Monitoring chilling injury: A comparison of chlorophyll fluorescence measurements, post-chilling growth and visible symptoms of injury in Zea mays

Changes in chlorophyll fluorescence emission from maize ( Zea mays L. cv. Northern Belle) seedlings chilled at 1.5°C in the dark for 3–30 h were compared with the ability of plants to resume growth in the immediate post-chilling period and with the development of visible symptoms of injury to the leaves. During chilling, the maximal rate of increase of the induced chlorophyll fluorescence rise. F_R, was measured on secondary leaf tissue. F_R decreased exponentially, at approximately the same rate in plants grown and chilled in hydroponic pots, in leaves detached from similar plants and in plants that were removed from the hydroponic pots and laid on wet filter paper adjacent to the detached leaves. The half-fall time for F_R in the 3 treatments was 7.8 ± 1.3 h, 8.6 ± 0.6 h and 8.8 ± 1.0 h, respectively. Following seedling removal from 1.5°C and return to 25/15°C, relative growth rates were determined from daily measurements of plant fresh weight gain. Compared with non-chilled seedlings, plants chilled for 3 h and longer showed depressed rates of growth. Inhibition of growth in the immediate post-chilling period (0–27 h) was linearly related to the duration of the chilling period and had a high positive correlation with the decrease in chlorophyll fluorescence (linearly related to log F_R) sustained during the chilling exposure. Visible symptoms of chilling injury developed during the post-chilling period on seedlings chilled for longer than 3 h. The decrease in log F_R during chilling was also linearly correlated with the severity of visual symptoms of chilling injury expressed in the post-chilling period. It is concluded that the extent of chilling injury in maize can be rapidly and non-destructively assessed from measurements of chlorophyll fluorescence.     

Effect of acetaldehyde, arsenite, ethanol, and heat shock on protein synthesis and chilling sensitivity of cucumber radicles

Exposure to a chilling temperature of 2.5°C for 96 h inhibited the subsequent growth of cucumber seedling radicles at 25°C by 92%. Exposing seedling with 5 ± 1 mm long radicles to acetaldehyde vapour (275 µl l⁻¹) or to an aqueous ethanol solution (0.6  M ) for 2 h, or to 45°C for 10 min before chilling, increased chilling tolerance so that the chilling treatment reduced growth by only 47, 39 or 36%, respectively. All of these effective treatments induced the synthesis of a number of proteins, and suppressed de novo protein synthesis (i.e. the incorporation of [³⁵S]-methionine) by about 70%. In contrast, treatment for 2 h with an aqueous arsenite solution (100 µ M ) had no effect on chilling sensitivity or the incorporation of [³⁵S]-methionine, yet it induced the synthesis of a complement of proteins that were similar to that induced by the effective heat-shock treatment. A unique protein or set of proteins may be responsible for heat-shock-induced chilling tolerance, but none was detected. The ability of various abiotic stresses to suppress protein synthesis may be more important in increasing tolerance to chilling injury than their ability to induce the synthesis of specific proteins.     

Genotype-specific differences in chilling tolerance of maize in relation to chilling-induced changes in water status and abscisic acid accumulation

Four inbred maize lines differing in chilling tolerance were used to study changes in water status and abscisic acid (ABA) levels before, during and after a chilling period. Seedlings were raised in fertilized soil at 24/22°C (day/night), 70% relative humidity. and a 12-h photoperiod with 200 μmol m⁻² s⁻¹ from fluorescent tubes. At an age of 2 weeks the plants were conditioned at 14/12°C for 4 days and then chilled for 5 days at 5/3°C. The other conditions (relative humidity, quantum flux, photoperiod) were unchanged. After the chilling period the plants were transferred to the original conditions for recovery. The third leaves were used to study changes in leaf necrosis, ion efflux, transpiration, water status and ABA accumulation. Pronounced differences in chilling tolerance between the 4 lines as estimated by necrotic leaf areas, ion efflux and whole plant survival were observed. Conditioning significantly increased tolerance against chilling at 5/3°C in all genotypes. The genotypes with low chilling tolerance had lower water and osmotic potentials than the more tolerant genotypes during a chilling period at 5/3°C. These differences were related to higher transpiration rates and lower diffusive resistance values of the more susceptible lines. During chilling stress at 5/3°C ABA levels were quadrupled. Only a small rise was measurable during conditioning at 14/12°C. However, conditioning enhanced the rise of ABA during subsequent chilling. ABA accumulation in the two lines with a higher chilling tolerance was triggered at a higher leaf water potential and reached higher levels than in the less tolerant lines. We conclude that chilling tolerance in maize is related to the ability for fast and pronounced formation of ABA as a protective agent against chilling injury.     

