Em w e r e observed b e t w e e n the con t ro l and the chi l led m a i z e l eaves in response to e i t he r of t he se inhib i tors when Em was d e t e r m i n e d using 21 C buffer . Similar ly , no d i f f e rence in Em response was d e t e c t e d b e t w e e n con t ro l and chi l led o a t s . The Em of m a i z e leaf cel ls depo la r i zed to a s l ight ly g r e a t e r e x t e n t when t h e t i s sue was per fused wi th 8°C buffer conta in ing 1 mM potass ium cyan ide in t he dark (data not shown).

C. Effects of Glycine

Afte r t he addi t ion of 50 mM glycine in buffer a t 21°C the Em of bo th spec ies i m m e d i a t e l y depo la r i zed but began to r e c o v e r a f t e r 2-3 minu te s and r e c o v e r e d m o r e quickly a f t e r t h e g lyc ine was r e m o v e d (Fig. 2). No major d i f fe rences w e r e d e t e c t e d b e t w e e n t h e Em response of m a i z e and of o a t s to t h e addi t ion of 50 mM glycine a t 21 C . However , when m a i z e

L I G H T


FIGURE 2. Effect of 50 mM glycine on maize and oat leaf cell Em at 21° and 8°C.

leaf sec t ions w e r e per fused with 50 mM glycine in 8 C buffer , only a minor depo la r i za t ion o c c u r r e d followed by a sl ight r e c o v e r y , while in oa t leaf cel ls depo la r i za t ion and r e c o v e r y w e r e s imi lar to r e su l t s wi th Zl C buffer . The Em of oa t leaf cel ls was of ten found to be uns tab le in the dark and, consequent ly , many 50 mM glycine e x p e r i m e n t s we re also conduc ted to t a l ly in l ight . The r e su l t s of t hese e x p e r i m e n t s , however , w e r e comparab l e wi th those in t he dark a l though t h e Em t ended to be h igher . Similar e x p e r i m e n t s in the l ight w e r e also conduc ted wi th m a i z e a t 8 C, but t he r e su l t s w e r e comparab l e to those in t he dark .

D. Effects of Sucrose

Addit ion of 50 mM sucrose in buffer a t Z1°C caused an i m m e d i a t e depo la r i za t ion of t h e Em of bo th spec ies , fol lowed by a slow r e c o v e r y a f t e r Z-3 minu tes (Fig. 3). A rapid r e c o v e r y o c c u r r e d when the sucrose was r emoved , and u l t i m a t e l y r e s u l t e d in a hype rpo la r i za t ion . A s imi lar p a t t e r n was observed in o a t s a t 8 C but t h e amoun t of in i t ia l Em depo la r i za t ion was r educed approx ima te ly 50% c o m p a r e d to t h e Zl C t r e a t m e n t . If e x p e r i m e n t s a t 8 C w e r e conduc ted in l ight , however , in i t ia l Em depo la r i za t ion in oa t s was c o m p a r a b l e to t h e dark e x p e r i m e n t s


5 0 m M

M9 h t - 1 3 8 m V I

t 2 mi n

FIGURE 3. Effect of 50 mM sucrose on maize and oat leaf cell Em at 21°C and 8°C.

a t Z1°C. Light had no a p p a r e n t e f fec t on r e su l t s wi th 50 mM sucrose in m a i z e a t e i t he r 8 C or 21 C In this spec ies a t 8 C, t h e in i t ia l Em depo la r i za t ion wi th sucrose was subs tan t i a l ly r e d u c e d and very l i t t l e r e c o v e r y could be d e t e c t e d even a f t e r r e m o v a l of suc rose .

IV. DISCUSSION

Since one componen t of Em a p p e a r s to be a t l eas t p a r t i a l l y dependen t on a m e t a b o l i c ene rgy supply, it is no t surpr is ing t ha t ce l ls of t i s sue exposed to low t e m p e r a t u r e s would depo la r i ze as a resu l t of d e c r e a s e d r e sp i r a t i on and consequen t ly ox ida t ive phosphory la t ion . Our r e su l t s h a v e shown t h a t this t e m p e r a t u r e e f f ec t on Em depo la r i za t i on is r ead i ly and rapid ly r eve r s ib l e wi th bo th m a i z e and o a t s . At 21 C t h e r e was cons i s t en t ly no d i f f e r ence in Em response b e t w e e n t i s sues grown a t 24 / 2 4 C and 24 / 8 C of bo th spec i e s . Thus, when Em was m e a s u r e d during l o w - t e m p e r a t u r e t r e a t m e n t of leaf t i s sue , a depo la r i za t ion was observed in bo th o a t s and m a i z e in t h e da rk . However , when t h e t i ssue was exposed to l ight , o a t leaf ce l ls w e r e able to r e - e s t a b l i s h an Em a t 8 C c o m p a r a b l e to s imi lar t i s sue a t 21 C in t h e l ight w h e r e a s m a i z e was appa ren t ly unable to u t i l i ze light ene rgy to r e c o v e r i t s Em a t 8 C . This chil l ing e f fec t wi th m a i z e is no t surpr is ing s ince Taylor et al. (18)


found t h a t seve ra l key e n z y m e s y s t e m s in m a i z e involved wi th photoconvers ion of l ight to chemica l energy w e r e seve re ly inhib i ted during chil l ing. In addi t ion, Novacky and Kar r (10) found t h a t suppress ion of Em responses to l ight was a common pa tho log ica l a l t e r a t i o n in some p lan t d i seases .

Amino acid u p t a k e was c o r r e l a t e d wi th d e g r e e of Em depo la r i za t ion in oa t co leop t i l e s (Z) and in oa t l eaves (14), while u p t a k e of seve ra l sugars , including sucrose , was c o r r e l a t e d wi th Em depo la r i za t ion in Lemna gibba (11). The change in Em in bo th o a t s and m a i z e following addi t ion of g lycine or of sucrose was, t h e r e f o r e , mos t l ikely caused by u p t a k e of t hese m a t e r i a l s ac ross t he p l a sma m e m b r a n e . The major d i f f e rence b e t w e e n t h e e f f ec t s of g lyc ine and sucrose on Em of m a i z e a t Zl C and a t 8 C probably ind ica t e s a s t rong inhibi t ion of u p t a k e of t h e s e m a t e r i a l s a t 8 C. The minor d i f f e rence b e t w e e n Em response a t Zl C and 8 C in o a t s sugges ts t h a t u p t a k e of g lycine and sucrose w e r e only sl ightly inhibi ted a t 8 ° C .

Novacky et al. (11) found a s t rong co r r e l a t i on b e t w e e n Em and in t r ace l lu l a r ATP c o n c e n t r a t i o n in L. gibba ce l l s . These r e s e a r c h e r s also found t h a t hyperpo la r i za t ion of Em, following sucrose addi t ion , was r e l a t e d to inc reased ATP produc t ion from metabo l i sm of this sugar . Leaf cel ls of oa t s a t 8 C w e r e able to r e c o v e r Em in sucrose and to hyperpo la r i ze Em following sucrose r e m o v a l , while those of m a i z e could no t , even though cel ls of m a i z e w e r e able to r e c o v e r Em in sucrose and to hype rpo la r i ze Em a f t e r sucrose r e m o v a l in s imi lar e x p e r i m e n t s a t Zl C. Chil l ing sens i t iv i ty in m a i z e , t h e r e f o r e , may be due to bo th suppressed u p t a k e and the inabi l i ty to rapidly m e t a b o l i z e compounds t h a t a r e t r a n s p o r t e d into the ce l l . Using Em we have been able to show a d i f f e rence in response b e t w e e n r e p r e s e n t a t i v e s of chil l ing and non-chil l ing sens i t ive p lan t spec ie s . The in i t ia l r e sponse in the r e s p i r a t o r y energy g e n e r a t i n g sys tem to low t e m p e r a t u r e was s imi lar in t h e two grass spec ies used in this s tudy . However , t he energy g e n e r a t i n g sys t em as soc i a t ed wi th pho tosyn thes i s is appa ren t ly very sens i t ive to chil l ing in ma ize and v i r tua l ly u n a f f e c t e d in o a t s , a t l eas t in t e r m s of m a i n t e n a n c e of Em.

We a n t i c i p a t e t h a t fur ther s tudies of t h e p h o t o s y n t h e t i c sys tem could be prof i tab ly pursued using var ious m e t a b o l i c probes in conjunct ion wi th Em m e a s u r e m e n t s to d e t e r m i n e t he site(s) a f f e c t e d by low t e m p e r a t u r e exposure . In addi t ion , g e n e t i c d i f fe rences might also be employed to help e l u c i d a t e chill ing injury m e c h a n i s m s , again using Em as a moni tor of p lan t r e sponse . The use of Em might also provide a new and useful app roach to sc reen ing of g e n e t i c m a t e r i a l for chil l ing r e s i s t a n c e in a p lan t b reed ing p r o g r a m . To fur ther e l a b o r a t e on the mechan i sm of chil l ing sens i t iv i ty , Em could be d e t e r m i n e d over a r a n g e of t e m p e r a t u r e s which spans the chil l ing sens i t ive r a n g e wi th compar i sons m a d e b e t w e e n chill ing and non-chi l l ing sens i t ive p lan t m a t e r i a l . O t h e r app roaches will undoubtedly develop to use Em in chil l ing injury s tud ie s .


V. R E F E R E N C E S

1. C r e e n c i a , R. P . and Bramlage , W. J . Plant Physiol. 47, 389-392 (1971).

2. E t h e r t o n , B. and Rubins te in , B. Plant Physiol. 61, 933-937 (1978). 3 . Fa r i s , J . A. Phytopath. 16, 885-891 (1927). 4 . Gradmann , D. Planta 93, 323-353 (1970). 5. Gradmann , D. and Ben t rup , F . W. Naturwisenshaften 57, 46-47

(1970). 6. Hig inbo tham, N. Plant Physiol. 24, 25-46 (1973). 7. Hoagland, D. R. and Arnon, D. I. Calif. Agric. Expt. Sta. Cir. 347,

pp . 1-32 (1950). 8. Ichino, K., Ka tou , K., and O k a m o t o , H. Plant and Cell Physiol. 14,

127-137 (1973). 9. Lyons, J . M. Plant Physiol. 24, 445-466 (1973). 10. Novacky , Α., and Kar r , A. L. In "Regula t ion of Cel l M e m b r a n e

Ac t iv i t i e s in P lan t s . " (E. M a r r e and O. C i fe r r i , eds.) , pp . 137-144, E l sev i e r /Nor th Holland, A m s t e r d a m (1977).

11 . Novacky , Α., Ul l r i ch-Eber ius , C. I., and L u t t g e , U. Planta 138 263- 270 (1978).

12. Poole , R. J . Plant Physiol. 29, 437-460 (1978). 13. Raison , J . K., Lyons, J . M., Mehlhorn, R. J . , and Ke i th , A. D . J.

Biol. Chem. 246 4036-4040 (1971). 14. Rubins te in , B. and T a t t a r , T. A. Plant Physiol. 61 (Suppl.) 107

(1978). 15. Sel lschop, J . P . F . and Salmon, S. C. J. Agric. Res. 37, 315-338

(1928). 16. Slack, C . R., Roughan , P . G., and Bas se t t , H. C . M. Planta 118,

57-77 (1974). 17. Slayman, C. L. In "Membrane Transpor t in P lan t s . " (U.

Z i m m e r m a n n and J . Da in ty , eds.) , pp . 107-119, Spr inger-Ver lag (1974).

18. Taylor , A. O., Slack, C. R., and McPherson , H. G. Plant Physiol. 54, 696-701 (1974).

19. Wade, N . L, Bre idenbach , R. W., Lyons, J . M., and Ke i th , A. D. Plant Physiol. 54, 320-323 (1974).



TEMPERATURE SENSITIVITY O F ION-STIMULATED ATPases ASSOCIATED WITH SOME PLANT MEMBRANES

Edward J. McMurchie

Plan t Physiology Unit CSIRO Division of Food R e s e a r c h and School of Biological Sciences

Macquar i e Univers i ty Nor th Ryde , N.S.W., Aus t r a l i a

I. INTRODUCTION

The hypothes i s t h a t changes in t h e molecu la r order ing of m e m b r a n e lipids a r e t he in i t ia l even t s in chil l ing injury (7, 13) has c o m e p r imar i ly from r e su l t s ob ta ined using i so la t ed p lan t mi tochondr i a (8, 13)· These s tud ies have shown tha t m e m b r a n e - a s s o c i a t e d r e s p i r a t o r y e n z y m e s of mi tochondr i a i so l a t ed from chi l l ing-sens i t ive p l an t s undergo a change in a p p a r e n t a c t i v a t i o n ene rgy (E ) a t some c r i t i c a l t e m p e r a t u r e . This change in Ε is no t obse rved in mi tochondr i a i so la ted from chi l l ing-r e s i s t a n t p l a n t s (8). The change in Ε for m e m b r a n e - a s s o c i a t e d enzymes c o r r e l a t e s wi th a change in m e m b r a n e lipid o rder or f luidi ty as d e t e r m i n e d by spin label (13, 14) or f luorescen t p robe t echn iques (1Z). However , t hese t echn iques have not def ined the p r ec i s e n a t u r e of the molecu la r change in t h e m e m b r a n e lipids which is respons ib le for t he change in e n z y m e Ε . Al though a change in molecu la r order ing of m e m b r a n e lipid is observed a t the c r i t i c a l t e m p e r a t u r e for chi l l ing, i t s r e l a t ionsh ip to such p rocesses as lipid phase t r ans i t ions and /o r lipid phase s epa ra t i ons , which have been i m p l i c a t e d as in i t ia l even t s in the phenomenon of chil l ing injury (7, 13) is no t c l ea r .

In addi t ion to d i f fe rences in t h e t h e r m a l behaviour of mi tochondr ia l m e m b r a n e s from chi l l ing-sens i t ive and ch i l l ing- res i s t an t p l an t s , it is possible t h a t o t h e r ce l lu la r m e m b r a n e s undergo changes of a s imi lar n a t u r e . In this r e g a r d changes in Ε h a v e been r e p o r t e d for some m e m b r a n e - a s s o c i a t e d p h o t o s y n t h e t i c a c t i v i t i e s of ch lo rop las t s i so la ted from chi l l ing-sens i t ive p l an t s (17). D i f f e r ences in t he t h e r m a l behaviour of p l a s m a m e m b r a n e s from chi l l ing-sens i t ive and ch i l l ing- res i s tan t p l an t s h a v e not been r e p o r t e d , a l though changes in t h e ion p e r m e a b i l i t y of some p lan t t i ssues subjec t to chil l ing t e m p e r a t u r e s , have been

Copyright · 1979 by Academic Press, inc. 163 All rights of reproduction in any form reserved

ISBN 012-460560-5

164 Ε. J. M c M u r c h i e

observed (2, 19). Such changes in ion p e r m e a b i l i t y may r e f l e c t changes in the t e m p e r a t u r e sens i t iv i ty of i on - s t imu la t ed ATPases a s soc ia t ed wi th t h e p l a sma m e m b r a n e , as these ATPases h a v e been i m p l i c a t e d in t h e ion t r anspo r t p rocess (3, 4 , 18). Thus t e m p e r a t u r e - i n d u c e d changes in the p l a sma m e m b r a n e and a s soc ia t ed enzymes may also play a ro le in chil l ing sens i t iv i ty .

In this s tudy t h e t e m p e r a t u r e sens i t iv i ty of i o n - s t i m u l a t e d ATPase was examined using a pos t -mi tochondr i a l m e m b r a n e p r e p a r a t i o n i so la ted from t issues of chi l l ing-sens i t ive t o m a t o and cucumber and from ch i l l ing- res i s tan t caul i f lower . Changes w e r e observed in t h e Ε of ion-s t i m u l a t e d ATPase which may ind i ca t e t ha t the p l a sma m e m b r a n e undergoes changes which a r e s imi lar to those observed for mi tochondr ia l m e m b r a n e s . As a means of co r r e l a t i ng t he se changes in e n z y m e Ε with the possible p r e s e n c e of phase t r ans i t ions in the m e m b r a n e , m e m b r a n e s and m e m b r a n e lipid e x t r a c t s w e r e examined by d i f fe ren t ia l scanning c a l o r i m e t r y .


A. Membrane Isolation

Green t o m a t o fruit (Lycopersicon escalentumfvar. Red China) , caul i f lower f lo re t s (Brassica oleraceae) and cucumber fruit (Cucumis sativus) w e r e ob ta ined from c o m m e r c i a l sources . For t o m a t o and cucumber , only t h e p e r i c a r p t i ssue was used. Tissues w e r e homegen i zed in a c o m m e r c i a l j u i ce e x t r a c t o r using an i ce -co ld medium of 300 mM sucrose , 0 .5% (w/v) polyvinylpyrol idone, 1 m g / m l bovine se rum albumin, 3 mM EGTA, 25 mM Tris , 2 mM Hepes , 4 mM d i th io th re i t o l , all adjus ted to pH 7.4 wi th HC1. A solut ionr t i ssue r a t i o of 2:1 (v/w) was used. The bre i was ma in t a ined a t pH 7.2 wi th NaOH during t h e e x t r a c t i o n . The bre i was then s t r a ined through 4 l aye r s of cheesec lo th and cen t r i fuged a t 13,000 g for 15 min. The s u p e r n a t a n t was then poured th rough 4 l aye r s of cheesec lo th and cen t r i fuged a t 135,000 g for 30 min. The pe l l e t was washed t w i c e in TM buffer (25 mM T r i s ' H C l , 1.5 mM M g S 0 4, pH 7.5) by resuspens ion and cen t r i fuga t ion a t 135,000 g for 30 min. The final pe l l e t was resuspended in TM buffer a t a c o n c e n t r a t i o n of 3 to 6 mg p ro te in pe r ml . This m a t e r i a l was used i m m e d i a t e l y for ATPase assays .

B. Assays

1. ATPase a c t i v i t y was measu red in a 1.0 ml J i n a l vo lume conta in ing 25 mM Tr is -HCl , 1.5 mM M g S 0 4, pH 8.0 (Mg

+A T P a s e ) or

t h e s ame buffer conta in ing 40 mM KCf adjus ted to pH 8.0 (K s t i m u l a t e d Mg ATPase ) . The r e a c t i o n con ta ined 300 to 600 y g m e m b rane p ro te in and was s t a r t e d by the addi t ion of T r i s

eA T P at a final

T e m p e r a t u r e Sens i t iv i ty of Ion S t i m u l a t e d A T P a s e s 165

c o n c e n t r a t i o n of 3.0 mM. The r e a c t i o n t i m e for any se t of assays was chosen so tha t no more than 20% of the s u b s t r a t e was c o n v e r t e d to inorganic phospha t e during t h e course of t h e assay . The r e a c t i o n was t e r m i n a t e d by t h e addi t ion of 50 μ 1 of 70% (v/v) pe rch lo r i c acid and inorganic phospha te was d e t e r m i n e d using t h e p rocedu re +of Rosen tha l and Matheson (15).^+The t e m p e r a t u r e sens i t iv i ty of Mg ATPase and Κ s t i m u l a t e d Mg ATPase was d e t e r m i n e d using a shaking t h e r m o g r a d i e n t a luminum block capab le of ma in ta in ing 14 s e p a r a t e assay t e m p e r a t u r e s a t any a c c u r a c y of ± 0.Z C over the e x p e r i m e n t a l per iod . Both e n z y m e a c t i v i t i e s w e r e d e t e r m i n e d s imul taneous ly using dup l i ca t e assays . Assays con ta ined iden t i ca l a m o u n t s of m e m b r a n e p ro t e in from the s a m e m e m b r a n e p r e p a r a t i o n . Final r a t e s of ATPase a c t i v i t y have been c o r r e c t e d for possible e f f e c t s of t e m p e r a t u r e on the blank va lue and the r a t e of non -enzymic ATP hydrolys is .

2. M e m b r a n e p ro te in was d e t e r m i n e d by t h e m e t h o d of Lowry et al, (6).

C. Lipid Extraction

M e m b r a n e p r e p a r a t i o n s suspended in TM buffer w e r e e x t r a c t e d overnight wi th 21 vol. ch lo ro fo rmrmethano l (2:1) con ta in ing 1 mg b u t y l a t e d hydroxy to luene as an t iox idan t . The e x t r a c t was then p a r t i t i o n e d aga ins t 0.2 vol . of 0 .73% (w/v) NaCl and the aqueous layer r e m o v e d . The lipid e x t r a c t was dr ied , t a k e n up in chloroform and s to r ed a t - 2 0 ° C .

D. Electron Microscopy

M e m b r a n e p r e p a r a t i o n s in TM buffer w e r e cen t r i fuged (135,000 g, 30 min) and r e suspended in 50 mM potass ium phospha t e buffer , pH 7.5 conta in ing 4 % (v/v) g l u t a r a l d e h y d e . Af te r 30 min the m e m b r a n e s w e r e cen t r i fuged as above and s ec t i oned spec imens w e r e p r e p a r e d by s t anda rd p r o c e d u r e s .

E. Differential Scanning Calorimetry

Dif fe ren t i a l scanning c a l o r i m e t r y (DSC) was p e r f o r m e d using a Pe rk in -E lme r DSC-2 c a l o r i m e t e r . M e m b r a n e p r e p a r a t i o n s in TM buffer w e r e cen t r i fuged a t 135,000 g for 30 min and the pe l l e t was loaded in to a 20 μΐ c a p a c i t y gold pan and then sea led . M e m b r a n e lipid samples in chloroform w e r e dr ied and then suspended in 25 mM T r i s ' H C l , 10 mM EDTA, pH 8.0 by vor t ex ing followed by a 2 min sonica t ion per iod . The lipid suspension was cen t r i fuged and loaded in to t h e pan as desc r ibed for the m e m b r a n e samples . Dry weigh t s of the samples w e r e d e t e r m i n e d a t


t he comple t ion of t he e x p e r i m e n t . Scans w e r e p e r f o r m e d a t a r a t e of 10°C/min with an i n s t rumen t ope ra t ing sens i t iv i ty of O.Z m e a l / s e c . Trans i t ion en tha lp ies we re d e t e r m i n e d from the a r e a s of the e n d o t h e r m i c or e x o t h e r m i c peaks using Indium as a s t anda rd .

III. RESULTS

A. Ion-Stimulated ATPase Activity

The p rocedu re used for m e m b r a n e isola t ion y ie lded m e m b r a n e ves ic les from all t h r e e t issues which w e r e s imi lar in a p p e a r a n c e to those shown for cauliflower (Figure 1). These m e m b r a n e p r e p a r a t i o n s con ta ined Mg dependen t ATPase a c t i v i t y which could be fur ther s t i m u l a t e d by monova len t ca t ions . ATPase a c t i v i t y could be a lmos t comple t e ly inhib i ted by Ag ions and p r o t e c t e d aga ins t such inhibi t ion by d i t h io th re i t o l . These p r o p e r t i e s a r e all cons i s t en t wi th t h e known p rope r t i e s of p l a sma m e m b r a n e - a s s o c i a t e d ATPases (3). As well as conta in ing p la sma m e m b r a n e s , t h e m e m b r a n e p r e p a r a t i o n would be e x p e c t e d to conta in m e m b r a n e f r a g m e n t s de r ived from t h e tonoplas t and endoplasmic r e t i c u l u m . As an e s t i m a t e of t h e level of mi tochondr ia l ATPase , t he t o t a l ATPase ac t i v i t y of t h e m e m b r a n e p r e p a r a t i o n was inhib i ted by no m o r e than 15% in t h e p r e s e n c e of 50 y g / m l ol igomycin (resul ts not shown). _ ^

2+ + 2+ Arrhenius p lo t s of Mg ATPase and Κ s t i m u l a t e d Mg ATPase ac t i v i t y from t o m a t o a r e shown in F igure 2. Both p lo ts exhib i ted a d i scont inu i ty a t about 20 C and they r e m a i n e d non- l inear below this t e m p e r a t u r e . However , no c lea r ass ignment of a c r i t i c a l t e m p e r a t u r e a t about 10 C, which would be in a g r e e m e n t wi th t h e c r i t i ca l t e m p e r a t u r e observed for mi tochondr ia l r e s p i r a t o r y a c t i v i t y of t o m a t o (8), could be m a d e from e i the r p lo t . 2 + + ^ +

Arrhenius p lo t s of Mg ATPase and Κ s t i m u l a t e d Mg ATPase a c t i v i t y from cucumber each exhib i ted a d i scont inu i ty a t about 10°C (Figure 3). This t e m p e r a t u r e is in a g r e e m e n t wi th the t e m p e r a t u r e a t which changes in m e m b r a n e - a s s o c i a t e d r e s p i r a t o r y a c t i v i t y a r e observed in cucumber mi tochondr i a (8). +T h e d i scont inu i ty in t he Arrhen ius plot was less p ronounced for the Κ s t i m u l a t e d ATPase which may ind i ca t e t ha t this e n z y m e is less sens i t ive to low t e m p e r a t u r e s when assayed in t he p r e s e n c e of Κ .

It has previously been shown tha t for mi tochondr i a i so la ted from chi l l ing- res i s tan t p l an t s , m e m b r a n e - a s s o c i a t e d r e s p i r a t o r y a c t i v i t i e s do not undergo abrupt changes in Ε in t h e t e m p e r a t u r e r a n g e from 0 to 25 C (8). However i on - s t imu la t ed ATPase a c t i v i t y a s soc i a t ed wi th m e m b r a n e s i so la ted from the ch i l l ing- res i s tan t caul i f lower did no t display l inear Arrhenius k ine t i c s , i .e . , a cons t an t Ε when assayed over t h e r ange 3 to 28°C (Figure 4). The Arrhenius p l o t ^ f Mg

+A T P a s e was

t r iphas ic wi th d iscont inu i t ies occur r ing a t about 8° and 15°C. In


FIGURE 1. Electron micrograph of a section of a pelleted membrane fraction isolated from cauliflower buds. (Electronmicrograph, courtesy of Dr. J. Bain, CSIRO Division of Food Research).


2_FIGURE 2. Arrhenius plots of Mg ATPase and Κ stimulated Mg ATPase activities associated with membranes isolated from green tomato fruit.

c o n t r a s t , only on,e d iscont inui ty a t about 15°C was observed for t he Κ s t imulated Mg ATPase a c t i v i t y . At a l H e m p e r a t u r e s , t he sens i t iv i ty of t he Mg ATPase to s t imula t ion by Κ was far g r e a t e r than t ha t observed for e i the r the t o m a t o or the cucumber A T P a s e s . In addi t ion , t he sens i t iv i ty of t h e caul i f lower enzyme to t e m p e r a t u r e s below 8 C was g r ea t l y d e c r e a s e d when the enzyme was assayed in t h e p r e s e n c e of Κ (Figure 4) or Rb (resul ts not shown).

The d a t a for caul i f lower and t o m a t o ATPases was r e p l o t t e d as t h e Κ dependen t ATPase ac t iv i ty , +which r e p r e s e n t s tlê d i f f e rence in r a t e b e t w e e n t h e Κ ^s t imulated Mg ATPase and t h e Mg A T P a s e . Arrhenius p lo t s of t h e Κ dependen t ATPase a c t i v i t y for t o m a t o and caul i f lower m e m b r a n e s a r e shown in F igure 5. Apar t from the 10-fold d i f f e rence in t h e specif ic a c t i v i t y of t h e r e s p e c t i v e ATPases , t h e Arrhenius p lo t s a r e r e m a r k a b l y s imi la r . Both p lo ts exhib i ted a d i scont inu i ty a t a s imi lar t e m p e r a t u r e and the values of t he Arrhenius a c t i v a t i o n energy when c o m p a r e d e i the r above or below the d iscont inu i ty w e r e a lmos t i den t i ca l .

B. Differential Scanning Calorirnetry

The t h e r m a l behaviour of m e m b r a n e s and m e m b r a n e lipids from t o m a t o , cucumber and caul i f lower was i nves t i ga t ed using d i f fe ren t ia l

T e m p e r a t u r e Sens i t iv i ty of I o n - S t i m u l a t e d A T P a s e s 169

gflGURE 3. Arrhenius plots of Mg ATPase and Κ stimulated Mg ATPase activities associated with membranes isolated from cucumber. Error bars represent the limits obtained using duplicate ATPase assays.

scanning c a l o r i m e t r y (DSC). This t echn ique has been used to d e t e c t t h e r m o t r o p i c lipid phase t r ans i t ions in a v a r i e t y of biological m e m b r a n e s (11).

Lipids i so la t ed from t o m a t o m e m b r a n e p r e p a r a t i o n s exh ib i t ed a r eve r s ib l e phase t r ans i t ion as shown in F igure 6. The t e m p e r a t u r e l imi t s of t h e t r ans i t ion d i f fered depending on t h e scanning mo d e . However , t he en tha lp ies of the t r ans i t ions ob ta ined using e i t he r mode w e r e a p p r o x i m a t e l y t h e s ame (Table I). No t r ans i t ion was d e t e c t e d in t he t o t a l m e m b r a n e lipids of caul i f lower over t h e t e m p e r a t u r e r a n g e 52 to - 5 C, (DSC scan not shown). For m e m b r a n e s i so la ted from caul i f lower and cucumber a smal l e x o t h e r m i c t r ans i t ion was ev ident by DSC in bo th m e m b r a n e p r e p a r a t i o n s (Figure 7). These t r ans i t ions w e r e s imi lar both in r e g a r d to the i r t e m p e r a t u r e l imi t s and the i r t r ans i t ion en tha lpy (Table I). No t r ans i t ion was d e t e c t e d in t h e m e m b r a n e p r e p a r a t i o n s from t o m a t o when examined under s imi lar condi t ions , (DSC scan no t shown).

The t h e r m a l behaviour of m e m b r a n e s from chi l l ing-sens i t ive t i ssues ο could be a l t e r e d by hea t i ng t h e m e m b r a n e sample a t 60 C for 3 min as shown in F igure 8. For c u c u m b e r m e m b r a n e p r e p a r a t i o n s , hea t i ng t h e


TABLE I. DSC Analysis of Cauliflower, Cucumber and Tomato Membrane Preparations.

Membrane preparation Transition range (°C) Δ Η (cal/gm lipid)

Cauliflower 9.0° to 2.0° (exotherm) 0.04 " 9.1° to 18.8° (endotherm) 0.04 " (denatured) N.D. " (total lipids) N.D.

Cucumber 10.5° to 0.4° (exotherm) 0.04 " (denatured) 21.3° to 0° (exotherm) 0.47

Tomato N.D. " (denatured) 27.9 to 0° (exotherm) 0.66 " (total lipids) 12.7° to -6.1° (exotherm) 0.15 " (total lipids) 3.2 to 22.4° (endotherm) 0.13

N.D. - Transition not detected.

sample r e su l t ed in an a p p r o x i m a t e doubling of t h e t e m p e r a t u r e r ange of the t r ans i t ion and a ten-fo ld inc rease in the en tha lpy of the t r ans i t ion (Table I). Whereas no t r ans i t ion was observed for t o m a t o m e m b r a n e s before hea t ing , a t r ans i t ion of s imilar en tha lpy to t ha t of the h e a t e d cucumber m e m b r a n e s , was d e t e c t e d a f t e r hea t ing (Table I). For t he t o m a t o , the r ange of the t r ans i t ion in the h e a t e d m e m b r a n e s exceeded t ha t observed for t he corresponding m e m b r a n e lipids and t h e t e m p e r a t u r e l imi t s of the t r ans i t ion were c o m p a r a t i v e l y h igher . In c o n t r a s t to the m e m b r a n e s from c u c u m b e r and t o m a t o , caul i f lower m e m b r a n e s did no t exhibi t a t r ans i t ion a f t e r h e a t i n g (Figure 8). Indeed the small t r ans i t ion evident be fore d e n a t u r a t i o n (Figure 7), was no longer d e t e c t a b l e .

IV. DISCUSSION

The t empera tu reôbse rved for the change in t he E & for Mg ATPase and Κ s t i m u l a t e d Mg ATPase a c t i v i t i e s i so la ted from cucumber a r e in c lose a g r e e m e n t with t he c r i t i ca l t e m p e r a t u r e for chill ing injury in this t i s sue . Such a g r e e m e n t is not observed for t he t o m a t o when the ATPase


2+ + 2fIGURE 4. Arrhenius plots of Mg ATPase and Κ stimulated

Mg ATPase activities associated with membranes isolated from cauliflower buds. Error bars are as described in Figure 3.

a c t i v i t i e s a r e p r e s e n t e d as Arrhenius p lo t s . However , when t h e d a t a is p r e s e n t e d in the forjn shown in F igure 5 (i .e. , as the d i f f e rence in the a c t i v i t y of t h e Mg ATPase due to t h e p r e s e n c e or absence of Κ ), changes in the t o m a t o ATPase a r e observed a t t e m p e r a t u r e s which a p p r o x i m a t e t h e c r i t i c a l t e m p e r a t u r e for chil l ing. It is l ikely t h a t t h e e f fec t of t e m p e r a t u r e on the ion s t imu la t i on of the ATPase may be an i m p o r t a n t physiological f ac to r to consider p a r t i c u l a r l y in r e g a r d to those ion t r anspor t p rocesses such as Κ u p t a k e , which a r e probably m e d i a t e d by m e m b r a n e - a s s o c i a t e d ATPases (3).

1 7 2 Ε. J. M c M u r c h i e

0-3H

0 ^

Ό)

ε

Q l" 0-1 -(Λ α> ο

• (Λ α> ο 0 0 8 -Ε a . 0 0 6 -> 4 -> Ι Ο 3 -

0) (Λ (Ό

Ω.

( 1 5) T O M A T O

• —

Ν . ( 7 5 )

I I 1

C A U L I F L O W ER (13 ) ^ ·

Λ . ( 7 7 )

ο ο ο Ο ο ° ~ 3 0 2 5 2 0 15 10 5 C

I Ι Ι . ι . 1 1. I 3 3 3 4 3 5 3 6

1 0 4/ Κ

FIGURE 5. Arrhenius plots of the Κ dependent ATPase activity associated wi^th membranes isolated from tomato fruit and cauliflower buds. The Κ dependent ATPase activity is the difference between the-Mg * ATPase activities measured in the presence and absence of 40 m Μ KCl. Numbers in brackets are the Arrhenius activation energy (k J/mole) for each linear segment of the Arrhenius plot.

Unlike t he s i tua t ion observed for mi tochondr ia l r e s p i r a t o r y a c t i v i t y in chi l l ing-sens i t ive and ch i l l ing- res i s tan t p l an t s (13), t he changes in the t e m p e r a t u r e sens i t iv i ty of i on - s t imu la t ed ATPases a r e not confined solely to the chi l l ing-sens i t ive p l an t s , but a r e observed in the chi l l ing-resistant caul i f lower . For t h e ^ caul i f lower Arrhenius p lo t s of Mg ATPase and Κ s t i m u l a t e d Mg ATPase a r e non l inear , and the Arrhenius plot of t he Κ dependen t ATPase a c t i v i t y (Figure 5) is r e m a r k a b l y s imilar to t ha t ob ta ined for t he t o m a t o . The Κ dependen t ATPase may be r e f l e c t i ng t ha t componen t of the e n z y m e a c t i v i t y which


FIGURE 6. DSC scans of a total lipid extract from tomato membrane preparations. Endo and exo refer to the endothermic or exothermic peaks obtained from heating or cooling scans respectively.

end o I

t HEAT FLOW

Cauliflower

00 1 mcal/sec l

1 ex o

Cucumbe r

2 0 10 0 TEMPERATURE (° C )

- 1 0

FIGURE 7. DSC cooling scans obtained for membrane preparation from cauliflower and cucumber.


HEAT FLOW

end o

ex o Cucumbe r

3 0 2 0 1 0 TEMPERATURE ( ° C )

0 - 1 0

FIGURE 8. DSC cooling scans obtained for heat-denatured membranes from cauliflower, tomato and cucumber. Membranes were heat-denatured at 60 C for 3 min.

is involved in t h e a c t i v e t r anspo r t of KT ions ac ross t he p l a sma

m e m b r a n e . If this is so, changes in t e m p e r a t u r e may a f f ec t ion t r anspo r t in some chi l l ing-sens i t ive and ch i l l ing- res i s tan t p l an t s in a s imilar manne r . Indeed t h e observa t ion t h a t Rb u p t a k e in to roo t t i s sue undergoes an i nc r ea se in Ε a t t e m p e r a t u r e s below about 1Z C in both corn (chil l ing-sensi t ive) and* bar ley (ch i l l ing- res is tan t ) , (1) is cons i s ten t wi th this proposa l .

The t h e r m a l response of m e m b r a n e p r e p a r a t i o n s from t o m a t o and cucumber w e r e d i f fe ren t to those of caul i f lower , when examined by DSC, but only under c e r t a i n condi t ions . This may r e l a t e to d i f fe rences in t h e chil l ing sens i t iv i ty b e t w e e n these two groups of p l a n t s . Phase t r ans i t ions w e r e only observed in t h e e x t r a c t e d m e m b r a n e lipids of the chi l l ing-sens i t ive t o m a t o and cucumber , and these t r ans i t ions occu r r ed within the chill ing t e m p e r a t u r e r ange for these t i ssues . However , t h e values ob ta ined for the en tha lpy of the t r ans i t ions i nd ica t ed t ha t less than 10% of t he m e m b r a n e lipids w e r e ac tua l ly involved in t h e t r ans i t ion . F u r t h e r m o r e , the t r ans i t ions were g r ea t l y enhanced in the n a t i v e m e m b r a n e s when the samples w e r e h e a t e d a t 60 C for 3 min. This sugges ts tha t a l though some of the m e m b r a n e lipids of ch i l l ing-sens i t ive p l an t s a r e capab le of undergoing a phase t r ans i t ion , some form of assoc ia t ion b e t w e e n the lipids and the in t r ins ic m e m b r a n e p ro te ins may p r e v e n t much of t h e lipid from undergoing a phase t r ans i t ion . Lipid-p ro te in i n t e r a c t i o n s which could accoun t for t he above s i tua t ion have

T e m p e r a t u r e Sens i t iv i ty of I o n - S t i m u l a t e d A T P a s e s 175

been observed in r e c o n s t i t u t i o n s tud ies (5). In t he se s tudies it has been d e m o n s t r a t e d t ha t lipids can b e c o m e s e q u e s t e r e d around m e m b r a n e p ro te ins to form an immobi l ized , d i so rdered boundary l ayer , which might no t undergo a phase t r ans i t i on . P ro t e in d e n a t u r a t i o n might abolish this i n t e r a c t i o n , and thus allow t h e bulk of t h e lipids to undergo a phase t r ans i t ion .

In compar i son to t h e r e l a t i ve ly g r e a t e r t r ans i t ions observed a f t e r h e a t i n g , the t r ans i t ions which o c c u r r e d in u n h e a t e d caul i f lower and cucumber m e m b r a n e s had an e x t r e m e l y low en tha lpy va lue and involved no more than 1% of- the m e m b r a n e l ipids . As these t r ans i t ions w e r e observed in t he m e m b r a n e s of bo th types of p l an t s , thei r possible ro le in t h e chil l ing r eponse is u n c e r t a i n .

It is c l ea r from the p r e s e n c e of e x o t h e r m i c / e n d o t h e r m i c t r ans i t ions t ha t some of the m e m b r a n e lipids of ch i l l ing-sens i t ive p l an t s a r e capab le of forming a gel or c rys ta l l ine phase within t he r a n g e of t e m p e r a t u r e s a t which chil l ing injury is obse rved . The p ropor t ion of ge l -phase lipid which ex is t s a t t he c r i t i ca l chill ing t e m p e r a t u r e , may be smal l in p ropor t ion to the amount of lipid in the fluid or l iqu id-c rys ta l l ine p h a s e . However , the p r e s e n c e of some ge l -phase lipid in t he m e m b r a n e may be suff ic ient to cause a l a t e r a l s epa ra t ion of the two lipid phases wi thin the m e m b r a n e , and lead to changes in m e m b r a n e funct ion . The p r e s e n c e of ge l -phase lipid a t physiological t e m p e r a t u r e s has been observed in mic rosomal m e m b r a n e s of bean co ty ledons by X- ray d i f f rac t ion s tudies (9). In these m e m b r a n e s t he amoun t of ge l -phase lipid was inf luenced by the p r e s e n c e of bo th p ro te in and n e u t r a l lipid (10). Whereas ge l -phase lipid was not d e t e c t e d in the i so la ted phospholipids of bean co ty ledon mic rosomal m e m b r a n e s a t t e m p e r a t u r e s g r e a t e r than 5 C, t h e addi t ion of e x t r a c t e d n e u t r a l lipid was able to induce the fo rmat ion of ge l -phase lipid in the i so la ted phospholipids up to t e m p e r a t u r e s of 40°C (10). In t h e p r e sen t s tudy, as bo th n e u t r a l lipids and phospholipids w e r e p r e s e n t t o g e t h e r in t h e m e m b r a n e lipid e x t r a c t s examined by DSC, some form of l ipid-lipid i n t e r a c t i o n , s imi lar to t ha t desc r ibed above , may c o n t r i b u t e to the phase t r ans i t ion (and ge l -phase lipid), which ex tends in to t h e chill ing t e m p e r a t u r e r a n g e . F u r t h e r m o r e , the absence of phase t r ans i t ions in m e m b r a n e s from ch i l l ing- res i s tan t p l an t s may be t he resu l t of a changed n e u t r a l l ipid-phospholipid i n t e r a c t i o n , r a t h e r than to a change pecu l ia r to t h e phospholipids .

No d i f f e rence in t he t h e r m a l behaviour of t h e u n t r e a t e d m e m b r a n e s from the two groups of p l an t s was observed by DSC. Indeed the smal l t r ans i t ion which involved less than 1% of t h e lipid was not observed with m e m b r a n e s p r e p a r e d from t o m a t o . As t r ans i t ions we re only c lea r ly seen a f t e r h e a t i n g of t h e m e m b r a n e s or in t h e e x t r a c t e d m e m b r a n e l ipids, it follows t ha t t h e change in t he of m e m b r a n e - a s s o c i a t e d i on - s t imu la t ed ATPase which was observed in u n t r e a t e d m e m b r a n e s from both groups of p l an t s , is probably no t d i r ec t ly r e l a t e d to t h e p r e s e n c e of phase t r ans i t ions in the bulk m e m b r a n e l ipids. In this r e g a r d a number of o the r m e m b r a n e - a s s o c i a t e d enzymes have been shown to undergo changes in Ε which a r e independen t of changes occur r ing in t h e surrounding m e m b r a n e lipids (16).



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(1977). 10. McKers ie , B. D. , and Thompson, J . E. Biochim. Biophys. Acta. 550,

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205-238 (1976). 12. P ike , C , Raison, J . K., and Berry , J . This volume (1979). 13. Raison , J . K. In "Mechanisms of Regu la t ion of P l an t Growth . " (R.

L. Bieleski , A. R. Ferguson , and M. Cresswel l , eds.) Ball. 12, pp . 487-497. The Royal Socie ty of New Zea land (1974).

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Acta 292, 152-161 (1973). 18. Sze, H., and Hodges , Τ. K. Plant Physiol. 59, 641-646 (1977). 19. Wheaton , Τ. Α., and Morr is , L. L. Proc. Am. Soc. Hort. Sci. 91,

529-533 (1967).


MEMRANE LIPID TRANSITIONS: THEIR CORRELATION WITH THE CLIMATIC DISTRIBUTION OF PLANTS

John K. Raison, Elza A. Chapman and Lesley C. Wright


Macquar i e Univers i ty Nor th Ryde , N.S.W. 2113, Aus t ra l i a

S. W. L. Jacobs

Royal Botan ic Ga rdens Sydney, N.S.W. 2000, Aus t r a l i a

I. INTRODUCTION

For sens i t ive p lan t s a very good co r r e l a t i on has been es tab l i shed b e t w e e n physiological d isorders which occur a t chil l ing t e m p e r a t u r e s and changes in both t h e s t r u c t u r e and funct ion of mi tochondr ia l and ch loroplas t m e m b r a n e s (4, 8). This has led to the hypothes i s tha t a t e m p e r a t u r e - i n d u c e d a l t e r a t i o n in t h e molecu la r order ing of m e m b r a n e lipids is the p r i m a r y even t in chil l ing injury (4, 8). The m e t a b o l i c d i sorders , such as the i m b a l a n c e b e t w e e n r e sp i r a t ion and glycolysis (13), which a r e observed when sens i t ive p lan t s a r e exposed to chil l ing t e m p e r a t u r e s a r e cons idered secondary even t s following the p r i m a r y change in m e m b r a n e lipid s t r u c t u r e . The magn i tude of these secondary e v e n t s depends on both t he age of t h e p lan t , t he t i s sue , t h e d e g r e e of chil l ing and the t i m e of exposure (4).

The c r i t i ca l t e m p e r a t u r e for t h e p r i m a r y change in m e m b r a n e lipid s t r u c t u r e has been r e f e r r e d to as Τ , b e c a u s e , by analogy wi th pure phosphol ipids , it was thought to r e p r e s e n t the t e m p e r a t u r e for the comple t ion of t h e l iqu id-c rys ta l l ine to gel t r ans i t ion (9). T^ was cons idered the t e m p e r a t u r e for t he in i t i a t ion of this t r ans i t i on . However from d a t a using t h e change in f luo rescence of pa r ina r i c ac id (Figure 7 and P ike et al. this volume) , Τ is m o r e l ikely t he t e m p e r a t u r e of t he in i t i a t ion of this t r ans i t ion , if a me l t ing t r ans i t ion is involved a t a l l . T g

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178 J. Κ. R a i s o n et al.

has in most i n s t ances been d e t e r m i n e d as t he point a t which t h e r e is a change in the t e m p e r a t u r e coef f ic ien t of mot ion of a spin label i n t e r c a l a t e d in to e i t he r m e m b r a n e lipids or in to ves ic les m a d e from m e m b r a n e polar l ipids. Τ can also be d e t e c t e d as a change in t h e Arrhenius ac t i va t i on energy (Ea) of some m e m b r a n e - a s s o c i a t e d e n z y m e s such as s u c c i n a t e oxidase of mi tochondr ia (5, 6) and pho to reduc t ion of NADP by ch lorop las t s (1Z). All ch i l l ing-sens i t ive p lan t s examined by t he se me thods show the changes in m e m b r a n e s t r u c t u r e and funct ion in t h e t e m p e r a t u r e r ange of about 9 to 15 C. For ch i l l ing- res i s tan t p l an t s no such changes a r e observed in t he t e m p e r a t u r e r ange 0 to 25 C but a s t r u c t u r a l change occurs a t or below 0 C (10). This c o r r e l a t i o n b e t w e e n the t e m p e r a t u r e response of m e m b r a n e lipids and the abi l i ty of p l an t s to w i th s t and chill ing has been observed mainly wi th c e r e a l and v e g e t a b l e c rops . It is possible t ha t s e l ec t i ve b reed ing of t hese crops might have favoured deve lopmen t of a m e m b r a n e with specif ic physical p r o p e r t i e s favourab le to a p a r t i c u l a r env i ronmen t . To d e t e r m i n e w h e t h e r the s a m e co r r e l a t i on exis ts in n a t i v e p lant spec ies , whe re n a t u r a l s e l ec t ion would d o m i n a t e , values for T g and T^ have been ob ta ined for the leaf polar lipids of p l an t s from d i f fe ren t c l ima t i c r eg ions . The r e su l t s show tha t in genera l Τ is r e l a t ive ly high (9 to 17 C) for p l an t s from t rop ica l reg ions and low for w in te r growing p lan t s from t e m p e r a t e r eg ions . Thus a good co r re l a t ion exis ts b e t w e e n Τ and the c l i m a t e of t h e p lan t ' s h a b i t a t .

II. METHODS

Leaves we re r e m o v e d from growing p lan t s , f rozen i m m e d i a t e l y in liquid n i t rogen and kep t f rozen unt i l e x t r a c t e d wi th ch lo ro fo rm:methano l as desc r ibed (7). The polar lipids w e r e s e p a r a t e d and i so la ted by c h r o m a t o g r a p h y on ac id -washed florisi l . The polar lipids were d ispersed in 0.1 Μ t r i s - a c e t a t e buffer , pH 7.Ζ conta in ing 5 mM EDTA a t a c o n c e n t r a t i o n of 15 mg l ip ids /ml . The t e m p e r a t u r e s of Τ and T^ w e r e usually d e t e r m i n e d by i n t e r c a l a t i n g a spin label in to the lipid dispersion and measur ing t h e r o t a t i o n a l co r r e l a t i on t i m e τ as previously descr ibed (3). The spin labels used we re t he me thy l e s t e r s of 1Z- and 16-n i t rox ide s t e a r a t e (lZNSMe and 16NSMe), 5- and 9-ni t roxide s t e a r i c acid (5NS and 9NS), 11-ni t roxide hene icosanane (11NZ1) and 5-ni t roxide d e c a n e (5N10).

m. RESULTS

Before descr ib ing the r e su l t s of t h e survey of n a t i v e p l an t s it is i m p o r t a n t to emphas i ze t he r a t i o n a l e under lying t h e use of Arrhen ius p lo t s of e n z y m e ac t i v i t y and the spin- label l ing me thods for de t e rmin ing Τ .

s

M e m b r a n e Lipid T r a n s i t i o n s 179

FIGURE 1. Changes in the succinate oxidase activity of maize root mitochondria as a function of temperature. The mitochondria were isolated from 6 day-old roots by the method described (9). Measurements were made on four preparations of mitochondria each at six different temperatures. The composite plot was made by normalizing the rates of the preparations at 25°C. The straight lines were fitted by regression analysis (9). The Ea above Tf (27°C) is 1 kcal/molef above Ts(12°C) 10 kcal/mole and below Τ 25 kcal/mole.

ο

For example , F igure 1 shows the change in r a t e of succ ina t e -ox idase a c t i v i t y of mi tochondr i a from m a i z e - r o o t t i ssue as a funct ion of t e m p e r a t u r e . Since t h e r e a r e abrupt v e r t i c a l d i s p l a c e m e n t s in r a t e s a t about 27 C and 12 C t h e d a t a is bes t desc r ibed by t h r e e , s t r a igh t l ines , which d e l i n e a t e t h r e e d i f fe ren t Ea va lues for this r e a c t i o n . As shown in F igure 2, changes in t he t e m p e r a t u r e coef f ic ien t of mot ion occur a t two t e m p e r a t u r e s ; a t 27 C d e t e c t e d by 12NSMe and a t 12 C d e t e c t e d by 16NSME. The t e m p e r a t u r e s ob ta ined for T^ and T g using t h e p lo t s of e n z y m e a c t i v i t y and f i t t ing s t r a igh t l ines a lways co inc ide with those ob ta ined from spin- label mot ion even when t h e r e is no ind ica t ion of non-i n t e r s e c t i n g d i scon t inu i t i es in t h e ox ida t ive p lo t s . This " s t ra igh t - l ine" app roach for r e p r e s e n t i n g t h e change in t h e r a t e of an e n z y m e r e a c t i o n wi th t e m p e r a t u r e thus provides a c o m p a r a t i v e m e a s u r e of t he t e m p e r a t u r e s a t which s t r u c t u r a l and funct ional changes occur and al lows the fo rmula t ion of a working hypothes i s to desc r ibe chill ing injury in molecu la r t e r m s . While it might be s t a t i s t i c a l l y m o r e c o r r e c t to fit polynomial curves to t h e po in t s in F igures 1 and 2 as Bagnall and Wolfe (1) did wi th the i r r e su l t s , it does no t p rov ide in fo rmat ion of a s s i s t ance in explaining chil l ing injury a t t he molecu la r l eve l . D a t a r e l a t i ng t h e t e m p e r a t u r e response of t h e secondary e v e n t s which follow from t h e p r imary even t might be m o r e a c c u r a t e l y desc r ibed in t e r m s of curves and


TABLE I. The Temperatures Γ § and Tf of Leaf Polar Lipids for Native Plants Sampled in their Natural Habitat and for Crop Plants Grown under Glasshouse Conditions.

Region Name Ts

(°C)

Tf

(°C)

TEMPERATE Hordeum vulgare -1 29 (Winter Triticum aestivum 0 30

grown) Atriplex nummularia 1 28 Atriplex vesicaria 1 28 Beta vulgaris 0 30 Rhagodia spinescens 0 27 Suaeda australis 1 32 Pisum sativum 0 26 Trifolium repens 2 30 Solanum tuberosum 0 30 Helianthus tuberosus (dormant tubers) -5 9

TEMPERATE Triodia irritans 1 30 ( Summer Zostera capricorni (A) 15 28

grown) Zoster a capricorni (B) 9 23 Zostera muelleri 1 30 Phaseolus vulgaris 14 0 Helianthus tuberosus (mature plant) 3 27

SUB Eragrostis phillipica 1 TROPICAL Hemarthria uncinnata 6 30

Themeda australis 11 30 Zea mays 12 30 Vigna radiata cv. Mungo 15 28 Vigna radiata cv. Berken 10 23 Cucumis sativus . 11 25 Passiflora edulis 2 26 Lycopersicon esculentum* 12 28 Lycopersicon esculentum 7 23

TROPICAL Saccharum officinarum 10 28 Themeda arguens 16 -Strongylodon sp. , 17 32 Passiflora flavicarpa 10 27

*From (7). cv. Grosse lisse (10007D, Yates).


gradual i nc reases in t h e t e m p e r a t u r e coef f ic ien t wi th dec reas ing t e m p e r a t u r e as sugges ted by Bagnall and Wolfe (1). The r a t e s of r e a c t i o n s a s soc i a t ed wi th t he secondary even t s a r e however t i m e dependen t and it is l ikely t ha t the k ine t i c s of t hese p roces ses a r e a l t e r e d to a g r e a t e r or lesser e x t e n t wi th t i m e of exposure to chil l ing. The k ine t ics can b e c o m e even more c o m p l i c a t e d s ince some p rocesses i nc r ea se the i r r e l a t i v e r a t e s a t low t e m p e r a t u r e as a resu l t of t h e chill ing injury (5). Consider ing these f a c t o r s , t he inabi l i ty to fit s t r a i g h t l ines to Ar rhen ius - type p lo t s of physiological even t s and t h e a b s e n c e of d i s t inc t v e r t i c a l d i scon t inu i t i e s canno t be used as "ev idence" (1) to r e f u t e the e x i s t e n c e of a t e m p e r a t u r e - i n d u c e d change in t h e molecu la r order of m e m b r a n e lipids in ch i l l ing-sens i t ive p l a n t s . Indeed sharp changes occur in t h e c u r v a t u r e of t h e graphs express ing growth r a t e s as a funct ion of t e m p e r a t u r e and these a r e ev ident a t t e m p e r a t u r e s which a p p r o x i m a t e Τ for t he p l an t s examined by Bagnall and Wolfe (1). F u r t h e r m o r e this t e m p e r a t u r e is "c r i t i ca l " for t h e j j lant , b e c a u s e , as n o t e d by these a u t h o r s , morning glory grown a t 28 C and t r a n s f e r r e d to below 9.8 C died w h e r e a s p l an t s t r a n s f e r r e d to 10.8 C showed apprec i ab l e gain in dry we igh t .

0 -

ι ι ι ι ι 32 33 34 35 36

104/K

FIGURE 2. The effect of temperature on the motion of spin labels intercalated into the membranes of mitochondria from maize root tissue. The spin label 16NSMe was used in the temperature range 0° to 30°C (O) and 12NSMe in the range 20° to 50°C (Φ). Τ was calculated as described (3) from first derivative spectra and straight lines were fitted to the data by regression analysis as previously described (9). The points of intersection of the straight lines using 16NSMe give Τ (12°C) and using 12NSMe, Tf(27°C).


FIGURE 3. Effect of temperature on the motion of spin labels intercalated into membrane lipids from wheat mitochondria. The labels used were 16NSMe (Φ) between-11°C and 10°C and 12NSMe (O) between 10° and 40°C indicating a Τ of 0°C and a Tf of 30° respectively.

8 ° C

15° C

FIGURE 4. First derivative esr absorption spectra for 5NS intercalated into the membranes of wheat mitochondria. The separation of the outer extrema is shown as 2T1:̂ for spectra at 8°C and at 15°C. [Data from Raison, et al., (10)].

M e m b r a n e Lipid T r a n s i t i o n s 1 8 3

FIGURE 5. Plot of 2T u as a function of temperature for 5NS in wheat mitochondrial membranes. The sample was in 50% v /v ethylene glycol for data in the temperature range -10° to 20 C (Φ) and in the aqueous medium (O) for the range 1° to 50°C. The lines drawn were fitted by regression analysis and indicate changes (T ) at 0°C and(Tf)at30°C.

FIGURE 6. The change in the partition coefficient (f) of 5N10 as a function of temperature for a suspension of wheat mitochondrial membranes. The inset shows the line heights used to calculate the partition coefficient, f. 7\. is the point of departure from the linear relationship between f and 1/T at 3(TC. [Data from Raison, et al (10)].

184 J. Κ. R a i s o n et al

A r e c e n t rev iew has been c r i t i ca l of me thods used in spin label l ing (11). For the d a t a shown in Table I t he polar lipids w e r e ana lyzed by the spin- label m e t h o d and T^ and T g w e r e d e t e c t e d as t h e t e m p e r a t u r e s a t which the t e m p e r a t u r e coef f ic ien t of spin- label mot ion changed . This is shown in F igure Ζ wi th mi tochondr ia l m e m b r a n e s of m a i z e . Th^ co r r e l a t i on t i m e for t he labels used in this s tudy w e r e b e t w e e n 8 x 1 0 sec and 1 x 1 0 s e c , an order of magn i tude f a s t e r than the 5 n i t rox ide s t e a r a t e label used by Schre ier et al. (11) as an example of t he p rob lems e n c o u n t e r e d in the ca lcu la t ion of mot ion p a r a m e t e r s . Using these m o r e mobi le labels , co r r e l a t i on t i m e s a r e less likely to be a r t i f i c ia l ly i nc reased as the lipids b e c a m e more o rde red a t low t e m p e r a t u r e s . However , in some p lo ts r e l a t i n g spin- label mot ion to t e m p e r a t u r e , it is diff icult to d e t e r m i n e a c lea r change in s lope, especia l ly in m e m b r a n e s or in lipids from those p l an t s whe re t h e r e is a 30C d e g r e e t e m p e r a t u r e r a n g e b e t w e e n the two t r ans i t i ons as shown in F igure 3 for m e m b r a n e s from w h e a t . In samples such as t he se it is possible to m e a s u r e the hyperf ine sp l i t t ing of a spin label which is undergoing an iso t rop ic mot ion . The m e a s u r e m e n t is m a d e as shown in F igure 4 and is the s epa ra t ion , in gauss , b e t w e e n the ou te r e x t r e m a of first de r iva t ive s p e c t r a . As shown in F igure 5 this also shows a change a t about 0 and 30 C and does not depend on Ar rhen ius - type p lo t s . The d e t e r m i n a t i o n of T g and T^ does however depend on f i t t ing s t r a igh t l ines to the d a t a . Ano the r p a r a m e t e r which can be used to d e t e c t changes in order ing is t h e p a r t i t i o n coef f i c i en t . This m e a s u r e s t he r e l a t i v e p ropor t ions of spin label in the lipid and aqueous phases in m e m b r a n e p r e p a r a t i o n s or lipid suspensions. It is d e t e r m i n e d from t h e r e l a t i v e he igh t s of the peaks in the high-f ield l ine of a first d e r i v a t i v e s p e c t r u m as shown in F igure 6. Al though t h e p a r t i t i o n coef f ic ien t should be ca l cu l a t ed from the r e l a t i v e c o n c e n t r a t i o n of label in the two phases this would involve de t e rmin ing line width as well as he igh t . In most i n s t ances the line width for the peak cor responding to label in lipid is not reso lved and t h e r e is probably less e r ro r in r e l a t i n g l ine he igh t s , especia l ly in a t e m p e r a t u r e r ange above the liquid to gel t r ans i t ion (11). Using this t echn ique , a change in t he pa r t i t i on coef f ic ien t is observed wi th lipids from whea t mi tochondr ia a t 30 C (Fig. 6) cor responding wi th T f as observed by the o the r me thods . When Τ is n e a r 0 C it c anno t be d e t e c t e d by this me thod because subs t ances used to p r even t ice fo rmat ion i n t e r f e r e with t he pa r t i t i on ing .

Since all t h r e e me thods ind ica t e t he s ame t e m p e r a t u r e s for T^ and Τ these t e m p e r a t u r e s we re most ly d e t e r m i n e d by p lo t t ing t h e logar i thm of spin- label mot ion agains t the r e c i p r o c a l of abso lu te t e m p e r a t u r e and by e s t i m a t i n g t h e poin ts for changes in slopes of s t r a igh t l ines .

F u r t h e r m o r e , based on a t echn ique using t h e f luorescence of pa r ina r i c acid, changes in t he molecu la r order ing of polar lipids from some p lan t s h a v e been d e t e c t e d a t t e m p e r a t u r e s co inc iden t wi th those shown by t h e esr m e t h o d s . An example is shown in F igure 7 for lipids of Phaseolus vulgaris w h e r e f luorescence in t ens i ty i nc rea se s below about 15 C which is app rox ima te ly t he t e m p e r a t u r e of Τ (14 C) d e t e r m i n e d by esr spec t roscopy (Table I).


10^/T

FIGURE 7. The change in the logarithm of relative fluorescence intensity of parinaric acid in polar lipids from bean as a function of temperature. (From data of Pikelet αϊ, this volume). The increase in the coefficient of intensity below 15 C is indicative of the formation of gel phase lipids.

Using t h e me thods out l ined we d e t e r m i n e d T f and Τ for m e m b r a n e polar lipids of e igh teen spec ies of p l an t s n a t i v e to Aus t r a l i a and co l l e c t ed from var ious c l i m a t i c reg ions . The r e su l t s for some of t h e s e a r e shown in Table I t o g e t h e r wi th s imi lar d a t a from a number of c e r e a l and v e g e t a b l e c rops . The p l an t s h a v e been grouped accord ing to t he c l i m a t i c condi t ions of the h a b i t a t in which they we re c o l l e c t e d . "Tropical" means a cont inuous warm c l i m a t e with no f ros t s , " sub- t rop ica l" , s l ight ly cooler than t rop ica l wi th no f rosts and " t e m p e r a t e " , cool wi th f ros t s . It is a p p a r e n t t h a t for t he win te r growing p l an t s from t h e t e m p e r a t e reg ions T^ is low and t h e t r ans i t i on r a n g e b e t w e e n T g and T^ l a rge , ( approx imate ly 30C deg rees ) . For s u m m e r growing p l an t s from this reg ion Τ is s o m e t i m e s h igher than ze ro as shown by Helianthus tuber-OSUS. This spec ies also provides an i n t e r e s t i n g example in t ha t Τ for t h e m a t u r e p l an t , which grows in s u m m e r , is about 8C d e g r e e s higher than t h a t of t h e d o r m a n t tuber in w in te r . Not only is Τ lowered but t he t r ans i t ion r ange is r e d u c e d . The lower ing of Τ for lipids of t h e tuber is p rogress ive , s t a r t i n g a t about 3 C a t m a t u r i t y , dec reas ing to - 5 C in m i d - d o r m a n c y and r e tu rn ing to 3 C a t t i m e of sprout ing in spring (2). For t h e p l an t s from t h e sub - t rop ica l and t rop ica l c l i m a t e s T g is r e l a t i ve ly high and the t r ans i t ion r a n g e smal l (24 to 13 C degrees ) .

The seag ras ses Zostera spp. p rov ide an i n t e r e s t i n g example of t he co r r e l a t i on b e t w e e n the t e m p e r a t u r e of Τ and t h e t e m p e r a t u r e of t he p lan t s ' h a b i t a t . These p l an t s grow in shallow sandy reg ions along the

186 J. Κ. R a i s o n et al

coas t of Aus t ra l i a from l a t i t u d e 10° (Cape York Peninsula) to l a t i t u d e 44° (southern Tasman ia ) . Z. capricorni shows pheno typ ic , cl inal v a r i a t ions along t h e coas t of New South Wales wi th a m a r k e d change a t about l a t i t u d e 34° (cen t ra l coas t region of New South Wales) . Sample A, (T of 15

UC) , was from this a r e a whe re oceanograph ic r e c o r d s i nd i ca t e a m e a n

win t e r sea t e m p e r a t u r e of 13 C . Sample Β, (T of 9 C), was from t h e sou the rn l imit for this spec ies ( l a t i tude 36°) and fhe lower Τ r e f l e c t s t he lower sea t e m p e r a t u r e of this reg ion . The closely r e l a t e d spec ies Z. muelleri (T o f - 1 ° C ) grows a t a l a t i t u d e of 4 4 ° in t he south Tasman Sea.

P l an t s wi th a Τ in t he region of 10 C a r e no t found growing in t e m p e r a t e c l i m a t e s . P l an t s wi th a Τ n e a r 0 C could however grow in t rop ica l c l i m a t e s and Helianthus tuberosus. (T of 3°C) is a good example as this p lan t is found growing successful ly in t rop ica l r eg ions .

This co r r e l a t i on b e t w e e n Τ and the t e m p e r a t u r e of t he p lan t ' s h a b i t a t ind ica tes t ha t the t e m p e r a t u r e response of t h e m e m b r a n e lipids is a major f ac to r in l imi t ing t h e d is t r ibu t ion of p lan t spec ie s . It also ind ica t e s t ha t this p r o p e r t y would be i m p o r t a n t in de t e rmin ing the lower t e m p e r a t u r e l imi t for the s t o r a g e and t r anspo r t of t rop ica l f rui ts and v e g e t a b l e s .


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zymology" (S. F le i sche r , L. P a c k e r and R. Es tabrook , eds.) , Vol. 32, pp . 258-262 (1974).

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8. Raison, J . K. In "Mechanisms of Regu la t ion of P lan t Growth" Bullet in 12, The Royal Socie ty of New Zealand, Well ington (R. Bieleski , A. Ferguson , and M. Cresswel l , eds.) , pp . 487-497 (1974).

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11 . Schre ier , S., Po lnaszek , C. F . , and Smith, I. C. P . Biochim. Biophys. Acta 515, 375-436 (1978).

12. Shneyour, Α., Raison, J . K., and Smill ie, R. M. Biochim. Biophys. Acta 292, 152-161 (1973).

13. Watada , A. E., and Morris , L. L. Proc. Amev. Soc. Hort. Sci. 89, 368-380 (1966).


THE USEFUL CHLOROPLAST: A NEW A P P R O A C H FOR INVESTIGATING CHILLING STRESS IN PLANTS

Robert M. Smillie

Plan t Physiology Unit CSIRO Division of Food R e s e a r c h , and School of Biological Sciences

P . O . Box 52, Nor th Ryde Sydney, N.S.W. 2113, Aus t r a l i a

I. INTRODUCTION

The r e s e a r c h desc r ibed in t h e pape r deals wi th e f f e c t s of chil l ing t e m p e r a t u r e s on the ch lorophyl l -con ta in ing inner m e m b r a n e s (thylakoids) of t h e ch lorop las t . The in t en t ion is no t so much to l ea rn m o r e about how t e m p e r a t u r e may a f f ec t t he funct ion and biogenesis of ch loroplas t m e m b r a n e s , as to use t h e ch loroplas t m e m b r a n e sys tem as an e x p e r i m e n t a l tool for i nves t iga t ing the d ive rse responses of p lan t spec ies to chil l ing s t r e s s e s , the i r g rowth and survival a t chil l ing t e m p e r a t u r e s , and the g e n e t i c a d a p t a t i o n s involved. The chloroplas t thylakoid m e m b r a n e s a r e espec ia l ly su i t ab le for this e x p e r i m e n t a l app roach as m o r e is known about the i r compos i t ion , s t r u c t u r e , funct ion and assembly than any o t h e r p l an t m e m b r a n e . F u r t h e r m o r e , t hese m e m b r a n e s con ta in high c o n c e n t r a t i o n s of co lored lipids and p ro t e in s t ha t may be used as in t r ins ic m e m b r a n e p robes for moni to r ing t e m p e r a t u r e - i n d u c e d changes .

In this s tudy, knowledge of t h e p r o p e r t i e s of ch loroplas t m e m b r a n e s has been applied to inves t iga t ing inhibi t ion of g rowth a t chil l ing t e m p e r a t u r e s , suscep t ib i l i ty to chil l ing injury, and possible ways of gene t i ca l ly modifying chil l ing sens i t iv i ty and r e s i s t a n c e .

Π. CHLOROPLAST ACTIVITY AND DEVELOPMENT AT DIFFERENT TEMPERATURES IN CHILLING-SENSITIVE AND RESISTANT PLANTS

Growth and deve lopmen t of p l a n t s or ig ina t ing in lowland t rop ica l c l i m a t e s is inhib i ted a t chil l ing t e m p e r a t u r e s . The inhibi t ion is

Copyright · 197Θ by Academic Press. Inc. 1 8 7 All rights of reproduction in any form reserved

ISBN σ 12-460560- 5

188 R Μ. Smil l ie

r eve r s ib le a l though prolonged exposure to low t e m p e r a t u r e s can lead to i r revers ib le ce l lu lar d a m a g e in these ch i l l ing-sens i t ive p l an t s (Section ΠΙ). The d i f fe ren t responses of ch i l l ing-sens i t ive and r e s i s t a n t p l a n t s to chil l ing t e m p e r a t u r e s have been i nves t i ga t ed by compar ing ch loroplas t p h o t o r e d u c t i v e a c t i v i t y (in i so la t ed chloroplas ts) and chlorophyl l synthes is as a funct ion of t e m p e r a t u r e in the two groups of p l an t s (1 , 11 , 1Z, 19, 20)· Two r e su l t s from these s tud ies will be h ighl ighted in this pape r . Both conce rn two bas ic t e n e t s pu t for th by Lyons and Raison (6, 7, 16) to explain t he phenomenon of chil l ing sens i t iv i ty , n a m e l y t h a t

1) t h e inhibi t ion of g rowth and me tabo l i sm a t chil l ing t e m p e r a t u r e s in ch i l l ing-sens i t ive p l an t s r e s u l t s from a t e m p e r a t u r e - i n d u c e d m e m b r a n e change below some ' c r i t i ca l ' t e m p e r a t u r e (usually in t h e p rox imi ty of 10 C) and,

Z) ch i l l ing- res i s tan t p l an t s do not show a t e m p e r a t u r e - i n d u c e d m e m b r a n e change around this t e m p e r a t u r e .

Their exp lana t ion is based mainly on an observed change in slope around 10 C, in t h e case of chi l l ing-sens i t ive but no t ch i l l ing- res i s t an t p l an t s , in Arrhenius p lo ts of membrane -bound a c t i v i t i e s of mi tochondr i a and ch loroplas t s and of t h e mot ion of spin labels p a r t i t i o n e d in to mi tochondr ia l and chloroplas t m e m b r a n e s . However , our s tud ies wi th severa l ch i l l ing- res i s tan t p l an t s have not p roduced t h e p r e d i c t e d l inear re la t ionsh ip wi th t e m p e r a t u r e . For e x a m p l e , Arrhenius p lo t s of p h o t o r e d u c t i v e a c t i v i t y of i so la ted bar ley (Hordeum vulgare) ch lo ro p l a s t s , using e i the r pho tosys t em 1 or^photosystem Ζ e l e c t r o n a c c e p t o r s , showed an i n c r e a s e in slope below 9 C (11). T e m p e r a t u r e coef f i c i en t s for the r a t e of chlorophyll synthes is in ba r ley and the r a t e of e longat ion of t h e p r i m a r y leaf i nc reased below 10 C (19).

The e x p e r i m e n t s wi th chi l l ing-sens i t ive p l an t s w e r e also r evea l ing . In cucumber (Cucumis sativus), for i n s t ance , t he Q . Q for both t h e r a t e of synthes is of chlorophyll in t h e co ty ledons and t h e r a t e of hypocoty l ex tens ion in dark-grown seedl ings i nc r ea sed marked ly around ZZ C (19, Z0). The d e c r e a s e in these r a t e s with dec reas ing t e m p e r a t u r e below ZZ C was such t ha t bo th g rowth p rocesses w e r e ba re ly d e t e c t a b l e once t e m p e r a t u r e s of 10 to 1Z C had been r e a c h e d . One is left wi th t h e ques t ion: Is it n ece s sa ry to p o s t u l a t e t h e e x i s t e n c e of m e m b r a n e changes below 10-1Ζ C (whether due to lipid or prote in) to explain inhibi t ion of g rowth a t chil l ing t e m p e r a t u r e s , when growth and deve lopmen t have a lmos t c e a s e d be fo re these t e m p e r a t u r e s a r e r e a c h e d ?

ΠΙ. CHILLING INJURY

A cont inuing problem for s tudies of chil l ing injury in p l a n t s has been the lack of su i tab le me thods for measur ing suscep t ib i l i ty to chil l ing s t r e s s e s . R e l i a n c e on visible s y m p t o m s of injury is genera l ly

T h e Useful C h l o r o p l a s t 189

u n s a t i s f a c t o r y , s ince these t end to be l a t e man i f e s t a t i o n s of ce l lu lar d a m a g e caused by chi l l ing. Func t iona l p r o p e r t i e s of t he ch loroplas t thylakoid m e m b r a n e s a r e known to change during chill ing (5, 9) and it was dec ided to see if t hese changes could be used to d e t e c t and m e a s u r e ce l lu la r injury well be fo re i r r eve r s ib le d a m a g e and visible s y m p t o m s of injury to t i ssues b e c o m e a p p a r e n t . Before assays for ce l lu lar chil l ing injury a r e descr ibed , i t is f i rs t n e c e s s a r y to examine t h e changes in ch loroplas t funct ion tak ing p l a c e in ch i l l ing-sens i t ive p lan t s a t low t e m p e r a t u r e s .

A. Changes in Chloroplast Function at Chilling Temperatures

In ch i l l ing-sens i t ive p l an t s , a n u m b e r of ch loroplas t funct ions d e t e r i o r a t e a t chil l ing t e m p e r a t u r e s ; the p a r t i c u l a r funct ions a f f e c t e d depend upon e x p e r i m e n t a l condi t ions and the spec ies of p lan t (Table I).

P h o t o r e d u c t i v e a c t i v i t y dec l ines in l eaves of bean , c u c u m b e r and t o m a t o a t t e m p e r a t u r e s n e a r 0 C in e i t he r dark or l ight . The lesion l ies on the oxygen-evolv ing s ide of p h o t o s y s t e m Ζ ( 8 ) . In i l l umina ted chi l led l eaves t h e reg ion of t h e e l e c t r o n t r ans fe r p a t h w a y b e t w e e n t h e two p h o t o s y s t e m s is also a f f e c t e d wi th the rap id loss of pho tophosphory la t ive and p ro ton u p t a k e c a p a c i t i e s . At ve ry high light i n t ens i t i e s chil l ing condi t ions also i n t e r f e r e wi th chlorophyll tu rnover in cucumber and b leach ing of t h e chlorophyll o c c u r s . The s i tua t ion is d i f fe ren t in l eaves of t h e t rop ica l golden pass ionfrui t (Passiflora edulis forma flavicarpa) in which pho tosys t em 1 and pho tosys t em Ζ a c t i v i t i e s dec l ine c o n c o m i t a n t l y . Pho tob leach ing a t chil l ing t e m p e r a t u r e s is no t obse rved .

TABLE I. The Effect of Chilling on Chloroplast Function

Reaction Conditions Plants References

Decline in capacity for oxygen evolution

Loss of photophosphoryl-ation and proton uptake

Decline in activity of photosystems 1 and 2

Light or dark

Light

Light or dark

eon

Cucumber

Tomato

Passionfruit

(9) (5) (8) (2) (4) (22)

(unpublished results)

Photobleaching of chlorophyll

High light Cucumber intensity

(25)

190 R . Μ . Smi l l ie

Β. Assays for Chilling Injury

A f e a t u r e common to t he ch loroplas t m e m b r a n e s of all chi l l ing-sens i t ive p lan t s spec ies i nves t iga t ed so far is the p rogress ive loss of c a p a c i t y for oxygen evolut ion a t chill ing t e m p e r a t u r e s (Table I). The dec l ine in this c a p a c i t y can be followed by measur ing Hill r e a c t i o n a c t i v i t y in i so la t ed ch lorop las t s . S p e c t r o p h o t o m e t r i c or f l uo rome t r i c m e a s u r e m e n t s can be used to moni to r changes in p h o t o s y n t h e t i c e l e c t r o n t r ans fe r a c t i v i t y in i n t a c t l e aves .

400 ι 1

FIGURE 1. Photoreductive activity of chloroplasts isolated from chilled leaves of the purple (P. edulis) and the yellow (P. flavicarpa) passionfruit. Leaves were detached and stored in darkness at 0 C for the times indicated. Chloroplasts were isolated and photoreductive activity [ μ moles ferricyanide photoreduced/hr /(mg chlorophyll) ] measured as described by Critchley et al (1).


1. Isolated ChloroplastS. An assay for chil l ing injury based on t h e dec l ine in p h o t o r e d u c t i v e a c t i v i t y in i so la ted ch lorop las t s has been desc r ibed by Smill ie and N o t t (ZZ) and used to c o m p a r e suscep t ib i l i ty t o chill ing injury in cu l t iva r s of t h e d o m e s t i c t o m a t o (Lycopersicon escu-lentum) and high and low a l t i tud ina l forms of a wild t o m a t o (L. hirsutum). T o m a t o l eaves w e r e chi l led a t 9 C and a f t e r var ious per iods of chil l ing, the ch lorop las t s w e r e i so la ted and oxygen evolving c a p a c i t y a s c e r t a i n e d by t h e pho to reduc t i on of f e r r i cyan ide in t h e p r e s e n c e of g ramic id in D, an uncoupler of pho tophosphory la t ion . p r e s e n c e of g ramic id in D, an uncoupler of pho tophosphory la t ion .

F igu re 1 shows this assay appl ied to l eaves of t h e ch i l l ing-sens i t ive golden or yellow pass ionfrui t (P. edulis forma flavicarpa) and l eaves of t h e purple pass ionfrui t (P. edulis), which is chil l ing r e s i s t a n t (13). T h e r e was l i t t l e change in a c t i v i t y of ch lo rop las t s i so la ted from leaves of t h e purp le pass ionfru i t , even a f t e r 36 days a t 0 C . In c o n t r a s t , t h e r e was a p rogress ive dec l ine in a c t i v i t y for l e aves of t h e yellow pass ionfrui t kept a t 0 C. F i r s t s y m p t o m s of mass ive chil l ing injury (loss of tu rgor in t h e l eaves ; changes in ch loroplas t m e m b r a n e s t r u c t u r e r e v e a l e d by e l e c t r o n microscopy) co inc ided wi th loss of p h o t o r e d u c t i v e a c t i v i t y , i .e . a f t e r 8 to 9 days a t 0 C . P h o t o r e d u c t i v e a c t i v i t y did no t dec l ine in l eaves of e i t he r pass ionfrui t held a t 10 C for 9 days . Thus t h e s e m e a s u r e m e n t s c l ea r ly dis t inguish b e t w e e n t h e spec ies suscep t ib le to chil l ing injury a t 0 C and t h e non-suscep t ib l e spec ie s . T h e r e a p p e a r e d to be l i t t l e or no lag in t h e onse t of t h e ch loroplas t m e m b r a n e changes which l ead to loss of p h o t o r e d u c t i v e a c t i v i t y in t h e suscep t ib le spec ies a t 0 ° C .

2. Intact Tissue. Changes in chlorophyll f luo rescence , t h e l igh t -dependen t abso rbance change a t 518 nm and the pho toox ida t ion and r educ t i on of c y t o c h r o m e f h a v e been used to follow deve lopmen t of chil l ing injury in i n t a c t g reen t i s sues . An example using c y t o c h r o m e changes is given in Sect ion IV, C. The chlorophyll f luo rescence m e t h o d will be desc r ibed h e r e s ince it is p robably t h e most v e r s a t i l e of t h e me thods deve loped and can be a d a p t e d to sc reen ing a r e l a t i v e l y l a rge n u m b e r of s amples .

F igu re Ζ shows changes in chlorophyll f l uo rescence in p e a n u t (Ara-chis hypogaea) and p e a (Pisum sativum) l e aves chi l led a t 0 ° C . The f luo rescence m e a s u r e m e n t s w e r e also m a d e while the l eaves we re a t 0 C; t h e r e was no r e - w a r m i n g of t h e l eaves be fo re m e a s u r e m e n t . Upon i l lumina t ion wi th r ed l ight an in i t ia l level of f luo rescence (F ) was es tab l i shed which did no t change apprec iab ly as chil l ing injury deve loped . In i n s t ances of m o r e s e v e r e d a m a g e to t h e ch loroplas t m e m b r a n e , as in t h e case of h e a t - i n d u c e d d a m a g e , F i nc r ea sed and has been used to follow t h e d e v e l o p m e n t of h e a t - i n d u c e d changes in ch loroplas t m e m b r a n e s and to c o m p a r e t h e h e a t sens i t iv i t i e s of p lan t spec ies (17, Zl , Z3, 24).

T h e r e was a fu r the r r i se in chlorophyll f l uo re scence (variabl chlorophyll f luorescence) to a new leve l ( F m a x) as a componen t of t he pho tosys t em Ζ complex , Q, b e c o m e s r e d u c e d . Q, which quenches

192 R. Μ. Smil l ie

C H L - F L U O R E S C E N CE D U R I NG C H I L L I NG T i me a t 0 ° C

P E A N U T L E AF PE A L E AF

FIGURE 2. Chlorophyll f l u o r e s c e n c e rise at 0 C in dark-adapted peanut and pea leaves held at 0 C for various periods of time. FQ

indicates the initial fluorescence level, F the maximum reached, h = , , , ' max hours; d - days.

chlorophyll f luorescence when in t h e oxidized s t a t e , is r e d u c e d as t h e resu l t of w a t e r sp l i t t ing and oxidized by componen t s of the e l e c t r o n t r ans fe r p a t h w a y l o c a t e d b e t w e e n pho tosys t em 2 and pho tosys t em 1. Thus f ac to r s which inhibit on the r educ ing side of pho tosys t em 2 (e.g. chill ing injury) would be e x p e c t e d to d e c r e a s e t h e r i se in f luo rescence above F , while f a c to r s which inhibit on t he oxidizing s ide of pho tosys t em 2 (e.g. DCMU) might be e x p e c t e d to i nc rease i t . F igure 2 shows t ha t both t he r a t e of t h e var iab le f luo rescence r i se and t h e e x t e n t of this r i se ^F ~ ^ 0 ' ^

ad d e c r e a s e d subs tan t i a l ly in peanu t leaf a f t e r

one day a t 0 smal l changes o c c u r r e d in f luo rescence in l eaves of t h e ch i l l ing- res i s tan t pea , even a f t e r 13 days a t 0 C.

In rou t ine m e a s u r e m e n t s only t he r a t e of t h e r i se in va r iab le chlorophyll f luo rescence was r e c o r d e d . Up to 96 samples of l eaves or leaf s e g m e n t s w e r e pos i t ioned on an a luminium block which was then se t in i ce con ta ined in a da rkened , insu la ted box. Using a p o r t a b l e f luo rescence sensor p robe (18), i t was possible to r e c o r d t he f luorescence r i se in the 96 d i f fe ren t samples of chi l led leaf t i s sue in 10 to 12 m i n u t e s . F ig . 3 c o m p a r e s t he t i m e course for t h e change in r a t e of t h e f luo rescence r i se in peanu t and p e a l eaves during s t o r a g e a t 0 C .


FIGURE 3. The rate of the rise in variable fluorescence at 0 C in dark-adapted leaves of pea and peanut stored at 0 C for various periods of time. The leaves were detached, positioned on an aluminium block, and kept in darkness for 15 min. The block was embedded in ice, except for the upper surface which was covered with insulation material. The first measurement of the rate of fluorescence rise was made about 1 hour later. Subsequent rates measured are expressed as a percentage of this first measured rate. Each point is the average value for 8 different leaf samples.

The chlorophyll f luo rescence m e t h o d can also be used to c o m p a r e the e f f ec t s of chil l ing on closely r e l a t e d spec ies , including in s t ances w h e r e both spec ies a r e suscep t ib le to chil l ing injury. F igu re 4 shows a compar i son b e t w e e n a t rop ica l guava (Psidium guajava) and a t e m p e r a t e spec ies of Psidium (P. cattleyanum). In Fig . 5 Capsicum annum is c o m p a r e d wi th C. rocoto, a spec ies also from t rop ica l l a t i t u d e s but found a t h igher a l t i t u d e s . Both a r e suscep t ib le spec ies but C. rocoto, on t h e basis of t he chlorophyll f l uo re scence m e a s u r e m e n t s , is less suscep t ib le than C. annum. Similar r e su l t s w e r e ob ta ined in a compar i son of t h e paw paw (Carica papaya) wi th t he mounta in paw paw (C. pubescens).


FIGURE 4. Development of chilling injury in leaves of two species of Psidium (chlorophyll fluorescence method). Conditions are as described in Figure 3.

The use of chlorophyll f luo rescence changes to moni to r t he progress ive d a m a g e to chloroplas t m e m b r a n e s caused by chil l ing t e m p e r a t u r e s has m a d e it possible to e m b a r k on a wide r ange of s tudies of chil l ing injury which w e r e h i t h e r t o not feas ib le . The rank ing of p l an t s for suscep t ib i l i ty to chill ing injury is being ex t ended to a wide r ange of spec ies . The k ine t ics of chill ing injury, i t s r evers ib i l i ty and the e f f e c t s of t r ans i en t r e - w a r m i n g a r e being s tud ied . By making m e a s u r e m e n t s a t d i f fe ren t t e m p e r a t u r e s , it should be possible to d e t e r m i n e t he for chill ing injury in d i f fe ren t p l a n t s . The e f f ec t s of env i ronmen ta l and o the r f a c t o r s (light, age , ho rmones , diurnal and seasonal changes , e tc . ) on t he deve lopmen t of chill ing injury can also be i nves t i ga t ed . D e t a i l e d analysis of t h e chlorophyll f luo rescence changes should yield new in format ion of mechan i sms involved in chill ing injury. The s impl ic i ty and rap id i ty of measur ing the change in induced f luo rescence makes it su i tab le for s tudies of a c c l i m a t i o n and a d a p t a t i o n to chil l ing t e m p e r a t u r e s , and perhaps coupled with in f ra - red pho tography , as a sc reen ing m e t h o d for the se lec t ion of chil l ing t o l e r an t p l an t s in p lan t b reed ing and m u t a t i o n p r o g r a m s .


C A P S I C U M

T i me a t 0 ° C , hr.

FIGURE 5. Development of chilling injury in leaves of two species of Capsicum (chlorophyll fluorescence method). Conditions are as described in Figure 3.

The chlorophyll f luo rescence m e t h o d also works well wi th t h e g reen pee l of f rui t , so t ha t m e a s u r e m e n t s of chil l ing injury can be ca r r i ed out using g reen fruit as well as l e a v e s .

IV. GENETIC MODIFICATION OF CHILLING SENSITIVITY

For many chi l l ing-sens i t ive p l an t s of c o m m e r c i a l i m p o r t a n c e , t h e r e is much i n t e r e s t in inc reas ing the i r r e s i s t a n c e to chi l l ing t e m p e r a t u r e s , in o rder to ex t end the growing season and r a n g e of c l i m a t e s in which crops can be r a i sed economica l ly and to make low t e m p e r a t u r e s t o r a g e of t h e fruit a feas ib le propos i t ion . G e n e t i c a l l y - c o n t r o l l e d responses of p l an t s to chil l ing t e m p e r a t u r e s may be a l t e r e d by changing e i t he r the g e n e t i c in fo rma t ion or t h e express ion of this in fo rma t ion . L i t t l e is known a t p r e sen t about r egu la t ion of gene express ion for chil l ing sens i t iv i ty by b iochemica l and env i ronmen ta l f a c t o r s . Of obvious i n t e r e s t is t h e ro le of t e m p e r a t u r e i tself as a p o t e n t i a l r e g u l a t o r of gene express ion and it is known t h a t some chi l l ing-sens i t ive p l an t s can be 'hardened

1 to w i th s t and m o d e r a t e l y low chil l ing t e m p e r a t u r e s .

196 R. Μ. Smi l l ie

W A V E L E N G T H , n m

FIGURE 6. Absorption spectra of leaves of barley mutants grown at different temperatures. Each spectrum was recorded for the region of the primary leaf 3 to 4 cm below the leaf tip. A. A temperature-dependent albino mutant originally produced at the Danish Atomic Energy Cgrgfnission's Research Establishment at Risjo. B. The mutant viridis-zj , from the stock of mutants at the Carlsberg Research Center.


G e n e t i c i n fo rma t ion d e t e r m i n i n g chil l ing sens i t iv i ty or r e s i s t a n c e may be a l t e r e d by m u t a t i o n or by the in t roduc t ion of new genes . Three r e c e n t s tud ies in this a r e a a r e out l ined be low.

A. Barley Mutants Chilling-Sensitive for Chloroplast Development

Can s e l e c t e d g rowth or d e v e l o p m e n t p roces ses in chil l ing r e s i s t a n t p l an t s be r e n d e r e d ch i l l ing-sens i t ive by m u t a t i o n of nuc l ea r genes? This ques t ion was explored by surveying a number of p h o t o s y n t h e t i c m u t a n t s of bar ley from among s tock held a t t he Ca r l sbe rg R e s e a r c h C e n t e r , Copenhagen , to s e e if any w e r e t e m p e r a t u r e - d e p e n d e n t for ch loroplas t d e v e l o p m e n t . Seedlings of m u t a n t p l an t s w e r e r a i sed in glass cy l inders i m m e r s e d in w a t e r b a t h s (24) held a t a r a n g e of t e m p e r a t u r e s s t a r t i n g a t 2 ° C . Several m u t a n t s w e r e iden t i f ied in which ch loroplas t deve lopmen t was t o t a l l y or s eve re ly inhib i ted a t chi l l ing t e m p e r a t u r e s a l though r a t e of g rowth of t h e young seedl ings was no t a f f e c t e d when c o m p a r e d wi th the wild t y p e . F igure 6 shows absorp t ion s p e c t r a of the p r i m a r y leaf of two of t hese m u t a n t s grown a t d i f fe ren t t e m p e r a t u r e s . In bo th m u t a n t s ch loroplas t deve lopmen t begins to be inhib i ted below 10 to 12 C. Chlorophyl l synthes i s was u n d e t e c t a b l e in the albino m u t a n t (Fig. 6A) a t s e v e r e ctnTl^g t e m p e r a t u r e s , while i t was g r e a t l y r e d u c e d in t h e m u t a n t viridis-zj (Fig. 6B). This m u t a n t is also h e a t - s e n s i t i v e for ch loroplas t deve lopmen t above 27 C (24). The loca t ion of t h e block in ch loroplas t deve lopmen t in t he se m u t a n t s is unknown. It occu r s a t a very ea r ly s t a g e in t h e p las t id b iogenesis and may possibly resu l t from l o w - t e m p e r a t u r e inhibi t ion of the synthes is of ch loroplas t DNA or i t s t r an sc r ip t i on during p las t id r ep l i ca t i on .

B. Interspecific Sexual Hybridization: Tomato Species

While m u t a t i o n can prov ide i n t e r e s t i n g m a t e r i a l for s tudies of chil l ing sens i t iv i ty , it does no t a t p r e s e n t appear to be a p r a c t i c a l way of inc reas ing t h e chil l ing t o l e r a n c e of sens i t ive spec ie s . A m o r e promis ing approach is the in t roduc t ion of new genes for chil l ing r e s i s t a n c e in to a d o m e s t i c spec ies by u t i l iz ing t h e g e n e t i c va r iab i l i ty t h a t o f ten ex i s t s in wild forms of the s a m e spec ies , or in c losely r e l a t e d spec ies wi th which it can by hybr id ized sexual ly . Y e t a n o t h e r app roach l ies in t h e in t roduc t ion of new genes for chil l ing r e s i s t a n c e from m o r e d i s t an t ly r e l a t e d spec ies by s o m a t i c hybr id iza t ion (Section IV, C) .

Dr . B. D . P a t t e r s o n in our l a b o r a t o r y is inves t iga t ing t h e feas ib i l i ty of inc reas ing t h e chil l ing r e s i s t a n c e of t h e d o m e s t i c t o m a t o (L. escu-lentum) by hybr id iza t ion wi th high a l t i tud ina l forms of t he wild t o m a t o (L. hirsutum) (14, 15). The f irst s t ep in this inves t iga t ion was to use a n u m b e r of me thods to es tabl i sh w h e t h e r some forms of L. hirsutum w e r e indeed m o r e chil l ing r e s i s t a n t than L. esculentum and w h e t h e r t he chill ing r e s i s t a n c e was c o r r e l a t e d wi th a l t i t u d e . F igure 7 c o m p a r e s suscep t ib i l i ty to chil l ing injury a t 0 C, using t h e chlorophyll f luo rescence


FIGURE 7. Development of chilling injury (chlorophyll fluorescence method) in leaves of potato and three altitudinal forms of wild tomato, L. hirsutum. The tomato plants are derived from seed collected at 30, 1500 and 3100 meters; the code letters MG, AC and AF, respectively, refer to the localities where the seed was collected [see Patterson et al., (14)] . Assay conditions as in Figure 3.

m e t h o d (Section ΠΙ, B, 2), of t h r e e forms of L. hirsutum r a i sed from seed original ly c o l l e c t e d from indigenous p l a n t s growing a t 30, 1500 and 3100 m e t e r s , in Ecuador or Pe ru . The low a l t i t u d e form co l l e c t ed nea r sea - l eve l was very suscep t ib le and was c o m p a r a b l e in this r e s p e c t to d o m e s t i c cu l t iva r s of L. esculentum. The form from 1500 m e t e r s was less suscep t ib le and t h e one from 3100 m e t e r s cons iderably less suscep t ib l e . These r e s u l t s a g r e e d wi th e s t i m a t e s of r e l a t i v e chil l ing sens i t iv i ty using o the r me thods (14). The p o t a t o (Solanum tuberosum), t he wild forms of which a r e indigenous to t he Sierras of Pe ru and Bolivia a t a l t i t udes above 2000 m e t e r s (3), was m o r e r e s i s t a n t aga in . The second s t a g e of t h e inves t iga t ion , to cross L. esculentum wi th t h e most ch i l l ing- res i s tan t forms of L. hirsutum, is in p rog res s . Hybrids of L. esculentum and L. hirsutum (AF, 3100 me te r s ) and first and second


FIGURE 8. Susceptibility of tomato-potato hybrids to chilling injury as measured by changes in reduction of cytochrome f. Leaves were detached and stored in darkness at 0 C or 18°C. Changes in photosynthetic electron transfer activity in individual leaves were assayed by rapidly warming the leaves to 23°C and recording the rate of dark reduction of cytochrome f (cytochrome-554) following a 30-second period of illumination with red light. The absorbance increase at 554 nm coinciding with cytochrome reduction was monitored using an Aminco DW-2a spectrophotometer operated in the dual wavelength mode (reference wavelength 541 nm). The values for the rate of cytochrome reduction are the Τ ± for the reduction in seconds and are expressed as a percentage of the initial rate obtained with freshly detached leaves. Changes in rate of cytochrome reduction were small in leaves stored at 18°C. A, potato; m , the tomato-potato hybrid 6a; · , tomato. The four somatic tomato-potato hybrids are identified as lb, 6a, 6b, and 7b; these designations refer to the protocols of the callus transfers and shoot regenerations used [Melchers et al., (10)].

2 0 0 R. Μ. Smillie

backc rosses of s e l e c t e d hybrids to L. esculentum have been p roduced and a r e cu r r en t ly being t e s t e d .

C. Intergeneric Somatic Hybridization: The Tomato-Potato Hybrid

Melchers et al. (10) have r e c e n t l y p roduced severa l s o m a t i c hybrid p lan t s of t o m a t o and p o t a t o r e g e n e r a t e d from fused t o m a t o and p o t a t o p r o t o p l a s t s . Fo r tu i tous ly , t h e new p lan t s a r e hybrids of a chi l l ing-sens i t ive p lan t and a ch i l l ing- res i s tan t one and it was of cons iderab le i n t e r e s t t h e r e f o r e to d e t e r m i n e the i r chill ing r e s i s t a n c e . Suscept ib i l i ty to chil l ing injury, as i nd ica t ed by changes in the dark r educ t ion of c y t o c h r o m e f in l eaves s t o r e d a t 0 C was m e a s u r e d in four of t h e s o m a t i c t o m a t o - p o t a t o hybrids (Fig. 8). All four showed enhanced r e s i s t a n c e c o m p a r e d wi th t o m a t o , but w e r e less r e s i s t a n t than p o t a t o . If g e n e t i c exchange b e t w e e n the t o m a t o and p o t a t o ch romosomes of the hybrid can be induced, e i t he r by homoeologous pai r ing fol lowed by cross ing-over or by r e c i p r o c a l t r ans loca t i ons , and subsequent s epa ra t i on of t he two genomes ob ta ined , t hese hybrids should be useful for t r ans fe r r ing genes for chil l ing r e s i s t a n c e in to the d o m e s t i c t o m a t o . Since the chlorophyll f luorescence assay for chil l ing injury is r e l a t i ve ly s imple and can be appl ied to smal l leaf samples , it should be of va lue in t h e iden t i f i ca t ion of ch i l l ing- res i s tan t r e c o m b i n a n t s in t he t o m a t o g e n o m e .

V. CONCLUSIONS

A change in slope in Arrhenius p lo t s of t he p h o t o r e d u c t i v e a c t i v i t y of i so la ted ch lorop las t s a t chil l ing t e m p e r a t u r e s does no t necessa r i ly p r ed i c t chil l ing sens i t iv i ty . Ch lo rop las t s i so l a t ed from bo th chi l l ing-sens i t ive and r e s i s t a n t p l an t s showed non- l inear Arrhenius p lo t s b e t w e e n 0 and 2 0 ° C

The r a t e of chloroplas t deve lopmen t in cucumber (Cu.CU .mis sati-VliS) co ty ledon and g rowth of cucumber hypocoty l d e c r e a s e rapidly wi th falling t e m p e r a t u r e s below 22 C and have a lmos t ceased be fo re t he chi l l ing-sens i t ive t e m p e r a t u r e r ange is r e a c h e d . The high Q i Q b e t w e e n 12 and 22 C is suff ic ient t o a ccoun t for t h e a p p a r e n t inhibi t ion of g rowth and deve lopmen t in cucumber by chil l ing t e m p e r a t u r e s .

The deve lopmen t of ce l lu lar chil l ing injury in l eaves and g reen fruit can be convenien t ly followed by m e a s u r e m e n t s of chlorophyll f luo rescence or oxidat ion of c y t o c h r o m e f. These me thods we re used to r ank p lan t s for suscep t ib i l i ty to chil l ing injury and in s tud ies a imed a t increas ing chill ing t o l e r a n c e in t o m a t o (Lycopersicon esculentum) via in t e r spec i f i c hybr id iza t ion wi th high a l t i t u d e forms of a wild t o m a t o

(L. hirsutum) and i n t e r g e n e r i c s o m a t i c hybr id iza t ion wi th p o t a t o (Sola-num tuberosum).

http://Cu.CU.mis


ACKNOWLEDGMENTS

I should l ike to thank Drs . B. D . P a t t e r s o n , W. G. Nolan, C . C r i t c h l e y and Miss R. N o t t who have c o n t r i b u t e d to var ious a s p e c t s of this work. The s tud ies desc r ibed in Sec t ions IV, A and IV, C a r e jo in t r e s e a r c h p r o j e c t s wi th t h e Ca r l sbe rg R e s e a r c h C e n t e r , Copenhagen , and t h e coope ra t i on and suppor t of Professor D . von W e t t s t e i n is g ra te fu l ly acknowledged .


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C h a p m a n & Hal l , London (1978). 4 . Kaniuga , Z., Sochanowicz , B., Zabek, J . , and Krzys tn iak , K. Planta

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818 (1972). 6. Lyons, J . M. Annu. Rev. Plant Physiol. 24, 445-466 (1973). 7. Lyons, J . M., and Raison , J . K. Plant Physiol. 45, 386-389 (1970). 8. Margul ies , Μ. M. Biochim. Biophys. Acta 267, 96-103 (1972). 9. Margul ies , Μ. M. and Jagendorf , A. T. Arch. Biochem. Biophys. 90,

176-183 (1960). 10. Melche r s , G., Sac r i s t an , M. D . , and Holder , A. A. Carlsberg Res.

Commun. 43, 203-218 (1978). 11 . Nolan, W. G., and Smill ie , R . M. Biochim. Biophys. Acta 440, 4 6 1 -

475 (1976). 12. Nolan, W. G., and Smil l ie , R . M. Plant Physiol. 59, 1141-1145

(1977). 13. P a t t e r s o n , B. D. , Mura t a , T., and G r a h a m , D. Aust. J. Plant

Physiol. 3, 435-442 (1976). 14. P a t t e r s o n , B. D. , Paul l , R. , and Smill ie , R . M. Aust. J. Plant

Physiol. 5, 609-617 (1978). 15. P a t t e r s o n , B. D. , Paul l , R. , and G r a h a m , D . This vo lume (1979). 16. Raison , J . K., and C h a p m a n , E. A. Aust. J. Plant Physiol. 3, 2 9 1 -

299 (1976). 17. Schre iber , U., and Berry , J . A. Planta 136, 233-238 (1977). 18. Schre iber , U., G r o b e r m a n , L. , and Vidaver , W. Rev. Sci. Instrum.

46, 538 542 (1975). 19. Smil l ie , R . M. In 'Gene t i c s and Biogenesis of Ch lo rop las t s and

Mi tochondr ia ' . (Eds Th. Bucher et al.) pp . 103-110. E l s e v i e r . A m s t e r d a m (1976).

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ZO. Smill ie, R. M. " T e m p e r a t u r e Regu la t ion of Chloroplas t Deve lopmen t and Pho tosyn thes i s . G e n e t i c , Biochemica l and Applied A s p e c t s . P r o c . XIV Int . Congr . G e n e t i c s , Moscow 1978,

In Press (1979). Z l . Smill ie , R . M. Aust. J. Plant Physiol. 6, 121-133 (1979). ZZ. Smill ie, R. M., and N o t t , R. Plant Physiol. 63, 796-801 (1979). Z3. Smill ie, R . M., and N o t t , R. Aust. J. Plant Physiol. 6, 135-141

(1979). Z4. Smill ie , R . M., Henningsen, K. W., Bain, J . W., C r i t c h l e y , C , F e s t e r ,

T., and von W e t t s t e i n , D. Carlrjy?,rg Res. Commun. 43, 351 364 (1978).

Z5. van Hasse l t , P . R. Acta Bot. Neerl 21, 539-548 (197Z).


LOW TEMPERATURE RESPONSE OF C H L O R O P L A S T THYLAKOIDS

M. P. Garber

Weyerhaeuse r Company Southern F o r e s t r y R e s e a r c h C e n t e r

Hot Springs, Arkansas

Low t e m p e r a t u r e is cons idered by many to be t h e single mos t l imi t ing f ac to r in t h e geograph ica l d i s t r ibu t ion of p l a n t s . Physiological dysfunct ions a r e e l i c i t ed p r imar i ly by two s e g m e n t s of t h e low t e m p e r a t u r e s p e c t r u m : (1) chil l ing t e m p e r a t u r e s (0 to a p p r o x i m a t e l y 1Z C), and (Z) subf reez ing t e m p e r a t u r e s (< 0 C) .

The physiological dysfunct ions a t chil l ing t e m p e r a t u r e s a r e co l l ec t ive ly r e f e r r e d to as "chill ing injury" and p l an t s which exhibi t dysfunct ions a r e r e f e r r e d to as "chi l l ing-sens i t ive" p l an t s , which includes many economica l ly i m p o r t a n t c rops . Al though mos t of the inves t iga t ions of chil l ing injury have involved chil l ing in t h e dark , t h e r e is subs t an t i a l ev idence sugges t ing tha t chi l l ing in the l ight is more damag ing to the p h o t o s y n t h e t i c a p p a r a t u s of ch i l l ing-sens i t ive p lan t s (3, 9> Zl) . Since under field condi t ions chil l ing t e m p e r a t u r e s a r e l ikely to occur in the p r e s e n c e of l ight , i n fo rma t ion pe r t a in ing to chil l ing in t h e l ight m a y provide a more a c c u r a t e exp lana t ion of r e d u c e d p lan t g rowth under chil l ing condi t ions . P e r t i n e n t ques t ions on l ight and chil l ing injury t h a t a r e addressed in this paper inc lude : (1) wha t a r e the s imi l a r i t i e s and d i f fe rences of chil l ing in t h e p r e s e n c e and a b s e n c e of l ight? (Z) a r e t h e r e q u i r e m e n t s for r e c o v e r y from light and chil l ing the s a m e as for da rk and chil l ing? and (3) is t h e r e d u c e d g rowth of f ie ld-grown chi l l ing-sens i t ive p l an t s m o r e a c c u r a t e l y p r e d i c t e d by l ight and chil l ing injury and t h e r e q u i r e m e n t of r e a c t i v a t i o n ?

The low t e m p e r a t u r e response of p l an t s will be discussed in r e f e r e n c e to thylakoids , t he ch loroplas t m e m b r a n e s y s t e m . Thylakoids have been u t i l i zed in t h e s tudy of low t e m p e r a t u r e injury due to the i r ea se of i so la t ion and many well def ined func t ions . Also, pho tosyn thes i s is a f f e c t e d by low t e m p e r a t u r e s and thylakoids play an i n t eg ra l ro le in t he p h o t o s y n t h e t i c p r o c e s s . However , t he u l t i m a t e u t i l i ty of t he i so la t ed thylakoid sys tem for t h e s tudy of low t e m p e r a t u r e injury may depend on how well thylakoids pa ra l l e l whole p lan t r e sponses . For i n s t a n c e , is the mechan i sm of i n a c t i v a t i o n of i so l a t ed thylakoids exposed to chill ing t e m p e r a t u r e s s imi lar to thylakoids t r e a t e d in situ?

Copyright · 1979 by Academic Press, inc. 2 0 3 All rights of reproduction in any form reserved

ISBN 012-4605605

2 0 4 Μ . P. G a r b e r

I. E F F E C T OF CHILLING TEMPERATURES ON C H L O R O P L A S T THYLAKOIDS

A. Chilling in the Dark

Most s tudies have observed chill ing injury of thylakoids in t h e absence of light and have genera l ly u t i l i zed one of two me thods of t r e a t i n g thylakoids : (1) leaf t i ssue is exposed to chil l ing t e m p e r a t u r e s and thylakoids subsequent ly i so la ted and m e m b r a n e funct ions measu red above t h e chill ing r ange — in situ t r e a t m e n t ; and (Z) thylakoids a r e first i so la ted from leaf t i s sue and then exposed to chil l ing t e m p e r a t u r e s wi th t h e m e a s u r e m e n t of m e m b r a n e funct ions a t or above t h e chil l ing r ange — in vitro t r e a t m e n t .

1. Thylakoid Response to in situ Chilling. Thylakoids from leaves of ch i l l ing-sens i t ive p lan t s chi l led in t h e dark exhibi t low Hill r e a c t i o n a c t i v i t y but good p h o t o s y n t h e t i c phosphory la t ion , as measu red by PMS-dependen t ATP synthes is (1Z), and t h e r educ t i on in PSII a c t i v i t y c o r r e l a t e d well wi th the r educ t i on in pho tosyn thes i s (9), sugges t ive t ha t PSII is t h e most sens i t ive p a r t of t h e thylakoid . Addi t ional ev idence for PSII sens i t iv i ty is the low f luo rescence y ie lds , even a t high l ight in t ens i ty , in t h e p r e s e n c e o f +DCMU and low donor -dependen t D C M U -sens i t ive r educ t i on of NADP (13). The r e d u c t i o n in PSII a c t i v i t y was a s soc ia t ed wi th a d e c r e a s e in loosely bound m a n g a n e s e and r e s t o r a t i o n of PSII a c t i v i t y was a s soc i a t ed wi th an i n c r e a s e in bound m a n g a n e s e (7, 13). Spinach, a ch i l l ing- res i s tan t p lan t , did no t lose Hill a c t i v i t y a f t e r chil l ing in the dark (9).

In t h e in Situ s tud ies , thylakoid funct ions , such as PMS-dependen t ATP synthes i s , w e r e m e a s u r e d a t 16-ZO C. T h e r e f o r e , t h e absence of a lesion in a m e m b r a n e function does not imply tha t the funct ion was u n a f f e c t e d by chil l ing t e m p e r a t u r e s but r a t h e r , any a l t e r a t i o n s t h a t occu r r ed a t low t e m p e r a t u r e s may be read i ly and c o m p l e t e l y r e v e r s e d a t above chill ing t e m p e r a t u r e s . Converse ly t h e a p p e a r a n c e of a lesion sugges ts t ha t the a l t e r a t i o n a t chil l ing t e m p e r a t u r e s is not r ead i ly r e v e r s e d . The re fo re , might a lesion such as d e c r e a s e d PSII a c t i v i t y be a r a t e - l i m i t i n g s t ep in t h e r e c o v e r y of ch lorop las t s from chil l ing injury?

2. Thylakoid Response to in vitro Chilling. The in vitro r e su l t s h a v e genera l ly been expressed as an Arrhenius p lo t , wi th a change in slope of the Arrhenius plot c o r r e l a t i n g wi th a change in m e m b r a n e f luidi ty (8). A change in t h e slope of t h e Arrhenius p lo t ( increased a c t i v a t i o n energy) for t h e pho to reduc t i on of NADP from w a t e r by ch loroplas t s from the chi l l ing-sens i t ive bean and t o m a t o occu r r ed a t 1Z C (18). A s imi lar change was no t observed for ch lorop las t s from ch i l l ing- res i s tan t p l a n t s . T e m p e r a t u r e - i n d u c e d phase changes w e r e observed by esr spec t ro scopy in thylakoids from chi l l ing-sens i t ive t o m a t o and corn , but not in thylakoids from ch i l l ing- res i s t an t p l an t s (17).

L o w T e m p e r a t u r e R e s p o n s e of C h l o r o p l a s t T h y l a k o i d s 2 0 5

The two t e m p e r a t u r e - i n d u c e d changes ( increased a c t i v a t i o n energy of e l e c t r o n t r a n s p o r t and m e m b r a n e phase change) o c c u r r e d a t a p p r o x i m a t e l y t h e s a m e t e m p e r a t u r e (12 C), which cor responds to t h e c r i t i c a l chi l l ing t e m p e r a t u r e of the p l a n t . The r e s u l t s for ch loroplas t thylakoids a r e cons i s t en t wi th t h e hypothes i s for mi tochondr i a t h a t t h e p r i m a r y e f fec t of chil l ing t e m p e r a t u r e s is a m e m b r a n e phase change (11).

The e x i s t e n c e of a phase change and i n c r e a s e d Arrhen ius a c t i v a t i o n energy has gene ra l ly been cons idered synonymous wi th ch i l l ing-sens i t ive p l an t s while ch i l l ing- res i s t an t p l an t s c h a r a c t e r i s t i c a l l y lack such a l t e r a t i o n s . T h e r e a r e , howeve r , s eve ra l s tud ies which ques t ion the concep t t h a t a m e m b r a n e phase t r ans i t i on is t he p r i m a r y mechan i sm of chil l ing injury in ch loroplas t thy lakoids . Al though a phase t r ans i t ion was no t d e t e c t e d in t o m a t o leaf thy lakoids , t h e r e w e r e a l t e r a t i o n s in t o m a t o thylakoid funct ions a t chi l l ing t e m p e r a t u r e s (7, 18), sugges t ive t ha t the lipid phase t r ans i t ion is no t a p r e r e q u i s i t e to m e m b r a n e dysfunct ion . The Ea for the pho to reduc t i on of DCIP and f e r r i cyan ide for ch i l l ing- res i s t an t and ch i l l ing-sens i t ive p l a n t s was c o n s t a n t in t h e chil l ing r a n g e . However , in the p r e s e n c e of an uncoupler of pho tophosphory la t ion thylakoids of bo th groups of p l a n t s exh ib i ted an i n c r e a s e Ea in t he chil l ing r a n g e . The inc reased Ea is cons idered to be a man i f e s t a t i on of a phase change of lipids but t h e r e s u l t s of Nolan and Smillie (14, 15) sugges t t h a t i t is no t possible t o dis t inguish ch i l l ing-sens i t ive and ch i l l ing- res i s t an t thylakoids on t h e basis of changes in Ea or phase t r ans i t ions in t he l ipids. Addi t ional ev idence for a lack of co r r e l a t i on b e t w e e n the phase t r ans i t i on and chil l ing injury comes from a compar i son of in vitro (18) and in situ s tud ies (9, 13). The in vitro s tudy showed a cons t an t Ea for PSII a c t i v i t y of ch i l l ing-sens i t ive p l an t s over t h e r a n g e of 3 to 25 C. However , PSII a c t i v i t y was t he mos t sens i t ive thylakoid funct ion under in Situ t r e a t m e n t and i t s inhibi t ion pa ra l l e l ed t h e loss of p h o t o s y n t h e sis . The loss of PSII a c t i v i t y was no t a secondary e f fec t of chil l ing inju ry or t h e resu l t of a l t e r e d me tabo l i sm s ince a ch loroplas t p r e p a r a t i o n from a m i x t u r e of fresh and cold s t o r e d l eaves had a spec i f ic a c t i v i t y about half t h a t of t h e sum of t h e two p r e p a r e d s e p a r a t e l y (12). Consequen t ly t h e a b s e n c e of a change in Ea for PSII a c t i v i t y did no t c o r r e c t l y p red ic t t h e response of PSII to in situ chi l l ing.

B. Chilling in the Light

Chil l ing in t h e l ight is m o r e damag ing to t h e p h o t o s y n t h e t i c a p p a r a t u s of ch i l l ing-sens i t ive p l a n t s than is chil l ing in t h e dark (3, 9, 21). Ini t ia l s tud ies emphas i zed e n z y m a t i c dysfunct ions (21, 22, 23) as opposed to m e m b r a n e con t ro l l ed p roce s se s .

I nac t i va t i on of thylakoids d i f fers in t h e p r e s e n c e and a b s e n c e of l ight . The r e d u c t i o n in pho tosyn thes i s under 0.5 C and light c o r r e l a t e d b e t t e r wi th t h e loss of cycl ic and non-cyc l i c phosphory la t ion than wi th t h e s lower loss of Hill a c t i v i t y (9). However , t h e au tho r s found t h a t t h e loss of pho tosyn thes i s due to chil l ing in t h e dark c o r r e l a t e d b e t t e r wi th

2 0 6 Μ. P. G a r b e r

t he loss of Hill a c t i v i t y than wi th PSI phosphory la t ion . Unfo r tuna te ly , PSII phosphoryla t ion was not m e a s u r e d . Pho tosys t em I a c t i v i t y was not a f f e c t e d by chill ing in t he dark (3, 9, 12) but was t h e most sens i t ive thylakoid function to chil l ing in l ight (Figure 1). The loss of cyc l ic phosphory la t ion p r e c e d e d t h e loss of cycl ic e l e c t r o n t r an spo r t and p ro ton u p t a k e . The ava i lab le ev idence sugges ts t ha t PSII is the thylakoid funct ion most sens i t ive to chil l ing in t h e dark w h e r e a s PSI phosphory la t ion is the thylakoid function most sens i t ive to chil l ing in the l ight . T h e r e is , however , need for a m o r e def ined c o m p a r a t i v e app roach to d e t e r m i n e the p h o t o c h e m i c a l funct ion of thylakoids most sens i t ive to chill ing in t he p r e s e n c e and absence of l ight . Addi t ional ev idence for a d i f f e rence in the inac t iva t ion of thylakoids due to dark and light chil l ing is t h a t t he ATPase a c t i v i t y of C F 1 is inhibi ted a t chil l ing t e m p e r a t u r e s in t h e p r e s e n c e of light but not in t h e dark (3).

Avai lable in fo rma t ion on t h e mechan i sm of light and chil l ing injury sugges ts t ha t t he i nac t iva t i on of thylakoids is p robably a pho to -ox ida t i ve p rocess s ince t h e r a t e of inac t iva t iop +of p ro ton u p t a k e (Figure 2), cycl ic e l e c t r o n flow (Figure 2), Ca - A T P a s e a c t i v i t y , and PMS-phosphory la t ion is d e c r e a s e d i n +t h e p r e s e n c e of a n d j n c r e a s e d in t h e p r e s e n c e of 0 ~ . The loss of Ca - A T P a s e a c t i v i t y a t 4 C in light is due

Control (percent)

Time (hours)

FIGURE 1. Cucumber thylakoids pretreated in vitro at 4 C in light. The rate of inactivation of PMS-phosphorylation exceeded that of cyclic electron transport and proton uptake, which were inactivated at the same rate. The control activities were: ΡMS-phosphorylatipn, 370μ moles Pi esterified/mg Chl/hr; proton uptake, 0.85 meq Η accum/mg Chi; cyclic electron transport (DAD/Ascorbate ^-methyl viologen), 1182 μ moles 0 9 consumed/mg Chl/hr.


Uptake (percent) Transport (percent)

Time (hours)

FIGURE 2. Isolated cucumber thylakoids exposed to 4°C in light in the presence of nitrogen (NJ, air, or oxygen (OJ. The thylakoid suspensions were bubbled with N^, oir, or O - The inhibition of proton uptake (A) and cyclic electron transport (B) is reduced in the presence of Ν2 and accelerated in the presence of 0~, suggestive that the inhibition is a photo-oxidative process.

to i nac t i va t i on of t he C F ^ e n z y m e as c o m p a r e d to a s imple r e l e a s e of C F j p a r t i c l e s from t h e m e m b r a n e . E l e c t r o n t r a n s p o r t , or t h e lack thereof , ha s been i m p l i c a t e d in t he deve lopmen t of l ight and chil l ing injury. vanHasse l t (25) sugges t s t h a t pho to -ox ida t ion of chlorophyll is due to the gene ra t i on of the ha rmfu l chlorophyll t r i p l e t s t a t e when ene rgy from absorbed light q u a n t a is not d e f l e c t e d by e l e c t r o n t r a n s p o r t . It was fur ther sugges t ed t h a t t he lag phase in chlorophyl l d e g r a d a t i o n is t h e t i m e r equ i r ed for inhibi t ion of e l e c t r o n t r a n s p o r t . It is unl ikely t h a t loss of e l e c t r o n t r anspo r t is the only p r e r e q u i s i t e to chlorophyll deg rada t i on s ince e l e c t r o n t r a n s p o r t is inhib i ted much sooner than the in i t i a t ion of chlorophyll d e g r a d a t i o n (3). However , t ha t non-cyc l i c e l e c t r o n t r anspo r t p lays a key ro le in p r e v e n t i n g pho to -ox ida t ive d a m a g e is d e m o n s t r a t e d by t h e t r e a t m e n t of thylakoids wi th NHÔH (Figure 3). The NHÔH t r e a t m e n t inhibi ts PSII a c t i v i t y y e t it a c c e l e r a t e d t h e loss of p ro ton u p t a k e , a pho tosys t em I funct ion .

D e t a i l e d inves t iga t ions in to t h e mechan i sm of l ight and chill ing injury of thylakoids of ch i l l ing-sens i t ive p l an t s would be f a c i l i t a t e d if in vitro t r e a t m e n t could be s u b s t i t u t e d for in situ t r e a t m e n t . The va l id i ty of using in vitro r e su l t s to m a k e in fe rences about in situ p h e n o m e n a has b e e n es tab l i shed for t h e s tudy of f reez ing injury (6). Ga rbe r (3) exposed cucumber thylakoids to 4 C and l ight , in situ and in vitro, and seve ra l m e m b r a n e funct ions or c o m p o n e n t s including, PMS-dependen t phosphory la t ion , p ro ton u p t a k e , o smo t i c response to sucrose , C a - A T P a s e a c t i v i t y and chlorophyll c o n t e n t w e r e mon i to red . The s equence of a c t i v i t i e s and c o m p o n e n t s lost during i nac t i va t i on was t h e s a m e with in vitro and in situ t r e a t e d thy lakoids . Also, t h e

2 0 8 Μ . P. G a r b e r

Control (percent)

Time (hours)

FIGURE 3. Cucumber thylakoids treated with NHÔH prior to in vitro exposure to 4°C in light. The NHÔH treatment specifically inhibits PSII but not PSI. Apparently non-cyclic electron transport can decrease the rate of inactivation of proton uptake, a PSI function.

mechan i sm of in vitro and in situ i n ac t i va t i on a p p e a r e d to be t h e s a m e . The addi t ion of DCCD to p a r t l y inhib i ted thylakoids did not s t i m u l a t e p ro ton u p t a k e desp i t e a r educ t ion in t h e C a - A T P a s e ac t i v i t y , sugges t ing t h a t t he C F j e n z y m e is i n a c t i v a t e d and not simply r e l e a s e d from the m e m b r a n e . In addi t ion , all thylakoids w e r e i n a c t i v a t e d to varying deg rees as opposed to t h e e x i s t e n c e of c o m p l e t e l y i n a c t i v a t e d or no rma l thy lakoids . The r e su l t s suggest t h e r e is va l id i ty in using in vitro r e su l t s to m a k e in fe rences about in situ p h e n o m e n a for c u c u m ber thylakoids exposed to 4 C in l ight . In s u m m a r y , i so la t ed thylakoids of chi l l ing-sens i t ive p lan t s can be used for t he s tudy of chil l ing injury. Also, t he man i f e s t a t i ons of chill ing injury of thylakoids a r e dependen t on t he p r e s e n c e or absence of l ight .

