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Chilling injury and changes in adenosine triphosphate of cotton seedlings.

Young Gossypium hirsutum L. seedlings chilled at 5 degrees showed a continual decrease in ATP concentration with time of chilling. Chilled plants returned to optimum conditions were able to restore the initial ATP concentration when chilled only 1 day, but not when chilled 2 days. The decrease in ATP with chilling was prevented by hardening the seedlings at 15 degrees for 2 days (14-hr-day-length) immediately before chilling. The ATP level of hardened plants was higher than of unhardened plants. When hardened plants were chilled at 5 degrees , the ATP level increased in the leaves but decreased in the roots.A mechanism of chilling injury is discussed in relation to the decrease in ATP concentration at low temperature.

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Ethylene Production by Chilled Cucumbers (Cucumis sativus L.).

Chilling at 2.5 C accelerated the synthesis of 1-aminocyclopropane-1-carboxylic acid (ACC) and C(2)H(4) production in cucumber fruit. Skin tissue contained higher levels of ACC and was more sensitive to chilling than was cortex tissue. Accumulation of ACC in chilled tissue was detected after 1 day of chilling and remained elevated even after C(2)H(4) production started to decline. These data suggest that ACC synthesis is readily stimulated by chilling, whereas the system that converts ACC to C(2)H(4) is vulnerable to chilling injury. Chilling-induced C(2)H(4) production was inhibited by amino-ethoxyvinylglycine, sodium benzoate, propyl gallate, 2,4-dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone, and cycloheximide. The utilization of methionine for ACC formation and chilling-induced C(2)H(4) biosynthesis was established using l-[3,4-(14)C]methionine. Chilled tissue had a higher capacity to convert l-[3,4-(14)C]methionine to ACC and C(2)H(4) than did nonchilled tissue.

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Impairment of photosynthesis by chilling-temperatures in tomato.

Chilling of attached tomato leaves (cv. Rutgers) in the dark for 16 hours at 1 C decreased both photosynthesis and transpiration. To separate the effects of chilling on stomatal CO(2) conductance from more direct effects of chilling on the chloroplasts' activities, measurements of photosynthesis and transpiration were made at atmospheric and saturating CO(2) levels. At atmospheric CO(2), the inhibition of photosynthesis was approximately 60%, of which about 35% was attributable to the impairment of chloroplast function and about 25% was attributable to decreased stomatal conductance. However, the affinity of the photosynthetic apparatus for CO(2) was not changed by chilling, since the dependence of the relative rate of photosynthesis on the intercellular CO(2) concentration was unaltered. The apparent quantum requirement for CO(2) reduction also was identical in chilled and unchilled plants. This observation contradicts the widely held notion that the chilling-induced inhibition of photosynthesis is caused by an impairment of the water oxidation mechanism. The impairment of chloroplast activity was not a consequence of an unfavorable water status within the leaf, since chilling caused only a small drop (1 bar) in water potential. A small loss of chlorophyll resulted as a secondary effect of chilling, but this loss of chlorophyll was eliminated as a cause of the inhibition of photosynthesis.No recovery of chloroplast activity occurred during the subsequent light period after chilling. The recovery seemed to be inhibited by light or to require both a light period and a dark period or to occur after a considerable lag period. After a period of both light and dark, restoration of stomatal conductance occurred more slowly than did the recovery of chloroplast activity.

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Protein synthesis and phospholipids in soybean axes in response to imbibitional chilling.

The responses of two cultivars of soybean (Merr.) to a chilling treatment (4 C for first hour of imbibition) were compared. The germination of cv. Biloxi was unaffected by the treatment, while the germination of cv. Fiskeby was reduced. The phospholipid fatty acids of dry axes of the two cultivars were very similar, and, thus, could not be correlated with their responses to chilling. The fatty acid composition of chilling-tolerant Biloxi did not change over a subsequent 23-hour warm incubation, but there was a marked reduction in the unsaturated fatty acids of chilling-sensitive Fiskeby after 12 hours, which may be a symptom of deterioration. Protein synthesis in both cultivars was reduced by the chilling treatment. Redrying of Biloxi axes up to 18 hours after the onset of imbibition had no effect on their germination upon rehydration. Germination of Fiskeby axes was reduced by redrying after 8 hours of imbibition. After 7 months of dry storage of intact seeds, the sensitivity of the axes to chilling was retested. Biloxi axes had become chilling-sensitive, while the germination of Fiskeby axes was reduced to zero by the chilling treatment. A hypothesis is presented that imbibitional chilling sensitivity is an indication of reduced vigor, axes with a high vigor can tolerate the stress, while those without cannot.

