Chilling temperatures (0-15°C) disrupt the metabolism of plal~ts, res~;!fir, g in abnormal growth and development and, often, in the death of the plant. ~ensitivity to chilling is a major constraint in the production and cold preservation of crops, bttt the metabolic origin of sensitivity to chilling is not clear and, consequently, the phenomenon is not easily controlled. However, recent data suggest that chilling may destabilize the assembly and function of biopolymers by disrupting macromolecule-water interactions. A similar explanation may account for the altered properties and quality of food crops that have been chilled.
Chilling temperatures lead to an array of metabolic abnormalities and injury in plants ~, manifested by sur- face pitting, discoloration, waterlogging, growth failure, impairment of development and, importantly, suscepti- bility to disease 2. Severe chilling stress may result in the death of the plant due to disease or to failure of tissue metabolism. Sensitivity to chilling is found in the vast majority of tropical plant species and in about half of the crops from temperate regions 3'4. In temperate regions, sensitivity of crops to chilling restricts the range of cultivable plant species, limits the production season of cultivated crops, and presents an economic risk in the event of intermittent chilling. Sensitivity to chilling frequently precludes the cold preservation of harvested commodities, thereby limiting the distribution and production of the majority of tropical crops.
Current explant4ions of sensitivity to chilling The study of chilling and the search for a molecular
explanation of sensitivity to chilling dates back more than a century ~'r'. At present, one hypothesis attributes sensitivity to chilling to the temperature-induced phase transition of membrane lipids from the fluid to the non- functional gel state 7 and, by extension of the hypothesis~ to the abundance of high-melting membrane lipids". This hypothesis implies that temperature sensing is due to the crystallization of lipid molecules and that the effect of chilling is transduced through the resultant membrane dysfunction (e.g. disruption of cellular cal- cium gradients)L An alternative hypothesis suggests that proteins and enzymes become labile on chilling, appar- ently by the weakening of hydrophobic interactions t° (inferring a wider range of non-specific, temperature- induced destabilizations of biomolecules). However, the fundamental question persists: how doe~ temperature modify the behavior of biological matrices.'?
*Publication D-12140-17-90 of the New Jersey Agricultural Experiment Station. Supported by State funds and by the Center for Advanced Food Technology (CAFT).
Chaim Frenkel is at the Department of Horticulture, Cook College, Rutgers - The State University, PO Box 231, New Brunswick, N] 08903, USA.
Disruption of
macromolecular
hydration - a possible
origin of chilling
destabilization of
biopo[ymers*
Chaim Frenkei
in addressing the effect of temperature on biological matrices, it is assumed, a priori, that the behavior of biomolecules reflects their organization, as clearly shown by the structure-function relationship of proteins and enzymes". This concept is implicit in the membrane hypothesis 7, which suggests that chilling induces ordering of membrane lipids from the random fluid to the non- functional crystalline gel state. However, this temperature effect is not readily applicable to the destabilization of enzymes and proteins by chilling L°. Furthermore, the mechanism of the temperature-induced molecular
i20"
E
100"
80
60-
40-
Liquid crystal
B ' ' |
Number of water molecules per lipid molecule
Fig. 1 Hydration and temperature dependency of the ;)hases of dipalmitoylphosphalidylcholine. Adapted from Crowe and Crowe 14.
reorganization may be indirect; chi'lling may disrupt macromolecular hydration and, subsequently, hydration- dependent assembly and function of biopolymers.
Importance of macromolecule-water interactions Folding and, consequently, function of proteins are
acknowledged to be inseparable from the interaction of the macromolecules with water. This perception was re- cently extended to the organization of nucleic acids ~2. Not surprisingly, the same hypothesis has been used to explain the assembly of lipid-rich biomembranes ~3. A hydration shell on the lipid hydrocarbon core in the membrane interior leads to the repulsion of neighboring lipid molecules, resulting in their mobility and, hence, in a disordered fluid state, Dehydration may abolish the in- termolecular repulsive forces, resulting in the coalescence of lipid molecules (due to van der Waals interactions), and their subsequent solidification ~3. Phase diagrams (Fig. 1) show that the temperature of the transition from the fluid to the gel state is lowered dramatically when
(a) ..:.v): .'.!..':;'~ ".7-: . ; :
I Partial dehydration
I :.~S : :
I Further dehydration
1 ...~ . . . , . . ,., . : , .