C. Recovery from Chilling Injury

The r e d u c e d growth of chi l l ing-sens i t ive p l an t s due to chil l ing injury will be t h e n e t resu l t of t h e e x t e n t of i nac t iva t i on and d e g r e e of r e c o v e r y . The prev ious sec t ion i nd i ca t ed t h a t t h e i nac t i va t i on of thylakoids is m o r e rapid in t he p r e s e n c e of light and t h a t t he mechan i sm of i nac t iva t i on differs from chill ing in t he dark . The possibi l i ty ex is t s t h a t t he r e q u i r e m e n t s for r e a c t i v a t i o n differ , and if so, may provide a basis for a m o r e a c c u r a t e exp lana t ion of r e d u c e d g rowth of chi l l ing-sens i t ive p lan t s under chill ing condi t ions .

The longer cucumber discs a r e exposed to 4 C in l ight t h e s lower is t h e r a t e of r e c o v e r y a t 20 C (Figure 4) which is in a g r e e m e n t wi th the r e c o v e r y of bean l eaves from chill ing in t he dark (1Z). The r a t e of r e c o v e r y of p ro ton u p t a k e was f a s t e r in t h e dark than in t h e l ight ,


20 C

80 - \ \

6 0 - \ \

4 0 - \ \

Dark

Light

0 v2 4 1 2

Time (hours) 3 4

FIGURE 4. Effect of the duration of exposure of cucumber discs to 4°C and light on the subsequent reactivation of proton uptake. Reactivation was carried out in the presence or absence of light.

sugges t ive t h a t l ight is inhibi tory to t h e r e c o v e r y of c u c u m b e r thylakoids from light and chil l ing injury. However , t he p o t e n t i a l for r e c o v e r y exis t s even in t h e p r e s e n c e of l ight . If c u c u m b e r thylakoids a r e a l lowed to r e c o v e r a t Z0 C in light and subsequent ly t r a n s f e r r e d to 20 C in dark , t h e r a t e of r e c o v e r y approaches t ha t of the dark conto l (Figure 5). In c o n t r a s t to r e c o v e r y from chil l ing in t he l ight , t h e r e c o v e r y from chill ing in t h e dark p roceeds f a s t e r in t h e p r e s e n c e of light (7, 12).

The enhanced thylakoid i nac t i va t i on due to l ight and chill ing condi t ions and the subsequent r e q u i r e m e n t s for r e c o v e r y sugges t s t ha t much of t h e chil l ing-injury a s soc i a t ed wi th f ie ld-grown chi l l ing-sens i t ive p l an t s can be m o r e a c c u r a t e l y expla ined in t e r m s of exposure to chil l ing t e m p e r a t u r e s in t h e p r e s e n c e of l ight . The inhib i ted p h o t o s y n t h e t i c a p p a r a t u s would r e c o v e r slowly during the dayl ight hours (even if t e m p e r a t u r e s r i se above the chil l ing range) wi th g r e a t e s t r e c o v e r y during the nex t da rk per iod , provided t e m p e r a t u r e s a r e suf f ic ien t ly high. The r e su l t s p rov ide an exp lana t ion for t he lack of r e c o v e r y from chill ing injury even though the day t e m p e r a t u r e s a r e above chi l l ing. A warm dark per iod would be r equ i r ed for op t imum r e c o v e r y from chil l ing injury.

D. Alterations in Thylakoids during Cold Acclimation

Chi l l ing-sens i t ive p l a n t s exhibi t a v a r i e t y of morphologica l s y m p t o m s when exposed to chil l ing t e m p e r a t u r e s , ye t a common under ly ing m e c h a n i s m of chil l ing injury has been proposed — a t e m p e r a t u r e induced physica l change in t h e lipids of m e m b r a n e s . If t h e ch i l l ing-sens i t iv i ty of p l a n t s is due to a common even t , t hen might t h e ch i l l i ng - re s i s t ance of p l an t s , such as spinach, also be t h e resu l t of a

2 1 0 Μ. P. G a r b e r

Control (percent)

100

80

60

40

20

0

4 C & light 20 C

1 2 3 4 Time (hours)

FIGURE 5. In vivo reactivation o £ proton uptake of cucumber thylakoids, following in vivo exposure to 4 C and light, as affected by the presence or absence of light. Reactivation occurred in the dark only, or in the light and then the dark.

common t r a i t ? A possibi l i ty with even g r e a t e r r ami f i ca t i ons is t h a t t h e ch i l l ing - res i s t ance and f r eez ing - r e s i s t ance ( acc l ima ted t issue) of sp inach be t he resu l t of a common p r o p e r t y of t h e ce l l . For i n s t a n c e , is the chill ing and f reez ing r e s i s t a n c e of the spinach p h o t o s y n t h e t i c a p p a r a t u s due to a " l o w - t e m p e r a t u r e - r e s i s t a n t " m e m b r a n e ?

Spinach seedl ings a r e known to undergo cold a c c l i m a t i o n in response to chil l ing t e m p e r a t u r e s , f ac i l i t a t ing the i r survival a t subf reez ing t e m p e r a t u r e s . The possibi l i ty of a l t e r a t i o n in thylakoids during a c c l i m a t i o n r e su l t ing in a more f r e e z e - r e s i s t a n t m e m b r a n e has been an issue of some con t rove r sy (4, 5, 19). Steponkus, et al., (Z0) ra i sed an i n t e r e s t i n g ques t ion in r ega rd to a l t e r a t i o n s in ce l lu lar o rgane l les , "if t h e p l a s m a l e m m a is t h e s i t e of f reezing injury and if during cold a c c l i m a t i o n p r o t e c t i o n of this m e m b r a n e is ach ieved , is it not r ea sonab le to assume tha t o the r m e m b r a n e sys t ems a c c l i m a t e , les t they b e c o m e t h e p r i m a r y s i t e of f reez ing injury?"

A l t e r a t i o n s in t h e hydrophobic region of thylakoids following a c c l i m a t i o n have been d e m o n s t r a t e d w^th t h e f r e e z e - f r a c t u r e t echn ique (Z). The re a r e 850 + 60 p a r t i c l e s / μ / m on the inner f r a c t u r e f ace of a c c l i m a t e d thylakoids c o m p a r e d to 1650 + 110 p a r t i c l e s for non-a c c l i m a t e d thylakoids . Also, t he inner f r a c t u r e f ace of n o n - a c c l i m a t e d thylakoids have two p a r t i c l e s ize groups, +100 A and + 165 A in d i a m e t e r while a c c l i m a t e d thylakoids have one s ize group, +140 A. If t h e u l t r a s t r u c t u r a l changes a r e causal ly r e l a t e d to t h e cold a c c l i m a t i o n p rocess and consequen t ly i nc reased f reez ing r e s i s t a n c e of t h e m e m b r a n e , then t h e f r eeze p r o t e c t i o n of thylakoids by sucrose should be d i f fe ren t for thylakoids i so la ted from a c c l i m a t e d and n o n - a c c l i m a t e d sp inach l eaves . In f ac t , a c c l i m a t e d thylakoids r equ i r e a Z5 mM sucrose solut ion for max imum p r o t e c t i o n of p ro ton u p t a k e whereas non-


a c c l i m a t e d thylakoids r e q u i r e 50 mM suc rose . The d i f f e rence in sucrose p r o t e c t i o n of p ro ton u p t a k e for a c c l i m a t e d and n o n - a c c l i m a t e d thylakoids sugges ts t h a t t h e f reez ing r e s i s t a n c e of a c c l i m a t e d thylakoids is a t l eas t in p a r t due to a m o r e f r e e z e - r e s i s t a n t m e m b r a n e .

A compar i son of in vitro and in situ chil l ing of sp inach thy la koids, in t h e l ight , wi th in vitro and in situ t r e a t e d cucumber thy la koids sugges t ed t h a t t h e r e s i s t a n c e of t h e sp inach p h o t o s y n t h e t i c a p p a r a t u s to chil l ing in t h e l ight is no t solely a funct ion of t h e thylakoid m e m b r a n e (Table I). The chil l ing r e s i s t a n c e of sp inach thylakoids may be due to t h e i n t e r a c t i o n of t h e thylakoids and c o m p o n e n t s of t h e s t r o m a s ince " in tac t " ch lo rop las t s r e suspended in an o s m o t i c u m w e r e i n a c t i v a t e d m o r e slowly than thylakoids washed f ree of s t r o m a l c o m p o n e n t s . However , t h e o smot i cum (sucrose) was no t e f f ec t i ve in p r o t e c t i n g washed thy lakoids . In s u m m a r y , t h e f reez ing and chil l ing r e s i s t a n c e of sp inach ch lorop las t s is appa ren t l y a funct ion of a "low-t e m p e r a t u r e - r e s i s t a n t " m e m b r a n e but t h e low t e m p e r a t u r e r e s i s t a n c e is only m a n i f e s t e d in t h e p r e s e n c e of s e l e c t i v e s t r o m a l c o m p o n e n t s . The f reez ing r e s i s t a n c e , but no t t h e chil l ing r e s i s t a n c e , of sp inach thylakoids is expressed in t h e p r e s e n c e of suc rose . The p reven t ion by sucrose of f reez ing injury but no t chil l ing injury may i m p l i c a t e a d i f fe ren t i a l s t r e s s for t h e f reez ing and chil l ing p roce s se s .

Π. E F F E C T OF SUBFREEZING TEMPERATURES ON SPINACH THYLAKOIDS

The d i f fe ren t i a l r e sponse of p l an t s to low t e m p e r a t u r e s has been c lea r ly def ined in s tud ies which d e m o n s t r a t e t ha t thylakoids of cucumber a r e i n a c t i v a t e d by chil l ing t e m p e r a t u r e s (3) whe rea s sp inach thylakoids a r e i n a c t i v a t e d by f reez ing t e m p e r a t u r e s but no t chil l ing t e m p e r a t u r e s (1,2). A c o m m o n s i t e of injury appa ren t l y ex is t s for bo th chil l ing and f reez ing . The ce l lu lar m e m b r a n e s h a v e been i m p l i c a t e d as t he p r i m a r y s i t e of chil l ing (10, 11) and f reez ing (5, 24) injury. However , no compar i son has been m a d e of m e m b r a n e lesions r e su l t i ng from chil l ing and f reez ing . The n a t u r e of chil l ing injury in c u c u m b e r thylakoids has been discussed above and a discussion of f reez ing injury in sp inach thylakoids fol lows. It should be m e n t i o n e d t h a t a p r i m a r y i m p e t u s for prev ious s tud ies on f reez ing injury in sp inach thylakoids was t h e r e a l i z a t i o n t h a t iden t i f i ca t ion of speci f ic m e m b r a n e a l t e r a t i o n s causa l to t h e cold a c c l i m a t i o n p roces s r equ i r e s e luc ida t ion of t h e p r i m a r y s i t e of f reez ing injury.

Garbe r and Steponkus (1) sub jec t ed i so la t ed thylakoids to a f r e e z e -thaw cyc le wi th t h e r a t e c o m p a r a b l e to n a t u r a l condi t ions . Two response reg ions to sucrose w e r e ev iden t sugges t ing t h e o c c u r r e n c e of a t l eas t two lesions in p ro ton u p t a k e . Nega t ive ly s t a ined thylakoids had a r e d u c e d number of C F ^ p a r t i c l e s when f rozen in t h e a b s e n c e or p r e s e n c e of low c o n c e n t r a t i o n s of sucrose , sugges t ive t h a t C F - is r e l e a s e d (1). P o s t - t h a w

2 1 2 Μ . P. G a r b e r

d e t e r m i n a t i o n of p ro ton u p t a k e and C a - A T P a s e a c t i v i t y on the s a m e samples conf i rmed t h a t t h e r e d u c e d p ro ton was due to C F - r e l e a s e (20). The p ro ton u p t a k e of thylakoids f rozen in 15 to 100 mM sucrose and r e c o n s t i t u t e d wi th C F j was only 50% of t h e con t ro l , sugges t ing a second lesion. Thylakoids f rozen in t h e p r e s e n c e of 100 μ Μ p las tocyan in fo rmed a p ro ton g rad ien t equal t o 3 3 % of t h e unf rozen con t ro l s sugges t ive t ha t p las tocyan in is r e l ea sed , a second lesion during f reez ing . The inabi l i ty of C F ^ to s t i m u l a t e t h e p ro ton g rad ien t of thylakoids f rozen in t h e absence or p r e s e n c e of 2 or 5 mM suc rose , and t h e inabi l i ty of D C C D to maximal ly s t i m u l a t e p ro ton u p t a k e below 15 mM sugges ted tha t a th i rd lesion c on t r i bu t ed to d e c r e a s e d p ro ton u p t a k e . If thylakoids w e r e frozen in 0 to 20 mM sucrose the major i ty of the thylakoids w e r e osmot ica l ly unrespons ive w h e r e a s , t h e major i ty w e r e osmot ica l ly respons ive if f rozen in sucrose c o n c e n t r a t i o n s g r e a t e r than 20 mM. The propor t ion of osmot ica l ly a c t i v e ves ic les pa ra l l e l ed t h e respons iveness of t he ves ic les to D C C D , sugges t ive t ha t t h e th i rd lesion in p ro ton u p t a k e is t h e loss of m e m b r a n e s e m i p e r m e a b i l i t y . If t h e t h r e e iden t i f ied les ions, loss of s emipe rmeab i l i t y , loss of C F ^ , and loss of p l a s tocyan in a r e t h e cause of r e d u c e d p ro ton u p t a k e then ame l io ra t i on of the lesions should resu l t in a p ro ton u p t a k e c o m p a r a b l e to t he unf rozen con t ro l . In f ac t , if thylakoids a r e f rozen in t h e p r e s e n c e of 100 μ Μ p las tocyan in , suff ic ient to p r e v e n t a n e t loss, and 100 mM sucrose , suff ic ient to p r e v e n t t he loss of s emipe rmeab i l i t y , and a f t e r thawing a r e r e c o n s t i t u t e d wi th C F ^ , they exhibit a p ro ton u p t a k e equal to the unf rozen c o n t r o l . The iden t i f i ca t ion of specif ic m e m b r a n e lesions, pa r t i cu l a r l y t h e r e l e a s e of a speci f ic p ro te in such as C F ^ , t a k e s us one s t ep c loser to t h e e luc ida t ion of t h e p r i m a r y s i t e of f reez ing injury in ce l lu lar m e m b r a n e s . A t t e n t i o n should now be d i r e c t e d to an unders tand ing of which m e m b r a n e component(s) r e t a i n C F ^ on t h e m e m b r a n e and a r e a l t e r e d by t h e f r e e z e - t h a w cyc l e . A l t e r a t i o n of such componen t s dur ing cold a c c l i m a t i o n could then be i m p l i c a t e d as causa l to t he i nc r ea sed f reez ing r e s i s t a n c e of a c c l i m a t e d sp inach thylakoids .

Iden t i f i ca t ion of f reez ing lesions in sp inach thylakoids p e r m i t s a compar i son wi th the mechan ism of l ight and chil l ing injury in cucumber thylakoids . In bo th t he f reez ing (5, 24) and chil l ing (3) s t r e s s e s , t he loss of ATP synthes is is a p r i m a r y lesion. However , t he loss of ATP synthes is due to f reez ing may be due to a l t e r e d m e m b r a n e p e r m e a b i l i t y (26) w h e r e a s under l ight and chil l ing condi t ions the a l t e r e d m e m b r a n e s e m i p e r m e a b i l i t y was d e t e c t e d subsequent to t he loss of ATP synthes i s (3). In addi t ion, f reez ing injury causes t h e r e l e a s e of C F - p a r t i c l e s (2) while chil l ing in t h e light r e su l t s in i nac t i va t i on of t h e C F ^ e n z y m e but not the r e l e a s e from the thylakoid (3). The f reez ing s t r e s s causes the uncoupling of ATP synthes is from e l e c t r o n t r anspor t w h e r e a s l ight and chil l ing inhibi ts ATP synthes is wi thou t s t imu la t i ng e l e c t r o n t r a n s p o r t . The ava i lab le in fo rmat ion ind ica t e s t h a t t he response of cucumber thylakoids to chil l ing injury and the response of spinach thylakoids to f reez ing a r e d i f fe ren t . The re fo re , t he s t r e s s during f reez ing is probably d i f fe ren t than t h e s t r e s s during chil l ing.

TABLE I. Pretreatment of Cucumber and Spinach Thylakoid In Vitro Cucumber and spinach thylakoids were exposed to 4 C in light (21,000 lux) or dark before measurement of membrane functions. The results are expressed as percent of the control (not exposed to chilling temperatures). The control activities for cucumber and spinacjh thylakoids are, respectively: 421 and 492 \imoles Pi esterified/mg Chl/hr\ 0.86 and 0.92 μ eg Η accum/mg Chi; 185 and 210 μ moles Pi liberated/mg Chl/hr and 200 μ g Chl/ml.

Time of Pretreatment (hr) Pretreatment

Sample Conditions ATP Proton uptake Ca -ATPase Chi Light Dark 1 2 3 4 5 6 2 4 6 8 4 8 12 16 24 8 16 24 32 40

Cucumber + 90 97 104 101 98 101 92 86 96 106 100 98 99 96 100 + 26 10 2 73 40 24 12 80 62 34 4 98 102 101 80 51

Spinach + 108 104 92 99 96 96 97 98 106 104 106 100 103 101 99 102 100 + 86 44 26 2 70 50 20 10 74 52 30 6 97 99 101 83 48

2 1 4 Μ. P. G a r b e r


1. Garbe r , Μ. P . and Steponkus, P . L. Plant Physiol. 57, 673-680 (1976a).

2. Garbe r , M. P . and Steponkus, P . L. Plant Physiol. 57, 681-686 (1976b).

3 . Garbe r , M. P . Plant Physiol. 59, 981-985 (1977). 4 . Heber , U. W. and Santar ius , K. A. Plant Physiol. 39, 712-719

(1964). 5. Heber , U. W. Plant Physiol. 42, 1343-1350 (1967). 6. Heber , U. W., Tyankova, L., and Santa r ius , K. A. Biochem. Bio

phys. Acta 291, 23-37 (1973). 7. Kaniuga , Z., Sochanowicz , B., Zabek, J . , and Krzys tyn iak , Κ. Ρ

Planta 140, 121-128 (1978). 8. Ke i th , A. D. and Melhorn, R. J . Chem. Phys. Lipids 8, 314-317

(1972). 9. Kislyuk, I. M., Vas'kovskii, M. D. Sov. Plant Physiol. 191, 688-692

(1972). 10. Lyons, J . M., and Raison, J . K. Plant Physiol. 45, 386-389 (1970). 11 . Lyons, J . M. Ann. Rev. Plant Physiol. 24, 445 -466 (1973) . 12. Margul ies , Μ. M., and Jagendorf , A. T. Arch. Biochem. Biophys.

90, 176-183 (1960). 13. Margul ies , Μ. M. Biochim. Biophys. Acta 267, 96-103 (1972). 14. Nolan, W. G. and Smill ie, R . M. Biochim. Biophys. Acta 440, 4 6 1 -

475 (1976). 15. Nolan, W. G. and Smill ie, R . M. Plant Physiol. 59, 1141-1145

(1977). 16. Raison, J . K., Lyons, J . M., Mehlhorn, R. J . , and Ke i th , A. D. J.

Biol. Chem. 246, 4036-4040 (1971a). 17. Raison, J . K. In "Mechanisms of Regu la t ion of P l an t Growth" (R.

L. Bieleski , A. R. Ferguson , and Μ. M. Cresswel l , eds . ) , pp 487-497 . The Royal Socie ty of New Zealand, Well ington (1974).

18. Shneyour, Α., Raison, J . K., and Smill ie, R. M. Biochim. Biophys. Acta 292, 152-161 (1973).

19. Steponkus , P . L. Plant Physiol. 47, 175-180 (1971). 20. Steponkus , P . L., Garbe r , M. P . , Myers , S. P . , and L ineberge r , R. D.

Cryobiolgoy 14, 303-321 (1977). 2 1 . Taylor , A. O. and Rowley , J . A. Plant Physiol. 47, 713-718 (1971). 22. Taylor , A. O., J epsen , Ν . M., and Chr i s t e l l e r , J . T. Plant Physiol.

49, 798-802 (1972). 23 . Taylor , A. O., Slack, C. R., and McPherson , H. G. Plant Physiol.

54, 696-701 (1974). 24. Ur ibe , E. G. and Jagendorf , A. T. Arch. Biochem. Biophys. 128,

351—359 (1968). 25. Van Hasse l t , Ph. R. Acta Bot. Neerl. 21, 539-548 (1972).


THE INFLUENCE O F CHANGES IN THE PHYSICAL PHASE OF THYLAKOID MEMBRANE LIPIDS

ON PHOTOSYNTHETIC ACTIVITY

David C. Fork

D e p a r t m e n t of P lan t Biology Ca rneg i e Ins t i tu t ion of Washington

Stanford, Cal i forn ia

I. INTRODUCTION

The ene rgy-conse rv ing r e a c t i o n s of pho tosyn thes i s , as well as t he ene rgy - l i be r a t i ng r e a c t i o n s of r e sp i r a t i on a r e found wi thin the confines of a lipid b i layer (51). C o m p o n e n t s e ssen t i a l to t h e p h o t o s y n t h e t i c p roces s a r e in many ca se s a s soc i a t ed wi th p ro t e in s , and a r e embedded or a t t a c h e d to t h e thylakoid m e m b r a n e . In this c a t e g o r y would be t he r e a c t i o n c e n t e r s of p h o t o s y s t e m s I and II a long wi th the i r r e s p e c t i v e a c c e p t o r s "Q" and t h e i ron-sulfur c e n t e r s . The highly l ipophil ic e l e c t r o n c a r r i e r , p las toquinone , s eems to be s e p a r a t e d vec to r i ly ac ross the m e m b r a n e from o the r c a r r i e r s such as c y t o c h r o m e f and p la s tocyan in . The p h o t o s y n t h e t i c a l l y - a c t i v e p i g m e n t s such as chlorophyl ls α and b,

t h e phycobi l ins and, to some e x t e n t , t h e ca ro t eno ids a r e also l o c a t e d in o r d e r e d p i g m e n t - p r o t e i n a r r ays so t h a t e f f ic ient ene rgy c a p t u r e and t r ans fe r can occu r . The e n z y m e causing ATP fo rma t ion by diss ipat ing t h e Η g rad i en t s fo rmed in t h e light ac ross p h o t o s y n t h e t i c m e m b r a n e s p r o t r u d e s from t h e su r f ace of t h e m e m b r a n e w h e r e i t is weakly held to a p ro te in subunit t ha t spans t he m e m b r a n e and is f i rmly bound within t h e m e m b r a n e (1). It would be e x p e c t e d t h a t some of t h e componen t s e s sen t i a l to pho tosyn thes i s would be a f f e c t e d by a l t e r a t i o n s in t h e physica l s t a t e of thylakoid m e m b r a n e lipids t ha t can occur wi th changes of t e m p e r a t u r e .

Carnegie Institution of Washington - Department of Plant Biology Publication No. 664.

copyright · 1979 by Academic Press, inc. All rights of reproduction In any form reserved

I S B N a i 2 46056a 5 2 1 5

2 1 6 D. C. F o r k

Π. THERMOTROPIC PHASE TRANSITIONS

M e m b r a n e lipids undergo a phase t r ans i t ion from a random or fluid phase a t high t e m p e r a t u r e to an o rde red c rys t a l l i ne (gel or solid) phase a t low t e m p e r a t u r e . The t e m p e r a t u r e (T ) a t which phase t r ans i t ions begin depends upon the kind of lipid, the leng th of the acyl hydrocarbon chains , the i r s a t u r a t i o n and the hydra t ion of t h e s amp le . Lipids in the fluid s t a t e can undergo r o t a t i o n a l i somer i za t ion about the C-C bonds of the f a t t y acid cha ins . In this s t a t e t he lipid molecu les can diffuse rapidly within the p lane of the bi layer (23) but not ac ross the m e m b r a n e (45). Lipid phase t r ans i t ions can occur over a very nar row or b road t e m p e r a t u r e r ange depending upon the pur i ty of the s y s t e m . In a model m e m b r a n e sys tem composed of 1,2 d ipa lmi toyl phosphat idyl chol ine , for example , t h e t e m p e r a t u r e s of onse t (T ) and comple t ion of me l t ing occur within severa l deg ree s (8). In a m e m b r a n e sys tem composed of two or more lipid types t he changes in s t a t e can occur a t two or m o r e t e m p e r a t u r e reg ions c h a r a c t e r i s t i c of the individual lipids or be fused to form one broad t e m p e r a t u r e region t h a t begins wi th t h e onse t and ends wi th t he comple t ion of me l t i ng .

A. Fluorescent Probes in Model Membrane Systems

Figure 1 shows a d i a g r a m m a t i c p r e s e n t a t i o n of phase t r ans i t ions in model m e m b r a n e s composed of two lipid spec ies . The upper p a r t of t h e f igures shows t h e resu l t e x p e c t e d using d i f fe ren t ia l scanning c a l o r i m e t r y (DSC) and the b o t t o m p a r t shows t he e x p e c t e d t e m p e r a t u r e d e p e n d e n c e of f luorescence yield using a f luorescen t p robe . In A t h e e n d o t h e r m i c t r ans i t ion occurs in two d i s t inc t ly s e p a r a t e d reg ions . In Β t h e phase t r ans i t ion occurs cont inuously over a wide r ange of t e m p e r a t u r e s . Above Τ. . , in both cases t he lipids a r e in t h e liquid c rys t a l l ine s t a t e and below ^low

t^

i e ^ P ^

s a re *

n t^

i e or s° l i d s t a t e . At t e m p e r a t u r e s b e t w e e n

T j q w and T^. ^ t h e lipids consis t of a m i x t u r e of t h e solid and liquid c rys ta l l ine s f a t e s . The lower p a r t of t he f igure r e p r e s e n t s d i a g r a m m a t i c a l l y wha t would be e x p e c t e d for t h e yield of f luo rescence e m i t t e d from f luorescen t p robes . In F ig . 1A two sigmoid curves would be seen . The beginning of t he first s t e ep f luorescence i n c r e a s e cor responds to t he onse t of t he s t e e p r i se of t h e first e n d o t h e r m i c t r ans i t ion . The peak of t he endo the rm and t h e mid-poin t of t he s t e e p f luo rescence r i se cor respond to t h e t e m p e r a t u r e w h e r e t h e t r ans i t ion occurs a t i t s max imum r a t e . The f luorescence max imum cor responds to t h e end of t h e gel to liquid c rys ta l l ine t r ans i t ion . In t he s i tua t ion i l l u s t r a t ed in Fig . IB t h e lipid compos i t ion is such t ha t only one b road endo the rm is seen . The f luorescence shows a broad minimum and max imum a t Tj and T, . ^ cor responding to t h e beginning and end of t he gel to liqu^F crystalline s t a t e s r e s p e c t i v e l y .

C h a n g e s in t h e P h y s i c a l P h a s e of T h y l a k o i d 2 1 7

FIGURE 1. Diagrammatic representation of phase transitions detected by calorimetric and fluorescent probe techniques in model membrane systems composed to two lipid species. See text for details.

B. Chlorophyll a - an Extrinsic and Intrinsic Fluorescent Probe

Colbow (9) found chlorophyll a to be a sens i t ive p robe of phase t r ans i t ion in phospholipid ves i c l e s . H e a t i n g l iposomes p r e p a r e d wi th chlorophyll and d ipa lmi toyl phosphat idyl chol ine (DPPC) p roduced a l a rge s igmoidal f l uo rescence i nc r ea se beginning a t 41 C, t he t e m p e r a t u r e of t r ans i t ion of D P P C from the gel to t he liquid c rys ta l l ine s t a t e . Colbow (9) and Lee (24) sugges t t ha t chlorophyll molecu les in the gel s t a t e of D P P C a r e so closely spaced t h a t a c o n c e n t r a t i o n quenching e f fec t r e su l t s in low f luo re scence . Increas ing t e m p e r a t u r e s give r i se to a dec reas ing quenching and a f l uo rescence i n c r e a s e s ince t he chlorophyll molecu les a r e no t as c losely p a c k e d in t h e la rger vo lume ava i lab le in t h e liquid c rys t a l l i ne s t a t e .

2 1 8 D. C. Fork

Another exp lana t ion of this e f fec t (32) used t h e analogy to t he behavior of a r t i f i c ia l f luorescent p robes such as l - a n i l i n o n a p h t h a l e n e - 8 -su l fona te or Ν -pheny l - 1-naphthalene t h a t a r e p a r t i t i o n e d in model m e m b r a n e s b e t w e e n t h e hydrophobic and t h e aqueous phase and f luoresce when they a r e bound to t he m e m b r a n e (41). In t h e gel s t a t e f luo rescence is low b e c a u s e t h e hydrophobic i ty is low and t h e number of bound probes is low. Trans i t ion to t h e m o r e hydrophobic liquid c rys t a l l ine s t a t e r e su l t s in m o r e bound probes and i n c r e a s e d f luo rescence . It is possible t ha t bo th of t he se ideas may be used to some e x t e n t to explain the f luorescence behavior of chlorophyll a when it is a s soc i a t ed with lipid m e m b r a n e s .

It appea r s t ha t chlorophyll a c t s as an in t r ins ic f luorescen t p robe in n a t u r a l m e m b r a n e s . This is f o r t u n a t e s ince chlorophyll is loca l ized exclusively in the thylakoid m e m b r a n e and added a r t i f i c ia l f luorescen t p robes have so far no t worked well in p h o t o s y n t h e t i c s y s t e m s (32). The use of chlorophyll as a n a t u r a l f luorescen t p robe r e s t s on the assumpt ion t h a t it behaves in t h e i n t a c t sys tem as in t he m e m b r a n e sys t ems desc r ibed above and on the co r r e spondence b e t w e e n phase t r ans i t ion t e m p e r a t u r e s d e t e r m i n e d by the f luo rescence t echn ique and those d e t e r m i n e d by s tud ies on X-ray d i f f rac t ion and d i f fe ren t ia l t h e r m a l ana lyses (N. Mura t a , persona l commun ica t i on ) . A fur ther co r re spondence was also n o t e d b e t w e e n phase t r ans i t ion t e m p e r a t u r e s as d e t e r m i n e d by f luorescence m e a s u r e m e n t s and those ob ta ined using t h e spin label 5-SAL tha t was added to thylakoid m e m b r a n e f r agmen t s p r e p a r e d from Anacystis nidulans grown a t 28 and 38°C (36).

III. THE E F F E C T OF PHASE TRANSITIONS ON PHOTOSYNTHETIC ACTIVITY

The s tudies descr ibed h e r e w e r e a imed a t ident i fying t e m p e r a t u r e induced changes in the physical phase of the thylakoid m e m b r a n e lipids by examining a wide v a r i e t y of p l an t s t h a t a r e capab le of ca r ry ing out photosynthes i s a t t e m p e r a t u r e s ranging from 75 C to nea r f reez ing and s tudying the e f fec t t hese phase t r ans i t ions have on p h o t o s y n t h e t i c funct ion.

A. Higher Plants

1. Phase Transitions Detected by Chlorophyll Fluorescence. A number of s tud ies have been d i r e c t e d a t ident i fying the phase t r ans i t ions of m e m b r a n e lipids in h igher p l a n t s using chlorophyll α as an in t r ins ic f luorescen t p robe . With t he excep t ion of t h e t h e r m o and xerophi l ic dese r t annual Tidestromia oblongifolia t h a t had a t r a n s i t ion nea r 5°C (33), no indica t ions of phase changes w e r e seen in t he reg ion from 0°C to about 22°C for sp inach (field grown) or for l e t t u c e


and t o m a t o grown a t 15 and 25^C (31). Ch lo rop las t s e x t r a c t e d from spinach and l e t t u c e l eaves and r e suspended in 50% e t h y l e n e glycol and 5 mM Mg showed broad f l uo re scence m a x i m a c e n t e r e d +a r o u n d -31 C. In t e res t ing ly , when t h e Mg was o m i t t e d and 5 mM Na s u b s t i t u t e d in t h e medium these m a x i m a shi f ted to h igher t e m p e r a t u r e s [ to - 2 0 ° C for sp inach and to -11 C for l e t t u c e (34) ] . This lower ing of t h e f reez ing point by d iva len t ca t ions is oppos i te to i t s e f fec t in model s y s t e m s (53) w h e r e addi t ion of Mg norma l ly r a i se s ^the t e m p e r a t u r e for t h e lipid phase t r ans i t ion p resumab ly b e c a u s e Mg s t ab i l i zes t h e gel phase by r emov ing t h e e l e c t r o s t a t i c repuls ion b e t w e e n polar head groups . Ca l c ium ions induced a phase s e p a r a t i o n of phospha t idy l se r ine and phospha t idy lchol ine m e m b r a n e s in to a solid phase of phospha t idy l se r ine br idged by ca lc ium che la t ion and a fluid phase of phospha t idy lchol ine molecu les (39).

Since lipids of thylakoid m e m b r a n e s from ch lorop las t s of h igher p l an t s and a lgae (with the excep t ion of some b lue -g reen and r ed algae) h a v e high c o n t e n t s of u n s a t u r a t e d lipids such as l inolenic ac id (16), it is r e a s o n a b l e to expec t t ha t the c o - o p e r a t i v e phase t r ans i t ions in t he se p l an t s would occur a t t e m p e r a t u r e s below f reez ing . Shipley et al., (49) used pur i f ied lipids from a h igher p lan t and found phase t r ans i t ions

a t - 30 C for monoga lac tosy ld ig lyce r ide (MGDG) and a t - 50 C for d iga lac tosy ld ig lyce r ide (DGDG).

Studies desc r ibed above t h a t used the f luo rescence of chlorophyll a of l eaves and ch lorop las t s and those done on pur i f ied lipids from higher p l an t s sugges t t h a t t he major phase t r ans i t ions of t h e bulk lipids occur in vivo in most p l an t thylakoid m e m b r a n e s a t ve ry low t e m p e r a t u r e s .

2. Electron Transfer Reactions. The Hill r e a c t i o n m e a s u r e d in l e t t u c e ch lorop las t s grown a t 25 C showed no b reaks in t h e Arrhenius plot of the in i t ia l r a t e of r educ t ion of 2 ,6-dichlorophenolindophenol (DCIP) as a funct ion of t e m p e r a t u r e over t he r a n g e from about 8 t o 33 C (35).

Inoue (20) found b reaks in Arrhen ius p lo t s of DCIP php^toreduction occur r ing a t -9 C in sp inach f r a g m e n t s (without adding Mg ) t h a t w e r e ob ta ined from p l an t s grown a t low and high t e m p e r a t u r e s . Ano the r b r e a k was seen a t 10 C but only in f r a g m e n t s p r e p a r e d from spinach grown in chil l ing t e m p e r a t u r e s . The 10 C b reak was no t cons idered to be p roduced by a m e m b r a n e phase t r ans i t i on s ince i t was s t i l l seen even a f t e r t h e m e m b r a n e was d i s rup ted by digi tonin t r e a t m e n t . D e t e r g e n t t r e a t m e n t is known (13) to abolish e f f ec t s t h a t r e q u i r e i n t a c t m e m b r a n e s such as t h e 515 nm c a r o t e n o i d shift (discussed l a t e r ) .

The t e m p e r a t u r e d e p e n d e n c e of e l e c t r o n t r an spo r t in l e t t u c e ch lorop las t s was also followed by measur ing the r a t e a t which oxidized P700 can be r e d u c e d in t h e dark upon e x c i t a t i o n of p h o t o s y s t e m Π with a 3 μ s ec flash of high i n t ens i t y r e d ac t i n i c l ight t h a t r e s u l t e d in t h e p roduc t ion of one e l e c t r o n (32). This e x p e r i m e n t avoids t h e need of adding exogenous e l e c t r o n c a r r i e r s and, unl ike t h e Hill r e a c t i o n wi th DCIP , involves all t h e e l e c t r o n t r an spo r t c a r r i e r s such as "Q", t h e

2 2 0 D. C F o r k

p r i m a r y a c c e p t o r of pho tosys t em Π, secondary a c c e p t o r s , R and p las toqu inone , c y t o c h r o m e f and p las tocyan in t h a t funct ion b e t w e e n p h o t o s y s t e m s I and Π. Over t he r ange from 9 to 23 C, t he Arrhenius plot also showed no b reaks or inf lec t ion po in t s . The d i f fe ren t por t ions of t h e e l e c t r o n t r anspo r t chain involved in t h e Hill r e a c t i o n wi th D C I P and t h e e l e c t r o n t r anspor t in Vi'vo seem to be r e f l e c t e d in t h e d i f fe ren t a c t i v a t i o n energ ies found - 10 k c a l / m o l e for t h e DCIP Hill r e a c t i o n and 28 k c a l / m o l e for t h e in Vi'vo P700 r educ t i on . R e d u c t i o n of DCIP may be r a t e l imi t ed a t t h e s t ep involving t h e dark oxida t ion of t h e l ipophilic e l e c t r o n t r anspor t c a r r i e r p las toqu inone while t h e in v i v o sy s t em, as expla ined above , involves all t h e e l e c t r o n c a r r i e r s and may have a d i f fe ren t r a t e - l i m i t i n g s t e p .

M e a s u r e m e n t s w e r e m a d e by Cox (10) of t h e r a t e s of da rk r educ t ion of c y t o c h r o m e f and P700 t h a t w e r e previously oxidized in t h e l ight . Breaks w e r e seen in t h e Arrhenius p lo t s nea r - 35 C for spinach ch lorop las t s suspended in a medium con ta in ing e t h y l e n e glycol and Mg This t e m p e r a t u r e cor responds to t h e phase t r ans i t ion seen a t -31 C in spinach ch lorop las t s as desc r ibed ea r l i e r .

The pho to reduc t ion of NADP also r equ i r e s t h e c o m p l e t e e l e c t r o n t r an spo r t s equence and t h e coope ra t ion of bo th p h o t o s y s t e m s . Shneyour et al., (50) using ch lo rop l a s t s j j r epa r ed from chil l ing sens i t ive p l an t s found a change in slope nea r 12 C in t he Arrhenius plot of t h e a c t i v i t y of f e r r edox in -NADP. By c o n t r a s t , Riov and Brown (44) using ch lorop las t s p r e p a r e d from ha rdened and non-ha rdened (chill sensi t ive) whea t and found no b reaks in t he Arrhenius p lo t for N A D P r e d u c t a s e a c t i v i t y .

3. Cation Induced Membrane Effects. Addi t ion of d iva len t c a t i ons to ch lorop las t s in a low sal t medium causes i nc r ea sed yield of chlorophyll α f l uo rescence appa ren t l y as a r e su l t of conf igura t iona l changes t h a t a l t e r t h e ef f ic iency of e x c i t a t i o n energy t r ans fe r b e t w e e n t h e two p igmen t s y s t e m s of pho tosyn thes i s (18, 29, 3 1 , 35). In both l e t t u c e and spinach ch lorop las t s t he r a t e of this ca t ion - induced f luo rescence i n c r e a s e was s t rongly t e m p e r a t u r e d e p e n d e n t . The Arrhenius plot for spinach and l e t t u c e (grown a t 15 or 25 C) gave no ev idence for d i scont inu i t i es in t he t e m p e r a t u r e r ange from about 5 to 35°C (36).

4. Pigment and Ion Permeability Changes in Thylakoid Membranes. The absorp t ion of light by the r e a c t i o n c e n t e r s of pho tosyn thes i s gives r i se t o n e g a t i v e cha rges on t h e ou t s ide and pos i t ive cha rges on the inside of t he thylakoid m e m b r a n e . The e l e c t r i c field t ha t is g e n e r a t e d by t he se cha rge sepa ra t ions can be of such magn i tude t h a t t h e absorp t ion m a x i m a of p i g m e n t s t h a t a r e embedded in t h e m e m b r a n e such as ca ro t eno ids and chlorophylls a r e shi f ted to longer wave leng ths in t h e light (55). In t h e dark t h e max ima r e t u r n to the i r or iginal pos i t ions . These abso rbance changes a r e l a rge and can easi ly be seen in all p h o t o s y n t h e t i c o rganisms so far examined (including t h e p h o t o s y n t h e t i c b a c t e r i a but excluding t h e b lue -g reen a lgae) .


Changes of this type a r e most commonly m e a s u r e d a t t he 515 nm pos i t ive max imum in the g reen a lgae and higher p l an t s and seem to be caused mainly by β - c a r o t e n e . The k ine t i c s of t h e 515 nm change (or "shift") a r e complex . The in i t ia l r i se in t h e light occur s within 20 nsec (56). But cont inuous i l luminat ion leads to a s t e a d y s t a t e pos i t ive 515 nm change t h a t appea r s to be p roduced by t h e fo rma t ion of a t r a n s i e n t diffusion p o t e n t i a l ac ross t h e m e m b r a n e as a r e su l t of t h e d i f fe ren t ia l p e r m e a b i l i t y of t h e thylakoid m e m b r a n e to c e r t a i n ions (21). P ro tons a c c u m u l a t e ins ide t he thylakoid s p a c e in t he light and a r e r e l e a s e d to t he ou t s ide medium in t h e dark (37, 47 , 48).

In i n t a c t l eaves t h e d e c a y of t h e 515 nm absorp t ion change is fas t , about 100 m s e c (33), p robably as a resu l t of t he diss ipat ion of t h e l igh t -induced Η g rad ien t t h a t is t igh t ly coupled to phosphory la t ion . The d e c a y of t h e 515 nm change in i so l a t ed ch lorop las t s can be much s lower [10 s e c , Wakama t su et al., (54)] s ince phosphoryla t ion would be e x p e c t e d to be less well coupled in ch lo rop las t s as c o m p a r e d to i n t a c t l e a v e s . The s a m e t r end in decay t i m e s is seen in ch lo rop las t s given flash i l lumina t ion . The d e c a y t i m e of t h e 515 nm change under non-phosphory la t ing condi t ions is a round 200 msec but is a c c e l e r a t e d up to about 5 t i m e s to 40 m s e c upon addi t ion of A D P , inorganic phospha te and Mg to p r o m o t e phosphoryla t ion (56).

The t e m p e r a t u r e d e p e n d e n c e of t h e dark decay of t h e 515 nm abso rbance change in l eaves of t h e chil l ing r e s i s t a n t p l an t s sp inach and l e t t u c e (grown a t 15 and 25 C) showed no d i scon t inu i t i e s in t he Arrhenius p lo t s in t h e t e m p e r a t u r e r a n g e from n e a r f reez ing to about 28 C (33). But l eaves of T ide s t romia die , however , show a b reak a t 5 C in t he Arrhen ius plot for t h e d e c a y of t h e 515 nm c h a n g e . This d i scon t inu i ty was seen bo th on dec reas ing and inc reas ing t h e t e m p e r a t u r e of t h e leaf. In t e re s t ing ly , t h e decay of t h e 515 nm change was monophas ic when t h e m e m b r a n e lipids w e r e in t h e liquid c rys t a l l i ne s t a t e . But below about 5 C, cor responding to t h e beginning of t h e phase s e p a r a t i o n s t a t e as r e v e a l e d by f l uo re scence m e a s u r e m e n t s , t h e decay of t h e 515 nm change b e c a m e biphas ic wi th t he a p p e a r a n c e of a n o t h e r , m o r e rap id phase .

Leaves from chil l ing sens i t ive p l an t s t o m a t o and bean w e r e also used (33). The Arrhen ius cu rve for t h e r a t e of t h e dark decay of t h e 515 nm

ο ο change ob ta ined using t o m a t o grown a t 15 C had a b reak n e a r 12 C. It was seen , p a r t i c u l a r l y in t he curves for bean and t o m a t o grown a t 25 C, t h a t a t t he b reak poin ts t h e l ines not only changed slope but also exh ib i t ed an abrup t d i s p l a c e m e n t to new pos i t ions on t h e graph .

So far no c l ea r ind ica t ions of phase t r ans i t ions h a v e been seen above 0 C using t o m a t o or bean in m e a s u r e m e n t s of t h e f luorescence of chlorophyll a. However , Ra ison (43) has sugges ted on t h e basis of s tud ies of epr wi th spin p robes t h a t phase t r ans i t ions do occur a round 10 C in chil l ing sens i t ive p l a n t s .

Since i t is known t h a t ions and smal l molecu les diffuse m o r e rapidly ac ros s model m e m b r a n e s t h a t a r e in t h e phase s e p a r a t i o n s t a t e (3, 4, 19, 38, 42), t h e ab rup t i nc r ea se of t h e decay r a t e of t h e 515 nm change below t h e phase t r ans i t ion t e m p e r a t u r e sugges t s t h a t t h e thylakoid m e m b r a n e

2 2 2 D. C. Fork

b e c o m e s leaky to ions in the p h a s e - s e p a r a t i o n s t a t e . If phosphoryla t ion is coupled to ion m o v e m e n t s ac ross m e m b r a n e s as sugges ted by the Mitchel l (26, 27) hypothes is then then it would appea r t h a t phosphoryla t ion would be g rea t ly impa i red a t low t e m p e r a t u r e s . Ono and M u r a t a (40) have r e c e n t l y shown t h a t phosphoryla t ion in Anacystis d e cl ines a t a t e m p e r a t u r e cor responding to the onse t of t h e phase t r ans i t ion from t h e liquid c rys ta l l ine to t h e phase s epa ra t i on s t a t e s . This obse rva t ion sugges t s t h a t e i the r t h e ion g rad ien t ac ross t he thylakoid m e m b r a n e and /o r t h e a c t i v i t y of C F _ , t he coupling f ac to r for photophosphory la t ion , is a f f e c t e d by this pnase t r ans i t i on .

The f luorescen t ind ica to r 9-amino ac r id ine (9AA) can be used to follow the r a t e of da rk decay of t h e pH g rad ien t p roduced previously ac ross t h e thylakoid m e m b r a n e in t h e l ight (47). When added to a chloroplas t suspension this l ipid-soluble amine can diffuse ac ross t he m e m b r a n e to es tabl i sh equil ibr ium b e t w e e n t h e inside and ou t s ide of t h e thylakoid s p a c e . P ro tons t ha t a c c u m u l a t e in t h e l ight within t h e thylakoid space combine with t h e f ree amine to form t h e non- f luorescen t p r o t o n a t e d form t h a t canno t diffuse ac ross t h e m e m b r a n e . Thus ch lorop las t s in t h e p r e s e n c e of 9AA show a f luo rescence d e c r e a s e upon i l luminat ion . F l u o r e s c e n c e inc reases again in t h e dark when t h e p r o t o n a t e d amine d i sassoc ia tes and the p ro tons a c c u m u l a t e d in t h e light and bound to 9AA a r e r e l e a s e d and diffuse out of t h e thylakoid s p a c e . This p ro ton r e l e a s e in t he dark can be followed as a f luo rescence i nc r ea se a t 500 n m . The in i t ia l r a t e s of f luo rescence i n c r e a s e w e r e t a k e n as a m e a s u r e of t h e r a t e of p ro ton efflux from chloroplas t p r e p a r a t i o n s ob ta ined from chill ing sens i t ive and chill ing r e s i s t a n t p l a n t s .

M e a s u r e m e n t s s t a r t i n g a t 10 C and inc reas ing t h e t e m p e r a t u r e in t he dese r t shrub Tic.€Stromia gave an Arrhenius p lo t having a s t r a igh t l ine up to about 27 C when a s t e e p i nc r ea se was seen (2). The b reak a t 27 C s e e m s not to be r e l a t e d to a phase t r ans i t ion but r a t h e r to an i r revers ib le i nac t iva t i on e f fec t s ince t h e poin ts above 27 C followed a cont inuous ly-upward sloping cu rve . D e c r e a s i n g t h e t e m p e r a t u r e from poin ts above 27 C did not p roduce t he d i scont inu i ty a t 27 C aga in . Ins tead , t he poin ts followed a s t r a igh t l ine t h a t r e p r e s e n t e d fa s t e r decay t i m e s than seen be fo re . If t h e t e m p e r a t u r e was ra i sed jus t to t h e 27 C point and then lowered i m m e d i a t e l y in t h e Tidestromia ch loroplas t f r agmen t s then the two l ines followed a lmos t t h e s a m e s lope. If t he s t a r t i n g t e m p e r a t u r e was around 8°C and was i nc reased only up to about 20 C and then lowered again , t h e poin ts of t h e Arrhen ius plot followed along t h e s a m e l ine . In this ca se changes in s lope of t h e l ine appea red n e a r 9.5 and 5 C . The 5 C point was observed in seve ra l o the r e x p e r i m e n t s wi th Tidestromia and s e e m s to be a r e f l ec t i on of a phase t r ans i t ion as r e v e a l e d by the f luo rescence m e a s u r e m e n t s desc r ibed above . Similar m e a s u r e m e n t s w e r e also m a d e using bean , an o th e r chil l ing sens i t ive p l an t . H e r e an anomoly was seen in t h e Arrhenius plot nea r 5 C w h e r e t h e l ine j u m p e d to a new posi t ion on t h e graph . Above about 18 C t h e po in ts for t he r a t e s of p ro ton efflux began to r i se s t eep ly . This t e m p e r a t u r e would seem to cor respond to t h e 27 C point desc r ibed above for Tidestromia.


M e a s u r e m e n t s of 9AA f luo rescence m a d e upon cooling chloroplas t f r agmen t s of a chil l ing r e s i s t a n t p lan t (spinach) from 17 to nea r 3 C showed no b r eaks in the Arrhenius p lo t . Also, no d i scon t inu i t i es w e r e seen upon inc reas ing t h e t e m p e r a t u r e again; t h e po in t s fell on t h e s a m e l ine as ob ta ined be fo re . D e c r e a s i n g the t e m p e r a t u r e from t e m p e r a t u r e s above ZO C gave poin ts following a s t r a igh t l ine t h a t r e p r e s e n t e d f a s t e r decay t i m e s than those observed in i t ia l ly . This r esu l t sugges t s an i r r eve r s ib le i nac t iva t i on of sp inach ch lorop las t s l ike t ha t desc r ibed above for Tidestromia.

These r e su l t s using 9 A A, and those desc r ibed above for t h e 515 nm change , sugges t t h a t below t h e phase t r ans i t i on t e m p e r a t u r e t h e m e m b r a n e b e c o m e s l eaky to ions and t h a t c l ea r d i f f e rences can be seen b e t w e e n chil l ing sens i t ive and chil l ing r e s i s t a n t p l an t s when measur ing t he se e f f e c t s .

B. Blue-Green Algae

1. Phase Transitions Detected by Chlorophyll Fluorescence. Probably t h e most e x t r e m e case of a chil l ing sens i t ive p lan t occur s in the ob l iga te the rmophi l i c b lue -g reen a lga Synechococcus lividus. This a lga has been r e p o r t e d (6, 7, ZZ) to pho tosyn thes i ze up to 75°C , t he h ighes t t e m p e r a t u r e s so far r e c o r d e d for this p r o c e s s . These a lgae a r e ob l iga te the rmoph i l e s (Z5). They will no t survive in t h e l ight a t room t e m p e r a t u r e but can be kep t in t h e dark a t th is t e m p e r a t u r e for some weeks (R. C a s t e n h o l z , pe rsona l commun ica t i on ) .

Phase t r ans i t ions w e r e m e a s u r e d (1Z) for a number of d i f fe ren t "s t ra ins" of th is a lga t h a t w e r e i so la t ed from seve ra l d i f fe ren t t e m p e r a t u r e n iches in n e u t r a l and a lka l ine hot spr ings . The curves for t h e t e m p e r a t u r e dependence of chlorophyll a f l uo rescence showed m a x i m a (or shoulders) from 40 to 4Z C on dec rea s ing and inc reas ing t h e t e m p e r a t u r e r e s p e c t i v e l y in cel ls t h a t w e r e grown a t t e m p e r a t u r e s rang ing from 55 to 65 C (Fig. ZD). Cel ls growing a t 55 C w e r e a d a p t e d to grow a t 38 C. The t e m p e r a t u r e d e p e n d e n c e of f luo rescence in these cel ls showed b reaks nea r ZZ and Z5 C upon dec reas ing and increas ing the t e m p e r a t u r e r e s p e c t i v e l y (15).

F igure ZA shows examples of t h e use of chlorophyll α f luorescence to d e t e c t phase t r ans i t ions in i n t a c t cel ls of Anacystis grown a t 38 C. In this c a s e a shoulder n e a r Z4°C and a b road max imum around 16 C w e r e seen upon cooling t h e ce l l s . R e h e a t i n g p roduced a b road max imum nea r Z1°C.

2. Phase Transitions in Phospho- and Glycolipids from Algae. Vesicles p r e p a r e d by suspending in buffer t h e phospholipids e x t r a c t e d from Synechococcus grown a t 55 C showed phase t r ans i t ions a t 39 and 38 C upon inc reas ing and dec rea s ing t h e t e m p e r a t u r e r e s p e c t i v e l y . Vesicles p r e p a r e d from t h e glycolipid f r ac t ion had m a x i m a a t 40 and 39 C upon inc reas ing and dec reas ing t h e t e m p e r a t u r e r e spec t i ve ly .

2 2 4 D. C. F o r k

Anacys+is

3 8 °C

Synechococcus

5 5 °C

10 2 0 3 0 4 0 5 0 6 0

Temperature, °C

FIGURE 2. Temperature dependence of chlorophyll a fluorescence in cells of the blue-green algae Anacys£is nidulans (grown at 38 C) (31), and Synechococcus lividus (grown at 55 C (15) and in vesicles made from lipids extracted from both types of algae.

Partially purified phospho- and glycolipids (mono and digalactosyldiglyceride and sulfoquinovosyl diglyceride from Anacystis that contained a trace of chlorophyll a were suspended in 10 mM phosphate buffer (pH 7.6), shaken with glass beads and sonicated for several minutes to form vesicles. Vesicles were made in a similar way


These phase t r ans i t ion t e m p e r a t u r e s in t h e model s y s t e m s a r e s o m e w h a t lower for bo th lipid c lasses c o m p a r e d to t h e i n t a c t ce l l s .

F igu re 2B and C shows t h a t chlorophyl l α-conta in ing ves ic les p r e p a r e d using ghospholipid and glycol ipids e x t r a c t e d from Anacystis grown a t 38 C showed phase t r a n s i t i o n s in t h e t e m p e r a t u r e r a n g e from 21 to 24 C s imi la r to t h e t e m p e r a t u r e s seen for whole ce l l s .

The ves ic les used in t he se s tud ie s w e r e p r e p a r e d from the phospho-and glycolipid f rac t ions ob ta ined from the whole ce l l s . P re sumab ly , the lipids o r ig ina t ed mainly from t h e thylakoids s ince in b lue -g reen a lgae t h e r e is an ex t ens ive thylakoid m e m b r a n e sys tem d ispersed throughout t h e cel l of which t h e c y t o p l a s m i c m e m b r a n e s m a k e up only a smal l f r ac t ion .

All t h e phase t r ans i t i ons i l l u s t r a t e d in F igu re 2 as observed by f luo rescence m e a s u r e m e n t s a r e r e v e r s i b l e . These t r ans i t i ons in the ves ic les (as well as i n t a c t cells) show a hys t e re s i s e f fec t l ike t h a t desc r ibed by Traub le and O v e r a t h (52) for biological and model m e m b r a n e s y s t e m s .

The f luo rescence curves shown in F igure 2 a r e l ike example Β of F igure 1 w h e r e the f l uo rescence max imum r e p r e s e n t s the beginning of t h e phase t r ans i t i on from t h e l iqu id-c rys ta l l ine to t he phase s epa ra t i on s t a t e . No min ima w e r e seen in t he cu rves shown in F ig . 2 (except pe rhaps for curves given in D). This sugges t s t h a t t he t r ans i t ion from t h e phase s epa ra t i on to the solid s t a t e s had not o c c u r r e d within the t e m p e r a t u r e r a n g e used. F l u o r e s c e n c e versus t e m p e r a t u r e curves of t h e type shown in A of F ig . 2 a r e no t c o m m o n . So far only t h e a lga Cyanidium caldari-um has b e e n seen to have this type of cu rve (11, 32).

3. The Effect of Growth Temperature Changes on Lipid Composition. The f a t t y ac id compos i t ion of Synechococcus is l ike t h a t of t h e r e l a t e d b lue -g reen a lga Anacystis nidulans in having only s a t u r a t e d and m o n o u n s a t u r a t e d f a t t y ac ids . P o l y u n s a t u r a t e d f a t t y ac ids

from the more purified lipids extracted from Synechococcus but in this case chlorophyll a extracted from sunflower leaves was added to give a chlorophyll: lipid ratio of 1:400.

Whole cells were treated with DCMU to inhibit photosynthetic electron transport reactions. The fluorescence of cells was excited with 430 nm light and measured at 684 nm. For the lipid vesicles the excitation wavelength was 433 nm and the fluorescence measured at 672 nm. The arrows indicate the direction of temperature change. The experiments with lipid vesicles were done in collaboration with G. van Ginkel.

2 2 6 D. C. F o r k

such as l inoleic and l inolenic a r e absen t from both of t he se the rmophi l i c a lgae . Synechococcus con ta ins only four major lipids (15) as do o the r b lue -g reen a lgae : Mono- and d iga lac tosy ld ig lycer ide (MGDG, DGDG), sulfoquinovosyldiglycer ide (SQDG) and phosphat idy lg lycero l (PG) (17, 46).

When Synechococcus growing a t 55 C was a d a p t e d to grow a t 38 C t h e r e was an i n c r e a s e of t h e m o r e fluid f a t t y ac ids (unsa tu ra t ed and shor t chains) in all t h e lipid types (15). In t he t o t a l lipids t h e r a t i o of u n s a t u r a t e d / s a t u r a t e d f a t t y acids wen t from 0.31 to 1.31 and the r a t i o of C to C f a t t y acids inc reased about 2 t i m e s . The n e g a t i v e l y - c h a r g e d lipids SQDG and PG ob ta ined from cel ls grown a t the lower t e m p e r a t u r e had more u n s a t u r a t e d f a t t y acids 16:1 and 18:1 and less s a t u r a t e d f a t t y ac id 18:0 while 16:0 did no t change . In the uncha rged lipids MGDG and DGDG only t h e u n s a t u r a t e d f a t t y acid 16:1 i nc r ea sed when the g rowth t e m p e r a t u r e was lowered and 18:1 r e m a i n e d the s a m e . However , bo th t h e s a t u r a t e d f a t t y acids 16:0 and 18:0 d e c r e a s e d in MGDG and DGDG a t t h e lower g rowth t e m p e r a t u r e .

4. The Effect of Growth Temperature Changes on Pigment Composition. Adap t a t i on of Synechococcus to grow a t 38 C in s t ead of 55 C not only p roduced d is t inc t a l t e r a t i o n s in t h e f a t t y ac id compos i t ion of t he lipids but also p roduced m a r k e d a l t e r a t i o n s in the i r p igmen t composi t ion (14). The d i f fe rence s p e c t r u m b e t w e e n t h e 38 and 55 C grown cells showed t ha t g rowth a t t he lower t e m p e r a t u r e p roduced m o r e of a p igment t ha t appea red to be a ca ro t eno id s ince peaks and a shoulder could be seen a t 480, 452 and 430 nm r e s p e c t i v e l y . The 38 C cel ls con ta ined less phycocyanin as well as was shown by the d e c r e a s e a t 618 n m . Di f fe rences in t h e region of chlorophyll absorp t ion could also be seen b e t w e e n the two c u l t u r e s . It appea r s , in addi t ion to the lipid changes desc r ibed above , t h a t a l t e r a t i o n of p i g m e n t composi t ion (par t icular ly of caro tenoids) is an i m p o r t a n t p a r t of the p rocess of a d a p t a t i o n to d e c r e a s e d growth t e m p e r a t u r e s .

5. Electron Transfer Reactions. In o rder to see if phase t rans i t ions in thylakoid m e m b r a n e s of Synechococcus can be c o r r e l a t e d with changes of p h o t o s y n t h e t i c funct ioning, m e a s u r e m e n t s w e r e m a d e on the t e m p e r a t u r e dependence of e l e c t r o n t r anspo r t b e t w e e n t h e two pho to sys t ems as well as on t h e t e m p e r a t u r e d e p e n d e n c e of a l t e r a t i o n s in t he s t a t e of p i g m e n t s in t he p h o t o s y n t h e t i c m e m b r a n e t h a t lead to changes in the d is t r ibu t ion of q u a n t a b e t w e e n the two p igmen t s y s t e m s of pho tosyn thes i s ( the so-ca l led p igmen t s t a t e 1 - s t a t e 2 shif t ) .

The t e m p e r a t u r e dependence of e l e c t r o n t r ans fe r can be followed readi ly in Synechococcus by measur ing t h e r a t e s of a t r ans i en t r educ t ion of t he f t ype c y t o c h r o m e t h a t funct ions as a c a r r i e r b e t w e e n t h e two light r e a c t i o n s of pho tosyn thes i s . The Arrhenius p lo t s showed d iscont inu i t i es nea r 43 and 26°C for cel ls grown a t 55°C and a t 37 and 27 C for cel ls grown a t 38 C (15). When t h e cel ls w e r e w a r m e d again t h e b reaks w e r e also seen again n e a r these s a m e t e m p e r a t u r e s (with a hys t e re s i s e f f ec t ) . The d i scont inu i ty observed a t 43 C in t h e curve for


e l e c t r o n t r anspo r t cor responds to t h e a p p e a r a n c e of a phase t r ans i t ion b e t w e e n the liquid c rys t a l l ine and the phase s epa ra t i on s t a t e s seen nea r this t e m p e r a t u r e in t h e f luo rescence versus t e m p e r a t u r e curve for ce l l s , phospho- and glycolipids (Fig. 2 D, E, F ) . So far no c lea r d i scon t inu i ty has been seen n e a r 37 C in t h e f l uo rescence cu rve ; a l though an ind is t inc t shoulder s o m e t i m e s appea red n e a r this t e m p e r a t u r e . Even though it has no t y e t b e e n c lea r ly d e m o n s t r a t e d , i t is a s sumed t h a t a phase t r ans i t ion occurs n e a r 37 C and is respons ib le for t h e 37 C b reak seen in t h e plot of e l e c t r o n t r an spo r t in t h e cel ls grown a t 38 C .

The c h a r a c t e r i s t i c poin ts seen n e a r 26 C for bo th t h e cel ls grown a t 55 and 38 C was no t r e f l e c t e d in a c l ea r way in t h e f luo rescence versus t e m p e r a t u r e cu rve ; a l though s o m e t i m e s a shoulder was seen a t this t e m p e r a t u r e . The origins of t h e s e c h a r a c t e r i s t i c po in t s a t t h e lower t e m p e r a t u r e s a r e st i l l be ing i nves t i ga t ed .

6. Pigment State Changes. I l luminat ion of a lgal cel ls wi th wave l eng ths of l ight t h a t cause unba lanced e x c i t a t i o n of t h e two p h o t o s y s t e m s of pho tosyn thes i s is o v e r c o m e to some e x t e n t by a kind of se l f - r egu la t ing sys tem tha t al lows t r ans f e r of l ight q u a n t a from the pho tosys t em t h a t r e c e i v e s too many q u a n t a to t he pho tosys t em t h a t r e c e i v e s too few (5, 28, 30). This mechan i sm p re sumab ly involves changes in o r i e n t a t i o n and /o r in d i s t a n c e b e t w e e n p h o t o s y n t h e t i c p i g m e n t s embedded in the thylakoid m e m b r a n e so tha t ene rgy t r ans fe r p robab i l i t i e s a r e a l t e r e d . I l lumina t ion of t h e b lue -g reen a lga Synechococcus wi th blue light t h a t e x c i t e s l a rge ly only pho tosys t em I gives r i se to a condi t ion ( t e rmed s t a t e 1) whe reby some of t h e excess b lue q u a n t a a r e t r a n s f e r r e d to pho tosys t em Π thus exc i t ing bo th p h o t o s y s t e m s m o r e evenly and causing a m o r e e f f ic ien t pho tosyn thes i s . If t hese ce l ls a r e suddenly sw i t ched to g reen light t h a t is absorbed p r e f e r en t i a l l y by pho tosys t em Π then a t r a n s i e n t high level of chlorophyll α f luo rescence is seen s ince t h e cel ls in s t a t e 1 cause q u a n t a to be t r a n s f e r r e d to or r e m a i n in sys t em Π and to be e m i t t e d as f l uo re scence . At 53 C th is t r a n s i e n t high f luo rescence d e c r e a s e s wi th a half t i m e about 0.1 sec for cel ls grown a t 55 C as t h e a lgae pass from s t a t e 1 to s t a t e 2. In s t a t e 2 t h e excess q u a n t a a r r iv ing in pho tosys t em Π a r e t r a n s f e r r e d to pho tosys t em I.

We found t h a t this s t a t e 1 to s t a t e 2 t r ans i t ion was highly t e m p e r a t u r e sens i t i ve . M e a s u r e m e n t of t h e r a t e s of change of this m e m b r a n e s t a t e as a funct ion of t h e r e c i p r o c a l of t h e abso lu te t e m p e r a t u r e for Synechococcus grown a t 55 and 38 C r e v e a l e d c l ea r d i scon t inu i t i e s a t 44 and 37 C r e s p e c t i v e l y (15). The l ine ob ta ined for t h e 55 C ce l l s had an abrup t change of slope and j u m p e d to a new posi t ion a t a t e m p e r a t u r e t h a t co r responded to t h e beginning of t h e t r ans i t ion from t h e liquid c rys ta l l ine to t h e phase s e p a r a t i o n s t a t e s . For cel ls grown a t 38 C a possible second b reak was seen n e a r 25 C; but this obse rva t ion r e s t s on only one d a t a po in t . The b reaks in t h e Arrhenius p lo t s w e r e a r e f l ec t i on of a r eve r s ib l e even t s ince they w e r e again seen n e a r these s a m e t e m p e r a t u r e s when t h e cel ls w e r e h e a t e d .

2 2 8 D. C. Fork


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FREEZE-THAW INDUCED LESIONS IN THE PLASMA MEMBRANE

Peter L. Steponkus

D e p a r t m e n t of Agronomy Cornel l Univers i ty I t haca , New York

Steven C. Wiest

D e p a r t m e n t of H o r t i c u l t u r e and F o r e s t r y Cook Col lege - R u t g e r s Univers i ty

New Brunswick, New J e r s e y

I. INTRODUCTION

As m e m b r a n e d a m a g e is commonly in fe r red to be t he p r i m a r y cause of f reez ing injury (see C h a p t e r 1), c h a r a c t e r i z a t i o n of m e m b r a n e lesions, espec ia l ly to t h e p l a sma m e m b r a n e is e ssen t ia l to a c o m p l e t e unde r s t and ing of t h e mechan i sm of f reez ing injury. A major i m p e d i m e n t to such a c h a r a c t e r i z a t i o n is t h e inabi l i ty to i so la te p lan t p l a sma m e m b r a n e s in bo th suff ic ient ly pu re form and in a s t a t e which can be shown to be iden t i ca l t o the i r s t a t e in VIVO. An a l t e r n a t i v e is to s tudy t h e p l a sma m e m b r a n e in situ. We have adop ted such an approach by s tudying t h e e f f ec t s of a f r e e z e - t h a w cyc le on i so la t ed p r o t o p l a s t s . Such s tud ies wi th i so l a t ed p r o t o p l a s t s p rov ide an oppor tun i ty to s tudy t h e r epe rcuss ions of f reez ing on the p l a sma m e m b r a n e under r e l a t i ve ly s t a n d a r d and s implif ied condi t ions w h e r e t h e complex i t i e s imposed by t i ssue o rgan iza t ion or cell walls a r e e l im ina t ed . These f a c t o r s and o the r cons ide ra t ions [see Wiest and Steponkus (48)] have p rec luded a d i r ec t compar i son of f reez ing of p lan t cel ls VS. m a m m a l i a n cel ls or m i c r o o rgan i sms . Mazur (23) has i nd i ca t ed t h a t t hese have been two d is t inc t fields in t he a r e a of cryobiology and l i t t l e e f for t has been m a d e to ". . . i n c o r p o r a t e t he r e su l t s in to more e f f ec t i ve c o n c e p t s of f reez ing injury."

Department of Agronomy Series Paper No. 1283.

Copyright ® 1979 by Academic Press, Inc. 2 3 1 All rights of reproduction in any form reserved

ISBN (> 12 46056O5

2 3 2 P. L. S t e p o n k u s a n d S. C. Wies t

In t eg ra t ion of t he observa t ions in t he s e p a r a t e a r e a s of p lan t and m a m m a l i a n cryobiology is of cons iderab le i m p o r t a n c e . Mammal i an cryobiology is well advanced in t h e de l inea t ion of phys i co -chemica l even t s occur r ing during f reez ing but is def ic ien t in speci f ic in fo rmat ion r ega rd ing the r e s u l t a n t ce l lu lar m e m b r a n e les ions . In c o n t r a s t , p lan t cryobiology is advanced in the l a t t e r a r e a but de f ic ien t in the fo rmer . Addi t ional ly , p lan t cel ls h a v e t he c a p a c i t y to i n c r e a s e in cold ha rd iness . I so la ted p r o t o p l a s t s provide a sys tem in which the repercuss ions of a f r e e z e - t h a w cyc le can be d i r ec t ly c o m p a r e d wi th those of m a m m a l i a n cel ls exposed to s imilar e n v i r o n m e n t s . If c o m m a n l i t i e s can be d e m o n s t r a t e d , i n t e g r a t i o n of t h e obse rva t ions in t h e two a r ea s will be enhanced cons iderably , and s ignif icant benef i t s could a c c r u e to bo th f ields.

A. The Freezing Process and Repercussions on the Cellular Environment

The phys ico -chemica l e v e n t s which occur during the f reez ing of a cel l suspension and the repercuss ions of t h e f reez ing p rocess on the ce l lu lar env i ronmen t have been discussed in t h e f irs t c h a p t e r of this vo lume. Since f reez ing r e su l t s in a m u l t i t u d e of s t r e s s e s , cons idera t ion of t he f reez ing p rocess as a sequen t ia l se r i es of po t en t i a l l y l e tha l s t r e s s ba r r i e r s appea r s espec ia l ly a p p r o p r i a t e . An in i t ia l s t r e s s ba r r i e r which must be o v e r c o m e is t he disequi l ibr ium b e t w e e n t h e chemica l p o t e n t i a l of w a t e r in t h e cell and t h a t of t h e surrounding medium following t h e in i t ia l ex t r ace l l u l a r nuc l ea t ion e v e n t . This may be ach ieved e i t he r by ce l lu lar dehydra t ion and con t inued ex t r ace l l u l a r i ce fo rma t ion or by in t r ace l lu l a r i ce fo rma t ion .

D i r e c t obse rva t ions of c e r e a l (rye) p r o t o p l a s t s exposed to var ied f r e e z e - t h a w r e g i m e s using an e l e c t r o n i c a l l y - p r o g r a m m a b l e c r y o m i c r o -scope capab le of p r ec i s e con t ro l of cooling r a t e s and t e m p e r a t u r e s (16) i nd i ca t e t ha t t he probabi l i ty of i n t r ace l lu l a r i ce fo rma t ion i nc rea se s if t he cooling r a t e exceeds 3.0 C /min (Fig. 1). However , t he inf luence of cooling r a t e on t h e probabi l i ty of i n t r ace l lu l a r i ce fo rma t ion is inf luenced cons iderably by t h e min imum t e m p e r a t u r e to which the cel l suspension is cooled (5). If cooled to t e m p e r a t u r e s b e t w e e n - 2 ° and - 5 ° C , t h e probabi l i ty of i n t r ace l lu l a r i ce fo rma t ion is e x t r e m e l y low— rega rd l e s s of t h e cooling r a t e (in the r ange of 2° to 120°C/min) . If cooled to t e m p e r a t u r e s b e t w e e n -5 and -20 C, the o c c u r r e n c e of i n t r ace l lu l a r i ce fo rmat ion is s t rongly inf luenced by the cooling r a t e (in t he r ange of 1 to 80 C/min . ) . The probabi l i ty is c lose to ze ro a t r a t e s less than 3 C /min . and inc rea se s as t h e cooling r a t e i n c r e a s e s . At cooling r a t e s g r e a t e r than 20 C/min . , t he probabi l i ty is g r e a t e r than 95 p e r c e n t . If cel ls a r e cooled to t e m p e r a t u r e s lower than -20 C, t he probabi l i ty of i n t r ace l lu l a r i ce fo rmat ion is g r e a t e r than 95 pe rcen t—rega rd l e s s of t he cooling r a t e s imposed (in t he r ange of 4 to 80 C/min . ) . A p re l imina ry i n t eg ra t i on and analysis of these r e su l t s i nd ica te s t h a t t h e probabi l i ty of

F r e e z e - T h a w i n d u c e d L e s i o n s in t h e P l a s m a M e m b r a n e 2 3 3

i n t r ace l l u l a r i ce fo rma t ion is high if t h e e x t e n t of supercool ing of the in t r ace l lu l a r solut ion is g r e a t e r than 10 d e g r e e s but is low if the e x t e n t is less than 3 deg ree s (15).

If cel ls a r e able to a c h i e v e equi l ib ra t ion by ce l lu lar dehydra t ion , s eve ra l consequences of dehydra t ion a r e e n c o u n t e r e d . Each may be cons ide red as a po t en t i a l l y l e tha l s t r e s s b a r r i e r . Lovelock (18) a l luded to th is possibi l i ty in cons ider ing the e f f ec t of f reez ing on r ed blood ce l l s . Mazur (26) has s imi lar ly proposed t h a t b e t w e e n t h e t i m e t h e cel ls f ace the f irs t e x t r a c e l l u l a r ice and the t i m e t h a t they a r e r e t u r n e d to pos t -thaw condi t ions , they m e e t a s equence of events—any one of which is po t en t i a l l y l e t h a l . He fur ther sugges t s t ha t the c o n c e n t r a t i o n of so lu tes (e lec t ro ly tes ) is t h e f i rs t such even t and is fol lowed by cell sh r inkage . While it is t r u e t ha t ex t r ace l l u l a r so lu te c o n c e n t r a t i o n mus t occur be fore ce l lu la r dehydra t ion and shr inkage , i t might be m o r e a p p r o p r i a t e to consider t h a t vo lume r e d u c t i o n is the f irs t po t en t i a l l y l e tha l s t r e s s ba r r i e r e n c o u n t e r e d . On t h e basis of r e l a t i v e changes in in t ens i ty , r e d u c t i o n s in cel l vo lume i n c r e a s e in in t ens i ty a t r e l a t i ve ly warm subze ro t e m p e r a t u r e s — b e f o r e i n c r e a s e d so lu te c o n c e n t r a t i o n s ach i eve apprec iab ly high l eve l s . This is because ce l lu la r vo lume d e c r e a s e s as an inverse funct ion of t e m p e r a t u r e while so lu te c o n c e n t r a t i o n (osmolali ty) i nc rea se s l inear ly wi th d e c r e a s e s in t e m p e r a t u r e (Fig. 2). In such a

RY E PROTOPLAST S

I 1 1 ι ι L 0 2 4 6 8 1 0

COOLIN G R A T E (°C/min )

FIGURE 1. The probability of intracellular ice formation in isolated rye protoplasts as a function of cooling rate.


FIGURE 2. A schematic representation of changes in cell volume, solute concentration and pH during freezing to demonstrate the sequential nature of potential stress barriers.

s c h e m e , changes in pH m a y be envis ioned to occu, abrupt ly—at some t e m p e r a t u r e whe re buffer ing s a l t s p r e c i p i t a t e . Wnether r educ t ions in cell volume r e a c h injurious leve l s be fo re c o n c e n t r a t i o n of e l e c t r o l y t e s b e c o m e s injurious r e m a i n s to b e reso lved , a l though Meryman (28, 30, 31) provides ev idence to suppor t th i s c o n c e p t .

If r e d u c t i o n s in cell vo lume a r e injurious, they may be m a n i f e s t e d in the c o n t r a c t e d s t a t e or m a n i f e s t a t i o n of a lesion may be d e f e r r e d unt i l cell expansion occurs dur ing thawing . H e n c e , t he expansion p rocess may also be cons idered as a p o t e n t i a l s t r e s s b a r r i e r which mus t be o v e r c o m e by t h e cel l on i t s r e t u r n to t h e osmot i c c o n c e n t r a t i o n s of t h e t hawed solut ion . Mazur (26) has p roposed tha t ce l ls a r e injured by d i f fe ren t s t r e s se s depending upon t h e f reez ing condi t ions : spec i f ica l ly , injury in the absence of c r y o p r o t e c t a n t s is due to causes d i f fe ren t than those which resu l t in injury in t h e p r e s e n c e of c r y o p r o t e c t a n t s . We would fur ther suggest t h a t if a sequen t i a l se r ies of s t r e s s ba r r i e r s is e n c o u n t e r e d during a f r e e z e - t h a w cyc le , i t is en t i r e ly app rop r i a t e to envision d i f fe ren t ce l lu la r les ions as a r e su l t of t h e d i f fe ren t s t r e s s e s . Thus, while injury may be caused by any one of severa l s t r e s s ba r r i e r s i t m a y be man i f e s t ed by any one of severa l po t en t i a l l y l e tha l s t r a in s and r e s u l t a n t les ions . E x p e r i m e n t a l d e m o n s t r a t i o n of mul t ip le , bu t

F r e e z e - T h a w - l n d u c e d L e s i o n s in t h e P l a s m a M e m b r a n e 2 3 5

0 .5 1.0 15 2.0 l/7r(osm-' )

FIGURE 3. Boyle van't Hoff plot of isolated rye protoplasts after 10-min. contraction in salt (equiosmolar CaCl9 + NaCl).

t empora l l y s epa rab l e , lesions in ch loroplas t thylakoid ves ic les which p rec lude man i f e s t a t i on of l igh t - induced p ro ton u p t a k e has been r e p o r t e d (8, 44). A s imi lar d e m o n s t r a t i o n of mul t ip le lesions in t h e p l a sma m e m b r a n e r e m a i n s to be p r e s e n t e d .

With t he above concep tua l f r amework , we would l ike to speci f ica l ly address t h e s t r e s se s a s soc i a t ed wi th ce l lu la r vo lume t r i c changes in p r o t o p l a s t s dur ing a f r e e z e - t h a w cyc le and t h e r e s u l t a n t les ions t h a t a r e speci f ica l ly m a n i f e s t e d as lysis of t h e p r o t o p l a s t . Such a de l inea t ion does no t infer t ha t o the r s t r e s se s which could resu l t in o the r po t en t i a l l y l e tha l lesions do no t exis t or a r e no t s igni f icant . Our ob jec t ive is me re ly to e x a m i n e t h e po t en t i a l l y l e tha l s t r e s s b a r r i e r s p r e s e n t e d by c o n t r a c t i o n and expansion and t h e r e s u l t a n t p l a s m a - m e m b r a n e - a s s o c i a t e d man i f e s t a t i on , lysis, which in tu i t ive ly is mos t l ikely to r e s u l t . The possibi l i ty and l ikel ihood of o the r m o r e sub t le lesions r e su l t ing from these or o the r s t r e s se s and m a n i f e s t e d as m e t a b o l i c dysfunct ions of t h e m e m b r a n e (32) a r e no t p rec luded .

B. Osrnometric Behavior During a Freeze-Thaw Cycle

P r o t o p l a s t s behave as ideal o s m o m e t e r s and exhibi t c h a r a c t e r i s t i c Boyle van ' t Hoff behav ior in solut ions b e t w e e n 0.35 and 2.75 osmola l , t he l imi t s of t h e r ange examined (48). A typ ica l p lo t for rye p r o t o p l a s t s is


The cell volume a t any given subzero t e m p e r a t u r e can be a p p r o x i m a t e d by assuming tha t the cel ls behave as o s m o m e t e r s and applying the Boyle-van ' t Hoff law (3). Thus, t he in t ens i ty of t h e f reez ing s t r e s s can be expressed as a funct ion of dehydra t ion r a t h e r than t e m p e r a t u r e . This would be especia l ly i m p o r t a n t if dehydra t ion r a t h e r than t e m p e r a t u r e is the p r imary s t r e s s r e su l t ing in injury and most i m p o r t a n t if the e x t e n t of ce l lu lar c o n t r a c t i o n is of i m m e d i a t e conce rn .

The above descr ip t ion of vo lume t r i c changes during f reez ing is a g rea t ly s implif ied po r t r aya l and a t bes t can only provide a qua l i t a t i ve descr ip t ion of ce l lu lar vo lume t r i c changes during a f r e e z e - t h a w cyc le . Numerous models have been p r e s e n t e d (11, 12, 13, 14, 2 1 , 22, 24, 25, 34, 39) and have cons idered var ious p a r a m e t e r s which will inf luence the fundamenta l behavior , most no tab ly the w a t e r pe rmeab i l i t y of the p l a sma m e m b r a n e , solut ion non- idea l i ty , diffusivi t ies of so lu tes in t h e i n t r a - and e x t r a - c e l l u l a r solut ion, and the su r face a r e a to volume r a t i o of t he ce l l . Mazur (25) acknowledges t ha t a major source of e r ro r can be a t t r i b u t e d to the i nco r r ec t e s t i m a t i o n of hydraul ic w a t e r pemeab i l i t y and i t s t e m p e r a t u r e coef f i c i en t . Unti l t h e s e p rob lems a r e reso lved , t he vo lume t r i c responses of cel ls in disequil ibr ium a t subzero t e m p e r a t u r e s can only be qua l i t a t ive ly p r e d i c t e d , however , t he p red ic t ions a r e valid under equil ibrium condi t ions .

Using a r e c e n t l y developed c ryomic roscope (16) we have d i r ec t ly observed the vo lume t r i c changes in p ro top l a s t s during a f r e e z e - t h a w cyc le . Video record ing of t he sequence al lows for t h e a c c u r a t e and p rec i s e d e t e r m i n a t i o n of ce l lu lar vo lume t r i c changes dur ing the f r e e z e -thaw cyc le . Reso lu t ion of 0.2 y m is possible as 2.5 mm on the video moni to r r e p r e s e n t s 1.0 \im in a c t u a l d imens ions . Rou t ine ly , c e r e a l

2 p r e s e n t e d in F igu re 3 . Volume va r i e s l inear ly (r = 0.94) wi th osmola l i ty . The minimum volume is equal t o t he o smo t i ca l l y - i nac t i ve volume of t h e cel l , V^. The volume a t any p a r t i c u l a r osmola l i ty may be desc r ibed by t h e equa t ion :

I = _ ° ( i -v ) + V ν ρ V b

ο w h e r e V = vo lume, Ρ = osmola l i ty , V^ = osmot ica l ly i n a c t i v e vo lume . The vo lume t r i c behavior is la rgely d e t e r m i n e d by t h e o s m o t i c a l l y - a c t i v e in t e rna l so lu te c o n t e n t .

During t h e course of ex t r ace l l u l a r i ce fo rma t ion and cel lu lar dehydra t ion , p ro top l a s t s will c o n t r a c t wi th t h e min imum volume ach ieved a funct ion of t h e lowest t e m p e r a t u r e e n c o u n t e r e d . Subsequent ly , they will expand to an e x t e n t d e t e r m i n e d by t h e osmola l i ty of t he suspending medium. The osmola l i ty of t he pa r t i a l ly f rozen solut ion ba th ing t h e cells a t any given subzero t e m p e r a t u r e can be a p p r o x i m a t e d as :

Τ m ~ " X 8 6


1.0

0.8

^ 0.6 Ld

o 0.4

ο 0.2

Start of "cooling * \ /

Ice formation • V e

-

_

• \ • *

• • • • t !

ι I 10 -10

TEMPERATURE (°C)

FIGURE 4. Chcgiges inQcell volume of an isolated rye protoplast during freezing to -8 C at 5 C/minute.

p r o t o p l a s t s a r e in t he r a n g e of 20 to 30 μ ιη in d i a m e t e r a t 0.54 osmola l . Al though p r o t o p l a s t s sub jec t ed to osmot i c manipu la t ion a t room t e m p e r a t u r e c o n t r a c t spher ica l ly in c o n c e n t r a t i o n s up to 2.75 osmola l , c o n t r a c t i o n during f reez ing m a y be i r r egu la r , espec ia l ly when t e m p e r a t u r e s lower than -5 a r e imposed . Beginning as spheroids , p r o t o p l a s t s m a y b e c o m e c o n t o r t e d in t h e i ce field, and an el l ipsoidal model is t hen r equ i r ed for t he ca l cu l a t i on of t h e vo lume . Such t r a n s f o r m a t i o n s , however , a r e only r equ i r ed a f t e r t h e major i ty of c o n t r a c t i o n has a l r eady o c c u r r e d . F r a c t i o n a l v o l u m e t r i c changes in a rye p ro top l a s t f rozen to -8 C a t 5 C /min a r e shown in F igure 4 .

C. Plasma Membrane Lesions Induced by Cellular Volumetric Changes

Previous ly , we h a v e d e m o n s t r a t e d s imula t ion of a f r e e z e - t h a w cyc le may be ach ieved by osmot i c manipu la t ion of p r o t o p l a s t s a t room t e m p e r a t u r e (48). The e x t e n t of injury (percen t lysis) i ncu r red by a popula t ion of cel ls sub jec t ed to a f r e e z e - t h a w cyc le can be q u a n t i t a t i v e l y a c c o u n t e d for by t h e e x t e n t of c o n t r a c t i o n and expansion t h e p ro top l a s t s unde rgo . Addi t ional ly , t h e k ine t i c s of f r e e z e - t h a w injury w e r e as p r e d i c t e d by o smot i c man ipu la t ion . Thus, bo th t h e e x t e n t and k ine t i c s of injury in p r o t o p l a s t s sub jec ted to a f r e e z e - t h a w cyc le a r e s imi lar to those sub jec ted to o smot i c man ipu la t ion . These f ac t s s t rongly


sugges t t h a t injury to p ro top l a s t s during a f r e e z e - t h a w cyc le and man i f e s t ed as cel l lysis is due to the s ame s t r e s s e s of c o n t r a c t i o n and expansion t h a t resu l t from osmot i c manipu la t ion in t he absence of i ce or low t e m p e r a t u r e s . However , o the r s t r e s s e s , mos t no tab ly e l e c t r o l y t e c o n c e n t r a t i o n , could have been responsib le for the injury observed . This possibi l i ty (with r e s p e c t to lysis as the man i f e s t ed injury) is d iminished, however , because s imi lar r e su l t s w e r e ob ta ined w h e t h e r t he o smo t i c manipula t ions or the f r e e z e - t h a w cyc le we re conduc t ed in an ionic (CaCl^ + NaCl) or non- ionic o smot i cum (sorbitol or manni to l ) . Addi t ional ly , if t he ce l ls w e r e exposed to var ied deg ree s of c o n t r a c t i o n , e i t he r by va r i ed c o n c e n t r a t i o n s of o smot i cum or var ied f reez ing t e m p e r a t u r e s , t he e x t e n t of lysis could be modif ied by a l t e r i ng the e x t e n t of expansion—by varying t h e osmola l i ty of t h e suspending medium during expansion or thawing . We submit tha t the s t r e s s e s of c o n t r a c t i o n and expansion, in t h e m s e l v e s , r esu l t in p o t e n t i a l l y - l e t h a l s t r a ins or m e m b r a n e lesions man i f e s t ed as lysis of t h e ce l l . Previous ly , Sca r th et al. (38) sugges ted t h a t injury to t h e p l a sma m e m b r a n e was r e l a t e d to p lasmolysis and deplasmolys is which o c c u r r e d during f reez ing and thawing and t h a t lysis o c c u r r e d during deplasmolys is .

Meryman (28) has proposed a "minimum c r i t i c a l volume" hypothes i s to accoun t for f r eeze - induced lysis in a wide v a r i e t y of ce l l t ypes . This hypothes is proposed ". . . t ha t m e m b r a n e injury from f reez ing is t he resu l t of o smot i c s t r e s s p roduced by the f reez ing-ou t of w a t e r and the a s soc i a t ed i nc rease in osmola l i ty of t h e suspending solut ion leading to a r educ t ion in cel l vo lume beyond a t o l e r a t e d min imum" (29). Such a minimum cell vo lume may be i n t e r p r e t e d in two ways : e i t h e r t h e o c c u r r e n c e of injury is s imply c o r r e l a t e d wi th the a t t a i n m e n t of a c e r t a i n cel l volume or t h a t t h e r e is, in f ac t , a physical minimum volume a t osmola l i t i e s less than infini ty beyond which the cel l canno t shrink, and t h a t t he a t t a i n m e n t of this physica l min imum volume c r e a t e s s t r e s se s which lead to hemolys i s . For the l a t t e r i n t e r p r e t a t i o n to be valid, dev ia t ions from ideal Boyle-van ' t Hof f o s m o m e t r i c behavior should occur a t osmola l i t i e s which resu l t in injury (with lysis subsequent ly occur r ing on di lut ion and expansion) . P r o t o p l a s t s do no t exhibi t any such dev ian t behavior in the region of osmola l i t i e s which r e su l t in subsequent lysis . R e c e n t analysis of ea r l i e r d a t a also sugges ts t h a t human e r y t h r o c y t e s do not exhibit such dev ian t behavior (51). On this bas is , we consider t ha t the "minimum c r i t i c a l volume" hypothes is should be v iewed from the p e r s p e c t i v e tha t injury is s imply c o r r e l a t e d wi th the a t t a i n m e n t of a c e r t a i n cell vo lume .

Several proposals h a v e been put for th to accoun t for t h e fac t t h a t injury occurs following exposure to hype r ton ic condi t ions when the cel l is s imul taneous ly exper ienc ing high so lu te c o n c e n t r a t i o n s (s t r ic t ly e l e c t r o l y t e s in some cases) and volume r e d u c t i o n s . Most expl ic t ly s t a t e or infer t ha t lysis will occur during cell expansion when the cel ls a r e r e t u r n e d to i so tonic (18, 19, 52) or hypotonic (33) condi t ions . Lovelock (18) proposed t ha t r ed blood cel ls b e c o m e p e r m e a b l e to Na and t h e u p t a k e of so lu tes causes the cel ls to subsequent ly p lasmolyze if r e suspended in " isotonic condi t ions ." Zade-Oppen (52) proposed t h a t


cel ls took up e l e c t r o l y t e s in hype r ton ic solut ions and burs t upon r e t u r n to i so tonic condi t ions due to the f ac t t ha t the cel l exceeded a max imum to l e rab le vo lume . Similar ly, Meryman (29) conc luded t h a t an influx of so lu te during hype r ton i c exposure inc reased the in t r ace l lu l a r so lu te c o n c e n t r a t i o n and t h a t t h e cell will t h e r e f o r e ". . . r e a c h hemoly t i c vo lume a t a h igher osmola l i ty ." Mazur (26) invoked a t r ans i en t h y d r o s t a t i c tension which p roduces a driving fo rce for a n e t influx of so lu te s .

The proposals differ in some r e s p e c t s , mos t no tab ly , Lovelock proposes t h a t t h e a l t e r e d p e r m e a b i l i t y is a t t r i b u t a b l e to t h e i nc r ea se in e l e c t r o l y t e c o n c e n t r a t i o n ; Meryman a t t r i b u t e s it to physical s t r e s se s a s soc i a t ed with a min imum c r i t i c a l vo lume; Mazur i nd i ca t e s t h a t i t appea r s r e l a t e d to i so t rop ic cel l sh r inkage . However , r ega rd l e s s of the r eason , all of t h e proposals sugges t t h a t so lu te influx occur s during t h e hype r ton i c exposure and lysis is a t t r i b u t e d to i nc reased volumes which occur on subsequent exposure to hype r ton ic so lu t ions . Impl ic i t is t h e no t ion of a fixed max imum volume which is me re ly ach ieved a t h igher osmola l i t i e s . If this w e r e t he c a s e , t h e cel ls would exhibi t a l t e r e d o s m o m e t r i c behavior dur ing di lu t ion. To d e t e r m i n e w h e t h e r a "loading" of p r o t o p l a s t s occu r s under hype r ton ic condi t ions , p ro top l a s t vo lumes w e r e d e t e r m i n e d in var ious hype r ton i c solut ions be fore and a f t e r vary ing e x t e n t s of di lut ion f romthese solut ions (Fig. 5). Had an i r r eve r s ib le

I 2 0

ε

>

ο

p 1 0 ο

5

' /Osmolalit y

FIGURE 5. Boyle van't Hoff plot of protoplast volume after 10-min. contraction in salt (equiosmolar CaCl2 + NaCl) followed by immediate addition of HÔ to induce protoplast expansion and obtain final osmolalities shown in the figure. Contraction was achieved in salt concentrations of 0.536 osmolal (Φ), 0.803 osmolal (O), 1.071 osmolal ( a ) , 1.338 osmolal (wi), and 1.607 osmolal (k). Regression coefficient (r = .951) is significant at the 1% level.


influx of so lu tes occu r red , p ro top las t vo lumes would d e v i a t e from a l inear van ' t Hoff p lo t . If m o r e so lu te influx was to have o c c u r r e d in p ro top l a s t s exposed to 1.6 osmolal solut ions than in those exposed to 1.0 osmola l , the vo lume of the fo rmer , following di lut ion to 0.8 osmola l , would be g r e a t e r than t h a t of t h e l a t t e r , which w e r e also d i lu ted to 0.8 osmola l . The l inea r i ty of the vo lume t r i c r e sponse and co inc idence of the vo lume t r i c responses for bo th c o n t r a c t i o n and expansion excurs ions d e m o n s t r a t e t h a t a so lu te influx and subsequent burs t ing upon di lut ion canno t explain the lysis of p ro top l a s t s observed following di lut ion.

Most proposals r e l a t e injury as a funct ion of vo lume t r i c r e sponses . Red blood cel ls a r e thought to possess a max imum c r i t i c a l vo lume (33). It should be emphas ized , however , t h a t a l though o s m o m e t r i c behavior is bes t c h a r a c t e r i z e d as a funct ion of volume and ex tens ive d a t a suggest t h a t a r educ t ion in cell volume per se is a s ignif icant s t r e s s imposed on a cell during hype r ton ic t r e a t m e n t ; lysis is t he resu l t of a dissolut ion of t h e p l a sma m e m b r a n e when a c r i t i ca l su r f ace a r e a of t h e p l a sma m e m b r a n e is exceeded . Thus, while t h e ce l lu lar response to hype r ton ic

τ 1 1 1 r

FIGURE 6. Protoplast survival plotted as a function of surface area of the protoplasts when contracted in varying salt concentrations and then expanded to varying extents by dilution of the osmoticumt as in Fig. 5. Numbers above each line represent the highest osmolality to which protoplasts were exposed before dilution. Surface areas were calculated from volumes given by the least squares line in Figure 5; osmolalities are represented by the same symbols as in Fig. 5.


t r e a t m e n t is a d e c r e a s e in cell vo lume, t he repe rcuss ions a r e man i f e s t ed as changes in the su r f ace a r e a over which t h e p l a s m a m e m b r a n e mus t be ex t ended . The change in su r f ace a r e a in response to vo lume t r i c changes will depend on the g e o m e t r i c a l shape of the ce l l . Red blood cel ls a r e concave discoids which a r e able to double in volume with l i t t l e or no change in su r face a r e a (6). P lan t p ro top l a s t s a r e spher ica l and must i nc r ea se in su r f ace a r e a during vo lume t r i c expansion.

Whether p ro top la s t lysis is or is not a s soc i a t ed wi th the a t t a i n m e n t of a max imum s ize as a function of su r face a r e a can be d e t e r m i n e d by observing survival as a funct ion of p l a sma m e m b r a n e su r f ace a r e a (Fig. 6). The spher ica l g e o m e t r y of p r o t o p l a s t s p e r m i t s a c c u r a t e d e t e r m i n a t i o n s of su r f ace a r e a from d i a m e t e r m e a s u r e m e n t s . P r o t o p l a s t s w e r e i so la t ed in 0.54 osmolal o smot i cum (CaCl^ + NaCl) and subsequent ly exposed to hype r ton i c solut ions before di lut ion of the o s m o t i c u m . F igure 6 d e m o n s t r a t e s t ha t 50 p e r c e n t of t h e p r o t o p l a s t s exposed to 0.54 osmolal solut ions and d i lu ted to varying e x t e n t s hajl lysed be fo re they ach ieved a su r face a r e a of a p p r o x i m a t e l y 3400 μ ιη . Addi t ional ly , 50 p e r c e n t of t h e p r o t o p l a s t s c o n t r a c t e d in 1.61 osmolal solut ions be fore di lut ion ^ysed before they ach ieved a su r face a r e a of a p p r o x i m a t e l y 1700 y m —a su r f ace a r e a less t han t h a t which they possessed in 0.54 osmolal so lu t ions . P r o t o p l a s t s which had been c o n t r a c t e d to i n t e r m e d i a t e values lysed when they we re expanded to su r face a r e a s i n t e r m e d i a t e b e t w e e n these two va lues . The d a t a d e m o n s t r a t e t ha t p r o t o p l a s t s do no t possess a fixed max imum su r face a r e a a t which lysis occu r s . R a t h e r , the su r f ace a r e a a t which lysis occur s is d e c r e a s e d if p ro top l a s t s h a v e previously been sub jec ted to c o n t r a c t i o n .

The s imi la r i ty of slopes of t h e curves fur ther sugges t s t ha t survival is a funct ion of t he abso lu te su r f ace a r e a i n c r e m e n t ach ieved during expansion, r ega rd l e s s of t he o the r condi t ions to which they w e r e exposed. For i n s t ance , 50 p e r c e n t survival o c c u r r e d when protoplasms i ncur red an abso lu te su r f ace a r e a i n c r e m e n t of a p p r o x i m a t e l y 900 μ m (Fig. 7a)— rega rd l e s s of w h e t h e r they we re in i t ia l ly exposed to 1.61 osmolal and d i lu ted to 0.81 osmolal or exposed to 0.54 osmolal followed by di lut ion to 0.36 osmola l . When survival is p l o t t e d as a function of volume change (Fig. 7b), it b e c o m e s appa ren t t ha t t he ce l lu lar volume a t which lysis of 50 p e r c e n t of the p ro top l a s t s occu r s va r i e s wi th the d e g r e e of c o n t r a c t i o n . The d a t a suggest t ha t a l though o s m o m e t r i c behavior of p r o t o p l a s t s is bes t desc r ibed as a funct ion of vo lume, the ly t ic lesion r e su l t ing from vo lume t r i c changes is bes t desc r ibed as a funct ion of su r f ace a r e a of t h e p la sma m e m b r a n e during expansion.

On t he basis of these obse rva t ions we have proposed t ha t f r e e z e -thaw injury to i so la t ed p r o t o p l a s t s is the re su l t of two major s t r a in s : a f r e e z e - or c o n t r a c t i o n - i n d u c e d m e m b r a n e a l t e r a t i o n which d e c r e a s e s the max imum c r i t i c a l su r f ace a r e a of the p l a sma m e m b r a n e and a t h a w -or expans ion- induced dissolut ion of t h e p l a sma m e m b r a n e which occurs when the maximum c r i t i c a l su r f ace a r e a is e x c e e d e d . These two s t r a ins i n t e r a c t during a f r e e z e - t h a w cyc le and resu l t in lysis of t h e ce l l . Thus, ce l lu lar v o l u m e t r i c changes during a f r e e z e - t h a w cyc le resu l t in injury


τ — ι 1 1 1 Γ

Chang e i n surfac e are a Tpm2 * I0~3]

FIGURE 7. Dependence of protoplast survival on the absolute increase in surface area or volume of the protoplasts. Conditions and symbols were as listed in Figures 5 and 6.

due to an a l t e r a t i o n in t he p ro top la s t r e s i l i ence—the capab i l i ty of a s t r a ined body to r e c o v e r i t s s ize and shape a f t e r de fo rma t ion caused espec ia l ly by a compress ive s t r e s s . While t h e a l t e r e d r e s i l i ence is t h e resu l t of an a l t e r a t i o n in the p l a sma m e m b r a n e which occurs during c o n t r a c t i o n , it is no t man i f e s t ed unt i l t h e p ro top l a s t s a r e induced to expand during thawing—when disrupt ion of i n t e r m o l e c u l a r forces in the m e m b r a n e causes p ro top la s t lysis .

As ear ly as 1939, Tornava (46) observed p ro top la s t d i a m e t e r s of severa l p lan t spec ies a t the m o m e n t of hypoton ic lysis . The gene ra l conclusion was t h a t p ro top l a s t s burs t when t h e su r f ace a r e a was app rox ima te ly double t he in i t ia l su r face a r e a , a l though it was added t ha t ". . .in many e x p e r i m e n t s ( the su r f ace a r e a a t t he m o m e n t of lysis, as a p e r c e n t a g e of t he or iginal su r f ace a rea) ha s been 110 p e r c e n t or less , and in severa l t r i a l s , w h e r e t he p ro top las t f irst has been m a d e to c o n t r a c t i tself or been p lasmolyzed , not even the original su r f ace a r e a has been r eached . " Sca r th et al. (38) also desc r ibed th is phenomenon in a q u a l i t a t i v e manne r : ". . . t he l imit (of expansion) va r ies g r e a t l y wi th such s imple t r e a t m e n t as p lasmolys is . The point of burs t ing of cel ls which have been s t rongly p lasmolyzed beforehand is lower than those weakly p lasmolyzed ." The r e su l t s p r e s e n t e d in this pape r and an ea r l i e r r e p o r t

F r e e z e - T h a w I n d u c e d L e s i o n s in t h e P l a s m a M e m b r a n e 2 4 3

TEMPERATURE

FIGURE 8. Changes in cell volume of an isolated protoplast (the same cell as in Figure 4) during a freeze-thaw cycle. Minimum t^jnperature attained was -8 C, cooling and warming rates were 5 C/min.

(48) a r e in c o m p l e t e a g r e e m e n t wi th t he se ea r l i e r obse rva t ions . R e c e n t l y , d i r ec t m e a s u r e m e n t s of ce l lu la r v o l u m e t r i c changes dur ing a f r e e z e - t h a w cyc le suppor t t h e conclusion t h a t cel ls which h a v e undergone c o n t r a c t i o n will undergo lysis a t a s ize smal l e r than the or iginal s ize (Fig. 8). The obse rva t ions fu r the r suppor t t h e con ten t ion t ha t p r o t o p l a s t s do no t possess a s ingle u n a l t e r a b l e su r f ace a r e a a t which lysis o c c u r s . In p rev ious proposals r ega rd ing t h e mechan i sm of injury, t h e basis for injury was a sc r ibed to a l t e r e d v o l u m e t r i c r e l a t i o n s due to so lu te influx. In p r o t o p l a s t s , we propose t h a t c o n t r a c t i o n d i r ec t l y a l t e r s t h e m e m b r a n e r e s i l i ence and the n e e d for a so lu t e influx to p rov ide a dr iving fo rce for lysis is no t r equ i red .

As was s t a t e d ea r l i e r , M e r y m a n (28, 29) has proposed t h e "minimum c r i t i c a l vo lume" hypothes i s to a ccoun t for f r e e z e - t h a w induced lysis which can be i n t e r p r e t e d in two ways . For t he hypothes i s to be appl icab le t o p lan t p r o t o p l a s t s , t h e i n t e r p r e t a t i o n t h a t injury is c o r r e l a t e d wi th t he a t t a i n m e n t of a c e r t a i n cel l vo lume r a t h e r than t h e a t t a i n m e n t of a phys ica l min imum volume mus t be m a d e . F u r t h e r m o r e , while v o l u m e / s u r f a c e a r e a r educ t i on does l ead to some m e m b r a n e a l t e r a t i o n which l imi t s the m e m b r a n e expansion p o t e n t i a l , th is c o n t r a c t i o n - i n d u c e d a l t e r a t i o n is a cont inuous (or nonreso lvab le d i sc re te ) funct ion of c o n t r a c t i o n r a t h e r than the e x i s t e n c e of some min imum c r i t i c a l vo lume .


FIGURE 9. A schematic representation of the "apparent" minimum critical volume of a protoplast.

As a r e su l t , any in fe r red min imum c r i t i c a l volume is only an "apparen t" vo lume, the man i f e s t a t i on of which depends on the e x t e n t of c o n t r a c t i o n r e l a t i v e to t h e e x t e n t of subsequent expansion. In o the r words , t h e man i fe s t a t ion of injury r e su l t i ng from c o n t r a c t i o n depends on the e x t e n t of expansion during subsequent hypotonic condi t ions . The "apparen t" n a t u r e of a minimum c r i t i ca l volume is i l l u s t r a t ed in F igure 9.

1. Contraction-Induced Lesion. When a p ro top la s t is exposed to a hype r ton ic solut ion and c o n t r a c t s in a uniform spher ica l conf igura t ion , componen t s of t h e p l a sma m e m b r a n e should b e c o m e more t igh t ly appressed . It is r ea sonab le to e x p e c t t h a t this would be a r eve r s ib l e p rocess and t h a t the m e m b r a n e should be able to r e t u r n to i t s or iginal su r face a r e a when the suspending medium is d i lu ted . However , as was previously d e m o n s t r a t e d , when a p ro top la s t is osmot ica l ly induced to expand from a c o n t r a c t e d s t a t e , lysis can occur be fo re i t r ega ins i t s or iginal su r face a r e a . Such observa t ions sugges t t h a t a c o n t r a c t i o n -induced m e m b r a n e a l t e r a t i o n l imi t s the expansion po t en t i a l of t he p l a sma m e m b r a n e . T h e r e a r e two obvious a l t e r n a t i v e s to a cco u n t for this possibi l i ty: a qua l i t a t i ve a l t e r a t i o n in t he duc t i l i t y of the m e m b r a n e or a q u a n t i t a t i v e r educ t ion in m e m b r a n e m a t e r i a l . S iminovi tch and L e v i t t (40) conc luded t h a t t h e su r face m e m b r a n e of p r o t o p l a s t s s t i f fened

Freeze-Thaw induced Lesions in the Plasma Membrane 245

when dehydrated osmotical ly and, as a result , ruptured more easily when subjected to tension. Meryman et al. (31) have suggested that lipid material is lost during plasmolysis .

To directly test this latter possibility, the plasma membrane of protoplasts was labelled with fluorescein conjugated Concanavalin A (Con A) (49). In order to minimize autof luorescence by chlorophyll, protoplasts were isolated from the achlorophyllous tissue of the crown region of wheat seedlings. If the protoplasts were subsequently contracted, f luorescence from the Con Α-fluorescein appeared aggregated and internalized in the cytoplasm. During expansion the f luorescent material remained in the interior of the cel l and there was no observable reincorporation of the material into the plasma membrane. Such observations suggest that invagination and endocytot ic vesiculation of the plasma membrane resulted during contraction. Preliminary experiments with thin sect ion e lectron microscopy indicate that vesiculation of the plasma membrane occurs in contracted protoplasts. Furthermore, this process did not appear to be reversible , as suggested by Meryman et al. (31). Evidence that the contraction-induced alteration is irreversible or only slowly reversible was obtained by varying the rate of expansion. If contracted protoplasts are expanded by direct osmotic manipulation, the amount of survival is similar in those which are expanded to the same extent in a s tepwise manner with 30 minute equilibration periods between success ive dilutions. Thus, the extent of injury is the same, whether expansion is immediate or sequential . The irreversible physical delet ion of components from the plasma membrane during contraction may, in part, account for the contraction-induced lesion which l imits the expansion potential of the plasma membrane.

2. Expansion-Induced Lesion. Cellular expansion during thawing is a potential ly lethal stress which is usually considered from the perspect ive of altered osmometric characterist ics . Tolerance of the membrane to the resultant strain, which is manifested as lysis of the cel l , is usually considered constant on the basis of the assumption that the expansion potential will remain constant. The expansion-induced dissolution of the plasma membrane which occurs when the maximum crit ical surface area is exceeded is a primary manifestation of freezing injury in protoplasts (Fig. 8). It is clearly a temporally separable strain and the environment during thawing could influence the extent to which this strain may be tolerated. Several observations suggest that biochemical influences on the plasma membrane may have a significant influence on freeze- thaw survival of protoplasts .

Both osmotic manipulation and freeze- thaw induced lysis of spinach protoplasts are influenced by the type of monovalent ions present in the suspending medium (45). The sensit ivity of sDinach +protonlasts +to a freeze- thaw cyc le follows the series: Li = Na < Κ = Rb = Cs and CI < Br < I . Further work has shown that the absolute surface area increment of the plasma membrane^ that r e s u l t e d +i n lysis was significantly less in the presence of Κ than when Na was present.


Addit ional ly , in t h e p r e s e n c e of an ionic o smot i cum (CaCl^+NaCl or CaCl^+KCl) , o smot ica l ly - induced lysis gradual ly i n c r e a s e d as t h e pH d e c r e a s e d below 6.0. Also, when p ro top l a s t s a r e f rozen in so lu t ions of varying r a t i o s of C a C l ?+ N a C l : sorb i to l , an op t imum in survival was observed . O t h e r work (50) i nd i ca t e s t ha t speci f ic a s p e c t s of t he ly t i c lesion and i t s i n t e r a c t i o n wi th t h e chemica l env i ronmen t va r i e s in p ro top l a s t s i so la t ed from d i f fe ren t spec ie s . Unlike p r o t o p l a s t s from spinach, lysis of whea t leaf p r o t o p l a s t s is no t s t rongly inf luenced by t h e monova len t c a t i ons p r e s e n t in the o smot i cum or by pH c h a n g e s . Also, speci f ica l ly in whea t p r o t o p l a s t s , expans ion- induced lysis , bu t no t ehe c o n t r a c t i o n - i n d u c e d a l t e r a t i o n , is t e m p e r a t u r e d e p e n d e n t . A given e x t e n t of expansion is m o r e injurious a t 20 C than a t 10 C or 4 C . Such a t e m p e r a t u r e in f luence was no t obse rved in sp inach p r o t o p l a s t s .

The obse rva t ions s t rongly sugges t t h a t t h e c h e m i c a l n a t u r e of t h e env i ronmen t dur ing a f r e e z e - t h a w cyc le may inf luence the t o l e r a n c e of t h e p l a sma m e m b r a n e to t h e s t r a ins i ncu r r ed during expansion. Such i n t e r a c t i o n s depend on the spec ies in ques t ion and a r e p re sumab ly due to d i f f e rences in p l a s m a m e m b r a n e compos i t ion . In t h e i n s t a n c e s w h e r e such chemica l i n t e r a c t i o n s a r e observed , the physical s t r e s s e s of c o n t r a c t i o n and expansion r e m a i n as t h e p r i m a r y s t r e s se s respons ib le for lysis , bu t t o l e r a n c e of the cel l to t h e s e s t r e s s e s m a y be q u a n t i t a t i v e l y a l t e r e d by t h e chemica l env i ronmen t . In some cases the i r in f luence may be min imal , as M e r y m a n et al. (31) h a v e i nd i ca t ed t h a t they ". . .find no ev idence t h a t b iochemica l mechan i sms a r e involved in e i t he r f reez ing injury or c r y o p r o t e c t i o n . . C lea r ly , fu r the r work is r equ i r ed to e l u c i d a t e the molecu la r n a t u r e of bo th the c o n t r a c t i o n - i n d u c e d a l t e r a t i o n and t h e expans ion- induced dissolut ion les ions to p rec i se ly d e l i n e a t e t h e i n t e r a c t i o n of t h e m e m b r a n e wi th i t s e n v i r o n m e n t .

D. Proposed Hypothesis of Freezing Injury

To r e i t e r a t e , f r e e z e - t h a w injury to i so la t ed p r o t o p l a s t s is t h e resu l t of two major s t r a i n s : a f r e e z e - or c o n t r a c t i o n - i n d u c e d m e m b r a n e a l t e r a t i o n which d e c r e a s e s t h e max imum c r i t i c a l su r f ace a r e a of t h e p l a sma m e m b r a n e and a t h a w - or expans ion- induced dissolut ion of t h e p l a s m a m e m b r a n e which occu r s when t h e max imum c r i t i c a l s u r f a c e a r e a is e x c e e d e d . These two s t r a ins i n t e r a c t dur ing a f r e e z e - t h a w cyc le and resu l t in lysis of t h e ce l l . Specif ical ly , p r o t o p l a s t s do no t possess a single max imum su r f ace a r e a a t which lysis occu r s (Fig. 6). The p r e c i s e su r f ace a r e a a t which lysis occu r s is a funct ion of two f a c t o r s : t h e e x t e n t of c o n t r a c t i o n incu r red and the abso lu te magn i tude of change in su r f ace a r e a which can be t o l e r a t e d be fo re lysis o c c u r s .

1. Practical Considerations. In a popula t ion of ce l l s , t h e abso lu te magn i tude of change in su r f ace a r e a t h a t r e s u l t s in lysis of 50 p e r c e n t of t h e cel ls appea r s t o be cons t an t and independen t of t h e e x t e n t of c o n t r a c t i o n (Fig. 7a) . We r e f e r to this cons t an t as t he To le rab le Surface


A r e a I n c r e m e n t and use TSAI™ to d e n o t e t h e t o l e r ab l e su r f ace a r e a i n c r e m e n t w h e r e 50 p e r c e n t of the cel ls lyse . Thus, t he max imum c r i t i c a l su r f ace a r e a a t which 50 p e r c e n t of t h e popula t ion will lyse is equal t o t h e min imum su r f ace a r e a plus t h e TSAI-^ . P r o t o p l a s t s will c o n t r a c t dur ing f reez ing a t slow cool ing r a t e s , wi th tne min imum su r f ace a r e a a funct ion of t h e lowest t e m p e r a t u r e a t t a i n e d . Subsequent ly , during thawing they will expand as a funct ion of t h e osmola l i ty of t h e suspending m e d i u m . Abso lu te vo lumes and cor responding su r f ace a r e a s will be a funct ion of the c h a r a c t e r i s t i c o s m o m e t r i c behavior and the in i t ia l cel l s i zes . If t h e e x t e n t of c o n t r a c t i o n is equal t o or less than t h e TSAI^^, 50 p e r c e n t or m o r e of t he popula t ion will be able t o subsequent ly expand to t h e or iginal s u r f ace a r e a wi thout lysing. A l t e r n a t i v e l y , if t h e e x t e n t of c o n t r a c t i o n equals or exceeds t h e TSAI^^, 50 p e r c e n t or m o r e of t h e cel ls will lyse be fo re r ega in ing the i r in i t ia l (p re -con t rac t ion) su r f ace a r e a . By d e t e r m i n i n g t h e o s m o m e t r i c behav ior and TSAI™, survival of i so la t ed sp inach p r o t o p l a s t s can be q u a n t i t a t i v e l y a c c o u n t e d for by the abso lu te m a g n i t u d e of change in su r f ace a r e a which t h e p l a s m a m e m b r a n e undergoes dur ing t he f r e e z e - t h a w - i n d u c e d c o n t r a c t i o n and expansion e v e n t s (48). F u r t h e r c o r r e c t i o n s for t e m p e r a t u r e in f luence on expans ion-induced lysis a r e r equ i r ed for s imi la r p r ed i c t i ons for w h e a t p r o t o p l a s t s (50).

It is r ead i ly a p p a r e n t t h a t TSAI™ is a powerful p a r a m e t e r for assess ing the c a p a c i t y of a popula t ion or ce l l s to surv ive a given f r e e z e -thaw cycê. This va lue va r i e s among spec ies examined to d a t e : TSAI^^ = 900 Mm for spinach p r o t o p l a s t s (48), 400 y m for whea t p r o t o p l a s t s (49), and 500 μ m for rye p r o t o p l a s t s (43). However , one canno t infer a rank ing of ha rd iness based on T S A I ^ ^ a l one . Because of t h e abso lu t e n a t u r e of T S A I ^ Q , a p a r t i c u l a r T S A I ^ ^ va lue mus t be v iewed in r e l a t i o n to the c h a r a c t e r i s t i c o s m o m e t r i c behavior of the ce l l s in ques t ion . This behav ior will be in f luenced by t h e i n t e rna l so lu te c o n c e n t r a t i o n and t h e or ig ina l ce l l s i z e . Smal ler ce l l s or ce l l s wi th h igher i n t e rna l so lu te c o n c e n t r a t i o n s or smal l e r o smot i ca l ly a c t i v e f rac t iona l cel l vo lumes will undergo sma l l e r changes in su r f ace a r e a pe r uni t change in e x t e r n a l osmola l i ty or f reez ing t e m p e r a t u r e . Thus , bo th t h e T S A I ^ Q and t h e o s m o m e t r i c behavior a r e r equ i r ed to p r e d i c t t he ce l l s c a p a c i t y t o w i t h s t a n d a given f r e e z e - t h a w c y c l e . In such an analys is , t h e o s m o m e t r i c behavior will de sc r ibe t he v o l u m e t r i c r e sponse of t he ce l l s to a given f r e e z e - t h a w cycle—or t h e i n t ens i t y of t h e s t r a in imposed , while t h e T S A I , - Q will desc r ibe t h e t o l e r a n c e of t h e cel ls to an imposed s t r a in . Thus, t he analys is af fords t he oppor tun i ty t o assess t h e con t r ibu t ion of f a c t o r s which inf luence t h e cel ls ' s ens i t iv i ty s e p a r a t e l y from those which in f luence t h e a c t u a l r e sponse t o a given f r e e z e - t h a w c y c l e .

2. Theoretical Considerations. The foregoing discussion has been based l a rge ly on obse rva t ions of popula t ion a v e r a g e s of ce l l s exposed to o s m o t i c man ipu la t ion or f r e e z e - t h a w c y c l e s . The obse rva t ions and i n t e r p r e t a t i o n s a r e useful to desc r ibe t h e responses of a popula t ion a v e r a g e of ce l l s and the discussion ha s solely addressed popula t ion a v e r a g e s . In o rde r t o p rov ide insight in to t h e speci f ic n a t u r e of t h e


p la sma m e m b r a n e a l t e r a t i o n s and t h e mechan i sm of injury, t h e s e obse rva t ions must be p ro j ec t ed to individual ce l l s . Unfo r tuna t e ly , i so la t ion of p ro top l a s t s from leaves r e su l t s in a popula t ion of cel ls of varying s i zes . Thus, we a r e able to infer the behavior of individual cel ls wi thin a popula t ion from popula t ion a v e r a g e s only by making seve ra l assumpt ions r ega rd ing the behavior of the p l a sma m e m b r a n e componen t s in cel ls of d i f fe ren t s ize r anges exposed to compress ive and tens i le s t r e s s e s . Der iva t ion of assumpt ions t ha t a r e physical ly r ea sonab le r equ i r e s us to d e l i n e a t e t he physical and /o r b iochemica l p rocesses which might occur dur ing c o n t r a c t i o n and expansion, and which lead to m e m b r a n e dissolut ion. At t h e p r e s e n t t i m e , we can only a t t e m p t to specu l a t e on the molecu la r mechan ism of lysis based on a r e l a t i ve ly l imi t ed knowledge of t h e i n t e r m o l e c u l a r i n t e r a c t i o n s of m e m b r a n e molecu le s .

F i r s t we m a k e the s imples t assumpt ion , t h a t in tens ive mechan i ca l p r o p e r t i e s of the p l a sma m e m b r a n e of in i t ia l ly smal l cel ls a r e iden t i ca l to the in tens ive mechan i ca l p r o p e r t i e s of t he p l a sma m e m b r a n e of in i t ia l ly l a rger ce l l s . We use the t e r m " in tens ive p r o p e r t i e s " in t he t h e r m o d y n a m i c sense - p r o p e r t i e s which a r e independen t of t h e amoun t of subs t ance cons idered . In the case of the s i tua t ion p r e sen t l y being cons idered , this assumpt ion will hold t r u e only if t he compos i t ion and topological a r r a n g e m e n t of m e m b r a n e c o n s t i t u e n t s is iden t i ca l in ini t ia l ly l a rge and ini t ia l ly smal l ce l l s . If e x p e r i m e n t a l findings a r e c o n t r a r y to this a ssumpt ion , our model will necessa r i ly be q u a n t i t a t i v e l y i n c o r r e c t . However , qua l i t a t ive ly our model could st i l l s e rve useful for the de r iva t ion of p a r a m e t e r s for individual c lasses of cel ls wi thin a mixed popula t ion (4).

Iden t i ty of in tens ive p rope r t i e s would fu r ther mean t h a t t he popula t ion surviving a con t r ac t ion -expans ion even t would have a s ize d i s t r ibu t ion cons i s t en t wi th t h e original popula t ion d i s t r ibu t ion . In addi t ion , ex tens ive mechan ica l p r o p e r t i e s would vary wi th cel l s i z e . One obvious example of an in tens ive p r o p e r t y in t h e model given l a t e r would be the energy of a t t r a c t i o n b e t w e e n ad jacen t m e m b r a n e componen t s , or t h e i n t e r m o l e c u l a r spacing b e t w e e n c o m p o n e n t s . An example of an ex tens ive p r o p e r t y will t hen be t he T S A I ^ . ^ va lue . Tha t is , T S A I - Q would vary as a d i r ec t funct ion of in i t ia l protorj last s i z e . These a s p e c t s of the problem will be app roached l a t e r as consequences of t h e mode l .