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Endogenous Gibberellin-Like Substances in Somatic Embryos of Grape (Vitis vinifera x Vitis rupestris) in Relation to Embryogenesis and the Chilling Requirement for Subsequent Development of Mature Embryos.

Endogenous gibberellin (GA)-like substances were examined in suspension cultures of somatic embryos of a hybrid grape (Vitis vinifera x Vitis rupestris) during embryogenesis, and in mature embryos chilled at 4 degrees C, and subsequently incubated at 26 degrees C with and without abscisic acid (ABA). The extract was separated into a nonpolar fraction (would contain GA-precursors); a fraction that would contain free GAs; and a highly H(2)O-soluble fraction (would contain GA glucosyl conjugates and very polar free GAs). Quantitation after SiO(2) partition chromatography was accomplished by microdrop and immersion dwarf rice bioassays. As embryogenesis developed, the free and highly H(2)O-soluble GA-like substances, expressed on a dry weight basis, decreased (however, they increased on a per embryo basis). Chilling at 4 degrees C for 1 week greatly increased activity of free GA-like substances (per g dry weight and per embryo), it then declined over the next three weeks of chilling. Activity (per g dry weight and per embryo) in the H(2)O-soluble fraction declined throughout chilling. Activity in the GA-precursor fraction, however, increased steadily with chilling (per g dry weight and per embryo). Incubation at 26 degrees C after chilling enhanced activity in the free GA and H(2)O-soluble fractions (per g dry weight and per embryo), but activity in the GA-precursor fraction dropped dramatically. Incubation at 26 degrees C with (+/-) ABA after chilling prevented germination and maintained high activity for GA precursors and less polar free GAs and low activity in the polar free GA and H(2)O-soluble fractions.Kaurene and kaurenoic acid were characterized in the GA-precursor fraction of chilled embryos by gas-liquid chromatography-mass spectrometry (GLC-MS). The existence of GA(4) and GA(9) in ABA-treated, chilled embryos was also confirmed by GLC-MS.

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Role of carbohydrates in diurnal chilling sensitivity of tomato seedlings.

Tomato seedlings (Lycopersicon esculentum Mill.) chilled starting at different times during the light/dark cycle were most chilling-sensitive at the end of the dark period (AI King, MS Reid, BD Patterson 1982 Plant Physiol 70: 211-214). Low-temperature tolerance was regained with as little as 10 minutes of light exposure. Low light intensities were less effective than high light intensities in reducing sensitivity, and the length of exposure to light directly influenced sensitivity. Seedlings kept at low night temperatures prior to chilling were also less injured following chilling. Light also restored chilling tolerance to seedlings whose roots were removed. Supplying cut shoots with sucrose, glucose, or fructose reduced chilling sensitivity and largely eliminated the diurnal difference in sensitivity. Endogenous carbohydrate content was correlated with changes in chilling sensitivity; starch and sugar content fell markedly during the dark period. Increased concentrations of sugars were detected 15 minutes after the start of the light period. This evidence all suggests that changes in chilling sensitivity over the diurnal period are regulated by the light cycle. It also suggests that increased sensitivity at the end of the dark period could be due to carbohydrate depletion, and that chilling tolerance following light exposure is likely due to carbohydrate accumulation or closely related events.

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A chilling sensitive mutant of Arabidopsis with altered steryl-ester metabolism.

