Temperature Preconditioning Increases Tolerance to Chilling Injury and Alters Lipid Composition in Zucchini Squash

J. PlantPhysiol. Vol. 140. pp. 229-235 (1992)

Temperature Preconditioning Increases Tolerance to Chilling Injury and Alters Lipid Composition in Zucchini Squash C. Y. WANG* 1, G. F. KRAMER** 1, B. D. WHITAKER1, and W. R. LUSBy2 1 2

Horticultural Crops Quality Lab., PQDI, ARS, U.S. Department of Agriculture, Beltsville, MD 20705, USA Insect Hormone Lab., PSI., ARS, U.S. Department of Agriculture, Beltsville, MD 20705, USA

Received July 21, 1991 . Accepted December 1, 1991

Summary Temperature preconditioning at 15°C for 2 d effectively delayed the development of chilling injury in zucchini squash (Cucurmta pepo L., cv.
Key words: Cucurmta pepo, chilling injury, fatty acids, lipids, postharvest physiology, preconditioning, storage, temperature, zucchini squash. Abbreviations: DGDG = digalactosyldiacylglycerol; FAME = fatty acyl methyl esters; FID-GC = flame ionization detector-gas chromatography; FS = free sterols; GC-MS = gas chromatography-mass spectrometry; MGDG = monogalactosyldiacylglycerol; PC = phosphatidylcholine; PE = phophatidylethanolamine; PG = phosphatidylglycerol; PI = phosphatidylinositol; SE = steryl esters; TLC = thin layer chromatography. Introduction

Most hypotheses on the mechanisms of chilling injury suggest that membranes are the probable site of the primary effects of chilling (Lyons et al., 1979; Raison and Orr, 1990). Lyons and Raison (1970) were the first to propose that a transition in the molecular ordering of membrane lipids is the primary event causing chillling injury. They postulated that a membrane-lipid phase transition from a liquid-crystalline to a solid-gel structure could lead to metabolic

* **

Corresponding author. Present address: Climate Stress Laboratory, U.S. Department of Agriculture, Beltsville, MD 20705, USA. © 1992 by Gustav Fischer Verlag, Stuttgart

imbalances, loss of cell compartmentation, increases in membrane permeability and leakage of ions, and stimulation of ethylene production and the respiration rate; resulting in the development of a variety of chilling injury symptoms (Lyons, 1973). This hypothesis has received wide acceptance and support during the past 20 years. However, it has also attracted much discussion and criticism. The main criticism is that it is unlikely that a heterogeneous mixture of plant membrane lipids containing a high percentage of polyunsaturated fatty acids would undergo an abrupt transition at a temperature above 0 °C (Bishop, 1986; Minorsky, 1985). During the past few years, disaturated and trans-monoenoic molecular species of phosphatidylglycerol in chloroplast membranes

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C. Y. WANG, G. F. KRAMER, B. D. WHITAKER, and W. R. LUSBY

were shown to undergo a phase change at temperatures well above O°C (Murata et al., 1982; Roughan, 1985). A freezefracture electron microscope investigation of the plasma membrane in chilled avocado fruit (Platt-Aloia and Thomson, 1987) and a differential scanning calorimetric analysis of polar lipids from mung bean (Raison, 1985) have also indicated that roughly 5 % of the membrane lipids undergo a phase transition at chilling temperatures. The presence of small areas of gel phase lipid in the plasma membrane may be sufficient to initiate the cascade of catabolic reactions that result in chilling injury. The fluidity of the lipid bilayer is determined, to a large extent, by the fatty-acid composition of the phospholipids. The relative proportion of saturated and unsaturated fatty acids in membrane glycerolipids may be associated with the flexibility of the membranes (Lyons et al., 1964; Lyons and Raison, 1970). Treatments that change the lipid composition of cell membranes have been shown to alter the tolerance of plant tissues to low temperature stress (Wang and Baker, 1979; Horvath and van Hasselt, 1985). Since temperature preconditioning treatment was found to be effective in reducing chilling injury of zucchini squash (Kramer and Wang, 1989 b), we have therefore undertaken the present study to determine if the observed reduction in chilling injury can be attributed to changes in the levels and composition of lipids.

