The effect of changing the composition of phosphatidylglycerol from thylakoid polar lipids of oleander and cucumber on the temperature of the transition related to chilling injury

The composition and phase behavior of some lipid classes and mixtures of thylakoid polar lipids were measured to investigate their role as determinants of the temperature of the transition associated with chilling injury. For Nerium oleander L., a plant which acclimates to growth temperature, a mixture of the phosphatidylglycerol (PG) and sulfoquinovosyldiacylglycerol (SQDG) showed transition temperatures of 22° and 10° C for plants grown at 45° and 20° C, respectively. This difference was similar to the 9 Celsius degrees differential in the transition of the polar lipids and indicated that the PG and-or the PG-SQDG mixture could be the major determinants of the transition temperature. Reconstitution of the PG-SQDG mixture from 20°-grown oleander with the galactolipids from 45°-grown plants, however, reduced the transition temperature by only 4 Celsius degrees. This indicates that some, low-melting-point lipids, which are structurally capable of forming a co-gel with the high-melting-point lipids, also play a role in determining the temperature of the transition and that the composition of these low-melting-point lipids also changes with growth temperature. More specific information on the role of PG was obtained using polar lipids from Cucumis sativus L., a chilling-sensitive plant. For this material the transition in the polar lipids was reduced from 9° to 5° and 4° C when the transition of the PG was reduced from 32° to 25° and 22° C. This was accomplished by reducing the proportion of disaturated molecular species in PG from 78 to 56 and 44 mol% by the addition of a fraction of the PG enriched in unsaturated molecular species. The data indicate that the transition temperature of the polar lipids of cucumber would be reduced to below 0° C, typical of a chillinginsensitive plant, when the transition temperature of PG was reduced to 15° C and this would occur at 21 mol% of disaturated molecular species. It is concluded that the transition in the thylakoid polar lipids, associated with chilling injury, involves both high- and low-meltingpoint lipids but can be reduced when the transition temperature of the high-melting-point component is reduced.Abbreviations DGDG digalactosyldiacylglycerol - MGDG monogalactosyldiacylglycerol - PG phosphatidylglycerol - SQDG sulfoquinovosyldiacylglycerol     

Phase transitions in liposomes formed from the polar lipids of mitochondria from chilling-sensitive plants

The thermal response of mitochondrial polar lipids from a variety of chilling-sensitive and chilling-insensitive plants was determined by differential scanning calorimetry. A phase transition was observed at 15°C for mitochondria from soybeam (Glycine max. cv Davis) hypocotyl, at 16°C for tomato (Lycopersicon esculentum cv Flora-Dade and cv Grosse Lisse) fruit, at 15°C for cucumber (Cucumus sativus L.) fruit, at 14°C for mung bean (Vigna radiata var Berken) hypocotyl, and at 15°C for sweet potato (Ipomea batatas L.) roots. The transition temperature was not significantly altered by the scan rate and was reversible. Changes in the temperature coefficient of motion for a spin label, intercalated with the polar lipids, occurred at a temperature slightly below that of the phase transition, indicating that the polar lipids phase separate below the transition. No phase transition was observed for mitochondrial polar lipids from barley (Hordeum vulgare) roots, wheat (Triticum aestivum L. cv Falcon) roots, and Jerusalem artichoke (Helianthus tuberosus L.) tubers. The results show that a phase change occurs in the membrane lipids of mitochondria a few degrees above the temperature below which chilling injury is evident in the sensitive species. Thus they are consistent with the hypothesis that sensitivity to chilling injury is related to a temperature-induced alteration in the structure of cell membranes.     

Temperature-dependent phase behavior of phosphatidylglycerols from chilling-sensitive and chilling-resistant plants

Seven major lipid classes were isolated from leaves of chilling-sensitive and chilling-resistant plants, and the temperature-dependent phase behaviors of their aqueous dispersions were studied by a fluorescence polarization method using trans-parinaric acid and its methyl ester. Phosphatidylglycerols from the chilling-sensitive plants went from the liquid crystalline state into the phase separation state at about 30°C in 100 mm NaCl and at about 40°C in 5 mm MgCl₂. In contrast, phosphatidylglycerols from the chilling-resistant plants went into the phase separation state at a much lower temperature. The other classes of lipids remained in the liquid crystalline state at all temperatures between 5°C and 40°C regardless of the chilling sensitivity of the plants, except sulfoquinovosyl diacylglycerol from sponge cucumber in which phase separation seemed to begin at about 15°C. Compositions and positional distributions of fatty acids of the lipids suggest that the phosphatidylglycerols from the chilling-sensitive plants, but no other lipids, contained large proportions of molecular species which undergo phase transition at room temperature or above. The thermotropic phase behaviors and the fatty acid compositions suggest that, among the major lipid classes from leaves of the chilling-sensitive plants, only phosphatidylglycerol can induce a phase transition. Since a major part of this lipid in leaves originates from the chloroplasts, phase transition probably occurs in the chloroplast membranes.     