Activity of enzymatic antioxidant defense systems in chilled and heat shocked cucumber seedling radicles

Chilling whole cucumber seedlings that had 10‐mm long radicles for 4 days at 2.5°C significantly inhibited subsequent radicle growth both by increasing the time it took the seedlings to recover from chilling and attain a linear rate of radicle growth, and by decreasing the subsequent rate of linear growth. Exposing cucumber seedlings to 45°C for up to 20 min had no effect on subsequent radicle growth, while longer exposures produced reductions in growth. A heat shock at 45°C for 10 min induced the optimal protection to 4 days of chilling at 2.5°C by reducing chilling inhibition from 60 to 42%. Two hours after being chilled, heat shocked or heat shocked and then chilled, there was no difference in protein content of the apical 1 cm of the seedling radicle among these treatments and the non‐heat shocked, non‐chilled control. Two days after treatment, the protein content was still similar in tissue that had been heat shocked or heat shocked and chilled, while it was significantly reduced in tissue that had been chilled. In general, 2 h after treatment, the activity of the 5 antioxidant enzymes examined in this study [superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.6), ascorbate peroxidase (APX; EC 1.11.1.11), guaiacol peroxidase (GPX; EC 1.11.1.7) and glutathione reductase (GR; EC 1.6.4.2)] were reduced by chilling and unaffected or increased by heat shock. When heat shock was followed by chilling, there was a consistent effect of the heat shock treatment on preventing the loss of enzyme activity following chilling. This protective effect of the heat shock treatment was even more pronounced after 2 days of recovery at 25°C for SOD, CAT and APX. In contrast, the activity of GR and GPX was substantially higher in chilled tissue than in tissue that had been heat shocked before being chilled. Elevated levels of GR and GPX therefore appear to be correlated with the development of chilling injury, while elevated levels of SOD, CAT and APX appear to be correlated with the development of heat shock‐induced chilling tolerance.     

Differential effects of chilling on the activity of C4 enzymes in two ecotypes of Echinochloa crus-galli from sites of contrasting climates

Five-week-old plants of Echinochloa crusgalli (L.) Beauv. from Mississippi and from Québec grown under controlled conditions were subjected to dark chilling for 10 h at 5°C or light chilling treatments for 14 h at 7°C under hight light (1 000 μmol m⁻² s⁻¹). The activities of four C₄ enzymes of Québec plants, measured 4 h after the completion of the cold treatment, were not affected by the chilling treatment in the dark. The activities of pyruvate, P_i dikinase (PPDK; EC 2.7.9.1) and NADP⁺-malic enzyme (NADP⁺-ME; EC 1.1.1.40), were significantly reduced in dark-chilled Mississippi plants. Chilling under high light conditions elicited significant levels of reduction in the activities of the four enzymes from both ecotypes but the reductions were significantly less severe for Québec plants. The recovery of activities of phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) and PPDK for both ecotypes was completed within 36 to 60 hours following the chilling treatment, but NADP⁺-malate dehydro-genase (NADP⁺-MDH; EC 1.1.1.82) and NADP⁺-ME activities of chilled Mississippi plants remained below that of control plants at the end of the 5-day monitoring period. PPDK was inactivated in vitro at 0 and 10°C and the rates of cold inactivation were significantly higher for PPDK extracted from Mississippi plants. The activity of PEPC of Mississippi extracts was slightly, but significantly reduced by a 60 min treatment at 0°C.     

Effect of heat shock on the chilling sensitivity of trichomes and petioles of African violet (Saintpaulia ionantha)

