{' HYSIOt. PL \:\T.\Rl"\ 1 tjj; 69:- 703. Copenbace n 19S6

Quantitative characterization of protoplasts and vacuoles fromsuspension-cultured cells of Catharanthus roseus

.I. P. Renaud in, S. C. Brown. H. Barbler -Bry goo and J. Guern

Renaudi n . J. P. . Brown . S. C . Barbier -Brveoo . H. •t G ucm. 1. 1'J~h_ O u.mntarivecbaracte nzauo n of proroplusts :..I nJ vacuol~~ rrom -us pension-cuiturcd .::::1" or Cl .thuranthus rUWI/S. - Phvsto i. Plunta rum eg: b95- "113

..\ simple and etficiem procedure for isolanon oi protoplasts and then vacuole-, fromcultured cells ,)[ Camaranshus roseus tL. J G . DIm is presented . Protopt.i sts were dis­rupted by an osmotic shock and the vacuoles vere purified bv fl otat ion on .J <ingic­step gradient..-\ cornp anso n o t the conten t ..t nJ concenrranon .)1 solu tes : pro teins.sugars, c rguruc acids. alkalords , mineral !C n S I IQ pro tc plasts and ..:d h ,h() ~\ ej th.nmassive and selective losses occur for most solu tes dun ng pro roplcst prcp urano n.Th ese are uu n bured to the osmonc ad justment and chan ges ,H' memb rane pcrmcab­dine s occurring J uring. ptasmoivsis. Dura concertung the -ize . ~"c :J aml puruc or theisola ted vacuoie s are discusseu . Bv ana!\'ils ot rsolureu vacuoie s . the vacuot.rr ccncen­rrano n and Iocaiizauo n 01 ~o l u tes · ·"" i t h i n protopiasts have been ue temu neu . The lim­its of this la tte r approach are stressed. however. Some evidence in 1;1\'(\l1r ,... f the ,e­lection or' a special class of vucuotcs dunng isolation is reported and Jb"::.l\...:J .

A dditional key wo rds - Alkaloids. mineral ions . orgur nc acids . ptcvmoly-rs , suga rs.

J. P. Renaudin treprmt req UI!StS } . S. C. Brown. H. Barbier-Brveoo wui J. Guern.l.a b. de PhyslOlugfe Cetlutuire Ve!(ttalt> C.\ RS-tS RA . BP .vo . t, F·o/!O() Gn- w r­} " ·I!rr t' . France.

Introduction

Th e vacuol e o f plant ce lls is a mul tifun ctional co mpa rt ­men t which ca n accumula te o r libe ra te rne tabo titcs andio ns (Many et ai. lYXO. Boiler 19S2. Wagner 19S2.Leigb 19~3 I. Our unde rst anding o f th is organe lle has in­creased significa nt lv since techniques fo r its iso lat ionwere discovered (reviewed by Wagne r 1983>' Da ta havebeen repo rted fo r t he co mpartmentanon of so lutes andmet abo lic path ways in vacuo les (Frc hnc r e t al. 1 98~ .

Yamuki I'iS4 . St ruck and Sha rma 1l.JS5) and . to a les se rexte nt. about transpo rt mecha nisms across the ton o­plast I Deus-Ne umann and Zen k !9S4. Mar tinoia e t JI.1 l)~5 . Tho rn and Murc tzki 1985). But progress has beenhamp e red by l J low or irrepmducible yie lds and fra g­ility o f vacuolcs . .:) inaccur acies in the de te rmi na tio n o fthe number and size o f isolat ed vacuo les , and hence indcti nuion of the vacuola r co mpa rtment t wngnc r 1982.Leigh IlJS31. J } co ntamina tion with in vacuol e pre pa ra ­nons (Holle r !lJS:!) and -I.) lack of an unambiguo us bio -

che m ica l marker of the to no plas t ( Burb ier- Brygoo et al.19S6).

O ur gro up has been inte rested in obtaining » c-latedvacuol es fro m suspensio n-cu ltured cell s o f Cuthurunthnsroseus fo r studie-, on a lka lo id accumula tio n in vacuolcs(Rena udin et al. 1985L ionic cha n nel s (Ta keda et 31.I Y~5 ) :.1 C).(.1 the e tecrroche rnical pot ent ial grad ient o f pro­to rts (Ba rb ier· Brygoo e t al. 1 ~85 . Kurk d jian et al. 1985)across the ton oplast. T here a re two recent repo rts o fvacuo le isol a tio n fro m C. roseus ce lls ( Dc us-Neurna nnand Zc nk IlJS.J. ~tango et al. 19S5>. but the cha racter­izunon o f these isola ted cucuo lcs was inco mple te .

tn the present pape r we des cribe a sim ple. effici entand reproducible procedure to r iso la tio n (If vacuoles byflota tio n fro m os moti ca lly burst pror opiasts. The e rn­phasls is upon char uctcnz.n ion. e .g. estimat ion o f theyield and pu rity t.l[ vucuolc s end an :J l ys i ~ of vacuole con­tent. Such qua nutauvc info rmat ion is use ful both torplann ing and inte rp re ting e xpe riments with isolatedvucuoles, bu r it is pa rt lv o r {Orally ab sent fro m numer­ous reports .

095

Th e first part o f the present repo rt shows that thecon te nt of pro topl asts (10 which isolated vacuole s arec1as~ H:;] J1 ~ compared ) is very diffe re nt from (hat of thecells Iro m which they are deriv ed . The second pa rt givesa uuunutu tive descriptio n of the: isolated vacuo lcs .

A bbreviations - EDTA . ethvlenedia rninctc traaceuc acid :HEPES. S-=·h~droxyethylr l perazmt:.~ · -:-t:thar.esulfonicacid .

~lateria ls and methods

Plant material

The cell line C:::O of Cathuranthus roseus (L. l G . DontApocynaceae }was grown at :::7"'( in B. medi um (Gam­borg et 31. 1%8) containing I uJ! :::A-d ichlo ro phenoxy­uceuc acid and 00 n,\1 kinetin . Cells were cultivated un­der co nti nuou s light (2.5 W rn" . Mazdafluor lncandia/lnc. } in 500 rnl Er len meyer flasks with ~no ml cultu remedi um . They wer e sub cultured every i days at the o n­set o f the stat ion ar y pha se . All expe rime nts wer e per ­form ed with 5- or e-d av-old suspensions.

Protoplast preparation

Cell s tea S g. fre sh weight ] were collected on a sinreredglass filter I.1U/90 urn I under partial vacuum and rinsed .5limes with 20 ml .55fJ mM sorbito l adj usted to pH 5.5with diluted KOH . The cells were [hen incubated for 2 hat 27c C in ~O ml of the sa me solution with 0.5 % {w/v]ce llulase Onozu ka RS (Yakult Hon sha l and 0.05%IW /v) pectolyase )'23 (Seishin Pharmaceutica l). Prow­plasts were filter ed throu gh 1U-t urn nylon cloth andwashed thrice with .10 ml of the protoplast medium con ­raining 550 mM sorb itol and 10 m.1f HEPES-KOH . pHi .-L by centrifuga tion at 200 g for 1.5 min at -r' C. Afterthe last washi ng the prot oplast medi um was added tothe pe llet in order to adju st the population density to3-6 ):; 10" proroplasts ml"".