It should be n o t e d a t this point t ha t all vo lumes and su r face a r e a s in the s tud ies r e p o r t e d w e r e c a l c u l a t e d on the basis of the a v e r a g e d i a m e t e r of t h e sampled popula t ion . Tha t is, t h e d i a m e t e r s of sampled p ro top l a s t s w e r e measu red , an a v e r a g e was t aken and from this a v e r a g e volumes and su r face a r e a s w e r e ca l cu l a t ed . This p r o c e d u r e was used b e c a u s e p ro top la s t d i a m e t e r s followed an appa ren t ly no rma l d i s t r ibu t ion . Since n=25 or 30 in these s tud ies , t h e C e n t r a l Limi t Theorem (42) was not appl icable to su r face a r e a or volume a v e r a g e s . Hence , it was d e e m e d a p p r o p r i a t e to use t h e d i a m e t e r a v e r a g e to c a l c u l a t e t h e a v e r a g e su r face a r e a and volume of the samples so t ha t the r e su l t ing means could be e v a l u a t e d wi th convent iona l s t a t i s t i c a l t echn iques and res idua l e r ro r s would on the a v e r a g e equal 0. This manipu la t ion of the d a t a is s imi lar in

F r e e z e - T h a w - l n d u c e d L e s i o n s in t h e P l a s m a M e m b r a n e 2 4 S

pr inc ip le to t h e t r a n s f o r m a t i o n of skewed d a t a to approach a p a r a n o r m a l d i s t r ibu t ion , e .g. , t he use of a g e o m e t r i c m e a n r a t h e r than an a r i t h m e t i c m e a n (42).

The fac t t h a t p r o t o p l a s t s su fc ic ien t ly c o n t r a c t e d a r e unable to r ega in the i r ini t ia l su r f ace a r e a wi thou t lysing sugges t s t ha t some a l t e r a t i o n occur s such t h a t t h e expansion p o t e n t i a l of t he p l a sma m e m b r a n e is d e c r e a s e d . Since t he su r f ace a r e a of t he p l a s m a m e m b r a n e can be r e d u c e d by m o r e than 50% of t h e or iginal a r e a during c o n t r a c t i o n (which t r a n s l a t e s to an a v e r a g e 29% d e c r e a s e in the l inear d i s t ance b e t w e e n ad jacen t molecu les ) , it is r ea sonab le to a s sume t h a t this a l t e r a t i o n is l o c a t e d within t he p l a sma m e m b r a n e i tself .

Cons ider ing m e m b r a n e s t r u c t u r e from a pure ly m e c h a n i s t i c p e r s p e c t i v e , i t is be l ieved t h a t in the "na t ive" s t a t e the m e m b r a n e is s t r u c t u r e d such t h a t t h e global f ree ene rgy is a t a min imum (41), or a t l eas t a t a local min imum or saddle point (9, 10, 20). The global f ree ene rgy r e p r e s e n t s t h e sum of individual i n t e r - and i n t r a m o l e c u l a r ene rg ie s of a t t r a c t i o n and repuls ion . Luke and Kaplan (20) h a v e c a l c u l a t e d t he family of spher ica l solut ions for t he f ree ene rgy of ves ic les as a funct ion of a r e a and h a v e shown it t o be an a p p a r e n t s ing le -min imum ene rgy s t r u c t u r e devoid of saddle po in t s . Tha t is , bo th compress ion and expansion will r e su l t in an i n c r e a s e in the global f ree energy , which may be unfavorab le from an e n e r g e t i c s t andpo in t . Some m e m b r a n e a l t e r a t i o n in s t r u c t u r e and /o r compos i t ion which r e d u c e s this i n c r e a s e in f r ee energy would be f avorab le . Such an a l t e r a t i o n could consis t of e i t he r a r e a r r a n g e m e n t of m e m b r a n e molecu les i n t r a -o r i n t e r m o l e c u l a r l y , a de le t ion of m e m b r a n e molecu les , or bo th . R e s u l t s of f luorescen t label ing of the p l a s m a m e m b r a n e (i .e. , t he appa ren t i n t e r n a l i z a t i o n of p l a sma m e m b r a n e - b o u n d f luoresce in -Concanava l in A following con t rac t ion ) a r e cons i s t en t wi th a p o s t u l a t e d de le t ion of mo lecu l e s . It a p p e a r s t h a t a m e m b r a n e a l t e r a t i o n does occur during c o n t r a c t i o n , and this a l t e r a t i o n cons is t s a t l eas t in p a r t of a de le t ion of m e m b r a n e c o m p o n e n t s . The possibi l i ty ex is t s t h a t this c o n t r a c t i o n -induced m e m b r a n e a l t e r a t i o n is suff ic ient to lower the global f ree energy of t h e c o n t r a c t e d m e m b r a n e to i t s p rev ious i so tonic va lue . This would then explain some of the p h e n o m e n a observed following p ro top l a s t expansion.

Cons ider t h e following mode l , which a t t e m p t s to desc r ibe t he phenomenon of p ro top l a s t lysis as a r e su l t of c o n t r a c t i o n followed by expansion. We p r e s e n t this model h e r e for severa l r ea son : 1) to aid t he r e a d e r in in tu i t ive ly unde r s t and ing the ly t ic phenomenon , and 2) to formal ly p r e s e n t one i n t e r p r e t a t i o n of t h e d a t a which, a l though h y p o t h e t i c a l , is t e s t a b l e .

A fundamen ta l p r inc ip le of ves ic le (or membrane ) m e c h a n i c s is t he r e l a t ionsh ip :

dw = Τ dA

w h e r e Τ is m e m b r a n e tens ion, A is su r f ace a r e a and w is t h e work done on t h e ves ic le by t h e env i ronmen t (7). In p r o t o p l a s t s , Τ mus t be r e l a t i ve ly


c o n s t a n t during both c o n t r a c t i o n and expansion in t h e r a n g e of osmola l i t i e s t e s t e d (0.35 to 2.75 osmolal) s ince p r o t o p l a s t s follow the Boyle-van ' t Hoff law. If Τ i nc r ea sed wi th an i n c r e a s e in cell s u r f a c e a r e a , t he m e m b r a n e would b e c o m e m o r e r e s i s t a n t to s t r e t c h i n g and would no t follow t h e Boyle-van ' t Hoff law (27). Genera l ly , t h e va lue of Τ in an uns t r a ined s t a t e has been assumed to be 0 (7), a l though no d i r ec t ev idence ex i s t s . Rand (35) has c a l c u l a t e d t h e ex i s t ence of a smal l , but n o n - z e r o , t ens ion . Assuming t h a t Τ has a r e l a t i ve ly c o n s t a n t , n o n - z e r o va lue in p r o t o p l a s t s , dw will be d i r ec t ly p ropor t iona l to dA. The work done on the m e m b r a n e is d i r ec t l y r e l a t e d to the i nc r ea se in i t s k ine t i c ene rgy (2). These re la t ionsh ips l ead to t h e concep t t h a t t h e d isrupt ion of i n t e rmo lecu l a r forces in the m e m b r a n e cause p ro top l a s t lysis . One can concep tua l ly envision the p rocess as follows. A m e m b r a n e is composed of many d i s t inc t componen t s whose assoc ia t ion is the resu l t of c h a r a c t e r i s t i c ene rg ies of a t t r a c t i o n (37). As the p l a sma m e m b r a n e expands , the above re la t ionsh ips d e m a n d tha t the k ine t i c ene rgy of the m e m b r a n e i n c r e a s e . Expe r imen ta l ly it has been shown tha t a t l eas t some of this k ine t i c energy is en t rop ica l ly t r a n s l a t e d in to inc reased m e m b r a n e f luidi ty of p lan t p r o t o p l a s t s as well as l iposomes (1). We s p e c u l a t e t h a t when this i nc reased k ine t ic energy exceeds the energy of assoc ia t ion of some p a r t i c u l a r s i te(s) , t he associat ion(s) will no longer be s t ab l e and m e m b r a n e d e n a t u r a t i o n (lysis of t he cell) will follow.

Such a concep t of m e m b r a n e disrupt ion can be used to consider t he observa t ion t h a t , upon di lut ion, lysis in a popula t ion of p ro top l a s t s is c o r r e l a t e d wi th t he a v e r a g e i nc r ea se in su r face a r e a (48). This impl ies t ha t d isrupt ion of the m e m b r a n e of a cel l of a given in i t ia l s ize r equ i r e s t h e same amoun t of t o t a l work (in a given type of osmot icum) r ega rd l e s s of the su r face a r e a of the cel l a t the t i m e of expansion. That is , r ega rd l e s s of t h e e x t e n t to which t h e p ro top la s t had been c o n t r a c t e d , the t o t a l i nc rea se in k ine t ic energy of the m e m b r a n e of a p ro top la s t a t the t i m e of lysis r e m a i n s c o n s t a n t . Thus, we t e n t a t i v e l y sugges t t ha t p ro top las t lysis occurs when the k ine t ic energy d iss ipa ted into the m e m b r a n e during expansion exceeds t he m a g n i t u d e of t h e weakes t of t he i n t e rmo lecu l a r fo rces joining the m e m b r a n e t o g e t h e r (i .e. , the weak link). This sugges t ion has r e l e v a n t consequences to t he n a t u r e of t h e c o n t r a c t i o n - i n d u c e d m e m b r a n e a l t e r a t i o n .

The specu la t ion m a d e above is a gene ra l i za t ion of t he m o r e spec i f ic , but eas ie r to c o n c e p t u a l i z e , idea t ha t lysis is r e l a t e d to the i n t e r m o l e c u l a r d i s t ance b e t w e e n adjoining molecu le s . Since this l a t t e r concep t is eas ie r to envision, the following discussion will c e n t e r about i t . However , in an effor t to s t r e s s t he point t ha t this discussion is pure ly h y p o t h e t i c a l , and a gross s impl i f ica t ion of t r u e m e m b r a n e p r o p e r t i e s , d imensionless number s will be used. In addi t ion , we a r e fo rced to l imit discussion to r e l a t i v e l y l a rge , homogeneous lipid ves ic les . Such ves ic les do c o n t r a c t uniformly, r ema in ing as spheres , when exposed to hype r ton ic so lu t ions , and they do lose the i r s e m i p e r m e a b i l i t y when the osmola l i ty is lowered (36). Assume tha t ves ic le lysis will occur a t an i n t e r m o l e c u l a r spacing ό =1.225 and t ha t an " isotonic" ves ic le has 6 = 1 . E m p e r i -


cal ly we obse rve t h a t 50% of t h e ves ic les lyse when t h e m e m b r a n e undergoes a s u r f ace a r e a i n c r e a s e T S A I ^ Q = 1 0 . Now le t us follow t h e h y p o t h e t i c a l ves ic le through a f r e e z e - t h a w cyc le t ha t r e s u l t s in 50% lysis . The ves ic le wi th an in i t ia l " isotonic" s u r f a c e a r e a of 20, is fo rced to c o n t r a c t t o a su r f ace a r e a of 10 dur ing f r eez ing . Af te r thawing , t he ves ic le a t t e m p t s to r e - e x p a n d to i t s in i t ia l s u r f a c e a r e a of 20. If al l t h e m e m b r a n e molecu les r e m a i n e d in t he ves ic le , t he i n t e r m o l e c u l a r d i s t a n c e in t h e c o n t r a c t e d s t a t e would be 0.707 and following expansion the i n t e r m o l e c u l a r d i s t a n c e would r e t u r n t o 1. However , i t was n o t e d t h a t 50% lysis occu r r ed , and this co r responds to 6 1.225. For t h e i n t e r m o l e c u l a r d i s t a n c e in this c a s e to r e t u r n to l .zZb, 6 in t h e cont r a c t e d s t a t e would h a v e to be 0.866. In o t h e r words , roughly 3 3 % of the or iginal ves ic le c o m p o n e n t s h a v e b e e n d e l e t e d or i n t r a m o l e c u l a r l y a l t e r e d so as to change the i r vo lume , and the r e m a i n i n g or a l t e r e d c o m p o n e n t s h a v e b e c o m e s o m e w h a t compres sed . It is useful t o m a k e t h e ana logy wi th a spr ing which b r eaks when i t s coi ls a r e a f ixed d i s t a n c e a p a r t . In o rde r t o sho r t en t h e spr ing we r e m o v e some of t h e coils and c o m p r e s s those r e m a i n i n g . Al though th is is a ve ry s impli f ied view of as complex an e n t i t y as t h e p l a sma m e m b r a n e , i t is in tu i t ive ly p leas ing when this c o n c e p t u a l model is e x t r a p o l a t e d back to t he or ig ina l , a b s t r a c t supposi t ion t h a t lysis occu r s a t a f ixed k ine t i c ene rgy of t h e m e m b r a n e . In an e n e r g e t i c sense , t h e two s y s t e m s m a y no t be ve ry d i f f e ren t .

Nex t cons ider t h e case of a ves ic le wi th in i t ia l su r f ace a r e a of 20 which is c o n t r a c t e d to 5. In this h y p o t h e t i c a l c a s e 7 5 % of the ves ic les lysed, which cor responds to 0 , ^ = 1 . 4 . In t h e c o n t r a c t e d s t a t e 6^=0.70, so t h a t this con t r ac t ed -ves i c l e con ta ins only 5 1 % of t h e in i t ia l a m o u n t of ves ic le m a t e r i a l . In this ca se lysis of 50% of the ves ic les should occur following expansion to a su r f ace a r e a of 15 (i .e. , when 6 = δ ς π = 1.225).

C

The values of as a funct ion of % lysis can be de r ived in t h e following m a n n e r . The equa t ions for t h e above e x a m p l e a r e :

δ%

= δ5 0 Ai

A + T S A I c n) c 50

1/2 ( 1 )

and [see Levin e t a l . , ( 1 7 ) ]

1 . 7 1 % S - 1 0 0 exp ( - 2 . 2 7 Δ A ) (2)

w h e r e 6 ^ is t h e r e l a t i v e d i s t a n c e b e t w e e n ad j acen t molecu les in survivors of a popula t ion t h a t u n d e r w e n t a given % lysis , δ is t h e d i s t a n c e a t 5 0 % lysis ( = 1 . 2 2 5 in this example ) , A . is in i t ia l ves ic le a r e a ( 2 0 in this example ) , A is c o n t r a c t e d ves ic le a r e a , T S A I ^ Q is t h e su r f ace a r e a i n c r e m e n t a t 5*0% surv iva l , % S is % survival and Δ A is

A . - A _i c

A . ι


The d i s t a n c e b e t w e e n ad jacen t molecu les in t h e c o n t r a c t e d s t a t e is

c %

A c

A. ι

1/2 (3)

The p e r c e n t a g e of t h e ini t ia l amoun t of ves ic le m a t e r i a l r ema in ing in t he c o n t r a c t e d ves ic le is

100A % m a t e r i a l r ema in ing = =- (4).

A. r L

ι c

In this manne r we q u a n t i t a t e how much m e m b r a n e m a t e r i a l mus t be d e l e t e d (or how much the p a r t i a l molar volume of the individual componen t s must be reduced) a t any given e x t e n t of c o n t r a c t i o n , in order to f a c i l i t a t e t e s t i ng of the mode l . It should be emphas ized t ha t a t the p r e sen t s t a g e in t he deve lopmen t of t he model , t h e above re la t ionsh ips a r e no t necessa r i ly q u a n t i t a t i v e l y a c c u r a t e , espec ia l ly s ince the p l a sma m e m b r a n e is no t homogeneous . N e v e r t h e l e s s , they do provide a basis for devising new e x p e r i m e n t s and for ex tend ing t h e theo ry .

A coro l la ry of t h e above concep tua l model may also be p r e s e n t e d . That is , s ince more m o l e c u l e s / m e m b r a n e a r e p r e s e n t in the c o n t r a c t e d s t a t e in t h e f irs t ca se (A = 20 •> 10) t han in t h e second (A = 20 -*· 5), and s ince t he abso lu te su r face a r e a i n c r e m e n t a t 50% survival is constant, it appa ren t ly t a k e s a lower amoun t of k ine t i c e n e r g y / m o l e c u l e to cause 50% of t h e m e m b r a n e s to lyse in the f irs t c a s e . This does , in f ac t , appea r to be the case in the above model s ince it should t a k e less energy to sp read the molecu les from 6 =.866 to 6=1.225 than to sp read them from 6 = . 7 0 t o 6=1.225 .

The above model makes no assumpt ions concern ing t h e behavior of cel ls within a given p e r c e n t i l e r ange of a popula t ion . The s imples t possible behavior of cel ls would be t he case of a single %S V S . 6 ~ su r f ace valid for all p e r c e n t i l e s fo t h e popula t ion . Tha t is , as men t ioned ea r l i e r , 6 ^ is an in tens ive p r o p e r t y and iden t i ca l for all cel ls in t h e popula t ion . This would m e a n t h a t TSAI^^ would vary wi th the original s ize of t he p ro top la s t , and the popula t ion d is t r ibu t ion of survivors of a f r e e z e - t h a w cyc le should be iden t i ca l to t he original d i s t r ibu t ion . P re l imina ry d a t a , wi th l imi t ed n u m b e r s of obse rva t ions , a r e cons i s t en t wi th this type of behavior (47). An a t e r n a t i v e type of behavior would be t ha t t he %S V S . 6 ^ su r f ace var ies with t h e ini t ia l s ize of t h e ce l ls , pe rhaps because of nonhomogene i ty of cell t ype in t h e popula t ion . For i n s t ance , if ini t ia l ly l a rge cel ls had a l a rge r δ _ Q t han cel ls t h a t w e r e ini t ia l ly smal l , t h e popula t ion d i s t r ibu t ion a f t e r a f r e e z e - t h a w cyc le would be skewed to t h e r ight of t h e or iginal d i s t r ibu t ion . The magn i tude of t h e d i f fe rence would be dependen t upon the m a g n i t u d e of t h e d i f fe rences in 6 L ikewise , if in i t ia l ly smal l cel ls had a l a rger δ ^ than cells t ha t w e r e in i t ia l ly l a rge , the resu l t ing d is t r ibu t ion of survivors would be skewed to t he left of t h e original d i s t r ibu t ion . In these cases equa t ion 4 would be a sum of t h e individual c h a r a c t e r i s t i c s of e a c h c lass


of t h e popula t ion . F u r t h e r work wi th l a rge n u m b e r s of obse rva t ions of the in i t ia l popula t ion and of survivors of a f r e e z e - t h a w cyc le could r e so lve t h e behavior of individual cel ls within a popula t ion . C u r r e n t l y , this app roach and d i r ec t obse rva t ions of individual ce l ls exposed to a f r e e z e - t h a w cyc le a r e being i m p l e m e n t e d .

Π. R E F E R E N C E S

1. Borochov, A. and Borochov, H. Biochim. Biophys. Acta 550, 546-549 (1979).

2. C r o m e r , A. H. "Physics for t h e Life Sciences ." McGraw-Hi l l , N.Y. (1974).

3 . Dick, D. A. T. In " In te rna t iona l Rev iew of Cyto logy" (G. H. Bourne and J . F . Danie l l i , eds.) , Vol. 8. pp . 387-448. A c a d e m i c P res s , New York (1959).

4 . Dick, N. P . , and Bowden, D. C . Biometrics 29, 781-790 (1973). 5. D o w g e r t , M. F . , and Steponkus , P . L. Plant Physiol. 63, (Abst rac t )

(1979). 6. Evans , Ε. Α. , and LeBlond, P . F . Biorheology 10, 393-404 (1973). 7. Evans , Ε. Α., and Waugh, R. J. Coll. Interf. Sci. 60, 286-298

(1977). 8. G a r b e r , M. P . , and Steponkus , P . L. Plant Physiol. 57, 673-680

(1976). 9. Helf r ich , W. Z. Naturforsch 28C, 693-703 (1973). 10. Jenk ins , J . T. SIAM J. Appl. Math. 32, 755-764 (1977). 11 . Levin, R . L., C rava lho , E. G., and Huggins, C. E. Cryobiology 13,

415-429 (1976). 12. Levin , R . L., C rava lho , E. G., and Huggins, C . E. J. Heat Transfer

Trans. ASM Ε 99, 322-329 (1977a). 13. Levin, R. L., C rava lho , E. G., and Huggins, C . E. J. Biomechanical

Engineering Trans. ASM Ε 99, 65-73 (1977b). 14. Levin , R . L. , C rava lho , E. G., and Huggins , C . E. Biochim. Bio

phys. Acta 465, 179-190 (1977c). 15. Levin , R. L., Ferguson , J . F . , D o w g e r t , M. F . , and Steponkus , P . L.

Plant Physiol. 63, (Abst rac t ) (1979a). 16. Levin, R . L. , S teponkus , P . L. , and Wiest , S. C. Agronomy

Abstracts p . 80 (1978). 17. Levin, R. L., Wiest , S. C , and Steponkus , P . L. P r o c . 10th Ann.

Modeling and S t imula t ion C o n f e r e n c e . P i t t sbu rgh , (in press) . (1979b).

18. Lovelock, J . E. Biochim. Biophys. Acta 10, 414-426 (1953). 19. Lovelock, J . E. Proc. Roy. Soc. 147, 427-433 (1957). 20. Luke, J . C . and Kaplan , J . I. Biophys. J. 25, 107-111 (1979). 2 1 . Mansoor i , G. A. Crybiology 12, 34-45 (1975). 22. Mazur , P . J . Gen. Physiol. 47, 347-369 (1963). 23 . Mazur , P . Ann. Rev. of Plant Physiol. 20, 419-448 (1969).


24. Mazur , P . Science 168, 939-949 (1970). 25 . Mazur , P . Cryobiology 14, 251-272 (1977a). 26. Mazur , P . In "The F r e e z i n g of M a m m a l i a n Embryos" p p . 19-48.

CIBA Founda t ion Symp. London (1977b). 27. Mela , M. J . Biophys. J. 7, 95-110 (1967). 28 . Meryman , Η. T. Nature 218, 333-336 (1968). 29. Meryman , Η. T. Cryobiology 8, 488-500 (1971). 30 . Meryman , Η. T. Ann. Rev. Biophys. 3, 341-363 (1974). 3 1 . Meryman , Η. T., Wil l iams, R . J . , and Douglas , M. St . J . Cryobiolo

gy 14, 287-302 (1977). 32 . P a l t a , J . P . , and Li, P . H. In "P lan t Cold Hard iness and F r e e z i n g

S t r e s s -Mechan i sms and Crop Impl ica t ions . " (P. H. Li and A. Sakai , eds.) , p p . 93-115 . A c a d e m i c P r e s s , New York (1978).

3 3 . Ponder , E. Protoplasmatologia 10 (Pt. 2), 1-123 (1955). 34 . Pushkar , N . S., Etk in , Υ. Α., Brons te in , V. L. , Gord iyenko , Ε. Α. , and

Kozmin , Υ. V. Cryobiology 13, 147-152 (1976). 35 . Rand , R . P . Biophys. J . 4, 303-316 (1964). 36 . R e e v e s , J . P . , and Dowben, R . M. J. Cell Physiol. 73, 49-60 (1969).

(1969). 37 . Sa lem, L. Can. J. Biochem. Physiol. 40, 1287-1298 (1962). 38 . Sca r th , G. W., L e v i t t , J . , and Siminovi tch , D . Cold Spg. Harbor

Sym. 8, 102-109 (1940). 39 . Si lvares , Ο. M., C rava lho , E. G., Toscano , W. M., and Huggins, C . E.

J . Heat Trans. ASME 97, 582-588 (1975). 40 . Siminovi tch , D. , and L e v i t t , J . Can. J. Res. 19, 9-20 (1941). 4 1 . Singer, S. J . , and Nicolson, G. L. Science 175, 720-731 (1972). 4 2 . Snedecor , G. W., and Cochran , W. G. Statistical Methods Iowa

S t a t e Univ. P r e s s , A m e s , Iowa (1967). 4 3 . S teponkus , P . L. , D o w g e r t , M. F . , and Levin, R . L. Plant Physiol.

63, (Abst rac t ) (1979). 4 4 . S teponkus , P . L, G a r b e r , M. P . , Myers , S. P . , and L inebe rge r , R . D .

Cryobiology 14, 303-321 (1977). 4 5 . S teponkus , P . L., Wiest , S. C , and Levin, R. L. Agronomy Ab

stracts p . 85-86 (1978). 46 . Tornava , S. R . Protoplasma 32, 329-341 (1939). 47 . Wiest , S. C . P h . D . Thes is . Corne l l Univ. , I t h a c a , N .Y. (1979). 4 8 . Wiest , S. C , and Steponkus , P . L . Plant Physiol. 62, 599-605

(1978a). 4 9 . Wiest , S. C , and Steponkus, P . L. Plant Physiol. 61 (Supple.), .32

(1978b). 50. Wiest , S. C , and Steponkus , P . L . Agronomy Abstracts, p . 88

(1978c). 5 1 . Wiest , S. C , and Steponkus, P . L. Cryobiology 16, 101-104(1979) . 52. Zade-Oppen , Α. Μ. N . Acta Physiol. Scand. 73, 341-364 (1968).


MEMBRANE STRUCTURAL TRANSITIONS: PROBABLE RELATION TO FROST DAMAGE

IN HARDY HERBACEOUS SPECIES

ο 3 C. Rajashekar, L. V. Gusta and M. J. Burke

D e p a r t m e n t of H o r t i c u l t u r e Colorado S t a t e Univers i ty

F t . Col l ins , Co lo rado

I. INTRODUCTION

T h e r e is a mp le ev idence in t h e s e p roceed ings t h a t t e m p e r a t u r e dependen t m e m b r a n e s t r u c t u r a l t r ans i t i ons occur in a wide v a r i e t y of chi l l ing sens i t ive p l a n t s . These s t r u c t u r a l t r ans i t i ons l ead to a l t e r e d a c t i v i t i e s of m e m b r a n e a s s o c i a t e d funct ions t h a t even tua l ly cause p lan t injury. It is an a t t r a c t i v e hypothes i s t ha t t e m p e r a t u r e dependen t m e m b r a n e s t r u c t u r a l t r ans i t i ons also occur a t subf reez ing t e m p e r a t u r e s in i n t e r m e d i a t e l y frost ha rdy p l an t s , and t h a t such t e m p e r a t u r e dependen t m e m b r a n e s t r u c t u r a l t r ans i t i ons a r e respons ib le for frost injury.

Before discussing t h e s imi l a r i t i e s b e t w e e n chil l ing and frost injury m e c h a n i s m s it mus t be n o t e d t h a t t h e r e a r e m a r k e d d i f f e rences b e t w e e n chil l ing and frost injury. Chil l ing injury in p l a n t s occu r s a t t e m p e r a t u r e s above 0 C, i t does no t involve t h e p r e s e n c e of i c e and t h e t i s sue is no t

1 These studies were supported in part by grants to M. J. Burke from

the Petroleum Research Fund (PRF 9702-ACl,6), the Research Corporation, the Horticultural Research Institute, and the Colorado Experiment Station and by grants to L. V. Gusta from the National Research Council of Canada (A-9661). Scientific Journal Series Paper #245(j of the Colorado Experiment Station.

Visiting Scientist from the Crop Development Centre, Crop Science Department, University of Saskatchewan, Saskatoon, Saskatchewan S7NOWO.

Present address, Department of Fruit Crops, University of Florida, Gainesville, Florida 32611.

2 5 5 Copyright

β 1Θ79 by Academic Press. Inc.

All rights of reproduction in any form reserved ISBN α 12 4β056Ο5

2 5 6 C. R a j a s h e k a r et al

seve re ly d e h y d r a t e d due to t h e g rowth of i ce c ry s t a l s . Chil l ing of ten r e su l t s from me tabo l i c dysfunct ions which a r e l ikely to be ve ry min imal in f rozen and d e h y d r a t e d t i ssues , and t h e r e f o r e of negl ig ib le i m p o r t a n c e in frost injury. Chil l ing sens i t ive p lan t s mus t of ten be given prolonged exposure below the i r c r i t i c a l t e m p e r a t u r e s for d a m a g e to develop and f requent ly r e c o v e r from the d a m a g e if exposed to warm t e m p e r a t u r e s . In c o n t r a s t , injury during f reez ing s t r e s s develops a f t e r brief exposure below a c r i t i c a l t e m p e r a t a u r e . It is c a t a c l y s m i c and the d i r ec t d a m a g e is i r r eve r s ib le and l e t h a l .

The re a r e many s imi la r i t i e s , however , b e t w e e n chill ing and frost injury. In bo th types of injury it is c lea r t ha t the m e m b r a n e s a r e of p ivo ta l i m p o r t a n c e . Chil l ing injury has been a s soc i a t ed wi th m e m b r a n e s t r u c t u r a l t r ans i t ions (16). Af te r frost injury has occu r r ed , one of the first signs of d a m a g e is t he loss of m e m b r a n e s e m i p e r m e a b i l i t y (15). In bo th cases t h e r e is a c r i t i c a l t e m p e r a t u r e involved and injury is observed only a f t e r exposure below this c r i t i c a l t e m p e r a t u r e . For frost ha rdy Kharkov win te r whea t c rowns , no injury is observed above -22 C, but a brief exposure below this t e m p e r a t u r e leads to frost killing (10). For t he chil l ing sens i t ive t o m a t o , pro longed exposure below 10 C is r equ i r ed for visible s y m p t o m s of injury to deve lop , w h e r e a s exposure above 10 C causes no injury.

T h e r e a r e severa l d i f f icul t ies in s tudying frost injury. Conven t iona l b iochemica l and biophysical me thods a r e no t easi ly appl ied to sy s t ems encased in i c e and a t low t e m p e r a t u r e s . These e x p e r i m e n t a l p rob lems make it diff icult to d e t e r m i n e the m o m e n t or t i m e the frost injury is i n i t i a t e d in a s amp le . This is a c lass ica l ques t ion discussed in de t a i l by Lev i t t (14, 15). To d e t e r m i n e the mechan ism of frost injury, the m o m e n t in t i m e t h a t injury is i n i t i a t ed during f reez ing or thawing must be known. There is only scan t ev idence w h e t h e r injury is i n i t i a t ed during f reez ing , i m m e d i a t e l y passing below a c r i t i ca l t e m p e r a t u r e , during thawing or a f t e r thawing . The e x p e r i m e n t a l r e su l t s sugges t ing a t wha t point during t h e f r e e z e - t h a w cyc le injury i n i t i a t e s a r e based on q u a l i t a t i v e ev idence [for i ao re discussion see Lev i t t (14)] , p r imar i ly from microscop ic obse rva t ions (19), or from f luorescen t changes in cel ls under f rozen condi t ions (13). These r e su l t s suggest t h a t f reez ing injury is s o m e t i m e i n i t i a t e d i m m e d i a t e l y on passing below a c r i t i c a l t e m p e r a t u r e , t he frost injury t e m p e r a t u r e .

The ob jec t ive of this s tudy is to es tabl i sh t h e m o m e n t and s i t e of f reez ing injury. The r e s u l t s conclus ively es tab l i sh in the sy s t ems s tud ied h e r e t ha t injury occu r s during f reez ing i m m e d i a t e l y upon pass ing through a c r i t i c a l t e m p e r a t u r e and sugges t t ha t the p r i m a r y s i t e of injury is the p lasma m e m b r a n e . He rbaceous p l an t s wi th wel l -def ined killing t e m p e r a t u r e s (lower than -15 C) w e r e chosen as model sy s t ems for t he s tudy . At the killing t e m p e r a t u r e of t hese spec ies (winter w h e a t , Ken tucky b luegrass and cac tus ) , l i t t l e or no change in cel l dehydra t ion occu r s . Thus the e f fec t of dehydra t ion can b e t t e r be s e p a r a t e d from low t e m p e r a t u r e e f f e c t s .

M e m b r a n e S t ruc tu r a l T r a n s i t i o n s 2 5 7

- 5 - 1 5 - 2 5 - 3 5 - 4 5 - 5 5 T ( ° C )

FIGURE 1. Freezing curves (closed symbols) and electrolyte loss curves (open symbols) for Kharkov winter wheat (Triticum aestivum L.) leaf segments (·, 0). Kentucky bluegrass (Poa pratensis L.f Merion) leaf segments (m ,cs ) and cactus (Opuntia polyacantha Haw.) stem (k) . LT

is the liquid water content at temperature Τ in gm liquid water per gm dry tissue. On the relative electrolyte loss scale, a value of 1 is the electrolyte loss for a sample held at 80 C for 30 minutes. All electrolyte loss measurements were made on the thawed samples that had been slowly cooled to and warmed from the temperature indicated on the ordinate. Note absence of anomalies in the freezing curves at the inflections in the electrolyte loss curves. The nmr procedures for measurinq liquid water content are described by Gusta et al. (9).

Winter wheat was cjrown in a greenhouse for 3 weeks prior to moving to a cold chamber at 3 C and 12 hour dark period where it was held for 4 additional weeks or more. Kentucky bluegrass plugs were obtained from the field during December as and when required. Leaf segments were 1 cm in length and were taken from the middle portion of leaf blades. Cactus was grown at 23 C and cylindrical plugs 1 cm long and 0.7 cm diameter from stem blades were used for the experiment. The cooling rate was 3 Cfiiour or less.

2 5 8 C. R a j a s h e k a r et ai

Π. FREEZING OF WATER AT THE KILLING TEMPERATURE

In severa l t i ssues s tudied , w a t e r f r eezes l ike an ideal sa l t solut ion and usual ly does no t supercool apprec iab ly (5, 9, 11). Under slow f reez ing condi t ions , mos t t i ssue w a t e r f r eezes above - I O C (Fig. 1) r e su l t ing in no major change in frost dehydra t ion below this t e m p e r a t u r e . N e v e r t h e l e s s , many he rbaceous p l an t s a r e kil led well below -10 C as ev idenced by t h e sharp i n c r e a s e in e l e c t r o l y t e l eakage for whea t l eaves a t -17 C and for K e n t u c k y b luegrass l eaves a t -38 C (Fig. 1). The inf lec t ions in t h e e l e c t r o l y t e loss cu rves a r e sharply def ined over t h e na r row t e m p e r a t u r e r a n g e . Unfo r tuna te ly , e l e c t r o l y t e loss cu rves do not p rov ide in fo rmat ion as to t h e m o m e n t when t h e e l e c t r o l y t e l e akage is i n i t i a t e d during t h e f r e e z e - t h a w c y c l e . Each point on t h e e l e c t r o l y t e l eakage curve r e p r e s e n t s t h e f rac t iona l l eakage m e a s u r e d a f t e r t h e s ample was cooled to t he s t r e s s t e m p e r a t u r e ( indica ted on t h e absc issa in F ig . 1) and t h a w e d . F rom such r e su l t s , i t is not known if t h e e l e c t r o l y t e l e akage was i n i t i a t ed during f reez ing to t h e t e s t t e m p e r a t u r e , a t t h e t e s t t e m p e r a t u r e or during thawing from t h e t e s t t e m p e r a t u r e .

The f reez ing curves in f igure 1 a r e ob ta ined using nuc l ea r m a g n e t i c r e s o n a n c e (nmr) r e l a x a t i o n t i m e d i f f e rences b e t w e e n liquid w a t e r and ice as desc r ibed in de t a i l by G u s t a et (ll. (9). The curves ob ta ined during f reez ing and thawing a r e i d e n t i c a l . No anomalous behavior in t he f reez ing or thawing cu rve could be d e t e c t e d in t h e t e m p e r a t u r e reg ion of the major e l e c t r o l y t e loss . F u r t h e r m o r e , d i f fe ren t i a l t h e r m a l analys is could no t d e t e c t anomalous h e a t s n e a r t h e t e m p e r a t u r e s of t he e l e c t r o l y t e loss in f lec t ions . In these e x p e r i m e n t s , f r e e z i n ^ i n l eaves of whea t and Ken tucky b luegrass was i n i t i a t ed a round -3 C wi th i ce nuc lea t ion and d i f fe ren t ia l t h e r m a l analysis was c a r r i e d out using a cooling r a t e of 1 C /min . R e s u l t s from these and from o t h e r ex tens ive n m r ana lyses (3, 5, 9) sugges t absence of f reez ing poin ts a s soc ia t ed wi th t h e frost killing t e m p e r a t u r e .

ΠΙ. NUCLEAR MAGNETIC RESONANCE STUDIES

One of t h e few d i r ec t m e a n s for c h a r a c t e r i z a t i o n of w a t e r in a pa r t i a l l y f rozen sample is to s tudy the n m r r e l a x a t i o n p r o p e r t i e s of w a t e r p r o t o n s . The two r e l a x a t i o n p rocesses can be desc r ibed by the longi tudinal (T J and t r a n s v e r s e (T^) r e l a x a t i o n t i m e s and they have been used ex tens ive ly to s tudy w a t e r in p lan t t i s sues (1 , 2, 4, 22). These r e l a x a t i o n t i m e s a r e sens i t ive to i n t e r a c t i o n s b e t w e e n w a t e r and var ious ce l lu lar c o n t e n t s and t h e r e f o r e r e f l e c t , to some d e g r e e , t he env i ronmen t t h e w a t e r expe r i ences .

The nmr r e l axa t i on p r o p e r t i e s of w h e a t leaf s e g m e n t s in f igure 2 a r e typ ica l of those found in p lan t t i ssues (4, 21) and in model p ro te in sy s t ems (12). T h e r e a r e two exponent ia l ly decay ing c o m p o n e n t s for T ?

M e m b r a n e S t r u c t u r a l T r a n s i t i o n s 2 5 9

I I 1 L J 1 1 1 1 1 I I 1 I I I I I I I I I I I I ι l ι

2 0 4 0 6 0 8 0 10 0 12 0 2 0 4 0 6 0 8 0 10 0 12 0 T I ME (msec )

FIGURE 2. Echo amplitude decay in a Carr-Purcell-Meiboom-Gill experiment for measuring Τ^ nmr relaxation. See Farrar and Becker (6) forQmore details on the nmr methods. All measurements were made at 25 C on wheat leaf segments. The relaxation for live samples ( Φ ) is analyzed using two first order reaction rate processes. For wheat leaf segments (A) the major component, comprising 90% of the nmr signal, had a time constant, 7\> of 105 msec and the minor component had a time constant of 8 msec. Tne frosfo killed sample ( Ο ) , which had been slowly cooled and warmed from -70 C, had only one component with a Τ2 of 33 msec. Experiments in Β are identical to those in A except that in part B, the leaf segments were equilibrated in a 0.031 molar MnSO. solution for about 30 minutes prior to the nmr measurements. Note the drop in Τ2+ relaxation after frost death, which is more pronounced in the Mn treated sample.

r e l a x a t i o n , one accoun t ing for 90% of t h e w a t e r p ro tons wi th a T^ of 105 m s e c and t h e o t h e r f a s t e r r e l ax ing componen t wi th a T~ of 8 m s e c . The T ' s for t h e s e two c o m p o n e n t s a r e 276 m s e c (85%) a n d 39 m s e c (15%), r e s p e c t i v e l y . T h e r e is usual ly about a 5% d i sc r epancy in the p ropor t ion of t h e two f r ac t ions depending on if they a r e m e a s u r e d by t h e T~ or T^ p r o c e s s . These two Relaxatio n t i m e s a r e only s l ight ly a f f ec ted D y t he add i t ion of t h e Μη , (Fig. 2B), sugges t ing t h a t t h e Mn does ng t p e n e t r a t e to t h e bulk of t h e t i s sue w a t e r . P a r a m a g n e t i c ions l ike Mn when added to w a t e r cause a subs t an t i a l r e d u c t i o n in bo th T^ and T j r e l a x a t i o n t i m e s . If w h e a t leaf s amples a r e ki l led by slow cooling to - 7 0 C, two major changes in t h e n m r r e l a x a t i o n t i m e s a r e obse rved . F i r s t , t h e T^

fs shor ten^s igni f icant ly . The shor ten ing of T^ is much m o r e

p ronounced in t h e Mn con ta in ing samples (Fig. 2B). This i nd i ca t e s t h a t

2 6 0 C. R a j a s h e k a r et al.

T C C)

> l I I ι l J I i ι 1 I I L_J I 1 1 I I ι ι 1 0. 1 0 . 2 0 . 3 0. 1 0 . 2 0. 3 0. 1 0 . 2 0 . 3

- l / T ( ° C )

FIGURE 3. Freezing curves for the two components of Τ^ relaxation described in figure 2. Components I and II are the major and minor fractions of tissue water respectively. The data are plotted as suggested by Gusta et al (9). LnJLo is the liquid water of component I or II at temperature Τ divided by the total water of the sample. In the equations for linear regression, Y is Lj/Lo and X is 1/T ( C). Δ Tm is the melting point depression of the tissue solution when thawed and is obtained by dividing the slope of the line by the total water of the component, r is the coefficient of determination. The results suggest that both Τ2 nmr components freeze as ideal solutions, with melting point depressions between -1.5° and -3.1 C.

TABLE I. Nmr Relaxation Times for Live and Frost Killed Kharkov Winter Wheat Leaf Segments, Kentucky Bluegrass Leaf Segments and Cactus Stern. The Killing Temperature Was 17 C for Wheat, -38 C for Kentucky Bluegrass and -24 C for Cactus.

Treatment0

(lowest temperature Relaxation time (msec) in cooling-warming T2 T l

Sample cycle) (°C) Long (I) Short (II) Long (I) Short (II)

Wheat -9.6 11.0 (0.72)b 2.5 (0.28) 1750 (0.64) 30 (0.36) (measurements -14 11.0 (0.69) 2.4 (0.31) 1600 (0.66) 34 (0.34) at -9.6 C) -20 6.0 (0.66) 1.5 (0.34) 1490 (0.38) 45 (0.62)

-70 4.7 (0.71) 1.3 (0.29) 1580 (0.31) 55 (0.69)

Kentucky bluegrass -15 7.2 (0.66) 1.4 (0.34) 2100 (0.71) 40 (0.29) (measurements -30 7.3 (0.63 1.4 (0.37) 2035 (0.68) 50 (0.32) at -15 C) -40 4.1 (0.69) 0.3 (0.31) 2300 (0.38) 47 (0.62)

-70 3.8 (0.68) 0.2 (0.32) 2020 (0.40) 40 (0.60)

Cactus -4.8 131.0 (0.77) 12.0 (0.23) 1200 (0.66) 110 (0.34) (measurements -23 126.0 (0.75) 12.0 (0.25) 1220 (0.64) 116 (0.36) at -4.8°C) -29 48.0 (0.70) 2.0 (0.30) 1300 (0.52) 130 (0.48)

-70 43.0 (0.71) 1.8 (0.29) 1340

aThe treatments consist of a slow cooling to the test temperature (-9.6 C, wheat; -15 C, Kentucky bluegrass; -4.8 C, cactus) where the first relaxation time measurements were made. The samples were then cooled to the next temperature indicated and then warmed back to the test temperature and relaxation times again measured. This process was repeated on the same sample for each succeeding temperature.

The value in parenthesis refers to the proportion of total nmr signal decaying with this relaxation time.

2 6 2 C. Rajashekar et al

FIGURE 4. Echo amplitude decay in a Carr-Purcell-Meiboom-Gill experiment forT~ determination. All measurements were made at

-9.6 C. First, tne echo amplitudes were obtained for wheat leaf segments slowly cogled £o -9.6 C (O). The same sample was cooled subsequently to -14 C (3 C above the killing temperature) and warmed to the test temperature, -9.6 C, again to measure the echo amplitudes (Δλ This sample was then cooled to -20°C (3°C below the killing temperature) and rewarmed to -9.6 C to make a similar measurement (Φ). Final gcho amplitude measurement (et) was made on the sample cooled to -70 C and rewarmed to -9.6 C. These changes^ in echo amplitude decay occur in a sample not warmed above -9.6 C. Similar results for Kentucky bluegrass and cactus are presented in table I.


t h e Mn read i ly p e n e t r a t e s t h e bulk of t h e t i s sue w a t e r a f t e r frost injury. Second, the long and shor t r e l ax ing c o m p o n e n t s b e c o m e one componen t a f t e r frost injury (Fig. 2) in whea t leaf s e g m e n t s . Similar r e s u l t s a r e observed wi th Ken tucky b luegrass leaf s e g m e n t s and c a c t u s s t e m .

The two nmr re lax ing c o m p o n e n t s a r e also observed a t subfreez ing t e m p e r a t u r e s and f reez ing of e ach of t hese c o m p o n e n t s can be mon i to red (Fig. 3). The d a t a in f igure 3 a r e p l o t t e d as sugges ted by G u s t a e t al. (9) and show the typica l ideal f reez ing behavior observed with he rbaceous and some woody p l a n t s . The f reez ing cu rves can be desc r ibed wi th two p a r a m e t e r s , t h e me l t ing point depress ion of t h e aqueous componen t , Δ Τ , ob t a ined from t h e slope of t he se l ines and a p a r a m e t e r b , t h e i n t e r c e p t of these l ines , which is the f rac t ion of the componen t t ha t is no t f r e e z a b l e . These p a r a m e t e r s a r e given in t h e f igure 3 .

Various a t t e m p t s have been m a d e to ident i fy t h e two nmr re lax ing componen t s in p lan t and model p ro t e in s y s t e m s (12, 21). In f rozen p ro t e in solut ions and in p ro te in c ry s t a l s , t h e two exponent ia l ly decay ing componen t s of nmr r e l a x a t i o n a r e a t t r i b u t e d to the m a g n e t i z a t i o n t r ans fe r b e t w e e n p ro te in and w a t e r and a d i s t r ibu t ion of r o t a t i o n a l co r r e l a t i on t i m e s for the unf rozen aqueous f rac t ion of the f rozen p ro t e in s y s t e m s . In t e re s t ing ly , Stout e t al. (22) have proposed t h a t t h e two n m r re lax ing componen t s c o m e from two physical ly s e p a r a t e d aqueous f r ac t ions of t h e p lan t t i s sues . They conc luded t ha t t he componen t wi th a shor t T ? r e l a x a t i o n t i m e (component Π here) is a s soc i a t ed wi th t h e e x t r a c e l l u l a r w a t e r . The componen t wi th long T^ r e l a x a t i o n t i m e (component I here) is a s soc i a t ed wi th t he i n t r ace l lu l a r w a t e r , p re sumab ly t he vacuo la r w a t e r which c o n s t i t u t e s t he l a rges t f r ac t ion of ce l lu la r w a t e r and has a min imal i n t e r a c t i o n wi th m a c r o m o l e c u l e s . However , for t he se t i ssues it has not been possible to d e t e r m i n e t h e origin of t h e two c o m p o n e n t s of n m r r e l a x a t i o n t i m e s .

The T^ and T^ r e l a x a t i o n t i m e s w e r e m e a s u r e d a t subf reez ing t e m p e r a t u r e s (Table I). Typical d a t a for whea t leaf s e g m e n t s a r e in f igure 4 . In this e x p e r i m e n t , whea t leaf s e g m e n t s w e r e slowly cooled to - 9 . 6 ° C . was d e t e r m i n e d from t h e echo a m p l i t u d e from a CPMG pulse s e q u e n c e . If t h e sample was subsequent ly cooled slowly to -14 C (3 C above t h e killing t e m p e r a t u r e ) and w a r m e d to -9 .6 C t h e r e was no change in t h e T ? va lues . However , when t h e s a m e leaf s e g m e n t s w e r e cooled to -20°CT!3

OC below the i r killing t e m p e r a t u r e ) and w a r m e d again

to -9 .6 C, a cons iderab le d e c r e a s e in T^ was observed . This T^ hys t e re s i s is fu r ther i n c r e a s e d if t h e s amp le is cooled to -70 C and r e w a r m e d to ο -9 .6 C . These r e su l t s s t rongly sugges t t ha t an i r r eve r s ib le change in T^ occur s only on pass ing below t h e kill ing t e m p e r a t u r e and in t h e absence of any app rec i ab l e thawing of t h e t i s sue w a t e r . Similar r e su l t s a r e obse rved in K e n t u c k y b luegrass leaf s e g m e n t s and c a c t u s s t e m , which a r e kil led a t - 3 8 ° C and -24 C r e s p e c t i v e l y .

To es tab l i sh t he m o m e n t t h e hys t e re s i s i n i t i a t e s , T^ was m e a s u r e d during cooling and warming in whea t and Ken tucky b luegrass l eaves and c a c t u s s t em t i ssue (Fig. 5). The T? hys t e re s i s is only observed on warming t h e s ample from below t h e kill ing t e m p e r a t u r e and is genera l ly i n i t i a t e d in t he a b s e n c e of s ignif icant thawing of t h e f rozen s a m p l e .

FIGURE 5. Temperature dependence of Τ^ during slow cooling and warming. The samples used are similar to those described in figure 1. The arrows indicate cooling and warming curves. All samples were cooled at the rate of 2 CAiour just below their respective killing temperatures and warmed at the rate of 3° to 4°CAiour. The hysteresis generally became greater with progressive warming for component 1(0) and II (Φ).


100 h-

o ω CO

Ε

0.3 3.75 3.80 .85

n 3 3.90

/ T ( ° K ) 3.95 4 . 0 0

FIGURE 6. Temperature dependence of T^+ dugjng cooling of Kharkov winter wheat leaf segments containing Mn . The sample was equilibrated in 0.031 molar MnSO .solution for about 15 minutes prior to the nmr measurements. The τ2 f°

r s^

ow relaxing component

(component I) is presented here. Note the anomalous shift in the curve at about -17 C, the killing temperature of these lgpf segments. In repeated experiments it was noted that addition of Mn decreased the values of Τ2 for unfrozen samples, but during freezing they were relatively higher tnan those for samples untreated with Mn .

2 6 6 C. Rajashekar et al

It was n o t e d in f igure 2 t h a t addi t ion of Mn in low c o n c e n t r a t i o n s to w h e a t leaf s e g m e n t s m a d e r e l a x a t i o n t i m e s m u c h m o r e sens i t ive to changes induced by frost d a m a g e . For i n s t a n c e , l ive whea t s e g m e n t s had a T? of 88 m s e c for t h e major w a t e r f r ac t ion but when f r e e z e kil led to -70 C, t h e va lue dropped to 6 m s e c , a 14 fold r educ t i on . The d e c r e a s e in T^ sugges t s t h a t frost injury r e s u l t s in loss of w a t e r c o m p a r t m e n t a l i z a t i o n .

TABLE II. Time Constants (t) for Live and Freeze Killed Kharkov Winter Wheat Crowns Supercooled to -4 C. The Freezing Rates Followed First Order Reaction Kinetics. The Plants Used Here are Described in Figure 1 with the Tender Samples Obtained ^Directly from the Greenhouse (Killing Temperatures, -23 C for Hardened and -5°C for Tender).

Tissue Treatment t (min)

Hardened Alive 14.0 Liquid N2

Q

b 11.8 Slow Freeze (-60 C) 12.0

Tender Alive 9.0 Liquid N9 5.5 Slow Freeze (-60 C) 5.4

In this treatment samples were plunged directly into liquid nitrogen. Q

In this treatment samples were slowly cooled (3 C/hr) to -60 C.

TABLE III. Dependence of Time Constants for Freezing on Degree of Supercooling. Tissues Used were Tissue Culture Cell Aggregates of Manitou Spring Wheat and Kharkov Winter Wheat.

t (min) Temperature Manitou Kharkov (°C) Tender Hardened Tender Hardened

-3 12.5 12.2 12.2 10.6 -5 8.2 7.8 7.1 7.0 -7 4.7 4.7 4.7 4.7 -10 2.5 2.4 2.5 2.7

M e m b r a n e S t r u c t u r a l T r a n s i t i o n s 2 6 7

T - v a l u e s can readily be determined for tissue in the frozen s ta te . twofom decrease in the long T^ for wheat leaf t issue incubated in Mn was observed the moment the t issue was cooled below its ^critical temperature (Fig. 6). This supports the above finding that Mn gains access to intracellular unfrozen water in the tissue precisely at the killing temperature. This establishes that injury of the tissue occurs during the freezing process and not during the thawing process .

IV. FREEZING AND THAWING RATE STUDIES

The kinet ics of freezing and thawing in leaf segments , crowns and tissue culture cel l aggregates of winter cereals have been measured using nmr techniques. The experiments are of two general types . In the first, supercooled samples are inoculated with i ce at different subfreezing temperatures and the rate of freezing measured under isothermal conditions (Table Π and ΙΠ). In the second type of experiments , samples are cooled to - 2 C, inoculated with i c e and al lowed to reach equilibrium overnight. The samples are cooled slowly to either -3 C or - 4 C, ensuring equilibrium freezing. Generally, over 60% of the sample water is frozen at - 4 C. The freezing and thawing rates are then measured during a rapid temperature change, b e t w e e n - 4 C and -8 C for crowns and -3 C and -5 C for cel l cultures. Freezing and thawing rates can be obtained for samples slowly cooled and warmed from various temperatures above and below the killing temperature without warming above -3 C. Typical results are in figure 7 and table 3 .

Freezing of supercooled samples always fol lowed first order kinetics and could be described by a single t ime constant, t ( i .e. , the reciprocal of the first order rate constant) . On the average, the t ime constant for freezing of hardened winter cereal crowns supercooled to - 4 C was approximately 14 minutes as compared to 9 minutes for tender crowns (Table Π). The t ime constants for frost killed crowns were reduced sl ightly. The dif ference in freezing rates for hardened and tender crowns is reduced substantially if the crowns are vacuum infi ltrated with water . There is no dif ference in the freezing rates of hardened and tender cereal cel l suspensions supercooled to - 3 , - 5 , -7 and -10 C. However, the rate of freezing did show strong dependence on the temperature of i ce initiation (Table ΙΠ). Overall there are no dramatic dif ferences be tween living and frost killed, or hardy and tender samples . The rate of freezing in supercooled samples is dependent upon many things including at least the following: the rate of water efflux (membrane permeabil ity) , the degree of supercooling, the rate of i ce front growth, and the rate of heat removal . The role of membranes in freezing can be best determined in

ο ο samples prefrozen to -3 C or - 4 C. If the temperature is changed quickly, the primary factor l imiting freezing would be the contribution of the membrane.


WHEAT 1.0 —

0. 7 — \ —

t r 0. 5 ^ y . —

UJ > a L I V E ( t = 14 5 s e c ) <

0. 3 ο

σ 0. 2 \ \ o _j

\ D E A D (t = 6 8 s e c ) 0.1

I I 1 1 1 1 1 1 1 1

2 3 4

T I M E ( m i n )

FIGURE 7. Freezing rates for Kharkov winter wheat leaf segments vacuum infiltrated with water. The sample was slowly cooled to -4°C and allowed to reach equilibrium. The sample temperature was then dropped to -8 C with the following of liquid water content as a function of time (Live tissue, 0). The liquid water follows first orderkineîcs with time constant t. The sample was then slowly cooled to -25 C(8 C below the killing temperature) and rewarmed to -4°C. The freezing rate was again determined for the frost killed tissue (Φ) as described above.

M e m b r a n e S t ruc tu ra l T r a n s i t i o n s 2 6 9

In e x p e r i m e n t s w h e r e t h e fast t e m p e r a t u r e change p r o c e d u r e desc r ibed above was used, t h e r e w e r e subs t an t i a l d i f f e rences in the r a t e of f reez ing and thawing b e t w e e n living and frost kil led samples (Fig. 7). In t he case of w h e a t leaf s e g m e n t s t h e t i m e cons t an t for f reez ing b e t w e e n -4 C to -8 C was 145 seconds . If t h e sample was slowly cooled to - 2 5 C (8 C below t h e killing t e m p e r a t u r e ) , and slowly w a r m e d to -4 C, t h e t i m e c o n s t a n t for f r eez ing b e t w e e n - 4 C to -8 C was r e d u c e d to 68 seconds . In con t ro l e x p e r i m e n t s w h e r e t he samples w e r e cooled only to -14 C (3 C above t h e killing t e m p e r a t u r e ) , t h e f reez ing t i m e cons t an t r e m a i n e d unchanged . Similar behavior b e t w e e n l ive and frost ki l led leaf s e g m e n t s was observed for t h e thawing t i m e c o n s t a n t s b e t w e e n -8 C and -4 C . The t i m e cons t an t for thawing of frost kil led samples was about a th i rd as l a rge as t h a t of living s amp le s .

TABLE IV. The First Order Time Constants of Freezing and Thawing of Puma Rye Crowns and Brome Grass Tissue Culture Cell Aggregates.

Killing 5(sec) Species Temperature Freezing Thawing

Winter Rye -4°C to -8°C -8°C to -4°C

Control SC -40°C

a

FC -60°CD

Heat killed0

-30°C 160 241 85 100 75 93 70 87

Brome Grass -3°C to -5°C -5°C to -3°C

Tender SC -40°C Hardened SC -60 C

-11°C

-22°C

135 165 75 120

146 210 75 116

^Sample slowly cooled and warmed from -40 C (2 C/hour). Sample rapidly cooled and warmed frorr^-60 C (60 C/min).

cHeat killed samples were warmed to 80 C for 30 minutes prior to

making measurements.

2 7 0 C. R a j a s h e k a r et al.

• 3 0 ° A

Μ f <4r {

5°

# *

' / l

// 1

Β

/ / / / 1

, i t

\ ' / ^

1 / 1

\ '/ \ '/ I ' / \ / /

1

MAGNETIC FIELD — MAGNETIC FIELD-

Τ CO

i.oo V

3. 2 3. 4 3. 6 3. 8 4 . 0

I03/T(

ek)

FIGURE 8. A) Electron spin resonance spectra of TEMPO in macerated potato leaf at two temperatures. The partitioning of TEMPO between the lipid and aqueous leaf fractions is determined using the high magnetic field band on the right. B) The calculated high magnetic field band in a TEMPO spectrum resolved into two lines. The amplitude of the line attributed to TEMPO in lipid is indicated by a 1 and the comparable aqueous TEMPO line by an a. See text for further description. C) van't Hoff plots for the amplitude ratio, (1/a) of macerated tomato leaf, i; macerated potato leaf, j ; and ffhole wheat lea£, k. The bends for tomato, potato and wheat are at +11 , + 3 and -11 C, respectively. In these experiments 1/a was raised to near 0.5 for the highest test temperature by controlled sample dehydration. Further experiments w^re conducted on wheat leaf and no additional bends were found up to 30 C, the highest test temperature with wheat. In a given experiment, measurements were made proceeding from high to low temperatures.


In l ive crown t i ssue , two f rac t ions of w a t e r w e r e observed during f reez ing and thawing . However , if the t i ssue was frost killed only one f rac t ion of w a t e r was observed , which had a m a r k e d t i m e cons t an t r e d u c t i o n for bo th f reez ing and thawing (Table IV). F r e e z i n g r a t e s in fast t e m p e r a t u r e change e x p e r i m e n t s , such as t h e s e , a r e thought to be l imi t ed by the r a t e of w a t e r efflux from within the cel ls to ex t r ace l l u l a r s i t e s con ta in ing i c e . The p l a sma m e m b r a n e is l ikely to be a major r e s i s t a n c e to the m o v e m e n t of w a t e r . The r e su l t s sugges t t ha t the r e s i s t a n c e to w a t e r m o v e m e n t is r e d u c e d s igni f icant ly a f t e r t h e samples a r e cooled below the i r killing t e m p e r a t u r e s . This migh t be caused by p e r m a n e n t r u p t u r e of t h e p l a sma m e m b r a n e and /o r fo rma t ion of i ce wi thin t he ce l l s .

V. ELECTRON SPIN RESONANCE STUDIES

The e l e c t r o n spin r e s o n a n c e spin label , TEMPO (2,2,6,6-t e t r a m e t h y l p i p e r i d i n e - l - o x y l ) has been used ex tens ive ly in model s y s t e m s and in lipids e x t r a c t e d from biological t i ssues (8, 17, 20). Changes in t e m p e r a t u r e dependen t pa r t i t i on ing of TEMPO provide ev idence of m e m b r a n e s t r u c t u r a l t r ans i t i ons . The a d v a n t a g e of using TEMPO in l iving sy s t ems is i t s solubi l i ty in bo th lipids and w a t e r which al lows i t s d i s t r ibu t ion th roughout t h e living t i s sues . TEMPO was i nco rpo ra t ed in to m a c e r a t e d t o m a t o and p o t a t o leaf t i ssue and in to whole C h e y e n n e win te r whea t l eaves (frost killing t e m p e r a t u r e -12 C) . The de ta i l s of TEMPO label ing r e s u l t s a r e r e p o r t e d e l s ewhe re (7).

F igu re 8A shows t h e t h r e e band TEMPO s p e c t r u m for p o t a t o leaf wi th typ ica l t e m p e r a t u r e d e p e n d e n c e of the high field band. The bands a r e s o m e w h a t b roade r than found in model m e m b r a n e s tud ies (8, 17, 20). Cons i s t en t wi th o the r inves t iga t ions (17, 20), the high field band is the sum of two l ines . Of t he two l ines t h e lower field l ine is from TEMPO dissolved in lipid or possibly o t h e r hydrophobic m a t e r i a l s , and the higher field l ine is from TEMPO in an aqueous e n v i r o n m e n t . In this analys is , t h e a m p l i t u d e of e a c h of t he two l ines is a s sumed to be p ropor t iona l t o t he TEMPO c o n c e n t r a t i o n in t h e lipid or aqueous env i ronmen t . The ampl i t udes w e r e ob ta ined from t h e m e a s u r e d s p e c t r a by a cu rve f i t t ing p r o c e d u r e . The lipid and aqueous l ines in t h e high field band w e r e c a l c u l a t e d as L o r e n t z i a n curves (Fig. 8B) and for conven ience t h e l ine wid ths for t h e two curves w e r e a s sumed equal and a l lowed to vary with t e m p e r a t u r e . The observed s p e c t r u m was then m a t c h e d wi th t he c a l c u l a t e d s p e c t r a ob t a ined by varying t h e l ine wid ths , and the l ine amp l i t udes . The bes t fit was chosen by visual compar i son . The t e m p e r a t u r e d e p e n d e n c e of a m p l i t u d e r a t i o s (i .e. , 1/a in F ig . 8B) ob ta ined in this way a r e p l o t t e d in f igure 8.


The equi l ibr ium cons t an t for pa r t i t i on ing of TEMPO b e t w e e n lipid and aqueous leaf f rac t ions Κ , is t he r a t i o of t h e TEMPO c o n c e n t r a t i o n s in t he lipid and aqueous frac^rons and follows:

w h e r e i' and a' a r e t h e c o n c e n t r a t i o n s of TEMPO in t he lipid and aqueous env i ronmen t s , r e spec t i ve ly , and L and A a r e the vo lumes of the lipid and aqueous env i ronmen t s of t he leaf, r e s p e c t i v e l y . In t h e absence of f reez ing , A/L is a cons t an t and i ' /a ' is e x p e c t e d to be p ropor t iona l to the m e a s u r e d ampl i tude r a t i o , i / a . The re fo re , Κ should be p ropor t iona l to t h e ampl i t ude r a t ion , i / a . For this r eason figure 8Cîs a van ' t Hoff g lo t .

van ' t Hoff p lo t s ob ta ined have bends n e a r 11 C in t o m a t o , 3 C in p o t a t o and -11 C in whea t l e aves . Similar anomalous behavior of TEMPO par t i t i on ing in o the r m e m b r a n e sy s t ems has been i n t e r p r e t e d to suggest m e m b r a n e phase s epa ra t i ons (8, 17, 20). In addi t ion , chill ing sens i t ive t o m a t o is known to have a phase s epa ra t i on n e a r 11 C (18). This fu r ther lends va l id i ty to this t echn ique for finding m e m b r a n e phase s epa ra t i ons . The bend nea r 3 C in p o t a t o is cons i s t en t wi th t h e o c c u r r e n c e of s t o r age injury to p o t a t o tube r s a t this t e m p e r a t u r e (23) and sugges ts t ha t a m e m b r a n e phase s epa ra t i on may be respons ib le . The change of slope for t h e supercooled whea t leaf is oppos i te to t h a t of t h e o the r p l an t s and is nea r -11 C . Supercooled samples of t hese l eaves survive exposure to below -11 C; however^ when f rozen t h e killing t e m p e r a t u r e of t hese samples was nea r -11 C. The bend in t h e van' t Hoff p lot for whea t sugges ts t h a t a m e m b r a n e s t r u c t u r a l change occur s a t t he killing t e m p e r a t u r e . Possibly such m e m b r a n e s t r u c t u r a l change a t -11 C in combina t ion wi th t h e s e v e r e frost dehydra t ion t h a t occur s when such t i ssues f r eeze , leads to frost injury observed a t -12 C in these leaf s e g m e n t s .

VI. CONCLUSIONS

The p lan t t i ssues s tud ied h e r e have frost killing t e m p e r a t u r e s whe re ve ry l i t t l e or no f reez ing of w a t e r t a k e s p l a c e . The re fo re , it is unlikely t h a t t he f r e e z e induced dehydra t ion a lone can accoun t for t h e injury observed . These t issues have sharply def ined killing poin ts as ev idenced by t h e rapid e l e c t r o l y t e loss a t t hese t e m p e r a t u r e s . It is very typica l in t i s sues , wi th a high to m o d e r a t e w a t e r c o n t e n t (such as leaves) to lose most of t he ce l lu lar e l e c t r o l y t e s as a resu l t of cel l lysis following frost injury. The ques t ion ar i ses as to t he mechan i sm tha t could accoun t for t h e lysis of cel ls over a nar row range of t e m p e r a t u r e . This could be app roached by es tabl ishing the possible s i t e and t i m e or m o m e n t t he frost injury is i n i t i a t ed .

M e m b r a n e S t ruc tu ra l T r a n s i t i o n s 2 7 3

The inf lec t ion in the e l e c t r o l y t e loss cu rves shows d a m a g e or lysis of p l a sma m e m b r a n e on pass ing below the l e tha l t e m p e r a t u r e s in a f r e e z e -thaw cyc l e . Unfo r tuna t e ly , t h e r e su l t s do not show when during the f r e e z e - t h a w cyc le the lysis o c c u r s . Ne i t he r f reez ing anomol ies a t the killing t e m p e r a t u r e nor hys t e re s i s in t he t e m p e r a t u r e dependen t f reez ing and thawing could be found using e i t he r nmr or d i f fe ren t i a l t h e r m a l ana lys is . The r e su l t s d e m o n s t r a t e t ha t t hese t i ssues f r eeze l ike an ideal solut ion. Dur ing a slow f r e e z e - t h a w cyc le , nmr T^ r e l a x a t i o n t i m e s a r e found to exhibi t hys t e re s i s which is i n i t i a t ed a f t e r pass ing below the killing t e m p e r a t u r e and is obse rved well be fo re any s ignif icant t i ssue thawing o c c u r s . This s t rongly sugges t s i r r evers ib le ce l lu lar changes a t t h e killing t e m p e r a t u r e while t he t i ssue is sti l l f rozen. In addi t ion , an anomalous drop in T^ could be observed p rec i se ly a t t he killing t e m p e r a t u r e in samples con ta in ing ^ ln ions. This occurs during cooling and sugges t s the p e n e t r a t i o n of Mn ions through t he p l a sma m e m b r a n e a t this l e tha l t e m p e r a t u r e . The acce s s of Mn ions to t he liquid w a t e r of the f rozen t issue could e i t he r be due to ice p e n e t r a t i o n of the cel ls or possible m e m b r a n e lysis . Cons i s t en t wi th r e su l t s impl ica t ing m e m b r a n e d a m a g e a r e t he f reez ing and thawing r a t e s which subs tan t i a l ly i nc r ea se on frost injury. It is a s sumed h e r e t h a t a r e s i s t a n c e to f reez ing is t h e p l a sma m e m b r a n e , in t ha t the cel l w a t e r mus t pass through the p l a s m a m e m b r a n e be fo re it can f r e e z e in t h e e x t r a c e l l u l a r space s . The inc reased f reez ing and thawing r a t e s a r e due to the m e m b r a n e d a m a g e . D i r e c t ev idence to t he t e m p e r a t u r e dependen t m e m b r a n e s t r u c t u r a l t r ans i t ions s imi lar to those observed in chil l ing sens i t ive p l an t s has been found by e l e c t r o n spin r e s o n a n c e me thods a s soc i a t ed wi th t he killing t e m p e r a t u r e in win te r whea t l e aves .

These s tud ies sugges t t h a t a m e m b r a n e s t r u c t u r a l t r ans i t ion , caus ing m e m b r a n e d a m a g e a t the kill ing t e m p e r a t u r e , r e su l t s in a ghange in t h e env i ronmen t of unf rozen ce l lu la r w a t e r , a l lows Mn ion p e n e t r a t i o n in to the cel ls and r e d u c e s r e s i s t a n c e to w a t e r m o v e m e n t during f reez ing and thawing . It is c l ea r ly d e m o n s t r a t e d t ha t l i t t l e or no thawing is nece s sa ry for observing t h e s e e f f e c t s .

ΥΠ. R E F E R E N C E S

1. Burke , M. J . , Bryan t , R. G., and Weiser , C. J . Plant Physiol. 54, 392-398 (1974).

2. Burke, M. J . , G e o r g e , M. F . , and Bryan t , R. G. In "Water R e l a t ions of Foods" (R. B. Duckwor th , ed.) , pp . 111-135. A c a d e m i c P re s s , New York (1975).

3 . Chen , P . M., Burke, M. J . , and Li, P . H. Bot. Gaz. 137, 313-317 (1976).


4. Chen, P . M., Gus ta , L. V., and Stout , D. G. Plant Physiol. 61, 878-882 (1978).

5. Chen, P . M., Li, P . H., and Burke, M. J . Plant Physiol. 59, 236-239 (1977).

6. F a r r a r , T. C , and Becker , E. D . "Pulse and Four ie r Transform NMR." A c a d e m i c P res s , New York (1971).

7. Fey , R. L., Workman, M., Marce l los , H., and Burke, M. J . Plant Physiol. (In Press) (1979).

8. G r a n t , C . W., Wu, S. H., and McConnel l , Η. M. Biochem. Biophys. Acta 363, 151-158 (1974).

9. Gus ta , L. V., Burke, M. J . , and Kapoor , A. C. Plant Physiol. 56, 707-709 (1975).

10. Gus ta , L. V., and Fowler , D. B. Can. J. Plant Science 57, 213-219 (1977).

11 . Harr i son , L. C , Weiser , C . J . , and Burke, M. J . Plant Physiol. 62, 899-901 (1978).

12. Hsi, E., Mason, R. , and Bryant , R. G. J. Phys. Chem. 80, 2592-2597 (1976).

13. K r a s a v t e v , O. A. Fiziol. Rastenii 9, 359-367 (1962). 14. L e v i t t , J . "The Hardiness of P lan t s . " A c a d e m i c P res s , New York

(1956). 15. L e v i t t , J . "Responses of P l an t s to Env i ronmenta l S t resses ."

A c a d e m i c P re s s , New York (1972). 16. Lyons, J . M. Ann. Rev. Plant Physiol. 24, 445-466 (1973). 17. McConnel l , Η. M. In "Spin Label ing: Theory and Appl ica t ions" (L.

J . Ber l iner , ed.) , pp . 525-560. A c a d e m i c P res s , New York (1976). 18. Raison, J . K. In "Mechanisms of Regu la t ion of P lan t Growth" (R.

L. Bieleski , A. R. Ferguson , and Μ. M. Cresswel l , eds.) , Bull. 12, pp . 487-497 . The Royal Socie ty of New Zealand, Well ington (1974).

19. Salcheva, G., and Samygin, G. Fiziol. Rastenii 10, 65-72 (1963). 20. Shimshick, E. J . , and McConnel l , Η. M. Biochem. 12, 2351-2360

(1973). 2 1 . S tout , D . G. "A Study of P lan t Cell P e r m e a b i l i t y and of t he Cold

Acc l ima t ion P rocess in Ivy Bark." Ph .D. Thesis , Cornel l Univers i ty , I t h a c a (1976).

22. S tou t , D . G., S teponkus , P . L., and C o t t s , R . M. Plant Physiol. 62, 636-641 (1978).

23 . Workman, M., Kerschner , E., and Harr i son , M. Amer. Potato J. 53, 191-204 (1976).


POSSIBLE INVOLVEMENT OF THE TONOPLAST LESION IN CHILLING INJURY OF CULTURED PLANT CELLS

S. Yoshida, T. Niki and A. Sakai

The I n s t i t u t e of Low T e m p e r a t u r e Sc ience Hokkaido Univers i ty

Sapporo, J a p a n

I. INTRODUCTION

A g r e a t deal of in fo rmat ion has been a c c u m u l a t e d so far concern ing the mechan i sm of chil l ing injury in p lan t ce l l s , mainly from b iochemica l and biophysical point of view (7). A vas t number of e x p e r i m e n t s have been focused on the i m p a i r m e n t (24, 25, 26, 27) or depress ion of ox ida t ive a c t i v i t y of mi tochondr i a during chil l ing (1 , 8, 13, 14, 21). On t h e o the r hand, only l imi ted s tud ies (5, 6, 12, 20) have been r e p o r t e d on the u l t r a s t r u c t u r a l r e sponses a s s o c i a t e d wi th chil l ing injury.

The p r e s e n t s tudy was des igned to improve our unders t and ing of t h e mechan i sm of chil l ing injury in p l a n t s by u t i l iz ing cu l tu red ce l l s , which w e r e highly suscep t ib le to chil l ing. Emphas is was p l aced on the p r i m a r y u l t r a s t r u c t u r a l r esponses of the ce l ls to chil l ing s t r e s s and specia l a t t e n t i o n was focused on t h e possible invo lvement of t h e tonoplas t lesion in t he mechan i sm of chil l ing injury in cu l tu red p lan t ce l l s .


A. Cell Culture and Evaluation of Cell Viability

The cal lus der ived from cambia l a r e a of twig p i eces of Cormus Stolonif era w e r e used as t he m a t e r i a l s . They w e r e subcu l tu red a t 26 C in the dark on the agar medium of Murashige-Skoog con ta in ing NAA (3mg/ l ) with a slight modi f ica t ion . The flasks con ta in ing cal lus on t he 10th day of c u l t u r e we re cooled to 0 C and kept t h e r e for d i f fe ren t l eng ths of t i m e . The cel l v iabi l i ty a f t e r chil l ing was e v a l u a t e d bo th wi th t h e TTC r e d u c t i o n t e s t and t h e r e g r o w t h t e s t . For t he d e t e r m i n a t i o n of the r e g r o w t h r a t e a f t e r chi l l ing, cel l c u l t u r e was con t inued on fresh

Copyright * 1979 by Academic Press, inc. 2 7 5 All rights of reproduction in any form reserved

ISBN0-I2-46056O5

2 7 6 S. Y o s h i d a et al

medium at 26°C for f i f teen days and then the i nc r ea se in fresh weight was d e t e r m i n e d .

B. Electron Microscopy

For t ransmiss ion e l ec t ron microscopy, cal lus was sampled a t the des i red t i m e of chil l ing and fixed in 3 % g lu t a ra ldehyde in 1/15 Μ po ta s s ium-phospha t e buffer , pH 7.2 for 2 hr a t 0 C or 26 C. The spec imens were then pos t - f ixed in 2% osmium t e t r o x i d e solut ion for 2 hr a t 0°C be fo re dehydra t ion in an e thano l se r ies and finally in n -bu ty lg lyc idy le the r . The embedded spec imens in Spurr 's epoxy res in (19) we re sec t ioned and s t a ined with s a t u r a t e d uranyl a c e t a t e and then with Reynolds ' l ead solut ion (15),

For f r e e z e - f r a c t u r e e l ec t ron microscopy , cal lus was fixed in g lu t a ra ldehyde as desc r ibed above . The p re - f ixed spec imens we re success ive ly t r e a t e d in 10, 20, 30, and 3 5 % glycerol e ach for one hour a f t e r a brief washing in po t a s s ium-phospha te buffer , pH 7.2. The g l y c e r o l - t r e a t e d spec imens we re p l aced on an a luminum sample holder and rapidly f rozen in Freon 22_gt -156 C. The frozen spec imens were f r ac tu r ed a t -96 C in 2-4 χ 10 mm Hg in a f r e e z e - e t c h i n g a p p a r a t u s , J E O L - 4 . Af ter minimum e tch ing , the f r ac tu red su r face was shadowed with both p la t inum and carbon a t -92 C for a few seconds . The rep l i cas were c leaned wi th a ch romic ac id solut ion and v iewed wi th an e l e c t r o n mic roscope , JEM-100 C.

C. Preparation of Crude Mitochondria

Ten g each of unchi l led or chil led cal lus was ground in a m o r t a r and a pes t l e wi th 10 ml of the grinding medium, 4 g of sea sand and 1.5 g of Polyclar AT. The grinding medium con ta ined 100 mM Tris-HCl buffer , pH 7.8, 0.5 Μ sorbi to l , 5 mM EGTA, 5 mM M g C l 2, 10 mM KC1, 0 .5% BSA and 20 mM c y s t e i n e - H C l . The ce l l - f r ee e x t r a c t s were success ive ly cen t r i fuged a t 300, 1,000 and 14,000 g for 5, 10 and 15 min, r e spec t i ve ly . The 14,000 g pe l l e t s were washed once wi th the grinding medium and suspended in t he r e a c t i o n med ium.

D. Sucrose Density Gradients

Ten g of unchi l led or chil led cal lus we re ground in a m o t a r and a pe s t l e wi th 10 ml of the grinding medium, 4 g of sea sand and 1.5 g of Polyc lar AT a t 0 C for 100 seconds . The grinding medium con ta ined 150 mM t r i s -HCl buffer , pH 7.8, 0.5 Μ sorbi to l , 5 mM EGTA and 20 mM c y s t e i n e - H C l . Af te r passing through two l aye r s of gauze and one layer of Mirac lo th , the ce l l - f r ee e x t r a c t s were cen t r i fuged a t 300 g for 5 min to r e m o v e cell debr is and then the s u p e r n a t a n t s we re sub jec ted to

T o n o p l a s t L e s i o n in Chill ing Injury of C u l t u r e d Cel ls 2 7 7

cen t r i fuga t ion a t 189,000 g for 30 min. The 189,000 pe l l e t s w e r e suspended and loaded onto a l inear sucrose g rad ien t (15-55%, w/w) in 5 mM Tris -HCl buffer , pH 7.6 and 1 mM EGTA, and cen t r i fuged a t 96,000 g for 2 h r . Ant imyc in Α- insens i t ive NADH cyt c r e d u c t a s e a c t i v i t y was assayed with an a l iquot of t h e f r a c t i o n a t e d samples (1.2 ml each) . The r e a c t i o n m i x t u r e con ta ined 100 μ moles po tass ium phospha te buffer , pH 7.2, 0.5 mg of cyt c (oxidized form), 5 μ moles of NaN^, 2 μ Μ of an t imyc in A and an a l iquot of t he e n z y m e p r e p a r a t i o n s . R e a c t i o n s we re p e r f o r m e d a t 25 C and followed the r educ t ion of cyt c a t 550 n m .

E. Determination of Respiratory Activities in vitro and in vivo

The ox ida t ive a c t i v i t y of mi tochondr i a in vitro was m e a s u r e d as oxygen u p t a k e by oxygen e l e c t r o d e . The r e a c t i o n m i x t u r e cons is ted of 0.5 Μ manni to l , 20 mM potass ium phospha te buffer , pH 7.23, 1 mM MgCl-, and 0 . 1 % BSA. The r a t e of oxida t ion of s u b s t r a t e (20 mM succ ina te ) was d e t e r m i n e d in t h e p r e s e n c e ( s t a t e 3) or absence ( s t a t e 4) of 100 μ Μ ADP wi th 1.0 mg of mi tochondr ia l p ro t e in in 2.0 ml of t he r e a c t i o n med ium.

R e s p i r a t o r y a c t i v i t y in vivo was assayed in 3 ml of t h e basal c u l t u r e medium wi th the oxygen e l e c t r o d e . The oxidat ion of added dopamine in VIVO was also d e t e r m i n e d by measur ing the i nc r ea sed u p t a k e of oxygen by oxygen e l e c t r o d e .

ΠΙ. RESULTS

A. Effects of Temperature on Cell Growth and Cell Lesion

The op t imum t e m p e r a t u r e for the g rowth of t h e cal lus u t i l i zed in t he p r e s e n t s tudy lay in the very na r row r a n g e b e t w e e n 20 and 26 C. No g rowth was observed below 10 C or above 28 C during c u l t u r e . When the cal lus on t he 10th day of c u l t u r e a t 26 C w e r e sub jec ted to t e m p e r a t u r e s below 10°C, they suf fered injury and lost the i r v iabi l i ty depending both on the t e m p e r a t u r e s and the t i m e du ra t i on . The lower the t e m p e r a t u r e , t he m o r e s e v e r e t he injury within a shor t per iod . As p r e s e n t e d in Fig . 1 A, TTC r e d u c t i o n r a t e of t h e cal lus sub jec ted to 0 C for 24 hr d e c r e a s e d to nea r ly s ix ty p e r c e n t of the unchi l led c o n t r o l . However , only a slight d e c r e a s e was observed in t he TTC r e d u c t i o n r a t e in the cal lus chi l led for 12 hr a t 0 ° C . The cal lus sub jec ted to 0 C for d i f fe ren t l eng ths of t i m e w e r e t r a n s f e r r e d to fresh medium and cu l t u r e was con t inued a t 26 C to d e t e r m i n e the capab i l i ty for r e g r o w t h (Fig. IB). The eva lua t ion of cel l v iabi l i ty with t h e TTC r educ t i on t e s t was c o r r e l a t e d wi th t he r e su l t s from the r e g r o w t h t e s t . Thus, the cal lus were observed to be very suscep t ib le to chill ing and injured within a r e l a t i ve ly shor t t ime of chil l ing a t 0 ° C .


TIME (hr)

FIGURE 1. TTC reduction rates (A) and regrowth rate (B) of callus subjected to 0°C for various lengths of time. Rates of TTC reduction and regrowth are expressed as percent of the control values. Regrowth was performed on fresh medium at 26°C for 15 days after chilling.

B. Changes in Cell Permeability during Chilling

As p r e s e n t e d in Fig . Z, only a p a r t i a l r e l e a s e of amino acids was observed in the ca l lus a f t e r chi l l ing for more than 48 hr , whe rea s l i t t l e or no l eakage was observed within Z4 hr of chil l ing. Abrupt p e r m e a t i o n and oxidat ion of added dopamine in vivo, however , w e r e observed in t he cal lus chi l led for m o r e than 48 hr (Fig. Z). The oxidat ion of the added dopamine by t h e cal lus was s t rongly inhib i ted by the addi t ion of phenyl th iourea which is a p o t e n t inhibi tor of polyphenoloxidases . The polyphenoloxidase was d e t e r m i n e d to be l o c a t e d in t h e cal lus in var ious ce l lu lar f r ac t ions (Table 1). About fifty p e r c e n t of t o t a l a c t i v i t y , however , was l o c a t e d in t he soluble s u p e r n a t a n t . When p ro top l a s t s p r e p a r e d from unchi l led cal lus were p r e i n c u b a t e d wi th dopamine and then washed, no u p t a k e of t h e s u b s t r a t e was observed . L i t t l e or no l eakage of the e n z y m e outs ide the cel ls was also observed even in the cel ls chi l led for 7Z hr a t 0 C. These r e su l t s may i nd i ca t e t h a t p l a sma m e m b r a n e s r e t a i n the i r no rma l funct ions unt i l ce l l s a r e seve re ly injured in t he l a t e r s t ages of chil l ing and t h e lesion of the m e m b r a n e s per se is by no means t he p r i m a r y r eason for the chil l ing injury of t h e cal lus , but follows t h e secondary ce l lu la r e v e n t s .

T o n o p l a s t L e s i o n in Chil l ing Injury of C u l t u r e d Cel ls 2 7 9

Ε Ο

g>150h

0 24 48 72

TIME (hr)

FIGURE 2. Amino acids leakage and oxidation of added dopamine after chilling for varioug lengths of time. The chilled cells were leached in distilled water at 26 C for one hr and the amino acids released were determined. The values are expressed as the percent of the killed cells. The permeation and oxidation of added dopamine was measured at 26°C with an oxygen electrode.

C. Changes in Respiratory Activity in vivo and in vitro

As p r e s e n t e d in Fig . 3, r e s p i r a t o r y a c t i v i t y in vivo was s l ight ly r e d u c e d a t t he t i m e of 12 hr of chi l l ing. However , i t r e t u r n e d a lmos t to t h e no rma l level upon pro longed chil l ing up to 24 hr . Af te r 24 hr , t he r e s p i r a t o r y a c t i v i t y was d e c r e a s e d fu r ther wi th the t i m e lapse of chil l ing. The enhanced r e sp i r a t i on ob ta ined by the addi t ion of uncoupler ( F C C P 1 μ M) in vivo was also revers ib ly d iminished a t t h e t i m e of 12 h r . Near ly t he s a m e r e s u l t s we re ob ta ined wi th r e s p i r a t o r y a c t i v i t y in c rude mi tochondr i a i so la t ed from chi l led ca l lus . The r e s p i r a t o r y con t ro l dec l ined t e m p o r a r i l y a f t e r 12 hr of chil l ing and then was r e s t o r e d nea r ly to t h e n o r m a l level upon pro longed chil l ing up to 24 hr . Af te r 48 hr of chi l l ing, a marked dec l ine of the r e s p i r a t o r y con t ro l was observed , sugges t ing an i r r eve r s ib l e dysfunct ion of t h e r e s p i r a t o r y con t ro l s y s t e m s .


300 Ο

C\J Ο -

^n ντνο

350

ε ο

1 300

ο

ο ε 250h

Β in vitro

20

10

c ο

4->

ΟΟ ο

1.4

1.1

1.0 24 48

TIME (hr)

72

FIGURE 3. Changes in respiratory activities in vivo (A) and in vitro (B) after chilling. The crude mitochondrial fractions were prepared from callus chilled for the indicated lengths of time. The oxygen uptakes bgth in vivo and in vitro were measured with an oxygen electrode at 26 C. Succinate (20 mM) was used as the substrate and 115 μ Μ of ADP was used for the measurement of state 3 respiration in vitro. FCCP was added in a final concentration of 1 μ Μ.

T o n o p l a s t L e s i o n in Chil l ing Injury of C u l t u r e d Cel ls 281

TABLE I. Cellular Localization of Polyphenoloxidase in Callus of Cornus stolonifera. Ten g of callus on 12th day of culture was ground as

described in text and the cell-free homogenate was subjected to differential centrifugations. The pellets were suspended in 5 mM of Tris-HCl buffer, pH 7.3. The reaction mixture consisted of 50 mM potassium phosphate buffer, pH 6.5 an aliquot of the enzyme preparations and 2 mM dopamine in a final volume of 2 ml. The oxidation of added dopamine was measured at 26°C with an oxygen electrode. The blank values without addition of the substrate were subtracted.

Total activity Specific activity Fraction Protein 02 nmoles/ 02 nmoles/mg/

(mg) 10 min (%) 10 min

300-1,000 g 6.7 549.1 20.0 81.9 1,000-14,000 g 12.1 549.0 20.0 45.3 14,000-189,000 g 5.9 287.0 10.9 48.6 189,000 g Sup 22.8 1248.0 47.3 54.7

Thus, obse rved changes in t h e r e s p i r a t o r y a c t i v i t y in VIVO during chil l ing may be asc r ibab le to a r eve r s ib l e or an i r r eve r s ib l e a l t e r a t i o n of t he mi tochondr ia l funct ions .

D. Ultrastructural Changes in Cells Associated with Chilling Injury

The u l t r a s t r u c t u r e s of unchi l led con t ro l cel ls on t he 10th day of cu l t u r e a r e p r e s e n t e d in Fig . 4A, Β in d i f fe ren t magn i f i ca t ions . In these cel ls a l a rge c e n t r a l vacuo le , rough ER and i r regu la r ly invag ina ted nucle i (Fig. 4A) we re c h a r a c t e r i s t i c a l l y observed . Prop las t ids showed an el l ipsoidal form conta in ing osmiophi l ic globules . Many r ibosomes we re observed in the cy top lasm (Fig. 4B). U l t r a s t r u c t u r a l changes we re first d e t e c t e d in p rop las t ids in t h e cel ls chi l led for 6 hr a t 0 C (Fig. 4C) . P rop las t ids showed an i n c r e a s e in the e l e c t r o n dens i ty of the m a t r i x and s o m e w h a t s t r e t c h e d and c o n s t r i c t e d shape which was a c c o m p a n i e d by folding. The osmiophi l ic globules in the p rop las t ids were s ca r ce ly visible in this ear ly s t a g e of t h e chil l ing t r e a t m e n t . In the u l t r a s t r u c t u r e of t h e o the r cel l o rgane l l e s , l i t t l e or no visible change was d e t e c t e d within 6 hr of chil l ing. As shown in F ig . 4D, on fu r the r pro longed exposure to 0 C up to I Z h r , a p a r t i a l d i la t ion followed by mic roves i cu la t ion of the rough ER m e m b r a n e s and r e l e a s e of r i bosomes from the m e m b r a n e s w e r e c lea r ly observed (Fig. 4D, a r rows in the lower left hand) . The no rma l form of


FIGURE 4A-D. Ultrastructures of urtchilled and chilled cells. Unchilled control cell in lower magnification (A), χ 4,000, and in higher magnification (Β), χ 8,000; cells chilled for 6 hr (C) and 12 hr (D), χ 8,000. Inset in D shows the initiation of dilated structures of rough ER (Arrow) at a higher magnification, χ 10,000. Ρ, proplastids contained osmiophilic globules; M, mitochondria; ER, rough endoplasmic reticulum; V, central vacuole; N, nuclei; CW, cell wall.

T o n o p l a s t L e s i o n in Chil l ing in jury of C u l t u r e d Cel ls 2 8 3

FIGURE 5 A-D. Ultrastructures of chilled cells. Chilled for 24 hr( A, χ 8,000; Β, χ 16,000), 48 hr (C, 8,000) and 72 hr (D, 16,000). We, vacuolated endoplasmic reticulum. Arrow in Β indicates a partial disruption of tonoplast.

2 8 4 S. Y o s h i d a et al.

rough ER was hardly d e t e c t e d in this s t a g e of chi l l ing. No d e t e c t a b l e change , however , was no t ed in the u l t r a s t r u c t u r e s of nuc le i , mi tochondr ia and p lasma m e m b r a n e s . Invagina t ion and infolding of t he tonop las t s was occas ional ly observed in this s t a g e . Numerous r ibosomes we re still visible in the cy top lasm.

In t he cells exposed to 0°C for 24 hr , more s t r ik ing u l t r a s t r u c t u r a l changes b e c a m e evident (Fig. 5A,B). The cy top lasm was occupied by v a c u o l a t e d ves ic les . The la rge vacuo l a t ed ves ic les appea red to be fur ther developed from the m i c r o - v e s i c u l a t e d ER observed in the 12 hr s t a g e (Fig. 4C, a r rows) . In the 24 hr s t a g e , a r e m a r k a b l e s t r u c t u r a l change also appea red in the tonop las t s . Tonoplas ts of ten exhib i ted i r regu la r ly infolded and engulfed s t r u c t u r e s . P a r t i a l disrupt ion of the tonop las t s was conspicuously visible in about th i r ty p e r c e n t of these cel ls (Fig. 5B, a r row) . Most of the mi tochondr ia sti l l r e t a i n e d the normal s t r u c t u r e , but qu i te a few b e c a m e swollen and less dense . Prop las t ids showed somewha t less dense s t r u c t u r e , but nuclei and p la sma m e m b r a n e s s t i l l r e m a i n e d unchanged . As c lea r ly shown in F ig . 5C, the ce l ls exposed to 0 C for 48 hr displayed an au to ly t i c deg rada t ion of t he cy top l a sm. Most of the cell o rgane l les such as mi tochondr ia , Golgi appa ra tu s and proplas t ids had d i sappeared . The tonoplas t was c o m p l e t e l y d i s rup ted and d i sappeared . Deg raded nuclei and mi tochondr i a s t i l l r e m a i n e d r ecogn izab l e . Af te r 72 hr of chil l ing, mos t of t h e cel ls had s t r u c t u r e s s imi lar to those in F ig . 5D.

The observed changes in t he u l t r a s t r u c t u r e of ER during the ear ly s t a g e of chil l ing, i .e . , t r an s fo rma t ion of rough ER in to v a c u o l a t e d smooth ER ves ic les , we re also conf i rmed by t h e changes in the sucrose dens i ty prof i les of an t imyc in Α- insens i t ive cyt c r e d u c t a s e as the p r e s u m e d marke r enzyme for ER m e m b r a n e s . As p r e s e n t e d in Fig . 6, 24 hr of chil l ing r e s u l t e d in a marked shift of the major a c t i v i t y peaks to a l igh ter por t ion , sugges t ing t he t r a n s f o r m a t i o n of rough ER to a vacuo la t ed smooth one . Upon fur ther chil l ing up to 48 hr , the a c t i v i t y peak wi th l igh te r dens i ty was shi f ted again to the heav ie r por t ion , sugges t ing fur ther d e t e r i o r a t i v e changes in those m e m b r a n e s .

Thus, sequen t i a l changes in t he u l t r a s t r u c t u r e s of cel l o rgane l les were observed to be closely r e l a t e d to the chil l ing injury of the cal lus sub jec ted to 0 C for d i f fe ren t l eng ths of t i m e .

To give some insights in to the i n t r a m e m b r a n o u s s t r u c t u r e s of ce l lu lar m e m b r a n e s , e x p e r i m e n t s were p e r f o r m e d ut i l iz ing the f r e e z e -f r a c t u r e t echn ique (11). No d e t e c t a b l e change in t he number and the d is t r ibut ion p a t t e r n s of the i n t r a m e m b r a n o u s p a r t i c l e s on both faces Ρ and Ε in severa l m e m b r a n e s could be d e t e c t e d within 6 hr of chill ing (data not shown). On fur ther chil l ing up to 12 hr , however , r e m a r k a b l e changes in t he d is t r ibut ion of t he p a r t i c l e s we re observed in the Ρ face of tonoplas t in cons iderab le number of ce l l s , while no d e t e c t a b l e change was observed in p l a sma m e m b r a n e s in this s t a g e (Fig. 7B). This fac t may suggest tha t in the 12 hr chil led ce l ls , changes in the n a t u r e of the tonop las t s p r e c e d e t he changes in t he p l a sma m e m b r a n e s . Upon fur ther prolonged chil l ing up to 24 hr , an aggrega t ion of the p a r t i c l e s on the Ρ


^ 1 0 - Β Φ Ε > \

FIGURE 6. Changes in the sucrose density profiles of membranes associated with antimycin A-insensitive NADH cyt c reductase. Α, Β and C correspond to unchilled, 24-hr chilled and 48-hr chilled callus, respectively. Mitochondria were banded at the indicated position (Mt).

2 8 6 S. Y o s h i d a et ai

FIGURE 7. Freeze-fracture electron micrographs of plasma membranes and tonoplasts in unchilled control and chilled cells. Fracture faces of the half membranes left frozen to the cytoplasm (P faces) are indicated. Unchilled control cellsiA, J . Chilled for 12 hr(B)f

24 hr(C 1 2) and 48 hr (D). PI, plasmamembrane;To, tonoplast χ 15,000.


f ace of p l a sma m e m b r a n e s was i n i t i a t e d in a smal l n u m b e r of t h e ce l ls , but most of the ce l ls gave the n o r m a l s t r u c t u r e of p l a s m a m e m b r a n e s as shown in F ig . 7 C A m a r k e d r educ t i on in t he p a r t i c l e n u m b e r s was also d e t e c t e d on the Ε face of tonoplas t and s e v e r e agg rega t ion of the p a r t i c l e s was observed on t h e Ρ f a c e of t he tonoplas t (Fig. 7C~).

The a l t e r a t i o n in t h e i n t r a m e m b r a n o u s s t r u c t u r e s of p l a s m a m e m b r a n e s b e c a m e qu i t e common and more s t r ik ing on fur ther pro longed chil l ing up to 48 hr . The p a r t i c l e s on t h e Ρ f ace of p l a s m a m e m b r a n e s w e r e severe ly a g g r e g a t e d and l a rge p a r t i c l e - f r e e a r e a s , forming p r o t u b e r a n t po r t ions , w e r e fo rmed in mos t of t h e cel ls (Fig. 7D). As a l r eady men t ioned , in this s t a g e of chi l l ing, a lmos t all of the cy top l a smic c o m p o n e n t s w e r e deg raded and t h e cel l v iabi l i ty dec l ined seve re ly .

IV. DISCUSSION

One of t h e most d r a s t i c ce l lu la r e v e n t s observed in t h e chi l led ca l lus was t h e abrupt d isrupt ion of cel l o rgane l l e s , including nuc le i , mi tochondr i a , p rop las t ids and r ibosomes , a f t e r 48 hr of chill ing a t 0 C. This was a ppa re n t l y followed by obse rvab le changes in t onop la s t s , i .e . , invag ina t ion , infolding and a p a r t i a l d is rupt ion , and a c o n c o m i t a n t d e v e l o p m e n t of the v a c u o l a t e d smoo th ER a f t e r 24 hr of chi l l ing. These f a c t s may sugges t t h a t t h e c ruc ia l even t lead ing to an i r r eve r s ib l e cel l d e c a y under chil l ing is the rup tu r ing of the t onop la s t s . A c o m p a r a b l e s e q u e n c e of ce l lu lar e v e n t s r ega rd ing t h e tonoplas t has been r e p o r t e d in l a t e s e n e s c e n c e of p lan t ce l l s (2, 16). Vacuoles h a v e been shown to be a lysosomal a p p a r a t u s in p l an t s , c o m p a r t m e n t a l i z i n g a v a r i e t y of ly t ic e n z y m e s (9, 10). In the ca l lus u t i l i zed in the p r e s e n t s tudy , an abrupt d e c r e a s e in DNA c o n t e n t o c c u r r e d a f t e r 24 hr of chi l l ing. This fac t also sugges t s t ha t the c o m p a r t m e n t a t i o n of DNAase in the ce l ls has been marked ly d i s tu rbed during t h e l a t e r s t a g e s of chi l l ing.

The u l t r a s t r u c t u r e s of ca l lus t h a t had been m o d e r a t e l y a l t e r e d by 12 hr of chil l ing w e r e a lmos t t o t a l ly r e s t o r e d wi thin 12 hr of warming a t 26 C . Even in t h e cel ls chi l led for 24 hr , t h e seve re ly a l t e r e d u l t r a s t r u c t u r e s pa r t i a l l y or c o m p l e t e l y r e s t o r e d a f t e r r e w a r m i n g , unless t h e tonoplas t had been seve re ly changed (Niki, T., 1979, in p r e p a r a t i o n ) .

The c u r r e n t t heo ry explains chil l ing injury in t e r m s of a t e m p e r a t u r e - i n d u c e d phase t r ans i t ion of l ipids from gel t o liquid c rys t a l in ce l lu la r m e m b r a n e s (see Lyons in this volume) and /o r a t e m p e r a t u r e -dependen t a l t e r a t i o n in t he hydrophobic n a t u r e of m e m b r a n e p ro t e in s (24, 25, 26, 27), espec ia l ly in mi tochondr ia l m e m b r a n e s . If i t w e r e also t he c a s e in our m a t e r i a l s , then we could e x p e c t to observe some visible changes in e i t he r t h e d i s t r ibu t ion or t h e n u m b e r of t h e i n t r a m e m b r a n o u s p a r t i c l e s in any sor t of ce l lu lar m e m b r a n e s i m m e d i a t e l y a f t e r lower ing t h e env i ronmen ta l t e m p e r a t u r e suf f ic ient ly below the c r i t i c a l t e m p e r a t u r e to induce t he gel- l iquid c rys t a l t r ans i t ion of m e m b r a n e

2 8 8 S. Y o s h i d a et ai

l ipids, with exclusion of t he p a r t i c l e s by t he o rde red lipid a r e a s . Tha t is, i n t r a m e m b r a n o u s p a r t i c l e s may be excluded from the o r d e r e d lipid domains , t he l a t e r a l d i sp l acemen t in to st i l l fluid regions producing p a r t i c l e aggrega t ion (4, 17, 22) or v e r t i c a l d i sp l acemen t producing an a p p a r e n t loss of p a r t i c l e number s (18, 23). In chi l led ca l lus , however , no visible change was d e t e c t e d both in the p a r t i c l e d is t r ibu t ion and the p a r t i c l e number in any sor t of m e m b r a n e s i m m e d i a t e l y a f t e r chil l ing to 0 C and even a f t e r 6 hr of chil l ing. No d e t e c t a b l e change in t he p a r t i c l e s in var ious m e m b r a n e s , excep t for tonop las t s , was observed unt i l 24 hr of chil l ing. In tonoplas t , an aggrega t ion and r educ t ion in t he number we re d e t e c t e d ea r ly on both Ρ and Ε faces a f t e r 12 hr of chi l l ing. Af te r 24 hr , an aggrega t ion of p a r t i c l e s on t he Ρ f ace of p l a sma m e m b r a n e s was in i t i a t ed in a l imi t ed number of ce l l s . F u r t h e r chil l ing up to 48 hr p roduced a s e v e r e aggrega t ion of p a r t i c l e s on p l a sma m e m b r a n e s in a lmos t all of the ce l l s . These changes in p l a sma m e m b r a n e s were co inc ident with t h e abrupt p e r m e a t i o n and oxidat ion of the added dopamine , and a p a r t i a l r e l e a s e of amino acids in t he ce l ls chi l led for 48 hr . These changes in p l a sma m e m b r a n e s observed in the l a t e r s t a g e of chil l ing, where most of the cel ls a r e seve re ly injured, may follow a secondary ce l lu lar even t . Suggest ing this not ion , the severe ly a g g r e g a t e d m e m b r a n e - p a r t i c l e s on p l a sma m e m b r a n e s from 48 hr chi l led cel ls we re found to be comple t e ly r e l o c a t e d to t he no rma l s t a t e by simrjly p re incuba t ing wi th po tass ium phospha te buffer , pH 7.2 a t 0 C i m m e d i a t e l y before f ixat ion in g lu t a ra ldehyde (11). Accordingly , the p a r t i c l e aggrega t ion on p lasma m e m b r a n e s during the l a t e r s t a g e of chil l ing might be a t t r i b u t e d to the a l t e r e d n a t u r e of t h e m e m b r a n e per se and /o r r educ t ion of pH in t he cytosol as a r e su l t of t he lesion of t he tonop las t s .

No u l t r a s t r u c t u r a l change in mi tochondr i a was d e t e r m i n e d wi th 24 hr of chil l ing. A t e m p o r a r y depress ion of the r e s p i r a t o r y a c t i v i t y , however , was no t ed in the 12 hr of chill ing and then it r e t u r n e d nea r ly to the no rma l s t a t e upon prolonged chil l ing up to 24 h r . Consider ing the observed changes in t he i n t r a - m e m b r a n o u s s t r u c t u r e s of tonop las t s and the c o n c o m i t a n t d i la t ion and vacuo la t ion of ER m e m b r a n e s in the ea r ly s t a g e of chil l ing, t he revers ib le changes in t he r e sp i r a t ion may be asc r ibab le to a r eve r s ib l e d i s tu rbance in the cy toso l ic env i ronmen t , i .e . , changes in t he pH, sal t concen t r a t i on , e t c . , p re sumab ly caused by a p e r m e a b i l i t y change in tonop las t . In Frankenia sa l t gland ce l ls , t he ves icu la t ion of rough ER has been r e p o r t e d to be r e l a t e d to the sa l t a ccumula t i on (3). If it we re the case in the cal lus u t i l i zed in the p re sen t s tudy, we could specu l a t e t ha t the ER m e m b r a n e s h a v e , more or less , the s imi lar funct ion to b a l a n c e t he cy top lasmic c o n c e n t r a t i o n of sa l t s and /o r p ro ton and so fo r th .

F u r t h e r e x p e r i m e n t s a r e c lea r ly needed to unde r s t and en t i r e ly the mechanism of chil l ing injury in cu l tu red p lant ce l l s . At the m o m e n t it s e e m s possible to conclude tha t the lesion of t h e tonoplas t in t he p r i m a r y s t e p in chil l ing injury of the cu l tu red p lant ce l l s , in a r eve r s ib le or an i r r evers ib le way, may subsequent ly lead to i r r eve r s ib le a l t e r a t i o n of cel l


funct ions . The r ea son for t he tonoplas t lesion per se under chil l ing r e m a i n s unsolved. Some e x p e r i m e n t s a r e under inves t iga t ion in our l a b o r a t o r y by u t i l iz ing i so la t ed vacuoles from the ca l lus .

Final ly , our hypothes i s for t h e mechan i sm of chil l ing injury in cu l t u r ed p lan t cel ls is s c h e m a t i c a l l y p r e s e n t e d in F ig . 8.

o r * Vacuol e Roug h E R

LOW

Mitochondri a

TEMPERATURE

Releas e o f salts,proto n etc .

Dilatio n Temporar y Releas e o f depressio n ribosome s o f functio n

12 h r Οι ? AT P

^ Declin e o f τ ι ATP leve l

Furthe r releas e

24 h r Ο ATP

Vacuolatio n Temporar y Reabsorptio n restoratio n o f salts,proto n o f functio n etc .

ADP ι l Disruptio n Releas e o f l y t i c enzyme s

Deterioratio n Dysfunctio n o f membran e an d destructio n

48 h r v z£s>

IRREVERSIBLE CELL DECA Y

FIGURE 8. A hypothetic mechanism of chilling injury of cultured plant cells (Cormus stolonifera). Some kinds of ATPase are presumed to take part in the process of salt-uptake and the vacuolation of ER in the reversible stage of chilling.

2 9 0 S. Y o s h i d a et al.


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FREEZING STRESS IN POTATO

P. H. Li J. P. Palta Η. H. Chen

L a b o r a t o r y of P l an t Hard iness D e p a r t m e n t of H o r t i c u l t u r a l Sc ience and L.A.

Univers i ty of Minneso ta St. Pau l , Minneso ta

The p o t a t oA is an ideal c rop to exploi t in an ef for t to m e e t t h e

inc reas ing food d e m a n d of the world because of i t s e conomic va lue among t h e world major food crops (9), high energy p roduc t ion pe r unit of land (36), and high n u t r i t i v e va lue c o m p a r e d wi th o t h e r food c rops (18). It p rov ides no t only a good sou rce of c a r b o h y d r a t e s , but also a high qua l i ty of p ro t e in s (19), mine ra l s and v i t a m i n s . More than 100 coun t r i e s a t p r e s e n t grow p o t a t o e s . However , i t has been p r imar i ly c u l t i v a t e d in t h e T e m p e r a t e Zone of Nor th A m e r i c a and Europe , and t rop ica l Andean highlands of South A m e r i c a w h e r e frost is o f ten a major f ac to r in r educ ing p roduc t ion or r e su l t i ng in c rop fa i lure (22). Breeding for b e t t e r a d a p t e d c lones to frost is p romis ing by g e n e t i c manipu la t ion of ex is t ing p o t a t o g e r m p l a s m s (8). A b e t t e r unde r s t and ing of p o t a t o frost r e s i s t a n c e (2), i t s ha rden ing c h a r a c t e r i s t i c s (1 , 5), and t h e e s t ab l i shmen t of a l a b o r a t o r y sc reen ing me thod (34) have co n t r i b u t ed to the r e c e n t successful b reed ing e f for t s (8).

F u r t h e r m o r e , t h e deve lopmen t of frost t o l e r a n t c lones will g r e a t l y expand to t h e a r e a s much of which is c u r r e n t l y marg ina l due to low t e m p e r a t u r e . By t h e s a m e token, t h e p o t a t o p roduc t ion can be i n c r e a s e d in t he p r e sen t l y c u l t i v a t e d a r e a s by simply ex tend ing t h e growing season.

'Potato1 in this paper is referred to the tuber-bearing Solanum

species in addition to the S. tuberosum potato.

Copyright « 1979 by Academic Press. Inc. 2 9 1 All rights of reproduction in any form reserved

ISBN0-12-460560-5

2 9 2 Ρ. Η. Li et al

I. FROST HARDINESS AND FREEZING INJURY

A. Frost Hardiness

Fros t hard iness is def ined as the r e s i s t a n c e of foliage to a f reez ing t e m p e r a t u r e above which no injury occur s in the t i s sue . During f reez ing , w a t e r moves from the cells (mainly vacuole) to t he i n t e r ce l lu l a r i ce t h a t r e su l t s in cel l dehydra t ion . As f reez ing p roceeds , ices cause ce l lu lar c o n t r a c t i o n . Thus, during f reez ing , bes ides the low t e m p e r a t u r e , a p lan t exper i ences mainly t h r e e types of s t r e s se s : a dehydra t ion s t r e s s due to ex t r ace l l u l a r i ce fo rmat ion , an osmot ic s t r e s s due to r emova l of w a t e r from vacuole and a mechan ica l s t r e s s due to i c e a ccumula t i on and cell c o n t r a c t i o n . The mechan i sms of frost hard iness in he rbaceous p l an t s have been r ev i ewed by Olien (24) and Lev i t t (21). Lev i t t (21) concluded t ha t the t o l e r a n c e of dehydra t ion and o the r s t r e s ses l i s ted above due to ex t r ace l l u l a r f reez ing and the avo idance of i n t r ace l lu l a r f reez ing a r e mechan i sms of frost ha rd iness . In genera l , he rbaceous p l an t s cannot w i th s t and t e m p e r a t u r e s below -20 C.

The d i f fe rence in frost hard iness b e t w e e n r e s i s t a n t and sens i t ive p o t a t o e s is only about 3 to 4 C. In some p lan t spec ies , a small va r i a t ion in t i ssue w a t e r c o n t e n t (g HÔ/g dry weight) could resu l t in such a d i f f e rence in a p lan t ' s c a p a c i t y to surv ive . A high cell sap c o n c e n t r a t i o n may inc rease sl ightly t he abi l i ty of t i ssue to supercool ; thus , t he t i ssue could survive g r e a t e r s t r e s s by avoiding f reez ing . However , t he t i ssue w a t e r c o n t e n t and the cell sap c o n c e n t r a t i o n have not c o r r e l a t e d to the frost hard iness in p o t a t o leaves (2).

Using f reezing curve analysis , Sukumaran and Weiser (35) found t ha t t h e leaf t i ssue of S. acaule p o t a t o could t o l e r a t e a g r e a t e r amoun t of i ce fo rmat ion than the sens i t ive spec ies , S. tuberosum. Nuc lea r m a g n e t i c r e s o n a n c e spec t roscop ic s tudies have shown tha t the r e s i s t a n t p o t a t o spec ies can t o l e r a t e m o r e f r eeze - induced dehydra t ion than t h e suscept ib le ones (2). For example , a t t he killing t e m p e r a t u r e s the amoun t of unfrozen t i ssue w a t e r ave raged to about 20 and 4 5 % of t o t a l liquid w a t e r in S. acaule and S. tuberosum, r e s p e c t i v e l y . Thus it appea r s t h a t t he major d i f fe rence in frost hard iness b e t w e e n r e s i s t a n t and suscep t ib le p o t a t o e s is due to t he abi l i ty of t he hardy p o t a t o to t o l e r a t e more f rozen w a t e r than the suscep t ib le one .

B. Freezing Injury

F re e z ing injury has been thought to involve cell m e m b r a n e s . One of t he most common signs of f reez ing injury is t he i n t e r ce l lu l a r space in f i l t e red with w a t e r , leading to a soaked a p p e a r a n c e and loss of cell tu rgor . F r e e z i n g injury also of ten causes l eakage of ions from the ce l l s . The efflux of ions and the in f i l t ra t ion with w a t e r following a f reez ing injury have been assumed due to t he breakdown of s e m i p e r m e a b l e p rope r t i e s of cell m e m b r a n e (21, 35). R e c e n t l y , P a l t a et al. (26, 27)

F r e e z i n g S t r e s s in P o t a t o 2 9 3

h a v e shown tha t during the p rogress of f reez ing injury ( revers ible or i r revers ib le ) , s e m i p e r m e a b l e p r o p e r t i e s (membrane lipids) r e m a i n i n t a c t w h e r e a s t h e a c t i v e t r anspo r t p r o p e r t i e s ( m e m b r a n e in t r ins ic proteins) a r e d a m a g e d .

TABLE I. Percent Ionic Leakage of Two Potato Species at Two Time Intervals after Freezing-thawing (Τ λ = 12 hr . , T£ = 60-120 hr.).

Freezing Temperature ( C) Τ -

Days in cold hardening conditions 7 14

1

Solanum tuberosum

-2.0 40, .5 20. ,2

-2.5 89. .0 57. ,7 45. 2 24, .7 16. .7 14, .7

-3.0 90 .9 68. .1 59. 0 38, .0 33, .1 37, .4

-3.5 92 .5 93. .0 54. .2 59 .5 55, .4 68 .1

Control 11 .3 8. .3 13. .7 7, .1 5 .6 4 .1

Solanum acaule

-4.0 23 .5 13 .7 10. .0 5 .6

-5.0 54 .0 41 .3 34, .6 21 .6 12 .5 9 .5

-6.0 93 .0 92 .9 90 .8 92 .6 52 .5 39 .1

-7.0 94 .0 92 .3 93 .0 93 .8 59 .3 70 .6

-8.0 93 .5 91 .9

Control 9. 5 6. 1 7. 1 5. 4 5. 9 4. 6

aThese data were presented at the Society for Cryobiology Annual

Meeting (28). Cold hardening conditions (1).

2 9 4 Ρ. Η. Li et al

1. Reversibility of Membrane Damage after Freezing. P l a n t s of S. acaule and S. tuberosum w e r e sub jec ted to cold harden ing condi t ions (for de ta i l s see Ref. 1). At 0, 7, 14 and Ζ 1-day i n t e r v a l s , l ea f l e t s of bo th spec ies w e r e f rozen from -Z C to -9 C a t a cooling r a t e of l ° C / h r . They w e r e then t hawed slowly over i c e . During t h e 60 to 1Z0 hours following t h e thaw, m e a s u r e m e n t s w e r e m a d e on t h e conduc t iv i ty of t h e e f fusa te . In t he unhardened p l an t s (0 day, Table I) t h e l e a f l e t s f rozen to -Z.0 and -Z .5°C for S. tuberosum, and -4 and - 5 ° C for S. acaule r e c o v e r e d from f reez ing injury during t h e p o s t - t h a w per iod , while a t t he lower t e m p e r a t u r e s , injury i nc r ea sed or r e m a i n e d unchanged wi th t i m e unt i l final d e a t h of t h e t i s sue . Af te r 14 days of ha rden ing , t h e l ea f l e t s f rozen to - 3 ° C in S. tuberosum and - 6 ° C in S. acaule also r e c o v e r e d during t h e p o s t - t h a w i n g per iods (Table I). In bo th spec ies , a r e c o v e r y in injury was followed by d i s a p p e a r a n c e of t h e in f i l t r a t ion wi th w a t e r , i nc rea se in tu rg id i ty and a d e c r e a s e in t h e conduc t iv i ty of t h e e f fusa te (Table I). The f reez ing t e m p e r a t u r e s a t which injury was r eve r s ib l e or i r r eve r s ib le va r ied by 0.5 or 1.0 C depending upon t h e age of p lan t m a t e r i a l s . These r e su l t s suggest t ha t t h e in i t ia l f reez ing injury may be to t h e a c t i v e t r anspo r t s y s t e m s of t h e cel l m e m b r a n e s b e c a u s e only t he r e c o v e r y of such p roces s can lead to an a c t i v e u p t a k e of e f fusa tes , such as Κ , aga ins t t h e cell g r ad i en t . These obse rva t ions a r e in a g r e e m e n t wi th ea r l i e r r e p o r t s (Z6, Z7).

2. Microscopic Observations of Freezing Injured Cells. As d is cussed, a f r e e z e injured cell may or may not have t he abi l i ty to r e c o v e r comple t e ly . In order to fu r ther s tudy t h e n a t u r e of injury, mic roscop ic obse rva t ions w e r e ca r r i ed out on injured cel ls (ZZ, Z9). L e a f l e t s of bo th r e s i s t a n t (S. acaule) and sens i t ive spec ies (S. tuberosum) w e r e s low-o ο ly f rozen down to -7 C from -Z C. I m m e d i a t e l y a f t e r slowly thawing , s e g m e n t s from t h e middle por t ion of t h e l e a f l e t s w e r e co l l ec t ed and fixed for u l t r a s t r u c t u r a l s tud ies . Al though t h e t i ssue b e c a m e soaked and f laccid var ious o rgane l le a p p e a r e d no rma l a t a low d e g r e e of f reez ing s t r e s s . Abnormal i t i e s a t t h e subcel lu lar level s e e m e d to s t a r t wi th swell ing of t he p ro top lasm (Z9). With increas ing s t r e s s , swell ing of t he mi tochondr i a and chloroplas t was a p p a r e n t . Separa t ion of p l a sma m e m b r a n e and cell wall (frost plasmolysis) and coagula t ion of t h e p ro top lasm w e r e observed in dead cel ls (ZZ). In t he se cel ls coagu la t ion of t h e p ro top lasm and disrupt ion of t h e tonoplas t and p l a sma m e m b r a n e w e r e observed . Possible s equence of f reez ing injury in p o t a t o leaf cel ls appea r s to i n i t i a t e wi th some d i s tu rbances in t he cel l m e m b r a n e leading to swell ing of p ro top la sm, and then swell ing of mi tochondr i a and ch lorop las t s , fol lowed by breakdown of t h e tonoplas t and p l a sma m e m b r a n e sys tem and coagula t ion of p ro top la sm resu l t ing in cel l d e a t h .

3. Membrane Permeability after Freezing Injury. In sp i t e of l eakage of ions and loss of tu rgor due to f reez ing injury, mic roscop ic observa t ions r e v e a l e d t ha t injured ye t l iving cel ls w e r e visually i n t a c t in cell m e m b r a n e . The l eakage of ions appea r s due to a l t e r a t i o n s in t he cel l


m e m b r a n e p r o p e r t i e s r a t h e r than the m e m b r a n e r u p t u r e . F u r t h e r m o r e , f r e eze injured cel ls could be p lasmolyzed in a hype r ton i c manni to l solut ion (0.8 osm), and they r e m a i n e d p la smolyzed for seve ra l days s imi lar t o the unf rozen con t ro l ce l l s (29). Even the i r revers ib ly injured cel ls t h a t showed a swell ing of t h e p ro top la sm could be p lasmolyzed in hype r ton ic manni to l solut ion jus t l ike the unf rozen con t ro l or r eve r s ib ly injured cel ls (29). These obse rva t ions i n d i c a t e t h a t t h e s e m i p e r m e a b l e p r o p e r t i e s of t hese cel l m e m b r a n e s a r e st i l l i n t a c t .

Dur ing t h e p o s t - t h a w per iod , an injured ye t l iving cell may have the abi l i ty to r e c o v e r from f reez ing injury ( revers ible injury) or may even tua l ly die ( i r revers ib le injury). In o rder to e x a m i n e the n a t u r e of a l t e r a t i o n s in m e m b r a n e p r o p e r t i e s , t he t r a n s p o r t of u r e a , m e t h y l - u r e a and po tass ium ac ross t he cel l m e m b r a n e w e r e s tud ied in f reez ing injured y e t a l ive ce l ls (29). A p l a s m o m e t r i c me thod desc r ibed by S tade lmann (33) was used in measur ing t h e cel l m e m b r a n e p e r m e a b i l i t y . In t he se e x p e r i m e n t s , t he p lasmolyzed p ro top l a s t s we re a l lowed to expand in response to t h e pass ive u p t a k e of p e r m e a b l e so lu te (equimolar solut ion of KC1, u r e a and m e t h y l - u r e a ) . The p ro top l a s t expansion was m e a s u r e d d i r ec t l y wi th a mic roscope wi th an e y e p i e c e m i c r o m e t e r .

The unf rozen cel ls did no t t a k e up Κ in two hours and r e m a i n e d p lasmolyzed a t a cons t an t vo lume . The injured ce l l s , on the o t h e r hand, expanded i m m e d i a t e l y upon t r ans fe r to KC1 solut ion and gradual ly dep lasmolyzed . The r a t e s of Κ u p t a k e var ied for individual cel ls in t he s a m e t i s sue b e c a u s e the e x t e n t of injury was d i f fe ren t in d i f fe ren t ce l l s . Al though f r e e z e injury r e s u l t e d in a rap id i n c r e a s e in t h e r a t e of Κ t r anspo r t ac ross the cel l m e m b r a n e s , no change in r a t e s was d e t e c t e d for n o n e l e c t r o l y t e s such as u r e a and m e t h y l - u r e a . The p e r m e a b i l i t y of a n o n e l e c t r o l y t e has long been known as a d i r ec t funct ion of i t s lipid solubi l i ty (6). The re fo re , a change in t h e n o n e l e c t r o l y t e p e r m e a b i l i t y should r e f l e c t the a l t e r a t i o n s in t h e lipid componen t of the m e m b r a n e . No change in t h e p e r m e a b i l i t y c o n s t a n t s of t he se n o n e l e c t r o l y t e s i nd i ca t e s t h a t the f reez ing injury does no t a l t e r the lipid por t ion of the cel l m e m b r a n e s . The finding t h a t w a t e r p e r m e a b i l i t y c o n s t a n t s r e m a i n unchanged during f reez ing injury fu r the r suppor t s this conclusion (26).