A chilling-sensitive mutant of Arabidopsis thaliana was isolated and subjected to genetic, physiological, and biochemical analysis. The chilling-sensitive nature of the mutant line is due to a single recessive nuclear mutation at a locus designated chs1. In contrast to wild-type plants, which are not adversely affected by low temperatures, the chs1 mutant is killed by several days of exposure to temperatures below 18 degrees C. Following exposure to chilling temperatures, the mutant displays two common symptoms of chilling injury-leaf chlorosis and electrolyte leakage. In these respects, the physiological response of the mutant to low temperatures mimics the response observed in some naturally occurring chilling sensitive species. The biochemical basis of chilling sensitivity was explored by examining the pattern of incorporation of (14)CO(2) into soluble metabolites and lipids in wild-type and mutant plants. The only difference observed between the mutant and wild type was that following low temperature treatment, the mutant accumulated 10-fold more radioactivity in a specific class of neutral lipids which were identified by a variety of criteria to be steryl-esters. The accumulation of radioactivity in the steryl-ester fraction occurs 24 hours before there is any visible evidence of chilling injury. These results suggest one of two possible explanations: either the mutation directly affects sterol metabolism, which in turn leads to chilling sensitivity, or the mutation affects another unidentified function and the accumulation of radioactivity in steryl-esters is a secondary consequence of chilling injury.

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Superoxide dismutase and chilling injury in Chlorella ellipsoidea.

The relationship between superoxide dismutase (SOD) and chilling injury was examined in chilling-sensitive and chilling-resistant strains of Chlorella ellipsoidea. The sensitive strain contained less SOD than the resistant strain. Moreover, all of the SOD in the sensitive strain was the H2O2-sensitive, iron-containing SOD, whereas most of the SOD in the resistant strain was the H2O2-resistant, manganese-containing SOD. Illumination further enhanced the disparity in SOD content between the sensitive and resistant strains since the SOD in the former declined during illumination, whereas the SOD in the latter strain did not. It was possible to elevate the SOD content of the sensitive strain and to increase the proportion of MnSOD by prior growth in the presence of 50 microM paraquat. The SOD content of the cultures after 5 h of illumination at 4 degrees C fell in the order sensitive strain less than paraquat-induced sensitive strain less than resistant strain. The resistance of these cultures to chilling injury was related to SOD content. This was the case whether resistance was assessed in terms of growth rate after chilling, bleaching of chlorophyll during chilling, or loss of viability during chilling. It thus appears likely that O2- is an agent of chilling injury.

Adaptation, Biological↗

Search for an endotherm in chloroplast lamellar membranes associated with chilling-inhibition of photosynthesis.

The phase transition of chloroplast lamellar membrane lipids has been proposed to be the underlying cause of chilling-induced inhibition of photosynthesis in sensitive plants. Differential scanning calorimetry has been used to search for any endotherms arising from lipid state changes in chloroplast lamellar membranes of the chilling-sensitive plants cantaloupe , kidney bean, domestic tomato, and soybean. For comparison, calorimetric scans of chloroplast lamellar membranes from the chilling-insensitive plants spinach, pea, and wild tomato were made. A large reversible endotherm, extending from below 10 degrees to nearly 40 degrees C, was observed in chloroplast membranes from tomatoes of both chilling-sensitive (Lycopersicon esculentum Mill. cv. Floramerica ) and chilling-insensitive (L. hirsutum LA 1361) species. A much smaller endotherm, approximately 5 to 10% of the area of that seen in the two tomato species, and extending over a similar temperature range, was detected in chloroplasts from chilling-insensitive spinach and peas, and also was generally observed in chloroplasts from chilling-sensitive cantaloupe , kidney bean, and soybean. The enthalpy of these smaller endotherms indicates that, if the endotherm arose entirely from a lipid transition, then it corresponded to the melting of less than about 10% of the total membrane polar lipid. On the basis of these data it is concluded that there is no correlation between chilling sensitivity of photosynthesis and the presence or absence of a phase transition of bulk membrane lipids of the chloroplast lamellar membrane at temperatures above 5 degrees C.

Chloroplasts↗

Different effects of chilling on respiration in leaves and roots of cucumber (Cucumis sativus).