Vacuole pre paration

One ml protoplast suspension was quickly added to 1.75ml of a solution of i .9 mM Na,· EDTA and 10 mMHE PES-KO H . pH 7.3 (final concentratio ns: 200 mMsor bitol . 5 mM EDTA . to m.\1 H EP ES: osmolali ty 2·.J2rrr Osm t. After 30 min at ~ C( with litt le or no shaking .intact vacuoles wer e pur ified by flot ation . T he suspe n­sion was mixed with 1O. ~.5 01 1of a rap idly ad de d solut ionof 3b(l mM so rbito l. 8.5% (w/v) .5-(N- 2.3-d ihyd roxy­propylacetamido j -2..t.6-tri iodo-:-:.:'" -bis(2.3-dihyd ro ­xypropyl lisopbthala rnide (Nycodenz. Nyegaard &. Co .•0 ,1". Norwav}. 5 mM Na.-EDTA and 10 m.lf HEPES·KO H . p H i.s (final co ncentrations: 325 mM so rbitol.o.70:-;" Nycodenz , 5 m.\! E DTA . 10 m.\! HEP ES : osmo­lalil Y ~6~ mrjsm : density 1.056 g 011-1) . One 011 proto­p last medium (osmo lality 607 O1 Osm : density 1.035 g

011-1) was layered above the la tter phase . vacuotcs were

recovered in the uppe rmost 0.7 ml aft er cent rifugatio nat 160 g for S min ai -r' C . Nycodcnz was used beca use ofit!" 10 1A' viscosity which allowed rapid migrat ion of thevacuo lcs d un ng centrifugat ion an d e nab led further ex­penmcn rs such as rnicroetecir opho resls. By slight di­lution with the lower phase of the gradient. the ac tualco ntent of Nycod enz in the vacuo le fraction was 1.3%.gwing a density of the solution of I. tJ-Wg 011- : . When re­lated to the volu me of the fracti ons. th is co rrespondedto a co ntaminati on of the vacuo le fraction by I '~o of the:sample zone .

Xum berin g and size measurement

Ce lls were counted after d issocia tion in lO u.~ chromicacid at iOCC for 15 min. Their mean volume was esti­mated from the num ber of cell s p.:or g fresh weight aftercorrect ion for the presen ce of extracellul ar medium infiltered cells . previo usly estimated to 1 2 . 5 °1~ . and as­suming a cell de nsity of l. 05 g rnl " ". Prot oplasts and vac­uolcs wer e co unted in a Mal assez huern atocyt orneter.vacuoles and the residual protoplasrs in vacuole prep­ar at ions were counted immedia te ly afte r their pu rifica­tion . with o nly gentl e mixing and no d ilution. The dia­meter s o f individual pro toplasts and vacuoles wer emeasu red with a calib rated ocular microme ter. Th eaverage volume of the pro roplasts and of the vacuo le,from o ne prepu runon was taken as the mean of 100 indi ­vid ual vo lume s. calcula ted from individual dia met ers.

Protein and enzyme OIssa~'

For the assay of proteins and enzymes . samples wer e di­lute d to final co ncentrat ions of 0.1% (v/\' ) Triton X-I IXJ ..5 mM MgCl:. 5 mM Na ;-E DTA. I mM potvethvleneglycol.O(KKl and 10 mM HEPES·K OH . pH 7.: . Proteinswer e extracted by grinding the ce lls (precoole d in liquidnitrogen) ~ times at ace in such a solution and cen ­tri fuging the extra ct for 10 min at 5 000 g.

Proteins wer e assayed by the Coo mas sie Blue G-250d ye-b indin g assay (Read and No rthco re 19S1) with bo­vine se rum albu min as a sta ndard . Pr eliminary pre ­cipitat ion by acet one of soluble proteins gave the sameresults as assay on crude hom ogenized samples . A mod ­ified Lowry met hod (Bensadoun and Weinstein 1970 igave simila r results.

c- Man nos ida se (Ee 3.2 . 1. 2~) . acid phosphatase (EC3. 1.3.2. ) and phosphodi esterase (E C 3. IA. l) wer e: as­saved at 37°C accordi na to Boile r and Kendc ( 1979) ex­cept that the buffer wa~s 65 mM ace tic acid-Na'G l-l, pH~ . 5 . Glucose -e-phospbate deh ydroge nase (Ee 1.1. 1.491and pe roxidase (EC 1.11.1.7 ) wer e assayed at 3(f C ac­cording to Lo hr and wallcr (1976) and at room tern­pc ratu re acco rding. to Staffo rd an d Bravinder-Bree(lQi2). respect ively. i':ADPH-cytochrom e c reductase(EC 1.6.: .-1) . NAD H-c\'lOchrome c reductase (EC1.6.9'1.3 ) and cytoc hrome c o xidase (E C 1.9.3. 1) cou ld

."

only be assuved a lter de sa lting , ;IS pre..io us tv ment ionedby Bollcr :.IoJ Kende (197°.1. These assays were per­forme d at roo m te mpe ra tu re accord ing. to Hedges andLeo naed ( l ~r;-1). ;";0 a tte mpts were made to dis tinguishbet ween anti mycin Acsensruvc (m itochondria l inne rmern brane l anti urui mvcin A-insensitive (mi rocbo nd nalo ute r memb rane . endo plasmic re ticulum I ~.-\ DH·cy !O­

ch ro me c reductase . Ca talase tEe l. 11.1.6) and fum ­arase l Ee -l .~ . l . ~ ) were assayed at roo m tempera tureacco rding to Ae bi ( 1474) and Cooper J.!1d Bee ve rsl l'lf)~l. respectivel y. They were also assayed in desa tte d<arnples . de void \)[ Nycod enz, since (his compound ab­so rbs st ro ngly around 2-1Unm. Desa lting W ::i.S pe rfo rmedw ith col umns prepacked with Sep hade x G-~5 ( PD- to.Phu rrnacia j. Enzyme samples (2.5 ml } were applied tothe co lumn. left to en te r the gel and eluted with 2.5 rnl':.5 m.\ / sodium ph osp hate . pH i .2. The act ual activitie s\If sampies were ca lculated fro m the activit ies in de­sa lted samp les and co rr ected by a facto r eq ua l to themean fraction of proteins and c-mannosidase wh ich wasrecovered .

~ I J lic acid and Ci tric acid were determined by the me th­ods 01 G utmann and Wah le reld ( 1 97~ ) and Mo llenngand Gruber {lYb61. resp ective ly. A jmalicinc and se r­penune were assayeu by high-perfo rmance liq uid chro­matogruphv with de tectio n by fl uo resce nce ( Re nuudinIlJSS" Sugars . i.e . glucose. glucose-e-pbosphate . fruct­ose . fruct csc-o-ph osphate and suc rose . were de te r­mined bv a spect ro pho to metric enzyme assay acco rd ingto Jones et al . ( 1977) and are exp res sed as hexose equiv­alcnrs. Ino rgan ic phospha te was assayed acco rdi ng toObnishi e t al. ( 19;5 ). K· was analyzed by emissionspectroscopy .100 \ lg> and Ca:- by atomic abso rptio nspectroscopy. NO ; was assayed colon merncat tv afte rreduct ion to NO ; in a co lumn filled with cadmium (T hi­baud and G rigno n 1 9~1 ).

Osmota line s we re measu red with a freez ing-po int de­pressio n microosmometer (Roebling). Densit ies of so­lutio ns were ca lcu lated fro m val ues in ha ndbooks.

Retatb e vacuolar content orone protoplast

The ex te nt of the vacuol a r local ization o f so lutes wit hinpretoplasrs ITab. ~ ) was calcula ted by using: a -man­nosidase as a vac uo lar marker . The relative amountA'~4 of a co mpound B in the vacuol e ts} fro m o ne proto­plast was calculated by the formula :

A% = (B/ B,) . ( M;~IJ ' lOO

where B :.1Od \ 1 are the amo unts o f the co mpound 8 andof c -manncsldase in Hr pro toplasts (subs cript p) andII r pur ified vacuoles (Subscrip t v). B. and ~ l . we re c:.II­culatcd alt e r co rrection for the presence o f a sm~1 1I frac ­tion of residu al pro Wplas lS in vacuole prepJrat ions . as-

~ 5 · Phn lOl. Pl;lnl;lrum NI. lll><tl

<urning those resid ual prc roptasts to have the sa me con­tent as stock pro to plas rs. A simila r p roced ure was usedto express the con ta rruna tion o r'vacu ole preparat ion-, byex trav acuula r biochemical ma rke rs (1".Jb. 3 ). In trnscase . the values O[ B JnJ \t .. we re not co rrected for thepresence o f residu al proro plasts in vacuole prepare­lio ns .