Materials and Methods

Plant materials Zucchini squash (Cucurbita pepo L. cv. Ambassador) fruit were freshly harvested from a local farm near Beltsville, MD, USA. One hundred and two fruit were selected for their uniformity of size (16 to 20 cm in length). After equilibration to room temperature (about 25°C), the fruits were randomly divided into two lots. The first group was placed in storage at 5 °C and served as control for the duration of the experiment. The second group was preconditioned at 15°C for the first two days of storage and then moved to 5 °C for the remainder of the study. The relative humidity of the storage rooms was maintained at 90 %. Three fruits were taken daily from each group throughout the storage period for evaluation of chilling injury and for analysis of lipids. The degree of chillling injury, as judged by the extent of surface pitting, was evaluated 5 h after transfer of squash from storage chambers to room temperature by rating on a scale of 0 to 4, with 0 = no abnormality, 1 = trace, 2 = slight, 3 = moderate, and 4 = severe chilling injury. After evaluation of injury, a 5.0-g sample of epidermal tissue was removed from various locations on each fruit daily and immediately frozen. Samples were stored at - 80°C prior to lipid analysis.

on a silica Sep-Pak (Waters Assoc., Milford, MA, USA) then steryl esters (SE) and free sterols (FS) were sequentially eluted with hexane: ether (10: 1, v/v) and hexane: ether (2: 1, v/v). Total phospholipid was quantified by the spectrophotometric assay of Ames (1966). The phospholipid and glycolipid (including MGDG and DGDG) fractions were further separated by thin-layer chromatography (TLC) on 20 x 2O-cm glass plates precoated with 0.25 mm silica G60 (EM Reagents, Darmstadt, FRG) and developed in a solvent mix of chloroform: methanol: acetic acid: water (85: 15: 10: 3.5, v/v). Individual phospholipids and galactolipids (MGDG and DGDG) were identified by co-chromatography with authentic standards (Sigma Chemical Co., St. Louis, MO, USA and Supelco, Bellefonte, PA, USA), and by spray reagents specific for phosphate (Dittmer and Lester, 1964) or hexose sugars (Kates, 1986). Individual phospholipid bands were scraped and eluted with chloroform: methanol (2: 1, v/v). Total phospholipid fatty acids were derivatized to fatty acid methyl esters (FAMEs) with BF3 in methanol. The FAMEs were analyzed by FID-GC using a 1.8-m glass coil column (2 mm i.d.) packed with 10 % SP 2330 on 100/200 mesh chromosorb (Supelco, Bellefonte, PA, USA). The N2 flow rate was 30 mL' min-I and the temperatures for oven, injector, and detector were 190,250, and 300°C, respectively. A known amount of n-heptadecanoic acid was included in all samples as an internal standard, and methyl heptadecanoate was used as an external standard. Individual FAMEs were identified by retention time comparison with authentic standards. Free sterols were precipitated from the neutral lipid fraction with digitonin (Sigma Chemical Co., St. Louis, MO, USA). Prior to digitonin precipitation, 20 IJ.g of cholesterol were added to each fraction as an internal standard. The digitonides were collected on a glass fiber filter (Reeve Angel, Clifton, N], USA) and washed with acetone/diethyl ether (1: 2, v/v). Free sterols were liberated from the digitonides by refluxing in 1 mL pyridine for 30 min at 100°C. After cooling, 1 mL deionized, distilled water was added and the free sterols were recovered by extraction with 2 mL of hexane. Individual sterols were quantified by FID-GC, using a 1.2-m glass coil column (2mm i.d.) packed with 3% SP 2100 on 100/200 mesh supelcoport (Supelco, Bellefonte, PA, USA). The N2 flow rate was 30 mL· min-I and the temperatures for oven, injector, and detector were 245, 300, and 350°C, respectively. The identification of individual sterols was confirmed by GC-MS analysis as described by Whitaker and Lusby (1989).