Phase transitions in thylakoid polar lipids of chilling-sensitive plants: a comparison of detection methods

The phase behavior of thylakoid polar lipids from plants sensitive to chilling injury was investigated by calorimetry, electron spin resonance spectroscopy of spin labels, and fluorescence intensity after labeling with trans-parinaric acid. The plants used were oleander (Nerium oleander), mung bean (Vigna radiata L. var Mungo), and tomato (Lycopersicon esculentum cv Grosse Lisse). For all plants the initiation temperature for the calorimetric exotherm was coincident (±1°C) with the transition determined by the increase in the temperature coefficient of spin label motion and fluorescence intensity of trans-parinaric acid. For oleander plants, grown at 45°C, the transition was at 7°C while for plants from the same clone, grown at 20°C, it was at −2°C. For mung bean and tomato the transition was between 9 and 12°C. The similarity in the transition detected by spin labeling and fluorescence intensity suggest that spin labels, like the fluorescent label trans-parinaric acid, preferentially partition into domains of ordered lipid. The coincidence of the temperature for initiation of the transition, determined by the three techniques, shows that each is a valid method of assessing a phase transition in membrane polar lipids.     

Physical Properties of Mitochondrial Lipids from Lycopersicon hirsutum

Mitochondrial lipids from Lycopersicon hirsutum undergo a broad thermal transition beginning well below 0°C and ending at approximately 25°C. Differential thermal analysis of mitochondrial lipids isolated from ecotypes of L. hirsutum that differ in chilling sensitivity indicates that these lipid preparations have physically similar properties. This was confirmed by electron-spin-resonance experiments, although this technique failed to detect the broad transition detected by differential thermal analysis. No quantitative differences were observed between the percentages of individual lipid classes (based on polar head group) or between the fatty acid compositions of mitochondrial lipids from the two ecotypes investigated. These results suggest that the observed differences between the responses of these ecotypes to prolonged exposure to 5°C may not be related to differences between the physical properties of their mitochondrial lipids.     

Sensitivity of Altitudinal Ecotypes of the Wild Tomato Lycopersicon hirsutum to Chilling Injury

The transition temperature of the leaf polar lipids and the critical temperature for chill-induced inhibition of photosynthesis was determined for three altitudinal ecotypes of the wild tomato Lycopersicon hirsutum. Photosynthesis was measured as CO₂-dependent O₂ evolution at 25°C after leaf slices were exposed to chilling temperatures for 2 hours at a moderate photon flux density of 450 micromoles per square meter per second. The transition temperature of the leaf polar lipids was detected from the change in the temperature coefficient of the fluorescence intensity of trans-parinaric acid. Chill-induced photoinhibition was evident in the three tomato ecotypes when they were chilled below a critical temperature of 10°, 11°, and 13°C, respectively, for the high (LA1777), mid (LA1625), and low (LA1361) altitudinal ecotypes. The temperature differential, below the critical temperature, required to produce a 50% inhibition was also similar for the three ecotypes. A transition was detected in the leaf polar lipids of these plants at a temperature similar to that of the critical temperature for photoinhibition. The results show that the three tomato ecotypes are similar with respect to their critical temperature for chilling-induced photoinhibition and the rate of their response to the chilling stress. They are, thus, similarly sensitive to chilling.     