Results and d iscussion

Cha r acte r iza tion orprotoplasts

Yield and size of proioo tastsFro m abo ut Ht ce lls t S g: fresh weigh t}. 7-9 x 10-proto­ptasrs were ro ut ine ly obtained. Protoptasrs were onlystab le in so rbito l co ncentra t io ns higher than ..LOOm.\ t . .-\co ncentrat io n of 550 m.\f so rbito l was opt imal fo r vac­uole isola tion since it favo ur ed [he lysis of nearly allpro toplasts d uring the subsequent osm otic shock for iso­Ia tio n of vacu o les. A conce nt ra tio n o f sorbi to l be low550 m.\/ Ied to ma ny more pro topiasts be ing resistant toos mo tic lySIS .

The mean volume of o ne cell was est imat ed to oJ. pI.Puri fied pnuopiasts had a mean vo lume o r only ISpi ( SE

~ ..l in ':0 experiments over I yeart . i.e . .:~ '~'O o f the mea nvo lume o f ce lls . Th ere was a broad dispersio n o f the in­dividu al volumes in protoplast suspe nsio ns (Fig. 1l.

Comparison vi (he C: Onft!nI of cells and vf protoplustsThe conte nt o f pro roplasrs was com pa red to thar o r cellsCrab . 1), The re was no diffe rence for proteins and sug­ars. Pro toplast s co nta ined :'0% less ~ Ig:> and serpcn-

150_ pr otoo last s Imean : 18 01 ~ o

~) va cve te s trnean e 9 61

100

\ 20~

u

•,sr•c-.

o1 " 11 28 35 11 ' 8

vct u m e l pll

Fi ~ . I , Volumes of prc roplasts and isola ted vncuoles from C.roscus cells . Data from J independe nt expen mcnts. rcpre­..cntmg :;50 pro toplasts and J50 vacuc lcs . have been cor nbmcdand .lssll!:ncd ttl9 classes ..... h...'se limi ts are ind icat ed on the X­axis. The poi nt I.:tlrrespondmg tu (he centcr \,,' '::..I,:h d :..lSs g.i\ esthe numt'lcr and frt:qu.:nc~ ,,:,( the: objects in that ..:lass.

till ;

Tab . I . Co nt e nt o f cell s an d protopla sts. Data a re mea ns with so In par enthesis ior S inde pe nden t expe rimen ts. Prot ein s arc es­pr essed as ll g- bovine serum albu min eq utvalenr s. E nzyme acuvinc s are In p kat. Suga rs are e xpressed as nmol hc xoses .

SI)IUH~

Cells Pro top tasts

Prot o plasts{"o o f ce lls)

Pro te ins ([HO

(l· Ma nno siJ ascAc id p ho sphatasePhosphodie steraseSUl!<HS\l;; iic acidCunc acidAi mal ici neScrpenuneK -M~: -

C;;,-; .

~O,-

3.1

10iO1515

3

"

~-I91 131)-1051651

:-195 (.~o2 )

715 C Q7 j3 100 t :1 00}1930 1·,n0 12ti H l51

0.336 (0.:6; \0.159 (0.09: )oSOO ( IOOO l:60 16(\\172 (45)-100 (.1tiO)

-1N C11 ~ )

:::1 7 10'; 1185.'1 3:0 )3 ~r, l l 191

3500 (311l1())()XOCOO )112 (44 )

0.155( O. r:: 10 .113 (0 .073)3:00 (7:'0 )

2111 19:' lM ll ~l

51 ( ~3)

105 C O)57 ( 131i\ (111-Itd I4 )

111.1csJ

..10 117 15..1 (::7)-1H1::\s; (I ll)J -;' (13\."j ll lH)J S (3)

I':( J)

Tab . 2 . Co nce nt ra tio n of solu tes in cell s a nd p ro topl ast s. Dataarc mea ns with so In parenthesis at the expe rime nts in Ta b. 1.In o rd er to pr eserve electroneut ralit v the co nce nt ration of anunkn ow n mono....alent ani on . which includes P, and th e ani oni csites of the p roteins. was calculated fro m th e sum o f the co n­ce nt rations o f the di fferent ionic sp eci es in each material. A tthe pH of the cell sap . near 6.0 . ma lic acid ha s 2 ne gativecharges . ci tric acid ha s 2.3 negativ e cha rges . an d the P, has 1negati ve charge .

tine than cell s. T he co nte nt of prot oplasts wa s half tha to f ce lls for ac id hydrotases. Ca-". K". mali c and citricacid . and ajrnaiicin e. The d iffe re nce was large st forNO~ . of which o nly 13% was recove red in protoplasts.

Since volume decreased mo re than co ntent. the con­cent ra tio ns o f sol utes were 2.4 times higher in p roto­ptast s than in cells (Tab. 2). the indi vidua l factors rang­in~ from 0 .7 for NO.~ to 4 .'7 for sugars . Th e data in Tab .2 are only overall va lue s that do not take in to accountthe cornpartmenta tio n of so lute s within cell s and pr oto ­plnsts. La rge variat ions in the ce llular leve ls ha ve beenobserved . mainly for sugar s. NO_~ and alka loid s. Ex­per iments ha ve be en pe rformed wit h 5- and o-day -oidcell suspensio ns . at a tim e when the inte rnal conte nt ofthe ce lls can ch ange dramat ically from o ne day to thenext (Merillon et al. 1983) . Moreover. there has been .

Co nce nt ra tion ( m M ) inSOIUh:

Su eursMalic acidCi tric acidA jmalicineSe rpen tineK·~1 g : ·

c i":·NO;A nio n

Tu! ;,;1

698

Cells

47 (3: ): 9 (0)~ . O C .: )

O .005~ 10.0(39)U.OO2-1 1O.0tIl4 1

109 ( 10)~ . 6 (1 .: ): .1 (0.-1)5.4 (5.5 )50

~5 1

Proto plasts

2 U ( 199 )..C 111)

6.6 I: .; )0.0086 (O.IX)7i)O.otli l /O . (X~4 )

1931 5S113.5 ( i .U)5.1i (0 .5 )3.':1 (";.0)113

604

over more than I year. a dec rease in th e production ofalka loids by the cell stra in. But the main po int IS thatth e re lat ive extent o f th e loss from the protop lusts hasbeen much less varia ble .

Th e total concentration of solutes was calculated asthe su m of the concentrations of each solute which hadbee n assayed plus that of an un kno wn ion req uired toscds fv e lec t rone urratlt v. Th e latt e r was found to be ananio n (o r seve ra l anio ns) whose co nce ntrat ion. as amon ovalen t species . was estimated a t 50 and 113 mAl incells and protopla sts. respect!vely. Likely candidateswo uld be Pi. HCO~ . 0 - and phenolics. T he total con­ce ntrat ion o f solutes was ~ 5 1 mAl in cell s and 004 mM inprotoplast s. T he va lue for cells ag reed with the meus­ured osmolali ty of the ce ll sap . 214 mO sm. and with theobservation that inci pient p lasmolysis occurr ed around~50 mM . The value for pr o top lasts was in acco rdancewith isotonic equilibrat ion of protoplasts wit h the sur­ro und ing med ium (560 mM solutes .. 607 mOsm) .

Why are solutes so mu ch lower in protoplasts?T he lower so lute content of protoplasrs relative to cell scan not be accounted for e ither by th e se lect ive iso lat ionof pr otoptasts with a low co nt ent (since the yield wasaro und SO% " or by th e death of a large proportion ofthe protoplasts . Viability te sts with ervthrosine Bandneutr al red accumulatio n demonstrated th at more than95% of th e pro topta sts were living. C rude mixtu re s ob ­wined af ter ma ce ration of ce lls in the presence of ee l­lulase and pectol vase co ntained the same amount of or ­ganic ac ids and alka loids as the initial ce lls. T his findin ge limina tes catabolism as a possib le reason for the de­crease in protoplast content .

The mo st like ly mechanism is a los s of solutes into theme dium . mainly d ue to the pla smolysis of the ce lls . Th isis de monst ra ted by the fac t tha t cell s processed as prot o­plasts in 550 mM so rbitol but in the ab sence o f cellulaseand pectolvase lost o rganic ac ids .. alkaloid s. K · andMg> into the extrace llula r medium whe re these ca m-

-;

"

pounds were det ecte d. The extent of the loss was nearlythe same as in pro topiasta. That losses were due to thehigh co nce nt ratio n o f os rno ticu m in the med ium was in­d ica te d by the find ing that p ro toptasts o r plasmc lvze dce lls p repa re d in J OO mJI so rbito l co n ta ine d ab o ut 20 %mo re: o f the ment ioned so lutes tha n tho se p re pa red in551.1 m .t f so rb ito l. Suc h a be ha viour agrees with the pre­viously descnbed increase in membrane pe rmeabi lityind uce d b~ plasmolysis t'Ta ylo r and Ha ll 197 6 . Smi th <,I

, 1. I ~~. Reed of 01. 1 ~'b J.