The effects of chilling on respiration (SHAM-resistant, cytochrome pathway and KCN-resistant, alternative pathway), temperature sensitivity, relative electrolyte conductivity, and degrees of oxidative stress (H(2)O(2) and malonaldehyde (MDA) contents) were separately examined in leaves and roots of cucumber (Cucumis sativus L.). After chilling at 8 degrees C for 4 days, both total respiration and KCN-resistant respiration in roots increased at different measurement temperatures. In contrast, SHAM-resistant respiration remained unchanged. In comparison, chilling significantly decreased the total respiration in leaves and this decrease was mostly due to a decrease in SHAM-resistant respiration. Chilling apparently decreased the sensitivity of KCN-resistant respiration to changes of temperature. The reduction levels of ubiquinone pool (UQr/UQt) increased both in chilled leaves and roots whilst pyruvate content increased only in chilled roots, but not in chilled leaves. Furthermore increases of H(2)O(2) and MDA contents were much greater in leaves than in roots. The same trend was also observed for ion leakage from tissues. Taken together, the results suggested that the higher chilling tolerance of roots was associated with their high total respiration and KCN-resistant respiration.

Cold Temperature↗

Hygiene aspects of modern poultry chilling.

An evaluation was made of six commercial poultry chilling systems in relation to factors affecting microbial-contamination of carcasses. These systems included water immersion chilling, air chilling and air chilling with evaporative cooling using water sprays. Samples of neck skin and body cavity were taken from carcasses, together with samples from the chilling environment. These were examined for total aerobic mesophilic microbes and counts of presumptive coliform bacteria and Pseudomonas spp. at specific points in the chilling process. Physical measurements included surface and deep-muscle temperatures of carcasses, water temperatures and chlorine concentrations in the immersion system and air speed and temperature during air chilling. The results obtained for water immersion chilling confirmed previous experience that the washing effect reduces microbial contamination of carcasses, although initially the numbers of pseudomonads tended to increase. The air chillers varied in design and mode of operation, but had little overall effect on microbial contamination of the skin. When a completely dry process was used, microbial numbers were reduced approximately ten-fold in the body cavity. However, the use of water sprays tended to increase contamination of the cavity, while relatively heavy spraying using non-chlorinated water, resulted in a substantial increase in the numbers of pseudomonads.

Animals↗

Chilling stress-induced changes of antioxidant enzymes in the leaves of cucumber: in gel enzyme activity assays.

To investigate the antioxidant defense system, chilling stress-induced changes of antioxidant enzymes were examined in the leaves of cucumber (Cucumis sativus L.). Chilling stress preferentially enhanced the activities of the superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR) and peroxidase specific to guaiacol, whereas it induced the decrease of catalase activity. In order to analyze the changes of antioxidant enzyme isoforms against chilling stress, foliar extracts were subjected to native PAGE. Leaves of cucumber had four isoforms of Mn-SOD and two isoforms of Cu/Zn-SOD. Fe-SOD isoform was not observed in this plant. Expression of Cu/Zn-SOD and Mn-SOD was preferentially enhanced by chilling stress. Expression of Mn-SOD-2 and -4 was enhanced after 48 h of the poststress period. Five APX isoforms were presented in the leaves of cucumber. The intensities of APX-4 and -5 were enhanced by chilling stress, whereas that of APX-3 was significantly increased in the poststress periods after chilling stress. Gel stained for GR activity revealed six isoforms in the plant. Activation levels for most of GR isoforms were higher in the stressed-plants than the control and poststressed-plants, but that of GR-1 isoform was significantly higher in the poststressed-plants than chilling stressed-plants. These results collectively suggest that chilling stress activates the enzymes of an SOD/ascorbate-glutathione cycle under catalase deactivation in the leaves of cucumber, but the response timing of enzyme isoforms against various environmental stresses is not the same for all isoforms of antioxidant enzymes.

Journal Article↗

Effects of chilling on the expression of ethylene biosynthetic genes in Passe-Crassane pear (Pyrus communis L.) fruits.

Passe-Crassane pears require a 3-month chilling treatment at 0 degrees C to be able to produce ethylene and ripen autonomously after subsequent rewarming. The chilling treatment strongly stimulated ACC oxidase activity, and to a lesser extent ACC synthase activity. At the same time, the levels of mRNAs hybridizing to ACC synthase and ACC oxidase probes increased dramatically. Fruit stored at 18 degrees C immediately after harvest did not exhibit any of these changes, while fruit that had been previously chilled exhibited a burst of ethylene production associated with high activity of ACC oxidase and ACC synthase upon rewarming. ACC oxidase mRNA strongly accumulated in rewarmed fruits, while ACC synthase mRNA level decreased. The chilling-induced accumulation of ACC synthase and ACC oxidase transcripts was strongly reduced when ethylene action was blocked during chilling with 1-methylcyclopropene (1-MCP). Upon rewarming ACC synthase and ACC oxidase transcripts rapidly disappeared in 1-MCP-treated fruits. A five-week treatment of non-chilled fruits with the ethylene analog propylene led to increased expression of ACC oxidase and to ripening. However, ethylene synthesis, ACC synthase activity and ACC synthase mRNAs remained at very low level. Our data indicate that ACC synthase gene expression is regulated by ethylene only during, or after chilling treatment, while ACC oxidase gene expression can be induced separately by either chilling or ethylene.