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Lipid extraction, fractionation, and analysis Frozen epidermal tissue samples of zucchini squash were lyophilized and homogenized in isopropanol (containing 4IJ.g 2,6di-t-butyl-4-methylphenol (BHT) mL -I). The three 5-g samples from individual treatments were combined prior to analysis. Total lipids were separated into neutral, glyco-, and phospholipid fractions by silicic acid column chromatography on 100 to 200 mesh Bio Sil A (Bio Rad Laboratories, Richmond, CA, USA), as described by Whitaker (1991). Neutral lipids dissolved in hexane were loaded

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Days of Storage Fig. I: Development of chilling injury with time of storage. The control samples were placed at 5 °C continuously from the beginning of the study. The preconditioned samples were held at 15°C for the first 2 days, then stored at 5°C.

Chilling Injury and Lipids of Zucchini Squash

231

Results

Effect of temperature preconditioning on chilling injury Storage of zucchini squash at 5 °C resulted in rapid development of chilling injury (Fig. 1). Symptoms of chilling injury such as surface pitting started to appear after 3 d of exposure to this temperature. By the 10th day of storage, most squash developed moderate or severe pitting. Temperature preconditioning treatment at 15°C for 2 d before storage at 5°C was effective in reducing chilling injury, not only in delaying the onset of chilling injury but also in reducing the rate of the development of injury symptoms. Surface pitting was not apparent on treated squash until 6 to 8 d of exposure to 5°C, and only slight pitting was noted on these squash after 16d of storage at 5°C.

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Effect of temperature preconditioning on polar lipids PC and PE were the predominant phospholipids in zucchini squash. Both PC and PE levels decreased steadily during storage at 5 °C (Fig. 2). The PC content declined from 199·kg- 1 dry weight to 7g·kg- 1 dry weight in 16 days of chilling exposure. The PE level was also reduced more than 50% after 10 days at 5°C. In temperature-preconditioned squash, the rates of decline of PC and PE after storage at 5 °C were much less than those in the control squash. PG and PI were also found in the extracts of epidermal tissue from zucchini squash. Although present in smaller amounts, PG and PI showed a trend similar to that of PC and PE in response to chilling and temperature preconditioning (Fig. 3). Tem-

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232

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perature-preconditioned squash maintained higher levels of PG and PI than control squash throughout the experiment. The major galactolipids MGDG and DGDG also decreased with time in storage at 5°C (Fig. 4). As observed for the phospholipids, the rate of decline of MGDG and DGDG during storage at 5 °C was slower in temperature-preconditioned squash than in control fruit. The total amount of MGDG and DGDG was consistently higher in temperatureconditioned fruit than in control fruit.

Effect of temperature preconditioning on fatty acid unsaturation Unsaturated fatty acids in the zucchini squash glycerolipids included oleic (C18: 1), linoleic (C18: 2), and linolenic (C18: 3), and the saturated fatty acids were palmitic (C16: 0) and stearic (C 18: 0). The ratio of unsaturated! saturated fatty acids in PC and PE decreased during chilling in both control and treated fruits (Fig. 5), but squash treated with temperature preconditioning maintained a higher degree of unsaturation in both PC and PE than control squash and the difference became more pronounced as storage progressed.

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Table 1: Free sterols isolated and identified from zucchini squash skin tissue sampled at harvest. Sterol identified Cholesterol A7-Campestenol A8.22-Stigmastadienol Spinasterol Sistosterol Stigmastanol A8(9l.Stigmastenol A 7.2s-Stigmastadienol A7-Stigmastenol A7-iso-Avenasterol A7-Avenasterol

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;:l8(9l.st igmastenol, ;:l7-campestenol, ;:l8.22-st igmastadienol, ~7_ avenasterol, sitosterol, and stigmastanoI. The sterol composition of zucchini squash found in our study agrees well with reports in the literature for fruit and vegetative tissues of Cucurbita maxima (Akihisa et al., 1986; Fenner et aI., 1989). The levels of free spinasterol and ~7-stigmastenol in control fruit increased slightly for the first 7 to 8 days during chilling exposure to 5 °C and then declined (Figs. 6 and 7). The concentration of spinasterol rose dramatically during temperature conditioning, while the level of ~7-stigmastenol declined steadily. Free sterols in the temperature preconditioned squash decreased less than those in the control squash during the later part of the storage period. However, the opposite was true for steryl esters. Steryl esters of spinasterol and ~7-stigmastenol remained slightly higher in control squash than in temperature preconditioned squash.