O n the other band . co mpounds part ly IIX~Hed in thece ll wall a re lost duri ng the hydro lysis su p o f proto p lastprepa ra tion. This is part icularly S'J for n-mu nnosidase ,acid phosphatase a nd phosphodiesterase locat ed bot h inthe: ce ll wall ( Mc ;";~ i l e t il l. 1 9S~ l a nd in the vacuole s.The same holds true fo r Ca" (Demarty et al. 198·0 .

T he degree of so lute loss fro m protoplasts d uri ngthe ir p repara tio n is conside rable . For mos t o f the sc lu­res studied. the exte nt o f the loss was :W~~ . But it wasno t the same tor a ll sol u te s: whereas for ~O; the lossreached 87~o . it was o n ly 20'l'O for so me so lu tes andeven ni l for suga rs. The last findin g is st rik ing in view o fthe major osmot ic co nt ribu tio n o f suga rs in the pla n tcel l sa p (Tab. 2), These d ifferences may be lin ke d to di f­fe re n t me cha nism s for tra nspo rt of the so lu tes th roughmembranes.

T he co n te nt I) r [he isola ted vacuole s ca n rh us be co m­pa re d with the content or the proroplasts bu t not withthat ut the ce lls. at least no t fo r the com pounds whic ha re lost durmg protopl ast p repara tio n. More ove r , thed ist rib ution of the sol u tes betwee n the cyto plasm a ndthe vac uotcs remai ns un o pe n question . It ma y d iffe r inpro roplus ts a nd in ce lls .

Characterization ur vacuotes

Purification vi vacuolesFro m several trials we dete rm ine d tha t the most effi­cic n t and simp le me thod was to use an osmo t ic sho ck tolibe rate the vucuoles fro m [he protoplasts, and the n topur ify the m by flotation o n J single -ste p grad ie n t. There co ve ry of vacuo tes by fl o ta tion ha s bee n o fte n usedt Bol ler and Kend e 19i9 . Mar ry et a l. 19S0. Mart inoi a eta l. l l;)Sl ). The aim of o ur p ro ce d ure was to re co ve r thevucuo les in :J. simp le medium and [0 min imi ze d ilut ionso r excessive ce nt r ifuga tio ns o f vacuotcs , whi ch couldle ad ( 0 thei r subd ivis io n (Wag ne r 19S: l.

Be st resu lts were o b taine d with fresh ly iso lat edpro top las rs (within 1 h) a t an in iria l de ns ity o f 3 -b X urpro to p lasts ml" l. v nd c r these conditio ns . after 30 minat ..1 ~ C . only I. S% ( SO 1.0 in 6 e xperiment s) of the pro w ­pl.ls ts remained int :lct. Proto pla st lysis wa s mUl:h de ­la ye d at pH abtw e i .5. a nd at pH bd o w 6 .9 the va cuol es~ en: bo unl1 ro o the r cdlu lar de bris. A p H o f 7.3 was,.:hos e n fo r the lysis mediu m . EDT.-\ was a dded to a fina l~I)m:c n t ra t i l ln of 5 m-l[ bec ause it pr o voked ag gr e gar ionLlf the debris itse lf bu t not to the vacuolcs . Fr o m vurda ta . the density o f the pur ifie d v:'l\.:uo le s wa s est imated

to be d os e to 1.04 g ml". T he la rges t vacu oles co uldev e n be reco ve red in the upp e r phas e wuhout cen­trifugation . a lte r 30 min a t I g. and with a yie ld at-outo ne -sixth o f tha t o bserv e d af te r ce ntrifugatio n.

Size, vieki and stability of tilt' ~'aclloles

The me an vo lu rne \11'o ne purifie d va cuole wa s 9 .0 pi ( 50

j .~ in 20 e xpe rime nts pe rfo rmed ove r 1 yea r ). Th is co r­responds to a mean diameter o f ::.S urn . The individua lvolumes ot vacuotes were more dispersed than those vipro topfusrs and spa nned o ve r a SO-fold ran ge IFig . I ).T hIS led to a sign ifica nt d iffe re nce be twee n the actua lmean vo lume of one vacuo le ca lcu lated as the mean ofindividual vo lu me s (9 ,6 pi ) and the mean vo lume catcu­la ted fro m the me an d iamete r (6.2 pl ), Due to the highconcentra tio n of vacuo les mr". ea 2.8 ;.< ICr ml" (so1.2L th e int e rn a l vo lume in the vacuo le suspension wasra ther hi gh . ea 2 . 6~~ (s o I .I L wh ic h was adequate forfut u re up ta ke stud ies involving flow dial ysis o r silico neoi l filt rat io n.

T he yie ld o f vacuoles alte r puri ficat ion can be ex­pre ssed e ithe r as num ber o t" pur ifie d vacuoles rela tive tonum ber of pro toplast, in it ially pre sent o r as the rate o fre covery of «-mannosidasc. a n e nzy me which in prow­pla sts is wh ol lv located in the vacuo le I Boiler an d Ken ­de L979. but ~e e di scu ssion be lo w ). From 20 muepend­e nt e xpe ru n e nrs the 2 valu es we re -C; ~~ (s o Il}l and ':0%rs o 7). respective ly . Th e difference be tween these val­ue s is du e to the fact tha t o ne pro toplas t can co nt ainmore tha n o ne vacuole. as sho wn by neutr a l red stain­ing . By combining in each ex periment th e two 'vclues fe ryie ld it was found [hat the vacuotes from o ne pro to p lastwe re recovered J S 2.0 ISO 0.7) isoiured vacuoles.

Iso lated vacuo les were sta b le. Thei r nu mb e r de­creas ed slowly a nd linea r ly to 7j "~ a lte r 5 h. Th e de­creas e was then slower since 00 °'0 rema ined aner : .:. h.T he res ults were the same at ..1"'C a nd at room te m­pe rature . Neit her 3 mJI ~tg:' with out EDT..1. nor I mgml " bo vine se rum al bumin im prove d the yie ld an d thestabili ty o f vacuotcs. The presence in the vacuole frac­tio n o f a sma ll a mo unt. estima te d to be 0.5 - 1 m.\! . ofEDTA co ming from the sam ple zo ne of the gradientco u ld as p revio us ly reporte d IKnuth et al. 19SJ ) con­tr ibu te to the st abil ity o r' the vacuo le s. T he o ve rall p ro­cedure or vac uole pr e pa rat ion took 35 min . It has beena pplied fo r more tha n I ye ar with good re prod ucib ilitya nd has bee n succe ssful ly adapted to [he iso la tion ofva cuo tcs fro m sus pe nsio n-cu ltu re d ce lls o f .-leer pseu­Joplacanus.

Purity vf l'acllv le preparmi01l5T he pur it y of va cuo le pre pa ra lio ns W JS es tim ate d in di f­fe re nt wa~ s. Bio\:'he mical m:.lrkc :"s as socia ted eith er withrhe va l:uo les o r with non-vacuo la r t.:o mp3 rt me n ts we reass :.l ~ ed bo th in proto plasts and in va cuo le prepa r::ui ons(Ta b. 3). Th e: rel.."O\ ery in the who le densi t~ g:radie ntswa s gener:.llly a ro und lOO% . enabling co mpan so n bt:·t\...·e en pro to p la sts a nd \3cuolcs. T he spe ciiil:' 31.."u vities

699

T<.J o. 3'. Purity of vacuole prepuranons. Means with so in parent hesis for N indepe ndent experimen ts. The recovery in grud ieru I"the aCII \lt~ found in the t\lo O parts o f the gradient (the vacuole phase and the rer uammg lo.... e r pna sct rel uuve to the aetl\ lty In theproroptas rs uti lized II)r the gradient. Pro te ins a rc expressed as ug bovine sc rum alb umin eq uivale nts . E nzyme acuvine s a re ev­r re, seu as pkar except for catalase ( ')' A:,a. rmn"! an d pe roxidase ( ~ .-\ .... min - I

) . A"g iv the acti\ i t ~ associ ated with the vacuolc -,from o ne protoplast rela tive to th e actl v i t ~ m one protoplast. It IS calcu lated ;.15 de scnbed in vt ate n a ls a nd methods by ul>ing ' 1 '

mannosrdase ;.I .. a vacuola r mark er and with out correction fur the residual proroplasts In vacuole pre paruno ns . Th e e nnch men t IS

tnc ratio 01 the specific acuvniev. retated to mg pro te in . in vacucle preparat ions \·s protoplast pr e purauo ns. The comarnmanon orvacuole pre puruuons by residual protoplasts IS expressed a.. the acuvu y due to thes e protoplusts rel auv e to rbe to ta l activll YIn \ '.1\.'­

uolc prcparuuo ns.