The i n c r e a s e in Κ p e r m e a b i l i t y provides ev idence t h a t the p ro t e in c o m p o n e n t s of the m e m b r a n e a r e possible t a r g e t s of f reezing injury. M e m b r a n e p ro t e in s a r e of two types , in t r ins ic and pe r iphe ra l . Some of t h e in t r ins ic p ro t e in s pass en t i r e ly th rough t h e b i layer (31). This type of in t r ins ic p ro t e in s has been p roposed as being s i t e s for a c t i v e t r an spo r t of ions (32). Suble thal f reez ing t e m p e r a t u r e can lead to t h e d e n a t u r a t i o n of m e m b r a n e p ro t e in s r e su l t ing in an i n a c t i v a t i o n of t h e a c t i v e t r anspo r t s y s t e m s . When i n a c t i v a t e d , t h e s e in t r ins ic p ro te ins could s e rve as channels for pass ive ion t r a n s p o r t , giving very high Κ p e r m e a b i l i t y va lues and swell ing of t h e p r o t o p l a s m . A l a rge pass ive efflux of ions could also occur th rough t h e s e channels in t h e d i r ec t ion of t h e c o n c e n t r a t i o n g rad ien t from t h e vacuo le to t h e e x t r a c e l l u l a r solut ion. A r epa i r of t h e i n a c t i v a t i o n leads t he t i s sue to r e c o v e r from f reez ing injury (reversible) and fa i lure to do so leads u l t i m a t e l y to t h e d e a t h of cel ls ( i r revers ib le) .

2 9 6 P. Η. Li et ai

Π. COLD HARDENING

Many he rbaceous p l an t s undergo inc rease in frost hard iness when sub jec ted to a specif ic env i ronmen ta l condi t ion such as low t e m p e r a t u r e s , short day leng th , changes in qual i ty and quan t i t y of l ight , r educ ing w a t e r supply, e t c . Some spec ies can ha rden ( increase in frost hardiness) ex tens ive ly in response to these env i ronmen ta l changes , while o t h e r s will ha rden only a few deg rees and some do not ha rden a t a l l .

A. Species, Varieties and Tissues

Prior to 1976, t h e r e was some d ispute as to w h e t h e r t he t u b e r -bear ing Solanum p o t a t o e s can be cold ha rdened . Hayden et al (14) i nd i ca t ed t ha t p o t a t o e s possess a s t ab le frost hard iness level and do not ha rden . Richardson and E s t r a d a (30) r e p o r t e d t ha t Ζ to 3 weeks of cool t e m p e r a t u r e s could d i f f e r e n t i a t e hardy from nonhardy p o t a t o e s . Chen and Li (1) conf i rmed t h a t some noncu l t i va t ed spec ies l ike Solanum acaule, S. chomatophilum, S. commersonii and S. multidissectum could ha rden , while the c u l t i v a t e d S. tuberosum (var ie t ies of "Red Pon t i ac , " "Kennebec ," "Norland" and "Norchip") fai led to ha rden .

R e c e n t l y , addi t ional t u b e r - b e a r i n g Solanum spec ies w e r e s c r e e n ed for thei r frost hard iness and cold hardening abi l i ty . Based on exis t ing in format ion , we can classify them in to four groups in t e r m s of the i r frost hard iness and cold hardening (Table II). These groups a r e (I) t he spec ies which possess frost hard iness (survive to -4 .0 C or colder) and a r e able to cold harden , (Π) t h e spec ies which possess frost ha rd iness but a r e unable to co ld Qharden , (ΠΙ) t he spec ies which possess no frost ha rd iness (survive to -3 .0 C or warmer) but a r e able to cold ha rden , and (IV) t h e spec ies which possess no frost hard iness and a r e unable to cold ha rden . Example spec ies for each group a r e l i s ted in Table Π. Solanum Tuberosum, t he commonly grown p o t a t o e s , falls in to t he las t c a t e g o r y .

The p o t a t o is a cool season grown crop and is usually chill ing r e s i s t a n t . We observed chill ing injury in S. trifidum (PI Z5541) when p lan t s w e r e grown in Z/Z C, day /n igh t , r e g i m e . It is possible t ha t addi t ional t u b e r - b e a r i n g Solanum spec ies , which a r e chill ing sens i t ive , ex is t .

S t e m - c u l t u r e d p lan t s of S. tuberosum and S. commersonii grown in agar medium showed s imilar hardening p a t t e r n s as t he p o t t e d p lan t s (5). Leaf cal lus t i ssues of S. tuberosum, "At l an t i c , " "Kennebec" and "Red Pon t i ac , " showed no hardening abi l i ty and we re kil led a t -3 C. The leaf cal lus t issues of S. acaule, however , can be ha rdened t o - 9 C a f t e r 15 days a t 3 C in darkness , a 3 C inc rease in frost hard iness (5). These r e su l t s ag reed with observa t ions from p o t t e d p lan t s previously r e p o r t e d (1).


TABLE II. Classification of Tuber-bearing Solanum Potatoes in Terms of Frost Resistance and Cold Hardening

Killing Temp (C°) Species Before After

Categories (examples) TreatmentaTreatment!

Group I: frost S. acaule -6. .0 - 9 . 0 resistance and (Oka 3885)° able to cold S. commersonii -4, .5 -11.5 harden (Oka 5040)

Group II: frost S. sanctae-rosae -5. ,5 -5.5 resistance but (Oka 5697) unable to cold S. megistacrolobum -5. .0 -5.0 harden (Oka 3914)

Group III: frost S. oplocense -3. .0 -8.0 sensitive but (Oka 4500) able to cold S. polytrichon -3. ,0 -6.5 harden (PI 184773)

Group IV: frost S. tuberosum -3. ,0 -3.0 sensitive and unable to cold S. stenotomum -3. ,0 -3.0 harden (PI 195188)

^Plants were grown in a regime of 20/15°C day/night, 14 hrs. ^Plants were grown in a regime of 2 C day/night, 14 hrs, for 20 days.

Identification number at Potato Introduction Station, Sturgeon Bay, Wisconsin.

B. Optimum Hardening Conditions

Low t e m p e r a t u r e s and shor t days a r e t h e two major env i ronmen ta l f a c t o r s in inducing frost ha rd iness . Some spec ies can be ha rdened by low t e m p e r a t u r e s wi th a suff ic ient amoun t of l ight , r ega rd l e s s of t h e pho toper iod (20). P o t a t o e s belong to this c a t e g o r y . Low t e m p e r a t u r e condi t ioning for max imum hard iness va r i e s from spec ies to spec ie s . For example , w in te r r a p e (17) and c a b b a g e (20) r equ i r ed f reez ing t e m p e r a t u r e during t h e ha rden ing to ach i eve t h e max imum hard iness . The op t imum harden ing t e m p e r a t u r e s for Solanum p o t a t o e s a r e about 1 to 2 C above f reez ing .

Cold harden ing of p o t a t o can be ach ieved by d i r ec t l y exposing p lan t s to cons t an t day /n igh t low t e m p e r a t u r e s (5) or s t epwise lower ing t h e t e m p e r a t u r e (1). The hard iness level which can be ach ieved is dependen t upon the t e m p e r a t u r e s used. Lower t e m p e r a t u r e s resu l t in g r e a t e r

2 9 8 P. Η. Li et al

Ε Ε ο υ (Λ

ι 3 to

1

<Λ

I 20/15 10/10 5/5 2 /2

DAY/NIGH T TEMPERATUR E (°C )

FIGURE 1. Changes in frost hardiness of three Solanum species at constant low day/night temperatures with 14 hr light for 2 weeks.

deg ree s of hard iness (Fig. 1). Under cons t an t low t e m p e r a t u r e s , p l an t s appea red to r e a c h thei r maximum hard iness a t about 15 days , while p l an t s t ha t w e r e sub jec ted to s t epwise hardening condi t ions con t inued to ha rden even a f t e r 20 days (5).

C. Growth Regulators

The app l ica t ion of g rowth r e g u l a t o r s to induce frost hard iness has long been cons idered but the r e su l t s have been c o n t r a d i c t o r y (21). Most s tud ies w e r e conduc ted by exogenous appl ica t ion to t he fol iage or t he soil . Fol ia r app l ica t ions ra i se c e r t a i n p rob lems b e c a u s e leaf su r faces h a v e a t endency to r e s t r i c t t he u p t a k e of exogenously appl ied chemica l s . Microbiological deg rada t ion causes p rob lems wi th t he soil app l ica t ion . For s tudying t h e e f fec t of g rowth r e g u l a t o r s on p o t a t o hard iness , Solanum s t e m - c u l t u r e d p lan t s grown in t he agar medium w e r e t h e r e f o r e used (5). P re l imina ry r e su l t s i nd i ca t ed t h a t t he exogenously appl ied abscis ic acid (ABA) can i nc r ea se p o t a t o frost ha rd iness . No i nc r ea se in frost hard iness was found wi th g ibbere l l ic ac id t r e a t m e n t s . The i nc r ea se


and change in ha rd iness wi th ABA t r e a t m e n t s a r e t h e s a m e in bo th warm and low t e m p e r a t u r e r e g i m e s . It appea r s t h a t the exogenous ABA may s u b s t i t u t e t h e funct ional ro l e of t h e low t e m p e r a t u r e in inducing frost ha rd iness . However , d i f fe ren t levels of ABA produced the s a m e r e s u l t s . As sugges ted by K a c p e r s k a - P a l a c z (17), ABA may se rve as a driving fo rce to shift t he me tabo l i c p a t h w a y in favor ing cold ha rden ing . Possibly higher levels of ABA h a v e an e f fec t s imi lar to t he lower levels once t h e endogenous ABA r e a c h e s the funct ional l eve l . Appa ren t ly , ABA can induce hard iness no t only in those spec ies which a r e able to ha rden but also in S. tuberosum which is a spec ies t h a t is unable to ha rden a t low t e m p e r a t u r e .

D. Biochemical Changes

Many s tud ies have been r e p o r t e d r ega rd ing b iochemica l changes in h e r b a c e o u s p l an t s during co ld-harden ing (7, 11 , 1Z). We also examined and c o m p a r e d some of t h e b iochemica l changes in l eaves of S. acaule and S. tuberosum during cold ha rden ing (4). The r e s u l t s a r e s u m m a r i z e d be low. Since DNA in m a t u r e d leaf cel l is m a i n t a i n e d a t a cons t an t leve l during g rowth (25) m e t a b o l i t e changes during harden ing would be b e t t e r expressed on unit DNA than on dry weight bas is . Fol lowing discussions r e l a t i v e to t h e changes in sugars , s t a r c h , nuc le ic ac ids , p ro t e in s and lipids a r e t h e r e f o r e based on unit DNA (same n u m b e r s of cel ls) .

1. Sugars and Starch. Sugar i n c r e a s e was found in bo th S. acule and S. tuberosum during ha rden ing . To ta l sugar c o n t e n t m o r e than doubled in bo th spec ies a f t e r 15 days ha rden ing . L e v i t t (21) r e v i e w e d t h e p r o t e c t i v e ro le of sugars aga ins t frost injury. However , such a ro l e in p o t a t o frost hard iness is ques t ionab le b e c a u s e S. tuberosum fails to h a r d e n even when i t s sugar c o n t e n t i nc r ea se s more than twofold during ha rden ing . Huner and Macdowal l (16) found t h a t t he cold adap t i ve rye p l an t s i nc reased the s t ab i l i t y of RuBP ca rboxylase in ch lo rop las t s under cold s t r e s s . This would enab le p l an t s to ma in t a in p h o t o s y n t h e t i c abi l i ty under cold ha rden ing condi t ion . T h e r e f o r e , sugars can a c c u m u l a t e in leaf t i s sues .

Under ha rden ing condi t ion , leaf s t a r c h c o n t e n t was also i n c r e a s e d in bo th S. acaule and S. tuberosum. H a t a n o (13) r e p o r t e d an i nc r ea se of s t a r c h in Chlorella during cold harden ing under l ight . Heldt et al (15) r e p o r t e d t h a t i so la ted sp inach ch lorop las t s from hardy win t e r m a t e r i a l s showed higher s t a r c h syn thes i s r a t e than from nonhardy win te r m a t e r i a l s . The d e c r e a s e in s t a r c h c o n t e n t during cold harden ing was a lmos t a lways found in nonpho tosyn the t i c t i ssues such as ba rk (23) and roo t (10) which possess ve ry l i t t l e or no p h o t o s y n t h e t i c a c t i v i t y . The cold harden ing is a p roces s requi r ing ene rgy . The ene rgy source in nonpho tosyn the t i c t i ssues s e e m s to be t h e s t o r e d food such as s t a r c h . On t h e o the r hand, l eaves which can ma in ta in the i r p h o t o s y n t h e t i c a c t i v i t y a t low t e m p e r a t u r e should be able to d i r ec t l y supply p h o t o s y n t h a t e as t he


energy source for harden ing . This may be an exp lana t ion for both s t a r c h and sugar inc reases in p o t a t o l eaves during cold harden ing .

2. Ribonucleic Acids. In S. acaule, p l an t s grown under h a r d e n ing env i ronment a lways ma in t a ined a t a higher level of rRNA than p l an t s grown under unhardening env i ronmen t . In S. tuberosum, t h e r e was no d i f fe ren t change in rRNA c o n t e n t b e t w e e n ha rdened p l an t s and con t ro l s . It has been r e p o r t e d t h a t the t e m p e r a t u r e had a marked inf luence on rRNA metabo l i sm while t he pho toper iod ic response was not as g r e a t in p o t a t o p lan t s (25). The higher level of rRNA in S. acaule p l an t s during hardening is pa ra l l e l ed with higher level of soluble p ro te in and the i nc rease of ha rd iness . The d i f fe rence in rRNA levels b e t w e e n S. acule and S. tuberosum in response to hardening sugges ts t h a t the hardening p rocess l ikely i n i t i a t e s a t t he t r ansc r ip t ion and /or t r ans l a t ion leve ls .

3. Proteins. In S. acaule, soluble p ro t e in s w e r e ma in t a ined a t a much hjgher level in cold ha rdened p lan t s than in con t ro l s . The soluble p ro te in f rac t ion also i nc reased a t a much higher r a t e in the fo rmer than in t he l a t t e r during harden ing . No s ignif icant p ro t e in changes we re observed in both cont ro l s and ha rdened p lan t s of S. tuberosum, which fai led to ha rden . Since t h e i nc r ea se in hard iness is a lways para l l e l wi th t he inc rease in soluble p ro te in c o n t e n t , severa l r e s e a r c h e r s have discussed the ro le of soluble p ro te in in cold harden ing (17, Zl) . Cox et al (7) concluded tha t only p lan t s t ha t we re able to conduct a c t i v e p ro te in synthes is a t low t e m p e r a t u r e had the capab i l i ty to ha rden . This conclusion was suppor ted by Hatano ' s work (13) in which cyc lohexamide , an inhibi tor for p ro te in synthes is , could inhibit t he hardening in Chlor-ella.

4. Lipids. During hardening , t o t a l lipid i nc reased in S. acaule and a m o r e or less c o n s t a n t level was ma in t a ined in S. tuberosum. The i nc rease in lipid in S. acaule during harden ing suppor ted t he EM observa t ions of lipid bodies accumula t i ons in ha rdened ch lorop las t s (3). The phospholipid c o n t e n t in ha rdened S. acaule p l an t s was much higher than in con t ro l s , while lower level of phospholipid was observed in S. tuberosum ha rdened p lan t s than in cont ro l s during hardening . Cold harden ing a l t e r s m e m b r a n e p rope r t i e s and inc reases i t s s t ab i l i ty during f reez ing as sugges ted (17). The deve lopmen t of hard iness in S. acaule is probably a s soc i a t ed wi th a l t e r a t i o n s in m e m b r a n e p rope r t i e s as ev idenced in phospholipid i n c r e a s e .

E. Electron Microscopic Observations

C o m p a r a t i v e u l t r a s t r u c t u r a l s tudies w e r e u n d e r t a k e n b e t w e e n hardy (S. acaule) and t ende r (S. tuberosum) p o t a t o e s grown under warm and cold t e m p e r a t u r e r e g i m e s . In less than 10 days during cold harden ing , t he hardy spec ies of S. acaule showed a d r a s t i c i nc rea se in s t a r c h grains in ch loroplas t s (Fig. ZB). Such an i n c r ea se , however , was


FIGURE 2. Electron microscopic observations of leaf cross-sections showing the accumulation of starch grains ( •> ) in chloroplasts after 8 days cold hardening (5/2°C day/night temp., 14 hr light). A,B: Solanum acaule; C,D; Solanum tuberosum; A & C: before hardening; Β & D: after hardening.


not observed in ch lorop las t s of t h e t ende r spec ies of S. tuberosum (Fig. ZD) grown under t he s a m e r e g i m e . It appea r s t h a t ene rgy sources for cold ha rden ing in p o t a t o c a m e d i r ec t l y from p h o t o s y n t h a t e r a t h e r t han from s t a r c h to sugar t r a n s f o r m a t i o n . Chen et al (3) have r e p o r t ed t he swell ing and i r r egu la r i t y of thylakoid m e m b r a n e s and l a rge p a t c h e s of s t r o m a in ch lorop las t s of h a r d e n e d S. acaule, while no s ign i f i can t changes w e r e observed in cold t r e a t e d S. tuberosum ch lo ro p l a s t s .

ΙΠ. SUMMARY

The d i f f e rence in frost ha rd iness b e t w e e n a r e s i s t a n t and sens i t ive type of p o t a t o is about 3 to 4 C . This d i f f e rence is no t due to t he avo idance of i n t r ace l lu l a r f reez ing because cel l sap c o n c e n t r a t i o n and t i ssue w a t e r c o n t e n t have shown no co r r e l a t i on to the va r i a t i ons of frost ha rd iness among d i f fe ren t spec ies ; r a t h e r , i t is due to t h e t o l e r a n c e of f r eeze - induced dehydra t ion (ex t race l lu la r f reez ing) . During the ini t ia l s t a g e of f reez ing injury, s e m i p e r m e a b l e p r o p e r t i e s of t h e cel l m e m b r a n e s r e m a i n i n t a c t . F r e e z i n g injury, due to ex t r ace l l u l a r ice fo rmat ion , r e su l t s in a l t e r a t i o n s of m e m b r a n e t r anspo r t p r o p e r t i e s , mos t l ikely the in t r ins ic m e m b r a n e p ro t e in s which involve in a c t i v e t r anspo r t s y s t e m s . F r e e z i n g injury can be r eve r s ib l e (leading to c o m p l e t e recovery) or i r r evers ib le (leading to d e a t h ) . The mechan i sms of frost hard iness and cold harden ing appea r to be independen t . Inc reases in r ibosomal RNA, soluble p ro t e in s and phospholipids a r e a s soc ia t ed wi th t h e i nc r ea se of frost ha rd iness during cold ha rden ing .


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138, Z76-Z85 (1977). 4 . Chen, Η. H. Mas te r P lan Β Pape r , Univers i ty of Minnesota , Saint

Paul (1978). 5. Chen, Η. H., G a v i n l e r t v a t a n a , P . , and Li, P . H. Botanical Gaz.

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H. Li and A. Sakai , eds.) . pp . 333-334, A c a d e m i c P ress , New York (1978).

9. Food and Agr icu l tu ra l Organ iza t ion . P roduc t ion Year Book 1974 (1974).


10. Gl ier , J . H., and Caruso , J . L. Cryobiology 10, 328-330 (1973). 11 . G r e a t h o u s e , G. Α., and S tua r t , N . W. Plant PhysioL 12, 685-702

(1937). 12. Green , D. G., and Ra tz la f f , C . D. Can. J. Bot. 53, 2198-2201

(1975). 13. H a t a n o , S. In "P lant Cold Hard iness and F r e e z i n g St ress ," (P. H. Li

and A. Sakae , eds . ) . pp . 175-196, A c a d e m i c P ress , New York (1978). 14. Hayden, R. E., Dionne, L, and Fensom, D. S. Can. J. Bot. 50 ,1547-

1554 (1972). 15. He ld t , H. W., Chon, C. J . , and Maronde , (1965). 1155(1977) . 16. Huner , Ν. P . Α., and Macdowal l , F . D. H. Biochem. Biophys. Res.

Comm. 73, 411-420 (1976). 17. K a c p e r s k a - P a l a c z , A. In "P lan t Cold Hard iness and F r e e z i n g

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FLUORESCENCE POLARIZATION STUDIES OF MEMBRANE PHOSPHOLIPID PHASE SEPARATIONS

IN WARM AND COOL CLIMATE PLANTS

2 3 4 Carl S. Pike ' , Joseph A. Berry, and John K. Raison

D e p a r t m e n t of P lan t Biology Ca rneg i e Ins t i tu t ion of Washington

Stanford, Cal i forn ia

I. INTRODUCTION

The s e a r c h for a p r i m a r y locus of p lan t responses to low t e m p e r a t u r e has focused on the phys ica l p r o p e r t i e s of the m e m b r a n e l ipids. At low t e m p e r a t u r e m e m b r a n e lipids may undergo a phase t r ans i t ion from a fluid, l iqu id-c rys ta l l ine s t a t e to a m o r e solid, ge l led s t a t e . When a lipid m i x t u r e is cooled, t h e lipids wi th t h e h ighes t me l t ing poin ts solidify first and undergo a l a t e r a l phase s epa ra t i on from the r ema in ing fluid lipid (19). The t e m p e r a t u r e d e p e n d e n c e of t h e physica l s t a t e of m e m b r a n e lipids (as s tud ied wi th e l e c t r o n spin r e s o n a n c e p robes in i n t a c t m e m b r a n e s or ves ic les p r e p a r e d from i so la ted lipid fract ions) has been c o m p a r e d to t he t e m p e r a t u r e d e p e n d e n c e of var ious b iochemica l p roces ses c a t a l y z e d by m e m b r a n e - b o u n d p r o t e i n s . These s tud ies , based la rge ly on crop p l an t s , h a v e sugges ted a s t rong co r r e l a t i on b e t w e e n the lipid phase s epa ra t i on t e m p e r a t u r e and an i n c r e a s e in t h e a c t i v a t i o n energy of var ious r e a c t i o n s (13, 14). An abrupt change in the t e m p e r a t u r e d e p e n d e n c e of spin label mot ion in mung bean lipids c o r r e l a t e d well wi th t he lower l imi t of seedl ing g rowth (16). Lipid phase changes in a se r ies of Passiflora s p e cies co r responded to the spec ies ' known t e m p e r a t u r e responses for

2CIW/DPB Publication Number 660 Permanent address: Department of Biology, Franklin and Marshall

Collie, Lancaster, PA 17604 Supported in part by an NSF Science Faculty Professional

Development Award and by Franklin and Marshall College Permanent address: Plant Physiology Unit, CSIRO Division of Food

Research and School of Biological Sciences, Macquarie University, North Ryde, N.S.W., 2113

Copyright β 197Θ by Academic Press. Inc. 3 0 5 All rights of reproduction In any form reserved

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3 0 6 C. S. P i k e et al.

growth (11). The physical s t a t e of t h e m e m b r a n e lipids is thus sugges ted to be a major d e t e r m i n a n t of the lower t e m p e r a t u r e l imi t for a p lan t ' s survival or g rowth .

R e c e n t work by Sklar et al. (23, 24, 25) has shown the usefulness of a pair of n a t u r a l l y - o c c u r r i n g f luorescen t po lyene f a t t y ac ids , CIS- and trans -par inar ic ac id (Fig. 1), as p robes of m e m b r a n e lipid p r o p e r t i e s . When b iosyn the t i ca l ly i nco rpo ra t ed in to m e m b r a n e lipids or m e r e l y added to a m e m b r a n e or lipid ves ic le p r e p a r a t i o n , t he se molecu les would be e x p e c t e d minimal ly to p e r t u r b t he lipid o r i en t a t i on , c o m p a r e d to a typ ica l ESR probe bear ing a bulky 2 .2-d imethyl -N-oxyloxizo l id ine r ing a t t a c h e d to a f a t t y acyl chain (2). The probes a r e very rapidly i nco rpo ra t ed in to lipid b i layers when added to a l iposome p r e p a r a t i o n . M e a s u r e m e n t s of t he t e m p e r a t u r e d e p e n d e n c e of t he f luorescence in t ens i ty can provide in fo rma t ion on lipid phase t r ans i t i ons , as shown in s tudies of s ing le -componen t phospholipid ves ic les (25), b a c t e r i a l m e m b r a n e s and e x t r a c t e d phospholipid ves ic les (26), and m a m m a l i a n m e m b r a n e and phospholipid p r e p a r a t i o n s (17). These probes h a v e been used to d e m o n s t r a t e the subs tan t i a l d i f f e rence in phase t r ans i t ion t e m p e r a t u r e s in m e m b r a n e lipids from an E. coli f a t t y acid auxo t roph fed f a t t y acids differ ing in chain length and s a t u r a t i o n (26). The two i somers differ in the i r pa r t i t i on ing behavior b e t w e e n solid and fluid phases in a mixed sys t em: t h e trans form has a s t rong p r e f e r e n c e for t h e solid phase , while t he CIS form has a very slight p r e f e r e n c e for t he fluid phase . As a r e su l t , trans -par inar ic acid is sens i t ive to a few p e r c e n t solid, but b e c o m e s insens i t ive to t he fo rma t ion of solid lipid above about 50% (29).

The f luorescence po la r i za t ion behavior of trans -par inar ic ac id - l a be led lipid p r e p a r a t i o n s can provide in fo rmat ion on f luidity behavior , in t h e s a m e manner as ESR probes [See Shini tzky and Barenholz (20) for review of f luorescence po la r iza t ion] . The po la r i za t ion r a t i o ( f luorescence in t ens i ty with emission and e x c i t a t i o n po l a r i ze r s o r i en t ed para l l e l to one ano the r divided by t h e in t ens i ty with t h e po la r i ze r s in pe rpend icu la r o r ien ta t ion) is used as an express ion of f luidi ty, with a higher r a t i o corresponding to lower f luidi ty (25).

FIGURE 1. The structures of cis-parinaric acid (I) and trans-parinaric acid (II).

F l u o r e s c e n c e Pola r iza t ion S t u d i e s 3 0 7

Most s tud ies on chil l ing sens i t iv i ty and r e s i s t a n c e have dea l t wi th crop p l an t s , which have undoubted ly been ex tens ive ly modif ied by a r t i f i c ia l s e l ec t i on . We h a v e chosen to e m p h a s i z e inves t iga t ions of wild p l an t s , s ince t he t e m p e r a t u r e r e sponses of the i r lipids might m o r e closely r e f l e c t t he cons t r a in t s of n a t u r a l s e l ec t ion ope ra t i ng in t h e n a t i v e e n v i r o n m e n t . P l an t s n a t i v e to D e a t h Valley and o the r a r e a s of the Mojave D e s e r t have been used for much of this s tudy . This region is a t e m p e r a t e d e s e r t wi th subs t an t i a l seasonal t e m p e r a t u r e f luc tua t ions ( the m e a n daily max imum and min imum a r e 4 5 C / 3 1 C in July and 18C/3C in J anua ry ) . Several annuals , cons idered to be p r imar i ly cool - or w a r m -season a c t i v e , w e r e i nves t i ga t ed . Whenever possible , p l an t s w e r e grown from seed a t the s a m e t e m p e r a t u r e , to allow us to focus on g e n e t i c , r a t h e r than env i ronmenta l ly - induced , d i f f e rences in lipid physical p r o p e r t i e s .

II. MATERIALS AND METHODS

The t e m p e r a t u r e condi t ions during growth of e a c h spec ies a r e ind ica ted in the t a b l e s . For p l an t s grown from seed , con t ro l l ed env i ronmen t c h a m b e r s w e r e used. Within t h e groups of g rasses and d i co t s , c h a m b e r - g r o w n p lan t s r e c e i v e d iden t i ca l t e m p e r a t u r e and l ight r e g i m e s ; chil l ing sens i t iv i ty (as assessed by growth) is dependen t on t h e s e f ac to r s (2). A few p lan t s w e r e c o l l e c t e d in D e a t h Valley; the mean daily max imum and min imum t e m p e r a t u r e s for t h e mon ths in which t h e co l l ec t ions w e r e m a d e a r e i nd i ca t ed be low.

Approx ima te ly 5 gm of leaf t i s sue was e x t r a c t e d for 5 min in 50 ml of boiling m e t h a n o l con ta in ing 1 mg of of b u t y l a t e d hydroxy to luene . Then 100 ml of chloroform was added, t h e t i ssue was ground in a Vir-Tis homogen ize r , and t h e e x t r a c t was f i l t e red through m i r a c l o t h . The e x t r a c t was p a r t i t i o n e d (6) 4 t i m e s wi th 0.55 Μ KC1, once wi th w a t e r , and once wi th 0.06 Μ KC1. The ch loroform layer was dr ied over Na^SO^, c o n c e n t r a t e d , and loaded on to a Bio-Sil A (Bio-Rad Labora tor ies ) column which was sequent ia l ly e lu t ed wi th ch loroform, a c e t o n e , and m e t h a n o l . The phosphol ip id-r ich me thano l f rac t ion was used in the f luo rescence s tud ie s . The p r e s e n c e of subs t an t i a l a m o u n t s of p i g m e n t s in t h e polar lipid f rac t ion (galactol ipids plus phospholipids , the me thano l e l u a t e ob t a ined following washes wi th chloroform and 10% a c e t o n e in chloroform) i n t e r f e r e d wi th the f luo rescence m e a s u r e m e n t s , so the phospholipid f rac t ion was used. Al iquots w e r e dr ied on to t he walls of a glass vial under N^, held in a vacuum d e s i c c a t o r , and then d ispersed by gen t l e son ica t ion in 0.1M Tr is -HCl buffer , pH 7.2, conta in ing 0.005M N a ?

EDTA. For f l uo re scence s tud ies , 400 μ g of lipid ves ic les was added to

buffer con ta in ing e i t he r 2 5 % or 3 3 % e thy l ene glycol (a c o n c e n t r a t i o n wi thout e f fec t on phase s e p a r a t i o n t e m p e r a t u r e s ) . I rans -pa r ina r i c a c id (0.7 p g in ethanol) was added and the sample s t i r r e d for 15 min a t room t e m p e r a t u r e The f l uo r ime te r (Perk in-Elmer MPF-3L) con ta ined a

3 0 8 C. S. P i k e et al

t e m p e r a t u r e - r e g u l a t e d c u v e t t e holder and a m a g n e t i c s t i r r e r ; t e m p e r a t u r e s we re measu red wi th a c o p p e r - c o n s t a n t a n t h e r m o c o u p l e . The exc i t a t i on beam (3Z0 nm) was passed through a polar iz ing pr ism (Karl L a m b r e c h t and Co.); the e m i t t e d l ight was passed through a p l a s t i c po la r i ze r (Edmund Scientif ic) and a 350 nm cut-off f i l te r , and t h e m o n o c h r o m a t o r was se t a t 4Z0 n m . All t e m p e r a t u r e scans w e r e m a d e in t he ascending d i rec t ion ; t h e r e w e r e a t l eas t 2 r e p l i c a t e s of e ach m e a s u r e m e n t . Phase t r ans i t ions of r e p l i c a t e samples usual ly did no t differ by m o r e than 1C d e g r e e .

III. PHASE SEPARATION PROCESSES

We will first examine d a t a from corn (Zea mays) as a gene ra l i l lu s t r a t ion . F igure 2 shows a plot of t he f luo rescence in t ens i ty m e a s u r e m e n t s and F igure 3, t he po la r i za t ion r a t i o . The change in slope of t h e in t ens i ty curve occurs a t 9C, in good a g r e e m e n t wi th ESR d e t e r m i n a t i o n s (3). Likewise , t he sharp d i scont inu i ty in t he po la r i za t ion r a t i o occurs a t 10C; in f ac t , in all p r e p a r a t i o n s s tudied the two p rocedu re s i nd i ca t e changes within about 1C d e g r e e of one an o th e r . The abrupt change in in tens i ty is a s soc ia t ed wi th a change in f luidity, and the f luidity change is more speci f ica l ly shown by the po la r i za t ion r a t i o . Al though we have con t inued the m e a s u r e m e n t s to 50C« we have not d e t e c t e d t he change usually seen around 25-35C wi th some ESR probes (14, 16).

At t e m p e r a t u r e s above the po la r i za t ion r a t i o s lope change , t h e low r a t i o i nd i ca t e s a fluid probe env i ronment (25). Analysis of model s y s t e m s has shown tha t this p robe is sens i t ive to t he a p p e a r a n c e of a few p e r c e n t solid (25). Thus we i n t e r p r e t t he slope change a t 9C in corn as t he beginning of the a p p e a r a n c e of d e t e c t a b l e solid as the t e m p e r a t u r e is lowered This i n t e r p r e t a t i o n is suppor ted by s tud ies in which the phase sepa ra t ion p rocess of Anacystis nidldans m e m b r a n e s was observed using f r e e z e - f r a c t u r e e l ec t ron microscopy and the p r o p e r t i e s of e x t r a c t e d phospholipids we re s tudied wi th trans -par inar ic ac id (1, 15). A s imi lar co r r e l a t i on is seen in s tudies of E. coli (7, 26). The idea t h a t chil l ing sens i t iv i ty c o r r e l a t e s wi th the f irst a p p e a r a n c e of solid lipid as the t e m p e r a t u r e is lowered is in c o n t r a s t to previous ESR work which sugges ted t ha t this point was the end of sol id i f icat ion (16). Tha t a ss ignment had been m a d e by analogy to model s y s t e m s (28). At p r e sen t we do not know wha t sor t of physica l change is d e t e c t e d by c e r t a i n ESR probes a t 25-35C; in model syst ems Sklar et al. (25) found t h a t some s t r u c t u r a l o rder may pers i s t 10C deg rees or m o r e above the t rans i t ion t e m p e r a t u r e .

In F igure 3 t he level l ing off of t he po la r i za t ion r a t i o of t he corn lipids a t about 2.0 (seen a t about -5C) does no t i nd i ca t e t h e comple t ion of t h e sol idif icat ion p roces s . In model sys tem s tudies Sklar et al. (25) showed t ha t levell ing-off o c c u r r e d a t about 50% solid; thus , t he trans

F l u o r e s c e n c e Polar iza t ion S t u d i e s 3 0 9

Temperature, °C 50 4 0 30 20 10 0 -10

ι 1 1 1 1 1 1 1 1 1 1 1 1 ι

ό a 9' ο d

Barley

32 3 3 3 4 35 36 37 38 39

l / T X I04, °K"'

FIGURE 2. Logarithmic plots of trans-parinaric acid fluorescence intensity of corn and barley phospholipid vesicles. The phospholipids were dispersed by gentle sonication in 0.1M Tris-HCl buffer, pH 7.2, containing 5 mM Na2 EDTA and 25% or 33% (V/V) ethylene glycol. A suspension containing 400\xg lipid per 3 ml of buffer was labeled with 0.7

μ g of trans-parinaric acid. Polarized light at 320 nm was used to excite the sample and fluorescence was measured at 420 nm, with the excitation polarizer parallel to the emission polarizer.

i somer is not su i t ab le to d e t e c t t he comple t ion of sol id i f ica t ion. The CIS i somer would be a b e t t e r i nd ica to r of t h a t po in t , which may no t occur in corn phospholipids unt i l below - 2 0 C .

The i n t ens i t y (Fig. 2) and po l a r i za t i on r a t i o (Fig. 3) p lo t s for ba r l ey (Hordeum vulgare) phospholipids a r e marked ly d i f fe ren t from t h e corn p lo t s . T h e r e a r e r a t h e r sl ight d i scon t inu i t i e s a t about - 6 C . The po l a r i za t i on r a t i o shows t h a t even below t h a t poin t t he lipids a r e st i l l no t very o rde red , c o m p a r e d to t he r a t i o found for corn . We have c o m p a r e d two l egume crops , ch i l l ing-sens i t ive beans (Phaseolus vulgaris) and ch i l l ing- res i s t an t peas (Pisum sativum), and found the phase s epa ra t i ons a t 9C and - 4 C , r e s p e c t i v e l y .


2.2

.2 2.0 V

O o o 0 0

1.2 - 2 0 -10 0 10 2 0 3 0 4 0 5 0

T e m p e r a t u r e , °C

FIGURE 3. Trans-parinaric acid fluorescence polarization of corn and barley phospholipid vesicles. Fluorescence emission was measured with the polarizer parallel (h .) and perpendicular (7. ) to the orientation of the excitation polarizer. The polarization raiio is J ι ι /I ι.

IV. PHASE SEPARATION TEMPERATURES IN WARM AND COOL CLIMATE PLANTS

A. Grasses

Table I shows t h e s epa ra t i on t e m p e r a t u r e s for seve ra l grasses genera l ly v iewed as cool - and w a r m - t e m p e r a t u r e a c t i v e . The warm t e m p e r a t u r e spec ies show sepa ra t ions seve ra l deg rees h igher than the c o o l - t e m p e r a t u r e p l an t s . In t he case of t h e crop p l an t s , t he s epa ra t i on t e m p e r a t u r e s c o r r e l a t e wi th observa t ions on chill ing sens i t iv i ty (corn) and r e s i s t a n c e (barley, oa t s ) .

B. Desert Dicots

Mojave D e s e r t p lan ts , chiefly annuals , w e r e s e l e c t e d to exempli fy t he win te r and s u m m e r - a c t i v e f lora (10, 22). As shown in Table Π, t he w i n t e r - a c t i v e spec ies exhib i ted phospholipid phase s epa ra t i ons a t 3C or less . These p lan t s would seem well a d a p t e d to t he typ ica l t e m p e r a t u r e r e g i m e in the D e a t h Valley a r ea , w h e r e t e m p e r a t u r e s a r e r a r e ly below OC (the m e a n daily minimum t e m p e r a t u r e in t he cooles t mon th is 3C). The s u m m e r - a c t i v e spec ies p r e s e n t a somewha t m o r e complex p a t t e r n . Ti-

F l u o r e s c e n c e Pola r iza t ion S t u d i e s 3 1 1

c

>

ω υ c ο (Ο ω Ι Ο 13

Grown a t 2 0

Grown a t 4 5 °

_ι L 6 0 5 0 4 0 3 0 2 0

Temperature, °C

FIGURE 4. Change in the relative intensity of chlorophyll fluorescence in whole, attached Nerium oleander leaves as a function of temperature. Leaves from plants grown at the indicated temperatures were heated at about 1C degree/min. The fluorescence from excitation by extremely low intensity light was measured continuously. The temperature of the fluorescence increase was determined as the point of intersection of the lines extending the two linear portions of the curves as shown. From Raison et al. (15). Reprinted by permission of John Wiley and Sons, Inc.

destromia oblongifolia is a w i n t e r - d o r m a n t pe renn ia l which shows op t ima l g rowth a t very high t e m p e r a t u r e s (about 45C/3ZC day /n igh t ) . In a 16C/11C r e g i m e , t h e r e is no n e t g rowth a l though the p l an t s a r e not ki l led (3). P l a n t s grown from seed a t 4 5 C / 3 2 C showed a phase s epa ra t i on a t 12C (cf. corn , beans , and Boerhaavia coccinea). Also, some leaves w e r e c o l l e c t e d in D e a t h Valley in D e c e m b e r , when t h e r e was probably l i t t l e n e t g rowth . The phase s e p a r a t i o n o c c u r r e d a t 7C; t h e p l an t s w e r e rou t ine ly exper ienc ing m u c h lower t e m p e r a t u r e s . Typical ly , Tidestromia loses i t s l eaves in w in t e r . The low s e p a r a t i o n t e m p e r a t u r e for Atriplex elegans elegans is an excep t ion , and may i n d i c a t e t h a t i t is no t n e c e s s a r y for a s u m m e r - a c t i v e spec ies t o h a v e a high phase s e p a r a t i o n t e m p e r a t u r e . Al though th is p lan t is p a r t of t h e s u m m e r f lora in t h e


TABLE I. Phospholipid Phas^ Separation Temperatures for Annual Grasses

Species Separation temperature^

Cool Climate

Avena fatua -9 Avena sativa -11 Bromus rigidus -10 Hordeum vulgare -6

Warm Climate

Chloris virgata 4 Digitaria sanguinalis 8 Panicum texanum 7 Zea mays 9

aAll plants were grown at constant 27C and a 16 hr

photoperiod. Determined as the average value from trans-parinaric

acid fluorescence intensity and polarization ratio plots of data from 2 samples.

Mojave D e s e r t , i t can also be found during t h e win te r (22). I ts phase sepa ra t ion behavior may r e f l e c t a g e n e t i c a d a p t a t i o n to the cooles t condi t ions t he p lant may expe r i ence . We have not observed any w i n t e r -a c t i v e p lan t s wi th high phase sepa ra t ion t e m p e r a t u r e s , so we suggest t h a t t h e r e is a d i rec t re la t ionsh ip b e t w e e n the o c c u r r e n c e of low t e m p e r a t u r e and n a t u r a l se lec t ion for lipids wi th a low phase s epa ra t ion t e m p e r a t u r e .

Some D e a t h Valley p lan t s a r e a c t i v e through much of t h e y e a r . We inves t i ga t ed t he phospholipids from 4 such spec ies c o l l e c t e d in J a n u a r y . In all cases the s epa ra t ion o c c u r r e d a t 1C or be low. Thus, t h e r e s e e m s to be a s t rong co r r e l a t i on b e t w e e n t h e abi l i ty of a p lan t (e i ther an e p h e m e r a l or a perennial) to grow a t low t e m p e r a t u r e s and t h e possession of a low phase s epa ra t ion t e m p e r a t u r e . The de se r t spec ies we have s tud ied co occur , y e t in genera l , n a t u r a l s e l ec t ion has appa ren t ly y ie lded lipid composi t ions wi th qu i t e d i f fe ren t phase s epa ra t i on t e m p e r a t u r e s in p lan t s with d i f fe ren t seasonal p r e f e r e n c e s .

A p lan t wi th a r e l a t i ve ly high phase t r ans i t ion t e m p e r a t u r e s e e m s r e s t r i c t e d from growth a t chil l ing or f reez ing t e m p e r a t u r e s . In t he case of wild spec ies , we base this s t a t e m e n t on t h e known seasonal a c t i v i t y of t h e p l an t s For crop p lan t s (such as corn and beans) , d i r ec t in fo rmat ion

F l u o r e s c e n c e Po la r iza t ion S t u d i e s 3 1 3

TABLE II. Phospholipid Phase Separation Temperatures for Desert Dicots

Species Growth0

Separation* conditions temperature C

Cool Climate

Boerhaavia annulata DV 19C/3C 2 Camissonia claviformis DV 22C/6C -3 Cryptantha angustifolia L 28C/21C 2 Eriogonum inflatum DV 22C/6C -4 Lepidium lasiocarpum L 28C/21C -1 Perityle emoryi L 28C/21C 3

Warm Climate

Atriplex elegans elegans L 28C/21C -1 Boerhaavia coccinea L 28C/21C 12 Pectis papposa L 28C/21C 13 Tidestromia oblongifolia L 45C/32C 12

DV 19C/3C 7

Active throughout the year

Atriplex hymenelytra DV 18C/3C None detectable to -IOC

Heliotropium curassavicum DV 18C/3C -2 Larrea divaricata DV 18C/3C -6 Psathyrotes ramosissima DV 18C/3C 1

For laboratory-grown (L) plants, the growth chamber day and night temperatures are given (16 hr photoperiod). For plants collected in Death Valley (DV)f the mean daily maximum and minimum temperatures for the month of collection are given.

Determined as in Table I.

on growth responses is ava i l ab le . Al though the p rec i se r ea son for this r e s t r i c t i o n is no t ye t e s tab l i shed , our s tud ies suggest t ha t t he complex of p h e n o m e n a known as chil l ing injury (9) r e l a t e s to t h e a p p e a r a n c e of solid phase l ipid. A p lan t growing in warm condi t ions need not have a high phase s e p a r a t i o n t e m p e r a t u r e ; we do not know how common the Atriplex elegans elegans s i t ua t i on might b e .

3 1 4 C. S. P i k e et al.

It is i m p o r t a n t to r e a l i z e t h a t f luorescen t p robe analys is , l ike o the r biophysical t echn iques , can only provide an a v e r a g e m e a s u r e of the bulk lipid p rope r t i e s (20). If some subf rac t ion of t h e l ipids, such as p ro t e in boundary layers (27), is c r i t i c a l to chil l ing sens i t iv i ty or r e s i s t a n c e , then these s tudies would no t speci f ica l ly i n v e s t i g a t e i t s p r o p e r t i e s . However , our r e su l t s and o t h e r s (11) provide a t l eas t qua l i t a t i ve a g r e e m e n t b e t w e e n bulk p r o p e r t i e s and p lan t t e m p e r a t u r e p r e f e r e n c e .

IV. ADAPTATION TO HIGH TEMPERATURE

Many of t he cold c l i m a t e p l an t s we h a v e s tud ied a r e unable to grow a t high t e m p e r a t u r e . The f luorescence m e a s u r e m e n t s do not i nd i ca t e any change in lipid physica l p r o p e r t i e s above t h e phase s e p a r a t i o n t e m p e r a t u r e as t he basis for this l im i t a t i on . Studies conduc t ed on Ne-rium oleander, a e v e r g r e e n capab le of a c c l i m a t i n g to a wide r a n g e of t e m p e r a t u r e s , p rov ide in fo rmat ion on t h e r e l a t i on of lipid p r o p e r t i e s and a c t i v i t y a t high t e m p e r a t u r e .

The a c c l i m a t i o n of c loned o leander p l an t s to g rowth in c h a m b e r s a t 4 5 C / 3 2 C (day/night) and 20C/15C is i nd ica t ed by the d i f f e rences in p h o t o s y n t h e t i c t e m p e r a t u r e op t ima : b e t w e e n 35 and 40C for t he w a r m -grown p lan t s and b e t w e e n 25 and 30C for the coo l -g rown. Also, the onse t of r eve r s ib l e t h e r m a l inhibi t ion of pho tosyn thes i s is a t 47 and 40C, r e s p e c t i v e l y (4). At any t e m p e r a t u r e b e t w e e n 14C (the lowest s tudied) and 40C, t h e p h o t o s y n t h e t i c r a t e of t h e cool-grown p l an t s exceeds t h a t of wa rm-g rown p l a n t s . Lipid phase s epa ra t i on t e m p e r a t u r e s of about 7C and -3C for w a r m - and cool-grown p lan t s w e r e d e t e r m i n e d wi th trans-pa r ina r i c ac id . Does this change in lipid p r o p e r t i e s seen a t low t e m p e r a t u r e have any r e l a t i on to t h e d i f fe rences in p h o t o s y n t h e t i c funct ion a t high t e m p e r a t u r e ?

The i n t a c t n e s s of t h e chloroplas t m e m b r a n e s can be r e v e a l e d by examining t h e f luorescence yield of chlorophyl l . In i n t a c t , undamaged l ame l l ae , chlorophyll f luo rescence is highly quenched by exc i t a t i on energy t r ans fe r to t he r e a c t i o n c e n t e r s . An i n c r e a s e in F chlorophyll f luo rescence sugges ts a d isrupt ion of this t r ans fe r (18). M e a s u r e m e n t s on i n t a c t o leander l eaves from w a r m - and cool-grown p lan t s showed an i nc r ea se in f luo rescence a t 53C and 43C, r e s p e c t i v e l y (Figure 4), t e m p e r a t u r e s which a r e qu i t e c lose to t h e point of i r r evers ib le inhibi t ion of whole- leaf pho tosyn thes i s (4). This resu l t sugges t s a 10C d e g r e e d i f f e rence in m e m b r a n e t h e r m a l s t ab i l i ty as a resu l t of d i f f e rences in g rowth t e m p e r a t u r e (15).

Chloroplas t m e m b r a n e polar lipid ves ic les from these p l a n t s w e r e s tud ied to see if t h e r e is a re la t ionsh ip b e t w e e n lipid physical p r o p e r t i e s and m e m b r a n e t h e r m a l s t ab i l i t y . F igure 5 shows t h e t e m p e r a t u r e dependence of t h e mot ion (expressed as r o t a t i o n a l t ime) of t h e spin label m e m b r a n e probe 12NS [3-oxazol idenyloxy-2-(10- ca rbme thoxydecy l ) -2 hexyl -4 , 4 - d i m e t h y l ] . The mot ion d e c r e a s e s as t e m p e r a t u r e i n c r e a s e s . The re a r e no changes in t h e t e m p e r a t u r e coef f ic ien t of mot ion over t he


FIGURE 5. The motion of a spin label incorporated into the polar lipids of chloroplast membranes from Nerium oleander as a function of temperature. Plants were grown at either 45C/32C day/night temperatures or 20C/15C. The lipids were suspended as described for Figure 2 and labeled with 12NS[3-oxazolidenyl-2-( 10 carbmethoxydecyl)-2-hexyl-4,4-dimethyl]. Motion was calculated as described by Raison and Chapman (16) from 5 spectra at each^foemperature. The standard ^fiviation for motion at 20C was + 0.2 χ 10 sec and at SOC, + 0.05 χ 10

sec for both samples.

r a n g e 20C to 50C, showing t h a t t h e r e is no change in t h e molecu la r o rder ing of t h e lipids in t h e reg ion m o n i t o r e d by this p robe (15). The s a m e conclusion was r e a c h e d using trans p a r i na r i c ac id . The lipids from both p l an t s showed a change in lipid o rde r wi th t h e p robe 5N10 a t about 3 0 C

At any given t e m p e r a t u r e t he mot ion of 12NS in t he polar lipids from t h e cool grown o leander is f a s t e r than the mot ion in the lipids from the w a r m - g r o w n p l a n t . Thus, s ince lipid v iscos i ty is r e l a t e d to spin label mot ion (8), t h e lipids from t h e cool -grown p lan t a r e m o r e fluid than those from t h e w a r m - g r o w n p l a n t . If m e m b r a n e t h e r m a l s t ab i l i t y is r e l a t e d to m e m b r a n e lipid v iscos i ty , then t h e i n c r e a s e in chlorophyl l f luorescence should occur a t a t e m p e r a t u r e which cor responds to t h e s a m e viscos i ty in t h e two lipid s amples . This is indeed the case (Fig. Jj)^ for lipids from a p lan t grown a t 20C/15C t h e mot ion a t 43C is 8.7 χ 10 sec a n ^ f o r lipids from a p lan t grown a t 4 5 C / 3 2 C t h e mot ion a t 53C is 8.8 χ 10 sec (15). We conc lude t h a t t h e t h e r m a l s t ab i l i t y of t h e s e m e m b r a n e s is r e l a t e d to t h e phys ica l p r o p e r t i e s of the i r l ipids.


The o leander e x p e r i m e n t s suggest t h a t a c c l i m a t i o n to widely d i f fe ren t g rowth t e m p e r a t u r e s r e su l t s in a t l eas t two changes in the m e m b r a n e l ipids. F i r s t , t h e r e is a shift of about IOC deg rees in t h e low t e m p e r a t u r e phase s epa ra t i on po in t . We have no t explored the chil l ing responses of o leander to see if t h e r e is a cor responding shift in t he sens i t iv i ty of any physiological funct ion . Second, the threshold for t h e r m a l d a m a g e shif ts by about IOC deg ree s , and this loss of funct ional i n t eg r i t y occurs a t a common lipid v iscos i ty , a l though t h e r e is no accompany ing d i scont inu i ty in the t e m p e r a t u r e d e p e n d e n c e of spin label mot ion . Studies of o the r w ide ly - to l e r an t spec ies should i nd i ca t e if t hese two se t s of changes genera l ly occur t o g e t h e r , which would suggest a common basis in a changed lipid compos i t ion .

V. CONCLUSIONS

F l u o r e s c e n c e in t ens i ty and po la r i za t ion m e a s u r e m e n t s wi th trans-pa r ina r i c acid a r e convenien t for inves t iga t ing p lan t phospholipid physical p r o p e r t i e s . Because t h e p robe p a r t i t i o n s p r e f e r e n t i a l l y in to solid phase lipid, i t can r e v e a l ve ry sharply t he f irs t a p p e a r a n c e of solid. If the physica l p r o p e r t i e s of lipids a r e of i n t e r e s t , they should be m e a s u r e d d i rec t ly , s ince an analysis of t h e f a t t y acid compos i t ion is not a lways a sure indica t ion of changes in phys ica l p r o p e r t i e s (14). The u t i l i ty of this p robe in surveys of p lan t lipids should be explored fu r the r .

Based on our work wi th trans -par inar ic ac id t h e phase s e p a r a t i o n seen a t about IOC in var ious typ ica l ch i l l ing-sens i t ive p l an t s appea r s to r e p r e s e n t the beginning of t h e a p p e a r a n c e of o r d e r e d (gel-phase) l ipids, a p rocess which is no t c o m p l e t e d unt i l well below OC. These p roces ses a r e shi f ted to lower t e m p e r a t u r e s in c o o l - c l i m a t e spec ies , including typ ica l ch i l l ing- res i s tan t p l a n t s . This proposed ass ignment of o rde r -d i so rde r t r ans i t ions ought to be ver i f ied by o the r t echn iques .

Our survey r e su l t s to d a t e suggest t ha t g rowth a t low t e m p e r a t u r e is a c c o m p a n i e d by (and may require) a low phospholipid phase s epa ra t ion t e m p e r a t u r e . Growth a t high t e m p e r a t u r e need not a lways r e q u i r e a higher phase sepa ra t ion t e m p e r a t u r e . F u r t h e r inves t iga t ions of highly a d a p t a b l e p lan t s under warm and cool condi t ions a r e in p rog res s .

The o rde r -d i so rde r t r ans i t ion ex tends over many deg ree s , b e c a u s e p lant m e m b r a n e s a r e complex m i x t u r e s . Our r e su l t s sugges t t h a t p l an t s normal ly growing in a c e r t a in env i ronmen t syn thes i ze a m i x t u r e of m e m b r a n e phospholipids which will not phase s e p a r a t e in the usual t h e r m a l r e g i m e .

It is not known what p r o p e r t y of t h e m e m b r a n e lipids is a c t e d on by n a t u r a l s e l ec t ion . P r e sen t t echn iques can only s tudy the bulk lipid p r o p e r t i e s . The re is no ev idence to d e t e r m i n e if t e m p e r a t u r e p r e f e r e n c e depends on the m a i n t e n a n c e of a p roper f luidity in the bulk phospholipids or if t h e bulk phase behavior is a chance r e f l ec t i on of some o the r p r o p e r t i e s of some cruc ia l f rac t ion . Indeed, d i f fe ren t f rac t ions might se t t he lower l imit for g rowth in d i f fe ren t p l an t s or a t d i f fe ren t


d e v e l o p m e n t a l s t ages in a given p l an t . If t h e phase s epa ra t i on s e t s a rough lower l imi t for n o r m a l funct ion, then it mus t be d e t e r m i n e d what physiological p roces ses a r e i ncompa t ib l e wi th t he so l id i f ica t ion . The impl i ca t ion t ha t changes in a c t i v a t i o n energy of e n z y m e s or g rowth a r e t h e resu l t of lipid phase changes (13, 16) has been ques t ioned (2). The rma l h a b i t a t p r e f e r e n c e in Passiflora c o r r e l a t e d wi th t h e loss of m e m b r a n e i n t eg r i t y as m e a s u r e d by ion l e akage (11) and wi th lipid p h a s e - c h a n g e t e m p e r a t u r e (12). However , no c o m p a r a b l e re la t ionsh ip ex i s t ed b e t w e e n chil l ing sens i t iv i ty or r e s i s t a n c e of t hese spec ies and the point of changes in t e m p e r a t u r e d e p e n d e n c e of t h e in vitro Hill r e a c t i o n (5). O t h e r examples of this lack of co r r e l a t i on a r e also c i t e d (5). This assay did c o r r e l a t e well wi th chil l ing behavior in seve ra l c rop p l an t s (21). Aside from seasona l i ty , we h a v e no physiological d a t a on the p l an t s s tud ied . It may be t h a t t h e r e is no t one single p roces s t h a t s e t s t h e lower l imit of g rowth for all spec i e s . A p lan t ' s t e m p e r a t u r e p r e f e r e n c e , which i n t e g r a t e s t he t h e r m a l p r o p e r t i e s of i t s var ious m e t a b o l i c s y s t e m s , may be a useful p a r a m e t e r for compar i son to lipid p r o p e r t i e s .

ACKNOWLEDGMENTS

We thank R. D . Simoni for t he pa r ina r i c acid, and M. Nobs and J . Ehle inger for seeds used in these s tud ies .


1. Armond, Ρ Α., and Staehel in , L. A. Proc. Nat. Acad. Sci. USA 76, 1901-1905 (1979).

2. Bagnal l , D . J , and Wolfe, J . A. J. Exp. Bot. 112, 1231-1242 (1978).

3 . Bjorkman, O. , Mahal l , B. , Nobs M., Ward, W., Nicholson, F . , and Mooney, H. Carnegie Inst. Wash. Year Book 73, 757-767 (1974).

4 . Bjorkman, O. , Badger , Μ , and Armond P . A. Carnegie Inst. Wash. Year Book 77, 262-276 (1978).

5. C r i t c h l e y , C , Smill ie , R . M., and P a t t e r s o n , B. D. Aust. J. Plant Physiol. 5, 443-448 (1978).

6 Fo lch , J . , Lees , M., and S loane-Stan ley , G. H. J. Biol. Chem. 226, 497 509 (1957).

7. G r a n t , C . W. M., Wu, S. H., and McConnel l , Η Μ. Biochim. Biophys. Acta 363, 151-158 (1974).

8. Ke i th , A. D. , and Snipes W. In "Magne t ic R e s o n a n c e in Colloid and I n t e r f a c e Science" (H. A. Res ing and C. G. Wade> eds.) , A m e r i c a n C h e m i c a l Socie ty , Columbus , Ohio (1976).

9. Lyons, J . M. Annu. Rev. Plant Physiol. 24, 445-466 (1973). 10. Mulroy, T. W., and Rundel , P . W. Bio Science 27, 109-114(1977) .


11. P a t t e r s o n , B. D. , Mura t a , T., and G r a h a m , D. Aus t . J . P l an t P h y siol . 3, 435-442 (1976).

12. P a t t e r s o n , B. D. , Kenr ick , J . R. , and Raison , J . K. Phytochemis-try 17, 1089-1092 (1978).

13. Raison, J . K., R. Soc New Zeal. Bulletin 12, 487-497 (1974). 14. Raison J . K., In "Biochemis t ry of P lan t s" Vol. 7 (P. K. Stumpf,

ed.) , A c a d e m i c P re s s , New York (1979). 15. Raison J . K., Berry , J . Α., Armond, P . Α., and P ike , C S In "A-

d a p t a t i o n s of P l an t s to Wate r and High T e m p e r a t u r e St ress" (P. K r a m e r and N. Turner eds.) , Wi ley- In te r sc ience , New York (1980).

16. Raison, J . K., and Chapman , E. A. Aust. J. Plant Physiol. 3, 2 9 1 -299 (1976).

17. Schroeder , F . , Holland, J . F . , and Vagelos, P . R. J. Biol. Chem. 251, 6739-6746 (1976).

18. Schre iber , U., and Berry , J . Planta 136, 233-238 (1977). 19. Shimshick E. J . , and McConnel l , Η. M. Proc. Nat. Acad. Sci. USA

71, 4653 4657 20. Shini tzky, M., and Barenholz , Y Biochim. Biophys. Acta 515,

367-394 (1978). 2 1 . Shneyour, Α., Raison , J . K., and Smill ie , R M. Biochim. Biophys.

Acta 292, 152-161 (1973). 22. Shreve, F . , and Wiggins, I. L. "Vege ta t ion and F lo ra of t h e Sonoran

Dese r t . " Stanford Univers i ty , Stanford, CA (1964). 23 . Sklar, L. Α., Hudson, B. S., and Simoni, R. D. Proc. Nat. Acad.

Sci. USA 72, 1649-1653 (1975). 24. Sklar, L. Α., Hudson, B. S., and Simoni, R D Biochemistry 16,

819-829 (1977). 25 Sklar, L. Α., Miljanich, G. P . , and D r a t z E. A. Biochemistry 18,

1707-1716 (1979). 26 T e c o m a , E. S , Sklar, L. Α., Simoni, R. D. , and Hudson, B. S.

Biochemistry, 16, 829-835 (1977). 27. Wolfe, J . Plant, Cell, and Environment 1, 241-247 (1978). 28. Wu, S. Η , and McConnel l , Η. M. Biochemistry 14, 847-854 (1975).


DIFFERENTIAL THERMAL ANALYSIS OF TOMATO MITOCHONDRIAL LIPIDS

Adam W. Dalziel and R. W. Breidenbach

Plan t Growth L a b o r a t o r y Univers i ty of Cal i forn ia


I. INTRODUCTION

Cons ide rab le ev idence now suppor t s t h e t heo ry t h a t t h e physical p r o p e r t i e s of biological m e m b r a n e s of chil l ing sens i t ive spec ies a r e a l t e r e d a t t h e c r i t i c a l t e m p e r a t u r e t h a t causes physiological d a m a g e (Lyons, 6). Studies have also i n d i c a t e d t h a t e x t r a c t e d m e m b r a n e lipids c losely r e s e m b l e i n t a c t m e m b r a n e s in physica l c h a r a c t e r i s t i c s (13, 12, 1, 2, 9, 8). This s tudy conce rns t he t h e r m o t r o p i c p r o p e r t i e s of mi tochondr i a l lipids from two e c o t y p e s of t h e t o m a t o Lycopersicon hirsutum.


The two e c o t y p e s used o r ig ina t ed from South A m e r i c a as p a r t of a co l l ec t ion m a d e by C . E. Vallejos. One was c o l l e c t e d in Ecuador a t an a l t i t u d e of 50 m e t e r s (Ecotype I), and t h e o t h e r (Ecotype Π) in Pe ru a t 3000 m e t e r s . The two e c o t y p e s differ cons iderably in the i r chil l ing sens i t iv i ty , Vallejos, th is vo lume (15).

We grew t h e t o m a t o p l an t s hydroponica l ly in a f if ty p e r c e n t modif ied Johnson n u t r i e n t so lu t ion (5) in a g reenhouse a t Davis . A v e r a g e m a x i m u m and min imum t e m p e r a t u r e s w e r e 21 .5°C and 18°C . Dai ly g rowth t e m p e r a t u r e s r a n g e d b e t w e e n 30 C and 16 C . Af t e r about 100 days t h e roo t s w e r e h a r v e s t e d and washed wi th dis t i l led w a t e r .

The roo t s w e r e chopped in to 1-cm s e g m e n t s and homogen ized in a Waring b lender (two 10-second bur s t s ) . The homogeniz ing buffer con t a ined 0.25 Μ suc rose , 50 mM po tass ium phospha t e , 5 mM EDTA ( e t h y l e n e d i a m e n e t e t r a a c e t i c ac id) , 0 .2% soluble PVP (polyvinylpyrooli-done) , 0 . 1 % f a t t y ac id poor bovine se rum a lbumin, and 5mM β- m e r -c a p t o e t h a n o l . The c rude h o m o g e n a t e was f i l t e red through four l aye r s of c h e e s e c l o t h and cen t r i fuged a t 750 χ g for 10 m i n u t e s . The s u p e r n a t a n t

Copyright · 1Q79 by Academic Press. Inc. 3 1 9 All rights of reproduction in any form reserved

ISBN 012-46056O5

3 2 0 A. W. Dalziel a n d R. W. Breidenbach

was cen t r i fuged a t 12,000 χ g for 15 m inu te s , and the c rude mi tochondr ia l pe l l e t was resuspended in the grinding medium (minus EDTA and PVP). This suspension was cen t r i fuged a t 12,000 χ g to wash t h e mi tochondr i a , and the pe l le t was suspended in a min imal vo lume of buffer . The pu r i ty of mi tochondr ia l p r e p a r a t i o n s was checked by e l e c t r o n mic roscopy which showed the p r e s e n c e of a smal l amount of c o n t a m i n a t i o n from endoplasmic r e t i c u l u m .

The mi tochondr ia l suspension was t r a n s f e r r e d to 50 ml of boiling isopropanol in o rder to i n a c t i v a t e any res idua l phosphol ipase a c t i v i t y . As wi th all t h e s t eps involved in lipid e x t r a c t i o n , t h e s ample was kep t under a n i t rogen a t m o s p h e r e to p r e v e n t ox ida t ive a l t e r a t i o n . Af te r boil ing for 5 minu tes t he sample was a l lowed to cool be fo re adding 400 ml ch lo ro fo rm.me thano l (2:1). The r e s t of the e x t r a c t i o n was by the me thod of Folch e t al (1957). The e x t r a c t was r e d u c e d to a smal l vo lume of chloroform in vacuo (35 C), and a sample was t r a n s f e r r e d to p rewe ighed a luminum cruc ib les . The r ema in ing chloroform was r e m o v e d , and t h e sample was dr ied for 12 hr over Ρ^Ος

ιη vacuo. The

sample was weighed and 20 μΐ of 50% e t hy l ene glycol buffer a t pH 7.2 was added (50% 2 mM HEPES, 10 mM NaCl , 0.1 mM EDTA; 50% e t hy l ene glycol) . When a pH 9 buffer was used it con t a ined 25 mM sodium b o r a t e i n s t ead of 2 mM HEPES. The sample was sea led h e r m e t i c a l l y in a n i t rogen a t m o s p h e r e and a l lowed to h y d r a t e overn ight a t room t e m p e r a t u r e .

A M e t t l e r TA 2000 was used to c a r r y out d i f fe ren t ia l t h e r m a l analysis (DTA) on these samples , with 50% e t hy l ene glycol as a r e f e r e n c e . The samples w e r e first p recond i t i oned by hea t i ng and cooling t h r e e t i m e s over t he t e m p e r a t u r e r ange of i n t e r e s t . Each scan a t a r a t e of 5 / m i n was r e p e a t e d a t l ea s t two t i m e s .

C a l o r i m e t r i c d i f fe ren t ia l t h e r m a l analysis d i f fers from convent iona l DTA in t h a t t h e signal observed a t t he d i f fe ren t ia l t he rmocoup le s is no longer i n t e r p r e t e d p r imar i ly as a t e m p e r a t u r e d i f fe rence , but r a t h e r , as a consequence of varying h e a t flows to t h e sample and r e f e r e n c e s ides .

Ts Sample t e m p e r a t u r e Tr R e f e r e n c e t e m p e r a t u r e Δ V Di f fe ren t ia l t h e r m o v o l t a g e o

S Sensi t iv i ty of measur ing sensor (S = 100 μ V/ C) R H e a t r e s i s t a n c e b e t w e e n the fu rnace wall and the c ruc ib le Ε C a l o r i m e t r i c sens i t iv i ty (E = R.S)

dH dt

Ts - Tr R

A V / S AV R Ε

T h e r m a l A n a l y s i s of T o m a t o Mi tochondr i a l L ip ids 3 2 1

ΙΠ. RESULTS AND DISCUSSION

F igure 1 shows a se r ies of t h e r m o g r a m s ob ta ined on mi tochondr ia l lipids e x t r a c t e d from Eco type I. The base l ine t h e r m o g r a m was ob ta ined by scanning over the r a n g e of i n t e r e s t wi thou t any sample pans in t he sample ho lder . The con t ro l s ample was rapidly cooled from 90 to -50 , w h e r e it was a l lowed to equ i l ib ra t e for 15 minu t e s . The t h e r m o g r a m s begin a t -33 .5 C and show t h e sample being h e a t e d to 90 C. A t h e r m o v o l t a g e of 2 y V cor responds to a h e a t flow of 0.15 mW. Compar ing t h e r e s u l t s wi th t h e base l ine r evea l s a broad e n d o t h e r m i c t r ans i t i on beginning around -3Ζ C . The t r ans i t ion may well begin below -32 C, bu t wi th t h e buffer used i t is no t possible to i n v e s t i g a t e this reg ion . The end point of this " t rans i t ion cannot be a c c u r a t e l y d e t e r m i n e d from a h e a t i n g scan , b e c a u s e of over lapping con t r ibu t ions from t h e e n d o t h e r m i c p roces s and the signal t i m e cons t an t for t h e i n s t r u m e n t . Even so, it can be e s t i m a t e d to l ie b e t w e e n 35 and 45 C. The shape of t h e t r ans i t ion i nd i ca t ed t h a t i t might be b iphas ic , wi th two b road over lapping t r ans i t i ons .

We found t h a t t h e shape of this e n d o t h e r m i c t r ans i t ion depends on t h e t h e r m a l h i s to ry of t h e s a m p l e . F igure 1 shows t h e e f fec t of incuba t ing th is s ample a t 10 C for 12 hr . In this c a s e , t he sample was rapid ly cooled from 90 C to 10 C, i ncuba t ed i so the rma l ly for 12 hr , and then rapidly cooled to -50 C, w h e r e it was a l lowed to equ i l ib ra te for 15 min . One can now see two pa r t i a l l y reso lved e n d o t h e r m i c t r ans i t i ons . The lower t ransi t ion^ again , begins around -32 C, and the upper one appea r s to begin a t 16 C . Af t e r h ea t i n g t h e sample to 90 , the con t ro l p r o c e d u r e was r e p e a t e d and a t h e r m o g r a m s imi lar to the con t ro l was ob ta ined . This i nd i ca t e s t h a t t he e f fec t of incuba t ing t h e s ample a t 10 C is c o m p l e t e l y r e v e r s i b l e . We then dec ided to t e s t t he e f f ec t of incuba t ing the sample a t o the r t e m p e r a t u r e s . This is d e m o n s t r a t e d by t h e th i rd t h e r m o g r a m (Fig. 1), which shows t h e e f fec t of incuba t ing t h e sample a t 20°C for 12 hr . The onse t of th is second t r ans i t i on had now shi f ted to 22.5 C . The d e p e n d e n c e of th is t r ans i t ion on the incubat ion t e m p e r a t u r e sugges ted tha t a phase s e p a r a t i o n may be occur r ing . It is possible t ha t lipids having a me l t ing point above the incubat ion t e m p e r a t u r e may s e g r e g a t e in to a s e p a r a t e pool and thus m e l t independen t ly from t h e r e m a i n d e r to t h e lipids p r e s e n t .

We then a t t e m p t e d to c h a r a c t e r i z e t h e k ine t i c s of this p roces s . The samples w e r e i n c u b a t e d a t 10 C for var ious per iods be fo re being rapidly cooled to -50 C . F igu re 2 shows r e su l t s ob ta ined wi th 5-m i n u t e , 30 -minu te , and 12-hr incuba t ions . It is c l ea r t h a t th is e f fec t occur s on a r e l a t i ve ly slow t i m e sca l e , t ak ing severa l hours to c o m p l e t e . C o m p a r e d wi th t h e types of phase s epa ra t i ons d e t e c t e d wi th s y n t h e t i c phospholipids, th is p roces s appea r s e x t r e m e l y slow (4, 8, 14).

3 2 2 A. W. Dalziel a n d R. W. Breidenbach

I ι 1 1 1 1 L _

- 4 0 - 2 0 0 20 4 0 6 0 8 0 TEMPERATURE (°C)

FIGURE 1. The effect of incubation temperature on the thermotropic properties of mitochondrial lipids isolated from Lycopersicon hirsutum f. glabratum (Ecotype I). The sample contained 11.7 mg of lipid and 20 μ Ζ 50% ethylene glycol buffer. The sample was either cooled rapidly to -50 C (control) or incubated at 10°C or 20°C for 12 hrs prior to cooling. The 2 μ V sccle corresponds to a heat flow of 0.15 mW.

I n t e r a c t i o n s among phospholipid polar head groups offers one possible exp lana t ion for this behaviour . P lan t lipids usually con ta in subs tan t i a l a m o u n t s of phospha t idy le thano lamine . This phospholipid r e s e m b l e s phospha t idy l se r ine in t ha t i n t e r a c t i o n s a r e possible b e t w e e n ad jacen t polar head groups when t h e amino group is p r o t o n a t e d ( -CH-CH^N H^). Also l ike phospha t idy l se r ine , it is less h y d r a t e d than phospha t iay lchol ine , so t ha t any a g g r e g a t e s fo rmed would probably p roduce anhydrous s t a c k s . Since such i n t e r a c t i o n s a r e possible only when t h e amino group is p r o t o n a t e d , we dec ided to t e s t t he e f fec t of pH on this s y s t e m .

Samples of mi tochondr ia l lipids from a second e c o t y p e of Lycopersicon hirsutum (Ecotype Π) w e r e p r e p a r e d a t two d i f fe ren t pH va lues . A de t a i l ed compar i son of t h e t h e r m o t r o p i c p r o p e r t i e s of these two

T h e r m a l A n a l y s i s of T o m a t o Mi tochondr i a l L ip ids 3 2 3

- 4 0 - 2 0 0 20 4 0 60 8 0 TEMPERATURE ( ° C )

FIGURE 2. The effect of incubation time on the thermotropic properties of mitochondrial lipids isolated from Lycopersicon hirsutum f. glabratum (Ecotype I). The sample contained 11.7 mg of lipid and 20 μ 1 50% ethylene glycol buffer. The sample was incubated at 10°C for 0 min (control) 5 min; 30 min; or 12 hrs before rapidly cooling to -50°C. 2 \iV Ξ 0.15 mW.

e c o t y p e s will be publ ished e l s e w h e r e . The d i f fe rences b e t w e e n Eco type I and Π shown in F igu re 1 and F igure 3 con t ro l s a r e la rge ly a c c o u n t e d for by d i f f e rences in sample s i ze . We h a v e m a d e no a t t e m p t to e s t i m a t e t he en tha lpy of the b road t r ans i t ions d e t e c t e d , because of the diff icul ty of drawing an a c c u r a t e base l ine for such b road t r ans i t i ons . ο

By incuba t ing a pH 7.2 con t ro l s ample a t 10 C for 10.5 hr , two pa r t i a l l y reso lved e n d o t h e r m i c t r ans i t ions were again d e t e c t e d . The second of t he se had an onse t t e m p e r a t u r e of about 15 C . Both t he pH 9.0 con t ro l and t h e pH 9.0 sample i n c u b a t e d a t 10 C for 10.5 hr had t h e r m a l c h a r a c t e r i s t i c s s imi lar to those of the cor responding samples a t pH 7.2.

3 2 4 A. W. Dalziel a n d R. W. B r e i d e n b a c h

l 1 1 1 1 __lJ

- 4 0 - 2 0 0 20 4 0 6 0 TEMPERATURE (

eC )

FIGURE 3. The effect of pH on the thermotropic properties of mitochondrial lipids isolated from Lycopersicon hirsutum (Ecotype II). The sample contained 5.0 mg of lipid and 20 μ I 50% ethylene glycol buffer^pH 7.2 or pH 9 (see text). TheQsamples were either cooled rapidly to -50 C (control) or incubated at 10 C for 10.5 hrs prior to cooling. 2 μ V Ξ 0.15 mW.

This leads us to be l i eve t ha t if a phase s epa ra t i on is in fac t tak ing p l a c e , it is not due p r imar i ly to t he phospha t idy le thano lamine polar head group.

Since p lant phospholipids a r e highly u n s a t u r a t e d , it is somewha t surpr is ing tha t a por t ion of the lipids discussed in this s tudy mel t a t r e l a t i ve ly high t e m p e r a t u r e s . It is possible t h a t d i s a t u r a t e d phospholipids a r e p re sen t in these p r e p a r a t i o n s , s imi lar to r e su l t s of Miljanich (11). A l t e rna t i ve ly , the phospholipids may be i n t e r a c t i n g wi th o the r lipid componen t s , a l t e r i ng the phase t r ans i t ion t e m p e r a t u r e (10). F u r t h e r s tudies will be requ i red to d e t e r m i n e which lipid componen t s a r e con t r ibu t ing to t h e observed t r ans i t ions .

In conclusion, we have used d i f fe ren t ia l t h e r m a l analysis to d e t e c t a b road e n d o t h e r m i c t r ans i t ion in mi tochondr ia l lipids from Lycopersicon hirsutum. We have found tha t the t h e r m o t r o p i c p r o p e r t i e s of t he se lipids a r e a f f e c t e d by incuba t ing t h e sample a t t e m p e r a t u r e s within t he r ange of t he e n d o t h e r m i c t r ans i t ion . Final ly , we w e r e unable to ob ta in

T h e r m a l A n a l y s i s of T o m a t o Mi tochondr i a l L ip id s 3 2 5

ev idence t h a t t h e phospha t idy l e thano lamine polar head group plays a s ignif icant ro l e in inf luencing the e f f ec t of incuba t ing the sample a t l i r e .

ACKNOWLEDGMENT

We would l ike to thank M e t t l e r I n s t r u m e n t Corpora t ion for the use of the ΤΑ 2000 t h e r m a l analys is s y s t e m . We would also l ike to thank Dr . R. Jones and E. C r u m m for the i r he lp wi th t h e e l e c t r o n mic roscopy .


1. Ashe, G. B., S te im, J . M. Biochim. Biophys. Acta 233, 810-814 (1971).

2. Blazyk, J . F . , S te im, J . M. Biochim. Biophys. Acta 266, 737-741 (1972).

3 . Fo lch , J . H., Lees , M., S loane-Stan ley , G. J. Biol. Chem. 226, 4 9 7 -509 (1957).

4 . Jacobson , K., Papahadjopoulos , D . Biochemistry 14, 152-161 (1975). 5. Eps te in . E. Mineral Nu t r i t i on of P l a n t s : Pr inc ip les and P e r s p e c t i v e s .

J . Wiley & Son, Inc . , C h a p t e r 3 . , 38-39 (1972). 6. Lyons, J . M. Ann. Rev. Plant Physiol. 24, 445 466 (1973). 7. Mabrey , S., Powis , G., Schenkman, J . B. , T r i t t on , T. R J. Biol.

Chem. 252, 2929-2933 (1977). 8. Mabrey , S., S t u r t e v a n t , J . M. Proc. Natl. Acad. Sci. USA 73, 3862-

3866 (1976). 9. Melchior , D . L. , S te im, J . M. Ann. Rev. Biophys. Bioeng. 5, 205-

238 (1976). 10. McKers i e , B. D . , Thompson, J . E. Biochim. Biophys. Acta 550, 4 8 -

58 (1979). 11 . Miljanich, G P . , Sklar, L. Α., Whi te , D . L., D r a t z , E. A. Biochim.

Biophys. Acta 552, 294-306 (1979). 12. R e i n e r t , J . C , S te im, J . M. Science 168, 1580-1582 (1970). 13 . S t e im , J . M., T o u r t e l l o t t e , Μ. E., R e i n e r t , J . C , McElhaney , R. N. ,

R a d e r , R. L. Biochemistry 63, 104-109 (1969). 14. Van Dijck, Ρ W. M., Kaper , A. J . , Oonk, H. A. J . , De Gier , J . Bio

chim. Biophys. Acta 470, 58-69 (1977). 15. Vallejos. C, E. This vo lume .



SOME PHYSICAL PROPERTIES O F MEMBRANES IN THE PHASE SEPARATION REGION AND THEIR

RELATION TO CHILLING DAMAGE IN PLANTS

Joe Wolfe0

D e p a r t m e n t of Applied M a t h e m a t i c s I n s t i t u t e of Advanced Studies

Aus t ra l i an Na t iona l Univers i ty C a n b e r r a , A C T 2601 Aus t r a l i a

The f in i te t e m p e r a t u r e r a n g e of b iological a c t i v i t y canno t easi ly be expla ined by s imple c h e m i c a l k ine t i c s . The inf luence of the f luidi ty of m e m b r a n e lipids has been i m p l i c a t e d in e n z y m e a c t i v i t y , and this pape r c o m p a r e s some models for this in f luence . The a c t i v i t y of l iving th ings , and t h e r a t e s of many biological r e a c t i o n s , have a r e m a r k a b l e t e m p e r a t u r e d e p e n d e n c e : they a r e nea r ly all t o t a l l y i nac t i ve below about 260-270 K, and above about 310-320 Κ (8). A va r i a t ion of only ΙΟΙ 5% in the t e m p e r a t u r e spans a r a n g e in which the r a t e s of a lmos t all b iological p roces ses i nc rease from ze ro to a f in i te va lue , r e m a i n cons t an t wi thin a few o rde r s of magn i tude and then r e v e r t to z e r o . This is r e m a r k a b l e b e c a u s e of t h e s t a t i s t i c a l d i s t r ibu t ion of ene rgy among p o t e n t i a l r e a c t a n t mo lecu le s : jus t below the observed min imum t e m p e r a t u r e some of t h e s u b s t r a t e molecu les should, one might e x p e c t , h a v e enough ene rgy to r e a c t . C lea r ly some th ing much m o r e c o m p l i c a t e d than t h e r e a c t i o n k ine t i c s of inorganic c h e m i s t r y is involved (see F ig . 1).

How can one explain such an enormous va r i a t ion in r e a c t i o n r a t e over such a smal l t e m p e r a t u r e ? Let ' s cons ider some poss ib i l i t ies :

Enzyme c o o p e r a t i v i t y canno t be t h e answer ; such a sys tem is much m o r e sens i t ive to s u b s t r a t e c o n c e n t r a t i o n than to t e m p e r a t u r e , and observed t e m p e r a t u r e dependences a r e no t nea r ly l a rge enough.

The d i f fe ren t i a l t e m p e r a t u r e d e p e n d e n c e of c o m p e t i n g r e a c t i o n s could p roduce a s t rong t e m p e r a t u r e d e p e n d e n c e in some c o m p l i c a t e d p roces s such as a g rowth r a t e . Chlorophyl l p roduc t ion in e t i o l a t e d p l an t s ,

Present address: Department of Agronomy, Cornell University, Ithaca, NY 14853.

Copyright β 1Q79 by Academic Press, Inc. 3 2 7 All rights of reproduction in any form reserved

ISBN CM 2 4 6 0 5 6 0 5

3 2 8 J. Wolfe

for example , i nc reases f a s t e r with t e m p e r a t u r e than i t s p h o t o -des t ruc t i on , and so t h e r e is some threshhold t e m p e r a t u r e above which a p lan t can p roduce chlorophyll , "green up" and grow, and below which it d ies . This threshhold t e m p e r a t u r e will , of course , depend on i r r ad i ance (2, 17). However , t h e chlorophyll p roduc t ion r a t e i tself va r ies very quickly wi th t e m p e r a t u r e and very l a rge i r r ad i ance changes a r e n e e d e d to a l t e r t he c r i t i c a l survival t e m p e r a t u r e by 5° or m o r e . So the "compe t ing r e a c t i o n s " app roach doesn ' t solve t he bas ic p rob lem, s ince individual r e a c t i o n s exhibi t t he s a m e "on or off" response to t e m p e r a t u r e .

Sharpe and De Michel le (21) addressed the problem by ex tending the model of Johnson et al. (9). They assign to the enzyme low t e m p e r a t u r e i nac t i ve , medium t e m p e r a t u r e a c t i v e , and high t e m p e r a t u r e i nac t i ve s t a t e s in to which enzyme molecu les p a r t i t i o n accord ing to Bo l t zmann s t a t i s t i c s . This is p i c tu r ed in F igure 2, w h e r e the η and nm equiva len t s t a t e s a t e ach energy level a ccoun t for t he "en t ropy of ac t i va t i on" n e c e s s a r y to p roduce the l a rge t e m p e r a t u r e d e p e n d e n c e . Unfo r tuna t e ly to p roduce t he observed behaviour bo th t h e number s of equiva len t s t a t e s , and the r e a c t i o n t i m e cons t an t s , mus t be (li terally) a s t ronomica l ly high; in fac t t r ans i t ions b e t w e e n these s t a t e s would neve r happen .

FIGURE 1. A comparison of the rates of reaction for a typical chemical reaction in the low density limit (solid line) and for the Hill reaction in mung bean. [Data from (18).]

M e m b r a n e s in t h e P h a s e S e p a r a t i o n R e g i o n 3 2 9

mx n equivalen t inactiv e s t a t e s

Τ . - J η equivalen t activ e s t a t e s

inactiv e s tat e

FIGURE 2. The model of Sharpe and deMichelle (21) requires the enzyme to portion in energy states as shown.

Membrane Lipid Fluidity

The t r ans i t ions b e t w e e n d i f fe ren t physica l phases of subs t ances a r e of ten abrupt and this sugges ted to Lyons and Raison (14) t h e possibi l i ty t h a t t he physica l s t a t e (solid or fluid) of t he lipids of cel l and organe l le m e m b r a n e s d e t e r m i n e d the a c t i v i t y of t he e n z y m e s embedded the re in , jus t as t he a c t i v i t y of a fish would depend on t h e physical phase of t h e surrounding w a t e r . Many enzymes a r e r e n d e r e d i nac t i ve , or less a c t i v e , by t h e r e m o v a l of the i r lipid env i ronmen t [e.g. J acobs et al. (1) ] , or by i t s r e p l a c e m e n t by s t ronge r s u r f a c t a n t s and this m a d e t h e hypothes is even m o r e r e a s o n a b l e . They and o the r r e s e a r c h e r s h a v e d i scovered t h a t many spec ies h a v e m e m b r a n e lipids of such a compos i t ion t ha t they f r e e z e over a r a n g e of t e m p e r a t u r e s cor responding roughly to t he t e m p e r a t u r e s in which the spec ies l ives (13). T h e r e is also ev idence t h a t t h e adap t ion to low or high t e m p e r a t u r e s by an organism involves inc reas ing or dec reas ing r e s p e c t i v e l y t h e f rac t ion of low mel t ing point lipids (6, 11 , 19, 20, 23), though o t h e r worke r s find t h a t this doesn ' t happen in some spec ies (4, 22). On the whole , t h e co inc idence of t h e r a n g e of t e m p e r a t u r e s in which the m e m b r a n e lipids of an organism might be e x p e c t e d to phase s e p a r a t e and the r a n g e of t e m p e r a t u r e in which it l ives s t rongly sugges t s t h a t t he physica l p r o p e r t i e s of t h e lipids in f luence biological a c t i v i t y . Severa l m e c h a n i s m s which could m e d i a t e such an in f luence h a v e been desc r ibed by Wolfe (24).

3 3 0 J. Wolfe

Croz ie r (3) had ea r l i e r proposed t ha t t he changing slope in Arrhenius p lo ts of biological r e a c t i o n s was due to the t r ans i t ion b e t w e e n r a n g e s of t e m p e r a t u r e in which d i f fe ren t r e a c t i o n s w e r e r a t e - l i m i t i n g , but this theory had diff icul ty in producing suff ic ient ly rap id changes from one r e g i m e to ano the r [see e.g. discussion in Johnson et al. (8)] . Lyons and Raison (14) we re able to o v e r c o m e this p roblem by proposing t h a t a c e r t a i n r a t e - l i m i t i n g r e a c t i o n had one a c t i v a t i o n energy when i t s enzyme was in a fluid m e m b r a n e and ano the r (higher) a c t i v a t i o n energy in t h e solid m e m b r a n e . Thus below some t e m p e r a t u r e (T ) an Arrhenius plot exhib i t s some cons t an t s lope, above ano the r t e m p e r a t u r e (T^) ano the r cons t an t s lope, and b e t w e e n the two (when the lipids phase sepa ra t e ) t he r e a c t i o n r a t e is t he sum of t he r a t e s of t he r e a c t i o n s occur r ing in t he d i f fe ren t phases (see Fig. 3). This approach , however , is necessa r i ly l imi t ed to descr ib ing only t he c e n t r a l p a r t of a typ ica l Arrhenius p lo t . Since Arrhenius

1 Law cannot reasonab ly be appl ied when the slope is

pos i t ive (corresponding to a n e g a t i v e a c t i v a t i o n energy) or n e g a t i v e and very l a rge (if t h e a c t i v a t i o n energy w e r e 100 k ^ T then the t i m e s c a l e of t he r e a c t i o n would be a s t ronomica l ) , some o the r approach must be employed to explain t he i nac t i v i t y a t high and low t e m p e r a t u r e s .

An a l t e r n a t i v e , s impler form of t h e genera l lipid f lu id i ty -enzyme a c t i v i t y theory p o s t u l a t e s an enzyme which can only form an a c t i v a t e d complex in a fluid lipid env i ronment (See F ig . 4) . Above some t e m p e r a t u r e T^ all t h e enzyme molecu les a r e a c t i v e and t h e r e a c t i o n follows Arrhenius ' Law. Below this t e m p e r a t u r e progress ive ly m o r e enzyme molecu les find themse lves in solid domains , and so t h e r e a c t i o n r a t e d e c r e a s e s m o r e rapidly wi th t e m p e r a t u r e . A p a r a m e t e r of such a model would be t he energy r equ i r ed to move an enzyme molecu le from a fluid to a solid domain [i .e. t h e d i f fe rence in solubil i ty in the two phases , as observed by Duppel and Dahl (5)] . Such a model explains the low t e m p e r a t u r e behaviour (and so is pe rhaps of i n t e r e s t to this conference) but not the r eve r s ib le or i r r evers ib le high t e m p e r a t u r e i nac t iva t ion , and so we must look e l s ewhe re .

Bilayer Lateral Compressibility

Linden et al. (12) sugges ted t h a t enzyme a c t i v i t y may r equ i r e a c e r t a i n minimum l a t e r a l compress ib i l i ty of t h e b i layer s ince the conf igura t ion of t he a c t i v a t e d s t a t e may r equ i r e displacing t h e ad jacent lipids a l i t t l e . Now the compress ib i l i ty of a lipid b i layer will be smal l for a solid, a few t imes l a rge r for a liquid, but o rders of magn i tude l a rger in t h e phase s epa ra t ion region (16). Thus t he f in i te t e m p e r a t u r e r ange of a c t i v i t y of an enzyme in a b i layer may be t h a t r ange over which the b i layer may easi ly be compressed in t he p l ane . Such a model p roduces qua l i t a t ive ly s imi lar r e su l t s to the hypothes is t ha t t he boundary layer of an a c t i v e enzyme must be of a l imi t ed r ange of mobi l i ty (25).

It is observed t ha t t r anspor t ac ross b i layers is also much g r e a t e r in t h e phase s epa ra t ion region than in e i the r fluid or solid region, and t h e r e is a s t r ik ing s imi la r i ty b e t w e e n the form of t h e t e m p e r a t u r e dependence


\ AO 2 0 0 (°

FIGURE 3. An Arrhenius plot predicted from the theory of Lyons and Raison (14) and the lipid fluid fraction, X on the same scale. Note that one of the constraints on the middle curved section is that it pass through the intersection of the two straight lines.

of t r a n s p o r t found by Wu and McConnel l (26) and t h e t h e o r e t i c a l compress ib i l i ty of a mixed lipid b i layer found by Marel ja and Wolfe (16). It has also b e e n a rgued t h a t subs tan t i a l t r a n s i e n t f luc tua t ions in t h e lipid a r e a s (and thus high l a t e r a l compress ib i l i ty) a r e r equ i r ed for t r anspo r t ac ross the m e m b r a n e (12).

Thus it can r easonab ly be a rgued t h a t r e a c t i o n s requi r ing e i the r t r anspo r t ac ross a m e m b r a n e , or reconf igur ing of an e n z y m e in a

3 3 2 J. Wolfe

Z>0 20 0(°C) I I I 1 I

s \

3 2 3-4 3-6

1/Τχ103 κ 1

FIGURE 4. An Arrhenius plot predicted from the modified form of the lipid fluidity-enzyme activity theory (see text). The difference in energy of solution is Ox, lx and 2x the thermal energy as indicated.

m e m b r a n e will be g rea t ly f a c i l i t a t e d by (and pe rhaps only possible with) the inc reased compress ib i l i ty in the phase s epa ra t ion r a n g e of t e m p e r a t u r e s . Now for a b inary m i x t u r e of lipids whose " ta i ls" differ only by two carbon a t o m s , the d i f f e rence b e t w e e n t e m p e r a t u r e s a t the e x t r e m e s of t he phase sepa ra t ion is small (' ~ 5 C) , and the r ange of biological a c t i v i t y is l a rger ( ~ 50°C) . A biological m e m b r a n e , however , can be e x p e c t e d to phase s e p a r a t e over a much wider r ange of t e m p e r a t u r e s than such a m i x t u r e . F i r s t it has severa l componen t lipids wi th a wide r ange of me l t ing t e m p e r a t u r e s and second, i t has "boundary layer" lipids a f f e c t e d by t h e p r e s e n c e of in t r ins ic p ro t e in s (10, 15). The hydrophobic region of an enzyme is c e r t a i n to be less o rde red than a f rozen lipid ta i l , and m o r e o rde red than a fluid lipid ta i l , and thus will p r e f e r en t i a l l y adsorb m o r e fluid or less fluid lipids than t h e bulk well below and above (respect ively) t h e e x p e c t e d phase s epa ra t i on r a n g e .


temperature

FIGURE 5. Lateral compressibility of a bilayer equally composed of DMPC and DPPC as a function of temperature, using the theory of Marelja and Wolfe (16) and the data of Albrecht et al. (1).

A q u a n t i t a t i v e analysis of such a sys tem is c u r r e n t l y being p r e p a r e d (Bates , Marel ja and Wolfe, unpublished d a t a ) . Since only a smal l f ree ene rgy change is a s s o c i a t e d wi th t he absorp t ion of boundary lipids over a wide r a n g e of t e m p e r a t u r e s , even in a one lipid componen t sys tem (15), t h e compress ib i l i ty will be high over a t e m p e r a t u r e r a n g e much wider than t ha t over which an u n p e r t u r b e d two lipid componen t b i layer phase s e p a r a t e s .

F rom t h e theory of Marel ja and Wolfe (16) it is possible to c a l c u l a t e t ha t t h e compress ib i l i ty of a b i layer fo rmed from an equal m i x t u r e of d ipa lmi toy l - and d imi r i s toy l -phospha t idy lcho l ine [for which lipids t h e r equ i r ed p a r a m e t e r s a r e well known (1) ] t h e compress ib i l i ty is_about 6 m.N in t h e fluid region, 1.5 m.N~ in t he solid, and about 50 mN in the phase s epa ra t i on (Fig. 5). Suppose t h a t t h e a c t i v a t i o n energy of some r e a c t i o n is Ε + EK w h e r e Ε is the chemica l ene rgy involved in a t t a c h i n g t h e s u b s t r a t e to t he e n z y m e , and E^. is t h e work done compress ing t h e

3 3 4 J. Wolfe

surrounding lipids to allow the fo rma t ion of t h e new conf igura t ion . If E.^ w e r e ~ 1.7 k Τ (1 k cal mole" ) in t he middle of t h e s epa ra t i on r a n g e , w h e r e i t would have l i t t l e e f fec t on t h e r e a c t i o n k ine t i c s , then it would quickly i nc rease to ~ 50 k^T in t h e solid r a n g e , whe re it would e f fec t ive ly " turn off" t h e r e a c t i o n , s ince t he c h a n c e of t h e r m a l e x c i t a t i o n providing t h a t much energy is negl ig ib le . Of course E ^ canno t be simply deduced from t h e slope of an Arrhenius plot s ince it is a funct ion of t e m p e r a t u r e .

I know of no independen t m e a s u r e m e n t s of E ^ so it would have to be an ad jus tab le p a r a m e t e r of the mode l . O t h e r p a r a m e t e r s such as p r o t e i n -lipid i n t e r a c t i o n energy and /or lipid compress ib i l i t i e s a r e also no t known wi th suff ic ient a c c u r a c y , and t h e r e f o r e , as an exe rc i s e in curve f i t t ing , such a model would be doomed to success . However , it is e l s ewhere sugges ted how these o the r p a r a m e t e r s could be independen t ly ob ta ined from e x p e r i m e n t (15, 16, 24), and so t he theory may in pr inc ip le be t e s t e d .

A cor ro la ry of t h e theory is t h a t t he low t e m p e r a t u r e l imi t of a biological r e a c t i o n depends not only on the p ropor t ion of sho r t - cha ined lipids in t he m e m b r a n e , but also on t h e f e a t u r e s of t he hydrophobic reg ions of t he e n z y m e , and how easi ly they adsorb a boundary layer of lipids (24)..

ACKNOWLEDGMENT

During t h e wr i t ing of this pape r I h a v e enjoyed useful discussions with Stjepan Marel ja .

R E F E R E N C E S

1. A lb rech t , O. , Gru le r , H. and Sackmann, E. J. de Physique 39, 3 0 1 -313 (1978).

2. Bagnal l , D . J . This volume (1979). 3 . C roz i e r J. Gen. Physiol. 7, 189-216 (1924). 4 . de la R o c h e , I. A. and Andrews , C. J . PI. Physiol. 51, 468-473

(1973). 5. Duppel , W. and Dahl , G. Biochim. et Biophys. Acta 426, 408-417

(1976). 6. Gerloff, E. D. , Richardson , T. and S tahmann , M. A. PL Physiol.

41, 1280-1284 (1966). 7. J a c o b s , Ε. E., Andrews , E. C , Wohlrab and Cunningham, W. In

"S t ruc tu re and Func t ion of C y t o c h r o m e s " , Univ. of Tokyo Press , Tokyo (1967).

8. Johnson, F . H., Eyring, H. and Pol issar , M. J . "The Kine t i c Basis of Molecular Biology", Wiley, New York (1954).



9. Johnson, F . H., Eyring, H. and Wil l iams, R. W. J. Cell Comp. Physiol. 20, 247-268 (1942).

10. J o s t , P . C , Gr i f f i ths , Ο. H., Capa ld i , R. A. and Vanderkooi , G. Proc. Nat. Acad. Sci. USA 70, 480-484 (1973).

11 . Kuiper , P . J . C . PL Physiol. 45, 684-686 (1970). 12. Linden, D. , Wright , K. L., McConnel l , Η. M. and Fox, C. F . Proc.

Nat. Acad. Sci. USA 70, 2271-2275 (1973). 13. Lyons, J . M. and Asmundsen , C . M. J. Amer. Oil. Chim. Soc. 42,

1056-1058 (1965). 14*. Lyons, J . M. and Raison , J . K. Plant Physiol. 45, 386-389 (1970). 15. Mare l ja , S. Biochim. et Biophys. Acta 455, 1-7 (1976). 16. Mare l ja , S. and Wolfe, J . Biochim. et Biophys. Acta (In press)

(1979). 17. McWill iam, J . R. and Naylor , A. W. PI. Physiol. 39, 262-268

(1967). 18. Nolan, W. G. and Smill ie , R . M. PI. Physiol. 59, 1141-1145 (1977). 19. P a t o n , J . C , McMurchie , Β. K., May, Β. K. and El l io t t , W. H. J.

Bact. 135, 754-759 (1978). 20. Raison , J . K., Berry , J . Α., Armond, P . A. and P ike , C. S. In "Adap

t ion of P l an t s to Wate r and High T e m p e r a t u r e St ress" , Wiley, New York. (In p repa ra t ion ) (1980).

2 1 . Sharpe , P . J . H. and DeMiche l l e , D . W. J. Theor. Biol. 64, 649-670 (1977).

22. S iminovi tch , D. , Singh, J . and de la R o c h e , I. A. Cryobiology 12, 144-153 (1975).

23 . Wil lemot , C . PI. Physiol. 55, 356-359 (1975). 24. Wolfe, J . Plant, Cell and Environment 1, 241-247 (1978). 25 . Wolfe, J . , Ph .D . Thesis , Aus t r a l i an Na t iona l Univers i ty , C a n b e r r a ,

Aus t r a l i a (1979). 26. Wu. S. H. and McConnel l , Η. M. Biochem. and Biophys. Res. Com.

55, 484-491 (1973).


LIPID PHASE OF MEMBRANE AND CHILLING INJURY IN THE BLUE-GREEN ALGA, ANACYSTIS NIDULANS

Norio Murata, Taka-Aki Ono and Naoki Sato

D e p a r t m e n t of Biology Col lege of Gene ra l Educa t ion

Univers i ty of Tokyo K o m a b a , Meguro-ku

Tokyo 153 J a p a n

I. ANACYSTIS NIDULANS

The b lue -g reen algal cel ls a r e composed of two kinds of m e m b r a n e s , the p l a sma m e m b r a n e and the thylakoid m e m b r a n e , but a r e devoid of a d i f f e r e n t i a t e d o rgane l l e of pho tosyn thes i s , t h e ch lo rop las t . The thylakoid m e m b r a n e is the s i t e of the p r i m a r y p rocesses of pho tosyn thes i s including t h e absorp t ion of l ight , and the p h o t o s y n t h e t i c e l e c t r o n t r a n s p o r t and phosphoryla t ion r e a c t i o n s . The p l a sma m e m b r a n e is t h e ba r r i e r b e t w e e n the cy top lasm and the ou t e r medium and also t h e s i t e of a c t i v e t r a n s p o r t .

Al though most b lue -g reen a lgae a r e r e s i s t a n t to chil l ing t r e a t m e n t , Anacystis nidulans is injured when exposed to chil l ing t e m p e r a t u r e s (Z, 6). Moreover , t h e ch i l l ing-sens i t iv i ty depends on t h e g rowth t e m p e r a t u r e (13). This a lga is also c h a r a c t e r i z e d by t h e fac t t h a t it con ta ins only s a t u r a t e d and m o n o u n s a t u r a t e d f a t t y ac ids (4, 14), while mos t o t h e r b lue -g reen a lgae con ta in p o l y u n s a t u r a t e d f a t t y ac ids (1).

We have been s tudying t h e e f f ec t s of t e m p e r a t u r e on pho tosynthes is in A. nidulans, and have r e v e a l e d t h e co r r e l a t i on b e t w e e n t h e physical phase of thylakoid m e m b r a n e lipids and t h e t e m p e r a t u r e d e p e n d e n c e of t h e p h o t o s y n t h e t i c p roces se s . Table I s u m m a r i z e s t he t e m p e r a t u r e d e p e n d e n c e of t h e m e m b r a n e lipid phase^ t h e pho tosynt h e t i c p rocesses and chil l ing injury in cel ls grown a t 38 and Z8 C.

3 3 7 Copyright © 1979 by Academic Press. Inc.

All rights of reproduction in any form reserved ISBN a i 2 4 6 0 5 6 0 5

3 3 8 Ν. M u r a t a et al

TABLE I. Temperatures for Characteristic Points in Cells Grown at 38° and 28°C

Measurement Material Temperatures Ref. for

characteristic points (°C)

Lipid phase transition of thylakoid membranes

Spin label Μ 13 24 (9) Chlorophyll a fluorescence C 13 24 (8) Thermal analysis Μ 14 *

Photosynthetic processes

Photosynthetic evolution //r>0-^ Dichlorophenolindophenol HÔ^ Ferricyanide Pnotophosphorylation Delayed fluorescence Pigment state 1 and 2 shift

c 13 24 (9) Μ 12 21 (12) Μ 7 13 (12) Μ 12 21 (12) C 14 22 (10) C 13 22 (9)

Chilling injury

Potosynthesis C 5 10 Fig. 2 H90++ Benzoquinone C 3 U **

*Ono, Murata and Fujita (unpublished) and Ono and Murata (unpublished). C and Μ correspond to "intact cells" and "thylakoid membranes", respectively. The numbers with and without underlines were obtained in cells grown at 38 and 28 , respectively.

Π. LIPID PHASE OF THYLAKOID MEMBRANE.

Thylakoid m e m b r a n e s we re p r e p a r e d by disrupt ing the l y sozyme-t r e a t e d cel ls by sonic osci l la t ion or F r e n c h p res su re t r e a t m e n t (11). Chemica l analysis ind ica ted tha t t he lipids a m o u n t e d to about 30% of t h e dry weight of t he thylakoid m e m b r a n e s . The physical phase of t he m e m b r a n e lipids was s tudied by EPR spec t ro scopy of a spin label (9), t he f luorescence yield of chlorophyll α (8) and c a l o r i m e t r i c d i f fe ren t ia l

Lipid P h a s e a n d Chil l ing in jury in A. nidulans 3 3 9

t h e r m a l analys is (Ono, M u r a t a and Fuj i ta , unpubl ished d a t a ) . These s tudies i nd i ca t ed t ha t the phase t r ans i t ion b e t w e e n the liquid c rys ta l l ine and the phase s epa ra t i on s t a t e s o c c u r r r e d a t about 24 or 13 C in cel ls grown a t 38 or 28 C, r e s p e c t i v e l y (Table I). The r e su l t s ob ta ined by Verwer et al. (15) by f r e e z e - f r a c t u r e e l e c t r o n m i c r o s c o p y of t h e thylakoid m e m b r a n e is c o m p a t i b l e wi th our f indings.

ΠΙ. TEMPERATURE D E P E N D E N C E OF PHOTOSYNTHESIS AND RELATED PROCESSES

The Arrhenius plot of p h o t o s y n t h e t i c oxygen evolut ion in i n t a c t cel ls a p p r o x i m a t e d two s t r a igh t l ines wi th a b r eak point a t 24 or 13 C in cel ls grown a t 38 or 28 C, r e s p e c t i v e l y (9).

T e m p e r a t u r e d e p e n d e n c e of t h e p h o t o s y n t h e t i c e l e c t r o n t r anspor t and phosphoryla t ion r e a c t i o n s was s tud ied in the thylakoid m e m b r a n e p r e p a r a t i o n (12). An Arrhen ius p lo t of t h e Hill r e a c t i o n wi th dichlorophenol indophenol r e v e a l e d a b r eak point a t 21 or 12 C in t h e m e m b r a n e from cel ls grown a t 38 or 28 C, r e s p e c t i v e l y . The Arrhenius plot of pho tophosphory la t ion m e d i a t e d by N-me thy lphenazon ium m e t h y l s u l f a t e r e v e a l e d a b r eak poin t a t 21° or 12°C in t h e m e m b r a n e from cel ls grown a t 38 or 28 C, r e s p e c t i v e l y .

It is n o t e d t ha t t h e abrupt change in t he a p p a r e n t a c t i v a t i o n energy of these r e a c t i o n s appea red a t the t e m p e r a t u r e reg ion of the lipid phase t r ans i t ion (see Table I). These findings suggest t ha t t h e lipid phase of t h e thylakoid m e m b r a n e has g rea t inf luence on the p h o t o s y n t h e t i c e l e c t r o n t r anspor t and phosphoryla t ion r e a c t i o n s .

The s i t e of p las toqu inone in e l e c t r o n t r an spo r t is mos t l ikely a f f e c t e d by the lipid phase , s ince the r a t e - d e t e r m i n i n g s t e p of e l e c t r o n t r an spo r t from HÔ to sys tem I is l o c a t e d a t t h e oxida t ion r e a c t i o n of r e d u c e d p las toqu inone . Moreover , p las toquinone is cons idered to be dissolved in t he lipid layer of t h e thylakoid m e m b r a n e and thus to be mos t sens i t ive to f luidi ty change in m e m b r a n e l ipids.

The e f fec t of t h e lipid phase on photophosphory la t ion may be bes t expla ined by the fac t t ha t the m e m b r a n e leaks ions when the m e m b r a n e lipids a r e in t h e phase s e p a r a t i o n s t a t e (5). In t h e c h e m i o s m o t i c mechan ism of phosphory la t ion , the g rad ien t of ions ac ross the thylakoid m e m b r a n e is t h e mo t ive fo rce to p roduce ATP (7).

In this r e s p e c t it is wor thwhi le no t i c ing t h e t e m p e r a t u r e dependence of de layed f luo rescence of chlorophyll α (10). F igure 1 shows the t e m p e r a t u r e d e p e n d e n c e of t h e yield of de layed f luo rescence in i n t a c t ce l l s . The change from t h e high to t h e low level of de layed f luorescence o c c u r r e d a t t h e t e m p e r a t u r e reg ion c e n t e r i n g a t 22 or 14 C in cel ls grown a t 38 or 28 C, r e s p e c t i v e l y . It is known tha t t h e de layed f luo rescence is marked ly inf luenced by t h e c o n c e n t r a t i o n of Η a c c u m u l a t e d inside t he thylakoid under i l l umina t ion +(3 ) . Thus, these r e s u l t s sugges t t h a t t h e thylakoid m e m b r a n e leaks Η from inside t he thylakoid when t h e m e m b r a n e lipids a r e in t he phase s epa ra t i on s t a t e .

3 4 0 Ν. M u r a t a et al.

FIGURE 1. Temperature dependence of delayed fluorescencegf chlorophyll a in intact cells of Anacystis nidulans grown at 38 and 28 C (10). The delayed fluorescence emitted at 0.05-1.75 msec after repetitive excitation flashes was measured with a Becquerel-type phosphoroscope. The steady state level of delayed fluorescence after 10 minutes of repetitive illumination was plotted against temperature.

On the o the r hand, t h e Hill r e a c t i o n wi th f e r r i cyan ide in t h e thylakoid m e m b r a n e responded to t e m p e r a t u r e in a d i f fe ren t manne r . The b reak point in t he Arrhenius p lo t appea red a t 13° or 7°C in t h e m e m b r a n e s from cel ls grown a t 38 or 28 C, r e s p e c t i v e l y . These t e m p e r a t u r e s we re much lower than the t e m p e r a t u r e s of lipid phase t r ans i t ion of t h e cor responding thylakoid m e m b r a n e s . It s e e m s r ea sonab le , however , to a s sume tha t t he se poin ts a r e r e l a t e d to the lipid f luidi ty, b e c a u s e they depended on t h e g rowth t e m p e r a t u r e . So far , we do not have any c lea r explana t ion for t hese c h a r a c t e r i s t i c poin ts in the Hill r e a c t i o n wi th f e r r i cyan ide .

IV. CHILLING INJURY

When the cel ls of A. nidulans a r e exposed to chil l ing t e m g e r a t u r e s , they lose p h o t o s y n t h e t i c a c t i v i t i e s . F igure Ζ shows t ha t 10 and 5 C w e r e c r i t i c a l t e m p e r a t u r e s for t h e cel ls grown a t 38 and Z8 C, r e s p e c t i v e l y . The e l e c t r o n t r anspo r t r e a c t i o n m e a s u r e d by t h e Hill r e a c t i o n wi th 1,4-benzoquinone also suf fered chil l ing injury in t h e | a m e t e m p e r a t u r e reg ion . Ano the r e x p e r i m e n t a l r e su l t i nd ica t ed t h a t Κ and Mg leaked from t h e cy top lasm when t h e cel ls w e r e t r e a t e d a t chill ing

Lipid P h a s e a n d Chil l ing in jury inA. nidulans 3 4 1

5°C •

1 / 28*C-grown/

/ / 38*C-grown

/ 1 Ο 10°C

ι ι

0 5 10 15

Temperature , ° C

FIGURE 2. Effect of chilling treatment on activity of the photosynthetic oxygen evolution in intact cells grown at 38 and 28 C. The cells were treated in culture medium at the designated temperatures for 1 Qour in the dark and then the photosynthetic activity was measured at 30 C. The rate of oxygen evolution at the maximum level was 200 or 300 μ moles O^mg chl/hour in cells grown at 38° or 28°C, respectively.

t e m p e r a t u r e s . It is n o t e d t h a t t e m p e r a t u r e s c r i t i c a l for chil l ing injury and ion l eakage w e r e much lower than t e m p e r a t u r e s for t he lipid phase t r ans i t i on of t h e thylakoid m e m b r a n e (Table I). This may sugges t t h a t t he lipid phase of the thylakoid m e m b r a n e is not involved in chil l ing injury of i n t a c t ce l l s . It is p lausible to a s sume t h a t t h e chil l ing injury is r e l a t e d to the lipid phase of the p l a s m a m e m b r a n e , and t ha t chi l l ing injury is induced by t h e l eakage of ions from t h e cy top lasm when the p l a sma m e m b r a n e is in the phase s e p a r a t i o n s t a t e . However , t he t e m p e r a t u r e d e p e n d e n c e of lipid phase in t h e p l a sma m e m b r a n e of A. nidulans has no t been s tud ied .

V. E F F E C T OF GROWTH T E M P E R A T U R E ON LIPID AND FATTY ACID COMPOSITIONS

The g rowth t e m p e r a t u r e - d e p e n d e n c e of t h e phase t r ans i t ion of thylakoid m e m b r a n e lipids led us to s tudy the e f fec t of g rowth t e m p e r a t u r e on t h e lipid and f a t t y ac id compos i t ion . Table Π shows t h a t the g rowth t e m p e r a t u r e did no t s igni f icant ly a f f ec t the r e l a t i v e compos i t ion of var ious lipid c l asses . Al though Sato et al. (14) r e p o r t e d a h igher c o n t e n t of DGDG (digalac tosyldiglycer ide) in cel ls grown a t 38 C, the i r r esu l t might have been p roduced by using t h e cel ls a t t h e l a t e c u l t u r e s t a g e s in which DGDG c o n t e n t was found to be abnormal ly high.

3 4 2 Ν

· M u r a t a et al.

TABLE II. Lipid Composition in Cells Grown at 38° and 28°C

Growth temperature

Lipid 38°C 28°C

MGDG (monogalactosyldiglyceride) 54% 57% DGDG (digalactosyldiglyceride) 14% 11% SQDG (sulfoquinovosyldiglyceride) 11% 11% PG (phosphatidylglycerol) 21% 21%

Cells were cultivated at constant temperatures for more than 7 days. Relative amounts of lipid classes were determined based on fatty acid contents. It is noted that no phosphatidylcholine was present in the blue-green alga.

TABLE III. Positional Distribution of Fatty Acids in the Four Lipid Classes in Cells Grown at 38° and 28 C.

Lipid MGDG DGDG SQDG PG

Growth temperature (°C) 38 28 38 28 38 28 38 28

Posi-tion 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2

14:0 1 1 1 2 1 1 1 1 1 0 1 0 1 0 0 0 14:1 3 0 7 0 4 0 6 0 1 0 3 0 1 0 2 0 16:0 3 45 1 43 3 45 2 41 13 50 5 49 4 49 3 45 16:1 41 2 40 5 40 3 40 7 34 0 41 1 39 1 44 3 18:0 0 2 0 0 0 1 0 0 0 0 0 0 0 0 0 0 18:1 2 0 1 0 2 0 1 1 1 0 0 0 5 0 1 2

Fatty acids bound to 1-position were hydrolyzed by lipase of Rhizopus delemar. The monoacylglyceride produced were purified by thin layer chromatography with silica gel and fatty acids bound to 2-position were analyzed. It is noted that there were no polyunsaturated fatty acids in this alga.

Lipid P h a s e a n d Chil l ing Injury in A. nidulans 3 4 3

Table ΠΙ shows the e f fec t of g rowth t e m p e r a t u r e on the pos i t ional d i s t r ibu t ion of f a t t y ac ids in the four lipid c lasses . Al though the va r i a t ion in f a t t y ac id compos i t ion was complex , t he following conclusions could be drawn when the g rowth t e m p e r a t u r e was lowered from 38° to 28°C; a t 1-position, 16:0++ 14:1 ( r ep l acemen t of 16:0 by 14:1) in MGDG (monogalac tosyld ig lycer ide) and DGDG, 16:0++ 16:1 and 16:0++ 14:1 in SQDG (sulfoquinovosyldiglyceride) and 18:1++ 16:1 in PG (phosphat idylglycerol) ; and a t 2-posi t ion, 16:0++ 16:1 and 18:0++ 16:1 in MGDG, 16:0++ 16:1 in DGDG and 16:0++ 16:1 and 16:0++ 18:1 in PG. The main changes in f a t t y ac ids wi th g rowth t e m p e r a t u r e can be s u m m a r i z e d as follows: t h e d e s a t u r a t i o n and shor ten ing of chain length a t t he 1-position and t h e d e s a t u r a t i o n a t t he 2-posi t ion. These va r i a t ions in f a t t y acid compos i t ion , though r e l a t i v e l y smal l , a r e compa t ib l e wi th t h e g rowth t e m p e r a t u r e d e p e n d e n c e of t h e phase t r ans i t ion t e m p e r a t u r e of m e m b r a n e l ipids, s ince t h e d e s a t u r a t i o n and t h e shor ten ing of chain l eng th in f a t t y acids a r e known to d e c r e a s e t he phase t r ans i t ion t e m p e r a t u r e of m e m b r a n e l ipids.

F igure 3 shows how t h e f a t t y ac id compos i t ion responded a f t e r t h e shift in g rowth t e m p e r a t u r e from 38 to 28 C. A most r e m a r k a b l e change was seen a t the 1-position of SQDG w h e r e 16:0 was c o n v e r t e d to 16:1 within 10 hours . Similar but smal le r changes w e r e found a t t h e 1-posi t ion of MGDG and PG. In addi t ion to this fast change in d e s a t u r a t i o n a t t he 1-position, two slow changes w e r e found to occur in seve ra l t ens of hours : t h e shor ten ing of chain l eng th a t t h e 1-position in all of t he lipid c lasses and t h e d e s a t u r a t i o n (mostly 16:0++ 16:1) a t t h e 2-posi t ion in MGDG, DGDG and PG.

Expe r imen ta l r e su l t s (not shown here) i nd i ca t ed t h a t f a t t y ac id synthes is was r e m a r k a b l y r e p r e s s e d within 10 hours a f t e r t h e shift in g rowth t e m p e r a t u r e . This sugges t s t h a t 16:0 bound to t h e 1-position of SQDG, MGDG and PG is d e s a t u r a t e d wi thout de novo syn thes i s of f a t t y ac ids .

IV. SUMMARY

The re la t ionsh ip of t h e lipid phase of m e m b r a n e s to t h e t e m p e r a t u r e d e p e n d e n c e of p h o t o s y n t h e t i c p rocesses and chil l ing injury was s tud ied in t h e b lue -g reen a lga Anacystis nidulans t h a t was grown a t d i f f e ren t t e m p e r a t u r e s . E x p e r i m e n t a l r e s u l t s of spin label , chlorophyll α f luores c e n c e and c a l o r i m e t r i c d i f f e ren t i a l t h e r m a l analys is i n d i c a t e d t h a t t h e t r ans i t ion of lipid phase of thylakoid m e m b r a n e b e t w e e n t h e liquid c rys t a l l i ne and t h e nhase s e p a r a t i o n s t a t e s o c c u r r e d a t about 24 or 13 C in cel ls grown a t 38 or 28 C, r e s p e c t i v e l y . Ar rhen ius p lo t s of e l e c t r o n t r a n s p o r t and phosphory la t ion r e a c t i o n s r e v e a l e d t h a t a b r eak point a p p e a r e d in t h e t e m p e r a t u r e reg ion of t h e lipid phase t r ans i t ion of t h e thylakoid m e m b r a n e . On t h e o t h e r hand, chil l ing injury of pho tosyn thes i s o c c u r r e d below 10 or 5 C in cel ls grown a t 38 or 28 C, r e s p e c t i v e l y . The lipid phase of p l a s m a m e m b r a n e s might be involved in t h e chil l ing

3 4 4 Ν. M u r a t a et al.

so 5 0 -

M G DG - ι—ι—ι—ι—ι—ι—ι—r-

1 6 : 1

S Q D G

1 6 : 1

c CO

i8:

0-

5 0

1 6 . 0 ' p * • — — =4-

1 6 : 0

14. 1

1 6 : 0

Γ18Ι1-

CM c co 16 M

— L" D G D G

oy r - i ih-so 5 0^ 16. 1

co 0

1 0 0

5 0

CM

C co

i 6 : o I 1411 · ,

0 4 0

1 6 . 1 ^

' ' //

1 6 : 1

Ρ G

1 6 : ι

1 6 1 0

1 6 : 1

8 0 / t_ " 2 00 (hr)

4 0

28"C 3 ^

8 0 20 0 (hr)

28 °C

FIGURE 3. Time course of fatty acid contents at two positions in lipid classes after the shift in growth temperature from 38° to 28°C. Before the shift of temperature, the cells were cultivated at 38°C for more than 7 days.

injury. It was found t h a t K+ and M g

2 + l eaked out from t h e cy top lasm a t

t h e t e m p e r a t u r e reg ion of chill ing injury. The algal cel ls responded to t h e g rowth t e m p e r a t u r e by varying the

f a t t y acid compos i t ion . When t h e cel ls grown a t 28 C w e r e c o m p a r e d wi th those grown a t 38 C, t h e d e s a t u r a t i o n was higher a t t h e 1- and 2-posi t ions and the chain length was sho r t e r a t t he 1-posit ion of t he g lycerol mo ie ty of l ipids. The va r i a t ion in f a t t y acid composi t ion a f t e r a

Lipid P h a s e a n d Chil l ing Injury in A. nidulans 3 4 5

rap id shift of g rowth t e m p e r a t u r e was composed of a fast change (desa tu ra t ion a t t h e 1-position) and two slow changes (desa tu ra t ion a t the 2-posi t ion and shor ten ing of chain l eng th a t t h e 1-position). These a l t e r n a t i o n s in f a t t y ac id compos i t ion a r e c o m p a t i b l e wi th the g rowth t e m p e r a t u r e d e p e n d e n c e of t h e t r ans i t i on t e m p e r a t u r e of lipid phase in t he thylakoid m e m b r a n e , s ince t he d e s a t u r a t i o n and the shor ten ing of chain l eng th in f a t t y ac ids a r e known to i n c r e a s e t h e f luidi ty and thus d e c r e a s e t h e t r ans i t ion t e m p e r a t u r e of lipid phase of m e m b r a n e .


1. Erwin, J . A. In "Lipids and B iomembranes of Eukaryo t i c Microorganisms ." (J. A. Erwin, ed.) , pp . 4 1 - 1 4 3 . A c a d e m i c P r e s s , New York (1973).