(c) !~;:~ ~'i~::: "~";
l Transition of phosphatidylethanolamine
to hexagonal phase
Fig. 2 Schematic diagram of the organization of biomembranes as a function of
hydration state. In hydrated membranes (a), the inner membrane components are separated and are in the fluid liquid-crystalline phase. With progressive
dehydration (b,c), some components coalesce, undergoing a transition to a rigid gel state. Progressive dehydration leads to lateral separation and exclusion of fluid components, and the membrane rearranges (b). On further dehydration,
other components enter the gel phase (c); in dehydrated systems, phosphatidylethanolamine tends to form an inverted hexagonal phase.
Adapted from Crowe and Crowe TM.
anhydrous lipids become even slightly hydrated ~4. Apparently, the hydration-induced intermolecular repul- sive forces mitigate the effect of decreasing temperature on the fluidity of lipids. Hence, the melting of lipids is not simply a function of temperature, since it is intimately related to hydration state.
The influence of water on the assembly and function of lipid-enriched membranes raises the possibility that temperature-induced changes in the aqueous micro- environment of macromolecules may mediate the chilling effect. A decrease in temperature is accompanied by alterations in the properties of water, including increases in viscosity and dielectric constant, and a decrease in the hydrogen ion concentration 15. An important feature is the development and strengthening of hydrogen bonds in the bulk solvenP 6. Because solvation, entailing the structuring of water at macromolecule-water inter- faces 17, occurs at the expense of hydrogen bonds in the bulk solven0 8 and, moreover, is energetically unfavor- able ~9, the chilling-induced strengthening of hydrogen bonds in the bulk solvent may restrict the apportioning of water for the solvation of non-polar substances. Hence, the lowering of temperature in a sense diminishes the hydration of hydrophobic substances, and leads to their partitionipg and rearrangement in a separate phase. In lipid-rich m=mbranes, anhydrous conditions and the resultant coalescence and crystallization of lipid molecules lead to gross and deleterious alterations in structure (Fig. 2), with subsequent dysfunction. By analogy, membrane lipids deprived of their hydration shell by chilling may interact and rearrange to a rigid gel structure, preventing appropriate assembly and membrane function.
In proteins, folding appears to be driven by hydro- phobicity u, which is generally perceived to originate from the structuring of water around apolar groups ~7. Changes in the hydration state of hydrophobic domains appear to be important in determining the stability of proteins 2°, and the lowering of temperature may, by altering the degree of hydration, lead to molecular destabilization zj. However, the relative importance of the roles of hydration forces and of other interactions in hydrophocity-driven protein folding and stability await further clarification 17.2o.2~.
Based on the suggestion that chilling induces a reduc- tion in hydration, it may be proposed that plants offset the effect of chilling by augmenting the hydrophilicity of temperature-sensitive biopolymers. It is well known that chilling leads to the accumulation of polar lipids (e.g. glycolipids) in membranes 22. Another feature as- sociated with chilling is the enrichment of membrane lipids with polyunsaturated fatty acids 22. Although the role of such changes is ascribed to lowering the melting point of lipids arid, hence, the avoidance of temperature- induced lipid rigidification ~, such compositional changes may relate more closely to adjustments in the affinity of iipids for water; unsaturation increases the polarity of lipids, and polyunsaturated fatty acids in the c/s con- formation form clefts that 'cage' water 23. Unsaturation also renders lipids susceptible to oxidation, which
40 Trends in Food Science & Technology February 1991
Table I. Putative changes in biopolymers resulting from chilling. induced disruption of hydration state
Biopolymer
Biomembrane lipids
Proteins
Nucleic acids
Polysaccharides
Presumed changes
Coalescence and rigidification of the lipid hydrocarbon core
Diminished solvation of hydro- phobic domains and refolding of the macromolecules
Not known
Increased crystallinily (retrogradation) in starch gels in vitro and, perhaps, in vivo
clearly increases their polarity. From this perspective, lipid oxidation associated with chilling may be viewed as an anti-stress mechanism, rather than as an origin of chilling stress ~.