Co mpound ,; Recover , Acuvuv In ..·\ cti\'ny in vacuole preparationsin gradien t II r

o0 prorc plnsts A "0 Enrich me nt Co ma minauon due

10 protoplusts. "'0

Proteins lOS . '" I llS) IO.O I3 ...q : 1 et 51u- Mannosidase 10 Si ~ l i (6'71 lOO 1l.J (..L~ ) 1.8(1 ,5 )Acrd phosp ha tase ; 11fI 1853 (3: U) I " C 31 IJ .Q (J.31 lA HI.5)-'Phosph odieste rase S IU.:! 320 ( 1'11,l ) IIN 119 ) IJ .o lJ .3 1 I.O ( l. ~ )

Glucosc-o- phosphate dehvdroccnase • 90 9J3 (J 9QI 8.3 (1).8 ) 0.0 (ll .";I 35 C~ l

Cvtoc nro me c oxidase 3 Il l() 11 ~5 C~o) 3.31 ':.7) 0.: 10. 11 70 (531FumJf Jse - 150 111Q Itl96 1 Q . .:' 15..:! ) 1). -: 10.3\ .:3 / 2111!'..;DH·..·...toch rome ( reductave 3 SJ. ISJ. IJl\ ) 3.1< 11t h) 0.3"'.1) 0; C S Il".-\OP H:cYl. ochromc c red uctase 3 p .:; 3: 1 Itli) 1': .· I.:',1) 1 u.s W.JI 3i1 is)Cata lase 3 1" J.l (0. $ 1 10.3 CS .UI n. 7 (O..JI .0 (: 3)--Peroxidase 3 J5- In lI t)) 8.31 5.7) O.fl II U ) 511 ( 101

Tab. J . Co nte nt and con ce ntration of solutes m pu nned vacu ole s. Da ta are mea ns with SOin pa re nthes is of :S; indepe nde nt expe n­me nt s performed over mo re than I yea r. The co nte nt o r vacuoles was calcula ted euc r co rrection fo r co nrum manon of vacuo lepreparanons ('IY residua l pnuoplasts. T he conte nt of the vacuolets I from one pro toplast. A% . relative to th e co nien1of one proro­plast . was calculated as descnbed in vlatcn a ts and methods by using c -ma nnosid asc as a vacuo la r marker. Pro toplusts co nrame d: 7S (12111 nmo i phosp hate t ltr protop tastsr", at a con cent ra tion ot 15.: 13.SI m.\l . For unit s a nd ot he r condit ions . see Ta bs 1 anJ

Proteinsc- Man no sidaseAcid pbos pna tasePhosphod iesteraseSucars~ I al ic acidCitri c acidAjmalicmeScrpennncK-M c>Cl~ '

SOcPhosnhateA ni,,'n

16583oo

HIHI;;5o6

In lI rvacuole s

: 3 (1(HlOol 3Ul

102..1 (317)1il (711

I.JOO t t}Olll595 C:7t1l

70 l:7)0.03810.Q.l1l 1O.ll'l l iON: 1t·no H601

93 136\JQ 11.J)~l OlJ)

IJ S (71)

9 / .1)101112::1:';11lJ6 , : 3)6: Ib l

113 ; 151se (tJ i

-l lf 19 \2 15 IS'))

OY t 5160 j~ \

1tl3 C ::).JS , n l93 lP I

Concent ration In

vucuoles ( m M )

1531 S4); 1 IW)

S.S (: .9 )O.fJ/)J. ) (fl.fM13310.01: :; CO .f1f jI.J9)

2: 3 (J5,14.1 15.3)0.9 C .O)2.5 {] .i l

1'+.0 (J .oJ)So

5$1

per mg protein o f vacuola r mar ke rs . c -rnannosidase.acid phosphatase and phosphodie ste rase , we re higherby a factor of 11 - 15 in vacu o le prepa ratio ns tha n inprotopt asrs (Ta b . 3). In con t rast . e xt ra-vacuo lar enzym emar kers a ll ha d a lowe r specifi c ac tivi tv per rng proteinin vac uol e pre parat ions than in pro lOplasts (Ta b . 3).

V:Icu ol e purity is cla ssicall y expressed as th e pa centage of no n-vacu o la r marke rs associa ted with th e va c·uolt.:'1s) l.k rived fro m one protoplas t re la tive to th e co n­t~n t of tht: p ro to plast. It was calcu la tc d hy using a ·

il ~)

mannovidase as a vacuola r mar ker as ex plained in Mate ­r ia ls and me thod s. T he lowest contamination rat es we refrom mitochondria (3.3% for cy toc hrom e c oxidase andb .5 ' ~·o fo r fuma ra se) . Th e- con ta m ina tion rate of 3 . 8~o

for ISA D H -cytoc hro me c reductase would ind icate thi se nzym e to be lo ca ted in mito chondria ra the r th;.ln in e n·dopl;Jsm ic ret icu lum since th e conwm inat io n fro m t helatt e r W ;;iS 13 . 7 ~o as indi cat ed b\' NADPH-nlochrome credu ;:tase . Con tam ina tio ns fro m c\' tos~1 (Q!ucose ·6-ph osphate de hydrogena se ) a nd fr~m micr~hodi es

-,

,

(caralase r were ,~ IO"~ . For peroxidas e the contomma­no n level was 3.3%. T he contamina tio n rates by extra ­vacuola r marke rs co rrespo nded to a 1l.1- .= .tJ"'r) contarn­inatio n of the vacuo le fracuon by the sample zone in thegra die n t. compar able to that calcula ted for Nycod enz in'-IJte-nah an d methods II wo ).

The vacuo le prepa ra tions still con tained J re sidualnum ber of pro roplasrs. 1. .J 0~ (s o 1.0 in : 0 exper iments Irela tive to the num ber o f purified vacuo les , Assumingthe ir co n te nt to be ide nt ica l to tha t of the stock prow­plasts , these few protoplasts accou nted for as muc h as.= 1" 0 of the proteins in vacuolar prepa rations . 3 (~35~~

1) ( giucose-e-phospbate de hydrogenase and NA D PH­cytochro me C red uctase . .J3-50oo of NADH~cyto.

chro me c redu ctase. ca talase , peroxidase and fumarase .and 70~'~ of cytochrome c oxidase (Tab.S). This unde r­noes the importance of disrupting 100% of the prow­plasts. if possible. during the osmotic shock 1)( o f elimin­cung pro roplas rs dur ing vacuol e purification. The con­tent o f the pu rified vacuoles analyzed below wassystematica lly co rre cted to r the prese nce o f the res idualpro roplasts.

Vacuole contentThe co nte nt of prot eins. sugars. organic acids. alkaloidsa nd mine ral ions in vacuo les is indica te d in T3b. .J. Fro mdia meter and hence vo lume measurements we co uld es­tima te the to tal concentra tio n of so lutes within the vac­uoles . Th is was calc ula ted [0 be 581 mM . including ;~6

meq . of one o r mor e unknow n anions (Tab. .J) . whichwus near ly ind en tical to the to tal concen tra tio n in thepro top lasts (Tab . 2 ). This de mo nst rates the iso to nicequilibra t io n o f [he vacuo les wit h their sur round ing me ­d ium. It also validates our co ncent ra tio n measure­ments.