Chilling at 6°C caused an immediate cessation of protoplasmic streaming in trichomes from African violets ( Saintpaulia ionantha ), and a slower aggregation of chloroplasts in the cells. Streaming slowly recovered upon warming to 20°C, reaching fairly stable rates after 4, 15, 25 and 35 min for tissue chilled for 2 min and for 2, 14 and 24 h, respectively. The rate of ion leakage from excised petioles into an isotonic 0.2  M mannitol solution increased after 12 h of chilling and reached a maximum after 3 days of chilling. A heat shock at 45°C for 6 min reduced chilling-induced rates of ion leakage from excised 1-cm petiole segments by over 50%, namely to levels near that from non-chilled control tissue. Heat-shock treatments themselves had no effect on the rate of ion leakage from non-chilled petiole segments. Protoplasmic streaming was stopped by 1 min of heat shock at 45°C, but slowly recovered to normal levels after about 30 min Chloroplasts aggregation was prevented by a 1 or 2 min 45°C heat-shock treatment administered 1.5 h before chilling, but heat-shock treatments up to 6 min only slightly delayed the reduction in protoplasmic streaming caused by chilling. Tradescantia virginiana did not exhibit symptoms associated with chilling injury in sensitive species (i.e. cessation of protoplasmic streaming in stamen hairs and increased ion leakage from leaf tissue).     

Uniconazole-induced thermotolerance in soybean seedling root tissue

Soybean [Glycine max(L.) Merr. cv. A2] seeds were germinated in 0 or 1 mg 1¹ (3.4 uM) uniconazole, after which seedling roots were excised and exposed to 22 or 48°C for 90 min. Prior to the temperature treatments there were few ultrastructural differences between uniconazole-treated seedling roots and the controls. Following exposure to 48°C, electron micrographs revealed near complete loss of normal ultrastructure in control epidermal root cells, whereas cellular integrity was maintained in treated roots, indicating that uniconazole conferred tolerance to high temperature. Total electrolyte, sugar and K⁺ leakage were all greater from control roots than treated roots during exposure to 48°C. Proline content in the roots was unaffected by uniconazole at 22°C but was 25–30% greater in treated tissue than in controls following exposure to 48°C. Malondialdehyde content was unaffected by uniconazole at 22°C but was nearly 20% less in treated tissue than in controls following high temperature exposure. This indicates that uniconazole decreased high-temperature-induced lipid peroxidation. Uniconazole elevated several antiox-idant systems in the roots, including water-soluble sulfhydryl concentration and catalase, peroxidase and superoxide dismutase activities. These findings are consistent with the hypothesis that uniconazole-induced stress tolerance is due, at least in part, to enhanced antioxidant activity which reduces stress-related oxidative damage to cell membranes.     

Heat treatment to decrease chilling injury in tomato fruit. Effects on lipids, pericarp lesions and fungal growth

Tomato fruits are sensitive to low temperature and develop chilling injury, while at nonchilling temperatures they ripen rapidly. Previously, a hot-air treatment was found to reduce the sensitivity of the fruit to low temperatures. In the present study hot air was compared to hot water and their effects on reducing chilling injury and fungal decay were investigated. Tomatoes ( Lycopersicon esculentum cv. Daniella) at the breaker stage were subjected to hot air, 48 h at 38°C, or various hot water dips, 30 min at 40°C or 2 min at 46, 48 or 50°C, before holding at 2°C. The unheated tomatoes developed chilling injury and fungal infections at 2°C, but not at 12°C. All the heat treatments reduced chilling injury and decay in tomatoes held for 3 weeks at 2°C. The outer pericarp tissue of heated tomatoes had higher phospholipid and lower sterol contents than unheated tomatoes. Heated tomatoes also had less saturated fatty acids than unheated tomatoes held at 2°C, but not at 12°C. Scanning electron micrograph observations showed that all the fruits had microcracks in their surface, but the unheated chilled tomatoes had also fungal growth in the cracks, while those of the heated tomato fruit did not. In the areas of chilling injury collapsed cells were present under the peel and could also support pathogen development. It is suggested that the heat treatment institutes a response to high temperature stress in the fruit tissue that leads to strengthened membranes. This prevents the loss of function and cell collapse which was found in the chilling-injured areas of affected fruit.     

Differential chilling sensitivity in cucumber (Cucumis sativus) seedlings

Cucumber ( Cucumis sativus L. cv. Poinsett 76) seeds were chilled at 2.5°C in a study of the chilling sensitivity and recovery of radicle tissue. The effect of chilling on radicle growth and the production of carbon dioxide and ethylene was measured. Chilling sensitivity of radicles increased as they grew from 1 to 7 mm in length. The length, not the age of the radicles, determined the level of chilling sensitivity. Apical tissue was most sensitive to chilling and slowest to recover from chilling, followed by subapical and basal tissue. Our data demonstrate that the chilling sensitivity of young seedling radicles differs along their length and that the rapid chilling-induced inhibition of elongation is probably due to an inability of meristematic cells to remain viable and active when chilled.     