Support for the hypothesized role of chilling in ",alter- ing the hydration state and, subsequently, the destabiliz- ation of biopolymers may also be found in the proper- ties of chilling-related proteins. Cold-regulated genes encode the synthesis of highly hydrophilic poly- peptides 2s and, similar to the chiUing-related com- positional changes in lipids, the role of such bydrophilic polypeptides may be to ret,~in the hydration state of chilling-sensitive macromole.~ules. Interestingly, the synthesis of cold-related proteins appears to be induced also by desiccation26; conversely, the production of another family of highly hydrophilic proteins that normally accumulate in seeds prior to desiccation 2~ can be induced by chilling 2". Apparently, cold- and desiccation-related proteins are produced interchange- ably in response to chilling or drought, suggesting that they have the common function of augmenting the water affinity of desiccation- or chilling-sensitive biopolymers (perhaps cell membranes).
Sugar accumulation ~ is apparently an analogous consequence of chilling. When undergoing dehydration, organisms produce large amounts of sugar, which pre- sumably occupies vacated water sites and prevents coalescence of anhydrous molecular domains ~3,'9. In chilled tissues, sugar accumulation may have a similar function: preservation of the structure of biopolymers with diminished hydration states.
In summary, molecular ordering of water due to the development and strengthening of hydrogen bonds in the bulk solvent may constitute a mechanism for sensing temperature changes. The ordering of water may be the process that leads to the destabilization of vital biopoly- mers by disrupting macromolecule-water interactions (Table 1). The proposed hypothesis may provide a uni- fying mechanism for the effect of temperature on cellu- lar matrices (including lipids, proteins and, perhaps, other biopolymers) that are reactive to chilling, and reconcile opposing views on the temperature-sensing process (lipid solidification TM versus hydrophobic inter- actions~°).
Disruption of water-macromolecule association, due to strengthening of the hydrogen bonds in the water, can also be achieved by hydrostatic pressure~6; high pressure
is known to alter the conformation of macromolecules, and is accompanied by loss of function in proteins and enzymes 3u. Accordingly, deep-ocean barophilic bacteria produce greater amounts of unsaturated fatty acids with increasing pressure 3', apparently to preserve the hydro- philicity of biomembranes subjected to dehydration by pressure. It may be interesting to examine whether deep-ocean organisms employ chilling-related metabolic strategies (e.g. production of highly hydrophilic poly- peptides). The insights obtained from studies of chilling and pressure may be used to understand the effects of such conditions on the hydration and organization of biological matrices.
Conclusion The s tudy o f ch i l l i ng - and pressure- induced d is rupt ion
o f macromolecule-water interactions may provide an understanding of the effects of such processes on mol- ecular assembly and on associated changes in the qual- ity of processed foods. For e~ ~mple, the cooling-related retrogradation of starch may arise from the temperature- induced hydration changes in the hydrophobic domains of starch 32, and subsequent increases in the interaction of polymer chains and in crystallinity 33. Similarly, the cooling-ioduced hydration changes and the associated molecular reorganization may influence the quality of other food matrices.
A c k n o w l e d g e m e n t l w~'dd like to acknowledge the support of the
Agency for International Development grant no. DDC- 5542-G-5105-00.