By using c -mannosidase as a vacuo la r marker . it waspossible to estimate the ex tent of the vacu olar local­c at ion of solutes within pro to plasrs. The accuracy ofsuch calcula tions dep ends strongly on the exclusive V:l C·

uo lar locatio n of o-m annosidase in prot oplasts . Becausev i its presence in the cell wall ( :-' fc~~ 1 1I et al. 1984), itseems like ly that secre to ry vesicles in the cyto plasm alsocontain this enzyme . However. we have o bserved thatthe vccuoles which wen: recovered after no tation for 30min at I g con tained exact ly the same amount of a -ma n­nosidase and had the same size as prorop lasts . Mor e­over . the ave rage volume (If vacuolcs iso la ted afte r ce n­trifuga tio n at It>Og was abo ut half [hat o f prorop las ts.which agrees with the: value of 2.0 vacuo les liberated perpro to plast. This suppo rts [he use or u-rna r mc sid ase as avacuo lar marker . However. one sho uld be: aware thatlarge unce rtainties exist as to the size of cyto plasmicpools calculated simply as the diffe renc e between the-:onte: nts of the protopl:Jst and the \"acuok . piln icularly(o r "lo lutc:s with a prdc:remi:.tl vJocuo lar locatio n l l eighj t)SJ L Finally. a sck l"tive loss o f so lute-s may also occurd uring the iso lation v( vacuole s. :.ts was rcpone:d by

l.eigh and De n Tomos lI QS3) fo r K - and Na " but notfor be tanin during the isolat ion o f red beet vacuc tes .

vacuo les o f C. roseus co ntained o nly small amo untsof pro teins. ea 23 ug ( ltf vacuolcs r'. Vacuola r pro teinsco nstitu ted 9°~ of the proteins in pro ropla sts. This vuiueis in the same range as thos e previ ou sly reported ino ther ma terial. be tween 5 and 16°~) IBo ile r and Kcnde1974. Maru no ia et al. 1981. Wagner 1983), Acid phos­phatase •md phosphodi este ra se had the same' localiza­tion as « -mun nosid asc . On ly abo ut 6'=oJ~ of the sugarsappeared to be loca ted in the vacuol cs . This is o nly anave rage value since significant differences have been re­ported in the cell ula r dist rib utio n of mono- and disac­cha rides (wagne r et al. 19831. Suga rs co ntributed mar k­edl y to the osmo tic potential o f the isolated vacuolessince their con centration was 150 mJ-t I this was a maxi­mu m value corre sponding to all sugars prese nt as hex­oses ).

Prelim inary assays ha d shown that malic a nd cit ricacid were the main ca rbo xylic acids in C. roseus cells.Th e bulk of malic acid in prot oplasrs W J S recovered invacuoles (l 13% ). and nearly all cit ric acid ,S6% l. inagreeme nt with previ ous results te .g . Yam aki 198-1 \.The concent ra tion of malic acid . i 1 m.tr , constit uted12'\ ) 01 the concentr atio n of total so lu tes in (he vacuole .The conce nnauon of Citri c acid was lowe r. around -i.Smoll .

Ca" and Pi were fou nd almost exclusively in vacuolesat concernrano ns of 6.9 and l-r .o mM . respectively.A bou t 68 ~"o of K· and .\1 g~ · we re in the vacuole s , atconcentranons of 223 and l.J.I m.\ / . The results for theminer al catio ns agree with those of Lin et al. (1977).The mean vacuo lar co nte nt of NO~ was .J5% of that ofprotoptasts. The ind ividual resul ts for ~O;- de pendedupon the Jge of the suspensio n. In 5 to o-day-o ld sus­pe-nsion s of our cell strain. intracel lula r .sO; is rapidlydecre-asing ( ~ I ~ ri llon et al. 19X3). Pro top lasts from 5­day-old suspe nsio ns contained II:! nmol :"0 ; t li fpro roplasts r" . of which 97°~ was in the vacuolet s}. At 6da ys. there was 38 nmol ~O; (Ur protoplasts}" and34"~ in vacuole s. ~O,- is prin cipally sto red in vocuoteswhe n present in large amounts {Martincia et al . 1981)but the vacuo lar pool decreases mo re rapidly than the­cyto plasmic one du ring starvanon .

The two alk aloids ajmalicine and se rpentine garve dif­ferent results. Wher eas only ..J I"'0 ajrnali cine was asso­cia ted with the vacuoles fro m one protoplast. as muchas 215% serpentine was vacuo lar . Losses from the vac­uo les are likely for ujmulicinc which ca n diffuse throu ghmem bran es ( Rcnuud in et al. IllS:' ). Fo r serpentine , thevalue or 2 1 5 ~/o (so SO) is apparen tly ab e rra nt. It was themean of to indepe ndent experirnerus pe rforme d over1.5 yea rs. with extre me ..alues be ing I ~j and 321% .

Is vacuole isolation and purification a selective proce­dure?The extrao rdinary levcl uf serp e-n tine in puri fied \"3':­

uuks rela tive to prot op lasts was no t d ue to the biosyn-

7tH

the... ic. of se rpentine durin g. the iso la tion of va cuoles.-incc in 10 experiments the recov e ry of se rpen tine inwhole gradie nt s was 49 °/0 ( SD 13l. The hypothesis thatthe pro ced ure o f pro toplas t lysis and vacuo le purifica­tion ha d sele cted a class of vacuoles was co nsid e red.

Indeed. we have already rep orted a la rge va ri a bilityot va cuol a r pa ramete r!' suc h ;IS p H or alk aloid co nte ntin po pulat ion s of cell s. prot oplusis and isola ted vncuolcs(Brown et al. IlJS·L Kurkdjian er al. 19x51. The range ofse rpe nti ne lev el s in a pop uianon of pro toplas ts has beer:sho wn III be ~5 · fo l d (Brown et al. JlJS~ ) . In the face ofs uch var iation the overa ll yiel d o f purified va c u o le s ,

':O'~~ . rais es th e po ssibi lity of th e sel ection of a spec ialc1:I,",S of vucuoles: th ose with i.I hig h se rpe nti ne co nten t.T he rclauonshrp be twe en the ...crpcnnne co nte nt andth e ab ility o f vac uo le- to susta in p:.r rificnuon remainsunknow n. It nl ;Jy he releva nt that the vacuolc s wi th ahigh ..e rpe nu nc conte nt have a n ac id ic pH (Brown e t al.Ilt;-.,~ I a nd tha t the pu rificat ion procedu re we used alsotavou r-, t h e' isolanon of the large r and less de nse \ <l.r ­uole s .

Dcu ..-v c um an n and Zc nk I IYSJ I re poned differe ntresult s to r serpentine in protoplasts and vacuoles of C.r O.\l' I I5 . T hev estim at ed t h ~11 o ne pu rif ied va cuol e co n­tamed Q(I~ o Ili the serpent ine of one pro top last . wit h 3p·prox ima te ly o ne vacuo le libe ra ted pe r prot oplast . Thelack ot indica tion s abou t the yield o r punfied vacuoles ,..IO U ma jo r diffe re nce... in the procedures t>l vacuo le im·lan on 11 .~ . vocuole-, we re recovered in a st ro n ~l y sa linemediu m . 550 m.\! S aC I. afte r sed ime ntatio n and the nflot at ion i hind e rs an y furt he r com pariso n 01 thei r re­su ns with o un-.

Cond usion

isoi a ted VJCUI) k'S a re goo d too ls fo r stu dyi ng th e pro·t~i n ::; a nd the trunspo Tl sys l em~ ot' the to noplast. a nd theenzymes wi th in the vJ.cuol es . For such use s the re is aneed fo r the recove ry o f a I<.lrge numhe r o f vacu oles withsa:isf:Jcto ry purity. Kno wledge of conte nts and co ncen·tr at ions for mo st solu te s a llows c:.llcula t ion of conct:n­tration t: rad icn t~ be tween vacuo les and th~j r sus pe ns ionml:diu m '.md provid es a solid bas is fo r th e study 0 1 t ra ns­pOri s~stcms . These diffe re nt aspects o f the sa me topich :..J \,I': heen eXlensi"dy a nalyze d at th e: quanti ta tive lcvdin tht: pres e: nt re po rt .