Thermal phase transitions in the polar lipids of plant membranes. Their induction by disaturated phospholipids and their possible relation to chilling injury

The phase behaviour of leaf polar lipids from three plants, varying in their sensitivity to chilling, was investigated by differential scanning calorimetry. For the lipids from mung bean (Vigna radiata L. var. Berken), a chilling-sensitive plant, a transition exotherm was detected beginning at $\text{10 ± 2°}\text{C}$. No exotherm was evident above 0°C with polar lipids from wheat (Triticum aestivum cv. Falcon) or pea (Pisum sativum cv. Massey Gem), plants which are insensitive to chilling. The enthalpy for the transition in the mung bean polar lipids indicated that only about 7% w/w of the lipid was in the gel phase at ?8°C. The thermal transition of the mung bean lipids was mimicked by wheat and pea polar lipids after the addition of 1 to 2% w/w of a relatively high melting-point lipid such as dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol or dimyristoylphosphatidylcholine. Analysis of the polar lipids from the three plants showed that a dipalmitoylphosphatidylglycerol was present in mung bean (1.7% w/w) and pea (0.3% w/w) but undetected in wheat, indicating that the transition exotherm temperature of 10°C in mung bean, 0°C in pea and about ?3°C in wheat correlates with the proportion of the high melting-point disaturated component in the polar lipids. The results indicate that the transition exotherm, observed at temperatures above 0°C in the membranes of chilling-sensitive plants, could be induced by small amounts of high melting-point lipids and involves only a small proportion of the membrane polar lipids.     

Growth temperature effects on thylakoid membrane lipid and protein content of pea chloroplasts

The lipid composition and level of unsaturation of fatty acids has been determined for chloroplast thylakoid membranes isolated from Pisum sativum grown under cold (4°/7°C) or warm (14°/17°C) conditions. Both the relative amounts of lipid classes and degree of saturation were not greatly changed for the two growth conditions. In cold-grown plants, there was a slightly higher linolenic and lower linoleic acid content for the glycolipids monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), and sulfoquinovosyldiacylglycerol. In contrast to thylakoid membranes, a non-thylakoid leaf membrane fraction including the chloroplast envelope, had a higher overall level of fatty acid unsaturation in cold-grown plants due mainly to an increase in the linolenic acid content of MGDG, DGDG, phosphatidylglycerol, and phosphatidylcholine. The most clear cut change in the thylakoid membrane composition was the lipid to protein ratio which was higher in the cold-grown plants.     

Changes in the Physical State of Membrane Lipids during Senescence of Rose Petals

Changes in the physical state of microsomal membrane lipids during senescence of rose flower petals (Rosa hyb. L. cv Mercedes) were measured by x-ray diffraction analysis. During senescence of cut flowers held at 22°C, lipid in the ordered, gel phase appeared in the otherwise disordered, liquid-crystalline phase lipids of the membranes. This was due to an increase in the phase transition temperature of the lipids. The proportion of gel phase in the membrane lipids of 2-day-old flowers was estimated as about 20% at 22°C. Ethylene may be responsible, at least in part, for the increase in lipid transition temperature during senescence since aminooxyacetic acid and silver thiosulfate inhibited the rise in transition temperature. When flowers were stored at 3°C for 10 to 17 days and then transferrd to 22°C, gel phase lipid appeared in membranes earlier than in freshly cut flowers. This advanced senescence was the result of aging at 3°C, indicated by increases in membrane lipid transition temperature and ethylene production rate during the time at 3°C. It is concluded that changes in the physical state of membrane lipids are an integral part of senescence of rose petals, that they are caused, at least in part, by ethylene action and that they are responsible, at least in part, for the increase in membrane permeability which precedes flower death.     

An assessment of phase transitions in soybean membranes

Phase transitions were measured in vesicles of phospholipids, alone and in various combinations, and in pelleted mitochondrial membranes, using thermal (DSC) and optical methods. The objective was to consider their possible involvement in chilling injury of soybeans (Glycine max [L.] Merr. cv Wayne 1977). Saturated phospholipids showed clear transitions in the temperature range of 50°C to near 0°C. When mixtures of two phospholipids were examined, there was a marked lowering and broadening of the transition peaks, and a shift in the transition temperatures to intermediate temperatures. The unsaturated phospholipids that occur naturally in soybeans showed no detectable phase transitions in this temperature range, alone or in combinations. Examination of the polar lipids from soybean asolectin revealed no transitions in the biological temperature range; the additions of cations such as Ca²⁺ and La³⁺ did not evoke a detectable phase transition in them. Mitochondrial membrane pellets likewise showed no transitions. The application of these two direct methods of examination of membrane components without the addition of foreign agents did not support the suggested occurrence of a bulk phase transition which could be related to chilling injury in soybeans.     