Chilling Injury and Lipids of Zucchini Squash "

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Fig.8: Effect of chilling and temperature preconditioning on the ratio of free sterol/phospholipid (FS/PL). The free sterol!phospholipid ratio increased steadily with storage at 5 °C (Fig. 8). This increase was more dramatic in control squash. Whereas, in the temperature preconditioned squash, only a slight increase in the ratio of free sterol!phospholipid was observed throughout the storage period. Discussion

Our study has shown that temperature preconditioning of zucchini squash at 15 °C for 2 d prior to storage at 5 °C effectively delayed the development of chilling injury in these

233

squash (Fig. 1). Temperature preconditioning treatment has been reported to be effective in reducing chilling injury in other chilling-sensitive plants (Hatton, 1990). Apparently this treatment induces an adaptive response to the chilling stress. Various physiological changes have been associated with temperature preconditioning treatments. Increases in sugar and starch, and decreases in RNA, protein, and lipidsoluble phosphate, were found in cotton plants during exposure to conditioning temperatures (Guinn, 1971). This preconditioning treatment also reduced leakage of metabolites and prevented subsequent chilling injury in cotton seedlings at 5 0c. Squalene and long-chain aldehydes were reported to increase with temperature conditioning of grapefruit (Nordby and McDonald, 1990, 1991). In the present study, temperature-preconditioning treatment of zucchini squash was found to maintain higher levels of the phospholipids PC, PE, PG, and PI, and the galactolipids MGDG and DGDG during chilling at SoC (Figs. 2, 3 and 4). Chilling has been shown to induce degradation of lipids in cucumber fruit (Parkin and Kuo, 1989), cucumber seedlings (Whitaker and Wang, 1987), and tomato pericarp (Nguyen and Mazliak, 1990). Our results showed that the loss of lipids could be reduced by temperature preconditioning treatment. It has also been found that increases in PC and PE that occur during temperature preconditioning of cucumber plants confer greater tolerance to chilling (Horvath et al., 1983). Zucchini squash treated with temperature preconditioning maintained a higher ratio of unsaturated to saturated fatty acids in PC and PE than the untreated squash (Fig. 5). This higher degree of unsaturation of fatty acids may have maintained greater membrane fluidity in the treated squash and hence lessened chilling injury (Lyons et al., 1964; Lyons and Raison, 1970). Temperature preconditioning at 12 °C also increased the degree of unsaturation of fatty acids in phospholipids and prevented chilling injury in leaves of Phaseolus vulgaris, Cucumis sativus, and Gossypium hirsutum (Wilson and Crawford, 1974). Temperature acclimation has also been reported to increase the levels of linoleic acid in PC and PE in the flavedo of grapefruit (Nordby et al., 1987). The decline in the ratio of unsaturated to saturated fatty acids during storage may reflect an accelerated degradation of unsaturated fatty acids. This observation is consistent with our previous findings of an increase in lipid peroxidation during low temperature storage of zucchini squash (Kramer and Wang, 1989 a). Temperature preconditioning inhibited this increase while enhancing the levels of polyamines (Kramer and Wang, 1989 a). Polyamines have been shown to inhibit lipid peroxidation (Kitada et al., 1979; Tadolini, 1988).

The ratio of free sterol/phospholipid increased dramatically in the control squash during chilling (Fig. 8). This increase was reduced by the temperature preconditioning treatment. It appears that treatments that diminish an increase in the free sterol!phospholipid ratio also tend to reduce chilling injury. This ratio has been reported to be closely associated with membrane viscosity and permeability (Demel and De Kruyff, 1976). It affects the fluidity of membranes and, in turn, influences the capacity of tissue to withstand chilling stress (Borochov et aI., 1982). In mung bean seedlings, choline treatment was shown to increase tolerance

234

C. Y. WANG, G. F. KRAMER, B. D. WHITAKER, and W. R. LUSBY

to chilling while enhancing phospholipid levels and thus reducing the increase in the free steroVphospholipid ratio (Guye, 1989). The effect of chilling on free steroVphospholipid ratio is similar to that of ripening or senescence (Legge et al., 1986; Whitaker, 1991). The increase in this ratio may contribute to the steady decrease in the fluidity of membranes during chilling or during senescence. In this respect, chilling injury can be regarded as an accelerated form of senescence. Taken together, our data show that temperature preconditioning treatment of zucchini squash decreases the rates that phospholipids, galactolipids, and fatty acid unsaturation decline with low temperature storage. This treatment also lowers the free steroVphospholipid ratio. All of these factors could contribute to maintaining high membrane fluidity and thus maintaining the membrane's normal functions during and after chilling exposure.