Reversible change in embryonic diapause intensity by mild temperature in the Chinese rice grasshopper, Oxya chinensis

The effects of temperature on maintenance and termination of embryonic diapause were investigated in Jining (35.4°N, 116.6°E) and Sihong (33.5°N, 118.2°E) strains of the Chinese rice grasshopper, Oxya chinensis Thunberg (Orthoptera: Catantopidae). Eggs of both strains entered diapause when incubated at 30, 25, or 20 °C. Chilling at 8 °C had an evident effect on diapause termination and almost all eggs chilled for 60 days ended diapause development. Chilling of eggs at 8 °C for only 20 days failed to result in any hatching at 20 °C, suggesting that such level of chilling was not enough to induce diapause termination. However, the treatment combining incubation of eggs at 30 °C for varying lengths of time with subsequent incubation to 20 °C had a distinct effect on the completion of diapause of the eggs. The results indicate that there were two temperature optima, that is, low temperature (chilling) and high temperature, for diapause development in this grasshopper species. Incubation of chilled eggs at 20 °C for 5–15 days followed by further incubation at 25 °C reduced termination of diapause significantly compared with the eggs only chilled at 8 °C. Exposure of eggs chilled at 8 °C to a pulse of 25 °C from 1 to 7 days, separated by a 20-day interval at 8 °C, resulted in a decrease in the percentage of successfully hatched eggs as the length of the pulse of 25 °C increased. The results suggest that diapause intensity may be restored at moderately high temperatures. This reversible change in diapause intensity would play an important role in maintaining diapause before winter.     

Influence of photosynthetic photon flux densities before and during long-term chilling on xanthophyll cycle and chlorophyll fluorescence quenching in leaves of tomato (Lycopersicon hirsutum)

A high-altitude ecotype of tomato ( Lycopersicon hirsutum f. typicum Humb. and Bonpl.) has previously been shown to resist further loss of photosynthetic function after three to four days of chilling stress. This study examined the influence of PPFD prior to, and during chilling on the development of protective zeaxanthin and energy-dependent quenching mechanisms in this ecotype. Five-week-old tomato plants were acclimated to either low PPFD (60 μmol m⁻² s⁻¹) or high PPFD (550 μmol m⁻² S⁻¹) at 25/20°C (day/night) for three days, and then exposed to a temperature of 5/5°C and a PPFD of either 60 or 550 μmol m⁻² s⁻¹ for three days. The plants acclimated to low PPFD had lower Chl a/b ratio, and lower level of total Chl per leaf area, total xanthophyll cycle pool and β-carotene. The capacity of their photosynthetic system to resist photoinhibition and to recover photosynthetic function was also lower compared to that of the plants acclimated at high PPFD but exposed to the same chilling stress. In the plants chilled at low PPFD, energy-dependent quenching preceded the formation of zeaxanthin on the first day of chilling and there was an overall reduction in the conversion of violaxanthin to zeaxanthin as compared to the plants chilled at high PPFD. During the last day of chilling-induced photoinhibition, energy-dependent quenching in any of the treatments did not increase, but zeaxanthin levels increased continuously throughout the three days of chilling. Our results suggest that light-acclimation before chilling affects the capacity of the plants to resist chilling-induced photoinhibition. In addition, photoinhibitory quenching appears to be a major component for quenching excessive energy at the latter stage of long-term chilling.     

A thermal time model for predicting time to aerial shoot elongation in Variegated Solomon's Seal

Aerial shoot development in Variegated Solomon's Seal (Polygonatum odoratum‘Variegatum’) was studied under warm (mean 18°C) conditions after dormant rhizomes had been stored at a range of temperatures. After chilling at 0.8–5.5°C for 21–77 days, all rhizomes produced elongated aerial shoots, with mean lengths from 33 cm to 44 cm. Exposure of rhizomes to 15°C or 20°C for 21–77 days resulted in 17% to 50% of buds emerging as shoots, but these either aborted or failed to extend beyond a rosette. The earliest aerial shoot elongation was observed after 7–13 days at 18°C in rhizomes that had been chilled at 0.8–2°C treatments for 59–72 days. The base, optimum and maximum temperatures during pre‐planting storage for subsequent aerial shoot elongation were derived respectively as ?1.5°C, 1.9°C and 15.8°C. A thermal time of storage was calculated from these cardinal temperatures and the rate of progress to normal aerial shoot clongation was shown to increase linearly with increasing thermal time to c. 150°Cd. The thermal time procedure for predicting time to aerial shoot elongation constructed from growth room/chamber data was validated using rhizomes that had been exposed to varying temperatures in shadehouse conditions during the winter in Taiwan.     