Growth Temperature-Induced Alterations in the Thermotropic Properties of Nerium oleander Membrane Lipids

The temperature boundary for phase separation of membrane lipids extracted from Nerium oleander leaves was determined by analysis of spin label motion using electron spin resonance spectroscopy and by analysis of polarization of fluorescence from the probe, trans-parinaric acid. A discontinuity of the temperature coefficient for spin label motion, and for trans-parinaric acid fluorescence was detected at 7°C and −3°C with membrane lipids from plants grown at 45°C/32°C (day/night) and 20°C/15°C, respectively. This change was associated with a sharp increase in the polarization of fluorescence from trans-parinaric acid indicating that significant domains of solid lipid form below 7°C or −3°C in these preparations but not above these temperatures. In addition, spin label motion indicated that the lipids of plants grown at low temperatures are more fluid than those of plants grown at higher temperatures.

A change in the molecular ordering of lipids was also detected by analysis of the separation of the hyperfine extrema of electron spin resonance spectra. This occurred at 2°C and 33°C with lipids from the high and low temperature grown plants, respectively. According to previous interpretation of spin label data the change at 29°C (or 33°C) would have indicated the temperature for the initiation of the phase separation process, and the change at 7°C (or −3°C) its completion. Because of the present results, however, this interpretation needs to be modified.

Differences in the physical properties of membrane lipids of plants grown at the hot or cool temperatures correlate with differences in the physiological characteristics of plants and with changes in the fatty acid composition of the corresponding membrane lipids. Environmentally induced modification of membrane lipids could thus account, in part, for the apparently beneficial adjustments of physiological properties of this plant when grown in these regimes.

     

Effect of temperature conditioning on chilling injury of cucumber cotyledons: possible role of abscisic Acid and heat shock proteins

Endogenous abscisic acid levels and induced heat shock proteins were measured in tissue exposed for 6 hours to temperatures that reduced their subsequent chilling sensitivity. One-centimeter discs excised from fully expanded cotyledons of 11-day-old seedlings of cucumber (Cucumis sativus L., cv Poinsett 76) were exposed to 12.5 or 37°C for 6 hours followed by 4 days at 2.5 or 12.5°C. Ion leakage, a qualitative indicator of chilling injury, increased after 2 to 3 day exposure to 2.5°C, but not to 12.5°C, a nonchilling temperature. Exposure to 37°C before chilling significantly reduced the rate of ion leakage by about 60% compared to tissue exposed to 12.5°C before chilling, but slightly increased leakage compared to tissue exposed to 12.5 or 37°C and held at the nonchilling temperature of 12.5°C. There was no relationship between abscisic acid content following exposure to 12.5 or 37°C and chilling tolerance. Five heat shock proteins, with apparent molecular mass of 25, 38, 50, 70, and 80 kilodaltons, were induced by exposure to 37 or 42°C for 6 hours, and their appearance coincided with increased chilling resistance. Heat shock treatments reduced the synthesis of three proteins with apparent molecular mass of 14, 17, and 43 kilodaltons. Induction of heat shock proteins could be a possible cause of reduced chilling injury in tissue exposed to 37 or 42°C.     

Chilling-Susceptibility of the Blue-Green Alga Anacystis nidulans: III. LIPID PHASE OF CYTOPLASMIC MEMBRANE

The lipid phase of cytoplasmic membrane was studied by freeze-fracture electron microscopy in the chilling-susceptible blue-green alga, Anacystis nidulans. At growth temperatures, intramembrane particles were distributed at random in the fracture faces of cytoplasmic membrane, whereas, at chilling temperatures, the fracture faces were composed of particle-free and particle-containing regions. These findings indicate that lipids of the cytoplasmic membrane were in the liquid-crystalline state at the growth temperatures and in the phase-separation state at the chilling temperatures. Temperatures for the onset of phase separation were 5 and 16°C in cells grown at 28 and 38°C, respectively.     