Kopka, M.L. and Dickerson, R.E. (1987) Science 238, 498-504 13 Crowe, J.H., Crowe, L.M., Carpenter, I.E and Aurell Wislrom, C. (1987
Biocbem. }. 242, 1-10 14 Crowe, J.H. and Crowe, LM. (1986) in Membranes, Metabolism and
Dry Organisms (Leopold, A.C., ed.), pp. 188-209, Comstock Publishing Association
15 Franks, F. 11983) What's New Plant Physiol. 14, 37-40 16 Walker, J S. and Vause, C.A. {1987) ScL Am. 26, 98-105 17 Dill, K.A. (1990) Science250, 297 18 Wolfe, j., Dowgert, M.F., Maier, B. and Steponkus, EL. (1986) in
Membranes, Metabolism and Dry Organisms (Leopold, A.C., ed.), pp. 286-305, Comstock Publishing Association
Trend~ in Food Science & Technology February 1991 41
22 Thompson, G.A. (1983) in Membranes in CellularAcelimation to Environmental Changes (Cossins, A.R. and Sheterline, P., eds), pp. 31-53, Cambridge University Press
23 Larsson, K. (1986) in The Lipid Handbook (Gunslone, ED., Ha~ood, I.L. and Padley, F.B., ecls), pp. 321-384, Chapman and Hall
24 Parkin, K.L. and Kuo, S-I. (1989) Plant Physiol. 90, 1049-1056 25 Lin, C., Guo, W.W., Eversuh, E. and Thomashow, M.F. (1990) Plant
Physiol. 94, 1078-1083 26 Hajela, R.K., Horvath, D.P., Gilmour, S.I. and Thomashow, M.F. (1990)
Plant Physiol. 93, 1246-1252 27 Duke, W.L., Crouch. M., Harada, I., Ho, T-H.D., Mundy, J.,
Quatrano, R., Thomas, T. and Sung, T.R. (1989} PlantMol. BioL 12, 475-486
28 Hahn, M. and Walbol, V. (1989) Plant PhysioL 91,930-938 29 Crowe, J.H., Crowe, L.M. and Chapman, D. (1984} Science 223,
The broad similarity between the milk clotting and blood
clotting processes has been apparent for many years.
Sequence homologies between fibrinogen and milk proteins,
and similarities between the actions of the blood-clotting
enzyme thrombin and the milk-clotting enzyme chymosin
may explain the basis of the parallels between the two
processes.
In the course of recent phylogenetic studies of proteins, it has become progressively clearer that there are fewer original, unrelated sequences than was commonly believed. Unexpected homology is frequently observed between what, at first sight, appear to be quite different protein sequences. Moreover, it is becoraing apparent
Pierre Joll~ is at the Laboraloire des Prot(~ines, UA CNRS 1188, Universil~ de Paris V, 45 rue des Saints-P~res, F 75270 Paris Cedex 06, France. Jacques P. Caen is at the Institui des Vaisseaux el du Sang (IVS), 8 rue Guy Patin, F 75010 Paris, France.
Viewpoint
that similar reaction pathways are responsible tbr a variety of important biological functions.
The coagulalion process The molecular basis of blood clotting has been inten-
sively studied for a long time, because of its biomedical implications. The formation of the thrombus by the con- version of soluble fibrinogen to insoluble fbrin by the enzyme thrombin (EC 3.4.21.5) is the terminal stage of the process. A structural relationship between the milk and blood coagulation processes was suggested as early as 1974 (Ref. 1). Indeed, it was demonstrated that certain amino acid sequences of bovine and ovine K-caseins (the substrates for chymosin (EC 3.4.23.4) in the milk-clotting process) are structurally related to the fibrinogen y chains'.
in 1985, Thompson et a l? also observed homology between the nucleotide sequences of the cDNA (comp- lementary DNA produced by reverse transcriptase from mRNA) of K-casein and the fibrinogen ychains.
The two coagulation processes also share the tbllowing features 4.
• Proteolysis is limited. Thrombin cleaves only Arg-Gly bonds, one in each of the two Aa chains and one in each of the two B~ chains of fibrinogen. Similarly, chymosin cleaves a unique Phe-Met bond (residues 105-106) in bovine It-casein.
• During both coagulation processes, short soluble peptides (fibrinopeptide A and fibrinopeptide B, or K-caseinoglycopeptides) are formed.
• The structures of all these peptides are highly variable from one species to another.
• Some amino acids (Cys and Trp) have never been found in any fibrinopeptides or K-caseinoglycopeptide.
• All fibrinopeptides carry a substantial negative charge; the K-caseinoglycopeptides are also acidic.
Milk proteins and, particularly, caseins have long been considered only as food proteins for young mam- mals. However, because of the structural homology described above, we have been tempted to con~ider