Stress and survival of small pike-perch Sander lucioperca (L.) after trawling and chilling

The mortality and stress responses (plasma cortisol concentration) of undersized (total length, L _T, <370 mm) pike-perch Sander lucioperca caught as by-catch in a trawl fishery were assessed. The effects of three different holding methods on pike-perch caught by trawl were compared: (1) fish that were transferred to ambient-temperature water (15·0–21·4° C) immediately after capture and not exposed to chilling water, (2) fish held in chilling water for 10 min and (3) fish exposed to chilling water for 2 h. The sample fish were held and monitored in individual plastic restrainers at ambient-water temperature for 48 h after each of the handling procedures. Total mortality was similar for fish held in chilling tanks for 10 min (27·2% total mortality) compared to fish that were only held in ambient-temperature water (28·2% mortality). Total mortality reached 91·3% when fish were held in chilled water for 2 h. Mortality and plasma cortisol concentrations correlated inversely with size (96–368 mm L _T) and directly with lake-water temperature (15·0–21·4° C) after treatment. In addition, the effect of chilling was observed in elevated cortisol concentration and delayed start of recovery. The results show that quickly removing (<10 min) undersized pike-perch from chilled water could markedly improve the survival of released fish. Due to delayed recovery, however, fish should be allowed to recover (30–40 min) in a deck tank before release.     

Differences in photosynthetic characteristics among northern and southern C4 plants

Both responses to short-term changes of temperature and to chilling under high light were analyzed in populations of Echinochloa crus-galli var. crus-galli (L.) Beauv. from Québec. North Carolina and Mississippi to improve the understanding of C₄ photosynthesis at low temperature. Comparison also included plants of Eleusine indica (L.) Gaertn. from Mississippi to provide for differences among species and populations. Plants were grown at two thermoperiods (28/22°C, 21/15°C). After transfer from cool (21/15°C) to warm (28/22°C) growth conditions, Echinochloa from Mississippi achieved the highest photosynthetic rates. Plants from Québec maintained the highest rates of CO₂ uptake upon transfer to cool conditions. Exposure to 7°C for 3 days at a photon fluence rate of 1000 μmol m−²s−¹ resulted in a reduction in the growth rates of all populations. This reduction was paralleled by a decrease in net photosynthesis and in stomatal conductance. Following chilling under hight light, the reduction in growth parameters was less important for plants from Québec than for the other populations. It suggests that, among other characteristics, northern plants had developed a certain tolerance to chilling under light.     

Leucine uptake by tomato leaf tissue under low temperature: Preincubation dependence of uptake reduction

The uptake of ³H-leucine by leaf fragments of Lycopersicon esculentum Mill. cv. Rutgers and L. hirsutum Humb. & Bonpl., a wild tomato, was studied. Two altitudinal races of L. hirsutum were used which differed in chilling tolerance. The temperature dependence of uptake was initially similar for all plant varieties. However, at temperatures below about 11°C, uptake progressively decreased in the more chilling-sensitive varieties ( L. esculentum , Low-altitude L. hirsutum ), but not in the more chilling-tolerant (high-altitude L. hirsutum ) with increasing preincubation time. More than 60 min preincubation was required for this effect, and it was greatest at the lower temperatures. When leaf fragments, chilled for short periods of time (>22 h), were returned to 22°C, initial rates of uptake were recovered within 2 h. The relationship between membrane lipid changes and membrane protein activity under chill stress is discussed.     

热激对水稻幼苗耐冷性及热激蛋白合成的诱导

萌发的水稻种子经42℃热激处理后其幼苗的耐冷性明显增强,膜伤害程度降低,脯氨酸含量增加,超氧物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POD)活性和抗氧化物质抗坏血酸含量增加,而膜脂过氧化的关键酶脂氧合酶(LOX)活性及其产物丙二醛(MDA)含量下降.并且热激诱导萌发的水稻胚合成78、70、64、60、46、38、24、17、16kD的热激蛋白(HSP),其中属于HSP70的内质网结合蛋白(BiP)的合成与水稻幼苗耐寒性的提高有关.     