Effects of temperature on the phase behavior and permeability of thylakoid lipid vesicles : relevance to chilling stress

Large unilamellar vesicles composed of thylakoid glycolipids, phosphatidylglycerol, and varying proportions of dipalmitoylphosphatidylglycerol (DPPG) have been examined for the temperature dependence of their permeability to ⁸⁶Rb⁺ and for the occurrence of liquid-crystalline to gel (L_α-to-L_β) phase separations. In vesicles in which the normal 12 mole percent of moderately unsaturated thylakoid phosphatidylglycerol was partially or completely replaced by DPPG, analysis by differential scanning calorimetry indicated that an L_α-to-L_β phase separation did not occur between 0 and 60°C. However, in similar vesicle dispersions that were first subjected to a freeze-thaw cycle, L_α-to-L_β phase separations were observed to occur between 17 and 53°C. The temperature and enthalpy of these phase separations were closely related to the proportion of DPPG in the original lipid mixture. In parallel experiments, large unilamellar vesicles were measured for their permeability to ⁸⁶Rb⁺ between 7 and 30°C. There were no systematic increases in permeability to ⁸⁶Rb⁺ as a function of DPPG content at the temperatures relevant to chilling stress in higher plants. It is concluded that (a) L_α-to-L_β phase separations do not occur in well-defined galactolipid vesicles containing ≤12 mole percent DPPG between 0 and 60°C and (b) these vesicles show no alterations in permeability to ⁸⁶Rb⁺ between 7 and 30°C that are relevant to chilling stress in higher plants.     

REVERSIBLE, THERMOTROPIC ALTERATION OF NUCLEAR MEMBRANE STRUCTURE AND NUCLEOCYTOPLASMIC RNA TRANSPORT IN TETRAHYMENA

We examine the effect of cooling upon the freeze-etch ultrastructure of nuclear membranes, as well as upon nucleocytoplasmic RNA transport in the unicellular eukaryote Tetrahymena pyriformis. Chilling produces smooth, particle-free areas on both faces of the two freeze-fractured macronuclear membranes. Upon return to optimum growth temperature the membrane-associated particles revert to their normal uniform distribution and the smooth areas disappear. Chilling lowers the incorporation of [¹⁴C]uridine into whole cells and their cytoplasmic RNA. Cooling from the optimum growth temperature of 28° to 18°C (or above) decreases [¹⁴C]uridine incorporation into cells more than into their cytoplasmic RNA; chilling to below 18°C but above 10°C causes the reverse. [¹⁴C]Uridine incorporation into whole cells and their cytoplasmic RNA reflects overall RNA synthesis and nucleocytoplasmic RNA transport, respectively. RNA transport decreases strongly between 20° and 16°C, which is also the temperature range where morphologically detectable nuclear membrane transitions occur. This suggests that the nuclear envelope limits the rate of nucleocytoplasmic RNA transport at low temperatures. We hypothesize that a thermotropic lipid phase transition switches nuclear pore complexes from an "open" to a "closed" state with respect to nucleocytoplasmic RNA transport.     

Phosphatidylglycerol and chilling sensitivity in plants

The hypothesis that molecular species of thylakoid phosphatidylglycerol containing two saturated fatty acids (disaturated phosphatidylglycerol) confer chilling sensitivity upon plants was tested by analyzing the fatty acid composition of phosphatidylglycerols isolated from leaves of a range of plants expected to have different sensitivities to chilling temperatures.

`Saturated' fatty acids (palmitate plus stearate plus hexadeca-trans-3-enoate) as a proportion of total phosphatidylglycerol fatty acids varied from 51 to 80 mole per cent in the plants analyzed but appeared to be rigidly fixed for a given plant species, being unaffected by leaf maturity or by environment.

Hexadeca-trans-3-enoate occurred only at the sn-2 position, whereas C-18 fatty acids occurred only at the sn-1 position of thylakoid phosphatidylglycerol. Therefore, the proportion of disaturated molecular species could be predicted accurately from the total fatty acids of phosphatidylglycerol.

Disaturated molecular species accounted for <25% of the total phosphatidylglycerol from leaves of chilling-resistant plants and for 50 to 60% of the phosphatidylglycerol in leaves from some of the most chilling-sensitive plants. However, not all chilling-sensitive plants contained high proportions of disaturated phosphatidylglycerol; solanaceous and other 16:3-plants and C₄ grasses may be important exceptions. Nonetheless, proportions of disaturated phosphatidylglycerol increased concomitantly with increasing chilling sensitivity of plants within a genus.