TWll new oh se rva tions ha' -e heen muoe In the: presentst ud~ . The: first arose from comparison of th e sol utecOn!l: nt of the pro top las t:-. nOl I)nly with ~ hat o f the \ '<l C­

lIolcs hUl a lso WIt h th e: content o f the ce lls from whichth ey de rived , This reve aled massive but sdect ivc losse sof so lut es fro m the pro toplasb during th::ir pr ep ara t io n .m;.t inl y dur in g pl;.t smo lysis. It clearly affected both pias­

m J lemma and to noplast. A pre\ iou s re po rt :lDo ut the1.)ss o f sul ule ... in isola ted vacul) lcs derh 'ed from who let i s~ u t"s (It.· i~ h ~IOJ Deri Tomo~ 14S.3 ) could be re la ted tothe sa me ph e n'Jmenon . out thc possibility of add ilionalan d selt:clivc lo ss. e ,g. o f a jm a licine , d urin g the iso-

lauon of vacuo le.. from pro ro plasrs remains ope n. Thesecon d o bservatio n is tha t so me se tectivuv co uld occu rdu ring vacuole iso la tio n and pu rifica tio n .

These two poin ts bo th indicat e tha t co nside rab le cau­t ion mu st be exe rcised in ex trapolating trorn the con­te nt s or iso la ted vacuoles and pro toplast s to g:a in insightinto sol ute co mpartme ruauon in pla nt ce lls.

A("k1lf Jl\ in/~flll('lIl- We thank P. M uller for excellent technicalassistance ,

References

Aebi . H. 11,1";'-1 . C:J I:J I :h~ . < ln Met hods 01 Enzvmauc AnalvsrsIH. U. Bergme j er . cd . r. vot . : . rr. 07:.;......0;\-1 . VcnagCherme. Weinncrm. ISB:-': 3·5':'-: 53'U· :\.

Bar bic r-Hrvcoo . H.. Gic ra r. R .. Re naudi n. J. P.. Brown . S.C. Pr:Jj r..: r. J . \ 1.. Griuncn . C S: G uem. J . Il,IS5 . Mem­f-ran c po tenna l di ffer ence of isol a ted plant vacuolcs : po si­me: o r n\.'I: ;1I1 \ I: ' 11. Com o...m son of measurem ent s wn h mi­croclcctr odc-, an o :.:IlIOI~ic probes. - Brocmm. Biopnvs.:\CI.1 ," I ~: : I:,-::J

Burtuc r-Brveoo . H.. Renaudm . J, P. ~\: G uern . J. 1':1":'0. Thevacuola r -mcmhrano: 0 1 plan! cells: a new-come r in (he: field01 bIOio!.!JC;l1 mcrnr-ranes. - Bioch irmc 01\: JIi-CS.

Bcnsad ou n . A . .,\: Wcmstctn . D , 1<)76 . Assav 01 prote ins In thenrescncc 1) 1 intertc rme molecules. - An al. Biochem. , 0::J J -~O. -

Bo llcr , T. IUS: . Enzvmanc equ ipme nt of plant vacuolcs . ­Ph\ sit11. Vee . ~ l l : :~ "7_':'':; ' '

- ...\: Ken de . H. 19-:' ''' . Hvdrolvnc enz vmes 10 the central vac­uo le III plant 'elk - Plan! ·P!'l\"si.)I.· fl,3: I l :~ I 13': .

Brown. S. C . Renaudm. J. P.. Prevor. C .s.: G uern . J. ] QSJ. .FI<m cvtomctrv and so ru na of plant prot o piasts: technicalpro blem.. and nh\ sioio aical results trom a "lUd ~ ' of pH andall...a io llh in C ifil ,lr.lIlIII1H cells, - Pl1\ SlOI. Vec . 22: :'-11 - 55.. ,

CI)t ' [I;: ~. T G . &: Beevcrs, H . l'!Ii'l . ~titochondna a nd alvoxv­~Jmc" rrom castor bean endospe rrn. Enzvrne consut uenrsand ..:at<.ll \tlC capac:!\". - J. Bioi. Che m . ~~ : 350i- 3513.

Dem~ rt \ . ~l ~ . ' tnr~'a n . ·C . &: Thellie r . \-1. 19.~ . Ca lcIUm and{he cell w.:I lI: a reVle" . - Pla nt Ce ll EmJr on . i : -Wl-1-l S.

Deu~- S~umann . B _& Zenk. M , H , l~~ . A hi£hlv selcct i\'e al·ka llJld upt<.lke syslem In vacu[)le~ of highe r ·p l ~ nts . - Planlalh: : 2:'O....~hll _

Fr~h ne r . \ 1.. "-d k r. F. & Wicntken . A . l Q~-I . LocaJiz:Jt iol1 offruet..n m l."ta boli~m In th e \ aCuOk s isol::ned Iro m prolO­pla<"ls (If Jcrus.:.J h:m J.nichol.:~ tubers' HcliiJ1ltilL/s woewsusL. 1. - J . Plant Ph \ SIOI. 110: IYi- 211S.

G am ho r;;. O . L. . ~ li ller. R. A . & Oj ima. K. 14hS. t"ul rie nt re­qui rcmc llls o i suspensIOn cull u re~ of ~('~b~J.n rOOI cells. ­E.x!" , Cd l Rl,,'s . 50: :::: I- SSt'! .

G Ulm'Jnn . 1, .s.: \\·uok k id. A . W 197.1 . L (- l ma im:: d::termi­nati on wilh malal e d r:hvdr ocen <.lse :Jnu :"AD -Ill ~1 e l hlld s

\)1 EnnmatlC A nal' sis " H . l.'. Ben!.ml."\'Cf. ~d . l. \ '01:., pp.15S5-1~:N . Vcr i OJ ~ Chc mie . W~mh~· im . ISBS 3-S:i-: 53";1l· \. . -

H \)J ;; r: ~ . T . K. & Leon;!Tli. R . T. : 1,\;.1. Purific.:l. t ion of r lasTa ;Jm~mhrane - r'ound :J d~ no~ i n l." triphosrh.:l.U~~ fro m plantroo lS. - " i l." thod.. Enz, -mo i. 32: 3Y:~()o.

J llncs.:--'1. G . K.. OUI lJ.\l. ." \\' . H . J r. 8.: Luwry. O . H . 1077. En ­l ym l": :Jssay 01 to ·· to Ill ' :' ml)k s of "UGO')" in pla nt (i ssue~ .

- Plant Ph\"siol. 60 : ;':'O-) S:' .KnUlh. :\1. E.: Ke ith. B.. G arl.: . C . Garcia · ~l ;,\ rti nez . J, l. &.

Rappar 0 rt . L. lYSY . SI3bilization and tran~rort capacity ofCO\\r ca :JnJ ba rley \.Jcuolc~ _ - Plan! Cdl Physio l. :.a :-1: 3­.a3:.

Kur j.,J jiJ.n , A . . Qui4u:Jmpoi x. H., Barbi C'r · B ry~oo . H .. Pb n.,\ 1.. ~la ni t::J u l t. P. &.. Gucrn . 1. IIJS5. Crilic.J1c\"alu:ui() :t ofm~lhod.. tu r es[im :J t i n ~ (hI." \':Jl.:uol:Jr pH t I t planl (,:db. -In

Bio c hcmist rv an d Function of Vacuol ar .A.TPa, e in FumnJ.nd P!..lnl') i B. vl.mn . ed.t . pp. 9:~- 1 1 3. Sp rmger-Verlae .Berlin . ISB:\ J·5JO-i5:h7- <.) .

Lcian . R..-\ . 1 t.,\ ~3 . vt ethods , progres s a nd poten tia l fo r the use»f iso iarcd vacu cles in studie s ot solu te tra nsport in t ughcrplant cell s. - Phvsio l. Plan ta rum 57: .}40- 390.

- &. Den Tomes . A . iYS3. A n attem pt !O use iso lated vac ­uolc s to de te rrmnc the d istri bution o f sodium and oota s­s.um In ce lls o f sto rage roo ts 01 red beet 1 8~ttJ l'fl fga .~is L. I.- Ptanta 15',): ":'h9-J.75.