Photosynthesis of Coffea arabica after chilling

Net photosynthetic CO₂ exchange of 1-year-old plants of Coffea arabica L. was studied after the above-ground parts had been exposed once or repeatedly to night temperatures in the chilling range. Chill-reduced rates of CO₂ uptake (measured at 24°C and at natural CO, level) were observed after a 12 h night exposure to about 6°C. After exposure to 4°C, activity was reduced to less than half of that of the controls, and after exposure to 0.5°C the leaves suffered visible necrotic injury and were no longer able to take up Co₂ If the leaves were not lethally injured, net photosynthesis recovered completely within 2 to 6 days. About 25% of chill-induced reduction of CO₂ uptake was due to reduced stomatal aperture and 75% to impairment of carboxylation efficiency.
Chilling on successive nights at 4–6°C reduced CO, uptake progressively on each day following treatment. After 10 nights, activity was decreased to less than 10% of initial performance. Conditioning at temperatures slightly above the chilling level (e.g. 15/I2°C) for 2 weeks led to almost complete impairment of photosynthetic activity without additional chilling stress instead of improving chilling tolerance.     

Chilling tolerance of cucumber (Cucumis sativus) seedling radicles is affected by radicle length, seedling vigor, and induced osmotic- and heat-shock proteins

Cucumber seedling radicles decrease in chilling tolerance as they increase in length or decrease in vigor. The protein content of the apical 5 mm of the radicle decreased with decreases in chilling tolerance ( R ² = 0.92). This general reduction in protein content was reflected in a decrease of six dehydrin-like proteins with apparent molecular weights of 13.0, 15.0, 16.8, 23.0, 26.8, and 33.5 kDa. The disappearance of naturally occurring dehydrin-like proteins in cucumber seedling radicles as they elongate or lose vigor was correlated with a loss of chilling tolerance. Exposure to an osmotic (0.6 M mannitol) or heat (2 min at 45°C) stress enhanced chilling tolerance. The osmotic-shock treatment induced both chilling tolerance and the appearance or strengthening of dehydrin-like proteins previously present in radicles. The heat-shock treatment also induced high levels of chilling tolerance and protein(s) that reacted with a 23 and 70 kDa antibody. However, these heat-shock protein (HSPs) did not cross react with the probe for dehydrin-like proteins. When organized into high, medium, and low chilling tolerance groups, radicle that were chilling tolerant contained either the 13.0 and 16.8 kDa dehydrin-like proteins, or the 15.0 and 23.0 kDa dehydrin-like proteins, or the 23 or 70 kDa HSP.     

The effect of low temperatures on the phloem transport of radioactive assimilates in the stolon of Saxifraga sarmentosa L.

Abstract. The movement of naturally assimilated ¹⁴C down the stolon of Saxifraga sarmentosa under the influence of localized cold treatments (0°-10°C) applied over lengths up to 10 cm was followed by finding the distribution of tracer at the termination of the experiment. Inhibition is slight at 10°C and very considerable at 0°C. It is very dependent on the length treated, but is definite even when this is only 0.8 cm. Cooling the daughter plantlet to 0°C has a smaller inhibitory effect than might be expected. This is difficult to understand if 'unloading' is metabolic; perhaps the concentration free energy of the sucrose or other solute can be utilized in the sink terminals on the lines of the chemi-osmotic hypothesis. If so the same source of non-respiratory free energy might be available to energize an active sieve-tube mechanism in mid-path chilling experiments.     

Oxygen-mediated cold-acclimation in cucumber (Cucumis sativus) seedlings

Cold acclimation of etiolated cucumber seedlings, consisting of cooling at 12°C for 48 h followed by a warming period at 25°C, led to tolerance to subsequent chilling at 2°C. Tolerance, as evidenced by freedom from chilling injury and continued growth, developed during the warming period in a time-course manner for 12 h but decreased with prolonged warming. A similar increase and subsequent decrease was also observed in the content of palmitic, linoleic and linolenic acids in total lipid fraction from cucumber hypocotyl tissue. During the warming period supra-ambient oxygen stimulated, whereas subambient oxygen inhibited, the increase in fatty acid content as well as development of chilling tolerance. A strong correlation between oxygen-mediated changes in fatty acid content and associated development of cold tolerance suggests that both these processes are interrelated. Cold acclimation, but not cold stress, led to an increase followed by a decrease in CO₂ evolution suggesting that a respiratory upsurge is yet another feature of cold acclimation in cucumbers.