     

“On-Off” Thermoresponsive Coating Agent Containing Salicylic Acid Applied to Maize Seeds for Chilling Tolerance

Chilling stress is an important constraint for maize seed establishment in the field. In this study, a type of “on-off” thermoresponsive coating agent containing poly (N-isopropylacrylamide-co-butylmethacrylate) (Abbr. P(NIPAm-co-BMA)) hydrogel was developed to improve the chilling tolerance of coated maize seed. The P(NIPAm-co-BMA) hydrogel was synthesized by free-radical polymerization of N-isopropylacrylamide (NIPAm) and butylmethacrylate (BMA). Salicylic acid (SA) was loaded in the hydrogel as the chilling resistance agent. SA-loaded P(NIPAm-co-BMA) was used for seed film-coating of two maize varieties, Huang C (HC, chilling-tolerant) and Mo17 (chilling-sensitive), to investigate the coated seed germination and seedling growth status under chilling stress. The results showed that the hydrogel obtained a phase transition temperature near 12°C with a NIPAM to MBA weight ratio of 1: 0.1988 (w/w). The temperature of 12°C was considered the “on-off” temperature for chilling-resistant agent release; the SA was released from the hydrogel more rapidly at external temperatures below 12°C than above 12°C. In addition, when seedlings of both maize varieties suffered a short chilling stress (5°C), higher concentrations of SA-loaded hydrogel resulted in increased germination energy, germination percentage, germination index, root length, shoot height, dry weight of roots and shoots and protective enzyme activities and a decreased malondialdehyde content in coated maize seeds compared to single SA treatments. The majority of these physiological and biochemical parameters achieved significant levels compared with the control. Therefore, SA-loaded P(NIPAm-co-BMA), a nontoxic thermoresponsive hydrogel, can be used as an effective material for chilling tolerance in film-coated maize seeds.     

Iron Superoxide Dismutase Protects against Chilling Damage in the Cyanobacterium Synechococcus species PCC7942

A strain of Synechococcus sp. PCC7942 lacking functional Fe superoxide dismutase (SOD), designated sodB⁻, was characterized by its growth rate, photosynthetic pigments, inhibition of photosynthetic electron transport activity, and total SOD activity at 0°C, 10°C, 17°C, and 27°C in moderate light. At 27°C, the sodB⁻ and wild-type strains had similar growth rates, chlorophyll and carotenoid contents, and cyclic photosynthetic electron transport activity. The sodB⁻ strain was more sensitive to chilling stress at 17°C than the wild type, indicating a role for FeSOD in protection against photooxidative damage during moderate chilling in light. However, both the wild-type and sodB⁻ strains exhibited similar chilling damage at 0°C and 10°C, indicating that the FeSOD does not provide protection against severe chilling stress in light. Total SOD activity was lower in the sodB⁻ strain than in the wild type at 17°C and 27°C. Total SOD activity decreased with decreasing temperature in both strains but more so in the wild type. Total SOD activity was equal in the two strains when assayed at 0°C.     

Low-Temperature Growth of Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 is a mesophilic bacterium with a maximum growth temperature of ≈35°C but the ability to grow over a wide range of temperatures, including temperatures near zero. At room temperature (≈22°C) MR-1 grows with a doubling time of about 40 min, but when moved from 22°C to 3°C, MR-1 cells display a very long lag phase of more than 100 h followed by very slow growth, with a doubling time of ≈67 h. In comparison to cells grown at 22°C, the cold-grown cells formed long, motile filaments, showed many spheroplast-like structures, produced an array of proteins not seen at higher temperature, and synthesized a different pattern of cellular lipids. Frequent pilus-like structures were observed during the transition from 3 to 22°C.     

Chilling-Induced Lipid Degradation in Cucumber (Cucumis sativa L. cv Hybrid C) Fruit

Chilling at 4°C in the dark induced lipid degradation in cucumber (Cucumis sativa L.) fruit upon rewarming at 14°C. Rates of ethane evolution by fruits rewarmed after 3 days of chilling were up to four-fold higher than those evolved by unchilled (14°C) fruits (0.02-0.05 picomoles gram fresh weight⁻¹ hour⁻¹). This potentiation of lipid peroxidation occurred prior to irreversible injury (requiring 3 to 7 days of chilling) as indicated by increases in ethylene evolution and visual observations. Decreases in unsaturation of peel tissue glycolipids were observed in fruits rewarmed after 3 days of chilling, indicating the plastids to be the site of the early phases of chilling-induced peroxidation. Losses in unsaturation of tissue phospholipids were first observed only after chilling for 7 days. Phospholipase D activity appeared to be potentiated in fruits rewarmed after 7 days of chilling as indicated by a decrease in phosphatidylcholine (and secondarily phosphatidylethanolamine) with a corresponding increase in phosphatidic acid. These results indicate that lipid peroxidation may have a role in conferring chilling injury.