2. F o r r e s t , H. S., van Baalen , C . and Myers , J . Science 125, 699-700 (1957).

3 . H a v e m a n , J . and Lavore l , J . Biochim. Biophys. Acta 408, 269-283 (1975).

4 . H i r a y a m a , O. J. Biochem. 61, 179-185 (1967). 5. Inoue, K. Biochim. Biophys. Acta 339, 390-402 (1974). 6. J a n s z , E. R. and Maclean , F . I. Can. J. Microbiol. 19, 381-387

(1972). 7. Mi tche l l , P . Ann. Rev. Biochem. 46, 996-1005 (1977). 8. Mura t a , N. and Fork , D. C . Plant Physiol. 56, 791-796 (1975). 9. Mura t a , N. , Troughton , J . H. and Fork , D. C. Plant Physiol. 56,

508-517 (1975). 10. Ono, T. and Mura t a , N . Biochim. Biophys. Acta 460, 220-229

(1977). 11 . Ono, T. and Mura t a , N . Biochim. Biophys. Acta 502, 477-485

(1978). 12. Ono, T. and Mura t a , N. Biochim. Biophys. Acta 545, 69-76 (1979). 13. R a o , V. S. K., Brand, J . and Myers , J . Plant Physiol. 59, 965-969

(1977). 14. Sa to , N. , Mura t a , N. , Miura, Y. and U e t a , N . Biochim. Biophys.

Acta 572, 19-28 (1979). 15. Verwer , W., V e r v e r g a e r t , P . H. J . T., Leunissen-Bi jve l t , J . and

Verklei j , A. J . Biochim. Biophys. Acta 504, 231-234 (1978).


MOLECULAR CONTROL OF MEMBRANE FLUIDITY

Guy A. Thompson, Jr.

D e p a r t m e n t of Botany The Univers i ty of Texas

Aust in , Texas

It is ve ry c l ea r from t h e casual obse rva t ions of l aymen as well as t h e work of s c i en t i s t s t ha t chil l ing t e m p e r a t u r e s h a v e a de l e t e r i ous e f fec t on many p lan t spec i e s . Ye t desp i t e vas t losses of c rop p l an t s due to chill d a m a g e , we sti l l have l i t t l e under s t and ing of why one spec ies can easi ly survive low t e m p e r a t u r e s while ano the r canno t .

One p r o p e r t y of t h e p lan t cel l known to be a f f e c t e d by low t e m p e r a t u r e is t h e physica l s t a t e of i t s m e m b r a n e s . Ear l i e r p a p e r s in this symposium h a v e discussed t h e concep t of " m e m b r a n e fluidity" and desc r ibed t h e l ipid-l ipid and l ip id-pro te in i n t e r a c t i o n s t h a t a f f ec t this p a r a m e t e r . It is known from a v a r i e t y of fundamenta l s tud ies t h a t t h e a c t i v i t i e s of m e m b r a n e - e m b e d d e d e n z y m e s p r o c e e d mos t expedi t ious ly when t h e f luidi ty of the i r m e m b r a n e env i ronmen t is wi thin an op t ima l r a n g e . Lower ing t h e cell 's t e m p e r a t u r e can r e d u c e m e m b r a n e f luidi ty (and mos t e n z y m a t i c ac t iv i t i es ) t o undes i rab le levels.,

It is r e m a r k a b l e t h a t many o rgan i sms a r e capab le of ove rcoming t h e r igidifying e f fec t of low t e m p e r a t u r e on m e m b r a n e s by e n z y m a t i c a l l y a l t e r i n g m e m b r a n e lipid compos i t ion so as to r e s t o r e a funct ional d e g r e e of f luidi ty . This pape r desc r ibes some of t h e molecu la r m e c h a n i s m s by which this " t e m p e r a t u r e acc l ima t ion" is ach ieved .

I. REQUIREMENTS FOR ACCLIMATION O F CELL MEMBRANES TO LOW TEMPERATURE

Two i m p o r t a n t c r i t e r i a mus t be m e t if an organism is to adap t i t s m e m b r a n e s to chil l ing t e m p e r a t u r e s wi thou t cel l d a m a g e . In t h e f i rs t p l a c e , i t mus t possess t h e b iochemica l c a p a c i t y to e f fec t lipid compos i t iona l changes t h a t will i n c r e a s e f luidi ty . Most commonly th is involves inc reas ing t h e number of double bonds in lipid f a t t y ac ids . Secondly, i t mus t be capab le of d i s t r ibu t ing t h e a l t e r e d lipids from t h e s i t e s of the i r e n z y m a t i c modi f i ca t ion to all o the r m e m b r a n e s of t h e ce l l .

Copyright · 1979 by Academic Press, inc. 34-7 All rights of reproduction in any form reserved

ISBN O- 12 46056O5

3 4 8 G. A. T h o m p s o n , Jr.

TOO FLUID

Ο

3

JUST RIGHT

TOO RIGID

FINAL INITIAL STATE STATE slo w

J coolin g ^ Γ

x 6 /

10 2 0 3 0

TEMPERATURE ( eC)

4 0

FIGURE 1. Representation of an organism's ability to maintain its membrane fluidity within an optimum range during a slow temperature reduction but not a fast one.

Accompl ishing t h e s e two s t eps , p a r t i c u l a r l y t h e l a t t e r one , r equ i r e s a c e r t a i n amount of t i m e , espec ia l ly in a s t r u c t u r a l l y complex ce l l . If t he t e m p e r a t u r e change is g radua l , t h e response of t h e cel l is usually quick enough to p r e v e n t m e m b r a n e f luidi ty from ever dec reas ing to a subopt imal level (Fig. 1). On the o the r hand, sudden chil l ing can d e c r e a s e m e m b r a n e f luidi ty so rapid ly t h a t lipid modi f ica t ion and in t r ace l lu l a r d i s semina t ion a r e ba re ly capab le (and s o m e t i m e s incapable) of r e s t o r i n g op t ima l condi t ions be fo re i r r eve r s ib le d a m a g e is done .

In t h e ensuing discussion I shall t r y to e m p h a s i z e no t only t h e n a t u r e of t he b iochemica l modi f ica t ions t h a t t a k e p l a c e but also t he t i m e involved in e f fec t ing t h e m .

Π. A COMPARISON OF TEMPERATURE ACCLIMATION MECHANISMS IN NATURE

I shall r e s t r i c t my c o m m e n t s to t h e s t r a t e g i e s employed by ae rob ic o rgan i sms , s ince anae robes , such as t h e b a c t e r i u m Escherichia C0lit

employ an e n z y m a t i c p a t h w a y not found in h igher p l an t s (18). But

M o l e c u l a r Cont ro l of M e m b r a n e Fluidi ty 3 4 9

No unsaturate d

F. A . o r F . A .

desaturas e presen t

a t 35°

Synthesi s o f F . A .

desaturas e induced ;

desaturatio n o f membran e

F. A . fol low s

FIGURE 2. Membrane response to low tempera aire in the aerobic bacterium Bacillus megaterium.

c u r r e n t under s t and ing sugges t s t h a t i t is p e r t i n e n t to consider t h e mechan i sm t h a t some ae rob i c b a c t e r i a h a v e for coping wi th low t e m p e r a t u r e . The l a b o r a t o r y of Fu lco has i n v e s t i g a t e d t e m p e r a t u r e a c c l i m a t i o n in Bacillus megaterium (4). When grown a t 3 5 ° , t he se cel ls con ta in no u n s a t u r a t e d f a t t y ac ids and no f a t t y ac id d e s a t u r a s e a c t i v i t y (Fig. 2). A v a r i e t y of e x p e r i m e n t s has shown t h a t a t e m p e r a t u r e drop leads to t h e induc t ion of f a t t y ac id d e s a t u r a s e synthes i s in a m o u n t s a d e q u a t e to modify m e m b r a n e lipids wi thin 1.5 hours .

Much less is known r ega rd ing t h e mo lecu la r mechan i sm for t e m p e r a t u r e a c c l i m a t i o n in h igher p l an t s , in sp i t e of t h e g r e a t economic i m p o r t a n c e of t h e p r o c e s s . In 1969 a r e m a r k a b l y s imple s c h e m e for t e m p e r a t u r e con t ro l of f a t t y ac id d e s a t u r a s e a c t i v i t y was proposed by Har r i s and J a m e s (6). It was based on t h e known p a r t i c i p a t i o n of mo lecu la r oxygen as a c o s u b s t r a t e in f a t t y ac id d e s a t u r a t i o n (Fig. 3). Ev idence was p roduced to show t h a t t h e i n c r e a s e d solubi l i ty of in p lan t cel l cv top lasm a t low t e m p e r a t u r e (O^ is 1.7 t i m e s m o r e soluble in w a t e r a t 10 C than a t 40°C) is suf f ic ient in i tse l f to i n c r e a s e t h e r a t e of r e a c t i o n s igni f icant ly dur ing a 5 hr incuba t ion . T h e r e st i l l r e m a i n s cons ide rab le doubt , however , as to w h e t h e r under no rma l env i ronmen ta l condi t ions ava i lab i l i ty ever b e c o m e s r a t e - l i m i t i n g in p l a n t s . If th is occu r s , i t would be e x p e c t e d only in non -pho tosyn the t i z ing t i s sues , which would be u t i l i z ing but no t g e n e r a t i n g O^ .

I shall d e v o t e most of t h e r ema in ing discussion to t h e p roces s of low t e m p e r a t u r e a c c l i m a t i o n in t h e c i l i a t ed p r o t o z o a n Tetrahymena pyri-formis. Cons ide rab le d a t a on t h e molecu la r con t ro l of a c c l i m a t i o n in

3 5 θ G. A. T h o m p s o n , Jr.

FIGURE 3. Membrane response to low temperature in higher plants. Because higher levels of the cosubstrate, Op, are found at a low temperatures (02is 1.7 times more soluble in water at 10° than at 40°), the fatty acid desaturation reaction is driven to the right.

Tetrahymena have r e c e n t l y been a c c u m u l a t e d in my l abo ra to ry and seve ra l o t h e r s , and t h e r e is good reason to be l ieve t ha t this cel l might s e r v e as a useful model sys tem for under s t and ing t e m p e r a t u r e a c c l i m a t i o n in m o r e advanced o rgan i sms .

Π. LOW TEMPERATURE ACCLIMATION IN TETRAHYMENA

A. Spatial and Temporal Membrane Relationships

In many r e s p e c t s , Tetrahymena is a typica l euka ryo t i c ce l l . It con ta ins the usual a s s o r t m e n t of i n t r ace l lu l a r o rgane l les (Fig. 4), and i t s me tabo l i sm is also s imi lar to t h a t of h igher o rgan isms (3), pa r t i cu l a r l y h igher p l an t s (7) (except t ha t i t is non -pho tosyn the t i c ) .

Each funct ional ly d i f fe ren t m e m b r a n e in Tetrahymena con ta ins i t s own c h a r a c t e r i s t i c p ropor t ions of s t r u c t u r a l lipids (19). These consis t mainly of t h e s t e ro l - l ike isoprenoid t e t r a h y m a n o l and t h r e e pr inc ipal phospholipids (Fig. 5). Because e a c h m e m b r a n e has d i f fe ren t p ropor t ions of t hese lipids and differ ing deg rees of unsa tu r a t i o n in i t s componen t f a t t y ac ids , t h e physical p r o p e r t i e s , i .e . , f luidi ty, of any one m e m b r a n e type might also be e x p e c t e d to differ from those of t h e o t h e r s . This has indeed been shown to be the case a t l eas t for t h e 5 - 6 funct ional ly d i s t inc t m e m b r a n e s t h a t have been t e s t e d (19). O n c e again , t h e r e is eve ry r ea son to be l i eve t h a t in this r e s p e c t , Tetrahymena r e s e m b l e s h igher p lan t ce l l s . We have examined the a c c l i m a t i o n of Tetrahymena to low t e m p e r a t u r e by measur ing seve ra l m e m b r a n e p r o p e r t i e s a f t e r shif t ing cel ls over a pe r iod of 30 min . from a g rowth t e m p e r a t u r e of 39° to a final t e m p e r a t u r e of 15° (11). This r educ t ion of 24° in cell

•

C H 3 ( C H 2) ?C H 2C H 2( C H 2) ? C — R » C H 3( C H 2) 7C H = C H ( C H 2) ?C — R

•

higher l e v e l s of the cosubstrate , C^, drive the reaction towards

the r ight . {0^ i s 1.7 X more soluble in water at 10° than at

4 0 ° ) .

Molecu l a r Cont ro l of M e m b r a n e Fluidi ty 3 5 1

FIGURE 4. Diagrammatic cross-section of Tetrahymena pyriformis.

t e m p e r a t u r e comple t e ly s tops cel l g rowth and causes a very abnormal physical s t a t e in t h e m e m b r a n e s . F ig . 6 i l l u s t r a t e s t h e l a t e r a l agg rega t i on of i n t r a m e m b r a n o u s p ro t e in s t ha t can be observed by f r e e z e f r a c t u r e e l e c t r o n mic roscopy of one p a r t i c u l a r m e m b r a n e .

By analyz ing var ious func t iona l ly -d i f fe ren t m e m b r a n e s , we w e r e able to es tabl i sh tha t t he in i t ia l lipid response to low t e m p e r a u r e occu r s in mic rosomal m e m b r a n e s . F ig . 7 r e c o r d s some of t he se changes . The solid l ine and squares r epo r t the t e m p e r a t u r e s a t which i n t r a m e m b r a n o u s p a r t i c l e - f r e e a r e a s could f irs t be d e t e c t e d by f r e e z e f r a c t u r e e l e c t r o n mic roscopy . This has been c o r r e l a t e d wi th m e m b r a n e f luidi ty. In addi t ion , m e m b r a n e f luidi ty, as judged by f luo rescence po la r i za t ion m e a s u r e m e n t s of t he e x t r a c t e d lipids (solid l ines and c i rc les ) , i nc reased e x t r e m e l y rapid ly in t he cel ls chi l led to 15 C, Final ly , t h e number of double bonds in t he phospholipid f a t t y ac ids (dashed l ines and Δ) inc r e a s e d from about 108/100 molecu les jus t a f t e r t h e shift to about 128/100 molecu les within 30 min. We have p l o t t e d these t h r e e p a r a m e t e r s t o g e t h e r to show t h a t t h e f luidi ty of t h e mic rosomal lipids i n c r e a s e s a t a r a t e t ha t is c losely c o r r e l a t e d wi th t h e i n c r e a s e in f a t t y ac id u n s a t u r a t i o n .

3 5 2 G. A. T h o m p s o n , Jr.

Ο

H p C - O - C - R

R - C - O - C H ο I I I

H o C - O - P - R c I

0"

R = 0 - C H 2C H 2- N +( C H 3) ;

= 0 - C H 2 C H 2 - N H 2

= CH2CH2-NH2

FIGURE 5. The three principal phospholipid classes of Tetrahymena membranes. R represents, from top to bottom, the polar head groups of phosphatidylcholine, phosphatidylethanolamine, and 2-aminoethyl-phosphonolipid. The major fatty acid constituents are myristic (14:0), palmitic (16:0), palmitoleic (16:1), oleic (18:1), linoleic (18:2), anda-linolenic (18:3) acids.

Signif icant ly, o t h e r m e m b r a n e s of t h e cell do no t exhibi t such a rap id response to t e m p e r a t u r e change . This is i l l u s t r a t ed in F ig . 8. H e r e t he f luorescence po la r i za t ion d a t a for mic rosomal lipids is c o m p a r e d wi th equiva len t d a t a for pe l l ic le lipids i so la ted in t h e s a m e e x p e r i m e n t s . Whereas t h e mic rosomal lipids show the i r mos t d r a m a t i c i n c r e a s e in f luidity in t h e f irs t few minu te s during and a f t e r chil l ing, t h e pe l l ic le lipids exhibi t l i t t l e change during th is per iod and b e c o m e m o r e fluid only a f t e r a long lag . This lag is r equ i r ed to t r anspo r t t h e newly d e s a t u r a t e d lipids from t h e s i t e of the i r d e s a t u r a t i o n in t h e mic rosomes to t he out ly ing m e m b r a n e s . Dis semina t ion of lipids to t he l eas t access ib le ce l lu lar m e m b r a n e , t ha t enclosing t h e ci l ia , r equ i r e s an even longer t i m e .

It is d a t a such as these t ha t have brought h o m e to us t h e i m p o r t a n c e of t i m e in t he t e m p e r a t u r e a c c l i m a t i o n p roces s . In Tetrahymena and o t h e r s t r u c t u r a l l y complex ce l l s , t h e lipid d i ssemina t ion s t ep is l ikely to be r a t e l imi t ing .

B. Molecular Mechanism for Desaturase Activation

While i t was easy to es tabl i sh t h a t low t e m p e r a t u r e causes a r i se in f a t t y acid d e s a t u r a s e a c t i v i t y , de t e rmin ing t h e p r ec i s e cause for this a t t h e molecu la r level was much m o r e di f f icul t . We have t e s t e d var ious poss ib i l i t ies . Le t us begin wi th t he c o n c e p t , m e n t i o n e d ea r l i e r , t ha t t h e d e s a t u r a s e c o s u b s t r a t e , Q ? , might be a r a t e - l i m i t i n g f a c t o r . We h a v e


FIGURE 6. The effects of rapidly decreasing temperature on the physical state of the Tetrahymena outer alveolar membrane (part of the pellicle), as visualized by freeze-fracture electron microscopy. A) pronounced lateral movement and aggregation of intramembranous protein particles in cells chilled from a growth temperature of 39.5° to 0.5°C immediately prior to fixation. B) the initial apoearance of particle-free regions in membranes chilled from 39.5° to 30 . C) control cells fixed at 39.5°. The 4 χ 10 nm frames may be used for quantifying the degree of aggregation. For details see Martin et al. (10).

3 5 4 G. A. T h o m p s o n , Jr.

FIGURE 7. Comparison of the changes of three independently measured properties of Tetrahymena microsomal nyembranes during temperature acclimation by cells chilled from 39.5 over a 30 min. period. Data from freeze-fracture electron microscopic observations of membrane particle redistribution ( ) , fluorescence polarization of diphenylhexatriene in membrane lipids (O), and the number of double bonds in phospholipid fatty acids (A) . The change in fatty acid unsaturation was due mainly to a decrease in 14:0 and 16:0 and an increase in 18:3. For details see Martin and Thompson (11).

ana lyzed t h e lipids of 39° -ce l l s and 15°-cel ls grown under var ious tens ions , as i l l u s t r a t ed in Fig . 9. In all cases t he f a t t y acid compos i t ion was c h a r a c t e r i s t i c of t he g rowth t e m p e r a t u r e , no t t h e tens ion (16).

A second possibi l i ty was tha t low t e m p e r a t u r e induces t h e synthes is of new f a t t y acid d e s a t u r a s e molecu les in a manne r s o m e w h a t analogous to t he s i tua t ion in Bacillus megaterium. This has been t e s t e d in var ious ways , mainly involving compar i sons of t e m p e r a t u r e - s h i f t e d cel ls i ncuba t ed with or wi thout inhibi tors of p ro t e in synthes is (5, 12, 17). It s e e m s c lear t ha t the pr inc ipa l changes in f a t t y acid composi t ion tha t a r e t r i g g e r e d by low t e m p e r a t u r e a r e obse rved no t only in n o r m a l ce l ls bu t also in cel ls incapab le of synthes iz ing p ro te ins (17). On the o the r hand Fukushima et al. (5) have r e c e n t l y r e p o r t e d ev idence for an i nc r ea sed

Molecu l a r Cont ro l of M e m b r a n e Fluidi ty 3 5 5

T I ME A F T E R REACHIN G 15 ° C (HOURS )

FIGURE 8. Time course of changes in diphenylhexatriene polarization in membrane lipids of 39.5°-acclimated cells following a shift to 15°C over a 30 min. period as in Fig. 7. At the times indicated, cells were fractionated, and total lipids from purified pellicles and microsomes were utilized for polarization measurements. The dashed lines show polarization values found in the two fractions from cells fully acclimated to 15°. For details see Martin and Thompson (11).

c o n t e n t of pa lmi toy l -CoA d e s a t u r a s e in cel ls shi f ted to 15 . The q u a n t i t a t i v e con t r ibu t ion of this appa ren t induct ion of e n z y m e synthes is r e m a i n s to be d e t e r m i n e d .

The bulk of our work wi th Tetrahymena favors a th i rd mechan i sm as a major fo r ce for cont ro l l ing f a t t y ac id d e s a t u r a s e a c t i v i t y (19). We be l i eve t h a t any env i ronmen ta l change which marked ly r e d u c e s m e m b r a n e f luidi ty causes an i n c r e a s e in f a t t y ac id d e s a t u r a s e a c t i v i t y , probably no t in abso lu te t e r m s but r a t h e r in compar i son to t h e r a t e of f a t t y ac id syn thes i s . Such an "ac t iva t ion" is p o s t u l a t e d to r e su l t from the impos i t ion of g r e a t e r c o n s t r a i n t s on t h e m e m b r a n e - e m b e d d e d enzymes by the m o r e rigid lipids surrounding t h e m . This i n t e r p r e t a t i o n was first sugges t ed as a poss ib le exp lana t ion for t h e d e s a t u r a t i o n a c t i v i t y in cel ls chi l led from 39 to 15 . But in those e x p e r i m e n t s it was ha rd to ru le out a d i r e c t e f f ec t of t e m p e r a t u r e on the d e s a t u r a s e e n z y m e s . In

3 5 6 G. A. T h o m p s o n , Jr.

10 Temperature ,

eC . ( )

2 0 3 0 4 0

II

10

9

8

Σ 7

CM ^

Ο 6

10 0 2 0 0 3 0 0 4 0 0

Cel l Dens i ty , c e , , 8

/ m l Χ I 03 ( )

FIGURE 9. Effect of cell density (lower abscissa) upon the concentration of dissolved On in Tetrahymena cultures grown at 15 and 39.5°C. Both cultures were m the logarithmic growth phase throughout the measurement period. The upper curve (broken line) indicates the concentration in air-saturated water at various temperatures (upper abscissa). The fatty acid patterns of phospholipids isolated from 39° cells at any 0~ tension resembled each other. Likewise, the fatty acid composition of 15° phospholipids, while different from the 39° pattern, did not vary over the observed range of O p tension (16).


Miniiir: increasin g

fluidity

d e c r e a s i n g

f l u i d i ty

wmm.. FIGURE 10. Diagrammatic representation of fatty acid desaturase

movement perpendicular to the plane of the membrane caused by decreased membrane fluidity.

co l l abora t ion wi th Nozawa and co l leagues a t Gifu Univers i ty , we have s tud ied a v a r i e t y of condi t ions in which the f luidi ty of mic rosoma l m e m b r a n e s was a l t e r e d i so the rma l ly (19). I shan ' t g ive all t h e de ta i l s of t he se s tud ies , but it is qu i t e a p p a r e n t from e x p e r i m e n t s in which Tetrahymena i n c o r p o r a t e d exogenous p o l y u n s a t u r a t e d f a t t y ac ids (8), b r a n c h e d chain (methoxy) f a t t y ac ids (9), or gene ra l a n e s t h e t i c s (13) in to i t s m e m b r a n e s t ha t an i n c r e a s e in m e m b r a n e f luidi ty can be coun ted on to r e d u c e f a t t y acid d e s a t u r a s e a c t i v i t y . We have found only one a p p a r e n t e x c e p t i o n , e t hy l a lcohol (14), which s e e m s to f luidize t he m e m b r a n e in a unique fashion t h a t is no t d i r ec t l y p e r t i n e n t to our p r e s e n t discussion.

3 5 8 G. A. T h o m p s o n , Jr.

activ e

increasin g fluidit y

decreasin g fluidit y

inactiv e

FIGURE 11. Diagrammatic representation of the fatty acid desaturase active site (hatched areas) being stabilized in a membrane of suboptimal fluidity.

Our hypothes is cal ls for f a t t y acid d e s a t u r a s e a c t i v i t y to be in a r ea l sense r e g u l a t e d by the f luidity of the m e m b r a n e env i ronmen t . No c o n c r e t e ev idence is ava i lab le to suggest how this r egu la t ion is ach ieved , but a number of h y p o t h e t i c a l models migh t be c o n s t r u c t e d . The re is some f r e e z e - f r a c t u r e ev idence t h a t in Tetrahymena m ic ro somes , unl ike t h e pe l l ic le m e m b r a n e , lipid phase s epa ra t i ons resu l t in a d i sp l acemen t of p ro te ins pe rpend icu la r to t he p lane of t h e m e m b r a n e r a t h e r than in a l a t e r a l d i r ec t ion (9, 20). Thus it is conce ivab le t h a t t h e f a t t y acid d e s a t u r a s e molecu les a r e fo rced in to a new posi t ion , as shown in F ig . 10, thus or ien t ing t h e a c t i v e s i t e more favorably wi th r e s p e c t to t h e s u b s t r a t e . This is in a g r e e m e n t wi th t h e b iochemica l and u l t r a s t r u c t u r a l obse rva t ions of Wunderl ich et al. (20). Ano the r equal ly p lausable but l a rge ly unsuppor ted possibi l i ty is t h a t a l iqu id-c rys ta l l ine lipid env i ronmen t would simply no t be cohes ive enough to ma in t a in t h e d e s a t u r a s e molecu les in the i r fully a c t i v e conf igura t ion (Fig. 11). We hope to t e s t some of these models using physical chemica l t echn iques , such as t he m i c r o c a l o r i m e t r i c app roach of Brand t s , et al. (1).


30i

2 5 h

0 5l 1 1 1 1 1 0 1 0 2 0 3 0 4 0

T E MP (C° )

FIGURE 12. The influence of temperature on diphenylhexatriene polarization in multibilayer vesicles of microsomal phospholipids from Tetrahymena grown at 39° and 15°C. Data from Dickens, et al. (2).

Although it is far from being proved , we feel t h a t t he concep t of f lu id i ty - r egu la t ed d e s a t u r a s e a c t i v i t y offers the most viable means of explaining t e m p e r a t u r e a c c l i m a t i o n in Tetrahymena. If i t is c o r r e c t , t hen a very exc i t ing gene ra l i z a t i on may be p o s t u l a t e d . Any e x t e r n a l or

3 6 0 G. A. T h o m p s o n , Jr.

i n t r ace l l u l a r f ac to r capab le of a l t e r ing m e m b r a n e f luidity will a f f ec t f a t t y ac id d e s a t u r a s e a c t i v i t y and induce an a c c l i m a t i o n response of s o r t s . It is t h e r e f o r e no t surpris ing t h a t we should observe an "acc l imat ion" of Tetrahymena to t h e p r e s e n c e of t h e unna tu r a l m e t h o x y - f a t t y ac ids (9); a n e s t h e t i c s (13); e l e v a t e d sal t c o n c e n t r a t i o n s (Mat tox & Thompson, unpublished observat ions) and a v a r i e t y of o t h e r f a c t o r s (15). How e legan t ly s imple it would be for a cel l to need only one d e t e c t i o n and response mechan i sm for such va r i ed types of env i ronmen ta l s t r e s s !

While ex t r apo l a t i on of this hypothes is to h igher p l an t s is c l ea r ly p r e m a t u r e , I shall p r o c e e d to do it wi th no fur ther apology. Imagine t he p r a c t i c a l bene f i t s of being able to "harden" a c o m m e r c i a l v e g e t a b l e or fruit c rop pr ior t o cold exposure by employing a c h e m i c a l a l t e r n a t i v e to low t e m p e r a t u r e or be ing ab le t o i m m e d i a t e l y c o u n t e r a c t t h e e f f ec t of chil l ing t e m p e r a t u r e s wi th a chemica l a l t e r n a t i v e to high t e m p e r a t u r e .

Such ideas a r e no t to be t a k e n ser iously a t t h e p r e s e n t t i m e . But ne i t he r can they be to t a l l y d i scoun ted . We p re f e r to le t t he se vague d r e a m s of glory provide mo t iva t i on for our con t inued bas ic inves t iga t ions of this e so t e r i c c r e a t u r e Tetrahymena.

C o r r e l a t i n g t h e a v e r a g e physical p r o p e r t i e s of m e m b r a n e lipids (as e s t i m a t e d by f luo rescence po la r i za t ion , e l e c t r o n spin r e s o n a n c e , X- ray d i f f rac t ion , e tc . ) wi th changes in f a t t y ac id d e s a t u r a s e a c t i v i t y sti l l falls far shor t of explaining p rec i se ly how the e f f ec t is ach ieved . We a r e cu r r en t l y devot ing cons iderab le ef for t t owards unders t and ing this r e la t ionsh ip in m o r e d e t a i l . Much of our work has focused on the mic rosomal m e m b r a n e s and the i r l ipids. In e x p e r i m e n t a l p lo t s of t e m p e r a t u r e vs . po la r i za t ion of the p robe d ipheny lhexa t r i ene in mic rosomal l ipids, we have observed two very reproduc ib le changes of s lope, or "break points" (2). F ig . 12 shows t h a t t h e b reak poin ts a t 17° and 32° in t he mic rosomal phospholipids i so la ted from 39°-grown cells a r e r e p l a c e d by s imi lar b reak poin ts a t 12° and 27° in mic rosomal phospholipids from 15°-grown ce l l s . P lo t s m a d e using t o t a l lipids (phospholipids and n e u t r a l lipids) gave , in each c a s e , b reak points a t s l ightly lower t e m p e r a t u r e s . P re l imina ry e x p e r i m e n t s wi th freshly i so la ted but s t i l l i n t a c t mic rosomal m e m b r a n e s i nd i ca t e t ha t the b reak poin ts occur a t t he s a m e c h a r a c t e r i s t i c t e m p e r a t u r e s observed in e x t r a c t e d lipid p r e p a r a t i o n s .

Unfo r tuna te ly , we have l i t t l e insight as to wha t t he b reak poin ts mean . By mixing increas ing p ropor t ions of 15° lipids with 39° l ipids, we were able to show tha t the higher t e m p e r a t u r e b reak point shif ted gradual ly from 32° down to 27° , ind ica t ing t h a t t h e s a m e p e r t u r b a t i o n t r igge r s the slope change in each c u r v e . The two low t e m p e r a t u r e b reak poin ts also seem to have a common origin.

Various r e l a t e d e x p e r i m e n t s , such as c a t a l y t i c hydrogena t ion of t h e mic rosomal lipids or mixing the n a t u r a l l ipids in di f fer ing propor t ions wi th a fully s a t u r a t e d s y n t h e t i c phospholipid - d ipa lmi toy lphospha t idy l -chol ine - sugges t ed t ha t t he b reak poin ts may signal t h e in i t i a t ion or t e r m i n a t i o n of a phase s epa ra t ion involving c e r t a i n d i s c r ee t phospholipid


FIGURE 13. A rationalization of the observed physical properties of 39 - and 15 -grown Tetrahymena microsomes and microsome-derived lipids. The left vertical axis depicts the physical behavior of preparations from 39°-grown cells while the riqht vertical axis represents the same features of preparations from 15 -grown cells. The lines connecting equivalent events on the two axes are not meant to imply that linear responses would necessarily be found in cells grown at intermediate temperatures. Wavy lines divide the system roughly into three regions, (a) Region of fully miscible lipids present in the liquid-crystalline phase; (b) region characterized by the presence of many relatively small liquid-crystalline and/or gel phase molecular assemblages that are immiscible in the bulk lipid phase. The dotted line connects those temperatures at which the first sizable discrete population of phospholipid molecular species begins undergoing a liquid-crystalline to gel transition upon cooling; (c) region featuring a rapid increase in the extent of highly ordered gel domains effecting a microscopically detectable reorientation of membrane integral proteins. The dotted line representing the second observed break point connects temperatures at which another discrete group of phospholipids commences undergoing a phase transition. Although the break points are detectable by fluorescence polarization, the impact of the phase transition on the overall structure of the membrane depends, as with the higher break point, upon the percentage of available phospholipid molecules that participate.

3 6 2 G. A. T h o m p s o n , Jr.

molecu la r spec ies , as proposed by Wunderl ich et al. (21) from X-ray d i f f rac t ion d a t a . One of severa l possible i n t e r p r e t a t i o n s of the d a t a is shown in F ig . 13. Accord ing to this s c h e m e , mic rosomal lipids m e a s u r e d in t e m p e r a t u r e r a n g e "a" exist en t i r e ly in a l iqu id-c rys ta l l ine s t a t e . As t he t e m p e r a t u r e is l owered towards t he g rowth t e m p e r a t u r e of t h e cel ls from which the lipids c a m e , smal l c lu s t e r s of highly s a t u r a t e d lipids a r e t r a n s f o r m e d in to m o r e rigid a g g r e g a t e s a t c h a r a c t e r i s t i c t e m p e r a t u r e s (upper wavy line a r e a "a") . F u r t h e r d e c r e a s e s in t e m p e r a t u r e cause a s t e a d y r i se in the s ize and number of gel phase domains (area "b"). If an espec ia l ly p r eva l en t phospholipid molecu la r spec ies r e a c h e s i t s phase t r ans i t ion t e m p e r a t u r e , then a r a t h e r p ronounced change in po l a r i za t i on (break point) might be n o t e d (dot ted l ines) . Area "c" r e p r e s e n t s t h a t t e m p e r a t u r e r ange in which phase s epa ra t i on is suff ic ient ly ex tens ive to p roduce a l a rge , e l e c t r o n microscopica l ly -v is ib le phase s epa ra t i on .

The purpose of this f igure is to i l l u s t r a t e t h a t po la r i za t ion b reak poin ts might well occur a t any s t a g e during the t e m p e r a t u r e - i n d u c e d phase t r ans i t ion of t he mic rosomal l ipids. We a r e cu r r en t ly employing t ime - r e so lved f luorescence m e a s u r e m e n t s to d e t e r m i n e how many d i s t inc t m i c r o - e n v i r o n m e n t s exist a t any c e r t a i n t e m p e r a t u r e . E x p e r i m e n t a t i o n along this line will hopefully shed addi t ional l ight on lipid i n t e r a c t i o n wi th t he f a t t y acid d e s a t u r a s e s and the reby , t h e molecu la r mechan i sm of low t e m p e r a t u r e a c c l i m a t i o n .

ACKNOWLEDGMENTS

Work from t h e au thor ' s l a b o r a t o r y was suppor ted by g ran t s from t h e Nat iona l I n s t i t u t e of Gene ra l Medical Sc iences , t he Nat iona l C a n c e r I n s t i t u t e , The R o b e r t A. Welch Founda t ion , and t h e Nat iona l Science Founda t ion .


1. Brand t s , J . F . , Tave rna , R. D. , Sadasivan, E., and Lysko, K. A. Biochim. Biophys. Acta 512, 566-578 (1978).

2. Dickens , B. F . , Mar t in , C. E., King, G. P . , Turner , J . S., and Thompson, G. Α., J r . , s u b m i t t e d for pub l i ca t ion .

3 . E l l io t t , A. M. In "Biology of T e t r a h y m e n a " , pp 508, Dowden, Hutchinson, and Ross , St roudsburg, Pennsylvania (1973).

4 . Fujii , D . K., and Fulco , A. J . J. Biol. Chem. 252, 3660-3670 (1977).

5. Fukushima, H., Nagao , S., and Nozawa , Y. Biochim. Biophys. Acta 572, 178-182 (1979).

6. Har r i s , P . , and J a m e s , A. T. Biochem. J. 112, 325-330 (1969). 7. Holz , G. G., J r . J. Protozool. 13, 2-4 (1966).


8. Kasa i , R., Ki ta j ima , Y., Mar t in , C . E., Nozawa , Y., Skriver , L. , and Thompson, G. Α., J r . Biochemistry 15, 5228-5233 (1976).

9. Ki ta j ima , Y., and Thompson, G. Α., J r . Biochim. Biophys. Acta 468 73-80 (1977).

10. Mar t in , C. E., H i r ami t su , K., Ki ta j ima , Y., Nozawa , Y., Skriver , L. , and Thompson, G. Α., J r . Biochemistry 15, 5218-5227 (1976).

11 . Mar t in , C . E., and Thompson, G Α., J r . Biochemistry 17, 3 5 8 1 -3586 (1978).

12. Nagao , S., Fukush ima, H., and Nozawa , Y. Biochim. Biophys. Acta 530, 165-174 (1978).

13. Nandin i -Kishore , S. G., Ki ta j ima , Y., and Thompson, G. Α., J r . Biochim. Biophys. Acta 471, 157-161 (1977).

14. Nandin i -Kishore , S. G., M a t t o x , S. M., and Thompson, G. Α., J r . Biochim. Biophys. Acta,551, 315-327 (1979).

15. Nozawa , Y., and Thompson, G. Α., J r . In "Biochemis t ry and Physiology of P r o t o z o a , 2nd Ed., Vol Π, (Levandowsky, M., and Hu tne r , S. H., eds.) A c a d e m i c P res s , New York, (in press) (1979).

16. Skriver , L. , and Thompson, G. Α., J r . Biochim. Biophys. Acta 431, 180-188 (1976).

17. Skriver , L., and Thompson, G. Α., J r . Biochim. Biophys. Acta 572, 376-381 (1979).

18. Thompson, G. Α., J r . "Regula t ion of M e m b r a n e Lipid Metabol i sm." C h e m i c a l Rubber Company , West Pa lm Beach , F lor ida (in press) (1979).

19. Thompson, G. Α., J r . , and Nozawa , Y. Biochim. Biophys. Acta, 472, 55-92 (1977).

20. Wunderl ich, F . , Rona i , Α., Speth, V., Seelig, J . , and Blume, A. Biochemistry 14, 3730-3735 (1975).

2 1 . Wunderl ich, F . , K r e u t z , W., Mahler , P . , Rona i , Α., and Heppe le r , G. Biochemistry 17, 2005-2010 (1978).



IN VITRO MEMBRANE LIPID RECONSTITUTION AND ENZYME FUNCTION

A. Waring and P. Glatz

Johnson R e s e a r c h Founda t ion D e p a r t m e n t of B iochemis t ry and Biophysics

Univers i ty of Pennsy lvan ia Phi ladelphia , Pennsy lvan ia

I. INTRODUCTION

The re l a t ionsh ip b e t w e e n t h e physica l s t a t e of m e m b r a n e phospholipids and changes in m e m b r a n e e n z y m e a c t i v i t y have been r e p o r t e d for a wide v a r i e t y of biological s y s t e m s . Lipid r e l a t e d inf luence on the funct ion of the mi tochondr ia l e l e c t r o n t r a n s p o r t chain is of specia l i n t e r e s t s ince t h e o rgane l le has a r e l a t i v e l y lipid poor m e m b r a n e sys tem (4, 13) which shows a p p a r e n t changes in e n z y m e mechan i sm as a funct ion of t e m p e r a t u r e (3,12). The ro le of phospholipid in t h e a l t e r a t i o n of e n z y m a t i c a c t i v i t y of the mi tochondr i a l e l e c t r o n t r anspo r t chain has been emphas i zed by t h e e l imina t ion of t e m p e r a t u r e dependen t changes in e n z y m a t i c a c t i v i t y using d e t e r g e n t p e r t u r b a t i o n of the m e m b r a n e env i ronmen t (10) or by modi f i ca t ion of t h e m e m b r a n e lipid compos i t ion upon a c t i v a t i o n of endogenous phosphol ipase a c t i v i t y (8, 11). However , t h e c o r r e l a t i v e s tud ies have not d e t a i l e d t h e n a t u r e of t h e t e m p e r a t u r e r e l a t e d change in m e m b r a n e phys ica l s t a t e nor have they c h a r a c t e r i z e d t h e speci f ic a l t e r a t i o n in e n z y m e mechan i sm induced by phospholipid.

The focus of t h e c u r r e n t s tudy has been an a t t e m p t to r e l a t e the d e g r e e of change in e n z y m a t i c a c t i v i t y t o the type of a l t e r a t i o n of the m e m b r a n e phospholipid physical c h a r a c t e r . For c o m p a r a t i v e purposes two types of phosphol ip id-pro te in s y s t e m s h a v e been examined . The first sys tem cons idered involves a t e m p e r a t u r e prof i le of c y t o c h r o m e oxidase a c t i v i t y in a h e t e r o g e n o u s l ipopro te in assembly (the i n t a c t inner mi tochondr i a l m e m b r a n e ) . The second sys tem cons is t s of t o m a t o mi tochondr i a c y t o c h r o m e oxidase r e c o n s t i t u t e d wi th a homogenous s y n t h e t i c phospholipid compos i t ion having a s ingle , well def ined, liquid c rys t a l t o gel phase t r ans i t ion in t h e physiological t e m p e r a t u r e r a n g e .

3 6 5 Copyright · 1979 by Academic Press, inc.

All lights of reproduction in any form reserved ISBN 012-46056O5

3 6 6 A. W a r i n g a n d P. Glatz

Π. METHODS

E t i o l a t e d t o m a t o seedl ings (Lycopersicon esculentum cv . VF 145-21-4, P e t o s e e d Co.) w e r e grown a t 25 C for six days be fo re use . Seedlings (100 gm seedl ing hypocoty ls plus cotyledons) w e r e r a z o r chopped in to 200 ml of 4 C grinding medium (0.3 Μ suc rose , 0 .2% w/v MOPS pH 7.4, 1 mM EDTA, 1 m g / m l BSA f rac t ion V, 0.1 m g / m l m e r c a p t o b e n z o t h i a z o l e ) . The s lurry was then homogen ized for 20 sec using an U l t r a t u r a x homogen ize r , and mi tochondr i a i so la t ed by d i f fe ren t ia l cen t r i fuga t ion (1), and osmot i c swell ing to r u p t u r e t he ou t e r m e m b r a n e . Mi tochondr ia used in f luorescen t p robe s tud ies and for lipid e x t r a c t i o n , w e r e fur ther sub jec ted to sucrose s t ep dens i ty g rad ien t cen t r i fuga t ion to r e m o v e mic rosoma l and mi tochondr i a l m e m b r a n e c o n t a m i n e n t s (1).

C y t o c h r o m e c oxidase a c t i v i t y in mi tochondr i a was m e a s u r e d po la rographica l ly wi th a YSI e l e c t r o d e in a 1.3 ml Gilson c h a m b e r and the t e m p e r a t u r e r e g u l a t e d using a Lauda K 2 / R r e f r i g e r a t e d c i r cu la t ing b a t h . The assay medium con ta ined 20 mM a s c o r b a t e pH 7.4, 10 uM TMPD, 10 uM c y t o c h r o m e c (sigma type VI) and 10 mM phospha te buffer pH 7.4 The r e a c t i o n was i n i t i a t ed by the addi t ion of e n z y m e and the m e a s u r e m e n t s c o r r e c t e d for oxygen consumpt ion wi thout e n z y m e . C y t o c h r o m e oxidase a c t i v i t y in r e c o n s t i t u t e d phospholipid ves ic les was also m e a s u r e d po la rographica l ly wi th 20 mM a s c o r b a t e , pH 7.4, 10 uM c y t o c h r o m e c, and 10 mM phospha t e buffer pH 7.4.

Mi tochondr ia l inner m e m b r a n e phospholipids w e r e e x t r a c t e d wi th c h l o r o f o r m / m e t h a n o l 2:1 con ta in ing 5% of 28% ammonium hydroxide (8), p a r t i t i o n e d accord ing to Folch et al.y (5), and flash e v a p o r a t e d to dryness a t 20 C.

Mi tochrondr ia w e r e dep l e t ed of lipid by d e t e r g e n t - a m m o n i u m su l fa te f r ac t iona t ion . Twenty volumes of phospha t e buf fe red (0.1 M, pH 7.4) 2% w/v cho l a t e was mixed with one volume of mi tochondr i a . The mix tu re was homogen ized and brought to 50% w/v wi th solid ammonium su l f a t e . The pH of t h e m i x t u r e was adjus ted to pH 7.4 wi th ammonium hydroxide and s t i r r e d one hour a t 4 C . The m i x t u r e was then cen t r i fuged a t 10,000 χ g for 10 min and the p r e c i p i t a t e sub jec ted to an addi t ional cyc le of f r ac t iona t ion . Lipid dep l e t ed mi tochondr ia l p ro t e in (3-5 ug lipid P / m g prote in) in 2% c h o l a t e was r e c o n s t i t u t e d wi th d imyr is toy l phosphat idylchol ine (2 mg phosphol ip id /mg mi topro te in) by sonica t ion (Branson Sonif ier -micro t ip - 20 sec) , fol lowed by dialysis aga ins t 10 mM phospha te buffer , pH 7.4, 4 C for 24 hours to r e m o v e cho la t e (16). Phospholipid l iposome ves ic les w e r e p r e p a r e d from e x t r a c t e d mi tochondr ia l phospholipid (0.1 mg PL/ml) or d imyr is toyl phosphat idylchol ine (0.1 mg PL/ml) in 10 mM phospha t e buffer pH 7.4 by sonica t ion (Branson Soni f ie r -micro t ip 60 sec) under a rgon. T r a n s -pa r ina r i c acid (TPnA) (Molecular P robes , Inc.) as added to mi tochondr ia l m e m b r a n e s or phospholipid l iposomes from an e thano l s tock solut ion (0.5 m g / m l ) . All solut ions we re deoxygena t ed wi th argon and the f luorescen t p robe to phospholipid r a t i o was app rox ima te ly 1:100.

In Vitro M e m b r a n e Lipid R e c o n s t i t u t i o n a n d E n z y m e F u n c t i o n 3 6 7

FIGURE 1. Arrhenius plot of cytochrome oxidase activity in mitochondria.

F l u o r e s c e n c e l i f e t ime m e a s u r e m e n t s of TPnA in l iposomes and m e m b r a n e s was m e a s u r e d by s ingle pho ton coun ' nig. A g a t e d nanosecond f lash lamp (Pho tochem. R e s . Assoc.) was used for t h e e x c i t a t i o n of t he s amp le s . The l a m p flash was op t ica l ly d e t e c t e d (RCA 1P28), and digi ta l ly p rocessed using fas t e l e c t r o n i c modules (Or tec 474, 473A). The f luo rescence was d e t e c t e d wi th a b lue -sens i t ive p h o t o t u b e (RCA 1P28) and p roces sed wi th O r t e c modules 454, 463 , 425 and 425A. The co inc idence of the l amp flash and the f luo rescence was ana lyzed using a O r t e c 457 t i m e - t o - p u l s e - h e i g h t c o n v e r t e r and a LeCroy 3001 mul t i channe l a n a l y z e r . The sample t e m p e r a t u r e was con t ro l l ed using a low t e m p e r a t u r e c u v e t t e holder (Pho tochem. R e s . Assoc.) c o n n e c t e d to a r e f r i g e r a t e d b a t h . The c h a m b e r was purged wi th dry n i t rogen gas which had been cooled by a h e a t exchange r i m m e r s e d in liquid n i t rogen which p r e v e n t e d condensa t ion on t h e op t ica l c o m p o n e n t s and q u a r t z ce l l .

3 6 8 A. W a r i n g a n d P. Gia tz

°C

2. 0 r

3.3 3 4 3 5 3 6

kpXlO 5

FIGURE 2. Arrhenius plot of cytochrome oxidase activity (nmoles OJmg protein/min) in DM PC-cytochrome oxidase vesicles (2 mg DMPC/mg protein).

ΠΙ. RESULTS

A. Temperature Dependence of Cytochrome Oxidase in Mitochondria and Phospholipid Vesicles

C y t o c h r o m e c oxidase a c t i v i t y in i n t a c t mi tochondr ia l m e m b r a n e showed a d iscont inui ty in the Arrhenius plot in t he te r r rpera ture r ange of 16 to 14 C (Fig. 1). At higher t e m p e r a t u r e s (+3Z C) mi tochondr ia l c y t o c h r o m e c oxidase a c t i v i t y also showed a sl ight dec l ine in a c t i v i t y .

In con t r a s t to c y t o c h r o m e oxidase ac t i v i t y in t h e mi tochondr ia l m e m b r a n e , a c t i v i t y and o x i d a s e - r e c o n s t i t u t e d wi th d imyr is toy l phosphat idylchol ine had no higher t e m p e r a t u r e dec l ine (+3Z C) . However , t h e r e was a d i s t inc t change in oxidase a c t i v i t y in the D M P C -oxidase ves ic les about Z4 C. The d iscont inu i ty observed for t h e i n t a c t ο ο m e m b r a n e sys tem around 16 to 14 C was not a p p a r e n t in t he r e c o n s t i t u t e d sys tem (Fig. Z).

3 0 2 1 12. 7 4. 7


Excitatio n

32(5 42 0 52 0

Wavelengt h (nm )

FIGURE 3. Uncorrected steady state excitation and emission spectra of TPnA in the mitochondrial membrane and phospholipid vesicle systems. Excitation wavelength 320 nm; emission wavelength 420 nm. Half-maximal band pass, 5 nm.

B. Fluorescent Probe Steady State Excitation and Emission Spectra

Exc i t a t i on s p e c t r a (Fig. 3) of TPnA conta in ing m e m b r a n e and ves ic le s y s t e m s r e v e a l e d a shoulder a t 294 n m , and two m a x i m a a t 306 nm and 320 nm wi th a val ley a t 312 n m . The in t ens i ty of t h e 320 nm e x c i t a t i o n peak was g r e a t e r than the 306 nm peak in the i n t a c t m e m b r a n e s y s t e m . In all o t h e r sy s t ems t h e 306 nm - 320 nm peaks w e r e equ iva len t .

Emission s p e c t r a of TPnA w e r e b road (Fig. 3), and showed a slight shift to t he longer wave leng ths (420 nm - 430 nm) in t he i n t a c t m e m b r a n e s y s t e m . T h e r e w e r e no app rec i ab l e shif ts in exc i t a t i on -emis s ion wave l eng th s when t h e sample t e m p e r a t u r e was lowered to +2 C . Mi tochondr ia l m e m b r a n e s and ves ic les of e x t r a c t e d mi tochondr ia l lipids had a smal l amoun t of endogenous f luo rescence (10% of t he t o t a l f l uo re scence wi th TPnA) around 380 nm to 450 nm which was not a l t e r e d by lower ing t h e t e m p e r a t u r e to 2 C.

C. Fluorescence Lifetimes of TPnA in Vesicles and Membranes

Examina t ion of t he f luo rescence decay of TPnA in DMPC-ox idase ves ic les , (Fig. 4), showed t h e p r e s e n c e of two c o m p o n e n t s . Above t h e t r ans i t i on t e m p e r a t u r e of DMPC (23 C) t h e r e was a fast componen t and

3 7 θ A. W a r i n g a n d P. Glatz

Oxidase/DMP C 34 °C Oxidase/DMP C 5° C

100 20 0 10 0 20 0 Time(ns ) Time(ns )

FIGURE 4. Fluorescence decay of TPnA in DMPC-oxidase (2 mg PL/mg protein). The shaded area under the curve shows the time domain of intensity integration (50 to 250 nsec) used for temperature scanning measurements. Excitation filter Perkin-Elmer UV2; emission filter Schott KV399.

a second s lower decay (Table I). Below t h e t r ans i t ion t e m p e r a t u r e of DMPC t h e r e was also shor t and long componen t s ev ident (Fig. 4), however t h e slow componen t had an enhanced l i f e t ime (Table I) and a higher r e l a t i v e i n t ens i ty .

In t h e mi tochondr ia l m e m b r a n e sys tem TPnA f luo rescence decay also app rox ima ted a double exponent ia l (Fig. 5). However , TPnA f luorescence l i f e t ime in t he mi tochondr ia l sys t em was unchanged for the s lower componen t over t he e n t i r e e x p e r i m e n t a l t e m p e r a t u r e r a n g e (Table I).

TABLET Summary of TPnA Fluorescent Lifetimes in DMPC-Oxidase and Mitochondria

Sample Temperature 1/k (ns) % 1/kJns) %

DMPC-oxidase 33°C 4 1+0 2 83 14 0+0 3 17 DMPC-oxidase 4 7+0 2 20 40 0+0 4 80 Mitochondria 34°C 2 5+0 2 86 15 2+0 3 14 Mitochondria 5°C 3 0+0 2 80 14 9+0 3 20

model curve: M(T) = A1 exp (-k^) + A^ exp (~k2t)

normalization = /°°M(t) dt = 100+2%

In Vitro M e m b r a n e Lipid R e c o n s t i t u t i o n a n d E n z y m e F u n c t i o n 371

Mito Membrane s 3 3 ° C Mit o Membrane s 5° C

10 0 2 0 0 10 0 2 0 0

Time (ns ) Tim e (ns )

FIGURE 5. Fluorescence decay of TPnA in mitochondrial membranes (1 mg/ml). The shaded area under the curve shows the time domain of intensity integration (50 to 250 nsec) used for temperature scanning measurements. Excitation filter Perkin-Elmer UV2; emission filter Schott KV399.

In t h e mi tochondr ia l m e m b r a n e sys tem TPnA f luo re scence decay also a p p r o x i m a t e d a double exponen t ia l (Fig, 5). However , TPnA f luo rescence l i f e t ime in t h e mi tochondr ia l sys t em was unchanged for t he s lower componen t over the e n t i r e e x p e r i m e n t a l t e m p e r a t u r e r a n g e (Table I).

D. Fluorescence Intensity Measurements

Trans -pa r ina r i c acid f luo rescence in DMPC ves ic les showed a sharp i n c r e a s e a t 23 C (Fig. 6), s imi lar to t h a t desc r ibed by Sklar et al., (14) and coinc ided wi th h e a t c a p a c i t y changes r e p o r t e d by Ladbrooke and C h a p m a n (9), for DMPC liquid c rys t a l t o gel phase t r ans i t ions using d i f fe ren t i a l scanning c a l o r i m e t r y . The enhanced f luo rescence pa ra l l ed changes in f l uo rescence l i f e t ime a s soc i a t ed wi th t he phospholipid in a "gel- l ike" s t a t e (14). Examina t ion of TPnA f luo rescence in t he D M P C -r e c o n s t i t u t e d oxidase ves ic le sys tem r e v e a l e d a t e m p e r a t u r e dependen t change in f l uo rescence a t 23 C s imi lar to t h e DMPC ves ic les (Fig. 6).

When t h e f luo rescence of TPnA in mi tochondr ia l m e m b r a n e s or in lipid ves ic les p r e p a r e d from e x t r a c t e d t o m a t o mi tochondr ia l lipids was m e a s u r e d as a funct ion of t e m g e r a t u r e t h e r e was no s ignif icant change over t h e t e m p e r a t u r e r ange 40 to 2 C (Fig. 6).

3 7 2 A. W a r i n g a n d P. Giatz

?*QoMotooQlo

t o

DMPC/Tomato Oxidase

β β ο· · ο ο ο # £

Tomato Mitochondrial Phospholipids

°0 § § · β _

Tomato Mitochondria

• • • ° § o § o t o t o o t o t o 0o ο β οΜ · 0 β § § β β

5 Ο 1 5 2 0 2 5 3 0 3 5 4 0 Temperatur e * C

FIGURE 6. Temperature profile of TPnA time domain of fluorescence intensity integration (50 to 250 nsec) in membrane and vesicle systems. Points represent the number of detected fluorescence flashes per 900,000 excitation pulses. Cooling scan O, heating scan · , at rates of 0.3 /min. Excitation filter, Perkin-Elmer UV2; emission filter Schott KV399.

IV. DISCUSSION

The f luorescence probe t r ans -pa r ina r i c ac id has been shown to moni to r liquid c rys ta l to gel phase t r ans i t ions per se in bo th a r t i f i c ia l (14) and biological m e m b r a n e sy s t ems (15, 18). The o c c u r r e n c e of enhanced l i f e t imes a t and below the t e m p e r a t u r e of t h e phase t r ans i t ion in DMPC and DMPC-ox idase ves ic les ind ica te s t h a t TPnA moni to r s t he


phospholipid phase t r ans i t ion and is no t apprec iab ly inf luenced by t h e p ro te in componen t . P robe behav ior in t o m a t o mi tochondr ia l m e m b r a n e s and ves ic les of e x t r a c t e d mi tochondr ia l phospholipids show no enhanced f luorescen t l i f e t ime implying t h a t l iquid c rys t a l t o gel phase t r ans i t ions do no t occur in t h e 40° to 2°C r a n g e for t he se s y s t e m s . R e c e n t inves t iga t ions of an imal mi tochondr i a using d i f fe ren t i a l scanning c a l o r i m e t r y (6) and TPnA f luo rescence (17, 18) i n d i c a t e t h a t a lipid phase t r ans i t ion occu r s a t subzero t e m p e r a t u r e (-5 to -20 C) . In c o n t r a s t , e x p e r i m e n t s using e l e c t r o n spin r e s o n a n c e n i tox ide p robe analys is have r e v e a l e d a change in p robe mot iona l p a r a m e t e r s for bo th e x t r a c t e d phospholipids and i n t a c t an imal mi tochondr ia l m e m b r a n e s around 20 C (12) and around 12 C for t o m a t o mi tochondr i a (8) sugges t ing t h a t t h e EPR obse rva t ions above ze ro c e n t i g r a d e may r e p r e s e n t "v i sco t rop ic -fluidity" e f f ec t s r a t h e r than phase t r ans i t ions in t h e m e m b r a n e phosphol ipids .

The a l t e r a t i o n of oxidase e n z y m a t i c a c t i v i t y by changes in phospholipid physical s t a t e as a funct ion of t e m p e r a t u r e in bo th i n t a c t m e m b r a n e s and ves ic le s y s t e m s is also of i n t e r e s t . Under t h e condi t ions of this s tudy , changes in oxidase a c t i v i t y (i .e. "breaks ," d i scon t inu i t i e s , or o t h e r nonl inear behavior in Arrhen ius plots) due to phase t r ans i t ions canno t be dis t inguished from those due to f luidi ty a l t e r a t i o n s . Enoch et al. (2) h a v e observed t h a t in a sys tem which has a close r a t i o of e n z y m a t i c p ro te in to phospholipid, t h e r a t e l imi t ing p a r a m e t e r is no t r e l a t e d to phospholipid dependen t t r ans l a t i ona l diffusion of r e a c t i n g m e m b r a n e c o m p o n e n t s . The c y t o c h r o m e oxidase complex is an analogous assembly in which t h e r a t e l imi t ing s t e p or s t eps may involve t h e phospholipid dependen t i n t r a m o l e c u l a r t r an spo r t of e l e c t r o n s b e t w e e n h e m e a componen t s (19, 20).

ACKNOWLEDGMENTS

The au tho r s wish to thank Dr . J . Vanderkooi for t he use of t h e t i m e -reso lved f luo rescence m e a s u r e m e n t a p p a r a t u s and her helpful sugges t ions . We a r e g ra te fu l to Dr . B. C h a n c e for his en thus i a s t i c discussions on r a t e l imi t ing s t eps in in t r ins ic m e m b r a n e c a t a l y t i c p r o t e i n s . The useful exchanges on f luorescen t p robe analysis wi th Dr . L. Bashford and Dr . J . Smith of t h e Johnson Founda t ion p robe group a r e g ra te fu l ly acknowledged .


1. Douce , R. , Mannel la , C. Α., and Bonner , W. D . Biochim. Biophys. Acta 292, 105-116 (1973).

2. Enoch, H. G., C a t a l a , Α., and S t r i t t m a t t e r , P . J. Biol. Chem. 251, 5095-5103 (1976).

3 . Erec inska , M. and C h a n c e , B. Arch. Biochem. Biophys. 151, 304-315 (1972).

3 7 4 A. w a r i n g a n d P. Gla tz

4. F le i sher , S., Klouwen, H., and Br ier ley , G. J. Biol. Chem. 236, 2936-2941 (1972).

5. Fo lch , J . , Lees , M., and S loane-Stan ley , G. H. J. Biol. Chem. 226, 497-509 (1957).

6. Hackenbrock , C . R. , Hochl i , M., and Chau , R . M. Biochim. Biophys. Acta 445, 466-484 (1976).

7. Ke i th , A. D. , Aloia, R. C. , Lyons, J . M., Snipes, W., and Pengel ly , E. T. Biochim. Biophys. Acta 394, 204-210 (1975).

8. Ke i th , A. D. , Bre idenbach , R. W., Lyons, J . M., and Waring, A. (unpublished data) (1976).

9. Ladbrooke , B. D. , and Chapman , D. Chem. Phys. Lipids 3, 304-367 (1969).

10. Raison, J . K., Lyons, J . M., and Thomson, W. W. Arch. Biochem. Biophys. 142, 83-90 (1971).

11 . Raison, J . K., and Lyons, J . M. Proc. Natl. Acad. Sci. U.S. 68, 2092-2094 (1971).

12. Raison, J . K., Lyons, J . M., Mehlhorn, R. J . , and Ke i th , A. D . J. Biol. Chem. 246, 4036-4040 (1971).

13. Schwer tne r , Η. Α., and Biale , J . B. J. Lipid Res. 14, 235-242 (1973).

14. Sklar, L. Α., Hudson, B. S., and Simoni, R. D . Biochemistry 16, 819-812 (1977).

15. T e c o m a , E. S., Sklar, L. Α., Simoni, R . D. , and Hudson, B. S. Biochemistry 16, 829-835 (1977).

16. Vik, S. B., and Capald i , R. A. Biochemistry 16, 5755-5759 (1977). 17. Waring, Α., C h a n c e , B., and G l a t z , P . Xlth International Cong, of

Biochem. Abstract (1979). 18. Waring, Α., G l a t z , P . , and Vanderkooi , J . M. Biochim. Biophys.

Acta (submit ted) (1979). 19. Yoshida, S., Ori i , Y., K a w a t o , S. and Ikegami , A. In " C y t o c h r o m e

Oxidase" (Τ. E. King e t a l . , eds.) pp . 231-240. E l s ev i e r /Nor th Holland Biomedica l P re s s , A m s t e r d a m , New York, Oxford (1979).

20. Yu, C. Α., Yu, L., and King, T. S. J. Biol. Chem. 250, 1383-1392 (1975).


THE INFLUENCE O F FATTY ACID UNSATURATION ON FLUIDITY AND MOLECULAR PACKING

OF C H L O R O P L A S T MEMBRANE LIPIDS

David G. Bishop, Janette R. Kenrick

Plan t Physiology Uni t , CSIRO Division of Food R e s e a r c h and School of Biological Sc iences

Macqua r i e Univers i ty Nor th Ryde , Sydney Ζ113, Aus t ra l i a

James H. Bayston, Athol S. Macpherson

CSIRO Division of C h e m i c a l Technology South Melbourne , 3Z05, Aus t r a l i a

Stanley R. Johns and Richard I. Willing

CSIRO Division of Applied Organ ic C h e m i s t r y Melbourne , 3207, Aus t r a l i a

I. INTRODUCTION

The ro le of m e m b r a n e lipids in t he suscep t ib i l i ty of c e r t a i n p l an t s to chil l ing injury a t t e m p e r a t u r e s b e t w e e n about 1 0 C and 15 C has been la rge ly deduced from d i scon t inu i t i e s in Arrhen ius p lo t s of biological p a r a m e t e r s , and of spin label mot ion in m e m b r a n e s and m e m b r a n e lipids (14, 15, 21). The use of d i scon t inu i t i e s in Arrhen ius p lo t s to d e t e r m i n e c r i t i c a l t e m p e r a t u r e s in biological s y s t e m s has been t h e subject of con t rove r sy for over fifty y e a r s . In the p r e s e n t c o n t e x t i t s use has been ques t ioned on t h e o r e t i c a l grounds (1 , 9> 30) and in a r e c e n t r ev iew, Schre ier et al., (25) h a v e drawn a t t e n t i o n to t h e possibi l i ty of a r t e f a c t u a l d i scon t inu i t i e s ar is ing in Arrhenius p lo t s of spin- label mot ion , due to t h e i ncompa t ib i l i t y of t h e e x p e r i m e n t a l m e a s u r e m e n t s and t h e formal equa t ions used to de r ive mot ion p a r a m e t e r s .

The f a t t y acid compos i t ion of an imal and p lan t m e m b r a n e lipids is such t h a t phase s e p a r a t i o n s and t r ans i t ions might no t be e x p e c t e d to

3 7 5 Copyright · 1979 by Academic Press, inc.

All rights of reproduction in any form reserved ISBN 0 1 2 4 6 0 5 6 0 5

3 7 6 D. G. B i s h o p et al.

occur above 0 ° . This is pa r t i cu l a r ly t r u e in ch lorop las t s of a lgae (except c e r t a i n Cyanophyta) and higher p l an t s , whose f a t t y acid compos i t ion is highly u n s a t u r a t e d (Z). Chlorop las t s also con ta in only very low levels of s t e ro l s (3) and the g r e a t bulk of chlorophyll molecu les a r e bound to speci f ic p ro te ins and canno t be cons idered as p a r t of t he bulk lipid (28). However , phase t r ans i t ions in m e m b r a n e lipids including those of the ch lo rop las t s h a v e been invoked as be ing respons ib le for t he chil l ing sens i t iv i ty of such p l an t s as m a i z e , t o m a t o (22) and mung bean (23). Much of this d a t a has been ob ta ined by m e a s u r e m e n t s of spin label mot ion in chloroplas t m e m b r a n e s and the i r l ipids, using the mot ion p a r a m e t e r s whose va l id i ty has been ques t ioned by Schre ier et al. (25).

This pape r desc r ibes an examina t ion of t h e t e m p e r a t u r e c h a r a c t e r i s t i c s of the ch loroplas t polar lipid from t h r e e spec ies of higher p l an t s , bo th chill ing sens i t ive and r e s i s t a n t , and c o m p a r e s the p r o p e r t i e s of g lvjol ipids wi th widely vary ing f a t t y ac id compos i t ion , by monolayer and C nuc lea r m a g n e t i c r e s o n a n c e t echn iques .

Π. EXPERIMENTAL

Chlorop las t s w e r e i so la t ed from greenhouse -g rown t o m a t o (Lycopersicon esculentum var. Floridade), m a i z e (Zea mays var. GH390), and peas (Pisum sativum var. Massey Gem) by me thods previously desc r ibed (3). The ch loroplas t polar lipid f rac t ion was p r e p a r e d by c h r o m a t o g r a p h y on columns of Kiese lgel 60 (Merck) (17), excep t t h a t t h e polar lipid f rac t ion was e lu t ed with C H C l ^ C H ^ O H , 1:1. F a t t y acid analysis was pe r fo rmed by gas liquid c h r o m a t o g r a p h y of me thy l e s t e r s (5).

Spin label mot ion was m e a s u r e d in a Varian E4 s p e c t r o m e t e r with con t ro l led t e m p e r a t u r e r egu la t ion as desc r ibed by Raison and C h a p m a n (23) a t a cons t an t temperance i nc rea se of 0.5 C /min . Spin label (to give a final c o n c e n t r a t i o n of 10 M) was added to a solut ion of lipid (2 mg) in CHC1- and the solvent r e m o v e d under Ν^· Then 0.2 ml of 20 mM Tr i s -a c e t a t e , 1 mM EDTA, pH 8.0 was added and the sample mixed for 2 min on a vo r t ex mixer in t he p r e s e n c e of glass beads . Th ree p a r a m e t e r s of spin label mot ion we re measu red , co r r e l a t i on t i m e ( τ ) as desc r ibed by Melhorn et al. (16), o rder p a r a m e t e r (S ) as desc r ibed by Gaffney (8) and pa r t i t i on as desc r ibed by Shimshick Ind McConnel l (26). The spin labels used we re 5NS-A (3-oxazol idenyloxy-2-(3 - ca rboxypropyl ) -2-t r idecy l -4 ,4 -d ime thy l ) , 12NL ( l -oc t adecanoy l -2 - [3 -oxazo l ideny loxy-2 -(10-carboxydecyl ) -2-hexyl -4 ,4-d imethyl ] -3sn-g lycery lphosphory lcho-line), — a gift from Dr . C. C. Cur ta in—, 12NS-Me (3-oxazol idenyloxy-2-(10-ca rbmethoxydecy l ) -2 -hexy l -4 ,4 -d imethy l ) , and 5N10 (3-oxazol ideny-loxy-2 -pen ty l -2 -bu ty l -4 ,4 -d ime thy l ) .

Di f fe ren t ia l scanning c a l o r i m e t r y was p e r f o r m e d in gold pans in a Perkin Elmer DSC-2 c a l o r i m e t e r , using samples of lipid (6-8 mg) h y d r a t e d with 10 μ 1 of 20 mM T r i s - a c e t a t e , 1 mM EDTA, pH 8.0

T h e I n f l u e n c e of F a t t y Acid U n s a t u r a t i o n o n Fluidi ty 3 7 7

con ta in ing 30% v/v e t h y l e n e glycol , as desc r ibed by van Dijck et al. (7). Both he a t i ng and cooling scans w e r e run a t 5 / m i n .

The isola t ion of highly pur i f ied ch loroplas t lipids for monolayer s tud ies and the t echn ique used for the m e a s u r e m e n t of f o r c e - a r e a curves will be inscribed in a fo r thcoming publ ica t ion (Bishop et al.in p r e p a r a t ion) . C-NMR m e a s u r e m e n t s w e r e p e r f o r m e d on a Varian CFT-20 s p e c t r o m e t e r as previously desc r ibed (12, 13).

m. RESULTS

A Fatty Acid Composition of Chloroplast Lipids

Chlorop las t m e m b r a n e lipids a r e composed p redominan t ly of glycol ipids, unl ike most o the r biological m e m b r a n e s , whose lipid c o m p o n e n t s a r e phosphol ipids . The major ch loroplas t lipids a r e MGG (monogalac tosyld iacylg lycero l ) and DGG (diga lac tosyld iacylg lycero l ) , wi th smal l e r a m o u n t s of SL ( sulfoquinovosyldiacylglycerol) and PG (phosphat idylglycerol) (4, 6). The f a t t y ac id compos i t ion of the t o t a l po lar lipid from ch lo rop las t s of t h r e e spec ies is shown in Table I. The compos i t ion is s imi lar in all spec ies a l though the chil l ing r e s i s t a n t spec ie s , pea , con ta ins a h igher level of s a t u r a t e d f a t t y ac ids and has a lower double bond index (DBI) than the two chil l ing sens i t ive spec ies , t o m a t o and m a i z e . The f a t t y acid compos i t ions give no ind ica t ion t h a t t h e bulk m e m b r a n e lipids would undergo a phase t r ans i t ion above 0 C. The p r e s e n c e of s ignif icant a m o u n t s of molecu la r spec ies which might undergo phase s epa ra t i ons from t h e bulk lipid, such as those con ta in ing two s a t u r a t e d ac ids , is also e l i m i n a t e d . Al though higher p lan t ch lorop las t SL may con ta in up to 50% of s a t u r a t e d f a t t y ac ids , mo lecu la r spec ie s con ta in ing two s a t u r a t e d ac ids a r e only ve ry minor c o m p o n e n t s (29).

B. Spin Label Motion in Chloroplast Lipids

The most c o m m o n p a r a m e t e r s of spin label mot ion which have been used to d e t e c t phase t r ans i t ions and sepa ra t i ons in m e m b r a n e lipids a r e t he o rder p a r a m e t e r , co r r e l a t i on t i m e and p a r t i t i o n p a r a m e t e r s . The l im i t a t i ons of t hese p a r a m e t e r s and the f ac t t ha t they may give r i se to a r t e f a c t u a l d i scon t inu i t i e s in Arrhen ius p lo t s has been po in ted out by Schre ie r et al. (25). F igure 1 shows t h e Arrhenius p lo t s of the p a r a m e t e r s S and τ m e a s u r e d wi th t h e spin label 5NS-A, and t h a t of τ m e a s u r e d wi th 1 S-Me, in t o m a t o ch loroplas t polar lipid. While t h e p?ot of S for 5NS-A is a p p r o x i m a t e l y a s t r a igh t l ine , those of τ for bo th 5 N # A and 12NS-Me obviously do no t fit a single s t r a igh t l ine , and it is diff icul t to dis t inguish w h e t h e r t h e d a t a fit a smooth cu rve , or two or t h r e e i n t e r s e c t i n g s t r a igh t l ines . It is also diff icult to r econc i l e t he

3 7 8 Ό. G. B i s h o p et al.

Fatty Acid Composition of Chloroplast Polar Lipids

Acid Tomato Maize Pea

% % %

16:0 9.2 7.2 19.2 16:1 0.8 2.8 5.1 16:2 - - 0.4 16:3 - - 8.7 18:0 1.5 0.8 1.9 18:1 1.9 0.3 1.4 18:2 12.8 3.0 12.0 18:3 73.7 85.9 50.9

Double bond index

0 2.48 2.67 2.10

aThe double bond index (DBI) was calculated as Σ (fatty acid %) ·

(number of double bonds in fatty acid) · 10~* for all unsaturated fatty acids in the molecule.

s t r a igh t l ine plot ob ta ined for t he m e a s u r e m e n t of S with 5NS-A with t h a t of t he τ p lot ob ta ined from t h e s a m e s p e c t r a l d a t a . Al though t h e n u m e r i c a l change in log S over t h e r a n g e 3 -36 C shown is only smal l , p lo t s of S or 2T aga ins t t e m p e r a t u r e (data not shown) gave no indica t ion §f d i scon t inu i t i e s . The va lues ob ta ined in pa r t i t i on e x p e r i m e n t s using 5N10 gave Arrhenius p lo t s which appea red to be cu rves , but these m e a s u r e m e n t s , which w e r e based on he igh t s of peaks , r a t h e r than a r e a s , may also give r i se to a r t e f a c t u a l d i scont inu i t i es in t h e p lo t s (25).

We have , however , a t t e m p t e d to fit s t r a igh t l ines to t h e Arrhenius p lo t s of spin label mot ion , to p roduce d i scont inu i t i es and to de r ive ' appa ren t t r ans i t ion t e m p e r a t u r e s ' whe reve r poss ible . The values ob ta ined a r e shown in Table II. While t he S values cons i s t en t ly p roduced s t r a igh t l ine p lo t s over t h e r a n g e , t h e r § did not appea r to be any cons i s t ency in t he a p p a r e n t t r ans i t ion t e m p e r a t u r e s der ived from τ ρ p lo t s , e i t he r within one spec ies , or b e t w e e n spec ies based on tfieir chil l ing sens i t iv i ty . It is n o t i c e a b l e however t h a t t he a p p a r e n t t r ans i t ion t e m p e r a t u r e for m e a s u r e m e n t s of τ wi th t he var ious spin labels a r e a lways in t he o rder 5NS-A > 12NL S 12NS-Me ind ica t ing t h a t t h e cho ice of spin- label may e x e r t an e f fec t on t he a p p a r e n t t r ans i t ion t e m p e r a t u r e . Such an e f fec t has previously been r e p o r t e d by O v e r a t h

TABLE I.


_ J I 1 L _

3 3 3 4 3 5 3 6

i o4

/ t ( ° K )

FIGURE 1. Arrhenius plots of spin-label motion in the polar lipid of tomato chloroplasts. The values for S and t q with 5NS-A have been derived from the same spectra. The straight lines drawn beside each set of points merely serve to assist in interpreting the shape of the lines created by the data points.

and Trauble (18) who found va r i a t ions of 7-8 C in t he t r ans i t ion t e m p e r a t u r e of t h e m e m b r a n e phospholipids of E. coil", depending on t h e spin label used. It should be s t r e s sed , however , t h a t a c c u r a t e f i t t ing of s t r a igh t l ines to d a t a which d e v i a t e as l i t t l e from a s t r a igh t l ine as those ob t a ined with 12NS-Me, is a diff icult unde r t ak ing .


TABLE II. Apparent Transition Temperatures Derived from the Motion of Spin Labels in Multibilayers of Chloroplast Polar Lipids

Spin label Parameter measured

Apparent transition temperature C

Tomato 5NS-A

12NL 12NS-Me

5N10

k τ Partition

None

21 17 12 17

Maize 5NS-A

12NL 12NS-Me

k τ

0 τ

0

None 14 12

8

Pea 5NS-A

12NL 12NS-Me τ

0

None 15

9 6

C. Differential Scanning Calorimetry

The t h e r m o t r o p i c p r o p e r t i e s of ch loroplas t polar lipid, m e a s u r e d by d i f fe ren t ia l scanning c a l o r i m e t r y a r e shown in F igure 2. In each c a s e , a smal l b road t r ans i t ion could be d e t e c t e d over t h e r a n g e from -20 to + 15 C. Entha lpy values (ΔΗ) for t he se t r ans i t ions , when c o m a p r e d to a pur i f ied phospholipid such as d io leylphosphat idylchol ine (ΔΗ, 14.5 c a l / gm) (7) sugges t t h a t less than 5% of t h e lipid is p a r t i c i p a t i n g in t h e t r ans i t ion . The possibi l i ty t ha t smal l amoun t s of p igmen t , st i l l r ema in ing in the lipid p r e p a r a t i o n s , could be responsib le for t he t r ans i t ion has no t y e t been e l imina ted . The fac t , however , t h a t t he t r ans i t ion is observed over t he s a m e r a n g e for all t h r e e samples , s e e m s to e l im ina t e it as a f ac to r in chill ing sens i t iv i ty .

D. Monolayer Experiments

Monolayer s tudies on m e m b r a n e lipids have in genera l been confined to s y n t h e t i c phospholipids wi th a c l ea r ly def ined f a t t y ac id compos i t ion . However , na tu ra l ly occur r ing lipids con ta in a d ivers i ty of f a t t y ac ids , a l though MGG and DGG of higher p lan t ch lo rop las t s a r e c h a r a c t e r i z e d by


τ 1 1 1 1 Γ

T o m a t o

l_ j ι ι ι ι i _ l

2 6 0 2 8 0 3 0 0 T e m p e r a t u re °K

FIGURE 2. Thermotropic properties of chloroplast polar lipid measured by differential scanning calorimetry. Approximate energy contents ( ΔΗ) of the transitions were : tomato, 0.4 cal/gm; maize, 0.5 cal/gm; pea 0.3 cal/gm.

high c o n t e n t s (80-95%) of α - l ino len ic ac ids (all ClS-9 ,12 ,15-octadeca-t r i eno ic ac id) . We have s tud ied t h e molecu la r pack ing p r o p e r t i e s of ch loroplas t polar l ipids, by i so la t ing individual lipids wi th widely di f fer ing f a t t y ac id compos i t ion , from d iverse p lan t and algal ch lo rop las t s , and compar ing the i r f o r c e - a r e a curves in monolayer e x p e r i m e n t s (Bishop et al., unpubl ished d a t a ) .

F o r c e - a r e a curves for s y n t h e t i c phosphat idy lchol ines conta in ing e i g h t e e n - c a r b o n f a t t y ac ids a r e shown in F igure 3 . The di-18:0 compound forms a very condensed mono laye r . More expanded monolayers a r e fo rmed as the d e g r e e of u n s a t u r a t i o n of the f a t t y acid chains is i nc reased , a l though the major change occu r s with t he in t roduc t ion of one double bond per f a t t y acid mo lecu le .

The f o r c e - a r e a cu rve of h y d r o g e n a t e d s i lver b e e t MGG (Fig. 4) is s imi lar to t h a t of di-18:0 phospha t idy lcho l ine , in being very condensed , and approach ing t h e l imi t ing a r e a occup ied by two long-chain s a t u r a t e d ac ids . The MGG from si lver b e e t , which con ta ins 94% t r i eno ic f a t t y ac ids (DB1, 2.9) does no t however form a monolayer as expanded as d i -18:3 phospha t idy lcho l ine , but r a t h e r , one which is s imi lar to di-18:1 phospha t idy lchol ine , ind ica t ing t h a t head group i n t e r a c t i o n s in t h e MGG molecu le a r e s t ronge r than in the phospha t idy lchol ine mo lecu l e . This is

3 8 2 D. G. B i s h o p et at.

FIGURE 3. Force-area curves of synthetic phosphatidylcholines, (a) di 18:0-PC, (b) di-18:l-PCt (c) di-18:2-PCt (d) di-18:3-PC.

d

5 0 100 150 2 0 0

molecul e

FIGURE 4. Force-area curves of chloroplast monogalactosyldiacyl-glycerols. (a) hydrogenated silver beet MGG, DBI, 0; (b) silver beet MGG, DBI, 2.9; (c) Ulva MGG, DBI, 3.3; (d) Synechococcus MGG, DBI, 1.0; (e) Anabaena MGG, DBI, 1.5.

T h e I n f l u e n c e of F a t t y Acid U n s a t u r a t i o n o n Fluidi ty

J L

5 0 100 150 ? 2 /

A / m o l e c u l e

FIGURE 5. Force-area curves of chloroplast digalactosyldiacylgly-cerols. (a) hydrogenated silver beet DGG, DBI, 0; (b) silver beet DGG, DBI, 2.6; (c) Ulva DGG, DBI, 1.9; (d) Synechococcus DGG, DBI, 1.3; (e) Anabaena DGG, DBI, 1.7.

conf i rmed by t h e fac t t h a t t h e fo rce a r e a cu rves of t h r e e o the r n a t u r a l l y occu r r ing MGG's, whose DBI's r a n g e from 1.0 t o 3 .3 , a r e s imi lar to those of s i lver b e e t MGG. Inc rease s in t h e d e g r e e of f a t t y ac id u n sa tu r a t i o n above m o r e than one double bond per mo lecu le h a v e l i t t l e e f f ec t on the pack ing p r o p e r t i e s of the MGG molecu le .

The fo rce a r e a cu rves of DGG's whose DBI's r a n g e from 0 to 2.6 a r e shown in F igu re 5. The fully s a t u r a t e d compound forms a monolayer which is m o r e expanded than t h a t of t h e cor responding MGG and which does not c o m p r e s s to t he s a m e e x t e n t . This no doubt r e f l e c t s the l a rge r s i ze of t he DGG h e a d group, but is also a p p a r e n t t h a t t he n a t u r a l l y occur r ing DGG's a r e also d o m i n a t e d by head -g roup i n t e r a c t i o n s .

The f o r c e - a r e a curves for t he four major po la r lipids of s i lver b e e t ch lo rop las t s a r e shown in F igu re 6. The cu rves for MGG and DGG a r e a lmos t iden t i ca l , de sp i t e t h e d i spa r i ty in s ize of the i r headgroups , and the only minor d i f f e r ences in the i r f a t t y ac id compos i t ion . Compar i son of t h e MGG and DGG curves from e a c h of t h e o t h e r spec ies (Figs. 4 & 5) conf i rms the s imi l a r i ty in the f o r c e - a r e a cu rves of the two ga lac to l ip ids from any one sou rce . Al though both SL and PG form m o r e condensed mono laye r s than the ga lac to l ip ids , they do not e x e r t any condensing e f f ec t on t h e ga lac to l ip ids , as t h e f o r c e - a r e a cu rve of a m i x t u r e of t h e

3 8 3


FIGURE 6. Force-area curves of silver beet chloroplast polar lipids (a) MGG, DBI, 2.9; (b) DGG, DBI, 2.6; (c) SL, DBI, 1.5; (d) PG, DBI, 1.3. The force-area curve of a mixture of the four lipids in the molar proportions in which they occur in the chloroplast (MGG:DGG:SL:PG, 50:33:13:4) is superimposable on the curves for MGG and DGG.

four lipids in t he p ropor t ions in which they occur in t he ch loroplas t , is i den t i ca l to t ha t of the two ga lac to l ip ids . (This cu rve is no t shown in F ig . 6 simply for c l a r i ty ) . Thus t he packing p r o p e r t i e s of t h e ch loroplas t m e m b r a n e polar lipid is d e t e r m i n e d by the MGG and DGG and is r e m a r k a b l y s imi lar for a v a r i e t y of spec ies with widely differ ing f a t t y acid compos i t ion .

E. ±KJ

C-Nuclear Magnetic Resonance

13 We have ex t ended our p rev ious s tud ies of ch loroplas t lipids by C -

NMR (6, 11 , 1Z, 13) by compar ing the m o t i o n a ^ p r o p e r t i e s of some of the MGG's used in t he monolayer e x p e r i m e n t s . C-NMR m e a s u r e m e n t s of si lver bee t DGG in me thano l p e r m i t the iden t i f i ca t ion of all t h r e e g lycerol ca rbon a t o m s , all twe lve sugar carbon a t o a m s and f i f teen of t h e e igh teen carbon a t o m s in the f a t t y acyl chain , on the basis of the i r chemica l shif ts , and t h e longi tudinal r e l a x a t i o n t i m e s (T^'s) of all t h e s e ca rbon a t o m s h a v e been m e a s u r e d (6, 1Z).

T h e i n f l u e n c e of F a t t y Acid U n s a t u r a t i o n o n Fluidi ty 3 8 5

TABLE III. Longitudinal Relaxation Time (Tj) of Individual Carbon Atoms of Hydrogenated Chloroplast Galactolipids in Methanol

Carbon atom 7 \ (Sec) MGG DGG

Fatty acyl chain 2 0.45 0.38 3 0.7 0.38 4 l . l

a 0.58

5 l . la 1.4

a

6 1.1? 1.4? 7-14 0.9° 1.2° 15 l . l

a 1.4°

16 2.9 3.5 17 4.1 4.6 18 3.3 5.0

Sugar molecules* 1" 0.45 0.21 2" 0.39 0.27 3" 0.35 0.21 4" 0.27 0.15 5" 0.42 0.21 6" 0.34 0.10 1 " - 0.26 2" - 0.23 3" - 0.21 4" - 0.20 5" - 0.26 6" - 0.26

Glycerol molecule* V 0.13 0.14 2' 0.26 0.27 3' 0.17 0.12

*,Sjee Fig. 2 in Bishop et al.f (6) for identification of carbon atoms. 1 Average value of unresolved peaks.

Table ΠΙ shows the T^'s of t h e individual ca rbon a t o m s in t he sugar head groups , g lycerol molecu les and f a t t y acyl chains for h y d r o g e n a t e d s i lver b e e t MGG and DGG. The T^'s of t h e sugar headgroups of h y d r o g e n a t e d DGG a r e lower than those of MGG showing tha t the

3 8 6 D. G. B i s h o p et ai

TABLE IV. Longitudinal Relaxation Time (TJ of Individual Carbon Atoms of Linolenic Acid Molecules in Chloroplast Glycolipids

Carbon atom ΤΊ (sec)

Silver Beet Ulva

MGG DGG SL MGG

2 0.50,0.47 0.30,0.25 0.29 0.52 3 0.72 0.53 0.44 0.76

4-6 0.84 0.72 0.67 0.79 7 1.1 0.91 0.86 0.80 8 1.2 1.5 1.0 1 A

h 9 1.9 2.1 1.2 1 J

b 10 1.9 2.1 1.2 2.4b

h 11 2.3 2.9° 2.6° 2.7

b

h 12 3.6 3.9 2.8 3.4

b

b 13 3.6 3.9 2.8 3.4

b

14 4.5 2.9° 2.6° 2.7b

15 7.4 7.6 4.6 6.6b

16 7.5 7.4 4.6 7.0b

JO. 4? 17 12.0 11.1 9.7 7.0

b

JO. 4? 18 8.6 8.9 6.5° 8.1

b

Average value of unresolved peaks.

headgroups a r e m o r e t igh t ly packed . Similar ly t h e TVs of t h e carbon a t o m s in the f a t t y acy l cha in , which a r e nea r to the headgroups (carbons 2-4) a r e sho r t e r in hyd rogena t ed DGG than in MGG b e c a u s e of t h e t i g h t e r pack ing of t he headg roup . However , t owards t he me thy l end of t he f a t t y acyl chain , t h e T^'s of t he DGG carbon a t o m s a r e longer than those in MGG because the l a rger s ize of the DGG headgroup p e r m i t s m o r e mot iona l s p a c e a t t h e me thy l end of t h e chain , even though DGG headgroups a r e m o r e t igh t ly p a c k e d . These r e s u l t s a r e in exce l l en t a g r e e m e n t wi th t he monolayer m e a s u r e m e n t s of t hese compounds .

The T ' s of carbon a t o m s in t he 18:3 f a t t y ac ids in s i lver b e e t MGG, DGG, SL and Ulva MGG a r e shown in Table IV. Silver b e e t MGG and DGG con ta in essen t ia l ly only 18:3 ac id (12), while SL con ta ins app rox ima te ly 50% 18:3 and 50% 16:0 (13) and Ulva MGG con ta ins 30%

T h e I n f l u e n c e of F a t t y Ac id u n s a t u r a t i o n o n Fluidi ty 3 8 7

18:3 wi th 57% t e t r a e n o i c ac ids (16:4 and 18:4). Compar i son of t h e T^'s of the f a t t y acyl ca rbon a t o m s in s i lver b e e t MGG and DGG show tha t overa l l mot ion is s imi lar in bo th compounds e x c e p t a t t h e carboxyl end of the cha in . This f ac t a g r e e s well wi th t he i n t e r p r e t a t i o n of the f o r c e - a r e a cu rves . The d i f f e rences in mot ion a t t h e m e t h y l end of the chains in hyd rogena t ed MGG and h y d r o g e n a t e d DGG is not appa ren t in the na tu r a l l y occur r ing compounds . In c o n t r a s t , mot ion of t h e ca rbon a t o m s in t he 18:3 chain of SL is cons iderab ly less than t h a t of the cor responding a t o m s in MGG and DGG, b e c a u s e of t h e p r e s e n c e in t h e SL molecu le of the 16:0 chain (13). However , a fu r ther i nc r ea se in the d e g r e e of u n s a t u r a t i o n of t h e companion ac ids in t h e acyl chains of MGG, as occu r s in Ulva MGG, c lea r ly does no t h a v e a m a r k e d e f fec t on t h e mot ion of t h e 18:3 chain (Table IV), even though e x a c t d e t e r m i n a t i o n of some T^'s is diff icul t b e c a u s e some averag ing wi th s ignals of ca rbon a t o m s from t h e t e t r a e n o i c ac id chains occu r s . Again this r e su l t is in exce l l en t a g r e e m e n t wi th t h e monolayer e x p e r i m e n t s (Fig. 4) in which it was found t h a t s i lver b e e t and Ulva MGG's have a lmos t iden t i ca l f o r c e - a r e a cu rves .

IV. DISCUSSION

The hypothes i s t h a t chil l ing sens i t iv i ty in h igher p l an t s is due to phase changes in p a r t or all of the m e m b r a n e lipids c a r r i e s an impl ic i t a s sumpt ion t h a t all m e m b r a n e s in a p a r t i c u l a r p l an t show t h e phase t r ans i t i on a t t he s a m e t e m p e r a t u r e (22, 23). The p r e s e n t r e s u l t s i n d i c a t e t h a t t h e componen t l ipids of t h e thylakoid m e m b r a n e s from a v a r i e t y of p h o t o s y n t h e t i c ce l l s , h a v e ve ry s imi la r phys ica l p r o p e r t i e s . On the basis of f a t t y ac id compos i t ion , spinlabel mot ion and d i f fe ren t i a l scanning c a l o r i m e t r y , we h a v e no t found it possible to d i f f e r e n t i a t e b e t w e e n the p r o p e r t i e s of ch loroplas t m e m b r a n e polar l ipids, wi th r e s p e c t to t h e d e g r e e of chil l ing sens i t iv i ty of the p l a n t . The va l id i ty of the major phys ica l t echn iques used to es tab l i sh t h e p r e s e n c e of phase changes , spin label mot ion and f luo rescence , has c o m e under ser ious ques t ioning (20, 25). A p p a r e n t d i scon t inu i t i e s in Ar rhen ius p lo t s h a v e been found above 0 in a v a r i e t y of p l an t s , bo th chil l ing sens i t ive and r e s i s t a n t (20). The high d e g r e e of u n s a t u r a t i o n of ch loroplas t m e m b r a n e polar lipids would i n d i c a t e t h a t s igni f icant phase changes would no t occur above 0 . D i f f e ren t i a l scanning c a l o r i m e t r y m e a s u r e m e n t s of h igher p lan t MGG and DGG h a v e i nd i ca t ed t h a t phase t r ans i t i ons occur a t abou t -30 and -50 C r e s p e c t i v e l y (27). The t r ans i t i on t e m p e r a t u r e s of dioleyl phospha t idy lcho l ine (-22 C) and s t ea ry l -o ley l -phospha t idy lcho l ine (3 C) (19), molecu les wi th a lower d e g r e e of u n s a t u r a t i o n than those found in ch loroplas t m e m b r a n e s a r e s t i l l below t h e r a n g e a t which chil l ing s y m p t o m s deve lop in sens i t ive p l a n t s . Al though head group compos i t ion can also e f f ec t t h e t e m p e r a t u r e of phase t r ans i t i ons , such e f f e c t s a r e diminished when u n s a t u r a t e d ac ids a r e p r e s e n t in the molecu le (7). It does no t appea r possible t h a t ch loroplas t m e m b r a n e lipids con ta in

3 8 8 D. G. B i s h o p et al

s ignif icant a m o u n t s of molecu la r spec ies of lipids con ta in ing two s a t u r a t e d f a t t y ac ids , as such ac ids a r e only minor c o m p o n e n t s of h igher p l an t MGG and DGG (10, 24, 29), but t h e s e two lipids appea r to es tab l i sh t he pack ing c h a r a c t e r i s t i c ^ of t he ch loroplas t m e m b r a n e lipid.

The monolayer and C-NMR s tud ies desc r ibed he re in show t h a t ga lac to l ip ids wi th a d iverse f a t t y ac id compos i t ion have very s imi lar physica l p r o p e r t i e s , provided t h a t they have a double bond index of one , or g r e a t e r . The ava i lab le ev idence wi th s y n t h e t i c phospholipids which conta in a t l eas t one m o n o u n s a t u r a t e d chain sugges t s t h a t such compounds will h a v e phase t r ans i t ions around 0 C or lower . Most na tu ra l l y occur r ing lipids have such a d e g r e e of u n s a t u r a t i o n or h igher .

We conc lude t h e r e f o r e t h a t on t h e ava i lab le ev idence phase t r ans i t ions or s epa ra t i ons would no t be e x p e c t e d to occur above 0 in t he bulk lipids in ch loroplas t m e m b r a n e s of e i t he r ch i l l ing-sens i t ive or ch i l l ing- res i s tan t p l an t s and t h a t ev idence a l r eady ob ta ined from Arrhenius p lo t s of spin label mot ion , or m e a s u r e m e n t s using f luorescen t labels mus t be t r e a t e d with g r e a t cau t ion . Cons ide ra t ion of t he m e m b r a n e lipid f a t t y acid compos i t ion of o the r m e m b r a n e s in higher p l an t s leads us to s imi lar conclusions in those cases a lso . Whether chil l ing sens i t iv i ty in higher p l an t s is due to t e m p e r a t u r e - i n d u c e d confo rma t iona l changes in m e m b r a n e p ro t e in s which i n i t i a t e changes in t h e molecu la r o rder ing of m e m b r a n e lipids r e m a i n s an open ques t ion .


1. Bagnal l , D. G. and Wolfe, J . A. J. Exptl. Bot. 29, 1231-1242 (1978).

2. Benson, A. A. In " S t r u c t u r e and Func t ions of Chlorop las t s" (M. Gibbs, ed.) pp . 129-148. Springer , Berlin (1971).

3 . Bishop, D. G. Arch. Biochem. Biophys. 154, 520-526 (1973). 4 . Bishop, D. G. Photochem. Photobiol. 20, 281-299 (1974). 5. Bishop, D. G. and Smill ie, R . M. Arch. Biochem. Biophys. 137,

179-189 (1970). 6. Bishop, D. G., Nolan, W. G., Johns , S. R . and Willing, R. I. In

"Light Transducing Membranes : S t r u c t u r e , Func t ion and Evolut ion." (D. W. D e a m e r , ed.) pp . 269-288. A c a d e m i c P res s , New York (1978).

7. van Dijck, P . W. M., de Kruijff, B., van Deenen , L. L. M., de Gier , J . and D e m e l , R. A. Biochim. Biophys. Acta 455, 576-587 (1976).

8. Gaffney, B. J . In "Spin Label ing Theory and Appl ica t ions" (L. J . Ber l iner , ed.) pp . 567-571 . A c a d e m i c P re s s , New York (1976).

9. Gray , B. F . , Gray , P . and Kirwan, N. A. Combustion and Flame 18, 439-459 (1972).

10. J amieson , G. R. and Reid , Ε. H. Phytochem. 10, 1837-1843 (1971).

11 . Johns , S. R., Les l ie , D . R., Willing, R. I. and Bishop, D. G. Aust. J. Chem. 30, 813-822 (1977a).


12. Johns , S. R. , Les l ie , D . R. , Willing, R. I. and Bishop, D. G. Aust. J. Chem. 30, 823-834 (1977b).

13. Johns , S. R., Les l ie , D . R. , Willing, R. I. and Bishop, D. G. Aust. J. Chem. 31, 65-72 (1978).

14. Lyons, J . M. Ann. Rev. Plant Physiol. 24, 445-466 (1973). 15. Lyons, J . M. and Raison , J . K. (This volume) (1979). 16. Mehlhorn, R., Snipes, W. and Ke i th , A. Biophys. J. 13, 1223-1231

(1973). 17. Nolan, W. G., and Bishop, D. G. Arch. Biochem. Biophys. 190, 4 7 3 -

473-482 (1978). 18. O v e r a t h , P . and Traub le , H. Biochemistry 12, 2625-2634 (1973). 19. Phi l l ips , M. C. , Hause r , H. and Pal tauf , F . Chem. Phys. Lipids 8,

127-133 (1972). 20. Quinn, P . J . and Wil l iams, W. P . Prog. Biophys. Molec. Biol, (in

press) (1979). 2 1 . Ra ison , J . K. J. Bio energetics 4, 285-309 (1973). 22. Raison , J . K. In "Mechanisms of Regu la t ion of P l an t Growth" (R.

L. Bieleski , A. R . Ferguson and Μ. M. Cresswe l l , eds . ) , p p . 487-497 . Royal Socie ty of New Zea land , Well ington (1974).

23 . Raison , J . K. and C h a p m a n , E. A. Aust. J. Plant Physiol. 3, 2 9 1 -299 (1976).

24. R u l l k o t t e r , J . , He inz , E. , and Tulloch, A. P . Z. Pflanzenphysiol. 76, 163-175 (1975).

25. Schre ie r , S., Po lnaszek , C. F . and Smith , I. C . P . Biochim. Biophys. Acta 515, 375-436 (1978).



TEMPERATURE REGULATION O F PLANT FATTY ACYL DESATURASES

Paul Mazliak

L a b o r a t o i r e de Physiologie Ce l lu la i re Un ive r s i t e P . & M. Cur ie

Pa r i s , F r a n c e

It has been r e p e a t e d l y obse rved t h a t t h e lipids from p l a n t s grown a t low t e m p e r a t u r e a r e genera l ly en r i ched in u n s a t u r a t e d f a t t y ac ids as c o m p a r e d wi th t h e lipids from t h e s a m e spec ies grown a t h igher t e m p e r a t u r e s (4). However , excep t ions to this gene ra l t r e n d have been s ignal led (1).

I. INCREASE IN FATTY ACID UNSATURATION AT LOW TEMPERATURE

The gene ra l ru le implying t h a t t h e m e a n u n s a t u r a t i o n of p l an t f a t t y ac ids d e c r e a s e s as t h e g rowth t e m p e r a t u r e i n c r e a s e s was i l l u s t r a t e d r e c e n t l y in our l abo ra to ry , in two se r ies of ana lyses concern ing flax s t e m and r a p e - s e e d lipids (Tables I and Π). At t h e lowest t e m p e r a t u r e s , lipids w e r e en r i ched in t h e most u n s a t u r a t e d f a t t y ac ids (l inolenic ac id in t h e ca se of flax and l inoleic plus l inolenic ac ids in t h e case of r a p e - s e e d s ) . If one a d m i t s t h e c lass ica l se r i es of d e s a t u r a t i o n s p o s t u l a t e d for l inolenic ac id b iosynthes i s :

Dl „

D2 „

D3 n

^ ^ ι η . ι — T T T ^ ^ ι η . -> τ τ τ ^ ^ ι '18:0 -2H 18:1 -2H 18:2 -2H " 1 8 : 3

w h e r e D^ , D^ and D^ r e p r e s e n t t h r e e d i f fe ren t acyl d e s a t u r a s e s , t h e p r eced ing r e su l t s m e a n t h a t low t e m p e r a t u r e s favour speci f ica l ly t h e funct ioning of t h e las t d e s a t u r a s e s of t h e chain (D^, l inoleyl d e s a t u r a s e and D^ , o ley l -desa tu rase ) while h igher t e m p e r a t u r e s p rovoke t h e r e l a t i v e a c c u m u l a t i o n of i n t e r m e d i a t e compounds in this chain (oleic and l inoleic ac ids) .

It is genera l ly thought t h a t t h e i n c r e a s e in u n s a t u r a t i o n of p lan t lipids a t low t e m p e r a t u r e f irs t conce rns m e m b r a n e lipids (16). The f luidi ty or v iscos i ty of t he se m e m b r a n e s is p r imar i ly d e t e r m i n e d by t h e

Copyright * 1979 by Academic Press. Inc. 3 9 1 All rights of reproduction in any form reserved

ISBN σ ΐ 2 4β056Ο5

3 9 2 P. Mazl iak

TABLE L Influence of Temperature on the Fatty Acid Compositions of Flax Stem Lipids The percentages given in this table are the means of results

given by three different samples. Each sample contained four stem apex (ca 300 mg) (from Moutot and Mazliak, unpublished).

Linum usitatissimum L. (flax)

Varieties % total fatty acid weight 3t

C14

C16

Ci 6

A C1 8

C1 8 : l

C1 8 : 2

C1 8 : 3

O c e a n

22°C 3 . 2 14 .6 3 . 2 1.6 1.7 1 5 . 1 6 1 . 3 27°C 3 . 8 1 9 . 1 3 . 4 2 . 2 2^4 2 0 . 6 4 8 . 6

He ra

22°C 3 . 1 1 5 . 4 3 . 0 2 . 0 2 . 2 16 .0 5 8 . 1 27°C 2 .9 14 .6 3 . 3 2 . 0 3^3 2 1 . 9 5 1 . 9

Prekul i 665

22°C 3 . 9 15 .7 2 . 9 1.7 1.3 1 2 . 2 6 2 . 2 27°C 2 .7 2 0 . 2 2 . 2 0 . 8 2 . 4 2 2 . 2 4 6 . 9

TABLE II. Influence of Temperature on the Fatty Acid Composition of Rape Seeds, Eight Weeks after Flowering Average data for two separate samples, from Tremolieres, et

al, (18).

Brassica napus L. var. Primor (Oerucic) (Rape seed)

% total fatty acid weight Daylength 16 hr Daylength 9 hr

Maturation temperature 12°

(BC)

17° 22° 27° 17° 22[

C16 8,2 9 9,5 7 7,6 8,5

C16:l 0,6 0 6 0,7 0,9 0,3 0,6

C18 1,6 2 2,5 2 2,1 0,6

C18:l 47,5 48,2 57,2 56 50 54

C18:2 28,5 26,5 19,5 21,5 26 24,7

c ^ 18:3 13,5 13,7 10 9 13,7 11,2

T e m p e r a t u r e R e g u l a t i o n of P l a n t F a t t y Acyl D e s a t u r a s e s 3 9 3

d e g r e e of u n s a t u r a t i o n of t h e acyl r e s idues p r e s e n t in lipid molecu le s . This f luidi ty a f f e c t s t h e a c t i v i t y of m e m b r a n e - b o u n d e n z y m e s and t h e mobi l i ty of m e m b r a n e p r o t e i n s . So, i t would be f i rs t to ma in ta in a c o r r e c t m e m b r a n e f luidi ty, to allow t h e p rope r funct ions of cel l m e m b r a n e s , t h a t t h e i n c r e a s e in f a t t y ac id u n s a t u r a t i o n induced by low t e m p e r a t u r e , would occur . As a m a t t e r of f ac t , t he e n r i c h m e n t of m e m b r a n e lipids in p o l y u n s a t u r a t e d f a t t y ac ids , a t low t e m p e r a t u r e , has been observed in some p lan t o rgane l l e s , for example mi tochondr i a (Table ΠΙ).

TABLE III. Influence of Growth Temperature on the Fatty Acid Composition of Isolated Wheat Motochondria. From Miller et al. (14).

Triticum aestivurn L. (wheat)

Growth moles percent Variety temperature

(°C) C16

C18

c ^18:1

c 18:2

c ^18:3

Marquis Marquis

2° 24°

26.1 23.1

1.9 1.3

2.6 5.8

16.9 39.9

52.8 29.6

Capelle Capelle

2° 24°

23.3 26.7

0.7 1.0

3.6 4.2

16.4 38.4

56.0 29.9

Rideau Rideau

% 24°

23.9 23.8

0.9 1.0

5.0 5.6

15.0 37.0

55.3 32.8

Kharkov Kharkov

2° 24°

22.8 24.4

1.0 1.7

4.9 6.2

17.2 37.6

54.2 30.1

However , in one single case in our l abo ra to ry , this i n c r e a s e in lipid u n s a t u r a t i o n of mi tochondr ia l m e m b r a n e s a t low t e m p e r a t u r e , has no t been observed : this was for mango f rui ts s t o r e d a t d i f fe ren t t e m p e r a t u r e s . In t h a t c a se , chil l ing injury deve loped over t h e f rui ts s t o r e d a t low t e m p e r a t u r e and we h a v e been able to show: 1) t h a t no i nc r ea se in lipid unsa tu r a t i on o c c u r r e d in mango mi tochondr i a (Table IV); and 2) t h a t t h e s u c c i n a t e oxida t ion c a p a c i t y of t h e o rgane l les was marked ly r e d u c e d , which could probably be r e l a t e d to t h e d e v e l o p m e n t of chil l ing injury (10).

3 9 4 P. Mazl iak

TABLE IV. Influence of Storage Temperature on the Fatty Acid Composition of Mango Mitochondria Isolated after 28 Days of Storage, from Kane et al.t (10).

Mangifera indica L., c v . Amelie (mango)

Storage moles percent temperature

C16

c ^16:1

C18

c ^18:1

c ^18:2

c ^18:3

4 34 24 3 14 15 10 8 34 26. 5 3 12.5 16 8

12 26 35. 5 4 15 13.5 6 20 26 37 2 15 12 8

II. REGULATION OF UNSATURATED FATTY ACID SYNTHESIS FROM C-ACETATE

To unde r s t and t h e p r e s u m e d adap t ive abi l i ty of many p l an t s to change thei r m e m b r a n e f luidity a t low t e m p e r a t u r e , i t was n e c e s s a r y to s tudy t h e r egu la t ion of p l an t f a t t y ac id d e s a t u r a s e s by t e m p e r a t u r e and o t h e r f a c t o r s .

F i r s t we h a v e checked , wi th labe led p r e c u r s o r s , t h a t t h e biosynthes is of po lyunsa tu r a t ed f a t t y ac ids was e i ^ c t i v e l y more a c t i v e a t low t e m p e r a t u r e . On the one hand, using C O ^

as a p r ecu r so r

pho tosyn thes i zed by flax s t e m s , we have found t ha t the b iosynthes is of l inoleic and, overa l l , l inolenic acids was rea l ly much m o r e in tense in t he se s t e m s a t 22 C, as c o m p a r e d wi th t h e b iosynthes is <j>|the s a m e acids a t 27 C (Fig. 1). On t h e o the r hand, using NH^- C - o l e a t e as a p recu r so r , we have observed t h a t exposing r a p e - s e e d s to low t e m p e r a t u r e s (12-17 C) during the m a t u r a t i o n of t h e seeds induced higher o l e a t e and l ino lea te d e s a t u r a t i o n in t he si l iqua (Table V).

A s imple exp lana t ion for the accumula t i on of p o l y u n s a t u r a t e d f a t t y acids in p l an t s , a t low t e m p e r a t u r e , was of fered t en y e a r s ago by Har r i s and J a m e s (8). Using 2- C - a c e t a t e as a lipid p recu r so r , t hese au tho r s showed t ha t t h e r e l a t i v e amoun t of u n s a t u r a t e d f a t t y acids s y n t h e t i z e d by the s l ices of var ious seeds (cas tor bean , flax, sunflower) i nc r ea sed wi th a d e c r e a s e in t h e t e m p e r a t u r e or wi th an i n c r e a s e in t h e 0 ?

c o n c e n t r a t i o n of cel l fluids. As it had been shown t h a t f a t t y acid dehydrogena t ions r equ i r ed O^ and r e d u c e d pyr id ine nuc l eo t ide as c o f a c t o r s , they concluded t h a t t h e fo rma t ion of u n s a t u r a t e d f a t t y acids by p lan t s was indeed con t ro l l ed by t h e O^ c o n c e n t r a t i o n in cel l s ap . Thus as t he t e m p e r a t u r e dec rea sed , t h e i nc r ea sed c o n c e n t r a t i o n of O - in solut ion would a c t i v a t e t he d e s a t u r a t i o n s y s t e m s . The 0 ? c o n t e n t of t i ssues , however , canno t be t aken as t h e only a spec t of t e m p e r a t u r e

T e m p e r a t u r e R e g u l a t i o n of P l a n t F a t t y Acyl D e s a t u r a s e s 395

0 6 2 4 0 6 2 4

h o u r s

FIGURE 1. Incorporatio^f C02 into the fatty acids of flax stems (variety Ocean). A pulse of C02 was given for 10 min to plants grown either at 22 C (02 J or 27 C (02γ). This pulse was followed by various periods of chase, me lengths of which are indicated in abscissa. The radioactivities of the different fatty acids present in one gram of fresh matter were determined by radio-gas chromatography at the end of each chase-period and plotted in ordinatesfcpm.g ) -T: total fatty acids - U : total unsaturated fatty acids - (from Moutot Jolivet and Mazliak, 1978, unpublished).

ac t ion on f a t t y ac id d e s a t u r a t i o n . Since all d e s a t u r a s e s r e q u i r e O^, i t is to be e x p e c t e d t h a t synthes is of all u n s a t u r a t e d f a t t y ac ids will be r e d u c e d if is p r e s e n t in l imi t ing a m o u n t s only. In f ac t , i t has been found in many t i ssues t h a t d e c r e a s e s in p o l y u n s a t u r a t e d f a t t y acids w e r e c o u n t e r b a l a n c e d by higher p ropor t ions of o le ic ac id i n s t e a d of s a t u r a t e d ones (for a rev iew see B e r i n ^ r (3) ( s ee also t h e d a t a of Tables I - II). Har r i s and J a m e s (8), using O a c e t a t e as a lipid p r ecu r so r , w e r e no t

3 9 6 P. Mazl iak

able to p rove d i r ec t l y t h a t t he a c t i v i t i e s of t he p lan t f a t t y acid d e s a t u r a s e s p roper w e r e dependen t on t e m p e r a t u r e and /o r O^, c o n c e n t r a t i o n . We dec ided to check this hypothes i s d i r ec t l y upon t r u e p lan t f a t t y acid d e s a t u r a s e s . We used for this s tudy seve ra l p lan t d e s a t u r a t i o n s y s t e m s t h a t previously we had been able to ge t funct ion in vitro, i .e . , 1) t h e o l e a t e - d e s a t u r a t i n g sy s t ems from var ious o leaginous seeds (6); and 2) t h e mic rosomal oleyl C o A - d e s a t u r a s e from p o t a t o tube r (2).

ΙΠ. REGULATION OF LINOLEIC ACID BIOSYNTHESIS ( C-OLEATE DESATURATION) IN OLEAGINOUS SEEDS

Sunflower seeds , r a p e seeds or flax seeds w e r e h a r v e s t e d t h r e e weeks a f t e r f lowering, i .e . a t t he t i m e of t h e g r e a t e s t p o l y u n s a t u r a t e d f a t t y acid b iosynthes is . Thin s l ices , cu t w i ^ a r a z o r b lade , w e r e i n c u b a t e d in aqueous solut ions of N H ^ - ( 1 - C ) - o l e a t e ^ t var ious temperatures or under var ious a t m o s p h e r e s (N^, O - or a i r ) . C- l inole ic and C-l inolenic acids fo rmed within t h e s u c e s a f t e r d i f fe ren t incuba t ion per iods w e r e ana lyzed by rad io -gas liquid c h r o m a t o g r a p h y (Fig. 2). F igu re Q3 shows t he k ine t i c s of o l e a t e ^ s a t u r a t i o n a t a fixed t e m p e r a t u r e (20 C) . The greater p a r t of t h e C - o l e a t e (given as a subs t ra te ) is d e s a t u r a t e d in to ^ C- l inole ic acid during t h e first 24 hours ; i t is only a f t e r t ha t t i m e t h a t C- l inolenic acid begins to appea r in smal l a m o u n t s .

F igure 4 shows t h e va r i a t ion of o l e a t e d e s a t u r a t i o n wi th t e m p e r a t u r e in sunflower seeds . Surprisingly, we could obse rve an op t ima l t e m p e r a t u r e for d e s a t u r a t i o n a t about 27 C and i t is c l ea r t h a t c o n t r a r y to what was e x p e c t e d , low t e m p e r a t u r e s (2 , 10 C) did not a c t i v a t e l inoleic ac id fo rma t ion . Similar r e su l t s w e r e ob ta ined wi th flax seeds (Fig. 5) and r a p e seeds (data no t shown).

F igure 6 shows t he va r i a t ion of o l e a t e d e s a t u r a t i o n in sunflower seed s l ices wi th O^ c o n c e n t r a t i o n in t he a t m o s p h e r e above t he s l ices ( then in cell saps) . As expected* no d e s a t u r a t i o n of o l e a t e was observed under N -but t he same d e s a t u r a t i o n in t ens i t i e s w e r e observed under air ( 2 1 % 0^7 and pure oxygen (100% Ο^)· The re fo re , t h e r e is no s imple re la t ionsh ip b e t w e e n O^ c o n c e n t r a t i o n and o l e a t e d e s a t u r a t i o n a c t i v i t y within sunflower seed . Exac t ly s imi lar r e su l t s w e r e found with flax or r a p e seeds (data not shown).

IV. REGULATION OF THE MICROSOMAL OLEYL-CoA DESATURASE FROM POTATO TUBER

This is an inducible sys tem which appea r s during aging of thin p o t a t o s l ices in an a e r a t e d humid medium ( 2 , 1 9 ) . The mic rosomal sys tem has been thoroughly s tud ied in vitro: 1 - C - o l e y l - C o A d e s a t u r a t i o n r e -


υ CD Ο Ό CD

Ί 8 :1 Ί 8 2 Ί 8 . 3

0 5 10 "τ— 15 m m

14 14 14 FIGURE 2. C-oleate desaturation into C-linoleate and C-linolenate by slices of rape-seeds fse| text for incubation conditions). The radio-gas chromatogram of C-fatty acid methyl esters is presented.

quires oxygen, r e d u c e d n i c o t i n a m i d e aden ine d inuc leo t ide (NADH) and bovine se rum albumin. The op t ima l pH is 7.Z; SH-complexing a g e n t s , cyan ide (9), heavy m e t a l s (13) and d e t e r g e n t s (1Z) inhib i ted o ley l -CoA d e s a t u r a t i o n . The l inoleyl r e s idue fo rmed by d e s a t u r a t i o n of o ley l -CoA appea r s a lways e s t e r i f i ed wi thin a phospha t idy lchol ine molecu le and the ques t ion has been r a i sed w h e t h e r o ley l -phospha t idy lchol ine was no t t he t r u e s u b s t r a t e of t h e d e s a t u r a t i o n sys tem (17). The mic rosoma l sys t em is p r e sen t l y envis ioned (7) as compr is ing t h r e e d i f fe ren t p ro t e in s embedded in t h e m e m b r a n e lipid m a t r i x (Fig. 7).

Dr s . Cher i f and Kade r (5), in our l a b o r a t o r y , have s tud ied in vitro t h e o ley l -CoA d e s a t u r a t i n g sys tem of p o t a t o m i c r o s o m e s and t h e e f f ec t s of varying t e m p e r a t u r e and oxygen c o n c e n t r a t i o n . F igure 8 p r e s e n t s t h e k ine t i c s of d e s a t u r a t i o n of o l e y l - c o e n z y m e A in vitro, a t var ious t e m -

3 9 8 P. Mazl iak

C1 8 : 3 p roduce d

incubat io n per io d ( h o u r s )

14 FIGURE 3. Kinetics of C-oleate desaturation by sunflower seed

slices, at 20 C (for incubation conditions, see text).

p e r a t u r e s . The highes t d e s a t u r a t i o n a c t i v i t y (34%) was ob ta ined a t 30°C; lower (ZO C) or h igher (35 C) t e m p e r a t u r e s d e c r e a s e d the in t ens i ty of d e s a t u r a t i o n . It is i n t e r e s t i ng to n o t i c e on the graph t h a t for t h e f irst 15 min of incubat ion , t h e slope of t he curves r e p r e s e n t i n g t h e d e s a t u r a t i o n a c t i v i t i e s is nea r ly s imi lar for these t h r e e t e m p e r a t u r e s . However , t he level of t h e p l a t e a u r e a c h e d a f t e r 30 min va r i e s accord ing to t h e t e m p e r a t u r e s , t he h ighes t level being observed a t 30 C. At low t e m p e r a t u r e s (Z and 10 C), t h e d e s a t u r a t i o n was s t rongly r e d u c e d . At 10 C for i n s t a n c e t h e slopes of t he curve r e p r e s e n t i n g d e s a t u r a t i o n a c t i v i t y aga ins t t i m e is n ine t i m e s lower than t h e slope a t 30 C, when m e a s u r e d for t he f irst 15 min. The p l a t e a u was r e a c h e d a t 10 C a f t e r a longer t ime of incuba t ion (90 min) than a t higher t e m p e r a t u r e s . At Ζ C, t h e p e r c e n t a g e of d e s a t u r a t i o n was a lmos t null ; a low d e s a t u r a t i o n a c t i v i t y (7%) was no t i ced a f t e r 90 min of incuba t ion a t this t e m p e r a t u r e .

Thus, con t r a r i ly to t h e hypothes is of Har r i s and J a m e s (8), t hese e x p e r i m e n t s show t h a t t h e mic rosomal o ley l -CoA d e s a t u r a s e of p o t a t o tube r p r e s e n t s i t s op t ima l a c t i v i t y a t m o d e r a t e d t e m p e r a t u r e s and approx imat ive ly follows c lass ica l enzymic k ine t i c s . At low t e m p e r a t u r e , a l though the solubil i ty of oxygen in t h e incuba t ion medium was g r e a t l y inc reased , o ley l -CoA d e s a t u r a s e a c t i v i t y was ve ry low.


i ncuba t io n p e r i o d ( h o u r s )

14 FIGURE 4. Variation with temperature of C-oleate desaturation

by sunflower seed slices incubated for dj.êrent periods of time as indicated in abscissa. Insets: variation of C-oleate-desaturation with temperature (abscissa) for 1 hour (left) or 24 hours (right) of incubation.

The d e s a t u r a t i o n k ine t i c s of A

^ C - o l e y l - C o A in vitro a t a f ixed t e m p e r a t u r e (30 C) and under a t m o s p h e r e s wi th var ious oxygen and n i t rogen c o n c e n t r a t i o n s a r e r e p r e s e n t e d in F igure 9. The r e su l t s show a c lea r co r r e l a t i on b e t w e e n t h e d e s a t u r a t i o n a c t i v i t y and t h e oxygen c o n t e n t of t h e a t m o s p h e r e only within t h e r a n g e 0 - 20% of oxygen. No va r i a t i on could be obse rved in t h e d e s a t a t u r a t i o n r a t e s for t h e f irs t t en m i n u t e s , b e t w e e n 5 and 20% of oxygen in t he a t m o s p h e r e , but a f t e r this t i m e a p l a t e a u was soon r e a c h e d , wi th t h e h ighes t level of d e s a t u r a t i o n cor responding to 20% of oxygen. No d e s a t u r a t i o n a t all was observed under p u r e n i t rogen . C o n t r a r y again to wha t was e x p e c t e d , no i n c r e a s e in o ley l -CoA d e s a t u r a s e a c t i v i t y was observed under pure oxygen, but ins t ead , some inhibi t ion o c c u r r e d as c o m p a r e d wi th t h e funct ioning of t h e sys tem under no rma l a i r . These r e su l t s conf i rm t h a t oxygen is a

4Ό Ο P. Mazl iak

i n c u b a t i o n p e r io d ( h o u r s )

FIGUFiE 5. Variation with temperature of C-oleate desaturation hy flax-seed slices incubated for different periods of t i m e , as indicated in abscissa.

l imi t ing f ac to r for the d e s a t u r a t i o n but this gas does not appear to be t h e sole or t he main cont ro l l ing f ac to r of t he d e s a t u r a t i o n ac t i v i t y .

VI. CONCLUSION

Based on the r e su l t s r e p o r t e d in this pape r , t he following conclusions may t e n t a t i v e l y be proposed:

1. Di f fe ren t t e m p e r a t u r e s during p lan t deve lopmen t may modify cons iderably the f a t t y acid composi t ion of p lan t l ipids. In gene ra l , lower ing t h e env i ronmen ta l t e m p e r a t u r e p roduces a h igher c o n t e n t of p o l y u n s a t u r a t e d f a t t y acids in cell m e m b r a n e s or r e s e r v e l ipids.