Lin. W .. waener . G . J. . Siezelmar m. H . W. ..\: H ind . G , 197; .Mcm orane-bou nd AT Pas-e- 01in tact vacuo lcs .JnlJ ronc o la ... rsisolated from ma ture plant [iss ue . - Biccbim. Bi..; phys.Act a -105 : 110-1 17.

Lobr. G . v.... & Wailer . H . D , 1976 . Gluc ose-o-pbosphate de­hvd roee nas e . - In vleth ods 0 1 Enzv rnanc An alvsis IH . U .Bergmevcr , ed. t. Vo l. ~ . pp. o.3~. Vc rl ag. Ch erme.Wemheim. ISBS 3·,5::!7'::S3iU-X.

\Ic~eli. \ 1.. Darv ill. A . G ._ Fr\.'. S. C. & Albersheim. P. A.1'olS-J . St ructu re and function o f the primary.. ..:e- IJ walls e tpla nts. - Ann u. Rev. Biocbern . 53: 025-663 .

Muriec . G .. Bouvssou . H . & Belkoura . ~t. 19S5. Vacuo lar ei ­tlux o r' malate and its in fluen ce on nitrate accumula tion in­Catha rasuhus roseus cel ls. - Plant Set. 39: >Ji- 103.

Maruno ia . E.. He ck . C . & Wicmke n. A . 19S1. Vacuole s assto rage com pa rtme nts fo r nit ra te in ba rley leaves. - Nature':s~ : :~>':~3 .

- . Fluese. F I.. Kaiser . G.. Heber. G. &:: Hek it, H. W.t 9~5 .- Enc rgy -depe nJe ne up tak e 0 1 mala te in to vacuolesrsoia ted tr o rn bur tev mc sophyt l protoplasts. - Biocb im. Bio­pnys. Acta ~Oh : '::- 11- 319.

~ l an y . F.. Branton . D. & L~igh . R..-\. !IJSO. Plant vJcuo les .­I" The Bio..:hemist ry 0 1 Planes. .-\ C .>mprehensi\e TreatIse( :--; . E . To lben . ed .1. Vo l. L pp. 0':::-055. Academic Pre ss.:--;ew Yo rk. (S8S IH ::-oi 5J.()I -: I \ . l) .

\ I~ nl ltm . J. .\ 1.. Che nieu l:. J. C. .t Ride:lu . \1. IllS3. Cin­clillUl: Je croisSJ OI.:c . ~ ·.'oi uuon Ju metabo lb me glucldo­azote ;: t J ccum uia uo n :.lkaioldique dans une su...penslon cei­lulJ.ire de l -,ltllarcmtlllts rouus. - P!anta \led. l i : 169-176 .

\ lullerin\.! . H . ..~ Gnihe r. w. 1906. De te rm ina tion of cit r:.Hewith ~i t ra te lvase . - A nal. 8 iOl.::hern . 1'j: 369-370.

O hnishl. T. . Gal l. R . S. &:. ~ laye r . \1. L. [1};5. A n im pro ve dass:lY o r ino rganic phosp h.Hc in the i' rese nce 01 extra la bllepho..ph;lt~ compo unds: Jpplic:ltion to the ATP:lse Jssay In

the presence or' phosphocrea tine . - Anal. Bioch ern. ()9 :2h l -:bi .

R~:.td . S. \1. &:. ~onhcote . O. H. 19S1. " linimiz:.l tion of van ­ation in the response to different protems of the Coo­massic ·hlue G dy::·bi ndi ng assay to r proteins . - A nal. Bio­che m. 110:5~.

R~.:d . R. H .. Richa rdso n. D . L. & Stl::war t. W. D . P. IOS6 . O s-

Ed it~d by P. :"lis~n

m.HICrcvponsev Ill' urucc llular blue ercen alcae i C vuno bac­tc rta t: cna nee s in cei l volumes and !n l rJ.cc !lu!.::. r -ojute , lcv­cts 10 re spo nse 10 hvpc rosmonc trea tme nt . - Plant C :!1 En­... iron. <,I : 25- 31.

Ren aucin. J. P, ! l).~5. Ex tractio n and Iluo rirne mc de tecti o n Jr'­ter hi ~h performance liuuid chromatogrupbv et mdole alka­1. ) j J.~ fro m cultu re d cells 01 Cutharanthus rosens, - Ph vsiot.V;:::. '=3: 3RI- 3:';S. .

- . Brown. S. c. &: G ue m . J. 19S5 . Cc mpart me ntanon o r' JI­caiouis in a cell suspensio n o f C.uharar uhus ro u:".s : A reap­p ra b J.1 0'- [he rol e o f pH grad ients. - In Pnmarv a nd Sec­o nda r-.. vteta oofi sm of Plant Cell Cultures [K. H . Xeu­mann . W . Barz and E . Reinhu rd. eds j. pp. 1 ':4- t 3~ .Spnnge r-Vertag. Be rlin. 158 ;-.; 3-540-15797-2.

Smith . .\1. A . L. .-P:lIta . J . W . &:. ~kCown . B. H . 19S-l. Themeasurement of Isotonicuv and mamtenunce o r' 0SmOI\Cbala nce In plant protop last ma rupulanons. - Plant Sci. Len .33: :J~:5S.

Stafford . H..--\ . & Bravinder -Bree . S. 197: . Peroxidase iso­~~~~~~~ first ir ue m od es 01 so rgh um. - Plant PhySIOI. l l} :

Struck. D. &: Sharrna. V. t 9~5 . Vacuo lar localizatio n of the en­zvmaric svnrh csrs o f h.....droxvcinn..emic acid esters 01 malicacid in p rotoplusts rrom Raphanus $(J. tl l'ilS leaves. - Physio l.Pluruarurn 1l5 : J5- 5tl.

Tak ed a . K . Ku r ~J j ia !l . A & Kac o . R. T . (''' 'is. Ionic en.m ­neb , ro n transport and ei.mt cell me mb ra nes: oo tenna i ap­pncan o n 01 the parchciam p technique . - Prot o piasrna I ~i :

Il7- 1()': .Tavlor . ..\,. R. D. .& Hall. J. L. 197h. Some phvsro logrcal prop­

.:ra es .)[ proco pias ls i ~olated from maIze J nJ lo bacco tlS­sue.:s. - 1. Exp . Bot. ~7 : 3:-i:-.Nl.

Th iba ud . J. B. &: G ril!no n. C. I t,J ~l. \1e chani sm of nur ate up ·tak e: in co rn roots ~ - P!~nc S..:i. L~~t. ~2 : ':':"4-: :'\9 .

Thorn . \ 1. &. .\ Iar..:tzki. A . 1 1:l~5 . G ro up tr:1nsl0Ca tio n as amcduOlsm {HT sucrose tr.:l ns te r into val.:uoks from "lllZ ar·can~ ..:e:ls. - Proc. :--;a[1. AC:Jd . Sel . CSA S: : .lil-J-:----4;oi.

Wa~ncr . G. J. t91'C . C",mpan ment.:u io n in pianI ;:.:lIs: .he roleo r' the ~ acuole . - Recent Ad\". Ph \toche m. 11'1 : t-15.

- t ~:'lJ . Highe r pla nt ....acuo les and con\)plas t. -In h o latlOn of\kmbr:mes ':1Od O Tlzane Hcs from Plant CdIs 11. L. H.:J II .ed .1. pp. ~3- 1 18 . ..\c:JJem k Press. Lonoon . ISI3:'-i 0-12­j l ~S20-:.

- _ Kclle r. F. & \Viemken . A . i983. Fructan meta bo lism ince reals : Inductio n in le 3.ves and co mpa n menIa tion in~~~top iasls :lnd ..acuo les . - Z . Ptlanze :1physioi. 1I:: 35~~ I _ .

Y:.lrnOl ki. S. t9SJ. Iso latio n of V:.lcuo les fro m imm;.J[ ure Jp p lc{ru lt 11e<. h ,1nd co mpart mc ntatio n u t' SUlw rs. o r'!:.lnic :.ldd'i.pht.:nolic compou nJs and am innacids . --Pbnt Ce!1 Physiol.::S: l :: l - l btl .

703