References A1uHlSA, T., N. SHIMIZU, P. GHOSH, S. THAKUR, F. U. ROSENSTEIN, T. TAMURA, and T. MATSUMOTO: Sterols of the cucurbitaceae. Phytochemistry 26, 1693-1700 (1987). AMES, B. N.: Assay of inorganic phosphate, total phosphate and phosphatase. Methods Enzymol. 8,115-118 (1966). BISHOP, D. G.: Chilling sensitivity in higher plants: the role of phosphatidylgiycerol. Plant Cell Environ. 9, 613-616 (1986). BOROCHOV, A., A. H. HALEVY, and M. SHlNITZKY: Senescence and the fluidity of rose petal membranes. Relation to phospholipid metabolism. Plant Physiol. 69, 296-299 (1982). DEMEL, R. A. and B. DE KRUYFF: The function of plant sterols in membranes. Biochim. Biophys. Acta 457, 109-132 (1976). DITIMER, J. C. and R. L. LESTER: A simple specific spray for the detection of phospholipids on thin-layer chromatograms. J. Lipid Res. 5, 126-127 (1964). FENNER, G. P., G. W. PATTERSON, and W. R. LUSBY: Developmental regulation of sterol biosynthesis in Cucurbita maxima L. Lipids 24,271-277 (1989).

GUINN, G.: Changes in sugars, starch, RNA, protein, and lipidsoluble phosphate in leaves of cotton plants at low temperatures. Crop Sci. 11, 262-265 (1971). GUYE, M. G.: Phospholipid, sterol composition and ethylene production in relation to choline-induced chill-tolerance in mung bean (Vigna radiata L. Wilcz.) during a chill-warm cycle. J. Expt. Bot. 40, 369-374 {1989}. HATTON, T. T.: Reduction of chilling injury with temperature manipulation. In: WANG, C. Y. (ed.): Chilling Injury of Horticultural Crops, pp. 269-280. CRC Press, Boca Raton, FL (1990). HORVATH, I. and P. R. VAN HASSELT: Inhibition of chilling-reduced photooxidative damage to leaves of Cucumis sativus L. by treatment with amino alcohols. Planta 164, 83-88 (1985). HORVATH, I., L. VIGH, P. R. VAN HAsSELT, J. WOL1}ES, and P. J. C. KUIPER: Lipid composition in leaves of cucumber genotypes as affected by different temperature regimes and grafting. Physiol. Plant. 57, 532-536 {1983}. KATES, M.: Techniques of Lipidology. p. 242. Elsevier Sci. Publishers, Amsterdam, The Netherlands (1986). KITADA, M., K. IGARASHI, S. HIROSE, and H. KITAGA.WA: Inhibition by polyamines of lipid peroxide formation in rat liver microsomes. Biochem. Biophys. Res. Commun. 87, 388-394 {1979}. KRAMER, G. F. and C. Y. WANG: Correlation of reduced chilling injury with increased spermine and spermidine levels in zucchini squash. Physiol. Plant. 76, 479-484 (1989 a).

Reduction of chilling injury in zucchini squash by temperature management. HortScience 24, 995 - 996 (1989 b). LEGGE, R. L., K. H. CHENG, J. R. LEPOCK, and J. E. THOMPSON: Differential effects of senescence on the molecular organization of membranes in ripening tomato fruit. Plant Physiol. 81, 954-959 (1986).

LYONS, J. M.: Chilling injury in plants. Annu. Rev. Plant Physiol. 24,445-466 (1973).