2. T e m p e r a t u r e a f f e c t s t he b iosynthes is of u n s a t u r a t e d f a t t y acids d i r ec t l y and not only by increas ing O^ c o n c e n t r a t i o n in t he cel l s ap . The fo rma t ion of t h e d i f fe ren t u n s a t u r a t e d f a t t y acids is c a t a l y z e d by d i f fe ren t d e s a t u r a s e s which might have d i f fe ren t t e m p e r a t u r e o p t i m a .


Thus, low t e m p e r a t u r e s would favour p o l y u n s a t u r a t e d f a t t y ac id a c c u m u l a t i o n while h igher t e m p e r a t u r e s would favour ole ic (or pa lmi to le ic ) acid a c c u m u l a t i o n .

3 . The molecu la r mechan i sm under ly ing these t e m p e r a t u r e e f f ec t s a r e no t p r e sen t ly unders tood , desp i t e cons iderab le s tudy . A s t imu la t i ng working hypothes is has been put for th r e c e n t l y by Nozawa , Thompson and co -worke r s (11, 15) ind ica t ing t ha t the m e m b r a n e f luidi ty i tself is the r egu la t i ng f ac to r cont ro l l ing t h e a c t i v i t y of t h e m e m b r a n e - b o u n d f a t t y ac id d e s a t u r a s e s . Fol lowing these v iews, c e r t a i n t e m p e r a t u r e changes provoking a l t e r a t i o n s of m e m b r a n e f luidi ty would be dec is ive for t r igge r ing the onset of f a t t y ac id d e s a t u r a t i o n in cel l m e m b r a n e s . As the work of N o z a w a and Thompson (11, 15) was con d u c t ed on t h e p r o t o z o a n Tetrahymena, new s tud ies a r e n e c e s s a r y to check t h e va l id i ty of the i r hypothes is as far as p lan t t i ssues a r e conce rned .

incubat io n p e r io d ( h o u r s )

Ο 14 FIGURE 6. Variation, with oxygen concentration, at 27 C, of C-

oleate desaturation by sunflower seed slices incubated for different periods of time, as indicated in abscissa.

4 0 2 P. Mazl iak

NADH

+ Η

NAD

oleoyl - l i n o l e y l -

2 F e

cytochrom e b> 5

2 F e

mixed-functio n oxidas e

1/2 0,

Η?0

FIGURE 7. The oleyl-desaturation system of plant microsomes.

T i me o f i n c u b a t i o n m in

FIGURE 8. Temperature effect on the activity of the microsomal oleyl-coenzyme A desaturcyse of potato tuber. The incubation temperature, varying from 2 to 35 C is indicated in the graph.


T i me o f i n c u b a t i o n , min

FIGURE 9. Effect of oxygen on the activity of microsomal oleyl-coenzyme A desaturase of potato tubers at 30°C. The oxygen concentration of the atmosphere, varying from zero (N9= pure nitrogen) to 100% is indicated in the graph.

TABLE V. 14

C-oleate Desaturation Activities at 22°C by Seeds from Rape Seeds (Winter Variety Primor (0 erucic) Grown at DiffeKtyit Temperatures during Four Weeks after Flowering NH4- C-oleate was injected in vivo, at 22 C, into different

samples of siliqua; 24 hours later, the seed fatty acids from the different samples were analyzed by radio-gas chromatography, from Tremolieres et al., (18).

% total fatty acid radioactivity Daylength 16 hr Daylenath 9 hr

Maturation Q

temperature 12 17° 22° 27° 17° 22°

^18:1 25.2 ^18:2 53.2

18:3 21.5

58.5 35.2

6.2

86.3 13.7 traces

84 16

79 93.3 15 6.7

5.5

4 0 4 P. Mazl iak

ΥΠ. R E F E R E N C E S

1. Appelqvis t , L. A. j n " R e c e n t Advances in t h e C h e m i s t r y and Biochemis t ry of P lan t Lipids" (T. Gal l ia rd and Ε. I. M e r c e r , edi tors) pp . 247-286, Acad . P res s , London (1975).

2. Ben Abde lkader , Α., Cherif , Α., D e m a n d r e C . and Mazl iak, P . Eu-rop. J. Biochem., 32, 155-165 (1973).

3 . Ber inger , H. In "Proceed ings of t h e 13th IPI - Colloquium of t he In t e rna t iona l Po t a sh In s t i t u t e " held in York, England, 123-133 (1977).

4 . Canvin , D. T. Can. J. Bot., 43, 63-69 (1965). 5. Cherif , A. and Kader , J . C. Potato Res., 19, 21-26 (1976). 6. Cherif , A. and Mazl iak, P . Rev. F. Corps Gras, 25, 15-20 (1978). 7. Fu lco , A. J . Ann. Rev. Biochem., 43, 215-241 (1974). 8. Har r i s , P . and J a m e s , A. T. Biochim. Biophys. Acta, 187, 13-18

(1969). 9. Kader , J . C . Biochim. Biophys Acta, 486, 429-436 (1977). 10. Kane , O. , Marce l l in , Ρ and Mazl iak , P . Plant Physiol., 61, 634-

638 (1978). 11 . Mar t in , C E., H i rami t su , K., Ki ta j ima , Y., Nozawa , Y., Skriver , L.

and Thompson, G. A. Biochemistry, 15, 5218-5233 (1976). 12. Mazl iak, P . C. R. Ac. Sc., 281, 1471-1474(1975) . 13. Mazl iak, P . , D e c o t t e , A. M. and Kader , J . C. Chem. Biol. Int., 16,

115-119 (1977). 14. Miller , R . W., de La R o c h e : I. and P o m e r o y , Μ. K. Plant Physiol.,

53, 426-433 (1974). 15. Nozawa , Y. and Kasa i , R. Biochim. Biophys. Acta, 529, 54-

66 (1978). 16. Raison, J . K. In " R a t e Con t ro l of Biological P roces ses - Symposia

of t he Socie ty for Expe r imen ta l Biology, XXVIT, pp . 485-512, Cambr idge Univers i ty Press (1973).

17. S t y m m e , S. and Appelqvis t , L. A. Europ. J. Biochem., 90, 223-229 (1978).

18. T remo l i e r e s , H., T remol i e r e s , A. and Mazl iak, P . Phytochemistry, 17, 685-687 (1978).

19. Wil lemot , C and Stumpf, P . K. Can. J. Botany, 45, 579-584 (1967).


CHEMICAL MODIFICATION OF LIPIDS IN CHILLING SENSITIVE SPECIES

Judith B. St. John

U.S. D e p a r t m e n t of Agr i cu l tu r e Sc ience and Educa t ion Admin i s t r a t i on

Agr icu l tu ra l R e s e a r c h Agr icu l tu ra l Env i ronmenta l Qua l i ty I n s t i t u t e

Weed Sc ience L a b o r a t o r y Bel tsv i l le , Maryland

Our bas ic s tud ies in to t he mechan i sm of he rb ic ide ac t ion sugges ted tha t s u b s t i t u t e d pyr idaz inones might be useful tools wi th which to a l t e r t h e f a t t y acid compos i t ion of p lan t m e m b r a n e l ipids. We ini t ia l ly r e p o r t e d on subs t i t u t ed pyr idaz inones and showed t ha t the e f f e c t s of Sandoz 6706 [4 -ch lo ro -5 - (d ime thy lamino) -Z- (a , a , a - t r i f luo ro -m- to ly l ) -3 (ZH)-pyridazionone] w e r e inhibi t ion of pho tosyn thes i s and i n t e r f e r e n c e wi th ch loroplas t p igmen t deve lopmen t (Z) and inhibi t ion of t h e fo rma t ion of t h e polar lipids (3). The inhibi t ion in t h e fo rma t ion of t h e polar lipids was r e l a t e d to an i nc rease in l inoleic ac id (18:Z) a c c o m p a n i e d by a d e c r e a s e in l inolenic acid (18:3) c o n t e n t of t h e polar l ipids. We sugges ted t h a t Sandoz 6706 might a c t by p r even t i ng t h e fo rma t ion of ch loroplas t m e m b r a n e s , r e su l t ing from inhibi t ion of t h e fo rma t ion of t h e polar l ipids, chlorophyl ls , and ca ro t eno ids n e c e s s a r y for ch loroplas t m e m b r a n e fo rma t ion . We subsequent ly d e m o n s t r a t e d t h a t t h e f a t t y acid compos i t ion of t h e major lipids of t h e ch loroplas t m e m b r a n e s , t h e mono-and d iga lac tosy l d ig lycer ides , can be definably a l t e r e d wi th var ious s u b s t i t u t e d pyr idaz inones (6). The ga lac to l ip id f a t t y ac id compos i t ion of whea t ( Triticum aestivum L. cv . 'Arthur ' ) can be a l t e r e d so t ha t t h e r e is a d e c r e a s e in l inolenic acid a c c o m p a n i e d by an i n c r e a s e in l inoleic acid wi thou t a shift in t h e r e l a t i v e r a t i o of s a t u r a t e d to u n s a t u r a t e d f a t t y ac ids . The f a t t y acid compos i t ion can be sh i f ted to a h igher p ropor t ion of s a t u r a t e d ac ids , and the f a t t y ac id compos i t ion of t h e monoga lac tosy l d ig lycer ides can be a l t e r e d in p r e f e r e n c e to t h e d iga lac tosy l d ig lycer ides .

Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.

Copyright · 1979 by Academic Press, Inc. 4Ό5 All rights of reproduction In any form reserved

ISBN α 12-460560- 5

4 0 6 J. Β. St. J o h n

S t r u c t u r e - a c t i v i t y compar i sons (9) r e v e a l e d t h a t inhibi t ion of t he Hill r e a c t i o n and pho tosyn thes i s by the subs t i t u t ed pyr idaz inones is r e l a t e d to t h e s t r u c t u r e of t h e p a r e n t compound, py razon [5 -amino-4 -chloro-2-phenyl -3(2H)-pyr idaz inone] . Tr i f luoromethy l subs t i tu t ion of t h e phenyl r ing of py razon , mono-me thy l subs t i tu t ion of t he amine , or subs t i tu t ions a t bo th posi t ions resu l t in inhibi t ion of ca ro t eno id b iosynthes is . Both subs t i tu t ions we re r equ i r ed for max imum e f f e c t . D ime thy l subs t i tu t ion of t h e amine of py razon is r e l a t e d to a d e c r e a s e in l inolenic acid accompan ied by an i n c r e a s e in l inoleic acid wi thout a shift in t he r a t i o of s a t u r a t e d to u n s a t u r a t e d f a t t y acids of t h e m e m b r a n e lipids; this subs t i t u t ed pyr idaz inone (Sandoz 9785 = BASF 13 338, h e r e i n a f t e r r e f e r r e d to as BASF 13 338) r e d u c e d levels of m e m b r a n e bound l inolenic ac id but did not i n t e r f e r e wi th chlorophyll or ca ro t eno id b iosynthes is . In addi t ion , of 49 analogs e v a l u a t e d (10), subs t i tu t ion of t h e phenyl r ing in the 2 posi t ion, halogen subs t i tu t ion in the 4 posi t ion, and var ious subs t i t u t ed amino groups in t he 5 posi t ion r e s u l t e d in compounds showing the h ighes t a c t i v i t y . Var ia t ion of subs t i t uen t s a t t hese posi t ions a l lowed shif t ing t he r a t i o of m e m b r a n e - b o u n d l inoleic to l inolenic acid from the con t ro l va lue of 0.50 to 6.7 for BASF 13 338 . The t r i f luo ro me thy l subs t i tu t ion of t h e phenyl r ing and mono-me thy l subs t i tu t ion of the amine a r e r e l a t e d to a shift t oward a higher p ropor t ion of s a t u r a t e d f a t t y acids of chloroplas t m e m b r a n e s .

Studies wi th h igher p l an t s a imed a t e luc ida t ing t he ro le of m e m b r a n e lipids and the i r componen t f a t t y ac ids in p lan t r e sponses to chill ing have re l i ed on approaches which yie lded ind i rec t c o r r e l a t i o n s . Numerous s tud ies have c o m p a r e d ch i l l ing- res i s tan t and chi l l ing-sens i t ive p lan t spec ies and d e m o n s t r a t e d t ha t ch i l l ing- res i s tan t p lan t spec ies conta in more u n s a t u r a t e d f a t t y ac ids , pa r t i cu l a r l y l inolenic ac id (18:3) (4). Secondly, as the t e m p e r a t u r e of g rowth d e c r e a s e s , lipid unsa tu r a t i on inc reases wi th the p redominan t i nc rea se being l inolenic ac id . When we d i scovered t ha t pyr idaz inones could be used to block l inolenic acid b iosynthes is , we r e a l i z e d tha t for the first t i m e it would be possible to chemica l ly man ipu l a t e t he l inolenic acid of p lan t m e m b r a n e s in a given p lan t spec ies and s tudy p lant response to t e m p e r a t u r e .

We have used BASF 13 338 to r e l a t e t he level of l inolenic ac id in t h e m e m b r a n e lipids of c o t t o n ( Gossypium hirsutum L.) roo t t ips to chil l ing r e s i s t a n c e in c o t t o n seedl ings under con t ro l l ed env i ronmen ta l condi t ions (9). Seeds w e r e g e r m i n a t e d a t 15, 20, 25, and 30 C. As t he t e m p e r a t u r e of g rowth d e c r e a s e d from 30 to 15 C, t h e l inolenic acid con t en t of the polar lipid f rac t ion of u n t r e a t e d roo t t ips i nc reased , as ev idenced by a shift in t he r a t i o of l inoleic to l inolenic acid (18:2/18:3) from 2.67 a t 30°C to 1.16 a t 15°C, T r e a t m e n t wi th BASF 13 338 r educed the l o w - t e m p e r a t u r e - i n d u c e d inc rease in l inolenic ac id , as ev idenced by an 18:2/18:3 r a t i o of 2.56 c o m p a r e d with t he cont ro l r a t i o of 1.16. BASF 13 338 lowered the levels of l inolenic acid a t all t e m p e r a t u r e s . Thus, BASF 13 338 also inhibi ts l inolenic acid synthes is in non -pho tosyn the t i c t i ssues in the absence of l ight and blocks the inc reased synthes is of l inolenic acid during chi l l -hardening . When hes i snon-hardened cont ro l

C h e m i c a l Modif icat ion of L ip ids in Chil l ing S e n s i t i v e S p e c i e s 4 0 7

and BASF 13 338 t r e a t e d seedl ings w e r e chi l led a t 8 ° C , t h e BASF t r e a t e d seedl ings wi l t ed within 24 hours . Af t e r 4 days a t 8 C, t he seedl ings w e r e r e t u r n e d to 30 C . The con t ro l seedl ings r e h y d r a t e d and r e s u m e d norma l g rowth , w h e r e a s the t r e a t e d seedl ings r e m a i n e d wi l t ed a f t e r 7 days and e i t he r died or con t inued to a p p e a r a b n o r m a l . The h a r d e n e d con t ro l and ha rdened BASF 13 338 t r e a t e d seedl ings showed fewer l a t e n t s y m p t o m s of chil l ing injury, a l though a g r e a t e r n u m b e r of abnorma l seedl ings occu r r ed in the l a t t e r . Thus, d e c r e a s e d levels of l inolenic acid in the m e m b r a n e s of r oo t t ips c o r r e l a t e d wi th i n c r e a s e d sens i t iv i ty to chil l ing in c o t t o n .

Wil lemot (11) subsequent ly obse rved t h a t when whea t p l an t s w e r e t r e a t e d wi th BASF 13 338 for 36 hours be fore frost ha rden ing , bo th the a c c u m u l a t i o n of l inolenic ac id and deve lopmen t of f reez ing t o l e r a n c e w e r e inh ib i ted . Wil lemot ' s work is d iscussed in de ta i l in an o th e r c h a p t e r of this vo lume .

We have r e c e n t l y ex t ended our s tud ies to t he e f f ec t s of BASF 13 338 on l inolenic ac id levels and hard iness of c e r e a l s under con t ro l l ed env i ronmen ta l and field condi t ions . BASF 13 338 was so i l - incorpora ted a t r a t e s of 0, Z.8, 5.2, or 11.2 kg /ha . Wheat ( Triticum aestivum L. cvs . 'Ar thur ' and 'Po tomac ' ) , , ba r l ey ( Hordeum vulgare L.), and rye (Secale cereale L.) w e r e sown in t h e fa l l . T r e a t e d p l an t s showed ea r ly s y m p t o m s of low t e m p e r a t u r e injury as ev idenced by frost banding . F ros t banding as a symptom of low t e m p e r a t u r e injury in c e r e a l s was f irs t r e p o r t e d by R icha rds (5); injury appea red to occur f irs t a t the soil su r f ace and the bands b e c a m e visible a f t e r leaf e longa t ion . Inc idence and seve r i ty of frost banding w e r e marked ly inc reased in BASF 13 338-t r e a t e d seedl ings . Sever i ty also i n c r e a s e d as c o n c e n t r a t i o n of t he chemica l i nc r ea sed . Bar ley appea red to be the most sens i t ive and rye the l eas t sens i t ive .

Analysis of f a t t y acid compos i t ion of m e m b r a n e lipids from shoots of seedl ings exposed to ha rden ing t e m p e r a t u r e s (as low as -1 .6 C) r e v e a l e d tha t seedl ings grown on soil i n c o r p o r a t e d wi th BASF 13 338 had s igni f icant ly lower leve ls of l inolenic ac id (Table I). R e d u c e d leve ls of l inolenic ac id w e r e r e l a t e d to r e d u c e d survival and r e d u c e d t i l l e r ing (Table Π, t i l le r ing d a t a only). Tables I and Π a r e t a k e n from St. John et al, (8).

In t h e a b s e n c e of BASF 13 338, all spec ies a c c u m u l a t e d l inolenic acid to the s a m e level and y e t spec ies and v a r i e t a l d i f f e rences in sens i t iv i ty to BASF 13 338 w e r e p ronounced . Ar thu r whea t was t h e mos t sens i t ive to the c h e m i c a l , fol lowed by P o t o m a c w h e a t , Monroe ba r ley and Abruzzi r y e . Abruzz i rye d i f fe red from t h e o the r spec ies in t h a t t he change in r a t i o of m e m b r a n e - b o u n d l inoleic to l inolenic ac id leve led off a t a t r e a t m e n t r a t e of 5.6 kg /ha . However , BASF 13 338 con t inued to r e d u c e t i le l r ing of Abruzz i r y e through a r a t e of 11.2 kg /ha . We be l ieve t h a t our combined d a t a i n d i c a t e t h a t i n c r e a s e d leve ls of m e m b r a n e -bound l inolenic ac id a r e a s soc i a t ed wi th the a d a p t a t i o n of p l an t s to func t iona l i ty a t low t e m p e r a t u r e s , but t h a t o the r f a c t o r s l imi t ha rden ing and possibly dis t inguish levels of sens i t iv i ty b e t w e e n cu l t iva r s and

4 0 8 J. Β. St. J o h n

TABLE I. Effects of BASF 13 338 Applied Preemergence on the Ratio of Membrane-Bound Linoleic (18:2) to Linolenic (18:3) Acid Under Field Conditions.

Β BASF 13 338 (kg/ha) Species 0 2 .8 5 .6 11.2

(18:2/18:3) (18:2/18:3) (18:2/18:3) (18:2/18:3)

Arthur wheat 0 .16a+

0. 68b 1. 29c 2. 30d Potomac wheat 0 .16a 0. 67b 1. 00c 1. 68d Monroe barley 0 .14a 0. 31b 0. 59c 0. 87d Abruzzi rye 0 .19a 0. 28b 0. 60c 0. 54c

Values across columns with common letters are are not significantly different at the 5% level by use of Duncan's multiple range test.

TABLE II. Effect of BASF 13 338 Applied Preemergence on Tillering of Winter Cereals During Winter of 1977-78.

BASF 13 338 (kg/ha)

Species 0 2.8 5.6 11.2

mean number of tillers/meter of row Arthur wheat 244a

+ 174b 124c lid

Potomac wheat 185a 173a 60b 5c Monroe barley 164a 138a 60b 3c Abruzzi rye 199a 133b 99b 9c

Means across columns with common letters are not significantly different at the 5% level by use of Duncan's multiple range test.

spec ie s . The d a t a do show tha t for a given spec ies , levels of l inolenic acid can ind ica t e suscep t ib i l i ty to chil l ing injury and might be used as a sc reen ing tool for i so la t ion of m o r e r e s i s t a n t l ines . However , t h e co r r e l a t i on b e t w e e n l inolenic ac id levels and chil l ing is no t c l ea r , and cau t ion is advised when crossing spec ie s .

C h e m i c a l Modif icat ion of L ip ids in Chil l ing S e n s i t i v e S p e c i e s 4 0 9

The c r i t i ca l ques t ion dea l ing wi th t h e ro le of m e m b r a n e lipids in p lan t r e sponse to chil l ing w e r e posed in t he i n t r o d u c t o r y c h a p t e r of this vo lume . It is c l e a r t h a t mo lecu l a r o rder ing of m e m b r a n e lipids can be a l t e r e d by va r i a t ions in f a t t y acyl compos i t ion in mic rob ia l s y s t e m s . Can t h e f a t t y acyl compos i t ion have any in f luence on t h e molecu la r o rder ing of the lipids in the m o r e complex m e m b r a n e s of h igher p l an t s? What is t h e re la t ionsh ip b e t w e e n f a t t y acyl compos i t ion of p l an t m e m b r a n e s and the t e m p e r a t u r e a t which lipid o rde r changes to a c r i t i ca l ly low leve l? We have shown t h a t s u b s t i t u t e d pyr idaz inones can be used to a l t e r t h e f a t t y acy l compos i t ion of m e m b r a n e s in h igher p l an t s and provide a tool for answer ing t h e s e ques t ions within a given p lan t spec ie s . We have also c o r r e l a t e d r e d u c e d levels of l inolenic ac id in m e m b r a n e s to i nc r ea sed sens i t iv i ty to chi l l ing. De La R o c h e (1) has r e c e n t l y sugges ted t h a t it is the inhibi t ion of pho tosyn thes i s by the pyr idaz inones , which we f irst r e p o r t e d (Z), t h a t is respons ib le for i nc r ea sed sens i t iv i ty to chi l l ing. Rega rd l e s s of the in t e rp l ay b e t w e e n pho tosyn thes i s and l inolenic ac id level (i .e. , is pho tosyn thes i s inh ib i ted by t h e pyr idaz inones b e c a u s e of d e c r e a s e d levels of l inolenic ac id in ch loroplas t m e m b r a n e ? ) the p rob lem of t he ro le of f a t t y acyl compos i t ion in mo lecu la r o rder ing of t h e lipids of p lan t m e m b r a n e s is s t i l l a m e n a b l e to a t t a c k using s u b s t i t u t e d pyr idaz inones .

R E F E R E N C E S

1. De La R o c h e , A. I. Plant Physiol. 63, 5-8 (1979). 2. Hi l ton, J . L., Scharen , A. L., St . John, J . B. , Moreland, D. E., and

Norr i s , Κ. H. Weed Sci. 17, 541-547(1969) . 3 . Hilt on, J . L., St. John, J . B., Chr i s t i ansen , Μ. N. , and Norr i s , Κ. H.

Plant Physiol. 48, 171-177 (1971). 4 . Lyons, J . M. Ann. Rev. Plant Physiol. 24, 455-466 . 5. R i c h a r d s , B. L. Proc. Utah Acad. Sci. Arts Lett. 11, 3-9 (1934). 6. St. John, Jud i th B. Plant Physiol. 57, 38-40 (1976). 7. St. John , Jud i th B. and Chr i s t i ansen , Μ. N. Plant Physiol. 57, 257-

259 (1976). 8. St . John , J . B. , Chr i s t i ansen , Μ. N. , Ashwor th , Ε. N. , a n d G e n t n e r , W.

Α., Crop Sci. 19, 6 5 - 6 9 ( 1 9 7 9 ) . 9. St . John , J . B. and Hi l ton , J . L. Weed Sci. 24, 579-582 (1976). 10. St. John , J . B., R i t t i g , F . R. , Ashwor th , Ε. N. , and Chr i s t i ansen , M.

N. In "Advances in P e s t i c i d e Sc ience" (H. Geissbuhler , ed.) P a r t 2, P e r g a m o n P r e s s . Oxford and New York, 272-273 (1979).

1 1 . Wi l lemot , C. Plant Physiol. 60, 1-4(1977).



CHEMICAL MODIFICATION OF LIPIDS DURING FROST HARDENING OF HERBACEOUS SPECIES

Claude Willemot

R e s e a r c h S ta t ion , Agr i cu l t u r e C a n a d a S te -Foy , Q u e b e c , C a n a d a

I. INTRODUCTION

It is genera l ly a c c e p t e d t h a t t h e main t a r g e t s of dehydra t ion d a m a g e caused by e x t r a c e l l u l a r i ce fo rma t ion a r e t h e cell m e m b r a n e s . It has t h e r e f o r e been an obvious app roach to look for changes in m e m b r a n e lipids and p ro t e in s c o r r e l a t e d wi th i n c r e a s e d frost t o l e r a n c e a t low harden ing t e m p e r a t u r e . These changes would m e e t t h e biophysical r e q u i r e m e n t s for m e m b r a n e s e i t he r to res i s t dehydra t ion s t r e s se s a t below f reez ing t e m p e r a t u r e s or to funct ion a t low above f reez ing t e m p e r a t u r e s and to allow t h e fu r ther changes which l ead to frost ha rden ing . The p r e s e n t discussion will be l im i t ed to those changes which occur dur ing low t e m p e r a t u r e a c c l i m a t i o n in m e m b r a n e lipids of h e r b a c e o u s p l an t s which acqu i r e i n t e r m e d i a t e levels of frost t o l e r a n c e in t h e r ange of - 1 5 ° C to - 3 0 ° C

II. CHANGES IN MEMBRANE LIPIDS DURING FROST HARDENING

Which changes l ikely to in f luence biophysical c h a r a c t e r i s t i c s of m e m b r a n e s h a v e been obse rved during low t e m p e r a t u r e a c c l i m a t i o n ? A s imple model of a m e m b r a n e is i l l u s t r a t e d in F igure 1. (1) Gene ra l i n c r e a s e in phospholipids and i nc r ea se in phospholipid tu rnover ; (Z) changes in polar heads of phospholipids; (3) i nc rea se in t he d e g r e e of u n s a t u r a t i o n of t h e f a t t y ac ids ; (4) changes in s t e ro l c o n c e n t r a t i o n and compos i t ion .

A. General Increase in Phospholipid Content

A gene ra l i nc r ea se in phospholipids has been observed amongs t o t h e r s in c e r ea l s (Z3), in w in t e r whea t (5, 3Z), and in a l fa l fa (11). This

Copyright β 1979 by Academic Press, inc. 4 1 1 All rights of reproduction in any form reserved

ISBN σ 12 4<3056O5

4 1 2 C. Willemot

MEMBRANE C O M P O N E N T S :

1 Polar h e a d s of phospho l ip id s 2 F a t t y a c i d s 3 S t e ro l s 4 P ro t e in s

FIGURE 1. Simple membrane model showing lipid components likely to influence biophysical characteristics of the membrane.

response to low t e m p e r a t u r e is analogous to t h a t of t r e e s (24, 36) (Fig. 2). P e r c e n t a g e dry weight g r ea t l y i nc reases in mos t ha rden ing t i s sues . The i nc r ea se in phospholipids was however shown to r e f l e c t a speci f ic i n c r e a s e in m e m b r a n e m a t e r i a l pe r ce l l , by e l e c t r o n mic roscopy (22) and by express ing it on a dry weight and a DNA basis (25) (Table I). It did no t mere ly r e f l e c t cel l division a t low t e m p e r a t u r e .

TABLE I. Augmentation of Phospholipid and Protein in Insoluble Homogenates of Summer and Winter Black Locust Bark Tissues*

Tissue Temperature at

LD50

Phospholipid DNA

Protein DNA

Summer Winter

-10°C -196 C

4.7 11.1

14.0 33.0

* From Singh et alt (25).

C h e m i c a l Modif icat ion of L ip id s d u r i n g F ros t H a r d e n i n g 4 1 3

SEPT OCT NOV DEC JAN FEB MAR APR MAY

FIGURE 2. Seasonal changes in frost hardiness and total amount of phospholipids in poplar cortex. Frost hardiness was expressed as the minimum temperature at which the cortex survived freezing without injury. -20 L and -30 L on hardiness scale: tissues were slowly grefrozen to -20 or -30 C prior to immersion in liquid nitrogen (-196 C). TG: triglyceride from cortex. From Yoshida (36).

In c e r e a l seeds g e r m i n a t i n g a t low t e m p e r a t u r e , t h e i n c r e a s e in phospholipids was shown to occur on a dry weight basis (5, 23). Spring and win t e r t ypes , which g r e a t l y d i f fe red in frost ha rd iness , showed the s a m e e x t e n t of i nc r ea se in m e m b r a n e m a t e r i a l a t low t e m p e r a t u r e (23) (Table Π). In t w e l v e day old win te r w h e a t p l a n t s , which w e r e beyond the ge rmina t i on s t a g e , phospholipids i n c r e a s e d during frost ha rden ing in roo t and crown on a fresh weigh t bas is , bu t d e c r e a s e d on a dry weigh t bas is (32) (Fig. 3). T h e r e was no d i f f e rence in lipid Ρ c o n t e n t b e t w e e n ha rdy Kharkov and less ha rdy C h a m p l e i n . The speci f ic i n c r e a s e in m e m b r a n e m a t e r i a l obse rved by Redshaw and Zalik (23) and de la R o c h e et al. (5) s e e m s t h e r e f o r e m o r e r e l a t e d to g e r m i n a t i o n a t low t e m p e r a t u r e , t han to frost ha rden ing . The gene ra l i n c r e a s e in dry we igh t , including phospholipids , obse rved by Wil lemot et al. (32) may be p r imar i ly a ^ w t e m p e r a t u r e r e sponse . When young win t e r whea t p l a n t s w e r e fed Ρ a f t e r var ious t i m e s of ha rden ing , phospholipid b iosynthes is was shown to be s t rongly s t i m u l a t e d in roo t and c rown, and this s t imu la t i on was much

4 1 4 C. Wi l l emot

TABLE II. Polar Lipid Phosphorus Content of Cereal Varieties at Different Stages of Vernalization*

Variety Weeks of

vernalization Polar lipid Ρ

mg/100 g dry sample

Sangaste fall rye 0 6.5 6 10.4

Prolific spring rye 0 6.4 6 10.3

Kharkov winter wheat 0 7.1 6 10.4

Red Bobs spring wheat 0 7.1 6 10.8

* From Redshaw and Zalik (23).

TABLE III. Lipid Ρ Content in Roots of Alfalfa Hardy Cultivar Rambler and Less Hardy Caliverde at Various Times of Hardening*

Days of \ig P/g fresh weight Variety hardening + SD

Rambler 0 76. 0 +4. .01 12 92. ,9 +2. ,25 24 87. 5 +2. .36 36 107. .3 ±1. .74

Caliverde 0 78. ,2 +2. .24 12 72. .9 +2. .68 24 68. .3 +2, .07 36 73, .1 +2, .34

* From Grenier and Willemot (11).

C h e m i c a l Modif icat ion of L ip ids d u r i n g F ros t H a r d e n i n g 4 1 5

30 0 -

~ 25 0 -σ

jo 20 0 Η

| 15 0

-σ loo 4

ϊ 5 0 H

KHARKOV

Y : 99 .04-0 .4 7 X

"4 - 2 _ _1

5 1 451 4<

30 0

25 0

20 0

150

2 10 0 ~ ~ 5 "

-

4

50

CHAMPLEIN

Υ:99 .29-0 .05 Χ

4 2

5

16 2 4 3 2 4 0 0 8 DAY S O F HARDENIN G

16 24 32 40

FIGURE 3. Lipid Ρ content (% of the mean on day 0) of root and crown of winter wheat after various times of frost hardening. The numbers refer to separate experiments. From Willemot et al., (32).

g r e a t e r in ha rdy Kharkov than in frost sens i t ive Chample in (29) (Fig. 4). This i n c r e a s e in phospholipid b iosynthes is c a m e l a t e r than the i n c r e a s e in frost t o l e r a n c e . P o t e n t i a l for frost ha rden ing c o r r e l a t e d wi th abi l i ty to syn thes i ze phospholipids a t low t e m p e r a t u r e , but phospholipid b iosynthes is was no t a p r e r e q u i s i t e for frost ha rden ing . It m a y be r equ i r ed to ma in t a in a high level of frost t o l e r a n c e , and was i n t e r p r e t e d as a r epa i r m e c h a n i s m .

Young al fa l fa p l an t s of t h e ha rdy cu l t iva r R a m b l e r showed an i n c r e a s e in lipid Ρ in the roo t on a fresh weight basis (11) (Table ΠΙ). This r e p r e s e n t e d however a d e c r e a s e on a dry weight bas i s . The change in lipid Ρ c o n t e n t c o r r e l a t e d wi th the ha rden ing abi l i ty of the cu l t i va r s , but t h e i n c r e a s e was nonspecific to m e m b r a n e m a t e r i a l . When young a l fa l fa p l an t s w e r e fed P , i nco rpora t ion in to t h e roo t l ipids d e c r e a s e d progress ive ly dur ing frost ha rden ing (11) (Fig. 5). The d e c r e a s e was g r e a t e s t in t he frost sens i t ive cu l t iva r C a l i v e r d e . Smolenska and Kuiper (26) showed no i n c r e a s e in lipid Ρ c o n t e n t of r a p e l eaves and roo t s a t low t e m p e r a t u r e .

F rom these d a t a i t is diff icult to conc lude what s igni f icance a gene ra l i n c r e a s e in phospholipids and m e m b r a n e m a t e r i a l has wi th r e s p e c t to frost ha rden ing a t low t e m p e r a t u r e .


FIGURE 4. Incorporation of ρ into lipids of root and crown of two cultivars of winter wheat, hardy Kharkov and less hardy Champlein, during 12 hours at 20°C and 24 hours at l°Cf after various times of frost hardening. From Willemot (29).

B. Changes at the Level of Polar Heads of Phospholipids

Few d a t a h a v e been g a t h e r e d on changes in p ropor t ions of phospholipid c lasses in p l an t s a t low t e m p e r a t u r e . In t h e bark of t h e poplar t r e e a l a rge i n c r e a s e in phospha t idy lchol ine (PC) and p h o s p h a t i d y l t h a n o l a m i n e (PE) was obse rved in t h e fal l , wi th l i t t l e change in t h e level of t h e r ema in ing phospholipids (36) (Fig. 6). These d a t a w e r e conf i rmed wi th Robinia (24, 25). Dur ing frost ha rden ing of a l fa l fa under con t ro l l ed condi t ions PC and PE i n c r e a s e d s igni f icant ly in t h e roo t s of t h e ha rdy cu l t iva r R a m b l e r , ^ L i t less in t ende r C a l i v e r d e (10) (Fig. 7). When a l fa l fa p l an t s w e r e fed C - a c e t a t e for a c o n s t a n t t i m e a f t e r d i f f e ren t per iods of ha rden ing , syn thes i s of PC and, t o a lesser e x t e n t , PE was s t rongly s t i m u l a t e d in ha rdy R a m b l e r , but no t in C a l i v e r d e (13) (Fig. 8). Specif ic a c t i v i t y of PC a lways m u c h g r e a t e r t han t h a t of PE . When al fa l fa p l an t s w e r e fed Ρ a f t e r d i f fe ren t t imes of ha rden ing , only PC showed a r e l a t i v e i n c r e a s e in label ing, and m o r e in R a m b l e r than in C a l i v e r d e . No d i f f e rence in phospholipid compos i t ion w e r e found in w in t e r whea t g e r m i n a t i n g a t 24 C and 1 C (6). When young p l an t s of w in t e r whea t w e r e fed P , l i t t l e change was observed in t h e

C h e m i c a l Modif icat ion of L ip ids d u r i n g F ros t H a r d e n i n g 4 1 7

FIGURE 5. Incorporation of Ρ into lipids of alfalfa roots during 12 hours at 22 C and 24 hours at 1 C after various times of frost hardening. From Grenier and Willemot (11).

p a t t e r n of phospholipids s y n t h e s i z e d in t h e roo t and crown (11). In t h e r a p e p l an t , low t e m p e r a t u r e c a u s e d an i n c r e a s e in PE and PC in t he h a r d e n e d leaf t i s sue , but a d e c r e a s e in t he se phospholipids in t h e unha rdened roo t t i s sue (26). The ava i l ab l e in fo rma t ion on changes a t t h e level of t h e polar heads of t h e phospholipids a t low harden ing t e m p e r a t u r e s is s t i l l too f r a g m e n t a r y to draw de f in i t e conclus ions . Excep t for w h e a t , mos t p l a n t s i n v e s t i g a t e d showed an i n c r e a s e in PC and PE dur ing frost ha rden ing . The t u r n o v e r of PC s e e m s to be higher than t h a t of o t h e r phospholipids a t low t e m p e r a t u r e .

C. Increase in Degree of Unsaturation of Fatty Acids

The a spec t of lipid m e t a b o l i s m which has been s tud ied t h e most in connec t ion wi th frost ha rden ing of h e r b a c e o u s p l an t s is t h a t of f a t t y ac id d e s a t u r a t i o n . In 1964 Lyons et al. (19) showed a r e l a t ionsh ip b e t w e e n d e g r e e of u n s a t u r a t i o n of t h e f a t t y ac ids of mi tochondr i a l m e m b r a n e s , f lexibi l i ty of t h e s e m e m b r a n e s , and chil l ing r e s i s t a n c e of severa l spec i e s . In t h e following y e a r s i n c r e a s e d u n s a t u r a t i o n of f a t t y ac ids a t low


TABLE IV. Fatty Acid Composition of The Polar Lipid Fraction of Cereal Varieties at Different Stages of Vernalization (% of Total Fatty Acids in the Fraction)*

Weeks Fatty acids Varieties vernalized 16. 0 18 :1 18. 2 18 •3

Sangaste 0 20 8 15 .3 58 5 5 4 (fall rye) 6 20 7 12 .2 40 2 26 9 Prolific 0 22 4 15 .2 56 9 5 5 (spring rye) 6 21 4 8 .8 39 4 30 4 Kharkov 0 18 0 13 .7 63 7 4 6 (winter wheat) 6 20 0 8 .1 60 2 11 7 Red Bobs 0 19 2 16 .3 61 0 3 5 (spring wheat) 6 22 9 7 .8 55 4 13 9

* From Redshaw and Zalik (23).

FIGURE 6. Seasonal changes in individual phospholipids from poplar cortex. The two lowest curves correspond to unidentified acidic phospholipids. From Yoshida (36).

Phosphatidylcholin e Phosphatidylethanolamin e Triglycerides

FIGURE 7. Total fatty acids of phosphatidylcholine, phosphatidylethanolamine and triglycerides in roots of alfalfa during frost hardening. From Grenier and Willemot (10).


RAMBLER.22C CALIVERDE, 2 2 C 8H

6H

4H

0 3 6 12 24 3 6 0 3 6 12

RAMBLER, 1C

6H

4H

2H

CALIVERDE.1C

SE - - ο - - —ο

PE

2 4 3 6 0 3 6 12 2 4 3 6

D a y s of h a r d e n i n g

14 FIGURE 8. C-acetate incorporation into phosphatidylcholine,

phosphatidylethanolamine and sterol esters of alfalfa roots during six hours at 22°C and during 12 hours at 1 C after various times of frost hardening. From Grenier et al.t(13).

C h e m i c a l Modificat ion of L ip ids d u r i n g Fros t H a r d e n i n g 4 2 1

to

9 u <

100-

8 0 -

6 0 -

4 0

2 0 ^

0 u_ λ φ —

K h a r k o v , Roo t s 18:3

18:2

100

8 0

6 0 \

4 0

16*0

18H

2 0 %

a a . 3 100

ο X Q- 8 0 to Ο

40H

1

Champlei n , R o o t s

« χ

18:2

1 6 : 0

2 0 4

0

4 0

100 -

8 0 -

2 4 1

Kharkov ,Leave s 6 0 -| 18:3

·«

18:2 4 0

1 6 : 0

18:1

I I 16:1-trans

Champlein , Leave s

„ ~ x " " * 1 8 : 3

1 8 : 2

ι ο 20 4 , , ,

·, ,· · · ,0· mO ·

~~1 16:1-trans I

W E E KS O F H A R D E N I N G

FIGURE 9. Phospholipid major fatty acids content, expressed as percentage of total fatty acids, in two cultivars of winter wheat during frost hardening. From Willemot et al., (33).

422 C. Wi l l emot

harden ing t e m p e r a t u r e was shown in a lmos t all spec ies inves t iga ted : in a l fa l fa in the field during the fall (9) and under con t ro l l ed hardening condi t ions (10, 1Z, 13), where the i nc r ea se , mainly in l inoleic acid (18:2), was g r e a t e s t in the hardy cu l t iva r s ; in pear t r e e s (16); in whea t and rye seeds ge rmina t ing at low t e m p e r a t u r e , whe re l inolenic acid (18:3) inc reased a t the expense of l inoleic ac id (18:2) (5, 23, 24); in young win te r whea t p l an t s beyond the ge rmina t ion s t age (32); and in the r ape p lan t (26). T e m p e r a t u r e had l i t t l e inf luence on f a t t y acid unsa tu ra t i on of p o t a t o tuber t i ssue , which has l i t t l e hardening po t en t i a l (8). With t he publ ica t ion of two rev iew papers on chil l ing injury by Lyons (17, 18) it b e c a m e increas ingly probable t ha t unsa tu r a t i on of f a t t y acids p layed an i m p o r t a n t ro le in low t e m p e r a t u r e a c c l i m a t i o n . In r e c e n t y e a r s , however , ev idence to the con t r a ry has been a c c u m u l a t i n g .

Several au tho r s have observed t ha t cu l t iva r s varying widely in frost t o l e r a n c e show the s ame inc rease in unsa tu ra t i on of the f a t t y ac ids a t low t e m p e r a t u r e . Redshaw and Zalik (23) showed s imilar i nc reases in l inolenic acid con ten t in fall and spring rye and in win te r and spring whea t ge rmina t ing a t low t e m p e r a t u r e (Table IV). de la R o c h e et al. (7) conf i rmed thei r observa t ions with 4 win te r whea t cu l t iva rs which d i f fered widely in hardening po ten t i a l (Table V). In 12 day old win te r whea t p l an t s , l inolenic acid inc reased to the same e x t e n t in the roo t and crown of hardy Kharkov and t ender Chample in (33) (Fig. 9). Poplar (36) and black locust (24, 25) bark t i ssue acqu i res e x t r e m e frost hard iness in t he fall , wi thout i nc reased f a t t y acid unsa tu r a t i on . Smolenska and Kuiper (26) showed the same inc rease in l inolenic acid con t en t a t low t e m p e r a t u r e in r ape leaves which acqu i re frost t o l e r a n c e and in roo t s which do not ha rden . The lack of d i f fe rences in f a t t y acid unsa tu r a t i on b e t w e e n hardy and less hardy cu l t iva r s did not p rove tha t it was not involved in frost ha rden ing . It could mean tha t i nc reased unsa tu ra t i on was a p re r equ i s i t e to frost hardening , but t ha t less hardy cu l t iva rs had their frost t o l e r a n c e l imi ted by o the r f a c t o r s . Two new e x p e r i m e n t a l approaches b e c a m e r e c e n t l y avai lable to t e s t this possibi l i ty: (1) Wilson (35) showed tha t in the chill sens i t ive Phaseolus vulgaris, in which the d e g r e e of unsa tu r a t i on of f a t t y acids i nc reased a t low t e m p e r a t u r e , hardening by drought was possible wi thout low t e m p e r a t u r e t r e a t m e n t and wi thout inc reased unsa tu ra t ion of t he f a t t y acids ; (2) St. John and Chr i s t i ansen (27) showed tha t t r e a t m e n t of ge rmina t ing co t t on seeds wi th pyr idaz inones r educed both the t o l e r a n c e of t he seedl ings to chil l ing and the low t e m p e r a t u r e induced inc rease in l inolenic acid c o n t e n t of t h e polar lipids in the roo t t ips .

Drought e x p e r i m e n t s w e r e p e r f o r m e d by de la R o c h e (2) wi th ge rmina t ing win te r whea t seeds (Table VI) and by Wil lemot and Pe l l e t i e r (3 1) with young win te r whea t p l an t s (Table VH). In bo th cases t he resu l t s conf i rmed those of Wilson (3 5). Significant frost ha rden ing was ach ieved wi thout low t e m p e r a t u r e t r e a t m e n t , and with a d e c r e a s e in t he level of l inolenic ac id .

T r e a t m e n t of young win te r whea t p l an t s with a pyr idaz inone (BASF 13-338) caused s imul taneous inhibi t ion of frost hardening and of synthes is of l inolenic acid in the roo t s a t low t e m p e r a t u r e (30) (Table

C h e m i c a l Modif icat ion of L ip id s d u r i n g Fros t H a r d e n i n g 4 2 3

TABLE VI. Fatty Acid Composition of Phospholipids from Epicotyls of Unhardened (24 C), Cold Hardened (2°C) and Desiccation Hardened (24 C) Seedlings of Winter Wheat, cv. Kharkov MC 22*

Fatty acids (mole %) Treatment LT

50 16:0 18:0 18:1 18:2 18:3

2fC 23.9 0.4 9.3 27.0 39.4 2°C -22

UC 22.2 0.6 7.2 17.8 52.2

90% R.H., 24°C -13°C 21.2 1.1 25.2 21.0 31.5

* From de la Roche (2).

TABLE VIII. Total (mg per g fresh weight) and Individual Fatty Acids (weight %) of Roots of Frost Hardening Winter Wheat Treated with BASF 13-338*

Treatment Control Treated

Days of hardening 0 14 0 14

Fatty acids 16:0 17.2 +0.2 17.6 +0.4 16.8 +0.3 18.8 +0.2 18:0 0.6 +0.1 0.5 +0.2 0.6 +0.2 1.1 +0.5 18:1 3.5 +0.5 4.4 +0.8 3.9 +0.5 4.8 +1.9 18:2 41.0 +0.3 32.1 +0.9 50.6 +0.5 49.4 +1.7 18:3 37.8 +0.6 45.3 +1.1 28.1 +0.8 25.9 +0.9

Total fatty acids 0.88 +0.22 1.28 +0.24 0.89 +0.14 0.72 +0.14

Ratio 18:3/18:2 0.92 1.41 0.56 0.52

LT5Q (°C) -5.0 -17.7 -5.0 -6.0

* From Willemot (30).

TABLE VII. Frost Resistance (LT ) and Fa t t y Acid Content (ma per g fresh weight) of 12 Day Old Winter Wheat Plants Grown Subsequently at 20 C or Hardened at 1 C, at Two Soil Mosture Levels, 40% (normal) or 10% (drought stressed)

Days of Temp of growing growing Moisture LT50 % total fatty acids Total fatty Ratio

and and level LT50

harden. harden. °C 16:0 18:0 18:1 18:2 18:3 acids 18:3/18:2

0 20°C 40 -7.2+0. .1 18.5 0.3 3.4 42.5 35.3 1.72+0.12 0.83 7 20°C 40 -5.9+0. .1 19.0 0.4 3.5 39.8 37.3 1.46+0.06 0.94

20°C 10 -10.9+0. .2 19.8 0.5 4.8 40.2 34.7 2.10+0.25 0.86 14 20°C 40 -6.8+0. .1 19.1 0.5 3.8 41.0 35.8 1.56+0.27 0.87

20°C 10 -9.6+0. .1 20.2 0.6 5.5 39.9 33.8 2.15+0.15 0.85 J o C 40 -19.5+0. .2 17.5 0.2 3.5 33.9 44.9 1.83+0.11 1.32 J o C 10 -23.0+0. .1 18.8 0.3 3.8 35.8 41.3 2.47+0.18 1.15

28 £C 40 -23.9+0. .2 17.7 0.2 3.1 28.2 50.7 1.90+0.10 1.80 1°C 10 -25.0+0. .3 19.4 0.4 4.0 30.1 46.2 2.58+0.11 1.54

From Willemot and Pelletier (31). v% of soil water holding capacity.

424

TABLE IX. The Effect of BASF 13-338 and Sandoz 6706 on the Fatty Acid Composition (weight %) and the Freezing Tolerance (LD^Q , C) of Kharkov Wheat Seedlings*

Lipid Fatty acid composition Treatment LD50 component 16:0 18:0 18:1 18:2 18:3

24°C, control -4 total lipids 17.6 0.5 8.3 23.7 49.9 phospholipids 23.1 0.7 9.6 31.1 35.5

2°C, control -23 total lipids 15.7 0.7 5.9 14.1 63.6 phospholipids 21.9 0.5 7.9 17.6 52.1

2°C, BASF 13-338 -23 total lipids 15.2 0.7 8.9 51.8 23.3 phospholipids 19.3 0.2 11.1 47.1 22.3

2UC, Sandoz 6706 -23 total lipids 14.7 0.4 10.4 38.4 36.1 phospholipids 18.5 0.6 12.6 35.3 33.0


425


VIII). Subsequent e x p e r i m e n t s showed t h a t pho tosyn thes i s , which is e ssen t ia l for frost ha rden ing of win te r whea t (Zl) was comple t e ly inhib i ted (34). de la R o c h e (3) r e p e a t e d this e x p e r i m e n t wi th whea t seeds ge rmina t i ng in t h e dark a t Ζ C. These seedl ings draw the i r c a r b o h y d r a t e s from the endosperm and do no t depend on pho tosyn thes i s for frost ha rden ing . His r e su l t s show unequivocal ly t h a t an i n c r e a s e in l inolenic acid is not a p r e r equ i s i t e to frost ha rden ing in win te r w h e a t (Table IX).

In s u m m a r y , excep t possibly for a l fa l fa , w h e r e i n c r e a s e d unsa tu r a t i on a t low t e m p e r a t u r e c o r r e l a t e s wi th ha rden ing abi l i ty of the cu l t iva r s , t he ev idence for major invo lvement of i nc reased f a t t y acid unsa tu r a t i on in frost ha rden ing a t low t e m p e r a t u r e is much less s t rong than it appea red only a few y e a r s ago . Inc reased u n s a t u r a t i o n is no t a p r e r e q u i s i t e to frost ha rden ing in win te r whea t and in e x t r e m e l y hardy t r e e s such as the b lack locust and poplar , and it is ap p a ren t l y no t r equ i r ed for chill hardening of Phaseolus vulgaris.

D. Changes in Sterol Content

The leas t s tud ied a spec t of lipid invo lvement in frost hardening is the f a t e of s t e ro l s a t low t e m p e r a t u r e . Davis and F inkner (1) showed l i t t l e e f fec t of t e m p e r a t u r e on t o t a l s t e ro l prof i le of one mon th old whea t p l an t s . T e m p e r a t u r e had no inf luence on the p ropor t ions of s t e ro l lipid c lasses in p o t a t o tuber (8). With a whea t embryo t o t a l m e m b r a n e f rac t ion , de la R o c h e (4) observed l i t t l e change in the s t e ro l to phospholipid r a t i o , but a s ignif icant d e c r e a s e in the r a t i o of c a m p e s t e r o l to s i to s t e ro l a t low t e m p e r a t u r e (Table X). This r a t i o d e c r e a s e d to the s a m e e x t e n t in 4 cu l t iva r s differ ing in frost ha rden ing p o t e n t i a l . In t he roo t and crown of young win te r whea t p l an t s of the hardy cu l t iva r Kharkov, l i t t l e change was shown during harden ing a t low t e m p e r a t u r e in t o t a l s t e ro l prof i le , p ropor t ions of s t e ro l con ta in ing lipid c lasses , s t e ro l prof i le within e a c h c lass , and lipid Ρ to s t e ro l r a t i o (Willemot, unpublished) . The few d a t a ava i lab le give l i t t l e ev idence for a s ignif icant modi f ica t ion of s t e ro l composi t ion under low t e m p e r a t u r e condi t ions .

ΠΙ. DISCUSSION

A. Involvement of Lipids in Frost Hardening

The ev idence for invo lvement of lipids in frost ha rden ing of he rbaceous spec ies is much less s t rong than it a p p e a r e d in i t ia l ly . Only in a l fa l fa a r e t he observed f ac t s ( increase in lipid P , in PC and PE, and in d e g r e e of u n s a t u r a t i o n of t he f a t t y acids) c o r r e l a t e d with hardening p o t e n t i a l of cu l t iva r s . G r e a t e r abi l i ty to syn thes i ze phospholipids a t low t e m p e r a t u r e was shown in the hardy cu l t iva r s of whea t and a l fa l fa . This

C h e m i c a l Modificat ion of L ip ids d u r i n g Fros t H a r d e n i n g 4 2 7

TABLE V. Major Fatty Acid Composition of the Total Membranes from Excise^ Embryos of Seedlings Germinated and Grown at 2 and 24 C for 5 Weeks and 50 Hours, Respectively*

Growth temperature LT

50 Fatty acid composition (mole %)

Cultivar (°C) (°C) 16:0 18:1 18:2 18:3

Kharkov 2 -18 21.4 4.9 30.9 42.0 24 -2 24.6 7.4 47.5 19.8

Rideau 2 -13 21.5 4.7 33.3 39.2 24 -2 24.6 6.8 49.8 18.3

Capelle-Desprez 2 -6 22.8 4.6 32.1 39.8 24 -1 24.7 5.5 48.8 20.2

Marquis 2 -5 22.0 4.5 32.3 40.5 24 -2 22.8 7.2 52.0 17.4

* From de la Roche et al. (7).

l ack of firm ev idence does not necessa r i ly m e a n t h a t lipids a r e not involved. But fur ther ev idence will have to inc lude , amongs t o t h e r s , compar i sons b e t w e e n ha rdy and t e n d e r cu l t iva r s , t h e d e m o n s t r a t i o n tha t specif ic inhibi tors of lipid t r a n s f o r m a t i o n s a t low t e m p e r a t u r e inhibit s imul taneous ly frost ha rden ing , and t h e biophysical d e m o n s t r a t i o n t ha t lipids fulfil the i r p roposed ro le in ha rden ing .

B. Role of Increased Fatty Acid Unsaturation at Low Temperature

It is diff icult to a c c e p t t h a t t he genera l ly obse rved inc rease in f a t t y acid uns a tu r a t i on a t low t e m p e r a t u r e would no t be benef ic ia l to the p l a n t s . Several au tho r s h a v e sugges ted t h a t even if lipid changes we re not involved in frost ha rden ing , they would help the p lan t to function a t low t e m p e r a t u r e . These two e v e n t s appea r however undissociable : frost ha rden ing a t low t e m p e r a t u r e p resupposes t ha t m e m b r a n e s function sa t i s f ac to r i ly a t low t e m p e r a t u r e , and f a c t o r s which favor m e m b r a n e funct ion a t low t e m p e r a t u r e should t h e r e f o r e improve frost harden ing .

Inc reased u n s a t u r a t i o n of f a t t y acids a t low t e m p e r a t u r e is not a p r e r e q u i s i t e to frost ha rden ing in win te r w h e a t . Is it then wi thout a ro le? Is it s imply t he resu l t of i nc r ea sed 0 ? solubi l i ty a t low t e m p e r a t u r e (14)? Is the exce l l en t co r r e l a t i on o b t a i n e a wi th a cu l t iva r of winter wheat b e t w e e n levels of frost t o l e r a n c e and of l inolenic acid t h e resul t of dependence on common f a c t o r s , low t e m p e r a t u r e and l ight , wi thout causal r e l a t ionsh ip (Willemot, unpublished)?

4 2 8 C. Willemot

TABLE X. Sterol Composition of Total Membrane from Four Cultivars of Wheat Differing in Freezing Resistance*

Cultivar

Growth temperature Sterol/

phospholipids

Sterol composition (mole %)

Campesterol Sitosterol

Kharkov 2 .14 21.0 79.0 24 .14 32.2 67.8

Rideau 2 .17 23.6 76.4 24 .14 29.9 70.1

Cappelle- 2 .16 23.4 76.6 Desprez 24 .16 33.5 64.5

Marquis 2 .16 23.8 76.2 24 .15 32.4 67.6


Anothe r i n t e r p r e t a t i o n of t h e d a t a , which would r e c o g n i z e a ro le for the inc reased unsa tu r a t i on of f a t t y ac ids a t low t e m p e r a t u r e , wi thout making it a p r e r equ i s i t e to frost ha rden ing , is t h a t when i t does not occur a t low t e m p e r a t u r e , the p lant uses a l t e r n a t e means to main ta in a funct ional level of m e m b r a n e f luidi ty. This could be ach ieved by a n e g a t i v e feedback con t ro l , as shown for Tetrahymena pyriformis by Mar t in et al. (ZO). If this w e r e t he ca se , one should be able to d e m o n s t r a t e b e t t e r con t ro l of m e m b r a n e f luidi ty in frost t o l e r an t than in t ende r cu l t iva r s a t low t e m p e r a t u r e . The ex i s t ence of a l t e r n a t e m e c h a n i s m s would c o m p l i c a t e the d e m o n s t r a t i o n of the i r invo lvement in frost hardening because none of t hese m e c h a n i s m s would be a p r e r e q u i s i t e to ha rden ing .

I n t e r p r e t a t i o n of t h e d a t a is fu r ther c o m p l i c a t e d by t h e f ac t t h a t t he b i o m e m b r a n e cont inuum may be broken up in to a number of l a t e r a l ly s e p a r a t e d liquid c rys ta l l ine domains a t equil ibr ium (15). This equil ibrium would shift wi th t e m p e r a t u r e . Analysis of individual m e m b r a n e domains may be r equ i r ed for t he biophysical i n t e r p r e t a t i o n of changes in compos i t ion .

C h e m i c a l Modif icat ion of L ip id s d u r i n g F ros t H a r d e n i n g 4 2 9

C. Conclusion

In conclusion, t h e ava i l ab le d a t a do no t show a s imple re la t ionsh ip b e t w e e n lipid compos i t ion of m e m b r a n e s and frost t o l e r a n c e of h e r b a c e o u s p l a n t s . They should no t be i n t e r p r e t e d as ev idence tha t m e m b r a n e lipids a r e no t involved, but r a t h e r t h a t t h e re la t ionsh ip is a complex one , involving possibly a l t e r n a t e m e c h a n i s m s and d i f f e r en t i a t ed m e m b r a n e domains , and w h e r e non-l ipid, e .g. p ro t e in , involvement may be m o r e i m p o r t a n t t han a n t i c i p a t e d .


1. Davis , D . L., and F inkner , V. C . Plant Physiol. 52, 324-326 (1972). 2. de la R o c h e , I. A. Acta Hort. 81, 85-89 (1978). 3 . de la R o c h e , I. A. Plant Physiol. 63, 5-8 (1979a). 4 . de la R o c h e , I. A. In " C o m p a r a t i v e Mechan i sms of Cold Adapt ion

in t h e A r c t i c . " (Symposium, 28th Annual Mee t ing , A.I.B.S.) (L. S. Underwood, Ed.) Acad . P re s s , N.Y. (In press) (1979b).

5. de la R o c h e , I. Α., Andrews , C . J . , and P o m e r o y , Μ. K. Can. J. Bot. 50, 2401-2409 (1972).

6. de la R o c h e , I. Α., Andrews , C . J . , and K a t e s , M. Plant Physiol. 51, 468-473 (1973).

7. de la R o c h e , I. Α., P o m e r o y , Μ. K., and Andrews , C. J . Cryobiol. 12, 506-512 (1975).

8. Gal l ia rd , Τ. , Berke ley , H. D. , and M a t t h e w , J . A. J. Sci. Fd. Agric. 26, 1163-1170 (1975).

9. Gerloff , E. D . , R icha rdson , T. , and S tahmann , M. A. Plant Physiol. 41, 1280-1284 (1966).

10. Gren i e r , G., and Wil lemot , C Cryobiol. 11, 324 -331(1974) . 11 . Gren ie r , G., and Wil lemot , C . Can. J. Bot. 53, 1473-1477 (1975). 12. Gren ie r , G., T r e m o l i e r e s , Α. , The r r i en , H. P . , and Wil lemot , C .

Can. J. Bot. 50, 1681-1689 (1972). 13. Gren i e r , G., Hope , H. J . , Wi l lemot , C , and Ther r i en , H. P . Plant

Physiol. 55, 906-912 (1975). 14. Ha r r i s , P . , and J a m e s , A. T. Biochem. J. 112, 325-330 (1969). 15. J a in , Μ. K., and Whi te , Η. B. In "Advances in Lipid R e s e a r c h " (R.

P a o l e t t i and D . Kr i t chevsky , eds . ) , Vol. XV, p p . 1-60. Acad . P re s s , N .Y. (1977).

16. K e t c h i e , D . O. Proc. Am. Soc. Hort. Sci. 88, 204-207 (1966). 17. Lyons, J . M. Cryobiol. 9, 341-350 (1972). 18. Lyons, J . M. Ann. Rev. Plant Physiol. 24, 445-466 (1973). 19. Lyons , J . M., Wheaton , Τ. Α., and P r a t t , Η . K. Plant Physiol. 39,

262-268 (1964). Z0. Mar t in , C . E., H i r ami t su , K., K i t a j ima , Y., Nozawa , Y., Skriver , L. ,

and Thompson, G. A. J r . Biochem. 15, 5218-5227 (1976). 2 1 . Paulsen , G. M. Crop Science 8, 29-32 (1968).


22. Pomeroy , Μ. K., and Siminovi tch, D. Can. J. Bot. 49, 787-795 (1971).

23 . Redshaw, E. S., and Zalik, S. Can. J. Biochem. 46, 1093-1097 (1968).

24. Siminovi tch , D. , Singh, J . , and de la R o c h e , I. A. Cryobiol. 12, 144-153 (1975).

25. Singh, J . , de la Roche , I. Α., and Siminovi tch, D. Nature 257, 669-670 (1975).

26. Smolenska, G. S., and Kuiper , P . J . C. Physiol. Plant. 41, 29-35 (1977).

27. St. John, J . B., and Chr i s t i ansen , Μ. N. Plant Physiol. 57, 257-259 (1976).

28. Thomson, L. W., and Zalik, S. Plant Physiol. 52, 268-273 (1973). 29. Wil lemot , C. Plant Physiol. 55, 356-359 (1975). 30. Wil lemot , C. Plant Physiol. 60, 1-4 (1977). 3 1 . Wil lemot , C , and Pe l l e t i e r , L. Can. J. Plant Sci. (In Press) (1979). 32 . Wil lemot , C , Hope , H. J . , P e l l e t i e r , L. , Langlois , J . , and Michaud,

R. Can. J. Plant Sci. 57, 555-561 (1977a). 3 3 . Wil lemot , C , Hope, H. J . , Will iams, R. J . , and Michaud, R. Cryo

biol. 14 87-93 (1977b). 34. Wil lemot , C , Hope, H. J . , and S t - P i e r r e , J . C . Can. J. Plant Sci.

59, 249-251 (1979). 35 . Wilson, J . M. New Phytol. 76, 257-270 (1976). 36. Yoshida, S. Contr. Inst. Low. Temp. Sci. Ser. Bll, 1-40 (1974).


CHILL-INDUCED CHANGES IN ORGANELLE MEMBRANE FATTY ACIDS

E. J. Bartkowski

Celpr i l Indus t r ies M a n t e c a , Ca l i forn ia

T. R. Peoples and F. R. H. Katterman

D e p a r t m e n t of P lan t Sciences Univers i ty of Ar izona

Tucson, Ar izona

Symptoms of chil l ing injury vary among p lan t and t i ssue types when they a r e exposed to var ious per iods of low nonf reez ing t e m p e r a t u r e s b e t w e e n 0 and 15C. C u l t i v a t e d c o t t o n (Gossypium hirsutum and G. barbadense) is one such t h e r m a l l y sens i t ive p l an t espec ia l ly dur ing seed ge rmina t ion and seedl ing e m e r g e n c e . Chr i s t i ansen (3) found t ha t chil l ing a t t he in i t i a t ion of c o t t o n s e e d hydra t ion r e s u l t e d in m e r i s t e m a t i c r ad ic l e abor t ion and a g rowth lag . Chil l ing t e m p e r a t u r e s for a shor t t i m e a f t e r t h e onse t of ge rmina t i on also caused a r educ t i on in g rowth r a t e fol lowed by an i ncac t i va t i on of the r ad i c l e c o r t e x due to ce l lu lar col lapse or d i s in teg ra t ion . Thus from t h e work of Chr i s t i ansen (3, 4) on shor t s t ap le c o t t o n (G. hirsutum) and r e s u l t s from r e c e n t work (Z) on long s t ap l e c o t t o n (G. barbadense) two per iods of chil l ing sens i t iv i ty w e r e desc r ibed for c u l t i v a t e d c o t t o n . The f i rs t pe r iod was e s t i m a t e d to be a t t h e beginning of ge rmina t i on (1 to 6 hrs) whils t t h e second per iod was from Z8 to 3Ζ hours l a t e r .

Since t he r ad i c l e t ip appea r s to be t h e mos t vu lnerab le s i t e of chil l ing d a m a g e , our nex t a r e a of inves t iga t ion was to s tudy t h e e f fec t of chil l ing on subcel lu lar o rgane l le m e m b r a n e s wi th r e s p e c t to a change in u n s a t u r a t e d f a t t y ac id compos i t ion .

I. MATERIALS AND METHODS

C o t t o n s e e d (G. barbadense) w e r e g e r m i n a t e d for 4Z hrs a t 34C and served as con t ro l s . A second lot was g e r m i n a t e d a t t h e s a m e t e m p e r a t u r e for 30 hrs then 6 hrs a t 5C and for a concluding 6 hrs a t 34C

Copyright · 1979 by Academic Press. Inc. 4 3 1 All rights of reproduction In any form reserved

ISBN012 460560-5

4 3 2 Ε. J. B a r t k o w s k i et ai

t o give a t o t a l ge rmina t ion t i m e of 42 h r s . Ten mm sec t ions of r ad i c l e t ips from both con t ro l and chill t r e a t e d p lan t s w e r e exc ised and ground in a s t anda rd t r i s - suc rose buffer a t 4C in a chi l led m o r t a r and p e s t l e . Nuc lea r , mi tochondr ia l , and mic rosomal en r i ched f rac t ions w e r e ob ta ined by d i f fe ren t ia l cen t r i fuga t ion t echn iques as desc r ibed e l s ewhe re (12). Af te r e x t r a c t i o n of t he o rgane l l es wi th a 2:1 ch lo ro fo rm-m e t h a n o l m ix tu r e , t h e organic e x t r a c t was fur ther pur i f ied by t h e Fo lch wash p r o c e d u r e (9) and then f r a c t i o n a t e d in to polar and n e u t r a l c lasses by si l icic acid c h r o m a t o g r a p h y (5). The f a t t y acid c o m p o n e n t s of t he polar lipid f rac t ion w e r e m e t h y l a t e d and assayed q u a n t i t a t i v e l y by means of gas liquid c h r o m a t o g r a p h y (1). The t o t a l double bond index (D.B.I.) for both con t ro l and chill t r e a t e d o rgane l les was d e t e r m i n e d by Eq. (1), s u m m e d over all 16 and 18 carbon f a t t y ac ids .

Eq. (1) Σ (# double bonds χ mole %) = D.B.I. 100

Π RESULTS AND DISCUSSION

R e s u l t s from Table I show t h a t t h e major i n c r e a s e in DBI due to chill ing t e m p e r a t u r e occurs in t h e mic rosomal m e m b r a n e s . The chi l led mi tochondr ia l m e m b r a n e s , however , show a gene ra l d e c r e a s e in u n s a t u r a t e d f a t t y ac id c o n t e n t mainly o le ic and l inoleic ac ids , as shown by a lower DBI. The nuc l ea r m e m b r a n e s exhibi t e s sen t ia l ly no change in f a t t y ac id compos i t ion when exposed to chil l ing t e m p e r a t u r e s . Addi t ional ly , the d a t a from Table I i nd i ca t e s t ha t the g r e a t e s t change in u n s a t u r a t e d f a t t y ac id level during r ad i c l e t ip chil l ing occur s wi th t h e l inoleic and l inolenic ac ids of the mic rosomal m e m b r a n e s . N o t e t ha t t h e r e is a d e c r e a s e in l inoleic ac id along wi th a cor responding i n c r e a s e in l inolenic ac id . The l a t t e r is cons idered to be fo rmed by s t epwi se d e s a t u r a t i o n of s t e a r i c acid (10). It has been sugges ted t h a t t h e higher levels of l inolenic ac id observed a t lower t e m p e r a t u r e s is a t t r i b u t a b l e to i nc r ea sed a m o u n t s of dissolved oxygen, a r equ i r ed c o f a c t o r , a t a fixed d e s a t u r a s e a c t i v i t y (10). Assuming tha t oxygen is no t l imi t ing in this s tudy, i nc reased l inolenic acid levels may be expla ined by d i f fe rences in d e s a t u r a s e a c t i v i t y as a funct ion of t e m p e r a t u r e . The inc reased levels of l inolenic acid in this s tudy a r e also c o m p a t i b l e wi th prev ious work on t o t a l lipids i so la ted from chi l led whea t and rye r ad ic l e s (8). In our s tudy, however , t he e f fec t of chil l ing was loca l ized in t h e mic rosoma l m e m b r a n e s .

The s ignif icant d e c r e a s e in chi l led mi tochondr ia l m e m b r a n e DBI pu t s for th (Table I) t h e possibi l i ty t h a t phospholipid t r ans fe r from t h e mic rosomal to t he mi tochondr ia l m e m b r a n e s by the phospholipid exchange p ro t e in s (PEP) (15) is d iminished by t h e lower t e m p e r a t u r e . If indeed t h e P E P w e r e not a f f e c t e d by exposure to chil l ing t e m p e r a t u r e , we would e x p e c t t o find t h e mic rosoma l and mi tochondr i a l m e m b r a n e s t o h a v e nea r ly s imi lar m e m b r a n e DBI's due to the i r mu tua l i n t e r a c t i o n (15).

Chi l l - Induced C h a n g e s in O r g a n e l l e M e m b r a n e F a t t y A c i d s 4 3 3

TABLE I. The Effect of Chilling Temperatures on Mole Percent Fatty Acid Composition of Microsomal, Mitochondrial and Nuclear Membranes Isolated from Gossypium barbadense Radicle Tips

Fatty Microsomal Mitochondrial Nuclear Acid Nonchill Chill Nonchill Chill Nonchill Chill

mole % mole % mole %

16:0 15.05 14.95 30.40 33.10 25.24 26.00 16:1 2.47 4.73 4.75 8.26 0.77 1.27 16:2 7.27 1.29 1.61 4.20 2.23 1.79 18:0 18.56 16.04 19.14 12.72 8.80 10.25 18:1 15.51 22.36 16.79 11.52 16.86 15.98 18:2 19.09 9.89 20.17 13.52 40.02 36.64 18:3 5.54 18.27 2.46 1.79 1.53 2.40

Double Bond Index 0.88 1.04 0.73 0.61 1.06 1.02

With a d e c r e a s e d t r anspor t of polar lipids to normal ly r e p l a c e deg raded mi tochondr ia l m e m b r a n e s , i t is possible t h a t t h e no rma l p roces s of β-oxida t ion of t h e u n s a t u r a t e d f a t t y acids from t h e deg raded m e m b r a n e f r a g m e n t s would then b e c o m e m o r e ev iden t as n o t e d by t h e r e d u c t i o n of o le ic and l inoleic acids in chi l led mi tochondr ia l m e m b r a n e s (Table I).

TABLE II. The Double Bond Index (DBI) and Specific Activity of DNA Isolated from Nuclei of a Chill-Resistant (#189) and Chill-Sensitive (#200) Seed Lot of Pima S-4 Cotton (Gossypium barbadense L.)

Pima S-4 DNA Specific Activity DBI

cpm/mg

Lot #189 582b* 1.01 n.s. Lot #200 417a 1.05

* Means followed by different letters are significantly different at the 5% level according to the Student Newman Keuls Test.

4 3 4 Ε. J. B a r t k o w s k i et al.

While f a t t y acid analysis and DBI's a r e useful for i n t e rp r e t i ng possible mechan i sm of chil l ing injury, they a r e not r e l i ab le for a c c u r a t e l y p red ic t ing suscep t ib i l i ty or r e s i s t a n c e to chill ing t e m p e r a t u r e s (14, 16). This fac t is emphas ized by the d a t a shown in Table II where in a compar i son is m a d e b e t w e e n t h e DNA p o l y m e r a s e a c t i v i t y and DBI of nucle i from G. barbadense P ima S-4 lo ts t h a t exhibi t r e l a t i v e deg rees of chill ing sens i t iv i ty (1). As can be no ted , t h e r e is no d i f fe rence in DBI b e t w e e n t h e chill sens i t ive lot ZOO and t h e chill r e s i s t a n t lot 189. However , t h e r e is a s ignif icant d i f f e rence b e t w e e n t h e DNA po lymera se ac t i v i t i e s of t h e two lo t s . Prev ious work from our l abo ra to ry (6, 7) has shown tha t DNA rep l i ca t ion in G. barbadense r ad i c l e t ips is dependen t upon a t t a c h m e n t to var ious s i tes of t h e nuc l ea r m e m b r a n e s and t h a t t he level of DNA rep l i ca t i on is a f f e c t e d by chil l ing t e m p e r a t u r e s . Use of t h e l a t t e r obse rva t ion enabled us to p r ed i c t a r e l a t i v e h i e r a r chy of chill ing r e s i s t a n c e in given lo ts of G. barbadense (7). As previously sugges ted by P a t t e r s o n , et al., (14) chill ing sens i t iv i ty may be r e l a t e d m o r e ef f ic ient ly to a physical change in t he m e m b r a n e s t r u c t u r e during t h e per iod of chil l ing. The l a t t e r even t can then be n o t e d in a p r o p o r t i o n a t e change of a c h a r a c t e r i s t i c b iochemica l r e a c t i o n (i .e. resp i ra t ion) as a f f e c t e d by a change in mi tochondr ia l m e m b r a n e s (11), a d i f f e rence in DNA rep l i ca t ion r a t e s as n o t e d above , and a change in the Hill a c t i v i t y of ch lorop las t s (13).

III. SUMMARY

Although fu r ther e x p e r i m e n t a t i o n is r equ i red , we feel t h a t t hese d a t a s t rongly suggest t h a t t he mic rosomal m e m b r a n e is t he focal point of con t ro l of chil l ing r e s i s t a n c e in p l a n t s . The abi l i ty of t h e mic rosome to d e s a t u r a t e f a t t y acids and ef f ic ient ly t r ans fe r t hese u n s a t u r a t e d f a t t y acids to o the r m e m b r a n e s may be t he r a t e l imi t ing s t ep in chil l ing r e s i s t a n c e in higher p l an t s and some lower o rgan isms such as Tetrahymena as r epo r t ed by Thompson (this vo lume) .


1. Bar tkowski , E. J . , Buxton, D. R., K a t t e r m a n , F . R. H., and Ki rche r , H. W. Agron. J. 69, 37 (1977).

Z. Buxton, D. R., Sprenger , P . J . , and Peglow, E. J . , J r . Crop Sci. 16, 471 (1976).

3 . Chr i s t i ansen , Μ. N. Plant Physiol. 38, 5Z0 (1963). 4 . Chr i s t i ansen , Μ. N. Plant Physiol. 42, 431 (1967). 5. Chr i s t i e , W. W., Noble , R. C , and Moore , J . H. Analyst. 95, 940

(1970). 6. Clay , W. F . , K a t t e r m a n , F . R. H., and Bar t e l s , P . G. Proc. Nat.

Acad. Sci. 74, 3134 (1975).

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7. Clay , W. F . , Buxton, D. R. , and K a t t e r m a n , F . R. H. Crop Sci. 17, 342 (1977).

8. F a r k a s , T., De r i -Had l aczky , and Bela, A. Lipids 10, 331 (1975). 9. Fo lch , J . , Lees , M., and S loane-Stan ley , G. H. J. Biol. Chem. 226

497 (1957). 10. Har r i s , P . , and J a m e s , A. T. Biochem. Biophys. Acta 187, 12

(1969). 11 . Lyons, J . M. and Raison , J . K. Plant Physiol. 45, 386 (1970). 12. Masca renhas , J . P . , L a t i e s , G. G., and Che r ry , J . H. Meth. Enz-

ymol., 31: 588-589 (S. F le i sche r & L. P a c k e r Eds.) (1974). 13. Nolan, W. G. and Smill ie , R. M. Plant Physiol. 59, 1141 (1977). 14. P a t t e r s o n , B. D. , Kenr ick , J . R., and Raison , J . K. Phytochem. 17,

1089 (1978). 15. Wir tz , K. W. A. Biochem. Biophys. Acta 344, 95 (1974 ) . 16. Y a m a k i , S. and Ur i t an i , I. Agric. Biol. Chem, 36 47 (1972).



DIFFUSION RELATIONSHIPS BETWEEN CELLULAR PLASMA MEMBRANE AND CYTOPLASM

Alec D. Keith, Andrea Mastro and Wallace Snipes

D e p a r t m e n t of B iochemis t ry & Biophysics The Pennsy lvan ia S t a t e Univers i ty

Univers i ty Pa rk , Pennsy lvan ia

The spin label app roach has been successful ly used by seve ra l r e s e a r c h e r s dur ing the pas t d e c a d e to c h a r a c t e r i z e s t r u c t u r a l and dynamic p r o p e r t i e s of model and biological m e m b r a n e s (3, 4, 6, 7). On a c o m p a r a t i v e sca l e knowledge of t he cy top lasm r e l a t i n g to s t r u c t u r a l and dynamic p r o p e r t i e s h a v e deve loped m o r e s lowly. The p r e s e n t t r e a t m e n t c o n c e n t r a t e s on the approach and ea r ly phases of using spin labels to c h a r a c t e r i z e t h e diffusional env i ronmen t p rovided by ce l lu la r cy top l a smic s p a c e s . This approach may be very useful in compar ing chil l ing sens i t ive and chil l ing r e s i s t a n t s t r a ins of ag r i cu l t u r a l c rop p l a n t s . It has been previously r e p o r t e d t h a t cy top l a smic s t r e a m i n g may be d i f fe ren t i a l ly modif ied in t h e two types of s t r a in s as a consequence of be ing t r e a t e d wi th low t e m p e r a t u r e s .

Typical spin label e x p e r i m e n t s allow four m e a s u r e m e n t s from the spin labe l s igna l . These four a r e , l ine he igh t (h), l ine wid th (W), hyper f ine coupling c o n s t a n t (A or Τ for t h e i so t rop ic componen t and tensor t e rms) and the g-va lue (g for the i so t rop ic or t ensor t e r m s ) . Some t imes pa r t i t i on ing b e t w e e n hydroca rbon r i ch zones and w a t e r r i ch zones yields a two componen t s ignal . F igure 1 shows most of t he d i r e c t m e a s u r e m e n t s t h a t can be - ^

ad e on spin label s ignals including t h e s p e c t r a l t ypes t h a t

occur from N.

I. ROTATIONAL MOTION RANGE

F igure 2 shows t h e l ine shapes of spin labe l s ignals r e su l t ing from solvent v iscos i ty changes . In t h e fas t tumbl ing r a n g e , in an i so t rop ic medium t h e s e m e a s u r e m e n t s can be t a k e n r e l a t i v e l y a c c u r a t e l y . As t h e v iscos i ty i n c r e a s e s and spin label r o t a t i o n a l mot ion slows, t h e individual N i t rogen hyper f ine l ines change in shape . In t h e fas t tumbl ing r a n g e (the mot ion r a n g e w h e r e t h e t h r e e Ni t rogen hyper f ine l ines h a v e a L o r e n t z i a n

Copyright β 1979 by Academic Press. Inc. 4 3 7 All rights of reproduction in any form reserved

ISBN 0 1 2 4 6 0 5 6 0 5

4 3 8 A. D. Keith et ai

FIGURE 1. Spin label spectra. The top spectrum is that of Τ EM PON Ε dissolved in octadecane. The second spectrum that of TEMPONE dissolved in octadecane with a small capillary of Ν TEMPONE dissolved in wojter inserted into the octadecane sample. The third spectrum is tljyit of Η TEMPONE dissolved in water containing a small capillary of Ν TEMPONE dissolved in water inserted into the

Η TEMPONE sample. The fourth signal is the midline of the H-T EM PON Ε signal amplified 10 times on the X axis to illustrate the

method of measurement of linewidths (W) and lineheights (h).

M e m b r a n e - C y t o p l a s m i c I n t e r r e l a t i o n s h i p s 4 3 9

FIGURE 2. Spin label motion series. Spectra on thq.left are for N-T EM PONE in glycerol and on the right are for N-TEMPONE in glycerol. The numbers beside the spectral lines at 7°, 12° and 23°C represent normalized values of the integrated intensities of different spectral lines. Values are normalized to 100. The small letters (a), (b), and (c) show asymmetry and non-averaged tensor contributions due to loss of motional averaging. The small letters (d) pjjid (e) ^)X )w comparative separation between two spectral lines of Ν and N-T EM PONE at the same state of motion. Small letters (f) and (g) point out how half lines may be used to measure the degree of line symmetry.


l ine shape b e t w e e n inf lec t ion po in t s and the i n t e g r a t e d in t ens i ty (I) of t h e t h r e e l ines a r e equal^ e.g. I j = IQ = I ^). The i n t e g r a t e d in t ens i ty can be expressed as I = kW h for a f irs t de r iva t i ve s p e c t r a l l ine . As so lvent v iscos i ty i nc reases t h e t h r e e Ni t rogen hyper f ine l ines b roaden d i f fe ren t ia l ly . At x-band m i c r o w a v e f requency , t h e o rder of l ine b roadening is W ^ > Ψ ^ > WQ . Because of t h e r e l a t i o n expressed above t he se l ines shape changes a r e mos t read i ly seen as l ine he ight changes . One of t h e convent iona l r o t a t i o n a l co r r e l a t i on t i m e (τ ) ca l cu la t ions ,

uses both W and h m e a s u r e m e n t s . Line he ight changes a r e m o r e read i ly seen because ,

I

or s imply t h a t h is p r o p o r t i o n a t e to 1/WU. As shown in F igure 2, as the

v iscos i ty i nc reases a l imi t ing condi t ion a r i se s . In s p e c t r a r e f l ec t i ng t h e i n t e r m e d i a t e mot ion r a n g e , t h e high field l ine (-1) loses i n t e g r a t e d in t ens i ty be fo re e i t he r t he low field (1) or mid field l ines (0). The n u m b e r s shown in F igure 2 show examples of i n t e g r a t e d in t ens i ty of d i f fe ren t r e sonan t l ines . The loss of i n t e g r a t e d in t ens i ty by the high field l ine r e su l t s b e c a u s e of i n c o m p l e t e t i m e - d e p e n d e n t ave rag ing of t he hyper f ine and g-va lue tensor e l e m e n t s . M e a s u r e m e n t s t a k e n for t he τ equa t ion shown above or for o the r τ equa t ions which depend on £ and /o r W m e a s u r e m e n t s , whe the r r e g a r d e d as numer i ca l ly valid or empi r i ca l , r esu l t in i n a c c u r a t e c a l c u l a t e d va lues . For t he condi t ion w h e r e I ^ > IQ t h e resu l t ing τ va lues a r e abnormal ly i nc rea sed . The exac t mot iona l s t a t e whe re this condi t ion ar i ses has some var iab i l i ty depending on t h e spin label used and t h e d e g r e e of h e t e r o g e n e r i t y in t h e local env i ronmen t s provided by t h e s amp le . An Arrhenius plot g e n e r a t e d from t h e s p e c t r a shown in F igure 2 is shown in F igure 3 . Glycero l under t h e condi t ions r e p o r t e d h e r e has no known or r e p o r t e d phase change in ^ . e t e m p e r a t u r e r ange whe re t h e Arrhenius plot shows a "break." The

N-TEMPONE s p e c t r a and τ va lues a r e inc luded to i l l u s t r a t e t h a t t he a p p a r e n t "break" point in Figujg 2 is a r t i f i c i a l . This b reak point occu r s b e c a u s e t he high field l ine of N-TEMPONE loses i n t e g r a t e d in t ens i ty be fo re t he mid-f ie ld l ine does .

Π. TRANSLATION AL DIFFUSION MOTION RANGE

Several exce l l en t t r e a t m e n t s for var ious a s p e c t s of He isenberg e l e c t r o n - e l e c t r o n spin exchange have been publ ished (1 , 2). The phenomenon of e l e c t r o n spin exchange io ) causes uniform broadening


l O O h

3 0 h

o χ I0|

I 1 1

/ i -

/ Αν / /

- jy t S t

S t s t

— χ t X / —

1 I l 3.0 3.2 3 .4

Κ"1 3.6

χ ΙΟ"3 °

FIGURE 3. Arrhenius plot for 14

N-TEMPONE data from Figure 2. Closed line is for N-TEMPONE and the dotted line is for

bN-

T EM PONE. The curve or break comes about because of loss of integrated intensity of the high field line in both cases.

of all t h r e e N i t rogen hyper f ine l ines as t h e coll ision f requency b e t w e e n spin labe ls or a spin label and s o m e o t h e r sp in -bear ing spec ies i n c r e a s e s . F igure 4 shows a s p e c t r a l se r ies w h e r e t h e spin label c o n c e n t r a t i o n in aqueous medium is i nc reased over a c o n c e n t r a t i o n r a n g e . The c o n c e n t r a t i o n r ange used shows t h e onse t of l ine b roaden ing , t h e gradual i nc r ea se in l ine width as the c o n c e n t r a t i o n dependen t l ine b roaden ing i n c r e a s e s , and finally t h e end re su l t of t h e t h r e e N i t rogen hyper f ine l ines merg ing in to a s ingle e l e c t r o n spin^ exchanged n a r r o w e d l ine . The s a m e c o n c e n t r a t i o n is also shown with N-TEMPONE to i l l u s t r a t e t h a t ^ n e shape changes smooth ly wi th no c e n t r a l l ine appea r ing unt i l t h e N -hyper f ine l ines m e r g e t o g e t h e r . F igure 5 shows a plot of c o n c e n t r a t i o n -dependen t l ine width (ΔΗ) aga ins t t h e spin label c o n c e n t r a t i o n (M). This p lo t shows the l imi t s of a c c u r a t e m e a s u r e m e n t . At t h e point in t h e p lo t

4 4 2 A. D. Keith et al.

FIGURE Λ. Concentration dependent changes of spin label linewidths. Ν on the left and N-TEMPONE on the right. The general pattern of electron-electron spin exchange line broadening and finally spectral narrowing is shown.


Molarit y

FIGURE 5 A. Concentration dependence of line broadening at four different viscosities. The four lines reading from left to right are: water, 65% aqueous glycerol, 75% aqueous glycerol, and 85% aqueous glycerol, τ values for each line are shown. The Δ Η values for spectra used for τ Measurements were less than 0.1G.

TEMPONE MOLARITY

FIGURE 5B. Concentration dependence for line broadening in confining volume. The P-notation refers to the minimum molecular weight in thousands excluded from that bead size. All beads were 100 mesh.

4 4 4 A. D. Keith et al

where t h e r a t i o ΔΗ/Μ, falls below 0.007 t h e va lues can no longer be a c c u r a t e l y measu red . The va lue

Δ Η -τ— = app rox ima te ly 0.2, (3) A

N

is a n u m e r i c a l approx ima t ion of t he upper l imi t of a c c u r a t e l ine width m e a s u r e m e n t s . V a r i e s g r e a t e r t han about 0.2 have too much over lap b e t w e e n ad jacen t N-hyper f ine l ines .

The genera l r e l a t i o n b e t w e e n t h e diffusion cons t an t (D), c o n c e n t r a t i o n dependen t l ine broadening (ΔΗ) and e l e c t r o n - e l e c t r o n spin exchange ^ 8 χ) h a v e been es tab l i shed and a r e shown below (5).

ω = 2 ΔΗ (4) ex

D = Κ ω ex Μ (5)

These r e l a t i ons hold for i so t rop ic media , low viscos i ty so lven ts , and i so t rop ic spin l abe l s .

O t h e r p a r a m a g n e t i c spec ies such as t h e 3d t r ans i t ion se r ies of m e t a l s and thei r c h e l a t e s can be used to e f fec t ive ly b roaden t h e Ni t rogen hyper f ine l ines of spin labe ls . These t echn iques have been deve loped for use on biological s y s t e m s using non- tox ic p a r a m a g n e t i c spin b roaden ing a g e n t s (5).

A. The Effect of Confining Space on Diffusion

Cons ide ra t ions based on t h e E ins te in -S tokes ' r e l a t i o n descr ib ing dif fusional even t s hold for i so t rop ic med ia . Consider ing a smal l spher ica l spin label so lu te , r o t a t i o n a l mot ion and t r ans l a t i ona l diffusion can be desc r ibed in t e r m s of e i t h e r . F rom

λ 3 4ΤΤΓ η

x c = 3kT , (6)

and


diffusion can be expressed in t e r m s of r o t a t i o n mot ion ,

= 0 . 2 2 r2

T c . (8)

This r e l a t i o n holds for mo lecu la r e n v i r o n m e n t s which a r e i so t rop ic and do no t con ta in b a r r i e r s to diffusion. For cy top l a sm, or po lymer fo rmed gels w h e r e "rigid" b a r r i e r s a r e d i spersed in an aqueous medium smal l mo lecu la r so lu tes may e x p e r i e n c e g r e a t e r r e s t r i c t i o n to diffusion than to mo lecu la r r o t a t i o n . For such cases t h e e f f ec t i ve diffusion cons t an t m e a s u r e d wi th spin labels (Dgr ) is d i s t a n c e d e p e n d e n t . The d i s t a n c e -dependency for D g ^ depends on t h e spac ing , s i ze , shape , and c o n c e n t r a t i o n of t h e po lymer field. For e x a m p l e , diffusion within t h e spaces provided by t h e po lymer n e t w o r k is m o r e rap id than diffusion b e t w e e n d i f fe ren t c o n t a i n e d - s p a c e s . T h e r e f o r e , diffusion values a r r ived a t by measu r ing t h e collision f requency io ) of a spin label p robe will be d i f fe ren t depending on w h e t h e r most o f

ef t i e col l is ional even t s occur

b e t w e e n molecu les res id ing in t h e s a m e conf ined s p a c e or b e t w e e n molecu les res id ing in ad jacen t conf ined s p a c e s . The m e a n f ree p a t h (p) b e t w e e n molecu la r coll is ions is given by

ρ , Μ . MW , 2 /3

sol sol sol

P SL M

S L M W

S L S

S L (9)

w h e r e MW is mo lecu la r weight of t h e so lvent (sol), and spin label (SL), Μ is mo la r i t y , pis buoyan t dens i ty and S is . the cubic l a t t i c e spac ing . The cubic l a t t i c e spac ing (S) va r i e s wi th (M ) by t h e express ion ,

c _ ( 1 02 7

A3/ N )

1 /3 ,

M l / 3 (10)

Where Ν is Avagadro ' s n u m b e r . A c h a r a c t e r i z a t i o n of spin label coll ision f requency in i so t rop ic med ia led to t h e equa t ion for diffusion (5).

D ω ex

SL , (11) SL

Where Κ is a proport ional i ty^ c o n s t a n t a r r i ved a t by using t h e m e a s u r e d va lue of D ob ta ined from a Η-spin label employing a cap i l la ry m e t h o d


Τ 1 1 Γ

0.1 0.3 1.0 3.0 10 l / r c χ 1 0 "

, 0 sec" '

FIGURE 6. The relation between translational diffusion (ΔΗ) where D - ΚΔΗ/2Μ and rotational motion as a rate process (l/τ ) . Solid circles represent relatively isotropic media (glycerol-water). Solid squares represent N-TEMPONE in polyacrylamide beads of different pore sizes.

(5). The va lue Dgy is equiva len t to D in i so t rop ic media ; however , in h e t e r o g e n e o u s media , Dgj^ b e c o m e s smal le r wi th dec reas ing spin label c o n c e n t r a t i o n . The re fo re , a p lot of

vs gives in fo rmat ion

r e l a t i n g to t h e s t r u c t u r e of the diffusion env i ronmen t . The d a t a shown in F igure 6 adds addi t ional c l a r i t y to this

cons ide ra t ion . P lo t t i ng (ΔΗ is p r o p o r t i o n a t e to DgjJ agains t 1 / T C

r evea l s , t ha t in i so t rop ic media , t he r a t e of molecu la r r o t a t i o n (1/τ var ies smooth ly with Dgj ; however , in a confining space env i ronmen t , t h a t is much m o r e a f f e c t e d than r o t a t i o n a l mot ion . Equat ions (11) and (12) show the r e l a t i on b e t w e e n Δ Η ^ χ and Dg^-

Δ Η = 2 ω ex

(12)


Open space Open-high viscosity

polymer barriers polymer barriers

in gel phase

FIGURE 7. Particle distribution in space. The relation between relatively open and confined space is illustrated.

Figure 7 i l l u s t r a t e s d i f fe ren t deg ree s of conf ined s p a c e . The upper left of F igure 7 shows one example of p a r t i c l e s r andomly d i s t r i bu ted over a uni t a r e a . With no b a r r i e r s , t he se p a r t i c l e s a r e f ree to diffuse abou t . The upper r ight i l l u s t r a t e s t he e f fec t of high v iscos i ty so lven t . The p roduc t (DgjJ

x (

T

c)> should be t he s a m e for bo th ca se s . The lower left i n t roduces i n c o m p l e t e po lymer b a r r i e r s . For this c a se t he p roduc t of ( D ^ ) χ (τ ) should be d e c r e a s e d . The lower r ight i l l u s t r a t e s diffusion b a r r i e r s c r e a t i n g r e l a t i v e l y i so la t ed s p a c e s . The p roduc t (DgjJ χ (l^) is l eas t for this c a s e .

F igure 8 i l l u s t r a t e s t he d i f fe ren t i a l e f f ec t of confining space on t h r e e d i f fe ren t po lymers wi th r e s p e c t to r o t a t i o n a l mot ion and t r ans l a t i ona l diffusion. All t h r e e po lymers i n t e r a c t wi th t h e P300 beads to fu r the r r e s t r i c t diffusion. It is of i n t e r e s t to n o t i c e t h a t BSA, t h e most spher ica l so lu te of t h e t h r e e , has t h e g r e a t e s t c o o p e r a t i v i t y wi th confining space , r e su l t ing in a l a rge d e c r e a s e in Ό ς τ .

4 4 8 A. D. Keith et al.

( I / T C) X I 0 1 0 see" 1

FIGURE 8. Polymer and confining space effect on diffusion. The solid line shows the ideal relation between D and(lAc). The open symbols show values for N-TEMPONE in preparations of the polymer shown. PVA, is 115,000 molecular weight polyvinyl alcohol at 10% in water. BSA is 10% Bovine serum albumin in water. DNA, is 4 mg/ml of high molecular weight calf thymus DNA in phosphate buffered saline (standard PBS solution). The closed symbols represent the same preparations with P300 polyacrylamide beads added to the aqueous medium and centrifuged in a capillary to insure that all spin label is inside the beads.

Figure 9 i l l u s t r a t e s t he e f fec t of d i f fe ren t c o n c e n t r a t i o n s of sucrose on molecu la r mot ion in f ree and confined s p a c e . The s t r a igh t r e f e r e n c e l ine shows the idea l r e la t ionsh ip b e t w e e n l / τ and D for p e r f e c t l y i so t rop ic med ia . The open c i r c l e s i lTustrate t h a t inc reas ing c o n c e n t r a t i o n s of sucrose i nc rea se s t he p roduc t (DgjJ χ (τ^). The c losed c i rc les i l l u s t r a t e t ha t confining s p a c e fu r ther l imi t s while essen t ia l ly having no e f fec t on τ ^ .


( l / r c ) x l 0 10 sec" 1

FIGURE 9. Sucrose effect on diffusion. Three concentrations of sucrose are shown. The left most open circle is 70% sucrose (W/W). The mid open and closed circle is 40% sucrose and the two circles on the right are 10% sucrose. Open circles represent measurements carried out in the medium alone. Closed circles represent the same preparations in the P300 beads.

One way to modify t h e spac ing b e t w e e n po lymer i c or m a c r o m o l e c u l a r diffusion b a r r i e r s inside cel ls is to vary t he o smo t i c p r o p e r t i e s of t h e ce l lu lar ba th ing m e d i u m . F igure 10 shows a p lot w h e r e de r ived D e v a l u e s a r e p l o t t e d aga ins t t h e r ec ip roca l of o s m o t i c p re s su re ( l / π ) . This d a t a was t a k e n from h u m a n embryon ic lung cel ls using TEMPONE as a spin labe l . The va lue d e c r e a s e d by a f ac to r of a lmos t t h r e e while τ changed only s l ight ly . These d a t a i n d i c a t e t h a t b iopolymers capab l e of r e s t r i c t i n g dif f usional p roces s a r e c rowded c loser t o g e t h e r as a popula t ion of cel ls a r e exposed to hype rosmo t i c s t r e s s . Reduc ing t h e vo lume of cy top la sm in this manne r e f f ec t ive ly i so la tes and i n c r e a s e s t h e p ropor t ion of i so l a t ed spin labe l molecu les and t h e r e f o r e r e d u c e s ω ^ ν and consequen t ly D Q T .

4 5 0 A. D. Keith et al

( Ι /7Γ) χ I0"3

FIGURE 10. Internal viscosity of cells as a function of osmotic strength. Human embryonic lung fibroblasts were treated with spin label, NiCl„ at 75 mM, and modulating concentrations of KCl. The extreme values for mildly hypotonic to hypertonic of both Tq and D values are shown. Under these conditions T C values change only slightly while D values change drastically.

Figure 11 shows the modifying e f fec t of Cytocholas in Β on BHK ce l l s . M e a s u r e m e n t s on cel ls t r e a t e d wi th Cytocholas in Β (closed circles) show a higher collision f requency for spin labels and consequen t ly t h e i n f e r ence is m a d e t ha t ba r r i e r s to diffusion in the cy top lasm have been r e d u c e d as a resu l t of Cytocholas in t r e a t m e n t .


FIGURE 11. Effect of Cytochalasin Β on intracellular viscosity. Open circles are BHK cells in growth medium at isotonic concentrations of ions. Closed circles are a split preparation that was treated with Cytochalasin B.


1. A b r a g a m , A. "Pr inciples of Nuc lea r Magne t i sm" Oxford: C la rendon P res s (1961).

2. Anderson, P . W. J. Phys. Soc. Japan 9, 316-339 (1954). 3 . Gr i f f i th , Ο. H. and Waggoner , A. S. Acc. Chem. Res. 2, 17-24

(1969). 4 . Ke i th , Α., Sharnoff, M. and Cohn, G. Biochim. Biophys. Acta (300,

379-419 (Biomem. Rev.) 1973). 5. Ke i th , A. D. , Snipes, W., Mehlhorn, R. J . and Gun te r , T. Biophys.

J., 19, 205-218 (1977). 6. McConnel l , Η. M. In "Spin Label ing: Theory and Appl ica t ions" (L. J .

Ber l iner , ed . ) . pp . 525-560, A c a d e m i c P re s s , New York (1976). 7. Smith , I. C P . In "Biological Appl ica t ions of ESR Spec t roscopy" (J.

R . Bolton and H. S w a r t z , eds.) , pp 483-539 , Wiley ( In te rsc ience) , New York (1972).



TEMPERATURE E F F E C T S ON PHOSPHOENOL PYRUVATE CARBOXYLASE FROM CHILLING

SENSITIVE AND CHILLING RESISTANT PLANTS

Douglas Graham, Denis G. Hockley and Brian D. Patterson

Plan t Physiology Unit CSIRO Division of Food R e s e a r c h , and School of Biological Sc iences

Macquar i e Univers i ty Nor th Ryde , N.S.W. 2113, Aus t r a l i a

I. INTRODUCTION

We have a t t e m p t e d to find e n z y m e s which a r e a f f e c t e d adverse ly by chil l ing t e m p e r a t u r e s , but which a r e no t m e m b r a n e - b o u n d or a s soc i a t ed wi th l ipids. P E P - c a r b o x y l a s e m a y sat is fy t he se c r i t e r i a . The t e m p e r a t u r e coe f f i c i en t , Q J Q *

or t h e a p p a r e n t Arrhenius a c t i v a t i o n

energy , Ea, of this e n z y m e has been shown to i n c r e a s e below about I O C for t rop ica l C4 p l an t s but no t for t e m p e r a t e - z o n e p l a n t s (7, 9). In i t ia l work sugges ted t h a t t h e d e t e r g e n t Tr i ton X-100 and t h e e n z y m e phosphol ipase C (7) e l i m i n a t e d "break" in a c t i v a t i o n ene rgy a t about I O C . This would imply an as soc ia t ion of t h e e n z y m e wi th lipids; m o r e r e c e n t work (13) has shown t h a t , wi th a highly pur i f ied P E P - c a r b o x y l a s e from m a i z e , t h e b reak in a c t i v a t i o n ene rgy was no t r e m o v e d by Tr i ton X-100.

The work quo ted r e f e r s pr inc ipa l ly to t h e e n z y m e from C4 p l an t s , w h e r e i t is c o n c e r n e d wi th p h o t o s y n t h e t i c C O - f ixa t ion . In C3 p l an t s , i t has an a n a p l e r o t i c ro le , rep len ish ing TCA cyc le i n t e r m e d i a t e s used for t h e synthes is of ca rbon ske l e tons . This impl ies an i m p o r t a n t r e g u l a t o r y ro le for the e n z y m e in C3 p l a n t s .

We have examined t h e e f f ec t of t e m p e r a t u r e on PEP-ca rboxy l a se from t h r e e groups of C3 p l an t s : t rop ica l (chi l l ing-sensi t ive) , t e m p e r a t e and alpine (both chil l ing r e s i s t a n t ) .

Copyright · 1979 by Academic Press, inc 4 5 3 All rights of reproduction in any form reserved

ISBN α 1 2 - 4 6 0 5 6 0 - 5

4 5 4 D. Graham et ai

Π. EXPERIMENTAL

T o m a t o (Lycopersicon esculentum, Mill cv. Ru tge r s ) , whea t (Tri-ticum aestivum L.), b road bean (Vicia faba L.) and purple pass ionfrui t (Passiflora edulis, Sims) w e r e grown in a con t ro l l ed t e m p e r a t u r e glasshouse (25°C d a y / 1 8 ° C n igh t ) . The Aus t ra l i an a lp ines , Cardamine sp . and Caltha intraloba L. co l l e c t ed from 2000m in t he Snovyy

Mounta ins w e r e ma in t a ined in a high light i n t ens i ty (160 w a t t m ), con t ro l l ed env i ronmen t , g rowth cab ine t ^12 h day /12 h n ight ; 15 C /5 C) .

Leaf m a t e r i a l was e x t r a c t e d a t 2 C by grinding in a pe s t l e and m o r t a r wi th sand and tw ice t h e t i s sue volume of 0.2 Μ Tr i s -HCl , pH 7.4, conta in ing 10 mM sodium d i e t h y l t h i o c a r b a m a t e (DIECA), 10 mM MgCl^? 2 mM sodium EDTA, 10 mM 2 - m e r c a p t o e t h a n o l and 1% Po lyc la r . The e x t r a c t was f i l t e red through two l aye r s of Mirac lo th and cen t r i fuged a t 12000 g for 30 min. The s u p e r n a t a n t was de sa l t ed (Sephadex G-25) in to 50 mM Bicine pH 7.6, conta in ing 5 mM MgSO^_, 1 mM sodium EDTA, and 2 mM DTT. The a p p r o p r i a t e e l u a t e was used wi thout fu r ther pur i f i ca t ion for assay of PEP-ca rboxy la se (E.C. 4.1.1.31 a c t i v i t y by following the oxidat ion of NADH a t 340 nm, in a coupled assay wi th m a l a t e dehydrogenase , accord ing to t h e m e t h o d of Ting and Osmond (12). Ma la t e dehydrogenase gave l inear Arrhenius p lo t s and t h e r e f o r e did no t a f fec t our r e su l t s for P E P ca rboxy lase . T e m p e r a t u r e was m e a s u r e d wi th a fine t he rmocoup le , l inked to a r e c o r d e r . Appa ren t Κ P E P values w e r e c a l c u l a t e d using a GENSTAT c o m p u t e r p rog ram b a s e < i on a me thod of Cle land (1), involving L ineweaver Burke p lo t s which we re l inear for t he p l an t s t e s t e d .

The leaf e x t r a c t s con ta ined a single PEP-ca rboxy l a se as shown by the p r e s e n c e of a s ingle peak of enzymic a c t i v i t y on e lu t ion from DEAE Sephacel and a single band of a c t i v i t y a f t e r e l ec t rophores i s on po lyac ry lamide g rad ien t ge ls .

ΠΙ. RESULTS

F i g u / e 1 shows an A rhenius plot of t he a c t i v i t i e s a t V for c rude iii ax p r e p a r a t i o n s of PEP-ca rboxy la se s e x t r a c t e d from leaves of the t e m p e r a t e p l an t , w h e a t , and the t rop ica l p l an t , t o m a t o , which bo th h a v e C 3 - t y p e pho tosyn thes i s . The r e su l t s for whea t r e p r e s e n t a s ingle e x t r a c t and give an app rox ima te ly s t r a igh t l ine re la t ionsh ip for log enzymic r a t e versus r e c ip roca l of abso lu te t e m p e r a t u r e over t he t e m p e r a t u r e r ange 1.5 to 38.5 C. The r e su l t s for t o m a t o a r e from seve ra l e x p e r i m e n t s in order to verify t he non- l inear n a t u r e of t he plot which shows an increas ing dec l ine in a c t i v i t y as t he t e m p e r a t u r e d e c r e a s e s below about 10-12 C. For t h e t o m a t o e n z y m e t h e r e is c lea r ly a dev ia t ion from the Arrhenius re la t ionsh ip a t bo th low and high (above 30 C) t e m p e r a t u r e s and the d a t a a r e f i t t ed well by a cu rve . The t e m p e r a t u r e coef f i c ien t , Q i n for t h e whea t enzyme is app rox ima te ly cons t an t a t about 2.1

T e m p e r a t u r e Effects o n P h o s p h o e n o l P y r u v a t e C a r b o x y l a s e 4 5 5

FIGURE 1. Arrhenius plots of wheat and tomato leaf PEP-carboxylase activities from crude preparations afêr Sephadex G25 treatment. Results have been normalized to 20 C to correct for different extractions of the enzyme. Both were measuredât 2 mM PEP. Enzymic rates are μ moles PEP (or NADH oxidized) min .

th roughout the t e m p e r a t u r e r a n g e w h e r e a s t he t o m a t o enzyme has a

Q-i Q of 1 .9 to 2 . 6 in t he t e m p e r a t u r e r a n g e 3 0 to 1 0 C . It i nc rea se s to g r e a t e r than 5 below 1 0 C . T h e r e is , t h e r e f o r e , a c l ea r d i s t inc t ion in t h e behaviour of t he enzymes from w h e a t and t o m a t o a t low t e m p e r a t u r e s .

The g e n e r a l i t y of this finding is i l l u s t r a t e d by F ig . 2 . The low t e m p e r a t u r e behaviour of t h e e n z y m e from Aus t ra l i an a lp ine p lan t s (Cardamine sp. and Caltha intraloba), t e m p e r a t e - z o n e p l an t s (wheat and broad bean) and t rop ica l p l a n t s ( t o m a t o and Passiflora edulis) is shown. R e l a t i v e enzymic a c t i v i t y a t 1 . 5 C is p l o t t e d as a p e r c e n t a g e of t h a t a t 2 0 C . The a lpine and t e m p e r a t e spec ies a r e very s imi lar in the i r behaviour and give app rox ima te ly l inear Arrhen ius p lo t s s imi lar to t h a t of whea t shown in F ig . 1 . The t rop ica l spec ies , however , show a r e l a t i v e l y much g r e a t e r dec l ine in enzymic a c t i v i t y a t low t e m p e r a t u r e s .

4 5 6 D. Graham et al.

Relative activity at 1.5 ° compare d to 20c

30 Alpin e Temperat e

Tropica l Whea t

o'

Tomat o Passiflor a

U FIGURE 2. PEP-carboxylase activities from Caltha intraloba,

Cardamine sp., Vicia faba (broad bean), wheat, tomato and Passiflora edulis at 1.5 C compared, as a percentage, to the activity at 20 C.

Our work, t h e r e f o r e , conf i rms t ha t a lp ine and t e m p e r a t u r e spec ies can main ta in r e l a t i ve ly higher r a t e s of PEP-ca rboxy l a se a c t i v i t y a t low t e m p e r a t u r e s than can t rop ica l spec ies .

We were i n t e r e s t e d in t he e f f ec t s of t e m p e r a t u r e a t l imi t ing s u b s t r a t e c o n c e n t r a t i o n s s ince this may b e t t e r approach the n a t u r a l m e t a b o l i c env i ronment of t he e n z y m e . Values for t he c o n c e n t r a t i o n of P E P in C3 leaves a r e less than 0.1 mM (5). Accordingly , the e n z y m e s from w h e a t and t o m a t o w e r e assayed over a wide r ange of s u b s t r a t e c o n c e n t r a t i o n s . In Fig . 3 we show how PEP-ca rboxy l a se a c t i v i t y changes wi th t e m p e r a t u r e a t two d i f fe ren t s u b s t r a t e c o n c e n t r a t i o n s . For t h e t rop ica l p lan t , t o m a t o (Fig. 3a), t he a c t i v i t y is r e d u c e d much m o r e below I O C when the s u b s t r a t e c o n c e n t r a t i o n is l imi t ing (^K PEP) than when i t is s a t u r a t i n g (>10 t i m e s Κ PEP) . However , t he r e su l t s in Fig . 3b show t h a t t h e e f fec t of low t e m p e r a t u r e s under l imi t ing s u b s t r a t e c o n c e n t r a t i o n is not nea r ly so a p p a r e n t for t h e e n z y m e from whea t , a t e m p e r a t e - z o n e p l an t . In all cases ini t ia l r a t e s w e r e used in de t e rmin ing t h e enzymic a c t i v i t i e s . These obse rva t ions a r e ampl i f ied by the t e m p e r a t u r e coef f i c ien t s , Q i n> c a l c u l a t e d for t he t e m p e r a t u r e r anges


FIGURE 3. Arrhenius plots comparing PEP-carboxylase activities at V (10 χ Κ PEP) and approximately Κ PEP (20°C) for a) tomato, cv. Rutgers, 6 mM and 0.125 mM PEP, and b) wheat, 2 mM and 0_.£25 mM PEP. Enzymic rates are μ moles PEP (or NADH oxidized) min .

4 5 8 D. Graham et al.

30° -20°C and 10° -0°C (Table I) which show a l a rge i n c r e a s e in Q 1 Q below 10°C for t he t o m a t o e n z y m e , but only a smal l change for t he whea t e n z y m e .

We w e r e unable to change the slope of t he Arrhenius p lo t s for t he t rop ica l spec ies by t r e a t m e n t of t h e c rude p r e p a r a t i o n s wi th Tr i ton ΧΙ 00. It is unlikely, t h e r e f o r e , t h a t t he m a r k e d dec l ine in enzymic a c t i v i t y a t chill ing t e m p e r a t u r e s is m e d i a t e d through lipids a s soc i a t ed wi th t he p ro t e in . In addi t ion , we could find no convincing ev idence for an abrupt i nc rea se in a c t i v a t i o n energy a t a de f in i t e t e m p e r a t u r e .

In o rder to d e t e r m i n e w h e t h e r t h e enzymic p ro te in undergoes any t e m p e r a t u r e - i n d u c e d changes we have examined t h e k ine t i c p a r a m e t e r , Κ P E P , which would ind ica t e whe the r t he aff ini ty of t h e e n z y m e for one

s u b s t r a t e s is a f f e c t e d by t e m p e r a t u r e . Changes in Κ a r e l ikely to r e f l e c t changes in t he o rde red s t r u c t u r e of t he p ro te in about t he a c t i v e s i t e . The re fo re , appa ren t Κ P E P was measu red a t t e m p e r a t u r e s from

ο π m o n 1.3 C to 38.5 C. The a p p a r e n t Κ P E P values a t 1.3 and 20 C for the enzymes from Caltha (alpine), whea t ( t empera t e ) and t o m a t o (tropical) a r e c o m p a r e d in Fig . 4 . The values for t he Caltha and w h e a t enzymes do not change s ignif icant ly a t t he lower t e m p e r a t u r e and r e m a i n b e t w e e n 0.1 and 0.2 mM. However , t he Κ P E P for t he t o m a t o enzyme inc reases n ine- fo ld ; from 0.1 mM a t 20°C ?o 0.9 mM a t 1.3°C. A minimum va lue for t he t o m a t o enzyme of 0.04 mM occur s a t 30 C, r e p r e s e n t i n g a 22.5 fold i nc r ea se a t t h e lower t e m p e r a t u r e .

TABLE I. Temperature Coefficients, Q.q, of PEP-carboxylase from Wheat and Tomato at Low and High Substrate Concentrations and Temperatures

« 1 0 Values

Plant PEP cone. 30°-20°C 10°-0°C

Wheat 2 mM 1.76 2.10 0.125 mM 2.09 3.67

Tomato 6 mM 1.89 3.50 0.125 mM 2.45 » 6.25


K m PE P a t 1.3 C Β an d 20° C •

Tomato Whea t Caltha

mM Γ1.0 Τ

m

FIGURE 4. Apparent Michaelis constant, ^mPEP, at 1.3° and 20°C for PEP-carboxylases prepared from tomato, c v . Rutgers, wheat and Caltha intraloba. Bars represent 95% confidence limits, derived from statistically analyzed Lineweaver-Burke plots.

We conc lude t h a t t he a c t i v e s i t e of t h e enzyme from this t rop ica l spec ies unde rgoes changes in i t s p r o p e r t i e s wi th low t e m p e r a t u r e and t h a t this r e su l t s in a changed aff ini ty of t he e n z y m e for one of i t s s u b s t r a t e s . This is possibly a d i r ec t e f f ec t of low t e m p e r a t u r e on the s t r u c t u r e of t h e p ro t e in .

IV. DISCUSSION

The r e su l t s i nd i ca t e t ha t t he a c t i v i t y of P E P - c a r b o x y l a s e from some t rop ica l (chi l l ing-sensi t ive) C3 spec ies m e a s u r e d over t he t e m p e r a t u r e r a n g e 1.3 - 38.5 C show non- l inear Arrhenius p lo t s for V below

ο rnax about I O C . Severa l a lp ine and t e m p e r a t e (chilling-resistantT spec ies show essen t ia l ly l inear Arrhenius p lo t s for t h e enzymic a c t i v i t y over t h e s ame t e m p e r a t u r e r a n g e . When s imi lar p lo t s a r e m a d e a t low s u b s t r a t e

4 6 0 D. Graham et al

c o n c e n t r a t i o n s , a round the appa ren t Κ for P E P , (i .e. a t app rox ima te ly in vivo concen t ra t ions ) t h e e f f ec t s of Eow t e m p e r a t u r e a r e a c c e n t u a t e d . These r e su l t s a r e s u b s t a n t i a t e d by the m e a s u r e m e n t s of a p p a r e n t Κ P E P for the a lp ine , t e m p e r a t e and t rop ica l spec ies which show t h a ? the t rop ica l spec ies have a subs tan t ia l ly i nc reased a p p a r e n t Κ P E P a t low t e m p e r a t u r e s . It is no t sugges ted t h a t this e f f ec t on ΚΓ is wholly respons ib le for the dec l ine in a c t i v i t y of the e n z y m e a t low t e m p e r a t u r e s s ince t he enzymic a c t i v i t y was genera l ly m e a s u r e d a t V . However , t he finding does i nd i ca t e t h a t low t e m p e r a t u r e has a d i r e c \

ae f f e c t on the

p ro te in , p re sumab ly in t he vic ini ty of t he a c t i v e s i t e . Similar e f f ec t s of t e m p e r a t u r e on the Κ of severa l e n z y m e s from an imals have been r e p o r t e d (4, 11). In these an imals t h e min imum va lue for appea r s to coincide wi th the no rma l env i ronmen ta l t e m p e r a t u r e of the organism and inc reases ou ts ide this r ange of t e m p e r a t u r e s .

Possible mechan i sms by which low t e m p e r a t u r e could e x e r t d i r ec t e f f ec t s on the p ro te in a r e via hydrophobic bonding (4) which is weake r a t low t e m p e r a t u r e , or hydrogen bonding and e l e c t r o s t a t i c i n t e r a c t i o n s which a r e s t ronge r a t low t e m p e r a t u r e . Hydrophobic bonding appea r s to be m o r e i m p o r t a n t in main ta in ing enzymic s t r u c t u r e (8). Such changes in hydrophobic bonding could inf luence the k ine t i c p a r a m e t e r s of the e n z y m e .

Similar e f f ec t s of low t e m p e r a t u r e on a n u m b e r of soluble enzymes such as m a l a t e , g l u t a m a t e , a lcohol , g lucose -6 -phospha te , N A D P -i s o c i t r a t e and g l u t a m a t e dehydrogenases , from t h e t rop ica l C3 spec ies , soybean (Glycine max (L.) Merr . ) , have been r e p o r t e d (2). However , it was concluded t h a t the sharp "breaks" in t he Arrhenius p lo t s w e r e probably due to lipid assoc ia t ion of t h e e n z y m e s . T h e r e is no ev idence to support such a conclusion in our s tud ies .

The findings r e p o r t e d in t he p r e s e n t pape r sugges t t h e possibi l i ty tha t the me tabo l i sm of t rop ica l , ch i l l ing-sens i t ive p l an t s may be d i s rup ted by d i rec t e f f ec t s of low t e m p e r a t u r e on the anap l e ro t i c e n z y m e P E P - c a r b o x y l a s e . P re l imina ry s tud ies in our l abo ra to ry ind ica t e t h a t p y r u v a t e me tabo l i sm is adverse ly a f f e c t e d by low t e m p e r a t u r e in the t rop ica l spec ies s tud ied . While not d i scount ing the possible i m p o r t a n c e of m e m b r a n e lipids in accoun t ing for chill ing injury in ch i l l ing/sens i t ive spec ies , the p re sen t r e su l t s sugges t an a l t e r n a t i v e , or addi t ional mechan i sm whereby the p lan t ' s me tabo l i sm could be d i s rup ted by low t e m p e r a t u r e s in the chil l ing r a n g e . An i m b a l a n c e in me tabo l i sm could resu l t from d e c r e a s e d a c t i v i t y of PE P -ca rb o x y l a se , espec ia l ly s ince the ope ra t ion of the TCA cyc le as a s y n t h e t i c p a t h w a y is dependen t on a con t inued supply of C4 acids which a r e pr inc ipal ly p roduced by this e n z y m e . Such an e f fec t would be a l t e r n a t i v e , or addi t iona l , to the m e t a b o l i c imba lances which w e r e previously sugges ted to be a consequence of chill ing (6, 10).



1. Cle land W. W. Adv. Enzymol. Relat. Areas Mol. Biol. 29, 1-32 (1967).

2. Duke , S. H., Schrader , L. E., and Miller , M. G. Plant Physiol. 60, 716-722 (1977).

3 . Hochachka , P . W. BUTChem. J. 143, 535-539 (1974). 4 . Hochachka , P . W., and Somero , G. N . Comp. Biochem. Physiol. 27,

649-668 (1968). 5. L a t z k o , E., Labe r , L., and Gibbs, M. In "Pho tosyn thes i s and P h o t o -

re sp i r a t ion" (M.D. H a t c h , C. Β Osmond and R. O. S la tyer , eds.) , pp . 196-201 . John Wiley, New York (1971).

6. Lyons, J . M. Annu. Rev. Plant Physiol. 24, 445-466 (1973). 7. McWill iam, J . R. , and F e r r a r , P . J . In "Mechanisms of Regu la t ion

of P lan t Growth" (R. L. Bieleski , A. R. Ferguson and Μ. M. Cresswel l , eds.) R. Soc. N.Z. Bull. 12, 467-476 (1974).

8. Oakenful l , D. , and Fenwick , D. E. Aust. J. Chem. 30, 741-752 (1977).

9. Phi l l ips , P . J . and McWill iam, J . R. In "Pho tosyn thes i s and P h o t o -r e sp i r a t ion" (M. D. H a t c h , C . B. Osmond and R. O. S la tyer , eds.) , pp . 97-104 . John Wiley, New York (1971).

10. Raison , J . K. Symp. Soc. Exp. Biol. 27, 485-512 (1973). 11 . Somero , G. N . Am. Nat. 103, 517-530 (1969). 12. Ting, I. P . , and Osmond, C. B. Plant Physiol. 51, 439-447 (1973). 13. Vedan, K., and Sugiyama, T. Plant Physiol. 57, 906-910 (1976).



CELL C U L T U R E MANIPULATIONS AS A POTENTIAL BREEDING TOOL

P. J . Dix

D e p a r t m e n t of G e n e t i c s Univers i ty of N e w c a s t l e upon Tyne

N e w c a s t l e upon Tyne Uni ted Kingdom

I. INTRODUCTION

P r o t a g o n i s t s of p l an t t i s sue and cell c u l t u r e me thods a r e now c o m m o n p l a c e . Appl ica t ions of t h e s e t echn iques can be found in nea r ly every field of p lan t s c i ence and they have r e s u l t e d in many va luable con t r ibu t ions to our knowledge of p r i m a r y and secondary m e t a b o l i s m , t h e cel l cyc le , t h e r egu la t ion of g rowth and d i f f e ren t i a t ion , and p l a n t / m i c r o - o r g a n i s m i n t e r a c t i o n s . F rom the ag r i cu l tu ra l point of view the obvious a t t r a c t i o n s of t i s sue c u l t u r e me thods for virus e r ad i ca t i o n and rapid clonal p ropaga t ion of c o m m e r c i a l v a r i e t i e s h a v e con t inued to draw a g r e a t dea l of a t t e n t i o n and the number of spec ies for which t h e bas ic c u l t u r e c r i t e r i a have e i t he r been m e t , or a r e under in tens ive s tudy, is now qu i t e ex t ens ive (43). I n t e r e s t in g e n e t i c manipu la t ions of p lan t cel l cu l tu re s has con t inued to i n c r e a s e , desp i t e t h e many obs t ac l e s in t he way of wide app l ica t ion of t he se m e t h o d s . It is t h e r e f o r e e x p e c t e d t h a t those i n t e r e s t e d in ove rcoming low t e m p e r a t u r e s t r e s s would also d i r ec t some a t t e n t i o n to t h e s e g e n e t i c man ipu la t ions .

This rev iew will consider t h e d i f fe ren t m e t h o d s for genome modi f ica t ion in cell cu l t u r e s and t h e p rob lems e n c o u n t e r e d in t h e app l ica t ion of t hese m e t h o d s . The e x t e n t to which t he se p rob lems h a v e been and can be o v e r c o m e will be e v a l u a t e d wi th p a r t i c u l a r r e f e r e n c e to e x p e r i m e n t s p e r f o r m e d wi th crop spec ies , and having a p o t e n t i a l for crop i m p r o v e m e n t . In addi t ion t h e l imi t ed p rogress which has been m a d e in b reed ing for chil l ing r e s i s t a n c e using t i s sue cu l tu re s will be cons idered .

4 6 3 Copyright ® 1979 by Academic Press, inc.

All rights of reproduction in any form reserved ISBNOI2 46056O5

4 6 4 P. J. DiX

Π. THE C U R R E N T STATE OF PLANT CELL CULTURE TECHNOLOGY

The bas ic cyc le of in i t i a t ion of u n d i f f e r e n t i a t e d c u l t u r e s , and r e g e n e r a t i o n of i n t a c t p lan t s from them has now been d e m o n s t r a t e d for a l a rge number of spec ies and in many of them finely d ispersed cell suspension cu l t u r e s , and p ro top las t i so la t ion , cu l t u r e and fusion, have ex t ende d the r ange of me thods which can be used to e f fec t g e n e t i c modi f ica t ion . In addi t ion a n t h e r c u l t u r e , f irst used by Guha and Maheshwar i (27) has r e s u l t e d in t h e ava i lab i l i ty of haploid m a t e r i a l in a far g r e a t e r r ange of spec ies than be fo re (50).

It r e m a i n s t r u e , however , t h a t for a l a rge number of va luable crop spec ies all t he e s sen t i a l cu l t u r e condi t ions have no t been worked out , and for many o t h e r s they have y e t to b e c o m e suff ic ient ly re f ined and r e p e a t a b l e as to provide a r ea l ly useful b reed ing too l . C e r t a i n fami l ies , mos t no tab ly Solanaceae, have p roved genera l ly more a m e n a b l e to cel l cu l t u r e manipula t ions than o t h e r s . T r e e spec ies and ce rea l s have t ended to p rove r e c a l c i t r a n t a l though t h e c o n c e n t r a t i o n of e f for t and r e s o u r c e s has begun to yield a m e a s u r e of success in severa l c e r e a l spec ies (43). Most commonly the r e g e n e r a t i o n of p l an t s from cell cu l tu re s is t he c r i t i ca l s t a g e .

ΙΠ. THE USE OF CELL CULTURES IN BREEDING

A. Exploitation of Existing Variation

Anthe r -de r ived haploid p lan t s and homozygous diploids de r ived from them by co lch ic ine t r e a t m e n t (28) provide a means of a c c e l e r a t i n g the exp lo i t a t ion of exis t ing va r i a t ion by convent iona l combina t ion b reed ing (39). This p rocedu re has a l r eady been successful ly used to develop new cu l t iva r s of t obacco (45, 9) and is c e r t a i n to be ex t en d ed to o the r c rop spec ies as the a n t h e r c u l t u r e p r o c e d u r e b e c o m e s more widely appl icable and re l i ab le .

B. Mutation and Selection

1. Selection Procedure. Se lec t ion in cel l cu l tu re s is only useful for c h a r a c t e r s which a r e l ikely to be expressed equal ly well in und i f f e r en t i a t ed cells and i n t a c t p l a n t s . Se lec t ion p rocedu re s usually involve t h e exposure of cal lus , cel l suspension, or p r o t o p l a s t , cu l tu res , with or wi thout a pr ior mu tagenes i s s t e p , to a su i tab le s e l ec t i ve t r e a t m e n t which kills or inhibi ts t h e division of n o r m a l ce l l s . Surviving cel ls give r i se to hea l t hy p ro l i f e ra t ing a g g r e g a t e s which can then be r e p e a t e d l y exposed to a cycle of se l ec t ion and r e g r o w t h , and p l an t s r e g e n e r a t e d from t h e m .

Cell C u l t u r e M a n i p u l a t i o n s a s a Po ten t i a l B r e e d i n g Tool 4 6 5

2. Problems

a. Mutagenesis. The number of cel ls p r e s e n t in a cu l tu re m a k e it r e a l i s t i c to s e l ec t m u t a n t s wi thou t r e c o u r s e to a m u t a g e n i c t r e a t m e n t , and this was t he case with many of t he va r i an t l ines descr ibed h e r e . Ef fec t ive use of m u t a g e n s has been desc r ibed in some cases (51, 42). It may be wise to r e s t r i c t t he use of m u t a g e n s as far as possible when a t t e m p t i n g to modify crop p l an t s .

b. Aggregation. Excep t in t h e case of p r o t o p l a s t s , cel l cu l tu re s do not exist p r edominan t ly of single ce l l s , but of a g g r e g a t e s of var ious s izes , and canno t t h e r e f o r e be r e g a r d e d as mic roo rgan i sms . Within an a g g r e g a t e cel ls may be of d i f fe ren t s izes and physiological s t a t e s , and in t e r ce l lu l a r connec t ions , t o g e t h e r wi th a v a r i e t y of g rad ien t s ac ross a g g r e g a t e s could i n t e r f e r e with t he se l ec t ion of some kinds of v a r i a n t .

C. Minimal cell density. Most cel l cu l tu re s r equ i r e a min imum cell dens i ty for g rowth which may pose p rob lems when t ry ing to se l ec t a few survivors from a l a rge popula t ion of dead ce l l s .

d. Chromosomal instability. A well known f e a t u r e of cel l cu l tu re s is t h e ch romosomal ins tab i l i ty induced by t h e cu l t u r e sys tem which over a per iod gradual ly gives r i se to polyploid or aneuploid cells (48, 11 , 1). This does not occur in all spec ies but appea r s to be pa r t i cu l a r l y p ronounced in haploid cu l tu re s . (48). Gross ch romosomal changes obviously do not favour t he use of cu l tu re s in b reed ing .

e. Loss of morphogenic potential. The p o t e n t i a l for t he in i t i a t ion of shoots or embryos in cu l t u r e is of ten r e d u c e d by an ex t en d ed c u l t u r e per iod , a phenomenon possibly a s soc i a t ed wi th d. This is the mos t commonly e n c o u n t e r e d problem in va r i an t s e l ec t ion in cel l c u l t u r e s . The major i ty of va r i an t cell l ines which have been descr ibed exis t only as cel l l ines. These two f e a t u r e s t o g e t h e r m e a n t h a t exped ien t use of freshly i n i t i a t e d cu l tu re s is l ikely t o r e m a i n des i rab le when crop i m p r o v e m e n t is the a im.

f. Loss of flowering or fertility. May be fu r ther conse quences of c u l t u r e induced inc iden ta l g e n e t i c changes . Loss of f lowering has been found in p l an t s r e g e n e r a t e d from a s t r e p t o m y c i n r e s i s t a n t cel l l ine of Nicotiana sylvestris (37).

g. Epigenetic variation. Pheno typ ic changes , r e su l t ing from causes o the r than m u t a t i o n (such as changes in gene expression) can of ten be found in cell cu l t u r e s . An example is cyc lohex imide r e s i s t a n c e (35). Some ep igene t i c v a r i a n t s may be very p e r s i s t e n t .

4 6 6 P. J. Dix

Many of t hese p rob lems can be avoided, or may not exis t for a p a r t i c u l a r sy s t em, and, in sp i te of them a number of va r i an t l ines have been s e l e c t e d in c u l t u r e . In some of them (38, 33 & 34, 3), sexual t ransmiss ion has been unequivocal ly d e m o n s t r a t e d . The re follows a brief survey of t he va r i an t l ines which have been s e l e c t e d for c h a r a c t e r s of p o t e n t i a l ag r i cu l tu ra l i n t e r e s t .

3. Disease Resistance. Car l son (5) p roduced t o b a c c o p lan t s r e s i s t a n t to in fec t ion by Pseudomonas tabaci by se lec t ion for r e s i s t a n c e to me th ion ine sulfoximine, an ana logue of t h e toxin, in haploid cell cu l t u r e s . Gengenbach and G r e e n (22) s e l e c t e d for r e s i s t a n c e to t he toxin of Helminthosporium maydis in Texas m a l e - s t e r i l e m a i z e cu l tu res to obta in p lan t s r e s i s t a n t to t h e pa thogen . Ce r t a in ly t h e most s ignif icant case is t he deve lopmen t of sugar c a n e cu l t iva r s r e s i s t a n t to four d i f fe ren t pa thogens (46).

4. Herbicide Resistance. Chaleff and Parsons (6) have d e m o n s t r a t e d t he sexual t ransmiss ion , as a dominan t a l le le , of p ic lo ram r e s i s t a n c e s e l e c t e d in t obacco cu l t u r e s . Several o the r cell l ines r e s i s t a n t to herb ic ides have r e c e n t l y been s e l e c t e d (26).

5. Environmental Stress Resistance

a. Low temperature. Dix and S t r e e t (13) ob ta ined cell l ines of Nicotiana sylvestris and Capsicum annuum wi th enhanced r e s i s t a n c e to exposure to -3 C and 5 C r e s p e c t i v e l y . Two types of r e s i s t a n t l ine of N. sylvestris d i f fe red marked ly in the i r level of r e s i s t a n c e . Unfo r tuna te ly p lan t s could only be r e g e n e r a t e d from some of t he l ines with a lower level of r e s i s t a n c e and cal lus der ived from the seedl ing progeny was sens i t ive (10). P l an t s could no t be r e g e n e r a t e d from r e s i s t a n t or sens i t ive C. annuum c u l t u r e s . It is conc luded t ha t t h e lower level of chill ing r e s i s t a n c e in N. sylvestris r e s u l t e d from a fairly s t ab le ep igene t i c change , p e r s i s t e n t th rough an indef in i te number of m i t o t i c divisions in cell cu l t u r e , but lost during the p l an t s sexual c y c l e . A l t e r n a t i v e exp lana t ions for t he loss of r e s i s t a n c e , such as cont inua l s eg rega t ion in cu l t u r e giving r i se to ch imera l p l an t s canno t be ru led ou t .

The se l ec t ion sys tem used in t he above work r e s u l t e d in many l ines which survived t h e first s e l ec t ion , but succumbed to subsequent exposure . This may r e f l ec t the physiological s p e c t r u m of cel ls p r e s e n t in t h e cu l tu re s imul taneous ly , and the p rob lems of va r i a t ions in a g g r e g a t e s ize a l r eady discussed. These f ac to r s may have a g r e a t e r i m p a c t on the se lec t ion of this kind of va r i an t than , for example , on se l ec t ion for r e s i s t a n c e to most drugs . This may be t he more so s ince we a r e looking for survival and growth subsequent to t he se l ec t ion p re s su re , r a t h e r than g rowth in t he p r e s e n c e of a s e l e c t i v e a g e n t . Cons iderab le r e f i n e m e n t of t h e se lec t ion p r o c e d u r e and a thorough examina t ion of a l a rge number of p u t a t i v e m u t a n t s should even tua l ly lead to cu l t iva r s wi th enhanced chil l ing r e s i s t a n c e . It is encouraging t h a t t he possibi l i t ies a r e being fur ther i nves t i ga t ed using cell cu l tu re s of t o m a t o (4) and r i c e (Xuan and Dix, unpublished).


b. High salinity. The se l ec t ion of cel l l ines wi th enhanced r e s i s t a n c e to g rowth inhibi t ion by sodium ch lor ide has been desc r ibed for Nicotiana sylvestris (56, 12), Nicotiana tabacum (44), Capsicum annuum (12), and Citrus sinensis (30). In t h e case of N. sylvestris a number of p l an t s h a v e been r e g e n e r a t e d from a r e s i s t a n t cel l l ine, and call i i n i t i a t ed from them r e t a i n thei r r e s i s t a n c e (Dix, unpublished) .

C. Aluminium. Mered i th (41) has r e p o r t e d t h e se l ec t ion of cell l ines of Lycopersicon esculentum wi th a s t ab l e r e s i s t a n c e to a luminium tox ic i ty .

6. Amino Acid Overproduction. A common mechan i sm for r e s i s t a n c e to amino acid ana logues is a r e d u c e d sens i t iv i ty of t he feedback con t ro l mechan i sm of t h e b iosyn the t i c p a t h w a y for t h e a p p r o p r i a t e amino acid, r e su l t ing in i t s ove rp roduc t ion . If th is could be t r a n s l a t e d in to improved levels of key amino acids in s t o r a g e organs or seed p ro te ins t h e p o t e n t i a l for crop i m p r o v e m e n t would be enormous . For this r eason m o r e a t t e n t i o n has been paid to se l ec t ion for amino acid ana logue r e s i s t a n c e in cell cu l tu re s than to any o t h e r c lass of m u t a n t , and seve ra l r e c e n t r ev iews h a v e cove red the subject (54, 55, 32). Many of t h e ana logue r e s i s t a n t l ines which h a v e been s e l e c t e d a r e indeed ove rp roduce r s of t he cor responding amino acids but in none of t he se has ove rp roduc t ion been shown in r e g e n e r a t e d p l an t s , or sexual t ransmiss ion been d e m o n s t r a t e d . S -2 -aminoe thy l - cys t e ine (AEC) r e s i s t a n c e s e l e c t e d in ba r ley embryos (Bright, persona l communica t ion) and me th ion ine sulfoximine r e s i s t a n c e s e l e c t e d in t o b a c c o cu l tu re s (5) w e r e sexual ly t r a n s m i t t e d as r ece s s ive t r a i t s , but t he mechan i sm of r e s i s t a n c e was not known in these ca se s .

The p o t e n t i a l of cell cu l tu re s for t he se l ec t ion of this kind of m u t a n t has been well i l l u s t r a t ed by Widholm (53). Using sequen t i a l s e l ec t ion for r e s i s t a n c e to four d i f fe ren t ana logues , he has ob ta ined a c a r r o t cel l l ine s imul taneous ly overproducing lysine, phenyla lan ine , me th ion ine and t ryp tophan .

C. Somatic Hybridization

1. Protoplast Isolation, Culture and Fusion. The main a t t r a c t i o n of s o m a t i c hybr id iza t ion l ies in t h e possibi l i ty of su rmount ing the compa t ib i l i t y ba r r i e r b e t w e e n spec ies , and t h e c r e a t i o n of novel hybr ids , but p r o t o p l a s t s may also p rove the most r e a l i s t i c v e c t o r for t he t r ans fe r of n u c l e a r and cy top la smic genes from one spec ies to a n o t h e r . The r ange of spec ies for which the i so la t ion and c u l t u r e me thods h a v e been m e t , has been cove red by severa l r e c e n t r ev i ews (21, 52, 19, 20). P r o t o p l a s t s can now of ten be ob ta ined from cal lus and cell suspension cu l t u r e s , as well as a wide r a n g e of p lan t o rgans . Fa i r ly genera l me thods for improving t h e f requency of fusion b e t w e e n p r o t o p l a s t s h a v e been developed, probably t h e most popular being t h e use of po lye thy lene glycol (PEG) in a me thod devised by Kao and Michayluk (29).

4 6 8 P. J. Dix

2. Hybrid Selection. The p r o d u c t s of p ro top la s t fusion a r e h e t e r o k a r y o n s which may then go on to form hybrids by nuc l ea r fusion. It is then neces sa ry to se l ec t out hybrid cells from the la rge major i ty of unfused ce l ls , and self - fused ce l l s . Most p r o c e d u r e s , well r e v i e w e d (7), involve c o m p l e m e n t a t i o n resu l t ing in p r e f e r e n t i a l g rowth of t h e hybrids under c e r t a i n s e l e c t i v e condi t ions . For this t he p a r e n t spec ies or cel l l ines must be careful ly chosen and m u t a n t cell l ines can be p a r t i c u l a r l y useful . C o m p l e m e n t a t i o n b e t w e e n two non-a l le l ic m u t a n t cel l l ines has been used, as in n i t r a t e r e d u c t a s e def ic ien t m u t a n t s of t o b a c c o (25), but a single m u t a n t l ine can also be combined wi th a visually d is t inc t one . For example , Ma l igae t al. (36) fused p ro top l a s t s of an albino kanamycin r e s i s t a n t l ine of Nicotiana sylvestris (14) wi th N. knightiana mesophyll p ro top l a s t s which divide a t a low f requency to give g reen colonies . Hybrids w e r e s e l e c t e d as g reen kanamyc in r e s i s t a n t colonies .

Where mesophyll p ro top l a s t s a r e fused to those of a cu l tu red cell l ine, which is normal ly unp igmented , h e t e r o k a r y o n s can of ten be visually ident i f ied and physical ly s e p a r a t e d using a m i c r o p i p e t t e (40). This may p rove a m o r e genera l p r o c e d u r e for t he i so la t ion of hybrids from a mixed popula t ion . Conf i rma t ion of hybr id i ty is genera l ly sought e i the r by compar i son with sexual hybrids (when sexual ly compa t ib l e spec ies have been used), by karyotyping , or by looking for i n t e r m e d i a t e morphological (17) or b iochemica l c h a r a c t e r i s t i c s , such as i soenzyme p a t t e r n s (36).

3. Somatic Hybrids

a. Intrageneric. Somat ic hybr id iza t ion within a spec ies or b e t w e e n closely r e l a t e d spec ies could be a very useful b reed ing tool . Hybr id iza t ion ία spec ies which take a long t ime to f lower could be a c c e l e r a t e d , s ince seedl ing p ro top la s t s could be used, and t h e r e may be incompa t ib i l i t y f a c to r s b e t w e e n qu i t e closely r e l a t e d spec ies . In the even t oi valuable m u t a n t cell l ines being ob ta ined , which have lost their c a p a c i t y for shoot r e g e n e r a t i o n or f lowering, in t r a spec i f i c , or i n t r a g e n e r i c p ro top la s t fusion may provide a means of u t i l iz ing the des i red pheno type . It may be possible to r e g e n e r a t e fe r t i l e p lan t s from the hybrid cells and e l imina t e ch romosome anomal ie s in subsequent sexual cyc les .

b. Intergeneric. All ind ica t ions a r e t ha t the possibi l i ty of producing novel hybrid p lan t s b e t w e e n d i s t an t ly r e l a t e d spec ies r e m a i n s r e m o t e . The biological ba r r i e r involved goes far beyond the physical ba r r i e r of t he cel l wal l . T h e r e is, however , no specia l p roblem in producing h e t e r o k a r y o n s and hybrid ce l ls , and the r ange of viable cel l hybrids p roduced in this way has been r e c e n t l y r ev i ewed by Cons tabe l (8) who also considers the f a t e of t he two s e t s of ch romosomes in the hybr ids . Genera l ly hybrid fo rmat ion is followed, during subsequent divisions, by ch romosome e l imina t ion , a long famil iar f e a t u r e of an imal cel l hybr ids . In some combina t ions , such as Vicia and Petunia (2) and Arabidopsis and Brassica (24), ch romosome e l imina t ion s e e m s to be nonspec i f ic , ch romosomes of e i t he r or both p a r e n t s being los t . In o the r


cases , however , t h e r e appea r s to be speci f ic e l imina t ion , of Petunia ch romosomes from ParthenociSSUS and Petunia cel ls (47) and Aego-podium ch romosomes from Daucus and Aegopodium cel ls (18 and persona l communica t i on ) . This las t is of p a r t i c u l a r i n t e r e s t s ince p lan t s could be r e g e n e r a t e d and a l though only Daucus ch romosomes could be d e t e c t e d , c e r t a i n f e a t u r e s of t h e p i g m e n t s p e c t r u m of Aegopodium w e r e d e m o n s t r a t e d . The impl i ca t ion , incorpora t ion of Aegopodium genes to Daucus ch romosomes , could c lea r ly be of g r e a t e r s igni f icance for b reed ing .

D. Other Methods of Genetic Modification

DNA has been in t roduced in to p lan t cel ls and p r o t o p l a s t s in a v a r i e t y of ways , such as using naked DNA (31), b a c t e r i o p h a g e (15), b a c t e r i a (23), o rgane l l e s (23), and an imal cel ls (16). Stable incorpora t ion and express ion of exogenous DNA in t roduced by any of t hese me thods has not been unequivocal ly d e m o n s t r a t e d , and it is diff icult to envisage any app l ica t ion to p lan t b reed ing in t he fo reseeab le fu tu re . More promis ing is the possibibi l i ty of t r a n s f o r m a t i o n using a well c h a r a c t e r i z e d p lasmid, such as t he Ti p lasmid of Agrobacterium tumefaciens, and th is sys tem has been fully r e v i e w e d (49).

IV. CONCLUDING REMARKS

To d a t e t he increas ing use of p l an t t i ssue and cell c u l t u r e me thods to ach i eve ag r i cu l tu ra l ob jec t ives has not been widely ex t en d ed to t he p roduc t ion of new cu l t iva r s a f t e r g e n e t i c modi f ica t ion of cel ls in c u l t u r e . I nc reased expe r i ence in t ack l ing t h e t echn ica l d i f f icul t ies has enhanced the possibi l i ty t h a t va luable con t r ibu t ions can be m a d e in this way. It is t h e view of this au thor t ha t t h e r e a r e two approaches mos t l ikely to be deve loped as va luable b reed ing tools used s e p a r a t e l y or in combina t ion : -

t h e d i r ec t se l ec t ion for des i rab le pheno types in freshly i n i t i a t ed cu l t u r e s , fol lowed by p lan t r e g e n e r a t i o n , and

t r ans fe r of p a r t s of genomes respons ib le for des i rab le t r a i t s using i n t r a - or i n t e r - g e n e r i c p ro top la s t fusion (chromosome e l imina t ion may p rove an a d v a n t a g e , r a t h e r than a p rob lem, for t h e app l ica t ion of this me thod ) .

With p a r t i c u l a r r e f e r e n c e to t he a r e a of i n t e r e s t to this m e e t i n g , I know of no ser ious ba r r i e r to t h e use of these t echn iques to ob ta in cu l t iva r s of c rop spec ies wi th enhanced r e s i s t a n c e to low t e m p e r a t u r e s t r e s s .

4 7 0 P. J. DiX


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153-190. The Univers i ty of Guelph, Guelph (1974). 29. Kao , Κ. N. , and Michayluk, M. R . Planta 115, 355-367 (1974). 30. Kochba , J . , Spiegel -Roy, P . , and Saad, S., IAEA publ . , (1978). 3 1 . Ledoux, L. "Gene t i c Modif ica t ions wi th P lan t Mate r i a l s . "

A c a d e m i c P re s s , New York (1975). 32. Maliga, P . In "F ron t i e r s of P lan t Tissue C u l t u r e 1978" (T. A.

Thorpe , ed.) , pp . 381-392. IAPTC 1978, Ca lga ry (1978). 3 3 . Maliga, P . , Sz-Breznovi t s , Α., and Mar ton , L. Nature New Biol.

244, 29-30 (1973). 34. Maliga, P . , Sz-Breznovi t s , Α., Mar ton , L. , and Joo , F . Nature 255,

401-402 (1975). 35 . Maliga, P . , L a z a r , G., Svab, Z., and Nagy, F . Molec. gen. Genet.

149, 267-271 (1976). 36. Maliga, P . , L a z a r , G., Joo , F . , H-Nagy , Α., and Mencze l , L. Mo-

lec. gen. Genet. 157, 291-296 (1977). 37. Maliga, P . , R . -Kiss , Zs. , Dix, P . J . , and Laza r , G. Molec. gen.

Genet. i 72 ,13 -15 , 1979). 39. Melchers , G. Plant Res. and Dev. 5, 86-110 (1977). 40 . Mencze l , L. , L a z a r , G. and Maliga, P . Planta 143, 29-32 (1978). 4 1 . Mered i th , C. P . Plant Sci. Lett. 12, 25-34 (1978). 42 . Muller , A. J . , and G r a f e , R. Molec. gen. Genet. 161, 67-76 (1978). 4 3 . Murashige , T. In "F ron t i e r s of P lan t Tissue C u l t u r e 1978" (T. A.

Thorpe , ed.) , pp . 15-26 IAPTC 1978, Ca lga ry (1978). 44 . Nabors , Μ. V., Danie ls , Α., Nadolny, L. , and Brown, C. Plant Sci.

Lett. 4, 155-159 (1975). 45 . N a k a m u r a , Α., Y a m a d a , T., Kado tan i , N. and I t agak i , R. In "Hap

loids in Higher P lan t s" (K. J . Kasha , ed.) , pp . 277-278. The Univers i ty of Guelph, Guelph (1974).

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Plant Sci. Lett. 5, 197-207 (1975). 48 . Sacr i s t an , M. D. Chromosoma 33, 273-283 (1971). 49 . Schi lperoor t , R. Α., Klapwijk, P . M., Hooykaas , P . J . J . , Kockman , B.

P . , Ooms , G., O t t e n , L. Α. Β. M., Wurzer -F igure l l i , Ε. M., Wullems, G. J . , and Rorsch , A. In "F ron t i e r s of P l an t Tissue C u l t u r e 1978" (T. A. Thorpe , ed.) , pp . 85-94. IAPTC 1978, Ca lga ry (1978).

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339-353 . The Univers i ty of Guelph, Guelph (1974).


GENETIC DIVERSITY OF PLANTS FOR RESPONSE TO LOW TEMPERATURES AND ITS POTENTIAL

USE IN C R O P PLANTS

C. Eduardo Vallejos

D e p a r t m e n t of Vege tab le Crops Univers i ty of Cal i forn ia , Davis

I. INTRODUCTION

G e n e t i c d ivers i ty for response to t e m p e r a t u r e s can be observed a t a lmos t all levels of o rgan iza t ion in the p lan t k ingdom. This d ivers i ty has probably evolved as a resu l t of t he c l i m a t i c va r i a t ion ex i s t en t on this p l a n e t . P l an t s , evolved to adap t to a c e r t a i n r a n g e of env i ronmen ta l condi t ions , a r e s t r e s sed if any env i ronmen ta l componen t ex t ends beyond t h a t r a n g e . S t ress then is a r e l a t i v e t e r m . For e x a m p l e , Tidestromia oblongifolia, a bush from D e a t h Valley, Cal i forn ia has an op t imum t e m p e r a t u r e for pho tosyn thes i s of 45 C, but a t ZO C or less pho tosyn thes i s s tops and d o r m a n c y is induced (5). On the o the r hand t h e r e a r e t h e a lpine and a r c t i c p l an t s such as Mimulus lewisii (Z5) and Oxyria digyna (Z) which a r e capab le of wi ths tand ing very low t e m p e r a t u r e s . So, low t e m p e r a t u r e s t r e s s has a d i f fe ren t mean ing depending on the group of p l an t s , and it is n e c e s s a r y to d i f f e r e n t i a t e b e t w e e n f reez ing (below 0 C) and chil l ing (low t e m p e r a t u r e s above 0 C) .

While cons iderab le in fo rmat ion has been r e p o r t e d in r e g a r d to t h e phenomenon of chil l ing injury, l i t t l e is known about the e x a c t s equence and t iming of e v e n t s a t t he ce l lu lar and molecu la r level t h a t lead to the injury and the subsequent express ion of the s y m p t o m s . One line of ev idence a c c u m u l a t e d in chil l ing sens i t ive spec ies sugges t s t ha t t he p r i m a r y e f f ec to r s of the d a m a g e a r e the ce l lu lar and subcel lu lar m e m b r a n e s (30). The physiological d isorder caused by low t e m p e r a t u r e s is a complex phenomenon . Severa l i m p o r t a n t f a c to r s a f f ec t the d e g r e e of injury including: a) t he t e m p e r a t u r e , b) t i m e of exposure , c) t h e organ of the p lan t or the t i ssue exposed, d) i t s physiological s t a g e , and e) the t e m p e r a t u r e a t which t h e organism has been growing.

Chil l ing sens i t ive spec ies a r e in gene ra l from t rop ica l and subt rop ica l origin and they can be found in t a x a as d iverse as Solanaceae (dicot) and Musaseae (monocot) . Var iabi l i ty in response to low

Copyright © 1979 by Academic Press. Inc. 4-73 All rights of reproduction in any form reserved

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4 7 4 C. Ε. Val le jos

t e m p e r a t u r e s , bo th chill ing and f reez ing , is found a t every level of t axonomic o rgan iza t ion in the p lan t kingdom, from within a phylum to within a genus such as Lycopersicon and a spec ies such as Zea mays, corn . Al though t o m a t o and corn a r e evolu t ionar i ly d i s t an t , they sha re a t r a i t or number of t r a i t s t h a t r ende r t hem chill ing sens i t ive .

It is also possible to see a s imilar kind of va r iab i l i ty from a d i f fe ren t p e r s p e c t i v e , even within a single chil l ing sens i t ive p l a n t . Thus va r i a t ion in sens i t iv i ty of d i f fe ren t t i ssues and /or o rgans can be observed in a p lan t of a given age when this p lan t is exposed to chil l ing t e m p e r a t u r e s (28); also va r ia t ion in sens i t iv i ty of a p a r t i c u l a r t i s sue /o rgan a t d i f fe ren t s t a g e s of deve lopmen t (30, 28). The las t and by no means t he l eas t i m p o r t a n t is the va r i a t ion in sens i t iv i ty of d i f fe ren t o rgane l les and o the r subcel lu lar s t r u c t u r e s within a cel l a t a given t i m e (see R. Ilker in this volume) .

Each s t r u c t u r e in a p lan t has a t l eas t one main physiological funct ion, a c e r t a i n physiological p rocess which in turn involves some b iochemica l r e a c t i o n s or p a t h w a y . The e x t e n t to which this p a t h w a y is sens i t ive to low t e m p e r a t u r e s r e l a t i v e to o the r p a t h w a y s will i m p a r t sens i t iv i ty to t he physiological p roces s and to t he t i ssue or organ in which this p rocess is t he most i m p o r t a n t .

These d i s t inc t levels of va r iab i l i ty men t ioned above resu l t from the fac t t h a t a p l an t , chil l ing sens i t ive or n o t , is a dynamic e n t i t y which is cont inuously growing and developing. Changes in sens i t ive s i t es r e p r e s e n t the dynamic n a t u r e of the p lant and the p rocess of d i f f e ren t i a t ion , which is an o rde red s equence of gene express ion and repress ion , and is r e g u l a t e d to some e x t e n t by the e n v i r o n m e n t . Thus, in a loose sense , var iab i l i ty in sens i t iv i ty within a p lan t (with t i m e and s t a g e of deve lopment ) can be cons idered a form of g e n e t i c va r iab i l i ty . This is a ve ry i m p o r t a n t concep t for s tudies in c o m p a r a t i v e physiology.

Π. THE GENETIC A P P R O A C H TO STUDY CHILLING INJURY

A s y s t e m a t i c s tudy of a s imple sys tem or mechan i sm will mos t l ikely involve d i r ec t m e a s u r e m e n t of i t s componen t s and the f ac to r s t ha t d i r ec t l y a f f ec t t h e s y s t e m . However , for m o r e complex s y s t e m s , espec ia l ly those wi th many unknown componen t s , a d i f fe ren t approach should be t a k e n . One l ikely approach is to vary the e x t e r n a l f a c to r s t h a t a f f ec t the sys tem and m e a s u r e the e f f ec t s of the changes tha t occur . Iden t i f i ca t ion of t h e unknown componen t s respons ib le for a p a r t i c u l a r r e sponse can be ach ieved by ana ly t i ca l compar i son of two sys t ems which differ only for t h a t response ; for example , two d i f fe ren t e co types from t h e s a m e spec ies .

To u t i l i ze gene t i ca l ly con t ro l l ed d i f fe rences for s tudies in c o m p a r a t i v e physiology in p lan t s y s t e m s , especia l ly of an unknown mechan i sm, one has to re ly on induced or n a t u r a l va r iab i l i ty . Al though many s tud ies have u t i l i zed v a r i a n t s induced by chemica l m u t a g e n s , it is

T h e C r o p P l a n t s R e s p o n s e to L o w T e m p e r a t u r e s 4 7 5

of ten no t possible to induce t h e kind of va r i a t ion des i red and in these cases the only a l t e r n a t i v e is to r e ly on n a t u r a l g e n e t i c va r i a t i on . The amoun t of va r iab i l i ty found in c u l t i v a t e d v a r i e t i e s of c rop p l an t s is usually minusc le c o m p a r e d to t ha t found in wild popula t ions or c losely r e l a t e d spec ie s . Many crop p lan t spec ies h a v e been d o m e s t i c a t e d by man to the point t ha t no wild forms of the spec ies ex is t , w h e r e a s o t h e r s do have wild forms or close i n t e r c ro s sab l e r e l a t i v e s growing in t he wild; it is in these wild forms tha t g r e a t va r iab i l i ty can be found. Al though a t t e m p t s wi th p lan t cel l suspension c u l t u r e have had some d e g r e e of success in r e c ove r ing useful v a r i a n t s , it will be a long t i m e before the t echn ique b e c o m e s a s t anda rd p r o c e d u r e to aid in gene t i ca l ly defining a physiological p roces s (see Dix in this volume) .

An exhaus t ive survey of va r iab i l i ty for t e m p e r a t u r e response in t he p lan t kingdom will yield a l a rge list of spec i e s . These spec ies can be class i f ied in t h r e e groups . F i r s t , those crop p l an t s for which no r e l a t i v e s from t h e s a m e genus can be found in t he wild such as Zea mays, corn or m a i z e . Second, crop p l an t s whose c lose r e l a t i v e s can be found in t h e wild, as in t h e case of t h e genus Lycopersicon, t o m a t o . And th i rd , wild spec ies l ike those belonging to t he genus Atriplex. While Atriplex has a

wor ldwide d is t r ibu t ion , corn and t o m a t o a r e spec ies from the new world . A geograph ica l reg ion in this con t inen t wi th a highly complex phys iography, such as Pe ru , provides exce l l en t condi t ions for t h e evolut ion of d ive rgen t g e n e t i c v a r i a n t s wi th speci f ic physiological m e c h a n i s m s a d a p t e d to d i f fe ren t env i ronmen ta l condi t ions .

It is in t h e geographica l c o n t e x t of Pe ru whe re I wan t to desc r ibe examples of geno typ ic var iab i l i ty within (corn) or b e t w e e n closely r e l a t e d spec ies ( tomato ) . A brief desc r ip t ion of t h e geograph ica l loca t ion and orographic sys tem of Pe ru h e r e will be helpful in o rder to unde r s t and t h e n a t u r e of i t s ecologica l d ivers i ty (Figure 1). I t s geograph ica l loca t ion is essen t ia l ly equa to r i a l , t he n o r t h e r n m o s t point is 0 2

1 S and t h e sou the rnmos t point is 18 12' S. The Andean Ranges of

moun ta ins (Cordil leras) run pa ra l l e l and very c lose to t h e d e s e r t i c Pac i f i c coas t (52). The orographic desc r ip t ion of t h e Pe ruv ian Andean sys tem p roceed ing from south to n o r t h is as follows: Two r anges of moun ta ins e n t e r from Chi le and Bolivia; t hese a r e t he "Cord i l l e ra Volcanica" (from Chile) and t h e "Cord i l l e ra Rea l " (from Bolivia), which conve rge south of Cuzco to form t h e Vi lcanota Knot . T h r e e "Cordi l le ras" , "Or ien ta l " , "Cen t r a l " , and "Occ iden ta l " surge n o r t h w a r d from t h e Vi lcanota Knot , m e e t i n g a t t h e Pasco Knot in C e n t r a l Pe ru . T h r e e new "Cordi l le ras" , "Or ien ta l" , "Cen t r a l " and "Occ iden ta l " p r o c e e d from this point n o r t h w a r d unt i l t hey join in t h e Loja Knot in Ecuador . The O c c i d e n t a l Cord i l l e ra , which has t he h ighes t peaks in Pe ru , is t h e Andean C o n t i n e n t a l Divide , i t s e p a r a t e s t h e Pac i f i c and A t l a n t i c w a t e r s h e d s . The coun t ry is divided by t h e Andes in to t h r e e main n a t u r a l r eg ions : t h e coas t , a long and na r row s t r ip of d e s e r t i c land b e t w e e n t h e Andes and the Pac i f i c Ocean , a number of r i ve r s t r a n s e c t t he coas t ca rv ing deep and pa ra l l e l va l leys in to t h e w e s t e r n s lopes; t h e h ighlands , compr is ing t h e moun ta ins wi th many i n t e r A n d e a n val leys and high p l a t e a u s ; and the jungle on t h e e a s t e r n slopes of t he Andes , compr is ing t h e lowlands with t rop ica l and


sub t rop ica l ra iny fores t (52). Th i r ty - f ive d i f fe ren t t rop ica l c l i m a t e s have been ident i f ied in Pe ru , a s soc i a t ed wi th d i f f e rences in mean t e m p e r a t u r e , annual p r e c i p i t a t i o n , humid i ty , and l a t i t u d e (52).

A. Zea mays

Corn is a crop p lan t from the new world. It deve loped under e x t r e m e l y d iverse env i ronmen ta l condi t ions found in Nor th , C e n t r a l , and South A m e r i c a . This is r e f l e c t e d in t he t r e m e n d o u s va r iab i l i ty found in this spec i e s . The re a r e 305 r a c e s of corn l i s ted in 11 d i f fe ren t vo lumes publ ished by the Na t iona l A c a d e m y of Sc iences - Na t iona l R e s e a r c h Counci l . They r e p r e s e n t a lmos t a c o m p l e t e inven tory of the new world m a i z e (31).

Pe ru has been p o s t u l a t e d to be an independen t c e n t e r of m a i z e d o m e s t i c a t i o n (23). At l eas t 42 r a c e s of m a i z e can be found in P e r u coming from a r ea s of d iverse ecologica l condi t ions having been p roduced by hybr id iza t ion and se lec t ion along wi th n a t u r a l l y occur r ing m u t a t i o n . Grobman et al. (2 >} h-.ve proposed t ha t d o m e s t i c a t i o n of corn in Pe ru o c c u r r e d in t h e low to middle a l t i t udes of t he Andes resu l t ing in the fo rma t ion of p r im i t i ve r a c e s and ex tens ion of t he original r ange of a d a p t a t i o n to h igher and lower a l t i t udes as well as more n o r t h e r n and sou thern l a t i t u d e s . The p r e s e n c e of g e n e t i c var iab i l i ty in corn a l lowed an e f f ec t i ve a r t i f i c i a l se lec t ion for a d a p t a t i o n during the d i f fe ren t s t a g e s of d o m e s t i c a t i o n of corn and h e n c e has ex t ended the r ange of geograph ica l d i s t r ibu t ion of m a i z e to h a b i t a t s wi th e x t r e m e d iverse condi t ions for p lan t g rowth and deve lopmen t , from sea level in the d e s e r t i c coas t to an e leva t ion of 4,000 m e t e r s in t he Andes . Grobman et al. (23) s t a t e d t ha t "corn popula t ions w e r e then sub jec ted to mul t ip le p ressu res of n a t u r a l se lec t ion i n t e r a c t i n g wi th fixed a r t i f i c i a l s e l e c t i o n -p re s su re , producing in the end a number of morphologica l gene t i ca l ly and adap t ive ly d i s t inc t popula t ions ." In these d i s t inc t popula t ions , morphologica l and physiological c h a r a c t e r s have a c e r t a i n d e g r e e of c o h e r e n c e ; however , they do not appear to be gene t i ca l ly l inked, for r e d u c t i o n s in r e c o m b i n a t i o n in hybrids of in te rpopu la t ion c rosses have not been observed . The c o n c o m i t a n t p r e s e n c e of these two se t s of c h a r a c t e r s in a given popula t ion will be ma in t a ined through cont inuous se l ec t ion . The deve lopmen t of these def ined ecologica l r a c e s was followed by expansion of d i s t r ibu t ion . In this p rocess c e r t a i n r ac i a l c h a r a c t e r i s t i c s were ma in ta ined a t a low level of var iab i l i ty , while o t h e r s had to vary in o rder to adap t to t he new env i ronmen t . It should also be men t ioned t h a t some of t he exis t ing r a c e s a rose from hybr id iza t ion .

Grobman et al. (23) classif ied t he exis t ing r a c e s of m a i z e from Peru in to six main groups accord ing to the i r chronological origin:

1) P r i m i t i v e R a c e s (of g r e a t e s t ant iqui ty) 2) Anc ien t ly Der ived or P r i m a r y R a c e s (derived from P r imi t i ve

Races) 3) La t e ly Der ived or Secondary R a c e s (derived from P r i m a r y

Races)


4) In t roduced R a c e s 5) Inc ip ient R a c e s ( r e s t r i c t ed geograph ic dis t r ibut ion) 6) I m p e r f e c t l y Def ined R a c e s

In t he following p a r a g r a p h s I will l ist some of t h e corn r a c e s from groups 1 and Z, br ief ly desc r ibe the i r c h a r a c t e r i s t i c s , origin and re la t ionsh ips . A discussion on the poss ibi l i t ies of s tudying t h e i n h e r i t a n c e of a d a p t a t i o n m e c h a n i s m s will follow in subsequent p a g e s . The c lass i f ica t ion of t h e Pe ruv ian R a c e s was based on a rchaeo log ica l ev idence for t h e p reh i s to r i c r a c e s and morphologica l and cy to log ica l c h a r a c t e r s of t he exis t ing r a c e s .

1. Primitive Races: All p r im i t i ve r a c e s a r e conf i t es or popcorns .

a) Confite Morocho. A shor t p l an t , ea r ly in m a t u r i t y . It is found a t i n t e r m e d i a t e a l t i t udes Z,500-3,000 m e t e r s above sea l eve l . This corn easi ly a d a p t s to t h e coas t showing r e s i s t a n c e to e x t r e m e env i ronmen ta l f luc tua t ions due to a lack of e n v i r o n m e n t a l spec i f i c i ty . It r e p r e s e n t s an evolved form of P r o t o Conf i t e Morocho, a p r eh i s to r i c r a c e .

b) Confite Puntiagudo. Also an ear ly m a t u r i n g shor t p lan t (117 cm. ) . C o l l e c t e d a t a l t i t u d e s of Z,500-3,500 m, i t is widely d i s t r i bu t ed along t h e Andes . Der ived d i r ec t l y from Conf i t e Morocho wi th s o m e in t rogress ion of Tripsacum p robably through a r a c e ca l led "Enano" from t h e lowlands eas t of t h e Andes .

c) Confite Puneno. The ea r l i e s t m a t u r i n g r a c e in t h e highlands and also t h e s h o r t e s t of all (56 cm) . This r a c e grows a t t h e h ighes t a l t i t u d e s in t h e world. It is found around Lake T i t i c a c a a t a l t i t ude s of 3,600 to 3,900 m. R a m i r e z et al. (45) r e p o r t e d t h e h ighes t e l eva t ion for this corn in Bolivia to be 3,993 m. It is p roposed t h a t this corn evolved when t h e p reh i s to r i ca l r a c e Conf i t e Chav inense was forced by ear ly f a r m e r s in to t he h igher lands around Lake T i t i c a c a .

d) Kculli. A ve ry shor t p l an t , (92 cm) found a t b e t w e e n Z,300 and 3,300 m. a l though t h e major i ty of typ ica l co l l ec t ions c o m e from above 3,000 m. It was ex tens ive ly c u l t i v a t e d in bo th t he coas t and t h e highland in p r e - C o l u m b i a n t i m e s . Thought to be o r ig ina ted from P r o t o Kcull i , an anc i en t popcorn r a c e of the Andes .

e) Enano. A shor t p lan t found in t he lowlands and jungle eas t of the Andes in t he sou the rn p a r t of Pe ru . This corn was c o l l e c t e d a t Z70 m. and can be found e a s t w a r d a t lower e l eva t ions . It is su spec t ed to have de r ived from "Conf i te Chavinense" and have Tripsacum i n t rogress ion .

It has been es tab l i shed t h a t of t he p r i m i t i v e r a c e s , "Conf i te Morocho" and "Conf i te Pun t i agudo" h a v e a wide r a n g e of a d a p t a t i o n . It would be i n t e r e s t i n g to know if a d a p t a t i o n of "Conf i t e Puneno" to t he h ighes t e l eva t ion and "Enano" to t he lowlands in t h e t rop ica l jungle r e s u l t e d in a loss of the i r ab i l i ty to s t and t h e oppos i t e e x t r e m e condi t ions .


FIGURE 1. Orographic map of Peru. It shows the distribution of 3 species of the genus Lycopersicon, and the Peruvian races of corn. Confite Morocho (CM); Confite Puntiagudo (CPo); Confite Puneno(CP); Kculli (K); Enano (E); Mochero (M); Huancavelicano (H); Piricinco (P).


2. Anciently Derived Races. These a r e r a c i a l s e l ec t ions from s imple as well as complex hybrid popula t ions t h a t r e s u l t e d from the in t e rc ross ing t h a t took p l a c e among t h e p r i m i t i v e popcorns .

a) Mochero. A shor t p l an t endemic to t h e no r th coas t a t a l t i t u d e s below 50 m. There is amp le ev idence poin t ing a t " P r o t o -Iqueno", a coas t a l p r eh i s to r i c popcorn , as t h e a n c e s t o r of "Mochero ."

b) Huancavelicano. P l an t s wi th an a v e r a g e he ight of 1.32 m. and a l t i t ud ina l d i s t r ibu t ion l imi t ed to lands b e t w e e n 2,200 to 3,500 m, a l though 80% of i t s co l l ec t ions c o m e from e leva t ions above 2,800 m. This r a c e is found in the sou the rn s i e r r a s of P e r u . The p r imi t i ve r a c e s "Conf i te Morocho" and "Kculli" a r e proposed to be t he a n c e s t o r s of th is r a c e .

c) Piricinco. A p lan t wi th a very long and s lender ea r , grows on the sub t rop ica l and t rop ica l lowlands eas t of the Andes a t a l t i t u d e s b e t w e e n 150 and 940 m. This r a c e is p roposed to be a hybrid b e t w e e n "Enano" (pr imi t ive r a c e from the t ropics) and a middle a l t i t u d e r a c e from t h e highlands which in turn is a de scendan t of Conf i t e Morocho . This r a c e shows s t rong t r ipsaco id c h a r a c t e r s ar is ing from in t rogress ion wi th Tripsacwn australe, a t rop ica l t r i p sacum from South A m e r i c a .

These r a c e s a rose from bo th hybr id iza t ion and se l ec t ion of o lder r a c e s so one e x p e c t s them to be b e t t e r a d a p t e d to c e r t a i n speci f ic e n v i r o n m e n t s . H e r e , i t will also be i n t e r e s t i n g to know if th is l a t t e r se lec t ion for m o r e speci f ic h a b i t a t s n a r r o w e d the r a n g e of adap t ab i l i t y displayed by t h e a n c e s t o r s of this g roup .

B. Lycopersicon

The 8-10 spec ies belonging to this genus have a n a t u r a l a r e a of d i s t r ibu t ion l imi t ed to w e s t e r n South A m e r i c a . This a r e a compr i ses n o r t h e r n Chi le , Pe ru , Ecuador , including t h e Ga lapagos Islands and sou the rn Colombia (47). The re is a vary ing d e g r e e of i n t e r c o m p a t i b i l i t y among t h e spec ie s . Some combina t ions a r e fully c o m p a t i b l e w h e r e a s hybrids of o the r combina t ions can be a t t a i n e d only wi th some di f f icul ty (48). All t h e spec ies have 12 pa i r s of c h r o m o s o m e s (2n=2x=24) and cy to log ica l s tud ies of hybrids have shown t h e r e is ve ry l i t t l e if any s t r u c t u r a l d i f f e rences b e t w e e n s e t s of c h r o m o s o m e s (48).

Holle et al. (26, 27) have r e c e n t l y publ ished a c a t a l o g of co l l ec t ions of g r een f ru i ted spec ie s . T h e r e a r e t h r e e g reen f ru i ted spec ies of i n t e r e s t (due to the i r wide r a n g e of a l t i t ud ina l d is t r ibut ion) found on t h e w e s t e r n slopes of t h e Andes : L. chilense, which inhab i t s one of t h e mos t ar id of t he world 's t e m p e r a t e d e s e r t s in sou the rn Pe ru and n o r t h e r n Chi le (47), wi th some popula t ions also found on t h e hi l ls ides a t h igher e l eva t ions ; L. peruvianum, a spec ies e x t r e m e l y po lymorphic both within


and b e t w e e n popula t ions (46), which has a r ange of d is t r ibu t ion from n o r t h e r n Pe ru to the n o r t h e r n m o s t coas t of Chi le , and i t s a l t i t u d e r ange is from sea level to 3,000 m; and L. hirsutum, which is n a t i v e to an a r e a t ha t ex tends from c e n t r a l Peru to n o r t h e r n Ecuador ; t h e a r e a of d i s t r ibu t ion is divided in 4 main reg ions , t h r e e on the w e s t e r n s ide of t h e C o n t i n e n t a l Divide and one on the e a s t e r n s ide . The a l t i tud ina l r a n g e in Pe ru ex tends from 500 m to 3,300 m*, t he h ighes t l imi t known for any t o m a t o spec ies . However , in Ecuador , a subspecif ic t axon f. glabratum inhabi t s coas t a l lowlands. The Ecuadorean form of hirsutum has a much smal le r corol la , s lender ca lyx and cons iderably less ha i r iness in l eaves and s t em than i t s Peruv ian c o u n t e r p a r t f. typicwn (49).

Popula t ions of L. hirsutum from t h e val leys in n o r t h e r n Pe ru the c e n t e r of d i s t r ibu t ion , a r e se l f - incompa t ib l e and highly va r iab le for a l lozymic and morphologica l c h a r a c t e r s , however , popula t ions from n o r t h e r n (Ecuador) and sou thern (Cent ra l Peru) l imi t s of d i s t r ibu t ion a r e s e l f - compa t ib l e and tend to be less polymorphic but ex tens ive ly d i f fe ren t from each o the r (49).

C. Atriplex

The numerous spec ies in this genus belong to two subgenera - Obione and Eua t r ip lex which have d iverged profoundly from each o the r dur ing evolut ion (41).

The spec ies of this genus a r e widely d i s t r ibu ted in Europe , Nor th A m e r i c a , and Aus t r a l i a . Some spec ies a r e typ ica l of cool coas t a l a r e a s whe rea s o t h e r s a r e from warm in te r io r val leys or warm d e s e r t s . In te res t ing ly t h e r e a r e some spec ies , such as A. lentiformis, t h a t have e c o t y p e s well a d a p t e d to bo th env i ronmen t s (5, 6, 43). Species with C^ me tabo l i sm a r e found in bo th t h e r m a l env i ronmen t s , t he s ame is t r u e for spec ies with C^ me tabo l i sm (41). A. lentiformis s e e m s to be t he most su i t ab le spec ies for c o m p a r a t i v e physiology and g e n e t i c s tudies of t e m p e r a t u r e a d a p t a t i o n . The spec ies of this genus , even within e a c h subgenus , have b e c o m e highly d i f f e r en t i a t ed gene t i ca l ly and t ha t poses a problem for in te r spec i f i c g e n e t i c analysis (41). Al though a c o m p l e t e unders t and ing of t h e g e n e t i c re la t ionsh ips among the spec ies has no t been a t t a i n e d , some in fo rmat ion is ava i lab le in t he l i t e r a t u r e . Within Euatriplex t h e spec ies A. rosea (from warm areas) and A. sabulosa a r e i n t e r c ros sab l e wi th some d e g r e e of success , bo th have C^ me tabo l i sm (41). It has been shown tha t A. vesicaria, a warm a r e a spec ies , can be cold t o l e r a n t (9). This spec ies is capab le of growing and

Seeds of L. hirsutum in this altitudinal range were collected by the author during an expedition in 1976.


pho tosyn thes iz ing when grown in a t e m p e r a t u r e r e g i m e 8UC d a y / 6 ° C

n igh t . A. vesicaria and A. confertifolia showed ev idence of some d e g r e e of p h o t o s y n t h e t i c a c c l i m a t i o n to g rowth t e m p e r a t u r e , mainly th rough some k ine t i c a d j u s t m e n t s . The a u t h o r s sugges ted t h a t cold t o l e r a n c e s t e m s m o r e from main ta in ing m e m b r a n e i n t e g r i t y a t low t e m p e r a t u r e s r a t h e r than t h e k ine t i c a d j u s t m e n t s during acc l ima t ion , this r e m a i n s to be d e t e r m i n e d .

ΠΙ. UNDERSTANDING SENSITIVITY, TOLERANCE, AND ADAPTATION TO LOW TEMPERATURE

The re is a bas ic need to unde r s t and the mechan i sms of sens i t iv i ty and r e s i s t a n c e to low t e m p e r a t u r e s t r e s s if one in tends to i n t roduce the l a t t e r t r a i t in to sens i t ive crop p l a n t s . Pu r suan t to this model , one needs s y s t e m s such as those wi th spec ies or g e n e r a t ha t have i n t e rb reed ing popula t ions a d a p t e d to e x t r e m e t h e r m a l env i ronmen t s (4). A few examples a r e men t ioned e l s ewhere in this c h a p t e r . The se l ec t ion of wild spec ies ( tomatoes) or p r im i t i ve forms of c u l t i v a t e d p l an t s (such as corn) has the a d v a n t a g e of providing g e n e t i c va r i ab i l i ty . As Bjorkman (4) po in ted out , t h e use of wild spec ies spa res us t he problem of deal ing wi th c u l t i v a t e d p l an t s which have been s e l e c t e d for un i fo rmi ty and b red for des i rab le c h a r a c t e r i s t i c s with t h e exclusion or a l t e r a t i o n of a d a p t a t i o n m e c h a n i s m s essen t ia l in s t r e s s e n v i r o n m e n t s .

Hiesey et al. (24) out l ined the bas ic pr inc ip les for s tud ies of c o m p a r a t i v e physiology of eco logica l r a c e s . An ex tens ive g e n e t i c , eco logica l , and physiological c h a r a c t e r i z a t i o n of t h e r a c e s or e co types from c on t r a s t i ng env i ronmen t s is r equ i r ed in a

1 1. . .p rogram a imed a t

d i scover ing bas ic physiological m e c h a n i s m s t h a t d e t e r m i n e t h e p e r f o r m a n c e of p l an t s in def in i te e n v i r o n m e n t s . . ."

G e n e t i c and ecophysiological s tud ies have been ca r r i ed out on a l t i t ud ina l r a c e s of Potentilla glandulosa by Clausen and Heisey (14) and of a l t i t ud ina l as well as l a t i tud ina l r a c e s of Mimulus spp . by Hiesey et al. (25). With both e c o t y p e s , c e r t a i n morphologica l and physiological c h a r a c t e r s we re gene t i ca l ly c h a r a c t e r i z e d for r a c e s from c o n t r a s t i n g env i ronmen t s (Stanford, T imber l ine) , and for the i r F , , F ^ and F^ p rogen ie s . In all c a se s , t he individuals we re diploid, f reely i n t e r f e r t i l e and wi th no rma l pa i r ing of c h r o m o s o m e s . C lea r morphologica l m a r k e r c h a r a c t e r s dis t inguishing wel l -def ined c o n t r a s t i n g e c o t y p e s w e r e chosen . Each morphologica l c h a r a c t e r and t h e abi l i ty to survive in c o n t r a s t i n g env i ronmen t s w e r e g raded from one to nine accord ing to the r e s e m b l a n c e of t he c h a r a c t e r to e i t h e r of t he p a r e n t s . Regress ions and co r r e l a t i ons w e r e ob ta ined for all possible combina t ions of two c h a r a c t e r s . Analysis of t hese r e s u l t s showed t h a t t h e r e was a p a r t i a l c o r r e l a t i o n b e t w e e n independen t non- l inked c h a r a c t e r s t h a t dis t inguish a spec ies , subspec ies , r a c e or e c o t y p e .


Anderson (1) and Mathe r (34) had previously r eco g n i zed the fac t t ha t most c h a r a c t e r s t ha t dist inguish spec ies and r a c e s a r e governed by sy s t ems of genes consis t ing of severa l c o m p o n e n t s r e f e r r e d to as polygenes which show complex seg rega t ion . Hiesey et al. (25) then coined t h e t e r m 'Gene t i c C o h e r e n c e ' * to def ine " the t e n d e n c y of and l a t e r gene ra t i on progeny to inher i t c e r t a i n combina t ions of c h a r a c t e r s of t h e p a r e n t s more f requent ly than would be e x p e c t e d on the basis of f ree random recombina t ion . " G e n e t i c c o h e r e n c e is e v a l u a t e d as t he r ange of co r r e l a t i on coef f i c ien t s wi th the h ighes t f requency . When clones of p a r e n t s and F ^ , F~ and F~ progenies w e r e grown a t t h r e e d i f fe ren t e l eva t ions (Stanford 30 m, Mather 1,400 m, T imber l ine 3,100 m) it was d i scovered in t he s eg rega t ing gene ra t i ons t h a t t h e r e w e r e s ignif icant co r r e l a t i ons b e t w e e n many of t h e morphologica l m a r k e r s and the abi l i ty of individuals to survive a t e a c h e l eva t ion . In o the r words , t h e r e was a m a r k e d t endency for t he types most r e sembl ing t h e p a r e n t a l e co types to be b e t t e r fit for survival , g rowth and deve lopmen t in t he env i ronmen t s normal ly occupied by the p u t a t i v e p a r e n t e c o t y p e .

The express ion of g e n e t i c c o h e r e n c e is enhanced when pa r t i a l incompat i l ib i ty ex is t s b e t w e e n the p a r e n t a l fo rms . Tha t is , the group of co r re l a t ion coef f i c ien t s wi th h ighes t f requency has a higher va lue . The enhanced g e n e t i c c o h e r e n c e is i n t e r p r e t e d as a r e su l t of the e l imina t ion of g a m e t e s and zygo tes differ ing marked ly in genie compos i t ion from the original p a r e n t a l combina t ion . Some of the i n t e r - and in t ra spec i f i c c rosses of Mimulus (25) showed h e t e r o s i s . P a r e n t a l e c o t y p e s unable to survive a t c e r t a in e l eva t ions y ie lded a vigorous progeny capab le of surviving a t those e l eva t ions . A case was also observed in which a hybrid b e t w e e n high and low a l t i t u d e yie lded a f irs t gene ra t i on hybrid with b e t t e r frost r e s i s t a n c e than the a lpine p a r e n t .

Based on responses ob ta ined for a l t i tud ina l r a c e s of Potentilla glandldosa a t con t r a s t i ng a l t i t udes , Clausen and Hiesey (14) concluded t h a t evolut ion of n a t u r a l r a c e s for con t r a s t i ng env i ronmen t s o p e r a t e s on two bas ic p r inc ip les . F i r s t , t he p r e s e n c e of a mechan i sm tha t enables e a c h r a c e to a t t a i n a c e r t a in d e g r e e of g e n e t i c c o h e r e n c e , which al lows it to exis t as a def ined biological e n t i t y capab le of surviving in i t s r e s p e c t i v e ecologica l n i che . Second, this g e n e t i c c o h e r e n c e is no t r igid enough as to p r e v e n t ex tens ive r ecombina t i on of t he g e r m p l a s m . The p o t e n t i a l for increas ing gene t i c d ivers i ty , th rough hybr id iza t ion of d i f fe ren t ecologica l e n t i t i e s , as a response to changes in s e l e c t i v e p ressu res , l ies in those two pr inc ip les .

All t h e bas ic concep t s and pr inc ip les on t h e gene t i c s and ecophysiology of a l t i tud ina l r a c e s p ionee red ea r l i e r by Anderson and Mathe r and l a t e r by Clausen and Hiesey (14) and Hiesey et al. (25) can be used to s tudy t h e gene t i c c h a r a c t e r s involved in t he phenomenon of

For a more extensive explanation of this term see Heisey et al. (25) pp. 78-82.


chil l ing injury. H e r e I will discuss t he advan t ageous a s p e c t s of using two s y s t e m s as working mode ls : A spec ies Zea mays (corn, Graminneae, a monoco t ) ; and a genus , Lycopersicon spp. , ( t oma to , Solanaceaef a d i -co t ) .

A working model to explain t h e bas ic mechan i sm in chil l ing injury has been proposed by Lyons (30), in which ce l lu lar and subcel lu lar m e m b r a n e s of chil l ing sens i t ive p l an t s a r e ass igned a major r o l e . H e r e I suggest t h e use of t he Pe ruv ian r a c e s of corn and t h e genus Lycopersicon as model s y s t e m s to u n d e r t a k e s tud ies t oward e luc ida t ing the mechan i sm for chil l ing injury. These two crop p l an t s sha re a number of c h a r a c t e r i s t i c s t h a t m a k e them useful for g e n e t i c and c o m p a r a t i v e physiological s tud ies of a d a p t a t i o n to d i f fe ren t e n v i r o n m e n t s . Both corn and t o m a t o show g r e a t g e n e t i c va r iab i l i ty : in corn it has been brought about by ex tens ive cross pol l ina t ion , wide d i s t r ibu t ion and se lec t ion for d i f fe ren t env i ronmen t s by man (39); w h e r e a s var iab i l i ty in t he t o m a t o is found in the n a t u r a l popula t ions of wild spec i e s . Both spec ies have r e l a t i v e l y shor t l ife cyc les and p roduce l a rge n u m b e r s of s eeds . The m a i z e g e n e t i c s t r a in s a r e ava i l ab le from the Maize G e n e t i c s C o o p e r a t i v e (39) and t h e t o m a t o access ions from the T o m a t o G e n e t i c s C o o p e r a t i v e (26, 27). These two spec ies a r e among the bes t gene t i ca l l y c h a r a c t e r i z e d in t h e p lan t k ingdom. For t o m a t o , m o r e than 1,100 genes have been ident i f ied and an ex t ens ive l inkage map has been deve loped (48).

In o rder to b e t t e r unde r s t and t h e p roces ses of n a t u r a l s e l ec t ion and a d a p t a t i o n it is n e c e s s a r y to have in fo rmat ion on the pheno typ ic express ion of genes or gene combina t ions over a r ange of env i ronmen t s (14) and es tab l i sh a d i r ec t r e l a t ionsh ip b e t w e e n the funct ional c h a r a c t e r s of an e c o t y p e and i t s n a t u r a l env i ronmen t (3).

A n u m b e r of c o m p a r a t i v e s tud ies (3, 4, 5, 6, 9, 22, 25, 35, 36, and 43) h a v e shown t h e op t ima l t e m p e r a t u r e for pho tosyn thes i s of p l a n t s n a t i v e to cold env i ronmen t s is lower than t h a t of the i r c o u n t e r p a r t s from w a r m e r e n v i r o n m e n t s . Bjorkman (3) r e c o g n i z e s t h r e e d i s t inc t ive p roces ses in pho tosyn thes i s : p h o t o c h e m i c a l , CO^-dif fusion, and b iochemica l ; and e a c h one can be a f f e c t e d d i f fe ren t ly by the d iverse f a c t o r s p r e s e n t in t he surrounding env i ronmen t . Since any of t he se t h r e e p rocesses can be l imi t ing under d i f fe ren t env i ronmen t s , p l an t s h a v e most l ikely evolved m e c h a n i s m s of a d a p t a t i o n to o v e r c o m e a p a r t i c u l a r l im i t a t i on . So far t he major conce rn in c o m p a r a t i v e s tud ies of a l t i tud ina l e c o t y p e s has been t e m p e r a t u r e as t he pr inc ipa l d e t e r m i n a n t . However , cau t ion should be t aken to consider o t h e r d e t e r m i n a n t s r e l a t e d to high a l t i t u d e s , e.g. t he lower p a r t i a l p r e s su re of C O ^ a t high e l eva t ions . In this r e g a r d Hiesey et al. (25) and Billings et al. (2) found t h a t e c o types from higher e l eva t ions w e r e very e f f ic ien t in absorbing C O ^ a t low c o n c e n t r a t i o n s and t h a t the i r s a t u r a t i o n point for C O ^ was lower than t h a t of low a l t i t u d e e c o t y p e s .

T e m p e r a t u r e can a f fec t t h e t h r e e p rocesses of pho tosyn thes i s in d i f fe ren t ways . Al though p h o t o c h e m i c a l r e a c t i o n s a r e independen t of t e m p e r a t u r e , a t l ea s t within t he r a n g e of t e m p e r a t u r e op t imum for l i fe , this p roces s may be a f f e c t e d by a l t e r a t i o n of t h e f luidi ty or molecu la r


order ing of thylakoid m e m b r a n e s w h e r e the two p h o t o s y s t e m s , t h e e l ec t ron t r anspo r t chain and the energy t r ansduc ing sys tem a r e imbedded (18, 37, 38). The diffusion of C O ^ from t h e a t m o s p h e r i c air to t he s i t e of ca rboxyla t ion is a f f e c t e d by t e m p e r a t u r e , for diffusion is a t e m p e r a t u r e dependen t p a r a m e t e r . Addi t ional ly t h e r e is the e f fec t of t e m p e r a t u r e on the r e s i s t a n c e s t ha t C O ? molecu les encoun te r on the i r way to t he s i t e of ca rboxy la t ion , espec ia l ly those r e s i s t a n c e s t ha t involve ce l lu lar m e m b r a n e s such as t he p l a s m a l e m m a of guard cel ls (55, 56) of mesophyl l cel ls and chloroplas t m e m b r a n e s . The r a t e of the b iochemica l r e a c t i o n s from t h e Calvin cyc le also depends d i r ec t ly on t e m p e r a t u r e and on C O ?

c o n c e n t r a t i o n a t the r e a c t i o n s i t e . Thus, it appea r s t ha t the e f f e c t s οϊ t e m p e r a t u r e on the t h r e e p rocesses involved in pho tosyn thes i s can be t r ansduced in one way or ano the r by ce l lu lar and subcel lu lar m e m b r a n e s .

Studies on a d a p t a t i o n of p h o t o s y n t h e t i c a p p a r a t u s of spec ies from c o n t r a s t i n g env i ronmen t s have led Bjorkman (4) to the conclusion t ha t a single geno type has a p o t e n t i a l for pheno typ ic ad jus tmen t of i t s p h o t o s y n t h e t i c c h a r a c t e r i s t i c s to changes in the env i ronmen t , but he po in ted out t ha t t he r ange of such ad jus tmen t is l imi t ed and r e f l e c t s a geno typ ic a d a p t a t i o n to the p a r t i c u l a r condi t ions preva i l ing in i t s n a t i v e h a b i t a t .

C o m p a r a t i v e s tudies of t o m a t o eco types and the r a c e s of corn from c o n t r a s t i n g env i ronmen t s should be most helpful to unde r s t and , as s t a t e d ear l i e r by Bjorkman (4), "The env i ronmen ta l and evo lu t ionary l imi t s of a d a p t a t i o n . . .and the physica l , s t r u c t u r a l and molecu la r mechan i sms involved."

What is t he i m p o r t a n c e of t he physical p r o p e r t i e s of m e m b r a n e s (as they a r e a f f e c t e d by t e m p e r a t u r e ) in the evolut ion and a d a p t a t i o n of the spec ies cons idered in this c h a p t e r ? In p re l imina ry e x p e r i m e n t s (53), bo th high a l t i t u d e (3,300 m) and low a l t i t i t u d e (100 m)* e c o t y p e s of L. hirsutum we re ^rown in hydroponic c u l t u r e in a g reenhouse and l a t e r t he adul t p l an t s we re t r a n s f e r r e d to a g rowth c h a m b e r with a 13 / 5 C day /n igh t t e m p e r a t u r e r e g i m e for a two-week per iod . At the end of t he e x p e r i m e n t t he low a l t i t u d e eco type had died whe rea s the o the r was not only a l ive but wi thout any s y m p t o m s of injury. It should also be men t ioned t h a t bo th eco types grow normal ly in t h e g reenhouse a t high t e m p e r a t u r e s up to 35 C. These r e su l t s suggest t h a t eco types from the reg ion of high var iab i l i ty in n o r t h e r n Pe ru (49) have g r e a t e r pheno typ ic p l a s t i c i t y in thei r abi l i ty to s t and a wider r ange of t e m p e r a t u r e s than the i r Ecuador ian c o u n t e r p a r t s which show less va r iab i l i ty . This could also be i n t e r p r e t e d as a g r e a t e r t h e r m a l s t ab i l i t y of m e m b r a n e s t r u c t u r e s in t he eco type from the region of h igher var iab i l i ty , but this needs to be p roven expe r imen ta l l y . Thus, t h e origin of t he Ecuador ian eco types might be expla ined in t e r m s of t he "Baldwin e f fec t " (ZZ) of organic se lec t ion , a p rocess in which pheno typ ic r e a c t i o n s p e r m i t a popula t ion to exist in an env i ronment to which it is no t well a d a p t e d and give it t i m e to

Seeds of this ecotype were kindly provided by Dr. C. M. Rick.


acqu i r e , by a m o r e or less r andom p roces s of gene m u t a t i o n s , geno types which a r e a d a p t e d to t h e new e n v i r o n m e n t . Loss of i t s ab i l i ty to survive a t low t e m p e r a t u r e s is probably the p r i c e this e c o t y p e had to pay in order to adap t to t he Ecuador ian t rop ica l fo res t . This can also be suppor ted by t h e ev idence a c c u m u l a t e d by Bjorkman (4), in s tud ies of geno typ ic a d a p t a t i o n of p l an t s from c o n t r a s t i n g e n v i r o n m e n t s . He found t ha t geno typ ic a d a p t a t i o n s which enab le t h e p lan t to pho to syn thes i ze wi th an unusual ly high e f f ic iency under one env i ronmen ta l e x t r e m e of l ight or t e m p e r a t u r e r equ i r e a spec ia l i za t ion of i t s p h o t o s y n t h e t i c mach ine ry which p rec ludes a high p h o t o s y n t h e t i c e f f ic iency a t t h e o the r e x t r e m e . On t h e o t h e r hand, t h e r e a r e t h e popula t ions from n o r t h w e s t e r n Pe ru which Rick (49) has found to be t h e reg ion of h ighes t va r iab i l i ty and g r e a t e s t ou tc ross ing which a r e c h a r a c t e r i s t i c of an a n c e s t r a l spec ies in t h a t reg ion of g r e a t ecologica l d ivers i ty . For corn a s imi l a r i ty could be drawn b e t w e e n the p r im i t i ve r a c e of Pe ruv ian corn and the popula t ions of L. hirsutum from n o r t h w e s t e r n Pe ru , bo th wi th a wide r a n g e of t e m p e r a t u r e a d a p t a t i o n on one hand t h e the marg ina l popula t ions of L. hirsutum from Ecuador and the modern and c l i m a t e spec ia l i zed r a c e s and se l ec t ions of corn, which have a d iminished r a n g e of a d a p t a t i o n .

Morphological and a l lozymic c h a r a c t e r s h a v e been r e c o r d e d ex tens ive ly for e c o t y p e s of L. hirsutum from c o n t r a s t i n g env i ronmen t s (49). Many of t hese c h a r a c t e r s can be used as g e n e t i c m a r k e r s to follow up in fu tu re g e n e r a t i o n s . Clausen and Hiesey (14) po in ted out such m a r k e r s t h e m s e l v e s may no t f a c i l i t a t e eco log ica l ad jus tmen t of the i r e c o t y p e s to t h e env i ronmen t but may be l inked wi th genes t h a t con t ro l the subcel lu lar s t r u c t u r e s and physiological c h a r a c t e r s of i m p o r t a n c e in d e t e r m i n i n g ad jus tmen t s to t h e env i ronmen t . Es tabl ishing such co r r e l a t i ons would be e x t r e m e l y helpful for sc reen ing purposes in the fu tu re .

It has been sugges ted in t h e pas t t ha t cold t o l e r a n c e is m a t e r n a l l y inhe r i t ed to some e x t e n t , a t l eas t for seed ge rmina t i on , but t e s t i n g t ha t c h a r a c t e r i s t i c tu rns out to be of some dif f icul ty using t h e Pe ruv ian and Ecuador ian e c o t y p e s due to the e x i s t e n c e of un i l a t e r a l i ncompa t ib i l i t y (33). The only successful way to m a k e the cross is to use t h e Ecuador ian type as t h e p i s t i l l a t e p a r e n t . The use of some e x t r e m e v a r i a n t s from the lowes t e l eva t ions in Pe ru (500 m) which a r e fully c o m p a t i b l e with t he popula t ion from the h ighes t a l t i t u d e (3,300 m) will he lp to e l u c i d a t e this p rob lem. Full compa t ib i l i t y can be found in the r a c e s of corn .

IV. THE SEARCH FOR "COLD TOLERANT GENES" IN C R O P PLANTS

Some effor t has been m a d e towards in t roduc ing and increas ing cold t o l e r a n c e of chill ing sens i t ive c u l t i v a t e d p l a n t s . In genera l t he a c c o m p l i s h m e n t s in t he se e f for t s have not been s p e c t a c u l a r . P l an t b r e e d e r s h a v e focused on seed ge rmina t i on a t low t e m p e r a t u r e s to enable t he ea r ly p lan t ing of c rops . Se lec t ion for seed ge rmina t ion a t low


t e m p e r a t u r e s had been m a d e in c o t t o n (1Z, 13, 15, 32), soybean (42), beans (16), corn (7, 8, 44) and t o m a t o (10, 17, 19, 40, 50). In a lmos t all ca ses , se lec t ion was appl ied to cu l t iva r s from each c rop . Once a cold t o l e r a n t l ine was s e l e c t e d , it was crossed wi th a sens i t ive l ine and from s tudy of thei r p rogen ies , conclusions we re drawn about the n a t u r e and complex i ty of the i n h e r i t a n c e for cold t o l e r a n c e . In gene ra l the r e s u l t s a r e qu i t e c o n t r a d i c t o r y . It has been c la imed t ha t cold t o l e r a n c e for seed ge rmina t ion is monogenic (10, 19). It has also been c l a imed polygenic in most cases and m a t e r n a l l y inhe r i t ed as well (12, 13, 32, 40). Some of t he p rob lems p lant b r e e d e r s have to face a r e se lec t ion c r i t e r i a and a meaningful way to quan t i t a t i ve ly m e a s u r e the response of b reed ing l ines in field t r i a l s .

Compar i sons and se lec t ions of cu l t iva r s for thei r t o l e r a n c e to low t e m p e r a t u r e s during d i f fe ren t s t a g e s of deve lopmen t (other than germinat ion) have been ob ta ined with a varying d e g r e e of success in: t o m a t o e s , during a whole life cyc le (28, 29) and fruit se t (11); corn , g rowth in genera l (7); and co t t on , g rowth , deve lopmen t and boll m a t u r a t i o n (20, 21). At the p re sen t t i m e e f fo r t s a r e being m a d e to i n t roduce cold t o l e r a n c e in to sorghum using r a c e s n a t i v e to t he highlands of Uganda and Ethiopia (54). The se lec t ion in c o t t o n , G. hirsutum, l ine M-8 is of i n t e r e s t s ince it s e e m s to be a l ine wi th a va r i an t for d e s a t u r a s e Δ15 , d i r ec t ly involved in t he synthes i s of l inolenic acid , which might be a c t i v a t e d a t low t e m p e r a t u r e s (51).

In all t h e examples l i s ted above , s e l ec t ion has been used only in c o m m e r c i a l cu l t i va r s . The re have not been any r e p o r t s of the use of wild spec ies or p r i m i t i v e forms of a spec ies as a sou rce of cold t o l e r a n c e . The main reason for the absence of this a t t e m p t is the t i m e involved in r ecove r ing the t r a i t in a commerc i a l l y a c c e p t a b l e t ype . Since mos t p re sen t day cu l t iva r s a r e the resu l t of a long per iod of se lec t ion for un i formi ty , high yields and a d a p t a t i o n to mild env i ronmen t s , t he probabi l i ty of finding s t r ik ing va r i an t s within these popula t ions is e x t r e m e l y low and a t bes t any a c c o m p l i s h m e n t s will be l imi ted .

Many useful c h a r a c t e r s from wild spec ies have been successful ly t r a n s f e r r e d to the c u l t i v a t e d t o m a t o (20, 21). At the p r e s e n t t i m e a jo in t ef for t is being m a d e with Dr . C. M. Rick, Dr . A. S tevens , Dr . R. Jones , S. D . Tanksley and the au thor to in t roduce cold t o l e r a n c e into the c u l t i v a t e d t o m a t o .

Accord ing to Mangelsdorf (31), t h e use of exo t i c ge rmplasm of corn in the USA has been given l i t t l e a t t e n t i o n . The reasons a r e , (a) the possibi l i ty for i m p r o v e m e n t of exis t ing c u l t i v a t e d va r i e t i e s is st i l l far from exhaus ted , and (b) the p r e s e n c e in exo t i c r a c e s of some undes i rab le c h a r a c t e r s such as some respons iveness to pho toper iod , luxurious g rowth , l a t e f lowering, e t c . Mangelsdorf sugges ts t ha t the most promis ing m e t h o d of ut i l iz ing exo t i c r a c e s in t he U.S. is by modifying one or m o r e inbred l ines . Hybrids of n a t i v e inbred l ines wi th exo t i c r a c e s (usually not well a d a p t e d to condi t ions of t h e U.S.) followed by two backc rosses wi th thei r inbred p a r e n t yield vigorous progeny wi th most of the undes i rab le c h a r a c t e r i s t i c s of t he exo t ic r a c e masked by t h e inbred . One of t h e


drawbacks of t h e cold t o l e r a n c e Pe ruv ian r a c e "Conf i te Puneno" is i t s high suscep t ib i l i ty to s m u t . The main conce rn of A m e r i c a n corn b r e e d e r s in t h e pa s t y e a r s has been yield; but today , wi th inc reas ing demands for food by a hungry world, t h e r e is a n e e d to conquer new land in marg ina l a r e a s for c rop p roduc t ion . Areas which a r e p r e sen t l y l imi t ed in use b e c a u s e of cool c l i m a t e s may s o m e day suppor t new v a r i e t i e s of ex is t ing crops b red to grow and develop no rma l ly in t he cold. This possibi l i ty r e p r e s e n t s a cha l lenge which has to be m e t wi th new g e n e t i c r e s o u r c e s .

ACKNOWLEDGMENTS

I express my g r a t i t u d e to Dr . Cha r l e s M. Rick, Dr . M. Allen S tevens , Dr . R icha rd Jones , S teve Tanksley, and Karen Koevary for r ev iewing this manusc r ip t as well as for the i r va luab le sugges t ions and c o m m e n t s . I also thank Dawn Nichols for typing the in i t ia l d r a f t s and Moira Tanaka for the a r t work.


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ADAPTATION TO CHILLING STRESS IN SORGHUM

J. R. McWilliam, W. Manokaran, and T. Kipnis

Univers i ty of New England Armida l e , N.S.W., 2351 , Aus t r a l i a

I. INTRODUCTION

Many annual p l an t s of t rop ica l and sub t rop ica l origin a r e now widely c u l t i v a t e d as s u m m e r crops in t e m p e r a t e e n v i r o m m e n t s . This has been ach ieved by adjust ing t h e phenology of t h e crops so t h a t the i r deve lopmen t t a k e s p l a c e during the per iod of favorable s u m m e r t e m p e r a t u r e s and the exposure to chil l ing t e m p e r a t u r e s in e i t he r air or soil is min imized .

D e s p i t e this long h is tory of t e m p e r a t e cu l t iva t ion t h e r e is l i t t l e ev idence t h a t any of these c rops have acqu i red a subs tan t i a l d e g r e e of ch i l l i ng - re s i s t ance and as a r esu l t t hey regu la r ly suffer from chill ing d a m a g e when spr ing-sown in t e m p e r a t u r e e n v i r o n m e n t s . Common s y m p t o m s inc lude fa i lure to g e r m i n a t e or r e t a r d e d ge rmina t ion , r ad ic l e inury or d e a t h , slow e m e r g e n c e of the p lumule or co ty ledons and chlorosis and accompany ing nec ros i s of newly fo rmed p h o t o s y n t h e t i c t i s sue (2, 4, 5, 7, 15, 18, 22). Var ia t ion in ch i l l ing-sens i t iv i ty has been r e p o r t e d in a number of crops of t rop ica l or igin. G e n e t i c d i f fe rences in g e r m i n a t i o n and ea r ly g rowth of inbred and hybrid corn grown under chil l ing t e m p e r a t u r e s have been r e p o r t e d (4, 5, 20). Also va r i e t a l d i f fe rences in the min imum t e m p e r a t u r e r equ i r ed for the ge rmina t ion of t rop ica l r i c e have been shown by Oka (17). P a t t e r s o n et al. (19) have shown a good co r r e l a t i on b e t w e e n t h e chil l ing t o l e r a n c e of Passiflora

spec ies and the t e m p e r a t u r e of t h e env i ronmen t in which they occur and have also r e c o r d e d va r i a t ion in g e r m i n a t i o n and green ing of e co types of wild t o m a t o which is c losely r e l a t e d to t h e a l t i t u d e of the i r origin in t he Andean region of South A m e r i c a . T h e r e is also ev idence t ha t t he m o v e m e n t of a number of t rop ica l g rass spec ies no r th and south in to

Permanent address: Agricultural Research Organization, The Volcani Centre, Bet Dagan, Israel

Copyright · 1979 by Academic Press, inc. 4 9 1 All rights of reproduction in any form reserved

ISBN0-12 46056O5

4 9 2 J. R. McWil l iam et al

m o r e t e m p e r a t u r e env i ronmen t s has been a c c o m p a n i e d by t h e acquis i t ion of a g r e a t e r d e g r e e of chil l ing t o l e r a n c e (1Z).

This paper provides a c o m p a r a t i v e analysis of t h e ge rmina t ion , ea r ly seedl ing deve lopmen t and chlorophyll synthes is of a number of sorghum spec ies a d a p t e d to con t r a s t i ng t h e r m a l env i ronmen t s , when exposed to a wide r ange of chil l ing t e m p e r a t u r e s . A b e t t e r unders tand ing of t h e e f f ec t s of chill ing s t r e s s on these i m p o r t a n t sequen t ia l s t eps in t h e p rocess of e s t ab l i shmen t and ev idence for va r i a t ion in t he se responses may suggest m o r e ef f ic ient ways of se l ec t ing for i nc reased chil l ing t o l e r a n c e in this and o the r i m p o r t a n t c rops of t rop ica l or igin.


A. Plant Material

For the major i ty of e x p e r i m e n t s t h r e e spec ies of Sorghum w e r e used (1) S. bicolor, a Nor th A m e r i c a n c o m m e r c i a l sorghum cu l t iva r with a Hegar i type background; (2) S. verticilliflorum, a t rop ica l wild sorghum (sha t t e r cane) now a widespread weed of t he t rop ics , co l l ec t ed ( la t i tude 17°S) in coas ta l no r th Queens land, and (3) S. leiocladum, a s u m m e r growing wild perennia l spec ies , n a t i v e of e a s t e r n and n o r t h e r n Aus t ra l i a , co l l ec t ed ( la t i tude 31 S) in n o r t h e r n New South Wales a t an e leva t ion of 1000 m. Al though Sorghum is p r imar i ly a genus of t rop ica l origin, the spec ies does occur na tu ra l l y and is grown in t e m p e r a t e env i ronmen t s . These t h r e e spec ies w e r e chosen to r e p r e s e n t m a t e r i a l wi th po ten t i a l ly con t r a s t i ng t h e r m a l a d a p t a t i o n s . In all e x p e r i m e n t s t he behaviour of t hese t h r e e chi l l ing-sens i t ive spec ies has been c o m p a r e d wi th bar ley (Hordeum vulgare cv Abyssinian), a chi l l ing - re s i s t an t t e m p e r a t e c e r e a l . All seed used was app rox ima te ly t he s ame age , co l l e c t ed within six months of unde r t ak ing t h e e x p e r i m e n t s . Seed viabi l i ty was high ( >95%) in all spec ies wi th t he excep t ion of S. leiocladum which was lower because of t he dif f icul ty of ident i fying e m p t y ca ryopses . All seed was t r e a t e d during s t o r a g e wi th a fungicide to min imize fungal con t amina t i on .

B. Temperature Control

Most responses w e r e m e a s u r e d over a t e m p e r a t u r e r ange from 2 4 ° -4 C, a l though t h e r e was l i t t l e measu rab l e a c t i v i t y observed in the sorghums below 8 C. Incuba to rs w e r e used to obta in the var ious t e m p e r a t u r e r e g i m e s . These w e r e m a i n t a i n e d in a cold room a t 4 C to min imize t e m p e r a t u r e f luc tua t ions which var ied within +0.25 C of t he s t i pu l a t ed t e m p e r a t u r e . In t he case of t he r e sp i r a t ion e x p e r i m e n t , t he w a t e r ba th t e m p e r a t u r e s for the m a n o m e t e r s was m a i n t a i n e d a t +0.1 C. In those e x p e r i m e n t s where young seedl ings w e r e exposed to l ight during

A d a p t a t i o n to Chil l ing S t r e s s in Sorghum 4 9 3

green ing a t chil l ing t e m p e r a t u r e s , r e f r i ge r a t i on was provided in i ncuba to r s to r e m o v e h e a t g e n e r a t e d by cool beam l amps . T e m p e r a t u r e was s t epped by Ζ C in t e rva l s b e t w e e n 24 and 1Z C and by 1 C in t e rva l s below 1Z C.

C. Experimental Procedures

1. Germination. Seeds w e r e g e r m i n a t e d a t t h e var ious t e m p e r a t u r e s in the da rk in p e t r i d ishes on mois t f i l te r p ad s . Seeds w e r e cons ide red to have g e r m i n a t e d when the length of t he r ad ic l e exceeded the smal l d i a m e t e r of the seed . The e x p e r i m e n t was t e r m i n a t e d a f t e r 50 days . R a t e s of ge rmina t i on w e r e based on t i m e in days to 50% ge rmina t i on (G^Q) c a l c u l a t e d by m e a n s of probi t analysis (8).

2. Respiration. Resp i r a t i on r a t e s of seed g e r m i n a t e d a t Z4°C w e r e measu red over the t e m p e r a t u r e r a n g e , using Warburg equ ipment (Z5). Oxygen u p t a k e (μΐ O^g h ) was m e a s u r e d on r e p l i c a t e samples of seedl ings of known weight a t a s imi lar s t a g e of ge rmina t i on ( e m e r g e n c e and a c t i v e ex tens ion of r a d i c l e and p lumule) . All seed was g e r m i n a t e d a t Z4 C and then equ i l ib ra t ed for Z4 h a t t he t e m p e r a t u r e a t which r e sp i r a t ion was to be m e a s u r e d . F ive seedl ings we re used for each m e a s u r e m e n t and a f t e r equi l ibra t ing for 30 min. in the f lasks, r e sp i r a t ion m e a s u r e m e n t s w e r e t aken over a per iod of two hours under s t e ady s t a t e condi t ions .

3. Extension. R a t e s of ex tens ion of t h e subcoleopt i le i n t e rnode (mesocotyl) we re measu red on seedl ings t ha t had been g e r m i n a t e d a t Z4 C and then grown wi thout exposure to l ight , on agar medium in single v ia ls . The mesoco ty l s w e r e p e r m i t t e d to e longa t e up through glass tubing wi th a d i a m e t e r s l ight ly l a rge r than the^ wides t d i a m e t e r of t he co leop t i l e . Extens ion m e a s u r e m e n t s (mm h ) w e r e m a d e over t he r ange of t e m p e r a t u r e s wi thout d is turb ing the seedl ings in g reen (safe) l ight a t 6 or 1Z hr i n t e rva l s depending on g rowth r a t e , using a b inocular m i c r o s c o p e . F ive success ive read ings w e r e t aken on e a c h of Ί 5 seedl ings of e a c h spec ies a f t e r a 1Z h equi l ibra t ion per iod a t the des igna ted t e m p e r a t u r e .

4. Arrhenius Plots. R a t e funct ions for ge rmina t ion , r e sp i r a t i on and ex tens ion g rowth a r e p r e s e n t e d in t he form of Arrhenius p lo t s by p lo t t i ng log r a t e aga ins t t h e r e c i p r o c a l of t h e abso lu te t e m p e r a t u r e . The s lopes of t h e curves w e r e ob ta ined by f i t t ing regress ion to po in t s de r ived from t h e a v e r a g e s of t h r e e r ep l i ca t i ons and se l ec t ing those giving t h e bes t f i t . Where d i scon t inu i t i es w e r e suspec ted , r egress ions w e r e f i t t ed to all combina t ions above and below the a p p a r e n t b reak and the p a r t i t i o n wi th t h e min imum sum of square was s e l e c t e d . Lines of bes t fit we re then drawn for e a c h p a r t i t i o n and from t h e i n t e r s e c t i o n of t he regress ions an e s t i m a t e of t h e b reak point ( t rans i t ion t e m p e r a t u r e ) was m a d e .

4 9 4 J. R. McWilliam et al

5. Carry-Over Effects. The e f f ec t s of exposure to chill ing t e m p e r a t u r e on subsequent deve lopmen t a t a favourab le t e m p e r a t u r e was examined in r e l a t i on to germinat ionând ex tens ion growth of S. bicolor. Seed was e i the r imbibed a t 8 for per iods up to 20 days or g e r m i n a t e d a t 24 C and then s to red as an e longat ing seedl ing a t 8 for up to 10 days . R a t e s of ge rmina t i on (Gj-J and r a t e s of mesoco ty l ex tens ion w e r e then m e a s u r e d as desc r ibed a t Z4 C to d e t e c t any ca r ry -ove r of t h e previous t e m p e r a t u r e s t o r a g e .

6. Chlorophyll Synthesis. The c a p a c i t y of young e t i o l a t e d seedl ings to syn thes ize chlorophyll in t h e l ight a t t he var ious t e m p e r a t u r e s was m e a s u r e d by exposing seedl ings , which had been grown to t he fir&J leâf s t a g e in the d a r k a t 24 C, to a light flux of e i t he r 250 or 25 μ Ε m s of PAR for 24 h. Pr ior to exposure , all seedl ings we re p re -cond i t ioned for 24 h a t the des igna ted t e m p e r a t u r e . Low vapour p re s su re def ic i t s we re ma in t a ined th roughout . The e x t e n t of g reen ing was d e t e r m i n e d by measur ing the t o t a l .chlorophyll (a+b) c o n c e n t r a t i o n of t he first t r ue leaf ( μ g chlorophyll g FW) s p e c t r o p h o t o m e t r i c a l l y (1) a f t e r e x t r a c t i o n in 80% (w/v) a c e t o n e .

7. Electron Microscopy. Leaf s e g m e n t s (1 cm) w e r e fixed in cold 3 % (w/v) g lu t a ra ldehyde in 0.01 Μ S0rensen's phospha te buffer , pos t fixed wi th osmium t e t r o x i d e and a f t e r dehydra t ion , embedded in Spurr 's low viscosi ty res in (24). Thin sec t ions we re cut on an LBK u l t r a m i c r o t o m e and examined under the e l e c t r o n mic roscope a f t e r con t r a s t i ng wi th uranyl a c e t a t e and lead c i t r a t e .

ΠΙ. RESULTS AND DISCUSSION

A. Germination and Early Seedling Development

The r a t e s of the t h r e e ge rmina t ion responses s tudied , ini t ia l ge rmina t ion , seed l ing- resp i ra t ion and mesoco ty l ex tens ion all dec l ined as the t e m p e r a t u r e was r educed from 24 down to 8 C. The r a t e of dec l ine , however , va r ied considerably b e t w e e n spec ies , espec ia l ly in t he lower p a r t of t h e t e m p e r a t u r e r ange below about 12 C (Fig. l a - c ) .

A single d iscont inui ty was observed in t he Arrhenius p lo t s for all t h r e e responses , bo th in t he chi l l ing-sens i t ive sorghum spec ies and in the ch i l l ing- res i s tan t ba r ley con t ro l . The p lo t s in all cases w e r e l inear above and below the b reak , which with one excep t ion (germina t ion of S. ver-ticilliflorum) o c c u r r e d a t a t e m p e r a t u r e ( t rans i t ion t e m p e r a t u r e ) around 12 C which lies in the t e m p e r a t u r e r ange (6-14 C) r e p o r t e d for most chi l l ing-sens i t ive p l an t s . Similar b r eaks in t he Arrhenius p lo t s of

Lambda Quantum Meter L185. Measures quantum flux density of PAR (400-700 nm).


4 9 6 J. R. McWil l iam et al.

24 2 0 1 6 1 2 8 1 r

1 ι

34 3 5 3 6 J / X10 4

FIGURE 1. Arrhenius plots for three processes involving the germination of chilling-resistant barley and three chilling-sensitive sorghum species adapted to different temperature environments. Q , q values derived from regressions are indicated for each slope, (aj Germination rat^ (reciprocal of days to 50% germination; (b) Respiration rate μ £ 0 ^ g h ) ; (c) Mesocotyl extension rate (mm h ; H.v. Hordeum vulgare; S.b. Sorghum bicolor; S.v. Sorghum verticilliflorum; S.l. Sorghum leiocladum.

var ious a s p e c t s of ge rmina t ion ac t i v i t y have also been r e p o r t e d in o the r chi l l ing-sens i t ive spec ies including cucumber and mung bean (22), cas to r bean (27) and r i c e (15). In the case of t rop ica l indica r i c e v a r i e t i e s , t he

ο ο b reaks occu r r ed a t a t e m p e r a t u r e of 16 -17 C which is in t he r a n g e found for the t rop ica l e co type of S. verticilliflorum used in this s tudy .

Discont inu i t i es in t he Arrhenius p lo t s of all t h r e e responses in ch i l l ing- res i s tan t bar ley , a l though not as p ronounced as in the sorghums, do r e p r e s e n t a s ignif icant change in the t e m p e r a t u r e coef f i c ien t s of these r e a c t i o n s . Similar b reaks in Arrhenius p lo t s for ch loroplas t deve lopmen t , Hill r e a c t i o n a c t i v i t y and leaf e longat ion of ba r ley a t a round the s ame t e m p e r a t u r e have been r e p o r t e d (16, 23) and also for r e sp i r a t ion in ch i l l ing- res i s tan t whea t and rye seedl ings (21).

A d a p t a t i o n to Chil l ing S t r e s s in Sorghum 49 7

20°C 14° C 8° C (a) GERMINATIO N

FIGURE 2. Relative rates of (a) germination, (b) respiration and (c) mesocotyl extension, of chilling-resistant barley and three chilling-sensitive sorghum species adapted to different temperature environments. Values are given for three temperatures, one above and two in the chilling range.

In t h e t h r e e ch i l l ing-sens i t ive sorghum spec ies , a l though in mos t cases the b reaks in the Arrhenius p lo t s occu r r ed a t or about the s a m e t e m p e r a t u r e (around 1Z C ) as in ba r l ey , t h e slopes of t he regress ions as m e a s u r e d by t h e t e m p e r a t u r e coe f f i c i en t s ( Q ^ ' s ) we re g r e a t e r espec ia l ly below the t r ans i t i on t e m p e r a t u r e . The enormous i nc rea se s in t h e Q , Q va lues below 1Z C in t he most ch i l l ing-sens i t ive sorghums ind ica t e e x t r e m e l y high a c t i v a t i o n energ ies and help explain the poor ge rmina t i on and low r a t e s of ex tens ion g rowth a t t h e s e t e m p e r a t u r e s . The r e l a t i v e d i f f e rences in the r a t e s of ge rmina t i on , r e sp i r a t ion and ex tens ion g rowth above and below the chil l ing t e m p e r a t u r e r ange a r e m o r e c l ea r ly i l l u s t r a t ed in F igu re Z. The sorghums as a group a r e r e l a t i ve ly m o r e dep res sed than ba r ley i m m e d i a t e l y above the chill ing t e m p e r a t u r e r ange ( Z O - 1 4 C ) and d r a m a t i c a l l y so below 1 0 C .

These r e su l t s sugges t t h a t a d i scon t inu i ty in t he Arrhenius p lo t s of t he se var ious ge rmina t i on p roces ses may no t be a unique or dist inguishing

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f e a t u r e of ch i l l ing-sens i t ive spec ies , but t h a t t he d i f fe rences in t he a p p a r e n t a c t i v a t i o n energ ies or t he Q J Q ' S above , and espec ia l ly be low, the t rans i t ion t e m p e r a t u r e may be a much more useful d iagnos t i c f e a t u r e , (Fig. l a - c ) . o

The r a t e s of Q I Qs °f t h e var ious responses below 1Z C may also

provide a m e a s u r e di the r e l a t i v e ch i l l i ng - to le rance within a group of chi l l ing-sens i t ive srjecies. The of t h e t h r e e sorghums spec ies used in this s tudy below 1 2 d i f fered qu i t e subs tan t i a l ly . In all cases t he Q -^ for t h e t rop ica l S. verticilliflorum was higher than S. bicolor, which is to be expec t ed , b e c a u s e of g r e a t e r ch i l l i ng - to le rance a s soc i a t ed wi th t he c o m m e r c i a l Hegar i type sorghums. The highes t QJQ> sugges t ing t he g r e a t e s t ch i l l ing-sens i t iv i ty , w e r e found for S. leiocladum which is an eco type a d a p t e d to a r e l a t i ve ly t e m p e r a t e env i ronmen t . This spec ies is a wild perennia l and i t s g r e a t e r ch i l l ing-sens i t iv i ty may r e p r e s e n t an a d a p t a t i o n to e s cape the e f f ec t s of low t e m p e r a t u r e in the spring and au tumn , as t he spec ies r ema ins d o r m a n t during this per iod and grows only during the w a r m e r s u m m e r mon ths .

In addi t ion to d i f fe rences in ch i l l ing-sens i t iv i ty in t he ge rmina t ion re sponse of d i f fe ren t sorghum spec ies , t h e r e is also some ev idence t ha t d i f fe rences exist within cu l t iva r s of S. bicolor. G^^ values for t h r e e groups of c o m m e r c i a l sorghum, one from U.S.A., one from t rop ica l Afr ica and a th i rd consis t ing of hybrids b e t w e e n t h e two , w e r e c o m p a r e d over the t e m p e r a t u r e r a n g e . The U.S. cu l t iva r s and the i r hybr ids , possibly because of the i r h i s to ry of s e l ec t ion for a d a p t a t i o n to cooler env i ronmen t s , had s ignif icant ly f a s t e r r a t e s of ge rmina t i on below 1Z C but not a t t e m p e r a t u r e s above the chil l ing r a n g e .

B. Carry-Over Effects of Chilling Exposure

The e f fec t of s tor ing e i t he r imbibed seed or g e r m i n a t e d seedl ings of S. bicolor for varying per iods a t a chil l ing t e m p e r a t u r e on the i r subsequent p e r f o r m a n c e a t a favourable t e m p e r a t u r e (Z4 C ) is i l l u s t r a t ed in F igure 3 . Storing imbibed seed for d i f fe ren t per iods up to ZO days a t 8 C caused a small de lay in subsequent ge rmina t i on r a t e (G^Q) but the d i f fe rences observed a f t e r d i f fe ren t per iods of chil l ing we re no t s ignif icant (Fig. 3a). The r a t e s of ge rmina t ion we re genera l ly enhanced by low t e m p e r a t u r e s t o r a g e by compar i son wi th the unchi l led con t ro l , but again t he d i f fe rences w e r e not s ignif icant and can be a c c o u n t e d for by t he e x t r a t i m e r equ i red to c o m p l e t e inhibi t ion in non-chi l led seeds .

The absence of a m a r k e d response to chill ing during imbibi t ion and the ear ly s t a g e s of ge rmina t ion in sorghum suppor t s the finding of Nish iyama ( 1 5 ) t h a t t h e w a t e r u p t a k e s t a g e in r i c e has a low Q^Q and is much less t e m p e r a t u r e dependen t than the subsequent ge rmina t ion phase . It is a t v a r i a n c e , however , wi th t h e r e su l t s of Wiles and Downs (Z8) who found tha t even shor t per iods of chil l ing w e r e harmful in c o t t o n , if given a f t e r t he seeds had begun hydra t ion .

Once ge rmina t ion is well advanced in sorghum t h e r a t e s of mesoco ty l ex tens ion a t a favourable t e m p e r a t u r e w e r e s ignif icant ly


I 1 ι ι ι » 0 2 4 6 8 1 0

DAYS AT 8° C

FIGURE 3. Carryover effects of storing either imbibed seed or germinating seedlings for periods up to 20 days at a chilling temperature of 8°C, on the subsequent (a) germinatioan rate (days to 50% germination) and (b) rate of mesocotyl extension (mm h ) of young seedlings. Both rates were measured at 24 C.

depressed by pr ior s t o r a g e of t h e g e r m i n a t e d seedl ings a t 8 C (Fig. 3b). The response was l inear and a f t e r 10 days , the max imum per iod of exposure to chill ing s t r e s s , t he ex tens ion r a t e was only Ζ 5% of t h e cont ro l r a t e . Similar r e su l t s have been r e p o r t e d in c o t t o n (3, 6).

These r e su l t s suggest t h a t t he ind i rec t e f f ec t s of chill ing s t r e s s a r e more ser ious in a c t i ve ly growing t i ssue which is undergoing cell ex tens ion and /o r cell division. The d a m a g e may be i n i t i a t e d in t h e cel l m e m b r a n e causing an i nc r ea se in p e r m e a b i l i t y and l eakage of cel l so lu tes and u l t i m a t e l y leading to m e t a b o l i t e d i s tu rbance and t h e accumula t i on of tox ic p r o d u c t s . All t hese lesions have been r e p o r t e d previously (10) but none w e r e speci f ica l ly ident i f ied in this e x p e r i m e n t .

5 0 0 J. R. McWil l iam et ai

0« 1 — • ι . ι . ι . ι 2 . ι 26 2 2 1 8 1 4 K )

TEMPERATURE °C

FIGURE 4. Greening of etiolated barley (H. vulgare) and sorghum (S. bicolor) seedlings produced at 24°C and then transferred to a range of temperatures from 26°-8°C and two levels of irradiance (400-700 nm)(a) 250 \iEm s , (b) 25 \iEm s .

D. Chlorophyll Synthesis in Etiolated Seedlings

The e x t e n t of greening of high t e m p e r a t u r e grown e t i o l a t e d leaf t i ssue of ba r ley and sorghum (S. bicolor) when exposed for 24 h to a r ange of t e m p e r a t u r e s from 26 to 8 C a t two levels of i r r ad i ance is i l l u s t r a t ed in F igure 4 . All values of chlorophyll have been expressed as a p e r c e n t a g e of t he c o n c e n t r a t i o n found a t 26 C.

The amoun t of chlorophyll syn thes ized in bo th spec ies dec l ined as t h e t e m p e r a t u r e was r educed , but below 17 in t he chill ing t e m p e r a t u r e r a n g e the abi l i ty of sorghum to a c c u m u l a t e chlorophyll was dependen t on


t h e level of i r r a d i a n c e . At t h e low flux dens i ty (25 p E m L

s A

) sorghum was able to syn thes i ze and a c c u m u l a t e chlorophyll down to 8 C but a t t he h igher flux (Z50 μ Ε m s , a p p r o x i m a t e l y 10% of full sunlight) v i r tua l ly no chlorophyll a c c u m u l a t e d in sorghum a t 16 C and below (Fig. 4a) . By c o n t r a s t , g reen ing in ba r ley was u n a f f e c t e d by the level of i r r ad i ance down to 15 C and whe re d a t a a r e ava i lab le a t low i r r a d i a n c e , t he r e l a t i v e c o n c e n t r a t i o n of chlorophyll was h igher than in sorghum.

These r e su l t s suggest t ha t chlorophyll syn thes i s in e t i o l a t e d t i ssue of a ch i l l ing-sens i t ive spec ies such as sorghum, which had deve loped a t high t e m p e r a t u r e , is possible a t t e m p e r a t u r e s down to 8 C under condi t ions of low light flux, but below 16 C a t h igher levels of i r r a d i a n c e t he r a t e of pho to -ox ida t ion or chlorophyll exceeds i t s r a t e of syn thes i s . Sensi t iv i ty t o pho to -ox ida t ion under t h e s e condi t ions appea r s t o be i n c r e a s e d if ch i l l ing-sens i t ive p l an t s a r e f irs t e t i o l a t e d a t lower t e m p e r a t u r e s (13), or if ch lorop las t deve lopmen t is d e f e c t i v e as in chlorophyll m u t a n t s of ba r ley (24) and m a i z e (14). Evidence from e l e c t r o n mic rographs of s o r g h u p ^ e a f t i s sue a f t e r 24 h exposure a t 15 C to e i t he r 25 or 250 μ μ Em s , i nd i ca t e t h a t t he fa i lure to develop chlorophyll a t t he h igher i r r ad i ance is due to the a r r e s t e d d e v e l o p m e n t of the m e m b r a n e sys tem of t he developing p las t ids which cons i s ted la rge ly of p r i m a r y l ame l l a l ayers wi th no ev idence of g rana deve lopmen t (Fig. 5a,b) . A s imi lar s i tua t ion has been desc r ibed in z a n t h a m u t a n t s of ba r l ey a t low t e m p e r a t u r e (24). Under the s ame condi t ion , but a t a lower i r r a d i a n c e , the sorghum ch lorop las t s w e r e m o r e c o m p a r a b l e to those deve loped a t t he h igher t e m p e r a t u r e (Fig. 5c,d) a l though the s t r o m a thylakoids and g rana w e r e less deve loped in t h e mesophyll ch lorop las t s and t h e r e w e r e no s t a r c h gra ins observed in t h e bundle shea th ch lo rop las t s .

The p r e s e n c e of chlorophyll and t h e abundance of s t a r c h gra ins in the mesophyll ch lorop las t s , howeve r , sugges t t ha t the ch lorop las t s we re fully func t iona l . This i nd i ca t e s t h a t t he pho to -ox ida t ion of chlorophyll and o the r leaf p i g m e n t s such as c a r o t e n e (11, 26) may p r e v e n t the deve lopmen t of a fully funct ional g r ana and s t r o m a thylakoid sys tem in the ch loroplas t and if pro longed, may d a m a g e m e m b r a n e s and resu l t in p e r m a n e n t chlorosis of leaf t i s sue as is commonly found in many crops and grasses of t rop ica l origin when grown a t chil l ing t e m p e r a t u r e s (9).

The sens i t iv i ty of e t i o l a t e d t i ssue to pho to -ox ida t ion in t h e l ight is a funct ion of the l ight flux and appea r s to be a gene ra l r e sponse in chi l l ing-sens i t ive p lan t s and may r e f l e c t a t e m p e r a t u r e induced dec l ine in the r a t e of a key enzyme(s) respons ib le for the synthes is of chlorophyll and i t s a s soc i a t ed p i g m e n t s . As t h e e x t e n t of g reen ing can be read i ly observed or measu red , it may be possible to use this r e sponse to ident i fy va r i a t ion in t he levels of ch i l l i ng - to l e rance in chi l l ing-sens i t ive crop p l a n t s .

As a p re l imina ry t e s t of this hypothes i s a r ange of known chi l l ing-t o l e r a n t and sens i t ive spec ies w e r e e t i o l a t e d as desc r ibed previously a t 24 C, then a f t e r 24 h equi l ib ra t iop ajt 17 C w e r e g r eened for 24 h a t 17 C a t an i r r a d i a n c e of 250 μ Ε cm s . The r e su l t s of this t r i a l a r e given in Table 1. The ch i l l ing- res i s t an t spec ies a r e i m m e d i a t e l y iden t i f i ab le ,

502 J. R. McWilliam et al

FIGURE 5. Chloroplasts of sorghum (S. bicolor) following exposure to different levels of irradiance at two temperatures. (a,b) Developing plastid&from sorghum exposed at 15 C to an irradiance of 250 \xE m s , containing only primary lamella layers, often in concentric rings. No grana development or even thylakoid extensions suggesting incipient grana development were present. 15000 and 21000 X respectively, (c) Mesophyll and bimdle sheath chloroplasts greened at 15 C at an irradiance of 25 \iE m s . Lamella system of mesophyll chloroplasts is less developed than at 24°C. Numerous small starch grains are present in mesophyll, but absent from bundle sheath chloroplasts. 15000 X. (d) Mesophyll and bungle sheath chlorqplqsts (showing prominent starch grains) greened at 24 C at 250 μ Em s . 21000 X.

as they syn thes ize apprec iab le amoun t s of chlorophyll under these condi t ions . Amongst the chi l l ing-sens i t ive group the r e l a t i v e a m o u n t s of chlorophyll syn thes ized do c o r r e l a t e reasonab ly well wi th t h e known or su spec t ed chi l l ing-sens i t iv i ty of these spec ies .


TABLE I. Chlorophyll content of the first leaf or cotyledons of etiolated seedlings of two groups of chilling-resistant and chilling-sensitive plants greened for 24 h at 17° C at an irradiance of 250 \iEm~ s~ . All values expressed as % of chlorophyll present in the tissue greened under similar conditions at 24 C.

Chilling-resistant % Chlorophyll Chilling-sensitive % Chlorophyll

Cauliflower 83 Pearl Millet 28 Cabbage 62 Rice (Indica) 23 Radish 60 Cantaloupe 22 Barley* Wheat*

57 Cotton 21 Barley* Wheat* 45 Sorghum 19 Oats* 43 Maize 19

*Spring varieties.

IV. CONCLUSIONS

Most p l an t s a d a p t e d to t rop ica l and sub t rop ica l env i ronmen t s respond in a s imi lar fashion when sub jec t ed to chil l ing t e m p e r a t u r e s and deve lop c o m p a r a b l e physiological d a m a g e when th is exposure is p ro longed . This is cons i s t en t wi th the hypothes i s t ha t the p r i m a r y even t in i t i a t ing t he se responses is a t e m p e r a t u r e induced physical change in the i r m e m b r a n e s .

O n e c o m m o n f e a t u r e of many responses of ch i l l ing-sens i t ive spec ies is a d i scon t inu i ty in the Arrhenius p lo t s which occur over a fair ly c h a r a c t e r i s t i c t e m p e r a t u r e r a n g e , as found for t he sorghum spec ies used in this s tudy . The e x i s t e n c e of such b reaks in a ch i l l ing- res i s t an t spec ies such as bar ley ind i ca t e s t h a t this phenomenon is no t unique to chi l l ing-sens i t ive spec ies and sugges t s t ha t the magn i tude of the t e m p e r a t u r e coef f i c i en t s above and below the b reak , or t r ans i t ion t e m p e r a t u r e , is more s ignif icant in ident i fy ing chi l l ing-sens i t ive spec ies than the b reaks per se, or t he t e m p e r a t u r e s a t which they occu r .

Var ia t ion in ch i l l ing-sens i t iv i ty b e t w e e n m e m b e r s of r e l a t e d spec ies and within e c o t y p e s of a s ingle spec ies was a p p a r e n t in sorghum and has been r e p o r t e d in a number of o t h e r c rop p l a n t s . This appea r s to be an a d a p t i v e response and under g e n e t i c con t ro l . One approach which appea r s to p rov ide a m e a n s of ident i fying such var iab i l i ty and one which could provide an index of ch i l l ing-sens i t iv i ty , is t he green ing response of e t i o l a t e d l eaves . A fu r the r s tudy has been i n i t i a t e d wi th a r a n g e of

5 0 4 J. R. McWill iam et al.

chi l l ing-sens i t ive crop spec ies using smal l leaf s e g m e n t s or co ty ledons to d e t e r m i n e if this t echn ique could be deve loped as a rapid mass sc reen ing p r o c e d u r e to assis t in se l ec t ing for g r e a t e r ch i l l i ng - to l e rance .

ACKNOWLEDGMENTS

We a r e g ra te fu l t o Mr. B. Ellem of t h e Division of M a t h e m a t i c a l S t a t i s t i c s , CSIRO for a s s i s t ance wi th probi t ana lyses and for a c c e s s to CIRONET t h e CSIRO c o m p u t e r , also to Dr . Haze l Har r i s and Dr . V. J . Bofinger, Univers i ty of New England, Armida le , NSW, for s t a t i s t i c a l adv ice and helpful c r i t i c i sm .


1. Arnon, D. Plant Physiol 25, 1 -15(1949. 2. Bre idenbach , R. W., Wade, N . L. and Lyons, J . M. Plant Physiol.

54, 324-327 (1974). 3 . Buxton, D. R., Sprenger , J . P . , and Pege low, E. J . Crop Sci. 16,

471-474 (1976). 4 . Ca l , J . P . and Obendorf, R. L. Crop Sci. 12, 369-373 (1972a). 5. Ca l , J . P . and Obendorf, R. L. Crop Sci . 12, 572-575 (1972b). 6. Chr i s t i ansen , Μ. N. Plant Physiol. 42, 431-433 (1967). 7. C r e e n c i a , R. P . and Bramlage , W. J . Plant Physiol. 47, 389-392

(1971). 8. F inney, D. J . "S ta t i s t i ca l Method in Biological Assay", 2nd Ed.,

Hafner , N.Y. (1964). 9. K a w a n a b e , S. Proc. Japan Soc. Plant Taxonomists 2, 17-20

(1968). 10. L e v i t t , J . In "Responses of P l an t s to Env i ronmenta l Stress" (Τ. T.

Kozlowski , ed.) pp . 27-43 , A c a d e m i c P res s , New York. 11 . McWill iam, J . R. and Naylor , A. W. Plant Physiol. 42, 1711-1715

(1967). 12. McWill iam, J . R. and F e r r a r , P . J . In "Mechanisms of Regu la t ion

of P lan t Growth" (R. L. Bieleski , A. R. Ferguson , Μ. M. Cresswel l , Eds.), pp . 467-476. Bullet in 12, The Royal Socie ty of New Zealand , Well ington (1974).

13. Millerd, A. and McWill iam, J . R. Plant Physiol. 43, 1967-1972 (1968).

14. Millerd, Α., Goodchild, D. , and Spencer , D. Plant Physiol. 44, 567-583 (1969).

15. Nish iyama, I. Plant and Cell Physiol. 16, 533-536 (1975). 16. Nolan, W. G. and Smill ie, R . M. Biochimica et Biophysica Acta

440, 461-475 (1976). 17. Oka, H. Jap. J. Breeding 4, 140-144 (1954).


18. Obendorf , R. L. and Hobbs, P . R. Crop Sci. 10, 563-566 (1970). 19. P a t t e r s o n , B. D. , Mura t a , T., and G r a h a m , D. Aust. J. Plant Phys

iol. 3, 435-442 (1976). 20. Pinnel l , E. L. Agron. J. 41, 562-568 (1949). 2 1 . P o m e r o y , Μ. K. and Andrews , G. J . Plant Physiol. 56, 703-706

(1975). 22. Simon, E. W., Minchin, Α., McMenamin , Μ. M. and Smith , J . New

Phytol. 77, 301-311 (1976). 23 . Smil l ie , R . M. In "Gene t i c s and Biogenesis of Chlorop las t s and

Mi tochondr ia" (Th. Bucher et al. Eds.) , pp . 103-110. Elsevier , Ams te rdam(1976) .

24. Smil l ie , R. M., Nie lsen , N. C , Henningsen, K. W., and von W e t t s t e i n , D. Aust. J. Plant Physiol. 4, 439-449 (1977).

25. U m b r e i t , W. W. In "Manomet r i c Techniques" (W. W. U m b r e i t , R. H. Burris and J . F . S tauf fer , Eds.) , pp . 1-17. Burgess Publ . Co . , Minneso ta (1964).

26. van Hasse l t , Ph . R. Acta Bot. Neerl. 21, 539-548 (1972). 27. Wade, N . L., Bre idenbach , R. W., and Lyons, J . M. Plant Physiol.

54, 320-323 (1974). 28. Wiles, E. L., and Downs, R. J . Seed Sci. and Tech. 5, 649-657

(1977).



CHILLING INJURY ASSAYS FOR PLANT BREEDING

R. E. Paull

D e p a r t m e n t of Botany Univers i ty of Hawaii a t Manoa

Honolulu, Hawai i

B. D. Patterson and D. Graham


Macquar i e Univers i ty Nor th Ryde , N.S.W., Aus t r a l i a 2113

I. INTRODUCTION

P l a n t s from t rop ica l and sub t rop ica l reg ions show cons iderab le g e n e t i c d ivers i ty in the i r abi l i ty to t o l e r a t e chi l l ing. The wild g r e e n -f ru i ted t o m a t o , Lycopersicon hirsutum Humb . & Bonpl. , is found from sea level in Ecuador under t rop ica l ra in fores t condi t ions to 3,300m in the Andes in n o r t h e r n Peru , well in to t he zone of chil l ing t e m p e r a t u r e s (65). For geograph ica l v a r i e t i e s of this spec ies , chil l ing t o l e r a n c e i nc rea se s wi th a l t i t u d e of origin (59, 60), Similar va r i a t ions a r e shown by c u l t i v a t e d spec ies of t h e genus Passiflora (58). The d ivers i ty in wild popula t ions may be asc r ibed to va r i a t i ons in the in t ens i ty of se lec t ion p res su res which r e m o v e the more chil l ing sens i t ive m e m b e r s from the popula t ion . There is cons iderab le i n t e r e s t in the deve lopmen t of a se l ec t ion p r o c e d u r e for d e t e r m i n i n g chil l ing t o l e r a n c e or r e s i s t a n c e for use in c rop b reed ing (18, 37, 66, 52). Much of the publ ished work on chil l ing injury does no t suggest me thods tor quant i fying smal l d i f fe rences in chi l l ing t o l e r a n c e , b e c a u s e i t c o n c e r n s compar i sons b e t w e e n u n r e l a t e d p l a n t s . The re fo r e , in this r ev i ew, s tud ies using closely r e l a t e d spec ies ,

aJournal Series #2369 of the Hawaii Agricultural Experiment Sta

tion.

5 0 7 Copyright « 1979 by Academic Press. Inc.

All rights of reproduction in any form reserved ISBN 0 1 2 46056&5

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Altitud e o f origi n (km ) FIGURE 1. Survival of altitudinal variants (L. hirsutum) after a

chilling stress. Seedlings were chilled for 7 days at 0°C after the appearance of the first true leaf. Survival was assessed after a further week at 18/22°C in a growth cabinet (60). cu l t iva r s or r a c e s within t he s ame g e n e r a have been emphas ized . Chill ing injury is usually man i fes t ed as visual symptoms which a r e difficult to quant i fy . This has m e a n t t h a t p rogress has been slow in developing q u a n t i t a t i v e measu re s of chil l ing t o l e r a n c e . It has of ten been n o t e d t h a t d i f fe ren t c r i t e r i a may m e a s u r e d i f fe ren t physiological p a r a m e t e r s a t d i f fe ren t deve lopmen ta l s t a g e s . This is t r u e for i n s t ance for bananas (57) and r i c e (9). At some s t a g e s p l a n t s can be ha rdened aga ins t chil l ing (79); this must also be t aken in to accoun t when measur ing sens i t iv i ty to chil l ing.

An assay for use in a breeding p rogram should be i) rap id , ii) r ep roduc ib le , iii) n o n - d e s t r u c t i v e to the whole p lan t , iv) q u a n t i t a t i v e or a t l eas t able to rank m a t e r i a l , and v) have a low labor cost per assay .

Π. SELECTION METHODS

A. Assays Under Natural or Semi-Natural Conditions

These t e s t s i n t e g r a t e the p lant ' s abi l i ty to survive , grow and develop under chill ing condi t ions . A p rogram based on field t e s t ing in a r e a s which

Chil l ing Injury A s s a y s for P l a n t B r e e d i n g 5 0 9

a r e marg ina l ly chil l ing is dependen t upon yea r to yea r va r i a t ion . However , u n p r e d i c t a b l e field condi t ions can be r e p l a c e d by con t ro l l ed condi t ions . Holber t et al. (31) des igned a p o r t a b l e r e f r i g e r a t e d c h a m b e r to s tudy cold r e s i s t a n c e of adul t corn p l an t s in t he f ield. The m e t h o d gave a r e l a t i v e ranking , but was slow. To o v e r c o m e some of t he se p rob lems wi th whole p l an t s , p a r t s of p l a n t s or seedl ings have been used. P a t t e r s o n et al. (60) t e s t e d seedl ings of t h e wild a l t i tud ina l co l l ec t ion of t o m a t o e s L. hirsutum by chil l ing a t 0 C . Survival was r e l a t e d to t he a l t i t u d e of origin (Figure 1 ,2 ) . In this type of s e l ec t ion p r o c e d u r e t he g e n e t i c m a t e r i a l whose t o l e r a n c e falls b e t w e e n t h e a r b i t r a r y t e m p e r a t u r e chosen and t e m p e r a t u r e below which injury normal ly occu r s (less than 10 C to 12 C) would be los t . However , i t could probably be used to s e l ec t t he most chil l ing r e s i s t a n t seedl ings from a gene t i ca l ly d iverse popula t ion .