LYONS, J. M. and J. K. RAISON: Oxidative activity of mitochondria isolated from plant tissues sensitive and resistant to chi11ing injury. Plant Physiol. 45, 386-389 (1970). LYONS, J. M., J. K. RAISON, and P. L. STEPONKUS: The plant membrane in response to low temperature: an overview. In: LYONS,J. M., D. GRAHAM, andJ. K. RAISON (eds.): Low Temperature Stress in Crop Plants: The Role of the Membrane, pp. 1- 24. Academic Press, New York (1979). LYONS, J. M., T. A. WHEATON, and H. K. PRATT: Relationship between the physical nature of mitochondrial membranes and chilling sensitivity in plants. Plant Physiol. 39,262-268 (1964). MINORSKY, P. V.: An heuristic hypothesis of chilling injury in plants: a role for calcium as the primary physiological transducer of injury. Plant Cell Environ. 8, 75-94 (1985). MURATA, N., N. SATO, N. TAKAHASHI, and Y. HAMAZAKI: Compositions and positional distributions of fatty acids in phospholipids from leaves of chilling-sensitive and chilling-resistant plants. Plant Cell Physiol. 23, 1071-1079 (1982). NGUYEN, X. V. and P. MAzUAK: Chilling injury induction is accompanied by galactolipid degradation in tomato pericarp. Plant Physiol. Biochem. 28, 283-291 (1990). NORDBY, H. E. and R. E. McDoNAW: Squalene, a natural inhibitor of chilling injury in grapefruit. Lipids 25, 807 -810 (1990). - - Relationship of epicuticular wax composition of grapefruit to chilling injury. J. Agr. Food Chern. 39,957 -962 (1991). NORDBY, H. E., A. C. PURVIS, and G. YELENOSKY: Lipids in peel of grapefruit and resistance to chilling injury during cold storage. HortScience 22, 915-917 (1987). PARKIN, K. L. and S. J. Kuo: Chilling-induced lipid degradation in cucumber (Cucumis sativa L. cv. Hybrid C) fruit. Plant Physiol. 90, 1049-1056 (1989).

PLATT-ALOIA, K. A. and W. W. THOMSON: Freeze fracture evidence for lateral phase separations in the plasmalemma of chill-injured avocado fruit. Protoplasma 136, 71- 80 (1987). RAISON, J. K.: Alterations in the physical properties and thermal response of membrane lipids: correlations with acclimation to chilling and high temperature. In: ST JOHN, J. B., E. BERLIN, and P. C. JACKSON (eds.): Frontiers of Membrane Research in Agriculture, pp. 383-401. Rowman & Allanheld, Totowa, NJ (1985).

RAISON, J. K. and G. R. ORR: Proposals for a better understanding of the molecular basis of chilling injury. In: WANG, C. Y. (ed.): Chilling Injury of Horticultural Crops, pp. 145-164. CRe Press, Boca Raton, FL (1990). ROUGHAN, P. G.: Phosphatidylgiycerol and chilling sensitivity in plants. Plant Physiol. 77, 740-746 (1985). TADOUNI, B.: Polyamine inhibition of lipoperoxidation. Biochem.

J. 249, 33-36 (1988).

WANG, C. Y. and J. E. BAKER: Effects of two free radical scavengers and intermittent warming on chi11ing injury and polar lipid composition of cucumber and sweet pepper fruits. Plant Cell Physiol. 20, 243-251 (1979). WHITAKER, B. D.: Changes in lipids of tomato fruit stored at chilling and non-chilling temperatures. Phytochemistry 30, 757 -762 (1991).

Chilling Injury and Lipids of Zucchini Squash WHITAKER, B. D.: Growth conditions and ripening influence plastid and microsomal membrane lipid composition in bell pepper fruit.]. Amer. Soc. Hort. Sci. 116, 528-533 {1991}. WHITAKER, B. D. and W. R. LUSBY: Steryllipid content and composition in bell pepper fruit at three stages of ripening.]. Amer. Soc.. Hort. Sci. 114, 648-651 {1989}.

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WHITAKER, B. D. and C. Y. WANG: Effect of paclobutrazol and chilling on leaf membrane lipids in cucumber seedlings. Physiol. Plant. 70, 404-411 {1987}. WILSON, J. M. and R. M. M. CRAWFORD: The acclimatization of plants to chilling temperatures in relation to the fatty acid composition of leaf polar lipids. New Phytol. 73, 805-820 {1974}.