TABLE I. Lag Time to 10% Germination at 15°C, Lowest Temperature at which 20% Rate Per Day of Germination was Attained and the Minimum Germination Temperature at which 50% Germination has Attained for an Altitudinal Collection of Lycopersicon hirsutum, L. esculentum and their Hybrids. For Details of the Altitudinal Varieties of L. hirsutum see (60).

Species Altitude Lag time Temperature Minimum of (days) (°C) (20%) germination

origin (to 10% rate of temperature (m) germination germination (°C) (50%

at 15°C) attained germination)

L. hirsutum

race - BG 400 8.3 17.0 15.2 PC 1000 5.0 18.2 13.0 AC 1500 4.4 12.2 8.8 LE 2000 5.6 10.4 10.0 EO 2100 2.7 21.8 7.1 CC 2650 5.7 13.9 9.2 AF 3100 3.6 11.4 5.9 Ric 3300 - - 7.4

L. esculentum

Rutgers 5.5 16.0 9.1 PI-341985 2.0 12.6 5.5 Rutgers χ BG 3.7 17.0 9.1 Rutgers χ AF 8.0 21.7 7.0

5 1 0 R. Ε. Paul le f ai

Tes t s based on seed ge rmina t ion a t marg ina l ly chill ing t e m p e r a t u r e s (10 to 1Z C) have been appl ied to crops such as : beans (18, 19, 38), l ima beans (75), c o t t o n (13, 4Z, 46) and t o m a t o e s (35). In a l t i t ud ina l v a r i e t i e s of Lycopersicon hirsutum t h e r e was cons iderab le va r i a t ion in lag t i m e before r ad i ca l e m e r g e n c e and the r a t e of ge rmina t i on , and the abi l i ty to g e r m i n a t e a t low t e m p e r a t u r e did no t necessa r i ly c o r r e l a t e with t h e abi l i ty to survive chill ing a t 0 C (Table I). The lag t i m e be fo re ge rmina t ion was more dependen t on t e m p e r a t u r e than was ge rmina t i on r a t e .

Methods based upon growth and deve lopmen t can be appl ied to p a r e n t a l se lec t ion and bulk b reed ing l ines . Se lec t ion me thods can be based on g rowth r a t e (1, 3, 35, 56, 70, 71), dry weight changes (45), roo t deve lopmen t (10) and abi l i ty of fruit to r ipen (4, 6). A c o m p l e t e l y d i f fe ren t ranking order was found b e t w e e n two d i f fe ren t c r i t e r i a of a d a p t a t i o n to low t e m p e r a t u r e : ge rmina t i on a t 8.5 C and fruit se t a t 4.5 C n igh t . Some bean l ines with high t o l e r a n c e to chill ing a f t e r seedl ing e m e r g e n c e appea red to lack t o l e r a n c e during ge rmina t ion (36). Resu l t s wi th t o m a t o also i l l u s t r a t e t h a t d i f fe ren t rankings of chil l ing t o l e r a n c e can be ob ta ined when d i f fe ren t c r i t e r i a (35) a r e used (Table Z). This ind ica tes t ha t chill ing sens i t iv i ty in the broad sense may not have a single cause .

TABLE IL Effects of Low Temperature on Seed Germination, Seedling Growth and Fruit Set of Tomato Varieties. The Numbers in Parentheses are the Order of Ranking. Note Different Ranking Order Obtained with Different Criteria of Chilling. Adapted from Kemp (35).

Germination Seedling growth Fruit set at 8.5°C 21°C/10°C at 4.5°C night

(%) (Scale 1 to 10) (%)

Earlinorth 64 (4) 3 (4) 58 (2) Rocket 69 (3) 3 (4) 28 (3) Borita 52 (5) 8 (2) 69 (1) Fireball 0 (6) 3 (4) 6 (7) Hilda 153 96 (1) 6 (3) 8 (6) Early Rutgers 0 (6) 2 (5) 0 (8) PI-280597 79 (2) 9 (1) 10 (5) Cold Set 52 (5) 6 (3) 20 (4)

Chill ing Injury A s s a y s for P l a n t B r e e d i n g 5 1 1

Much work has been r e p o r t e d on the d e t e r m i n a t i o n of s t o r a g e condi t ions of fresh fruit c o m m o d i t i e s . These r e s u l t s a r e genera l ly p r e s e n t e d as visual c r i t e r i a such as t he p e r c e n t a g e of d a m a g e d f rui t . Of ten cu l t iva r s of a spec ies differ in r e s i s t a n c e . For e x a m p l e , Yolo Wonder Capsicum was p a r t i c u l a r l y sens i t ive c o m p a r e d to some o the r cu l t i va r s (25). Similar va r i a t i ons h a v e been found for c u c u m b e r s (2) and bananas (57). Tes t s on t he h a r v e s t e d p roduc t involve cons iderab le t i m e and expense in ca r ry ing the crop to m a r k e t i n g s t a g e . It would be p r e f e r a b l e to be able to m a k e the se l ec t ion a t an ea r ly g rowth s t a g e .

B. Laboratory Measures

1. Tissue Culture. This t echn ique offers t he a d v a n t a g e of being able to subject l a rge n u m b e r s of cel ls to se l ec t ion a f t e r mu tagenes i s or in t roduc t ion of genes using p ro top l a s t fusion, which offers t he possibi l i ty of ove rcoming s t e r i l i t y b a r r i e r s b e t w e e n spec ies and g e n e r a . Dix and S t r e e t (20, 21) exposed cell cu l t u r e s of Nicotiana sylvestris and Capsicum annuum to chill s e l ec t i on wi th and wi thout m u t a g e n (see also Dix, this vo lume) . The deve lopmen t of this t echn ique and i t s subsequent app l i ca t ions will depend on the abi l i ty to r e g e n e r a t e p l an t s from the t i s sue c u l t u r e s .

2. Plant Tissue Tests. Chil l ing even tua l ly d i s rupts t he ce l lu lar i n t e g r i t y of p l a n t s . The subsequent l eakage of cel l c o n t e n t s , loss of a c t i v e t r an spo r t and changes in t h e levels of t i s sue m e t a b o l i t e s a r e possible to m e a s u r e and could be used to s e p a r a t e l ines of d i f fe ren t chil l ing r e s i s t a n c e .

L e a k a g e of cel l c o n t e n t s has been used to assay chil l ing injury. K a t z and Reinhold (33) found by d i r ec t obse rva t ion t h a t chi l led ep ide rma l cel ls of Coleus l e aves p l a smo lyze m o r e slowly than unchi l led ce l l s . An e f fusa t e conduc t iv i ty assay has been appl ied to corn (15), c u c u m b e r (74, 82), c o t t o n (12), and to a co l lec t ion of Passiflora spec ies (58). The r e s u l t s wi th t he Passiflora co l l ec t ion showed t h a t i t was possible to r ank Passiflora spec ies for chil l ing t o l e r a n c e on t h e basis of t i m e to 50% e l e c t r o l y t e loss a t 0 C . All of t h e above assays w e r e done following l eakage per iods of longer than one day . Ta t sumi and M u r a t a (74) showed t h a t p i t t i ng in cucumber frui t o c c u r r e d be fo re an i n c r e a s e in l eakage was a p p a r e n t . It t h e r e f o r e appea r s t h a t gross l eakage may not a lways be sens i t ive enough to d e t e c t t he changes which a r e visually a p p a r e n t . We s tud ied l eakage of leaf cel ls of t he t o m a t o spec ies L. hirsutum and L. esculentum. Using t h e r a t i o of t h e r a t e of l e akage a t 1 C to t h a t a t 20°C , the e c o t y p e of L. hirsutum from 3200 m a l t i t u d e showed a r e l a t i v e l y lower r a t e of l e akage a t 1 C than did t h a t from 30 m a l t i t u d e . For L. esculentum, t h e low t e m p e r a t u r e g e r m i n a t i n g se l ec t ion PI-341985 showed a lower r a t e a t 1 C than did t he v a r i e t y R u t g e r s (Table 3).

5 1 2 R. Ε. PauHef al

TABLE III. Ratio of Leakage Rate at 1°C and 20°C for the First Six Hours after Exposure to Temperature of Rubidium-86 Preloaded Leaf Cells of Lycopersicon spp. and Passiflora spp.

Altitude of collection Species (M) or chilling Ratio —

sensitivity 20°C rates

L. hirsutum race MG 30 2.2 BG 400 2.9 PC 1000 1.68 AC 1500 0.93 AF 3100 1.32 Ric 3300 0.6

L. esculentum Rutgers - 1.29 PI-341985 - 0.82

Organic so lu te loss from chi l led t i ssue has been cons idered as a se lec t ion p r o c e d u r e . Chr i s t i ansen (11), for example c o m p a r e d roo t exuda t ion from seve ra l g e n e t i c l ines of c o t t o n a t chil l ing t e m p e r a t u r e s and showed d i f fe rences which may be a basis for g e n e t i c s e l ec t ion .

An i n t e r e s t i n g va r i an t of t he conduc t iv i ty l eakage m e a s u r e is t ha t of K a t z and Reinhold (33) who found an i nc rease in the e l e c t r i c a l conduc t iv i ty of Coleus l eaves be fore d a m a g e was vis ible . This has not been followed up as a possible b reede r ' s se lec t ion me thod for chil l ing t o l e r a n c e .

Ac t ive t r anspo r t p r o p e r t i e s of p lan t cel ls a r e in t r ins ic m e m b r a n e p r o p e r t i e s . Paull et al (61) showed t h a t l euc ine u p t a k e by leaf f r agmen t s of the t o m a t o R u t g e r s was more sens i t ive than the low t e m p e r a t u r e ge rmina t i ng PI-341985. U p t a k e in to t h e low a l t i t u d e form of L. hirsutum was more sens i t ive to chil l ing than in to t he high a l t i t u d e fo rm. The assay was slow and h e n c e i m p r a c t i c a l as a mass se l ec t ion p r o c e d u r e . It could be simplif ied by using jus t the r a t i o of u p t a k e r a t e a t ZO C and 1 C. O t h e r a c t i v e t r anspo r t p r o p e r t i e s such as a d i r ec t m e a s u r e m e n t of e l e c t r i c a l po t en t i a l may be good c r i t e r i a but have not y e t been appl ied to chill ing s t r e s s .

The use of v i ta l s t a ins such as n e u t r a l r e d or dyes excluded by i n t a c t m e m b r a n e (e.g. Evan's Blue, f luorescein d i a c e t a t e ) t o d e t e c t uninjured, revers ib ly injured and dead cells has no t been deve loped in to an assay for chill ing injury. Widholm (80) t e s t e d a number of s t a ins and found f luorescein d i a c e t a t e for viable cel ls and phenosaf ran ine for dead cel ls the most r e l i ab le a f t e r f reez ing suspension cu l tu re s of a number of chil l ing sens i t ive p l a n t s . T o m a t o cu l t u r e cel ls held for 6 days below 1 0 C to 1Ζ C showed a l a rge i n c r e a s e in t h e number of cel ls which did no t s ta in wi th f luorescein d i a c e t a t e (5). These t e s t s a r e rap id , s imple and appl icab le to p l an t s which can be r e g e n e r a t e d from c u l t u r e s .

Chill ing Injury A s s a y s for P l an t B r e e d i n g 5 1 3

Chil l ing t e m p e r a t u r e s cause a d i sp ropo r t i ona t e d e c r e a s e in r e sp i r a t i on in i so la t ed mi tochond r i a from chil l ing sens i t ive spec ies (44). Sweet p o t a t o mi tochondr i a i so la t ed a f t e r five weeks of s t o r a g e a t 7.5 C showed a dec l ine in oxida t ion and phosphory la t ion a c t i v i t y when assayed a t Z5°C (40). Similar changes in r e sp i r a t i on have been found in whole t i ssues ; cucumber l eaves have a Q ^ Q of 5.7 below 1Z C, 1.7 above 1Z C (51). At non-chi l l ing t e m p e r a t u r e s t h e r a t e of C O ^ produc t ion for whole fruit d e c r e a s e s wi th s t o r a g e , w h e r e a s a t chi l l ing t e m p e r a t u r e s the r a t e inc reases with t i m e to a p l a t e a u fol lowed by a dec l ine . In c u c u m b e r frui t t he i nc reased r a t e occur s a t the s a m e t i m e as the onse t and deve lopmen t of chil l ing injury (Z3). These r e s u l t s point out t he need to specify t he s t a g e a t which the assay was p e r f o r m e d , o t h e r w i s e running a se l ec t ion m e t h o d based upon r e sp i r a t i on is i m p r a c t i c a l . An assay could possibly be deve loped using the r educ t i on of t r iphenyl t e t r a z o l i u m chlor ide (TTC) (7Z). Al though t h e m e t h o d r equ i r e s only smal l a m o u n t s of m a t e r i a l va r i ab le r e s u l t s wi th the TTC t e s t h a v e been r e p o r t e d (71) and it may not be p r a c t i c a l for l a rge n u m b e r s of s amples . This t e s t has been appl ied to t o m a t o fruit (14). Bre idenbach and Waring (5) used it on t o m a t o cel l c u l t u r e and found tha t r e d u c t i v e c a p a c i t y d e c r e a s e d below 10 C to 1Ζ C. The m e t h o d may be a rap id ind ica to r of ab i l i ty to r e s p i r e and could be deve loped as a s u p p l e m e n t a r y assay .

Pho tosyn thes i s is s eve re ly r e d u c e d by shor t exposures to chill ing t e m p e r a t u r e s (17). Though t h e r e is no c lea r r e l a t ionsh ip b e t w e e n chil l ing sens i t iv i ty of a spec ies and the p r e s e n c e or absence of a change in t h e t e m p e r a t u r e d e p e n d e n c e of the Hill a c t i v i t y a t low t e m p e r a t u r e s (16), an assay can be based on the r a t e of loss of ch loroplas t a c t i v i t y of d e t a c h e d l eaves of suscep t ib le spec ies s t o r e d a t 0 C (69). Af te r var ious per iods of chil l ing t h e i so la t ed ch lo rop las t s can be assayed a t Z3 C for pho to reduc t i on of f e r r i cyan ide in the p r e s e n c e of the uncoupler g ramic id in D. The r a t e of dec l ine in a c t i v i t y was t aken as a m e a s u r e of sens i t iv i ty .

P a t t e r s o n et al (63) desc r ibed a s imple assay based upon the min imum t e m p e r a t u r e of ch loroplas t d e v e l o p m e n t . This could be appl ied to p a r e n t a l and bulk beeed ing l ines . The seeds w e r e g e r m i n a t e d in t he dark and the seedl ings then exposed to dim r ed l ight to p r o m o t e yellow e t iop las t d e v e l o p m e n t . The yellow p lan t s w e r e then t r a n s f e r r e d to a t h e r m o g r a d i e n t ba r . Af te r t e m p e r a t u r e equi l ib ra t ion , the l eaves w e r e i l l umina ted wi th whi t e l ight for up to 6 days . The chlorophyll was e x t r a c t e d and d e t e r m i n e d or the seedl ings sub jec t ive ly r anked as to w h e t h e r or no t g reen ing had o c c u r r e d . This assay showed t ha t a high a l t i t u d e wild t o m a t o was b e t t e r able to g reen a t low t e m p e r a t u r e than low a l t i t u d e fo rms .

Chlorophyll f l uo re scence is an in t r ins ic p robe of m e m b r a n e s . M u r a t a and Fork (53) and Me lca rek and Brown (47) showed t ha t f l uo rescence moni to r ing of i n t a c t l eaves is a s imple m e t h o d to specify d i f fe ren t i a l r e sponses to chil l ing s t r e s s . The de layed f luo rescence yield of chil l ing sens i t ive p l an t s is r e d u c e d to a g r e a t e r e x t e n t than t h a t of chil l ing r e s i s t a n t spec ies (48). Chlorophyl l f l uo re scence has been used as a rapid sc reen ing t echn ique for p h o t o s y n t h e t i c m u t a n t s of h igher p l an t s

TABLE IV. Summary of Reports on the Inheritance of Chilling Tolerance in Sensitive Crops

Crop Character study ι Maternal Genetic effect References effect Number of Dominance

genes tolerance

Corn Low temperature seed germination yes several some Pinnell (63) yes multigenic — Helgason (29) yes — -- Ventura (78) — multigenic some Grogan (26) y e s multigenic some Pesev (62)

Imbibitional chilling injury yes — — Cal and Obendorf (7)

Cotton Low temperature seed germination y e s multigenic partial to Marani and Dag (46) complete

Imbibitional chilling injury y e s — — Christiansen 6c Lewis (13)

Tomato Low temperature seed germination — single gene recessive Cannon et al. (8) yes multigenic recessive Ng 6c Tigchelaar (55)

minimum 3 to 5

— multigenic additive El Sayed 6c John (24) 24 pairs

Fruit set — single gene recessive Kemp (34)

Beans Low temperature seed germination — multigenic low Dickson (18) heritability

Muskmelon Low temperature seed germination — multigenic dominant Kubicki (39)

Rice Flower sterility after low — multigenic dominant Toriyama 6c Futsuhara (76) temperature exposure

Chil l ing injury A s s a y s for P l an t B r e e d i n g 5 1 5

(49). The f luo rescence can be r e c o r d e d e i the r on pho tog raph ic fi lm, or by using a p o r t a b l e solid s t a t e f l uo rome te r (67) or wi th a light m e t e r . This t e chn ique has been appl ied to leaf discs of a l t i t ud ina l v a r i e t i e s of the wild t o m a t o L. hirsutum which w e r e exposed to 1 C for 2 days . It was possible to r ank the a l t i tud ina l forms visually and by the r a t i o of the peak to s t e a d y - s t a t e f l uo re scence in a s imi la r o rder to t h a t ob t a ined for t h e seedl ing survival shown in F igure 1. The t echn ique is rap id , having r e l a t i ve ly low labor r e q u i r e m e n t and could be a d a p t e d to handle l a rge n u m b e r s of s amples wi th a low equ ipmen t r e q u i r e m e n t . The t echn ique gave pos i t ive r e su l t s with t o m a t o , beans , egg p lan t , papaya , and caps i cum.

Chi l led t rop ica l p l a n t s show l a rge changes in t h e levels of c e r t a i n m e t a b o l i t e s . D i sp ropo r t i ona t e change in the ve loc i ty of the g lycoly t ic and r e s p i r a t o r y p a t h w a y s of me tabo l i sm (43) may be a t l eas t in p a r t respons ib le for such m e t a b o l i c i m b a l a n c e s . These changes i nd i ca t e ano the r possible basis for a s e l ec t ion p r o c e d u r e . S t a rch a c c u m u l a t e s in ch lo rop las t s of t he grass Digitaria decumbens moved from 30 C/30 C to 30 C /10 C (30). The d i f f e r ence in s t a r c h levels a f t e r two consecu t ive n igh t s a t 10°C was t en t i m e s t h a t a t 3 0 ° C . As y e t , this has no t been shown wi th o the r spec i e s . O the r m e t a b o l i t e s shown to change a t chil l ing t e m p e r a t u r e s in var ious p l a n t s a r e d e c r e a s e s in t o t a l sugars and ascorb ic ac id (50, 77), and inc reases in g l u t a m a t e , a s p a r t a t e and ch lorogenic ac id (22, 41); and a c c u m u l a t i o n of α - k e t o g l u t a r a t e , p y r u v a t e , e thano l , a ldehydes , and α - f a r n e s e n e (54, 68, 81). These changes a r e not un iversa l , for while ascorb ic acid d e c r e a s e d in chi l led p ineapple f rui t , b a n a n a and s w e e t p o t a t o (28, 40, 50) i t did no t in t o m a t o (14).

Tyros ine supplied to chi l led p ineapple fruit (73) in tens i f ied t he s y m p t o m s of endogenous brown spot , which is a chil l ing injury. The fo rma t ion of a s imi lar d isorder in l e t t u c e (russet spot t ing) was p r e c e d e d by an i nc r ea se in phenyla lan ine a m m o n i a lyase (PAL) w h e r e a s in the r e s i s t a n t v a r i e t i e s much less PAL was deve loped (32). Ano the r e n z y m e of the ch lorogenic ac id phenylpropanoid p a t h w a y , hydroxyc innanyl -t r a n s f e r a s e , i nc r ea se s in chi l led t o m a t o e s (64), T o m a t o t i ssue cu l tu re s visibly da rkened below 9 C p re sumab ly by oxida t ion of pheno l i c s .

This group of assays holds cons ide rab le p romise as a rap id assay for chil l ing t o l e r a n c e .

UI. GENETICS O F CHILLING RESISTANCE

Few r e p o r t s dea l wi th i n h e r i t a n c e of chil l ing r e s i s t a n c e . The r e su l t s of s o m e of the g e n e t i c s tud ie s a r e s u m m a r i z e d in Table 4 . For t he ge rmina t i on of c o t t o n and t o m a t o , m a t e r n a l e f f e c t s a r e i m p o r t a n t and for m a i z e , this has been a s s o c i a t e d wi th t he double g e n e t i c con t r ibu t ion from t h e f e m a l e s ide to t h e endosperm (63). This would no t be t h e c a s e for c o t t o n and t o m a t o , as the endosperm is a lmos t c o m p l e t e l y absorbed dur ing seed d e v e l o p m e n t . The m a t e r n a l e f fec t could be a t t r i b u t a b l e to t he m a t e r n a l l y de r ived non-nuc lea r g e n e s . With t he excep t ion of low t e m p e r a t u r e fruit s e t in t o m a t o e s , which was r e p o r t e d (34) to be

5 1 6 R. Ε. Paul le f al.

r e ce s s ive and s imply inher i t ed as was t o m a t o seed ge rmina t i on (8), chil l ing t o l e r a n c e appea r s to be an add i t ive , mu l t i gene r i c c h a r a c t e r . However , i t should be borne in mind t ha t d i f fe ren t g e n e t i c mechan i sms appa ren t l y con t ro l r e s i s t a n c e or t o l e r a n c e a t d i f fe ren t deve lopmen t s t a g e s (27).

IV. CONCLUSIONS

Many of t h e above assays c o r r e l a t e wi th t he final p e r f o r m a n c e of t h e p lant as a whole to chil l ing s t r e s s . A major problem wi th any assay is t h a t it may not i nd i ca t e the chill ing response a t a d i f fe ren t physiological or deve lopmen ta l s t a g e .

A number of assays show p romise for use in a p lan t b reed ing p rogram for chil l ing t o l e r a n c e . The final t e s t will be in the field, but l abo ra to ry m e a s u r e m e n t will be useful during the in i t ia l s e l ec t ion . Loss of f luorescence and changes in c o n c e n t r a t i o n s of c e r t a i n m e t a b o l i t e s and enzymes show promise as rapid , r ep roduc ib le and q u a n t i t a t i v e assays for chil l ing t o l e r a n c e .


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(1978). 46 . Maran i , Α., and Dag , J . Crop Sci. 2, 243-245 (1962). 47 . Melca rek , P . K., and Brown, G. A. Plant and Cell Physiol. 18,

1099-1107 (1977a). 48 . Melca rek , P . K., and Brown, G. A. Plant Physiol. 60, 822-825

(1977b). 49 . Miles, C. C , and Danie l , D . J . Plant Sci. Letters 1, 237-240 (1973). 50. Miller , Ε. V. Plant Physiol. 26, 66-75 (1951). 5 1 . Minchin, Α., and Simon, E. W. J. Exp. Bot. 24, 1231-1235 (1973).

5 1 8 R. Ε. Paul le f al

52. Mock, J . J . , and Bakri , A. A. Crop Sci. 16, 230-233 (1976). 53 . Mura t a , N. , and Fork , D. C. Plant Physiol. 56, 791-796 (1975). 54. Mura t a , T. Physiol. Plant. 22, 401-411 (1969). 55. Ng, T. J . , and T igche laa r , E. C . Jour. Amer. Soc. Hort. Sci. 98,

314-316 (1973). 56. Ormrod , D. P . , and Bunte r , W. A. Agron. J. 53, 133-134 (1961). 57. P a n t a s t i c o , Ε. B., Gr ie rson , W., and Soule, J . Amer. Soc. Hort. Sci.

Trop. Reg. Proc. 11, 82-91 (1967). 58. P a t t e r s o n , B. D. , Mura t a , T., and G r a h a m , D. Aust. J. Plant

Physiol. 3, 435-442 (1976). 59. P a t t e r s o n , B. D. , and G r a h a m , G. J. Exp. Bot. 28, 736-743 (1977). 60. P a t t e r s o n , B. D. , Paul l , R. E., and Smill ie , R . M. Aust. J. Plant

Physiol. 5, 609-617 (1978). 6 1 . Paul l , R . E., P a t t e r s o n , B. D. , and G r a h a m , D. Aust. J. Plant

Physiol. (In press) (1979). 62. Pesev , Ν. V. Theor. Appl. Genetics 40, 351-356 (1970). 6 3 . Pinnel l , E. L. Agron. J. 41, 562-568 (1949). 64. Rhodes , M. J . C. , and Wool tor ton , L. S. C. Phytochem. 16, 655-659

(1977). 65. Rick , C. M. In "Genes , Enzymes and Popula t ion" (A. M. Srb. ed.) ,

pp . 255-269, P lenum Pres s , New York (1973). 66. Robinson, W., and Kowalewski , E. Proc. XIX Inter. Hort. Congr. 18,

716 (1974). 67. Schre iber , U., G r o b e r m a n , L., and Vidaver, W. Rev . Sci. Instrum.

46, 538-542 (1975). 68 . Smagula , J . M., and Bramlage , W. J . HortScience 12, 200-203

(1977). 69. Smil l ie , R. M., and N o t t , R. Plant Physiol. 63, 796-801 (1979). 70. 70. Smith , O. F . J. Amer. Soc. Agron. 27, 467-479 (1935). 7 1 . S terg ios , B. G. and Howell , G. S. J. Amer. Soc. Hort. Sci. 98, 325-

330 (1973). 72. Steponkus , P . L., and L a m p h e a r , F . O. Plant Physiol. 42, 1423-1426

(1967). 73 . Sun, S-K. Plant Prot. Bull. (China) 13, 39-48 (1971). 74. Ta t sumi , Y., and Mura t a , T. J. Japan Soc. Hort. Sci. 47, 105-110

(1978). 7 5. Toole , V. K., Wes te r , R. E., and Toole , Ε. H. Proc. Amer. Soc.

Hort. Sci. 58, 153-159 (1951). 76. Tor iyama , K., and Fu t suha ra , Y. Jap. J. Breeding 10, 143-152

(1960). 77. Van Lelyveld, L. J . , and DeBruyn, J . A. Agrochemophysica 8, 65-68

(1976). 78. Ventura , Y. Diss. Abst. 20, 1985-1986 (1959). 79. Wheaton , Τ. Α., and Morris , L. L. Proc. Amer. Soc. Hort. Sci. 91,

529-533 (1967).

Chil l ing in jury A s s a y s for P l a n t B r e e d i n g 5 1 9

80. Widholm, J . M. Stain Technology 47, 189-194 (1972). 8 1 . Wills, R. Β. H., McBai ley, W., and Sco t t , K. Plant Physiol. 56, 550-

551 (1975). 82 . Wright , M., and Simon, E. W. J. Exp. Botany 24, 400-411 (1973).


SPECIAL TOPICS RELATED TO THE USE O F ARRHENIUS PLOTS

Much of t h e d a t a deve loped for s tudy of t e m p e r a t u r e r e l a t e d p h e n o m e n a in p l an t s , p a r t i c u l a r l y t h a t r e l a t e d to the " m e m b r a n e hypothes i s" has been p r e s e n t e d as Arrhen ius p l o t s . As discussed more fully in P A R T VI. EPILOGUE, it may no t be a p p r o p r i a t e to apply Arrhenius law to some of t h e e v e n t s occur r ing in response to t e m p e r a t u r e , bu t m o r e i m p o r t a n t l y the d rawing of l ines , and h e n c e conclus ions , mus t be app roached m o r e cau t ious ly than genera l ly ex is t s in t h e c u r r e n t l i t e r a t u r e .

The Seminar p r o m p t e d seve ra l to app roach this ques t ion of s t a t i s t i c a l t e s t s to d e t e r m i n e the "bes t - f i t " of s t r a i g h t l ines , s t r a i g h t l ine s e g m e n t s and curves to Arrhenius p lo t s and o the r d a t a . Severa l such ana lyses a r e p r e s e n t e d in this s ec t ion and they provide useful app roaches for cons ide ra t ion . T h e r e is some r edundancy and over lap in t h e p r e s e n t a t i o n s but r a t h e r than a t t e m p t i n g to syn thes i ze them in to one consensus , t hey a r e e a c h p r e s e n t e d i n t a c t to allow t h e r e a d e r to choose t he m e t h o d bes t su i ted to his or he r n e e d s . It was obvious from discussions in t he Seminar t h a t some s t a t i s t i c a l r igor should be appl ied to Ar rhen ius - type d a t a and the t e s t s desc r ibed h e r e provide t h a t oppor tun i ty .

5 2 1



BREAKS OR CURVES? A VISUAL AID TO THE INTERPRETATION OF DATA

Mary E. Willcox

CSIRO Division of M a t h e m a t i c s and S t a t i s t i c s Nor th Ryde , N.S.W. 2113, Aus t r a l i a

Brian D. Patterson

CSIRO Division of Food R e s e a r c h Nor th Ryde , N.S.W. 2113, Aus t r a l i a

The visual c o n t r a s t b e t w e e n smooth curves and i n t e r s e c t i n g s t r a igh t l ines in Arrhenius p lo t s can be emphas i zed by changing the angle b e t w e e n t h e χ and y axes . This has t h e e f fec t of compress ing t h e d a t a in a speci f ied d i r ec t ion .

F igure 1 shows g e n e r a t e d po in ts which lie along t h r e e i n t e r s e c t i n g s t r a igh t l ines (A) or on a smooth curve (B), a l though t h e d i f f e rence is no t obvious to t h e eye . F igure 2 shows t h e s a m e d a t a p l o t t e d wi th t he angle b e t w e e n t h e axes i nc reased to give a compress ion of t en t i m e s along t h e d iagonal , and then en la rged to h a v e t h e s a m e dimensions as t h e original d a t a . This t r e a t m e n t makes it obvious which se t of po in t s l ie on a smooth cu rve , and which se t l ie a long t h r e e i n t e r s e c t i n g s t r a igh t l ines . Table I shows a worked example of t h e m e t h o d .

In t h e p r eced ing pape r (1), F igu re 1 shows an Arrhenius plot of t h e t e m p e r a t u r e d e p e n d e n c e of spin label mot ion in t o m a t o l ipids. F igure 2 of t h e s a m e pape r shows the d a t a a f t e r compress ion . The d a t a a r e well r e p r e s e n t e d as a smooth curve tending to l i nea r i ty a t e i t h e r end.

N o t e t h a t t h e l inea r i ty of s t r a igh t l ines is m a i n t a i n e d during compress ion , while t h e s c a t t e r of d a t a po in t s i nc reases p r o p o r t i o n a t e l y as t he c u r v a t u r e is i nc reased .

Copyright · 1979 by Academic Press. Inc. 5 2 3 All rights of reproduction in any form reserved

ISBN σ ΐ 2 4β056Ο5

5 2 4 Μ. Ε. Willcox a n d Β. D. P a t t e r s o n

F i g . 1

A

X *

F i g - 2 A

X

y '

***

Β Β X

X Λ Χ

Κ χ

χ

y ' / /

χ* χ "

„.··

FIGURE 1. Points generated to lie on (A), three straight lines; (B), a smooth curve.

FIGURE 2. The data of A and Β in Figure 1 compressed and enlarged ten times. It is now obvious that A consists of three straight lines and that Β is a curve. When the data are transformed to lie between zero and 1, the formulae used for calculating the new coordinates are:—

x' = n[x-^(x + y)]

y = n\y-%±(x + y)]

where η is the degree of compression.

A V i s u a l Aid to t h e i n t e r p r e t a t i o n of D a t a 5 2 5

TABLE I. Worked Example of a Compression of Five Times (n - 5)

Original coordinates Coordinates transformed New coordinates for to lie between 0 and 1 η = 5

x-axis y-axis X y X' y

10, .0000 20.0000 0. .0000 0. .0000 0. .0000 0. .0000 10, .6000 20.5000 0, .1000 0. .0833 0. .1333 0. .0500 11,1000 21.0000 0, .1833 0. .1667 0. .2167 0. .1333 11, .6000 21.5000 0. .2667 0. .2500 0. .3000 0. ,2167 12, .2000 22.0000 0. .3667 0. .3333 0. .4333 0. .2667 12, .7000 22.4000 0, .4500 0. .4000 0. .5500 0. ,3000 13, .2000 22.8000 0. .5333 0. .4667 0. .6667 0. .3333 13, .6000 23.3000 0, .6000 0. ,5500 0. .7000 0. .4500 14, .0000 23.7000 0. .6667 0. .6167 0. .7667 0. .5167 14, .3000 24.1000 0, .7167 0, ,6833 0. .7833 0. ,6167 14, .6000 24,5000 0. .7667 0. ,7500 0. ,8000 0. ,7167 14, .9000 24.9000 0. .8167 0. .8167 0. ,8167 0. ,8167 15. ,4000 25.4000 0. .9000 0. .9000 0. ,9000 0. ,9000 16. ,0000 26.0000 1. .0000 1, .0000 1. .0000 1. .0000

The original coordinates were transformed to lie between 0 and 1, giving the values of χ and y to substitute in the equations of Figure 2. The new coordiantes x' and y

1 were then calculated for η = 5.

R E F E R E N C E S

1. P a t t e r s o n , B. D. , Paul l , R. , and G r a h a m , D. This vo lume . (1979).



STATISTICAL TESTS TO DECIDE BETWEEN STRAIGHT LINE SEGMENTS AND CURVES AS SUITABLE FITS

TO ARRHENIUS PLOTS OR OTHER DATA

Joe Wolfe

D e p a r t m e n t of Appl ied M a t h e m a t i c s R e s e a r c h School of Phys ica l Sc iences

Aus t ra l i an Na t iona l Univers i ty C a n b e r r a , A C T 2600, Aus t r a l i a

David Bagnall

Division of P l an t Indus t ry , CSIRO, C a n b e r r a , A C T 2601 , Aus t r a l i a

I. INTRODUCTION

A popular and powerful m e t h o d of e x t r a c t i n g in fo rma t ion from a se t of pa i r s of m e a s u r e m e n t s is to p lo t one funct ion of t h e va r i ab les aga ins t a n o t h e r , choosing two funct ions which, acco rd ing to t h e theo ry appl ied, a r e l inear ly r e l a t e d . The i n t e r c e p t and slope of a s t r a igh t l ine f i t t e d to this p lot a r e r e l a t e d to p a r a m e t e r s of t h e sys tem inves t i ga t ed . Thus, for e x a m p l e , a p lot of t h e l eng th of a pendulum aga ins t t h e squa re of i t s pe r iod should yield a s t r a igh t l ine wi th slope g / 4 7 T

Z, w h e r e g is g r a v i t

a t iona l a c c e l e r a t i o n . In c h e m i s t r y , Ar rhen ius (1 , 2) showed t h a t , in c e r t a i n ca ses , a p lo t of In ( reac t ion r a t e ) aga ins t t h e r e c i p r o c a l of t h e abso lu te t e m p e r a t u r e should yield a s t r a igh t l ine wi th slope -E / k w h e r e Ε is t h e a c t i v a t i o n ene rgy (per molecule) of t h e a c t i v a t e d complex and k g is Bol tzmann ' s c o n s t a n t .

In some cases t h e law which p r e d i c t s t h e l inear r e l a t i o n may hold only over a c e r t a i n r a n g e of va lues , and thus only p a r t of t h e plot r e s e m b l e s a s t r a igh t l ine , or a law may hold, but wi th d i f fe ren t va lues of t h e p a r a m e t e r s , over d i f fe ren t r a n g e s . In t h e l a t t e r c a se , two or more s t r a igh t l ines m a y be f i t t e d (by min imiz ing t h e sum of t h e squares of d i s p l a c e m e n t s from t h e r e l e v a n t line) over var ious reg ions on t h e p lo t . The coo rd ina t e s of a point of i n t e r s e c t i o n acqu i r e cons iderab le s ign i f icance s ince i t is a t this point p r e sumab ly t h a t t h e sys tem changes from one ope ra t i ng r e g i m e to a n o t h e r . 1

σ σ Copyright · 1979 by Academic Press. Inc. 5 2 7 All rights of reproduction in any form reserved

ISBN 0 1 2 4 6 0 5 6 0 5

5 2 8 J. Wolfe a n d D. B a g n a l l

When i n t e r s e c t i n g s t r a igh t l ines a r e f i t t ed , and when the angles b e t w e e n those l ines a r e smal l , it may be diff icul t to be sure t ha t the poin ts rea l ly a r e well r e p r e s e n t e d by s t r a igh t l ines r a t h e r than a cu rve , pa r t i cu l a r l y if the number of poin ts is smal l or the possible e r ro r in m e a s u r e m e n t is l a r g e . Con ten t ious examples a r e t h e f i t t ing of s t r a igh t l ines [usually t h r e e but s o m e t i m e s as many as five,] to Arrhen ius p lo t s of enzyme c a t a l y z e d r e a c t i o n r a t e s or o the r physiological r a t e s . C r o z i e r (7) proposed a theory to explain t h e e x i s t e n c e of such l ines, but this t heo ry has been c r i t i c i zed as s impl is t ic (4, 8, 9). Severa l au tho r s (3, 9, 16, 20, 21) have proposed t h a t the log of t h e r a t e of biological r e a c t i o n s should have a curv i l inear dependence on inverse t e m p e r a t u r e , but i n t e r s e c t i n g s t r a i g h t l ines appear on Arrhenius p lo t s of biological d a t a in many r e c e n t p a p e r s . Of ten it is no t c l ea r t h a t s t r a igh t l ines a r e an app rop r i a t e f i t , but we know of only one case whe re the app rop r i a t enes s was t e s t e d s t a t i s t i c a l l y , and in t ha t case curves w e r e found to fit t he d a t a equal ly well (11).

A t t e m p t s to fit a se r ies of s t r a igh t l ines to Arrhenius p lo t s of, for example , the r a t e of biological r e a c t i o n s a r e made only over a l imi ted t e m p e r a t u r e r a n g e . This r a t e is ze ro , and h e n c e i t s log is inf ini te ly n e g a t i v e , ou ts ide a f in i te r ange of t e m p e r a t u r e (usually about -5 C to 45 C) and Arrhenius ' law does not allow a ze ro r e a c t i o n r a t e a t a f ini te t e m p e r a t u r e . F u r t h e r , mos t au tho r s [some excep t ions being (6, 12)] decl ine to fit s t r a igh t l ines to t ha t region of t h e plot wi th pos i t ive s lope, and thus avoid the awkward concep t of n e g a t i v e a c t i v a t i o n ene rg i e s . The resu l t is t h a t only t h a t region of t he plot in which the slope changes slowly ^ cons idered and this , combined wi th the spread usual ly p r e s e n t in biological d a t a , explains why t h e r e is some ambigu i ty about w h e t h e r or no t s t r a igh t l ines or curves b e t t e r r e p r e s e n t t he d a t a .

To l imi t t h e r ange of appl icabi l i ty of t he fit to a c e r t a i n r ange impl ic i t ly employs two p a r a m e t e r s - t h e high and low t e m p e r a t u r e l imi t s of t ha t r a n g e . F i t t i n g Ν s t r a igh t l ines involves a fu r ther 2N independen t ly adjus table p a r a m e t e r s ( the i n t e r c e p t s and slopes of those l ines) . Of ten t he number of poin ts is not much l a rge r than 2N (e.g. Ref 5) and so t he fit is nea r ly p e r f e c t . Even when the number of d a t a poin ts is subs tan t i a l ly g r e a t e r than 2N, t he res idual sum of squares is of ten no l a rger than one would expec t from jus t t he inhe ren t sp read of t he d a t a . However , t h e s ame is t r u e of many a r b i t r a r y curves involving as many or fewer adjus table p a r a m e t e r s (e.g. A + <^/x+ £ e

X cosh(E + Fx) , or

equally a rb i t r a r i ly , A + Bx + Cx + Dx + Ex + Fx ). Bagnall and Wolfe (3) for i n s t ance , found t ha t 5th order and 3rd order polynomials gave smal le r res idues than 3 s t r a igh t l ines when f i t t ed to thei r Arrhenius p lo t s , but in all cases these res idues w e r e too smal l to r e j e c t the curve on t ha t basis a lone .

The re a r e , however , o the r c r i t e r i a which one can apply to dis t inguish b e t w e e n Ν s t r a igh t l ines and some smooth curve (not necessa r i ly a polynomial , of course) .

T h e U s e of A r r h e n i u s P lo ts 5 2 9

TABLE I. Table of values of b for sets of ρ points such that, when q is determined as described in the text, \ q - p/2| > - | allows the rejection of the hypothesis that the points are well fitted by straight lines at the indicated confidence level

* * No. of points (p) 10 20 30 40 50 60

Confidence level = 95% b = * 2*

* 4 5 6 7 Confidence level = 98% b = 3 4 5 6 7 8

* This test is not likely to be very useful for sets of 20 or fewer

data points. However, any attempt to determine six or eight parameters from a set of 20 or fewer points is likewise ambitious.

Table I gives va lues of b which a r e solut ions to this equa t ion for var ious va lues of ρ and a t conf idence levels of 9 5 % and 9 8 % . Thus one can t e s t t h e s t r a igh t l ine hypothes is a t t he se conf idence levels by eva lua t ion of ρ and q, then compar ing | q-p/21 wi th t h e va lue of b given in t h e a p p r o p r i a t e column of Table I. If | q -p /2 | > b one can r e j e c t t he hypo thes i s .

This t e s t can be m a d e m o r e sens i t ive by weight ing each point acco rd ing to i t s v e r t i c a l d i sp l acemen t from the l ines and i t s ho r i zon ta l d i s t a n c e from a boundary b e t w e e n reg ions I and Π. It can also be improved by choosing the boundar ies of reg ions I and II on t he basis of the d a t a , r a t h e r than t h e a r b i t r a r y division used h e r e . However , t he t e s t in the s imple form works qu i t e well and the possible i m p r o v e m e n t s would m a k e i t s i m p l e m e n t a t i o n much m o r e t ed ious .

Applying this t e s t to Arrhen ius p lo t s in t he l i t e r a t u r e one can in mos t cases r e j e c t t h e hypothes i s t h a t s t r a igh t l ines a r e a su i tab le f i t . However , fa i lure to r e j e c t t h e s t r a igh t l ine fit does no t imply t h a t it can be conf ident ly a c c e p t e d . One could employ a s imi lar t e s t to a t t e m p t to r e j e c t a spec i f ic cu rve fit a lso , but r e j e c t i n g only one cu rve does no t necessa r i ly imply t ha t a s t r a igh t l ine fit mus t be a c c e p t e d .


FIGURE 1. Two sets of data are shown fitted with straight line segments, and divided into regions for the application of test (A) (see text). At the 95% and 98% confidence levels, the straight line fit can be rejected for Β but not for A.

Π. TESTS

A. Is There a Pattern in the Displacement of Points from the Fitted Straight Lines?

Figure 1 shows 3 s t r a igh t l ines appropr i a t e ly and inappropr ia te ly f i t t ed to two se t s of po in t s . In t he case where t he poin ts a r e b e t t e r r e p r e s e n t e d by a curve , t h e poin ts t end to be below t h e l ines n e a r t h e ends of t he s e g m e n t s , and above the lines in t he middle . This d i sc r imina t ion p r o c e d u r e can be roughly quant i f ied as follows: Divide each s e g m e n t in to 3 sec t ions , t he middle tw ice t he length of t h e ends; t h e s e g m e n t middle sec t ions a r e ca l led regions (I) and the o the r reg ions (II). Suppose t ha t q is t h e number of po in t s which lie below the l ines in reg ions Π plus t he number of poin ts which lie above the l ines in regions I, and ρ is t h e t o t a l number of po in t s . If s t r a igh t l ines a r e an a p p r o p r i a t e f i t , then t h e most l ikely va lue for q is p / 2 , and t h e p robabi l i ty of any p a r t i c u l a r q is

PC (Vz)^.

Now t h e r e is a 9 5 % change t ha t q will lie in a rangef^ - b)<q<(^ +^b) e r e

p / 2 + b

Σ p c q ( y 2)p = 9 5 %

q = p /2 -b

T h e U s e of A r r h e n i u s P lo ts 5 3 1

B. Are the "Breaks" or Intersection Points Dependent on the Range over which the Fit is Considered Applicable?

If s t r a igh t l ines a r e a convincing f i t , t hen t h e i n t e r s e c t i o n s will occur a t t he s ame p l a c e if one d e l e t e s po in ts a t e i t he r end of t h e r ange and pe r fo rms a l eas t squares s e g m e n t fit on t h e r e m a i n d e r (see Fig . Z). If t h e i n t e r s e c t i o n s occur a t d i f fe ren t p l ace s , then the in fo rmat ion ob ta ined from such l ines is as much dependen t on t he r ange of t e m p e r a t u r e t he e x p e r i m e n t e r sought to i n v e s t i g a t e as it is on t he sys tem s tudied . Bagnall and Wolfe (3) found wi th the i r d a t a t h a t a s e g m e n t f i t t ing r o u t i n e p roduced s e g m e n t s of a p p r o x i m a t e l y equal length for any subset of t h e d a t a , t h a t is , t he "break poin ts" w e r e d e t e r m i n e d by t h e r a n g e chosen for t h e e x p e r i m e n t .

C. Is N, the Number of Straight Lines, Obvious and Unique?

If Ν s t r a igh t l ines is an a p p r o p r i a t e f i t , then (N + 1) s t r a igh t l ines f i t t ed will inc lude t he Ν l ines found previously , wi th one very shor t e x t r a s e g m e n t (see F ig . 3). F u r t h e r , t h e res idues from t h e Ν l ine fit and t h e (N + 1) l ine fit should be very nea r ly equal , w h e r e a s an (Ν - 1) l ine fit should give a very much l a rge r r e s idue . Bagnall and Wolfe (3) found for the i r d a t a t h a t t h e r e s idue d e c r e a s e d smooth ly wi th inc reas ing number s of l ines .

FIGURE 2. The data are the same as in Figure 1, but four points at either end (O) are not included in the fitting procedure (see Β in text).


FIGURE 3. The data are the same as in Figure 1, but four straight lines have been fitted (see C in text).

D. Is it Clear where the Straight Line Fit Ceases to Apply?

Since t he plot goes inf ini te ly n e g a t i v e a t f ini te values of 1/T, and consider ing t h e a r g u m e n t in (B), this is r a t h e r i m p o r t a n t .

E. When One Shows the Points Alone to the Person Employed to Make the Tea, How Does He or She Describe it?

It is e l s ewhere a rgued t ha t t h e r e is no t h e o r e t i c a l r eason to expec t Arrhenius p lo ts to be bes t f i t t ed by s t r a igh t l ines . However , in the p a r t i c u l a r case of t h e analysis of chil l ing suscep t ib i l i ty in p l an t s , i t is s o m e t i m e s a rgued tha t while t h e r e may be no t h e o r e t i c a l r eason for expec t ing s t r a igh t l ines , and while curves may fit t he d a t a a t l eas t as well as s t r a igh t l ines (11), the (Ν - 1) i n t e r s ec t i on po in ts or b r eaks give useful q u a n t i t a t i v e r e su l t s , i .e . one of t h e b reaks may r e p r e s e n t a "c r i t i ca l t e m p e r a t u r e " below which the p lan t will no t survive (10, 13, 14). This is no t so: F i r s t , t h e i n t e r s e c t i o n poin ts can be var ied by t h e design of t he e x p e r i m e n t . The inclusion of e x t r a poin ts a t a lower t e m p e r a t u r e will cause t h e lowest t e m p e r a t u r e "break" to occur a t a lower t e m p e r a t u r e . Second, t e m p e r a t u r e is no t the only "c r i t i ca l " env i ronmen ta l va r i ab l e . One canno t a sc r ibe to one of t h e "break" po in t s t h e low t e m p e r a t u r e l imi t to survival , s ince the l a t t e r is dependen t on i r r a d i a n c e , humid i ty and C O ? c o n c e n t r a t i o n (15, 18, 19).

T h e U s e of A r r h e n i u s Plo ts 5 3 3

m. CONCLUSION

Summing up, t h e r e is of ten suff ic ient s t a t i s t i c a l ev idence to dec ide t h a t Arrhenius p lo t s of biological r e a c t i o n r a t e s a r e highly unlikely to be r e p r e s e n t e d by s t r a igh t l ines . Breaks c a l c u l a t e d from such p lo t s a r e m a t h e m a t i c a l or b iochemica l a r t i f a c t s (17) and can be va r i ed by e x p e r i m e n t a l design. These "breaks" should no t be used to p red ic t a gene ra l c r i t i c a l survival t e m p e r a t u r e , s ince c r i t i c a l survival t e m p e r a t u r e s will be d i f fe ren t for d i f fe ren t condi t ions .


1. Ar rhen ius , S. Z. Physik. Chem. 4, 226-248 (1889). 2. Ar rhen ius , S. " Q u a n t i t a t i v e Laws in Biological Chemis t ry . " Bell,

London (1915). 3 . Bagnal l , D. J . and Wolfe, J . J. Exp. Bot. 29, 1231-1242 (1978). 4 . Bur ton, A. C. J. Cellular and Comp. Physiol. 9, 1-14 (1936). 5. Champigny , M. L. and Moyse, A. Biochim. Physiol. Pflanzen. 168,

575-583 (1975). 6. C r i t c h e l y , C , Smill ie , R. M. and P a t t e r s o n , B. D. Aust. J. Plant.

Physiol. 5, 443-448 (1978). 7. C roz i e r , W. J . J. Gen. Physiol. 7, 189-216 (1924). 8. Hei lbrunn, L. V. "An Out l ine of Gene ra l Physiology." Saunders ,

Phi lade lphia (1937). 9. Johnson, F . H., Eyhring, H. and Pol issar , M. J . "The Kine t i c Basis of

Molecular Biology." Wiley, New York (1954). 10. Lyons, J . M. A. Rev. PL Physiol. 24, 445-466 (1973). 11 . Nolan, W. G. and Smill ie , R. M. Biochim. Biophys. Acta 440, 4 6 1 -

475 (1976). 12. Nolan, W. G. and Smill ie, R. M. Plant Physiol. 59, 1141-1145(1977). 13. Raison , J . K. and Chapman , E. A. Aust. J. PI. Physiol. 3, 291-299

(1976). 14. Raison , J . K. and Lyons, J . M. PL Physiol. 45, 382-385 (1970). 15. Rowley , J . A. and Taylor , A. O. New Phytol. 71, 477-481 (1972). 16. Sharpe , P . J . H. and De Michele , D. W. J. Theor. Biol. 64, 649-670

(1977). 17. Silvius, J . B., R e a d , B. D. , and McElhaney , R. N. Science 199, 902-

904 (1978). 18. Taylor , A. O. and Rowley , J . A. Plant Physiol. 47, 713-718 (1971). 19. Wilson, J . M. New Phytol. 76, 257-270 (1976). 20. Wolfe, J . Plant, Cell and Environment 1, 241-247 (1978). 2 1 . Wolfe, J . This vo lume (1979).



MAXIMUM LIKELIHOOD ESTIMATION OF BREAKPOINTS AND THE COMPARISON OF THE GOODNESS O F FIT

WITH THAT OF CONVENTIONAL CURVES

J. F. Potter

C o m p u t e r Uni t , Welsh P l an t Breeding S ta t ion Plas Gogerddan , Nea r Abe rys twy th

Dyfed, Uni t ed Kingdom

G. J. S. ROSS

Sta t i s t i c s D e p a r t m e n t R o t h a m s t e d E x p e r i m e n t a l S ta t ion

Harpenden , H e r t s . , Un i t ed Kingdom

I. INTRODUCTION

The e s t i m a t i o n by the m e t h o d of max imum likelihood of b reakpo in t s in mu l t i -phase s t r a igh t l ine funct ions is d iscussed. On one e x t r e m e it is a rgued t h a t t he not ion of b r eakpo in t s is t enab l e and provides a b e t t e r fit to d a t a which might h a v e been h i t h e r t o desc r ibed by a cont inuous c u r v e . On t h e o the r e x t r e m e it is shown t h a t t h e r e is o f ten no r igorous s t a t i s t i c a l r e a son for suspec t ing mul t i -phase r e sponses . A gene ra l m a t h e m a t i c a l model for t he mu l t i -phase response is proposed , and t h e m e t h o d of e s t i m a t i n g i t s p a r a m e t e r s is desc r ibed by using t h e Maximum Likel ihood P rog ram (MLP).

In developing t h e theory of l eas t squa res , Gauss used the m e t h o d of max imum l ikel ihood. It is a very old and useful tool but lay d o r m a n t unt i l F isher (5) r e - i n t r o d u c e d i t .

If we t a k e minus t h e log of t he l ikel ihood dens i ty funct ion for a r e g i m e having norma l ly d i s t r i bu t ed e r ro r s , it can be shown t h a t min imiz ing the res idua l sum of squares is suff ic ient to m a x i m i z e t he l ikel ihood (Z). However , t he m e t h o d may p roduce unsa t i s f ac to ry r e s u l t s for smal l n u m b e r s of obse rva t ions (1Z). With t h a t r e s e r v a t i o n in mind, we find t h a t even wi th smal l n u m b e r s of d a t a (less than t en , say), if t h e e r ro r s a r e smal l and t h e change of g rad ien t a t a b reakpo in t l a rge and wel l -def ined , then the m e t h o d works wel l .

Copyright « 1979 by Academic Press. Inc. 5 3 5 All rights of reproduction in any form reserved

ISBN012 4605605

5 3 6 J. F. Po t te r a n d G. J. S. R o s s

A. The General Model

The m a t h e m a t i c a l model for a mul t iphase response having u phases and u-1 b reakpo in t s is given by:

Yi = *o + βι χί + 6 iW " Pi ) + · · · Vi W " p u - i } · •· · ( 1 )

where β^ is t he i n t e r c e p t on t he Y axis , β^ is the g rad ien t of t he l e f t most l ine and fur ther $s a r e g rad ien t c o r r e c t i o n s to t h e l e f t - ad jacen t l ines . Thus β^ is t h ^ g rad i en t c o r r e c t i o n of phase (u - 1) to p roduce phase u; p ^ is the k b reakpo in t . δ ^ is 1 if X > ρ , e lse 0. Hence , t he d iscont inuous response can be r e p r e s e n t e d by w fiat appea r s to be a cont inuous funct ion, ρ is a p a r a m e t e r i n t e rna l to t he model and is thus t r e a t e d as non- l inear , e problem is t h a t t he funct ion 6 qual i f ies both X and p ^ and is dependen t on p ^ .

II. METHOD

An op t imiza t i on a lgor i thm is r equ i r ed to min imize t h e n e g a t i v e log l ikel ihood funct ion or in this c a se t h e res idual sum of squares (RSS). On finding max imum likelihood e s t i m a t e s (MLEs) for t he b reakpo in t s , t w o -dimensional ' s l ices ' whose coord ina te s a r e ρ p a r a m e t e r s t aken in pa i r s , can be p l o t t e d through the u-d imens ional p a r a m e t e r s p a c e . Thus a compos i t e p i c t u r e of t he l ikelihood in p a r a m e t e r space can be buil t up . Examina t ion of this p i c t u r e might show t h a t an MLE cor responds to a false min imum. It may sit on a saddle point (an a r e a of the space where a min imum in one d i rec t ion is a max imum in a d i rec t ion a t r ight angles) . By su i t ab le r e - a d j u s t m e n t of the ini t ia l e s t i m a t e s the i t e r a t i v e p rocess is se t in mot ion again and the MLEs for t he b reakpo in t s should even tua l ly be found.

A. Two Straight Lines

Po^r.?bly t he c o m m o n e s t ca se is t he e s t i m a t i o n of a single a s sumed breakpoin t b e t w e e n two s t r a igh t l ines . Equat ion (1) b e c o m e s s implif ied to :

Υ. = β o + g j X . + δ β 2 (Χ. - ρ) . . .(2)

The a t t r i b u t e s t h a t a r e cons idered i m p o r t a n t in assessing the va l id i ty of a b reakpoin t a r e as follows.

M a x i m u m L ike l i hood E s t i m a t i o n of B r e a k p o i n t s 5 3 7

a) The mul t ip le co r r e l a t i on coef f ic ien t (R ). This is

Tota l sum of squares - RSS - „ „ ^ ^ . Ί ^

x 100%,

Tota l sum of squares o the rwi se known as t he p e r c e n t a g e va r i a t i on a c c o u n t e d for by t he f i t . This me re ly i nd i ca t e s w h e t h e r one model f i ts t he d a t a b e t t e r than ano the r .

b) The v a r i a n c e r a t i o of

Lack of fit m e a n squa re P u r e e r ro r m e a n squa re (3).

This is only possible whe re t h e r e is r e p l i c a t i o n . However , e a c h X va lue need not have t h e s a m e n u m b e r of obse rva t i ons . The within-X sum of squares gives a m e a s u r e of e r ro r unbiased by the fit of any mode l . The RSS cons is t s of this sum of squa res , added to a sum of squares r e p r e s e n t i n g the lack of fit of the mode l . Given the pu re e r ro r and the RSS, t he lack of fit sum of squares and t h e cor responding deg ree s of f reedom can be ob ta ined by s u b t r a c t i o n . The F r a t i o ,

Lack of fit m e a n squa re Pu re e r ro r m e a n squa re

can then be c a l c u l a t e d .

The problem with models t h a t a r e non- l inea r in t he p a r a m e t e r s is t ha t the F - t e s t is not s t r i c t l y app l i cab le . Bea le (1) worked out a t e chn ique for assess ing w h e t h e r t he non- l inea r i t y is ser ious or n o t . If Beale 's non- l inea r i t y c r i t e r ion is less than 0 .01 , l inear i n f e r ences (e.g. t he F - t e s t ) a r e not ser iously wrong. Whethe r this is so or no t , if t he v a r i a n c e r a t i o is less than 1 ( r emember we a r e looking for low values) , then it is safe to a s sume t h a t t he fit is a good one .

Methods a r e being cons idered whe reby t r u e conf idence l imi t s to t he posi t ion of the b reakpoin t can be a sc r ibed in a s imple way . H a r t l e y (7) has cons idered this problem for non- l inea r models in gene ra l . It may well be tha t d iscont inuous models p r e s e n t a fu r ther p r o b l e m . Almost c e r t a in ly any conf idence i n t e r v a l c a l c u l a t e d a round t h e b reakpo in t will be a s y m m e t r i c , owing to t h e r e being m o r e s c a t t e r on one s ide than on the o t h e r .

c) The number of d a t a po in t s suppor t ing each l ine

A ru le of t humb used is t h a t t h r e e po in t s in u n r e p l i c a t e d and two in r e p l i c a t e d d a t a give suff ic ient suppor t for a s t r a i g h t l ine s e g m e n t . The b reakpo in t should rea l ly belong to bo th l ines (or n e i t h e r ) . In t he m e t h o d desc r ibed h e r e i t belongs to the l e f t -hand l ine; but this is jus t an a c a d e m i c po in t . The word ' support ' used h e r e should no t be confused with t h e word as used by Edwards (4).

5 3 8 J. F. Po t te r a n d G. J. S. R o s s

d) The res idua l sum of squares

This is suff ic ient as a c r i t e r ion for improving t h e fit for a given se t of d a t a , but gives no idea of conf idence in t he e s t i m a t e .

e) The s ize of t he change in g rad ien t

If it is very smal l then the model would be d e e m e d o v e r d e t e r m i n e d .

f) The compar i son of a t t r i b u t e s a) , b) and d) above wi th those for f i ts of empi r i ca l l inear models (here r e s t r i c t e d to l inear and q u a d r a t i c polynomials) .

g) E s t i m a t e d va r i ances of p a r a m e t e r s

These a r e convent iona l ly c a l c u l a t e d from the inverse m a t r i x of second de r iva t i ve s of the log l ikel ihood funct ion. For the d iscont inuous models under cons ide ra t ion the i r use is no t r e c o m m e n d e d b e c a u s e t h e log l ikel ihood is d iscont inuous .

B. An Example of Satisfactory Breakpoint Estimation

In this example we a r e g ra te fu l to Dr . H. Thomas and Dr . J . L. S toddar t for supplying the d a t a on m e a s u r e m e n t s of the r a t e of change in c o n c e n t r a t i o n of c e r t a i n compounds in t he cell m e m b r a n e s of var ious crops inj-esponse^ to chi l l ing. The fami l iar Arrhenius t r a n s f o r m a t i o n , (1 /Temp Κ) χ 10 , was used for t he X-ax i s . The c rop used was win te r o a t s grown a t Z0 C pr ior to t he chil l ing p r o c e d u r e . The compound whose c o n c e n t r a t i o n change was m e a s u r e d was chlorophyl l , and t h e logjQ c o n c e n t r a t i o n was used for the p lo t . F rom this d a t a the following e s t i m a t e s w e r e m a d e : β , -16 .5128; 0 .5011; β ^ , -0 .2149; ρ, 34.8795.

TABLE Ι.

Straight line Quadratic Two straight lines

R 82.28 82.99 83.78 Lack of fit MS Pure error MS 0.9 0.9 0.8 RSS 0.4525 0.4344 0.4142

M a x i m u m L ike l i hood E s t i m a t i o n of B r e a k p o i n t s 5 3 9 LOGIO CHLOROPHYLL CONCENTRATION

0.6

0.9

1.2

34.5 35.0 35.5 (l/T K j x / O4

4 FIGURE 1. Log.Q chlorophyll concentration on (l/Temp.°K) χ 10 . The breakpoint is shown by V.

The a t t r i b u t e s for t he m e c h a n i s t i c and the empi r i ca l models a r e given in Table I.

F i f t e e n observed d a t a po in ts suppor ted e a c h l ine in t h e two s t r a igh t l ines model giving good suppor t to e a c h l ine . The change of g rad ien t a t t h e b reakpoin t being -0 .2149 gives good suppor t to t h e not ion of a b r eakpo in t . As r e g a r d s all t h r e e a t t r i b u t e s in Table I, the two s t r a igh t l ines model comes out be s t . Now, b e c a u s e t h e r e is pr ior belief, based on the e x p e r i m e n t e r s ' e x p e r i e n c e , in t he possibi l i ty of a b reakpoin t in this reg ion , t he two s t r a igh t l ines model can be a c c e p t e d . If it m a d e no sc ien t i f i c sense , the e x p e r i m e n t e r might e i t he r r e - t h i n k the mode l , or if s tubborn he might co l lec t m o r e d a t a . F igu re 1 shows t h e f i t t ed two s t r a i g h t l ines model through the s c a t t e r of observed d a t a po in t s . The re a r e t h r e e obse rva t ions pe r X va lue , but some a r e o v e r w r i t t e n on the graph owing to p rox imi ty of po in t s . The r e a d e r will n o t e wha t appea r s to be an ou t l i e r . Surprisingly, i t s e e m s to have l i t t l e e f fec t on t he posi t ion of t he b reakpo in t (as was d i scovered when it was removed) but the (3^ t e r m is a f f e c t e d qu i t e ser iously by i t .

Responses in n a t u r e t end to follow a s t r a igh t l ine re la t ionsh ip over a t l eas t some of the i r r a n g e . The higher o rder polynomials a r e p rogress ive a t t e m p t s to join t h e do t s , bu t , if successful , t h e sc ien t i f i c mean ing will be nil and e x t r a p o l a t i o n out of t he ques t ion , a l though empi r i ca l models can be useful if handled wi th r e s p e c t . However , a m e c h a n i s t i c model if i t f i ts r ea l ly well , m e a n s some th ing and can t h e r e f o r e be used, e.g. for e x t r a p o l a t i o n (2).

5 4 θ J. F. Po t t e r a n d G. J. S. R o s s

FIGURE 2. -Log likelihood plot (conditional) for values of ρ between 34.1 and 36.4.

C. The Likelihood Plot

Figure 2 shows the plot of minus t he log l ikel ihood over t he speci f ied r a n g e of va lues for p . For each va lue of ρ, β^, β^ and β~ a r e e s t i m a t e d by mul t ip le regress ion and the RSS is c a l c u l a t e d and p l o t t e d . This plot is i n f o r m a t i v e . F i r s t ly , i t can be seen if t h e MLE for ρ has been ach ieved , and in t h e example c i t e d it c l ea r ly has . The l ikelihood plot gives conf idence in t he MLE owing to i t s m a r k e d pa rabo l i c shape . In i l l -def ined example s , t he l ikel ihood p lo t can t a k e on qu i t e a b i z a r r e shape and may have severa l min ima ind ica t ing seve ra l possible so lu t ions . By examining a smal l por t ion of the l ikel ihood p lo t , and sca l ing it up , i t may be possible to d iscover t he MLE.

The work of Raison (9) and Fork et al. (6) a r e examples of a r a t h e r sub jec t ive approach to the f i t t ing of b r eakpo in t s . The c r i t i c i sm of Wolfe and Bagnall (13), t h a t if a s t r a igh t l ine s e g m e n t is no t i tself a l eas t squares fit then the b reakpo in t is spur ious , is p a r t i c u l a r l y ap t in the work of Fork et al. (6). A quick g lance shows t h a t in many cases t h e r e is m o r e s c a t t e r on one side of a s egmen t than on the o t h e r . In one graph, a s ingle point is a l lowed to imply a d r a m a t i c b reak . Even if th is is t he mean of a se t of r e p l i c a t e d obse rva t ions , it is no t enough suppor t for the l ine . In the work of both Raison (9) and Fork et al. (6), t h e r e a r e fair ly convincing c o m p l e t e b reakpo in t s w h e r e the l ines 'miss ' e a c h o t h e r . This kind of d i scont inu i ty canno t be desc r ibed in a single mode l . The two l ines a r e d i s t inc t s t r a igh t l ine models . But sti l l t h e l ikel ihood plot will show any t e n d e n c y for this to happen . F igure 3 shows such a p lo t . Where t he two p a r a b o l a e 'crash ' cor responds to where the two l ines 'miss ' . This sor t of thing is only cons idered convincing if t h e ' c rash ' occu r s a t t he s a m e low l ikel ihood as t h e two end a r ea s of t h e p lo t . Tha t is , it is found to be

M a x i m u m L ike l i hood E s t i m a t i o n of B r e a k p o i n t s 5 4 1

FIGURE 3. Plot of -log likelihood for a part of ρ parameter space, showing 'crashing' parabolae. Data obtained from spring oats grown at 5 C without Arrhenius transformation. Lines 'miss' at 13.56°C.

exceed ing ly unlikely t h a t t h e r e is a l ine a t th is point owing to t h e d i scovery t ha t i t is exceed ing ly unl ikely t h a t t h e r e is a conven t iona l b r eakpo in t .

D. The Maximum Likelihood Program

All t h e t echn iques used in this pape r w e r e p e r f o r m e d using MLP, a l a rge comprehens ive non- l inea r mode l - f i t t i ng p rog ram by G. J . S. Ross et al. (11). As well as t h e s t a n d a r d (l inear and non- l inear) cu rves and f requenc ies , i t enables one by m e a n s of a s imple i n t e r p r e t a t i v e l anguage to fit u se r -de f ined models such as t h e two s t r a igh t l ines mode l . The model is p r e s e n t e d to MLP by der iv ing va lues X - ρ above ρ and ze ros be low. This b e c o m e s t h e second X v a r i a t e . This and t h e or iginal X v a r i a t e a r e then f i t t e d by mul t ip le r egress ion on op t imiz ing t h e va lue of p . O p t i m i z a t i o n is a l a rge subjec t and is dea l t wi th a t l eng th in t he Numer i ca l A lgor i thms Group (NAG) Manual (8). Also, Ross (10) gives a full a ccoun t of funct ion min imiza t ion as a background to MLP.

m. CONCLUSION

It is c l ea r t h a t b reakpo in t s do occur in n a t u r e , but t h a t the i r e s t i m a t i o n by e x p e r i m e n t e r s has been s o m e w h a t cava l i e r . The model

5 4 2 J. F. Po t t e r a n d G. J. S. R o s s

given h e r e using t h e MLP program is cons idered to be a r igorous t r e a t m e n t of the sub jec t . It is hoped to p roduce soon a smal le r d e d i c a t e d p rogram which will deal specif ica l ly wi th this model f i t t ing t e chn ique . An increas ing number of c o m p u t e r c e n t r e s these days offer MLP as a p a c k a g e , and t h e manua l descr ibes fully how to e s t i m a t e b r eakpo in t s .

ACKNOWLEDGMENTS

The au thor s a r e g ra te fu l to Dr . H. Thomas and Dr . J . L. S toddar t (Biochemis t ry D e p a r t m e n t , Univers i ty Col lege of Wales, Welsh P lan t Breeding S ta t ion , Nr Abe rys twy th , Dyf ed, UK) and Dr . R. W. Bre idenbach (Plant G r o w t h Labora to ry , Univers i ty of Cal i forn ia , Davis , USA) for posing the p rob lem, co l lec t ing t he d a t a and c r e a t i n g t he demand . Thanks also due to Mrs. N . Wells for her cons t an t he lp .


1. Bea le , Ε. M. L. J . Royal Statist. Soc. (B) 22, 1, 41-88 (I960). 2. Box, G. E. P . and Hun te r , W. G. Technometrics 7, 23-42 (1965). 3 . D r a p e r , N . and Smith , H. "Applied Regress ion Analysis" , Wiley, New

York (1966). 4 . Edwards , A. W. F . "Likelihood", C a m b r i d g e Univers i ty P re s s (1972). 5. F isher , R. A. Mess. Math., 41, 155-160 (1912). 6. Fork , D. C , Mura t a , N . and Sato , N. In: Annual Report of the

Director of the Department of Plant Biology, S tanford, Cal i forn ia (Carnegie Ins t i tu t ion of Washington) , pp . 283-289 (1978).

7. H a r t l e y , H. O. Biometrika 51, 3 and 4, 347-353 (1964). 8. Numer i ca l Algor i thms Group . F O R T R A N Library Manual (1977). 9. Raison, J . K. In R a t e Con t ro l of Biological P roces se s . Society for

Experimental Biology Symposia 27, 485-512 (1973). 10. Ross , G. J . S. Appl. Statist. 19, 3, 205-221 (1970). 11 . Ross , G. J . S., Jones , R. D. , K e m p t o n , R. Α., Lauckner , F . B., Payne ,

R. W., Hawkins , Diana , Whi te , R. P . MLPrMaximum Likel ihood P r o g r a m . (Revised edi t ion) , R o t h a m s t e d Expe r imen ta l S ta t ion , Harpenden , H e r t s , UK (1979).

12. Wasan, Μ. T. " P a r a m e t r i c Es t ima t ion" , McGraw-Hi l l , New York (1970).

13. Wolfe, J . and Bagnal l , D. In The P roceed ings of t he US-Aus t ra l ian New Zealand C o o p e r a t i v e Sc ience P rog ram C o n f e r e n c e on Low T e m p e r a t u r e S t ress in Crop P l a n t s : The Role of t he M e m b r a n e (1979).


EPILOGUE

The semina r "Low T e m p e r a t u r e S t ress in Crop P l a n t s : The Role of the Membrane" was o rgan ized to examine c r i t i ca l ly the phys ica l , b iophysical and biological basis of p lan t r esponses to low t e m p e r a t u r e . Sc ien t i s t s wi th e x p e r t i s e in the field d iscussed and d e b a t e d the physica l and b iochemica l consequences of t e m p e r a t u r e e f f e c t s on ce l lu la r me tabo l i sm and how these e f f e c t s migh t be t r a n s l a t e d in to dysfunct ion and injury. Emphas is was p l a c e d on t h e molecu la r biology of cel l m e m b r a n e s and sc ru t iny of the hypothes i s t ha t the p r i m a r y molecu la r even t in t h e low t e m p e r a t u r e r e sponse is a t e m p e r a t u r e - i n d u c e d a l t e r a t i o n in the molecu la r o rder ing of m e m b r a n e l ipids. The final ques t ion posed for discussion was to consider g e n e t i c d ive rs i ty in c rop p l an t s for t o l e r a n c e to low t e m p e r a t u r e s and possible s t r a t e g i e s to exploi t t ha t p o t e n t i a l .

This shor t addi t ion is no t i n t ended to r e p r e s e n t a consensus of t he s emina r , but r a t h e r is provided by the ed i to r s as a means of focusing some a s p e c t s of t he genera l discussion t h a t o c c u r r e d . The r e a d e r is left to examine each paper and the accompany ing ques t ions and answers to a r r i ve a t his or her own conclus ions .

T h e r e w e r e a r ea s whe re some ambigu i ty and a p p a r e n t c o n t r a d i c t i o n in the l i t e r a t u r e was c lar i f ied and reso lved ; t h e r e w e r e also a r e a s whe re new ques t ions and d e b a t e was e l i c i t ed . The following br ings out some of those issues and sugges t s a r e a s of r e s e a r c h n e e d e d to a c c e l e r a t e the genera l field of s tudy .

A. Definitions Related to Chilling Injury

In t he discussion, it o f ten b e c a m e ev iden t t h a t some confusion and d i f f e rences of opinion evolved as a r e su l t of lack of common vocabu la ry or a t l eas t l ack of un i fo rmi ty in using t h e vocabu la ry . The following r e i t e r a t e s usage and def in i t ions which we would hope provide some c l a r i t y :

—chilling is t h e ac t of exposing p lan t m a t e r i a l to some low t e m p e r a t u r e (above 0 C);

—chilling injury is a d a m a g e to p l an t t i s sues , ce l l s , or o rgans which resu l t from exposure to some low t e m p e r a t u r e (above 0 C) for a per iod of t i m e suff ic ient to cause p e r m a n e n t or i r r eve r s ib l e d a m a g e ;

—dysfunction, a cco rd ing to Webs te r , is a "d isordered or impa i r ed funct ion of a sys tem or organs" ;

—primary response to temperature is t he p r i m a r y even t in t he cel l which senses t he low t e m p e r a t u r e and i n i t i a t e s some dysfunct ion (which dysfunct ion in t i m e will l ead to injury). The p r i m a r y response (or synonomously, t h e p r i m a r y sensor) may i n i t i a t e t h e dysfunct ion as an i m m e d i a t e r esponse but it may involve some t i m e per iod to cause t he injury.

Copyright * 1979 by Academic Press. Inc. 54-3 All rights of reproduction in any form reserved

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Β. Arrhenius Plots

Much a t t e n t i o n in the seminar was focused on the use or misuse of "Arrhenius" p lo ts in p r e s e n t i n g t e m p e r a t u r e dependen t d a t a . The issues a r e i m p o r t a n t s ince much of t h e concep tua l f r amework of t he " m e m b r a n e hypothes is" invoked as an exp lana t ion of the p r i m a r y response to low t e m p e r a t u r e in p l an t s revolves around the a p p e a r a n c e of "breaks" or d i scont inu i t i es in p lo ts of some r a t e funct ion p l o t t e d agains t t he r ec ip roca l of t he abso lu te t e m p e r a t u r e , which accord ing to Arrhenius theory should yield a s t r a igh t l ine .

D e b a t e evolved around severa l ques t ions . In d a t a p r e s e n t e d in t he l i t e r a t u r e as Arrhenius p lo t s , a r e they bes t r e p r e s e n t e d by two (or more) s t r a igh t l ines or a r e they bes t fit by a curve? Do t h e "d iscont inui t ies" , the "breaks" or the "kinks" r e p r e s e n t r e a l even t s? Views w e r e highly po la r i zed and some of t he a r g u m e n t s we re iden t i ca l to issues d e b a t e d in the l i t e r a t u r e in the 1920's shor t ly a f t e r Arrhenius theory was applied to biological s y s t e m s . The issues w e r e pe rhaps no m o r e conclusively reso lved a t this seminar than they w e r e 50 y e a r s ago; but t h e r e did seem to be some axioms tha t e m e r g e d and a r e wor th a r t i cu l a t i ng :

—plots of t h e logar i thm of r e a c t i o n r a t e vs . t h e r ec ip roca l of t h e abso lu te t e m p e r a t u r e can be a useful way to display t e m p e r a t u r e dependen t d a t a to s e a r c h for anomal ies ;

—such p lo t s may or may not be r e p r e s e n t i n g even t s which come within the concep t the Arrhenius function and h e n c e it may not be valid to c a l c u l a t e a c t i va t i on energ ies (Ea) from such p lo t s . The re is a l im i t a t i on to the r a n g e of slopes to which Arrhenius law can be appl icable and t h a t l imi t a t ion of ten exc ludes t he s lopes of some of t h e l ines appea r ing in the l i t e r a t u r e . For example , Arrhenius theory cannot deal with slopes t h a t go to ze ro as whe re t he d isassoc ia t ion of some low t e m p e r a t u r e labi le e n z y m e des t roys the po lymer i zed a c t i v a t i o n complex r equ i r ed for r e a c t i o n . It is i nappropr i a t e to c a l c u l a t e an Ea for t he t e m p e r a t u r e r a n g e below tha t where d isassocia t ion occu r s . H e n c e , cau t ion should be exerc i sed and only those s y s t e m s whe re Arrhenius theory can be appl ied dese rve to be ca l led "Arrhenius" p lo t s ; t he o t h e r s should be r e f e r r e d to as "plots of t he logar i thm of r e a c t i o n r a t e s vs . t h e r e c i p r o c a l of the abso lu te t e m p e r a t u r e " and from such p lo t s va lues of "apparen t" Ea's may be useful;

—no longer should such p lo ts be p r e s e n t e d in t he l i t e r a t u r e wi thout having been sub jec ted to app rop r i a t e s t a t i s t i c a l t e s t s to d e t e r m i n e the su i tab i l i ty of f i t t ing var ious l ines or curves to s e t s of d a t a . Addi t ional discussion and me thods of applying such t e s t s a r e p r e s e n t e d in P a r t V — Special Topics R e l a t e d toThe Use of Arrhenius P lo t s , this vo lume .

C. Characterization of Physical Phenomena in Plant Membranes

Much of t he knowledge on the molecu la r a r c h i t e c t u r e of biological m e m b r a n e s has been d i sce rned by e x p e r i m e n t a t i o n on model m e m b r a n e s ,

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bi layers and t h e r a t h e r s imple m e m b r a n e s from yeas t and s imi lar o rgan i sms . These r e s u l t s and t echn iques have been employed to address t h e ques t ion of m e m b r a n e a s s o c i a t e d e v e n t s in h igher p l an t s , p a r t i c u l a r l y those r e l a t e d to t e m p e r a t u r e s t r e s s .

The main ev idence p r e s e n t e d was der ived from e l e c t r o n spin r e s o n a n c e (ESR) spec t ro scopy using a v a r i e t y of spin label p robes , f luo rescence po la r i za t ion s tud ies employing t rans -paranar ic acid, and d i f fe ren t i a l t h e r m a l analysis (DTA) or d i f fe ren t i a l scanning c a l o r i m e t r y (DSC). The d a t a p r e s e n t e d a t f i rs t inspec t ion s e e m s to be c o n t r a d i c t o r y . ESR and most of the f luorescence d a t a i nd i ca t e a change in lipid o rder ing a t t e m p e r a t u r e s c o r r e l a t i n g with t he physiology of chill ing injury; some f luorescence d a t a and the DTA (or DSC) d a t a i nd i ca t e t h e r e is no e n d o t h e r m i c phase t r ans i t ion in t he bulk m e m b r a n e lipids in t he t e m p e r a t u r e r ange above z e r o .

One issue t ha t s e e m e d to r e f l e c t gene ra l a g r e e m e n t is t ha t a phase change from a " l iquid-crys ta l l ine s t r u c t u r e to a solid gel" in the bulk m e m b r a n e lipid does no t occur . This a g r e e m e n t would be suppor ted by the d a t a p r e s e n t e d on lipid analys is (acyl chain compos i t ion , polar head group analys is , unsa tu r a t i on of acyl chain , e tc . ) as it r e f l e c t s t he bulk m e m b r a n e lipid.

It is c l ea r from t h e f lou rescence po la r i za t ion and ESR s tud ies t h a t some change occurs in the molecu la r o rde r ing and f luidi ty of m e m b r a n e lipids of chil l ing sens i t ive p lan t s in response to low t e m p e r a t u r e s — a change t h a t c o r r e l a t e s wi th physiological p h e n o m e n a . This is one a r e a of r e s e a r c h t ha t dese rves i nc reased a t t e n t i o n to gain a b e t t e r unders tand ing of the event(s) which do occur in p lan t m e m b r a n e s a t t e m p e r a t u r e s which c o r r e l a t e wi th injury and which r e g u l a t e m e m b r a n e and m e m b r a n e a s soc i a t ed funct ions . F r e e z e f r a c t u r e t echn iques , expansion of the t echn iques using a v a r i e t y of new spin label p robes which d iscern even t s in the cy top lasm and the m e m b r a n e con t inuum, and o the r s imi lar powerful new techn iques must be employed to e l u c i d a t e these even t s t h a t a r e r ea l but might no t be d i sce rned by DSC or DTA.

An addi t ional point of c l a r i f i ca t ion on this sub jec t . The t e rmino logy deve loped from s tud ies of b inary m i x t u r e s of phospholipids and m e m b r a n e s of E. coli has been used in des igna t ing t h e change in physical s t a t e whe re t he de f lec t ion a t t he lower t e m p e r a t u r e is Τ and a t t h e h igher t e m p e r a t u r e , T^. These w e r e cons idered as t h e " s t a r t " and "finish" of a t h e r m a l phase t r ans i t i on in t he m e m b r a n e l ipids. Because of discussion on the subject it would appea r t ha t t he de f l ec t ion a t t he lower t e m p e r a t u r e in the t e m p e r a t u r e r e sponse of m e m b r a n e even t s in higher p l an t s should simply be r e f e r r e d to as T^ and a t t he h igher t e m p e r a t u r e Τ^, wi thou t any impl i ca t ion of deno t ing a " t h e r m a l phase t r ans i t ion" .

D. Similarities between Freezing and Chilling Injury

There of ten occurs discussion in t h e l i t e r a t u r e compar ing the mechan i sm of f reez ing and chil l ing injury, p a r t i c u l a r l y as it focuses on

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any hypothes is t h a t invokes possible changes in t he physical s t a t e of ce l lu lar m e m b r a n e s in response to low t e m p e r a t u r e s as the p r i m a r y even t . This then leads to analysis and a t t e m p t s to c o r r e l a t e changes in lipids and p a r t i c u l a r l y levels of unsa tu ra t i on in f a t t y ac ids wi th acc l ima t ion , condi t ioning and u l t i m a t e abi l i ty to wi ths t and the f reez ing p roces s . On the l a t t e r poin t , it is c lear in these s tud ies t h a t (as wi th t he chil l ing phenomenon) t h e r e is no cons i s t en t co r r e l a t i on b e t w e e n lipid analysis and suscep t ib i l i ty to f reez ing or chil l ing d a m a g e . That is , as many r e p o r t s of a n e g a t i v e co r r e l a t i on of a m o u n t of u n s a t u r a t i o n and suscept ib i l i ty to low t e m p e r a t u r e injury occur as those which show a pos i t ive co r r e l a t i on . As t he discussion a t this semina r brought out , it is now appa ren t t ha t lipid analysis to d a t e can only r e p r e s e n t a compos i t e p i c t u r e of t he bulk m e m b r a n e lipids: if lipids a r e involved in cont ro l l ing the molecu la r order of the m e m b r a n e lipids which p e r t a i n to f reez ing or chil l ing injury, this con t ro l might occur within d i s c r e t e lipid domains , no t in the bulk phase .

The re is some diff icul ty wi th t e rmino logy in t he f reez ing p rocess as t h e r e is wi th chi l l ing. That is , discussions of ten do not c lea r ly dis t inguish b e t w e e n the primary response to low t e m p e r a t u r e and the secondary even t s ; b e t w e e n the i m p a c t of low t e m p e r a t u r e on the phys ico -chemica l even t s in the m e m b r a n e and the t r ans l a t ion of those even t s in to a specif ic m e m b r a n e lesion and u l t i m a t e l y ce l lu lar injury.

A g r e e m e n t was not r e a c h e d amongs t t he p a r t i c i p a n t s on ev idence as to whe the r the m e m b r a n e lesion and injury was the r e su l t of the f reez ing or t he thawing p roces s . Con t r ibu t ing to this problem is t h e fac t t h a t t he specif ic m e m b r a n e lesion is most of ten i l l -def ined and only c o n j e c t u r e , and the fa i lure to consider t ha t a s p e c t r u m of m e m b r a n e lesions may resu l t from a single f r e e z e - t h a w p ro toco l .

E. The "Membrane Hypothesis"

The seminar was o rgan ized to assess c r i t i ca l ly t he ev idence t ha t has evolved around the proposed hypothes is on the mechanism of chil l ing injury. As original ly s t a t e d , "a single cont ro l l ing response is found in t he ev idence t ha t ce l lu lar m e m b r a n e s in sens i t ive p l an t s undergo a phys ica l -phase t rans i t ion from a no rma l f lexible l iqu id-crys ta l l ine to a solid gel s t r u c t u r e a t the t e m p e r a t u r e c r i t i ca l for chil l ing injury was sugges ted ." F u r t h e r m o r e , this hypothes is impl ied a ro le for t he lipids as t he cont ro l l ing spec ies in t he m e m b r a n e .

Discussion on this point was qu i t e po la r i zed and t h e probabi l i ty is high tha t the p a r t i c i p a n t s were no t pe r suaded to move too far from the posi t ion each held or iginal ly . Pe rhaps this r e f l e c t s t he fac t t ha t this p a r t i c u l a r field of s c i ence is r a t h e r juvena l — the young bird wi th i t s f irst p lumage of t r u e f e a t h e r s but y e t lacking adult c h a r a c t e r i s t i c s . The re was insuff ic ient ev idence or hard d a t a to reso lve t he opposing v iews. This con t rove r sy should, however , help to focus fu ture r e s e a r c h and hopefully develop some new insight and not simply plow old ground. Some

Epilogue 5 4 7

of t he se po in ts deve loped in t h e p r e s e n t a t i o n s and discussions as well as a r e a s of a g r e e m e n t inc lude:

—as original ly s t a t e d , t h e m e m b r a n e hypothes is assumed t h a t a change in molecu la r o rder ing (change in "s ta te") of the m e m b r a n e would br ing about a c o n t r a c t i o n , caus ing "c racks" or "channels" leading to i nc reased p e r m e a b i l i t y . Ev idence does now exist t ha t says this is no t the case and in fac t a decrease in m e m b r a n e p e r m e a b i l i t y is t h e i m m e d i a t e r esponse to t e m p e r a t u r e below the t r ans i t ion in molecu la r o rder ing . The " leakage" and i nc r ea se in p e r m e a b i l i t y only appea r s a f t e r m e m b r a n e d a m a g e has o c c u r r e d for some t i m e per iod;

—the concep t of a "physical phase change from a l iqu id-c rys ta l l ine to a solid gel" should be abandoned as far as descr ib ing e v e n t s in the bulk m e m b r a n e . Changes in f luidi ty and molecu la r order ing do occur but these a r e not d i sce rned as "mel t s " of t he bulk l ipids. More r e s e a r c h needs to be focused on descr ib ing even t s a t t h e molecu la r level in m e m b r a n e s in response to low t e m p e r a t u r e ;

—lipid analysis of t h e bulk m e m b r a n e would sugges t t h a t t h e r e is no basis for a phase change to occur a t physiological t e m p e r a t u r e s . While t he se ana lyses lead to t ha t conclusion, i t is also t r u e t h a t changes in f luidi ty and molecu la r o rder ing do occur and as we develop t echn iques to e x a m i n e t h e physical s t a t e of d i s c r e t e "domains" within t he m e m b r a n e , we mus t focus t he c h e m i c a l analys is on t h e s e s a m e domains . (We can probably l ea rn l i t t l e m o r e from bulk lipid analysis . ) ;

—the " m e m b r a n e hypothes is" ignores or dismisses p ro t e in s , (e i ther soluble or m e m b r a n e - a s s o c i a t e d ) , as a p r i m a r y t e m p e r a t u r e sensor . Ev idence was p r e s e n t e d and discussed t h a t d i r ec t e f f ec t s of t e m p e r a t u r e on e n z y m e p ro t e in s could be d i sce rned and t h a t a con fo rma t iona l change or po lymer i c s e p a r a t i o n of a p ro te in may be as p lausible an exp lana t ion for the p r i m a r y t e m p e r a t u r e sensor as invoking the m e m b r a n e lipid as the p r i m a r y sensor;

—the view was expressed by some tha t a r t i c u l a t i o n of a single p r i m a r y t e m p e r a t u r e sensor as med ia t i ng the chil l ing response should be abandoned . This view sugges ted t h a t imposing low t e m p e r a t u r e on chil l ing sens i t ive spec ies invokes a b road r a n g e of "pr imary" even t s which lead sequen t ia l ly to t h e observed injury;

—the view was expressed t h a t t h e p r i m a r y i m p a c t of low t e m p e r a t u r e on these sens i t ive spec ies was on the t e m p e r a t u r e coef f ic ien t or r a t e of g rowth and d e v e l o p m e n t .

Again, t he se poin ts in t he discussion should help focus r e s e a r c h d i rec t ions and provide a f r amework for a r e a s of endeavor requi r ing new knowledge .

F. Germplasm and Potential for Manipulation

It is of i n t e r e s t t ha t this s emina r inc luded r e s e a r c h t ha t has focused on deve lopmen t of new ge rmplasm and exp lo i t a t ion of na tu ra l l y occur r ing g e n e t i c d ivers i ty to address chill ing injury in crop p l an t s .

5 4 8 Appendix

Trad i t iona l b reed ing p r o g r a m s have long focused on f reez ing t o l e r a n c e and win te r hard iness in c e r e a l s bu t l i t t l e has been done wi th ho r t i cu l t u r a l c rops and chill ing injury. If ano the r seminar such as this we re to be held some t i m e in the fu ture we would a n t i c i p a t e the p a r t i c i p a n t s would be discussing many new advances in crop i m p r o v e m e n t as t he resu l t of g e n e t i c manipu la t ion . And much of this success will depend on a c c u r a t e unders tand ing of t h e mechanism(s) in low t e m p e r a t u r e s t r e s s so t h a t p r e s u m p t i v e and rapid sc reen ing me thods can be devised.


APPENDIX

Table of Temperature Coefficients of Various Plant Responses

This t ab le i n c o r p o r a t e s d a t a on t he t e m p e r a t u r e coef f i c ien t , of bo th physiological and physica l p a r a m e t e r s using whole p l an t s , seeds ,ce l l s , o rgane l les , m e m b r a n e f r a g m e n t s or e n z y m e s . Where t he au tho r s of t h e p a r t i c u l a r a r t i c l e have n o t e d a sudden change(s) in the t e m p e r a t u r e response or t e m p e r a t u r e coef f ic ien t t h e t e m p e r a t u r e a t which these occur h a v e been l i s ted as T^ and T^ for t h e higher and lower t e m p e r a t u r e s r e s p e c t i v e l y . Where m o r e than two t e m p e r a t u r e s we re n o t e d t he se have been inc luded. Where t h e d a t a shows an i n c r e a s e in t e m p e r a t u r e coef f ic ien t a t low t e m p e r a t u r e but the au tho r s have no t i nd i ca t ed a p a r t i c u l a r t e m p e r a t u r e t h e change is l i s ted as a "curve ." If no change is d e t e c t e d , i .e . , t he t e m p e r a t u r e coef f ic ien t is cons t an t over the t e m p e r a t u r e r ange examined it is l i s t ed as "ND" (none d e t e c t e d ) .

Abbrev ia t ions :

DCIP 2,6-dichlorophenol indophenol DAD 2 , 3 , 5 , 6 - t e t r a m e t h y l - p - p h e n y l e n e d i a m i n e TTC t r i p h e n y l t e t r a z o l i u m chlor ide

N o t e : D a t a on changes in t h e t e m p e r a t u r e coef f ic ien t of spin label mot ion and f luo rescence in t ens i ty in lipids from a number of p l an t s n a t i v e to Aus t ra l i a , de se r t p l an t s from D e a t h Valley in Cal i forn ia as well as c rop p l an t s , can be found in C h a p t e r s 13 and 21 of t hese p roceed ings .

5 4 9 Copyright · 1979 by Academic Press. Inc.

All rights of reproduction in any form reserved ISBN012 460560-5

Plant species Tissue/ organelle

Physical measurement

Tl T2 Physiological Tl (oc)

T2 Ref.

Apple Fruit Ethylene synthesis 5 (1) Malus domestica Fruit mito ESR 3 (2)

chondria

Avocado Fruit mito Succinate oxidation 9 (3) Persea americana chondria

Barley Root Rb+ uptake ND (4) Hordeum vulgare Root Rb uptake 10 Oxygen uptake ND ND (5) Hordeum vulgare

Chloroplasts Esr 28 10 DCIP photoreduction ND ND (6) DCIP Photoreduction with 29 20 9 (6) uncoupler

Washed DCIP photoreduction 29 ND 9 (6) chloroplasts

Bean Phaseolus vulgaris Chloroplasts Wide angle X-ray -30 (16)

microsomes (crystalline 23 (16) structure below)

Chloroplasts Decay of delayed light "15 (17) emission

Whole plant Water absorption "14 (8) Chloroplasts Reflection coefficient 11 (9)

Beet root Root-Beta vulgaris mitochondria Succinate oxidation ND (7) Beta vulgaris

Leaf Light-induced carotenoid 15 (10) shift

Chloroplasts H+ efflux 5 (11)

Bell pepper Capsicum Fruit tissue K+ leakage 10 (12) frut esc ens

Broad bean Vicia faba Mitochondria State 3:-

(hypocotyl) Oxidation of malate 30 18 (18) Oxidation of a-keto-

glutarate 30 18 (18) Oxidation of citrate 31 18 (18) Oxidation of Ν AD Η 30 10 (18) Oxidation of Succinate 30 10 (18)

550



T l h to c)

Physiological Tl (oc)

T2 Ref.

State 4:-Oxidation of malate 30 13 (18) Oxidation of a-keto-

glutarate ND 13 (18) Oxidation of citrate ND 13 (18) Oxididation of NADH ND 10 (18) Oxidation of Succinate ND 10 (18) Malic enzyme 30 ND (18) Malate dehydrogenase ND ND (18)

Capsicum Mitochondria NADH oxidation oft-(13) Capsicum from tissue Sensitive C Q line 9 (13)

annuum culture Resistant Cv34 line ND (13)

Castor bean Seedlings Respiration 11 (14)

Ricinus Mitochondria Succinate oxidation 9 (14) communis Glyoxysomes Isocitrate lyase ND (14)

Glyoxysomes Malate synthase ND (14) Glyoxysomes Malate dehydrogenase ND (14) Glyoxysomes 3 oxidation ND (14) Glyoxysomes Glycolate oxidase ND (14) Mitochondria ESR 11 (15) Glyoxysomes ESR 11 (15) Proplastids ESR 11 (15)

Cucumber (48) Cucumis sativus Seed Germination 14 (48)

Seed respiration NO (48) Fruit-

(7) Mitochondria Rty + uptake

Succinate oxidation 10 (7)

Root Rty + uptake 15 (4) Fruit tissue Κ leakage 10 (12) Microsomes Calorimetry 20 Κ -ATPase activity 10 (47)

Cauliflower Mitochondria Succinate oxidation ND (7)

Brassic 15 (47) oleraceae Microsomes Calorimetry ND Κ ATPase activity 15 (47)

551


Physical measurement X Physiological Τ,

(0 C) Ref.

Foxglove Digitalis Trichome Protoplasmic Curve (19) purpurea cells streaming

Jerusalem artichoke Tuber ESR (non-dormant Mitochondria tubers) 27 3 Succinate oxidase 27 (20)

Helianthus (dormant tubers) 9 3 (20) tuberosus

Lettuce Leaf Light-induced Lactuca sativa (grown 15° C) carotenoid shift ND (10)

Chloroplasts (grown 25°C) H20 - DCIP Hill reaction ND (21)

" cation-induced tl fluorescence

shift ND P700 reduction ND (21) Chloroplasts Chi a

fluorescence in: (22) 5 mM MgCl9 -31 100 mM NoCl -25

Maize Excised roots R* uptake 10 Oxygen uptake 10 (5) Zea mays Root

mitochondria ESR 27 12 (23) Amino acid uptake 13 (24) Succinate oxidation 27 12 (23)

Chloroplasts DCIP photoreduction 39 ND (25) " " uncoupled 39 11 (25)

Mango Mangifera indica Fruit Succinate oxidation

mitochondria measured at 25 8-12 (26) (stored at low temp.)

Morning glory Plants Growth rate (dry wt.) Curve (27) Leaf area increase Curve (27)

Pharbitis nil Chlorophyll formation ND 15 (27)

Mung bean Hypocotyl Growth rate 28 15 (28) Vigna radiata (dark)

Seeds Germination "14 (48)

552


Physical measurement toc)

Physiological Tl T2 (oc)

Ref.

Respiration of seeds ND (48) Hypocotyl-mitochondria ESR 28 15 Succination oxidation 28 15 (28) Plants Growth (dry wt.) Curve (27)

Leaf area increase Curve (27) Chlorophyll formation 15 (27)

Chloroplasts DC1P photoreduction 28 17 (25) DCIP photor eduction with uncoupler 28 ND (25)

Mustard Sinapis alba Seeds Germination ND (48)

Respiration of seeds ND (48)

Muskmelon Cucumis melo Rb+ uptake 16 (4)

Passionfruit Passiflora sp. Chloroplasts Photor eduction of 24 16 (29)

Fe(CN)~6a

P. quadrangularis Chloroplasts ESR 9 (30) P. flavicarpa (Polar lipids ESR 9 (30)

( Chloroplasts Photor eduction of Fe(CN)~J

10 (29)

P. edulis (Polar lipids ESR 3 (30) (Chloroplasts Photor eduction of 16 4 (29)

Fe(CN)'6J

Ρ. cincinnata (Polar lipids ESR 6 (30) (Chloroplasts Photor eduction of 15 ND

Fe(CN)'6J

P. caerulea Polar lipids ESR 2 (30) Chloroplasts Photor eduction of 19 7 (29)

Fe(CN)~J6

P. flavicarpa Polar lipids ESR 7 (30) χ cincinnata Chloroplasts Photor eduction of

Fe(CN)'0 16 1 (29)

553



T2 )

Physiological Τ,

<°c> T2 Ref.

Pea Chloroplasts Reflection coefficient ND (9) Pisum sativum Photoreduction of NADP ND (31)

Chloroplasts ESR ND (49) DCIP photoreduction ND ND (25) DCIP photoreduction with uncoupler 25 14 (25)

Chloroplasts Decay of delayed light emission ND (17)

Potato Tuber ESR mitochondria (non-dormant tuber) 25 3 (33)

Solanum (dormant tuber) 21 1 (33) tuberosum Succinate oxidation ND (7)

Tuber tissue K+ leakage ND (12)

Rice Seed Oryza sativa (japonica and

indica types) Germination 17 (35)

Roots Rb* uptake 9 (4)

Sorghum Sorghum vulgare Roots Rb+ uptake 7 (4)

Spinach Chloroplasts DAD reduction 5 (42) Methyl purple reduction -12 (42)

Spinacia oleracea Chloroplasts Light-induced change Methyl purple reduction

at 515 nm 15 (43) Light-induced change at 546 nm 15 (43)

Chloroplasts plants grown Photoreduction of DCIP ND -9 (34) at 10°to 30°C plants grown Photoreduction of DCIP 11 -9 (34) 0°to 10°C

Chloroplasts Reflection

Leaf coefficient ND (9)

Leaf Light-induced carotenoid (9)

absorption shift ND (10)

554



T l h to c)

Physiological T l T2

(oc) Ref.

Leaf Cation-induced fluorescence shift ND (36)

Chloroplasts Chi a fluorescence in:

5 mM MgClr. ND -31 (22) 5 mM NaCl* ND -20 (22)

Sweet potato Root mitochondria Succinate oxidation 10 (7)

Ipomoea batatas Root Mitochondria ESR 12 (32) Mitochondria Phospholipids ESR 12 (32)

Amino acid incorporation 13 (24) Tidestromia Leaf Light-induced 5 (10) oblongifolia chloroplasts carotenoid shift H+ efflux "7 (11) 555

5 5 6 A p p e n d i x

R E F E R E N C E S

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3 . Kosiyachinda , S., and Young, R. E. Plant Physiol. 60, 470-474 (1977).

4 . Zsoldos, F . Z. Pflanzenernaehr Bodenk. 119, 169-173 (1968). 5. C a r e y , R. W., and Berry , J . A. Plant Physiol. 61, 858-860 (1978). 6. Nolan, W. G., and Smill ie, R . M. Biochim. Biophys. Acta 440, 4 6 1 -

475 (1976). 7. Lyons, J . M., and Raison, J . K. Plant Physiol. 45, 386-387 (1970). 8. Wilson, J . M. New Phytol. 76, 257-270 (1976). 9. Nobel , P . S. Planta 115, 369-372 (1974). 10. Mura t a , N. , and Fork , D. C. Biochim. Biophys. Acta 461, 365-378

(1977). 11 . Avron, M., and Fork, D. C. Carnegie Inst. Year Book 76, 231-235

(1977). 12. T a t s u m i , Y., and Mura t a , T. J. Japan. Soc. Hort. Sci. 47, 105-110

(1978). 13. Dix, P . J . , and S t r e e t , Η. E. Ann. Bot. 40, 903-910 (1976). 14. Bre idenbach , R. W., Wade, N. L., and Lyons, J . M. Plant Physiol.

54, 324-327 (1974). 15. Wade, N. L., Bre idenbach , R. W., Lyons, J . M., and Ke i th , A. D.

Plant Physiol. 54, 320-323 (1974). 16. McKers ie , B. D. and Thompson, J . E. Plant Physiol. 61, 639-643 . 17. Ju r s in ic , P . and Govindjee . Phytochem. Phytobiol. 26, 617-628

(1977). 18. Marx, R. , Br inkman, K. Planta 144, 359-365 (1979). 19. P a t t e r s o n , B. D. , and G r a h a m , D. J. Exp. Bot. 28, 736-743 (1977). 20. Chapman , Ε. Α., Wright , L., and Raison, J . K. Plant Physiol. 63,

363-366 (1979). 2 1 . Mura t a , N. , Troughton , J . H. and Fork , D. C. Plant Physiol. 56,

508-577 (1975). 22. Mura t a , N., and Fork , D. C. Plant and Cell Physiol. 18, 1265-1271

(1977). 23 . Raison, J . K. Chapter 13, these proceedings (1979). 24. Towers , N. R., Ke l l e rman , G. M., Raison , J . K., and Linnane , A. W.

Biochim. Biophys. Acta 299, 153-161 (1973). 25. Nolan, W. G., and Smill ie, R. M. Plant Physiol. 59, 1141-1145

(1977). 26. Kane , O., Marcel l in , P . , and Mazl iak, P . Plant Physiol. 61, 634-

638 (1978). 27. Bagnal l , D. J . , and Wolfe, J . A. J. Exp. Bot. 29, 1231-1242 (1978). 28. Raison, J . K., and Chapman , E. A. Aust. J. Plant Physiol. 3, 2 9 1 -

299 (1976).

Appendix 5 5 7

29. C r i t c h l e y , C , Smill ie , R . M. and P a t t e r s o n , B. D. Aust. J. Plant Physiol. 5, 443-448 (1978).

30. P a t t e r s o n , B. D. , Kenr ick , J . R., and Raison , J . K. Phytochemistry 17, 1.089-1092 (1978).

3 1 . Shneyour, Α., Raison , J . K., and Smil l ie , R. M. Biochim. Biophys. Acta 292, 152-161 (1973).

32 . Raison , J . K., Lyons, J . M., Mehlhorn, R. J . , and Ke i th , A. D. J. Biol. Chem. 246, 4036-4040 (1971).

3 3 . Wright , L., and Raison , J . K. (Unpublished d a t a ) . 34. Inoue, H. Plant Cell Physiol. 19, 355-363 (1978). 35 . Nish iyama, I. Plant Cell Physiol. 16, 533-536 (1975). 36 M ura t a , N. , and Fork , D. C. Plant Physiol. 56, 791 796 (1975). 37. Bre idenbach , R. W., and Waring, A. J . Plant Physiol. 60, 190 192

(1977). 38 . Melca rek , P . K., and Brown, G. N . Plant and Cell Physiol. 18, 1099-

1107 (1977). 39. Waring, Α., and G l a t z , P . C h a p t e r 26, t hese p roceed ings (1979). 40 . Paul l , R. E., P a t t e r s o n , B. D. , and G r a h a m , D . C h a p t e r 37, t he se

p roceed ings (1979). 4 1 . Paul l , R . E. (Unpublished d a t a ) . 42 . Cox, R. P . Biochim. Biophys. Acta 387, 588-598 (1975). 4 3 . Y a m a m o t o , Y., and Nish imura , M. Plant Cell Physiol. 18, 55-66

(1977). 44 . Raison , J . K., C h a p m a n , Ε. Α., and Whi te , P . Plant Physiol. 59,

623-627 (1977). 4 5 . P o m e r o y , Μ. K., and Andrews , C. J . Plant Physiol. 56, 703-706

(1975). 46 . Miller , R. W., De La R o c h e , I., and P o m e r o y , Μ. K. Plant Physio-

I. 53, 426-433 (1974). 47 . McMurchie , E. J . C h a p t e r 12, t hese p roceed ings (1979). 4 8 . Simon, E. W., Minchin, Α., McMenamin , Μ, M., and Smith , J . M.

New Phytol.77, 301-311 (1976). 49 . Raison , J . K. In R a t e Con t ro l of Biological P r o c e s s e s . Symposia

for Socie ty of Expe r imen ta l Biology XXVII, p . 485-512 (1973).


I N D E X

A

Anacystis nidulans thylakoid membrane lipids, 337 EPR-spectroscopy, 338 fluorescence, 338 differential scanning calorimetry, 338

Arrhenius law, 330 range of validity, 528

Arrhenius plot apparent discontinuity, 387 barley chloroplast, 188 breaks, 26, 532, 535 activation energy (Ea), 178 "critical" temperature, 532 cytochrome C-oxidase activity, 368 dark reduction of cytochrome f, 220 DCIP-photoreduction, 219 germination rate, 495 Hill reaction

lettuce chloroplasts, 219 thylakoid membrane, 340

ion leakage chilling insensitive potato tuber, 143 chilling sensitive cucumber, 142

ion stimulated ATPase, 166, 171 intersecting straight lines, 523 PEP-carboxylase, 454 photosynthetic O 2 evolution, 339 respiration rate, 495 smooth curve, 523 spin label parameters, 378 succinate oxidase, 178 Tidestromia, 222 transition temperatures, 378

A trip lex species area of distribution, 480

Β

Barley fluorescence

intensity, 309 polarization ratio, 309

linolenic acid levels, 407 Blue-green alga, 337 Boundary layer lipids, 330

C

Callus tissue TTC reduction rate, 277

Calorimetric differential thermal analysis mitochondrial lipids, 320

Calorimetry membrane preparations from

tomato, 169 cauliflower, 169 cucumber, 169 , 3

C - N M R , 387 Capsicum annum

chlorophyll fluorescence, 193 Carrot, 143 Cauliflower

ion-stimulated ATPase, 166 Cell membranes

acclimation to low temperature, 347 Cell suspensions, 464 Cell suspension cultures, 454 Cell volume

changes in, during freezing, 234 Cellular dehydration

concentration of solutes, 233 Chayote, 142 Chilling

and water stress Phaseolus vulgaris, 54

5 5 9

5 6 0 index

effect on cell permeability, 278 chlorophyll fluorescence, 191 chloroplast function, 189 chloroplast photoreductive activity, 191 germination, 492 ion leakage, 142 membrane fluidity, 178 PEP-carboxylase, 460 respiratory activity, 279 root-cell plasmalemma, 121

ultrastructural changes in mesophyll cells, 98 microfilaments, 110 microtubules, 110 mitochondria, 284 plasmalemma, 110 plasma membrane, 284 tomato cotyledons, 98

Chilling injury acetaldehyde production, 83 acetate production, 83 alleviation by

antioxidants, 92 calcium, 93 carbon dioxide, 89, 90 chemical treatments, 92 controlled atmospheres, 89 gibberellic acid derivatives, 93 humidity control, 89 storage treatments, 86, 87, 89

Anacystis nidulans, 340 apple, 81 assays for, 190

field testing, 509 using plant tissue, 511

ATP levels, 59 banana, 81 chloroplast membranes, 8 critical temperature (Ts), 82 cucumber, 81 cytological responses, 6 definitions of, 3, 543 during cool-storage, 81 eggplant, 81 electrolyte leakage, 51 fatty acid changes, 58 genetic approach, 474 genetic diversity, 507 glycolysis, 83 grapefruit, 81 hypothesis, 289

ion leakage, 341 leaf respiration, 61 lime, 81 mango, 81 membrane fluidity, 11 mitochondrial membranes, 8 muskmelon, 81 orange, 81 peach, 81 papaya, 81 photosynthesis, 59 photooxidation of pigments, 59 pineapple, 81 physiological dysfunctions, 2 plasma membrane, 7 proposed mechanisms, 135, 188 recovery from, 208, 209 senescence, 81 translocation, 59 water loss

and water permeability, 54 and stomata, 54

Chill-hardening hormones

abscisic acid, 57 mechanisms, 57 root permeability, 57 stomata, 57 water stress, 58

Chilling resistance diurnal variation of, 34 genetics of, 515 in tissue cultures, 466 Lycopersicon esculentum, 25 L. hirsutum, 25 metabolism of, 34 Passiflora species, 26

Chilling sensitivity barley photosynthetic mutant, 197 effect of light intensity, 74 genetic modification of, 195 Sorghum bicolor, 67 S. halapense, 67 S. leiocladum, 67 tomato

interspecific hybridization, 197 Chilling treatment, and changes in fatty acid

composition, 432 Chlorophyll A

divalent cations and fluorescence, 220 extrinsic fluorescent probe, 217 intrinsic fluorescent probe, 217, 218

I n d e x 561

Chloroplast glycolipids Chloroplast lipids

, 3C - N M R studies, 384

Chloroplast membrane lipids fatty acid composition of, 377

spin label motion in, 377 Chlorophyll synthesis, 188 Chloroplast thylakoids

inactivation in absence of light, 205, 206 presence of light, 205

low temperature response of, 203 response to

in situ chilling, 204 in vitro chilling, 204

Chromosomal instability, 465 Cold-shock treatment and,

anomolous potassium uptake effect of pH on, 125

potassium efflux, 128 potassium uptake, 124, 128

content along roots, 130 calcium content, 130 uptake of ΝΗί, NO"3, H 2PO;, Γ, R b

+1 3 3 ,

41 3 4

Cold acclimation and thylakoid alteration, 210

Cold hardening biochemical changes during, 299, 300 Solanum species, 296 ultrastructural changes during, 300, 301

Cold tolerance and plant breeding, 486

Cormus stolonifera cell culture in, 275

Corn, races of, 484 Cotton

membrane lipids, 406 Critical temperature

correlation with plant habitat, 185, 186 relation to T sT f, 177

Cucumber chlorophyll synthesis, 188 hypocotyl extension, 188

ion-stimulated ATPase, 166 thylakoids, 206, 207

Cucumis melo, 147 Cucurbita pepo, 142 Cucumis sativus

ion leakage during chilling, 142 Cytoplasm, cytoskeletal elements, 16 Cytoplasmic structure

effect of chilling on, 30

D

Differential scanning calorimetry chloroplast polar lipids, 380

Differential thermal analysis tomato mitochondrial lipids, 319

Diffusional barriers as affected by osmotic properties, 449

Diffusional events effect of polymer and confining space, 448 in isotropic media, 445 particle distribution in space, 447

Draught-hardening fatty acid composition and, 53

Ε

Electron spin resonance TEMPO, 272 in plant tissues, 272

ESR-spectroscopy leaf polar lipids, 184 mitochondrial membranes, 184 partition coefficient, 184

Enzymes configuration of, 333 solubility of, 330 direct effect of temperature on, 451

Epigenetic variation, 465

F

Fatty acid biosynthesis, 394 desaturase activity, 352 desaturases, 397

variation with temperature, 400 desaturation

variation with oxygen concentration, 402, 403

unsaturation linoleic acid, 391 linolenic acid, 391 oleic acid, 391 synthesis of, 394

Fluorescence of parinaric acid, 184

Fluorescent probes model membrane systems, 216

Freezing membrane damage, 294 membrane permeability, 294 repercussion of the process, 232

Freezing injury, 4 effect on spinach thylakoids, 212

5 6 2 index

membrane permeability, 295 Freezing lesions, 212 Freezing and thawing winter cereals, kinetics

of, 267 Freeze-thaw cycle

electrolyte leakage, 258 isolated protoplasts

changes in cell volume, 243 plasma membrane lesions, 237 protoplasts as osmometers, 235 volumetric changes, 236, 237

Frost-hardening, 411 involvement of lipids, 426 winter wheat

changes in lipid content, 415 Frost-hardiness

as affected by growth regulators, 298 Frost injury, 255

moment of, 256

G

Genetic adaption, 28 Genetic diversity, 473 Genome modification

in cell cultures, 463 Germination, at low temperatures of

bean, 510 cotton, 510 lima bean, 510 tomato, 510

Gossypium barbadense chilling sensitivity of, 434 fatty acid composition of, 433 seed lots with differing chilling sensitivity,

433 Gossypium hirsutum, 431

membrane lipids, 407 Growth temperature, effect on fatty acid

composition, 342 lipid composition, 225, 341 pigment composition, 226

Guava, chlorophyll fluorescence, 193

Η

Haploid plants from anther cultures, 464

Intracellular ice formation probability of, 233

Ion leakage Capsicum annuum, 146 changes during storage, 145

during storage of potato tubers, 147

snap beans, 148 soy beans, 148

effect of detergents on, 144 Lycopersicon esculentum, 146

J

Jersualem artichoke, chilling of, 82

L

Leakage solutes from seeds, 38

Linoleic acid biosynthesis, regulation of, 396

Lipids fluidity of, 330 Lycopersicon esculentum, 26 L. hirsutum, 26

Lipid bilayer lateral compressability of, 330

Low temperature anomoly in potassium transport, 123

Low temperature stress effect on ion transport, 123 effect on phospholipid composition, 411

Lycopersicon esculentum cytochrome C-oxidase activity, 368 ultrastructural changes following chilling,

98, 109 ion leadage, 146 chilling resistance in, 25 spin-label motion in, 26

Lycopersicon hirsutum chilling resistance in, 197 ecotypes differing in chilling sensitivity, 319 lipids of, 26

Lycopersicon species leakage rate of leaf cells, 512 natural area of distribution, 479

Μ Mango

fatty acid composition of mitochondria, 394 Membrane fluidity

characterization of parameters, 12 Membrane lipids

changes during frost hardening, 412 molecular ordering of, 13 phase transitions, 305 physical properties of, 305

Membrane physical properties lipid fluidity, 329

index 5 6 3

chemical perturbors, 16 Membrane potential

A vena sativa, 154 effect of

glycine, 157 light, 156 metabolic inhibitors, 157 sucrose, 158

Zea mays, 154 Membranes

of dry seeds, 38 Tetrahymena, phospholipid classes of, 352 transport across, 331

Membrane structure global free energy, 249

Mesophyll cells, 100, 102 Metabolism, disruption by chilling

direct effect on enzymes, 460 Microscopy

and microtubule elements, 30 and vesicles, 30 phase contrast, 30

Microtubules, 98 Mitochondrial lipids, thermotropic properties

of, 322 Mitochondrial membranes

fluorescence of trans-paranaric acid, 369 fluorescence intensity, 372 fluorescent probe analysis, 369

Monolayer experiments force-area curves, 381, 382 phosphatidyl choline, 381

Morphogenic potential, 465 Mutagenesis, 465

Ν

Nerium oleander physical property of membrane lipids, 315

Nuclear magnetic resonance freezing curves, 258 longitudinal relaxation time, 258 relaxation properties of

cactus stem, 263 Kentucky bluegrass, 263

tansverse relaxation time, 258

Ο

Oleyl-CoA desaturase, regulation, 397 Onion, 143 Oriental pickling melon, 142 Oryza sativa, 140

Ρ

Parinaric acid, fluorescence polarization ratio, 308

Passion fruit, isolated chloroplasts of, 191 Pawpaw, chlorophyll fluorescence of, 193 Peanut, chlorophyll fluorescence of, 191 PEP-carboxylase in

Alpine plants, 456 temperature zone plants, 456 tropical plants, 456 C 4 plants, 453 temperature coefficients of, 458

Phaseolus vulgaris fluorescence of parinaric acid in polar

lipids, 185 chilling and water stress, 54 fluorescence polarization ratio, 309

Phase transitions algae glycolipids, 223 algae phospholipids, 223 as detected by cholorphyll fluorescence, 218

Phospholipid vesicles, fluorescence analysis of, 371

Phospholipids, polar head group changes at low temperature, 417

Physical properties of membrane lipids, 365 Anacystis nidulans, 308 chloroplast membranes, 314 correlation with changes in fatty acid

desaturase activity, 360 desert plants, 310, 312 Nerium oleander, 315

Phase separation, of membrane lipids, 310 Pisum sativum, fluorescence polarization

ratios, 309 Plant growth, at chilling temperatures, 187 Plant breeding

selection after mutagenesis in tissue culture, 511

Plasmalemma, 98 plasma membrane, critical surface area of,

240, 241 Poplar

total phospholipids and changes in frost hardiness, 413

Potassium uptake, in thermophylic species, 124

Potato frost hardiness in, 292 low temperature resistance in, 198

Proteins, direct effect of low temperature, 18, 451

5H4 index

Protoplasts as affected by freeze-thaw regimes, 232 culture of 464 isolation of, 464 minimum critical volume, 244 population average, 247

Pumpkin, 142 Pyridaziones, 405

R

Respiration pollen, 28 temperature effects on, 28 in sorghum species, 497 maize root mitochondria, 179

Root exudation as a result of environmental stress, 115 chilling induced, 116 effect of metabolic inhibitors, 117 role of calcium in, 117, 119, 121

Rye isolated protoplasts, 235 linolenic acid levels, 407, 408

S

Seed germination carryover effect of chilling, 499 Arrhenius plots of, 495 at low temperatures, 510

Solanum acaule, frost tolerance in, 292 Solanum tuberosum, frost tolerance in, 292 Somatic hybridization, 199, 467

intergeneric, 468 intrageneric, 468

Sorghum bicolor chilling sensitivity of, 67 leaf water potential, 76, 77 photosynthetic response, 68, 71 relative growth rates in, 68, 69 water stress in, 76, 77

Sorghum leiocladum chilling sensitivity in, 67 photosynthetic responses in, 68, 71 relative growth rates in, 68, 69

Sorghum halapense chilling sensitivity, 67

Sorghum species chilling sensitivity of, 496 effect of chilling on

chlorophyll synthesis, 500 growth extension, 497 rate of germination, 497 respiration, 497

Spinach thylakoid alterations, 211 Spin label motion, in lipids of Lycopersicon

esculentum, 26 L. hirsutum, 26

Spin label signals concentration dependency of, 443 rotational motion range, 437 translational diffusion motion range, 440

Statistical tests confidence levels, 530 estimation of a breakpoint, 536 likelihood plot, 540 maximum likelihood, program for, 542 straight line hypotheses, testing of, 529

Sterols, changes during frost hardening, 426 Summer squash, 142 Super cooling, 258

Τ

Temperature acclimation, mechanisms of, 348, 349

Temperature adaptation, 329 Temperature coefficients, table of, 549 Temperature responses, genetic diversity of,

473 TEMPONE, spin label spectra of, 438 Tetrahymena pyriformis

low temperature acclimation of, 350 membrane fluidity, 355

Thylakoid membranes ion permeability changes in, 221 pigment changes in, 220

Tomato different ecotypes, 484 ion-stimulated ATPase in, 166 isolated chloroplasts, 191 PEP-carboxylase, 455

Tomato/potato hybrids, 199 Trichomes, 30

V

Variant cell lines and aluminum resistance, 467 amino acid analogue resistance, 467 disease resistance, 466 environmental stress resistance, 466 herbicide resistance, 466 high salinity resistance, 467 kanamycin resistance, 468 nitrate reductase deficient, 468

Viscosity effect of Cytochalasine B, 451

Index 5 6 5

of cells, as a function of osmotic strength, mitochondria, fatty acid composition of, 450 393

W Ζ

Water, NMR relaxation properties of, 258 Zea mays Wheat temperature response of, 475, 476

linolenic acid levels of, 407

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Low temperature stress in crop plants: the role of the membrane: proceedings of an international...

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