Alkenes↗

Dark chilling effects on soybean genotypes during vegetative development: parallel studies of CO2 assimilation, chlorophyll a fluorescence kinetics O-J-I-P and nitrogen fixation.

The effects of dark chilling on CO2 assimilation, chlorophyll a fluorescence kinetics and nitrogen fixation were compared in two Glycine max (L.) Merr. genotypes. The aim was to elucidate the mechanisms by which photosynthesis was inhibited as well as identification of selection criteria for dark chilling tolerance. Seedlings were dark chilled (8 degrees C) for 9 consecutive nights but kept at normal day temperatures (28 degrees C). CO2 gas exchange analysis indicated that photosynthesis in Maple Arrow was inhibited largely as a result of stomatal limitation, while in Fiskeby V, it indicated inhibition of the mesophyll reactions. Increased intercellular CO2 concentration and decreased carboxylation efficiency suggested loss of Rubisco activity in Fiskeby V, although no effect on the KM (CO2) of Rubisco was observed. Quantification and deconvolution of the Chl a fluorescence transients into several phenomenological and biophysical parameters (JIP-test) revealed large genotypic differences in the response of PSII to dark chilling. These parameters differentially changed in the two genotypes during the progression of the chilling treatment. Among them, the performance index, reflecting several responses of the photochemical apparatus, provided the best preliminary overall assessment of the genotypes. In contrast, the quantum yield of primary photochemistry varphiPo (FV/FM) was quite insensitive. The recovery of most of the JIP-test parameters in Maple Arrow after 6 and 9 nights of dark chilling was a major genotypic difference. Genotypic differences were also observed with regard to the ureide response and N2 fixation appeared to be more sensitive to dark chilling than CO2 assimilation. The JIP-test provided information consistent with results derived from CO2 assimilation and N2 fixation studies suggesting that it can substitute the much more time-consuming methods for the detection of chilling stress and can well satisfy the requirements of a rapid and accurate screening method.

Journal Article↗

Investigation of hygiene aspects during air chilling of poultry carcases using a model rig.

1. An experimental rig, designed and built to simulate conditions found in commercial poultry chilling systems, was used to investigate the effects of varying air temperature and chilling duration, and the effect of chlorinated water sprays, on the microbial load present on the skin and in the body cavity of freshly eviscerated poultry carcases; deep muscle and skin temperatures were monitored during chilling at three different temperatures. 2. During dry chilling for 2 h, total viable microbe counts (TVC) and counts of coliforms and pseudomonads from the body cavity fell by between half and one log unit; smaller reductions were observed in samples from the breast skin. 3. The situation changed when chlorinated water sprays (50, 100 or 250 ppm available chlorine) were applied for the first hour of chilling; spraying carcases enhanced the reduction in numbers on the skin; the effect was most pronounced with 250 ppm chlorine; conversely in the body cavity, the general effects of sprays was to increase contamination by up to one log unit. 4. There was no evidence that sprays increased the rate of chilling. 5. When carcases were held overnight in the rig at 11 degrees C after chilling, microbe counts on dry-chilled carcases remained stable, but increased on carcases that had been sprayed with chlorinated water.

Animals↗

Genotypic variation of Rubisco expression, photosynthetic electron flow and antioxidant metabolism in the chloroplasts of chill-exposed cucumber plants.

Genetic improvement of agronomic crops is necessary to cope with chilling stress. To identify the physiological factors responsible for this genotypic difference in chill-induced inhibition of photosynthesis, leaf CO2 assimilation, the electron flux in the chloroplast and the antioxidant metabolism in isolated chloroplasts were examined in two genotypes of cucumber (Cucumis sativus) plants with distinct chilling tolerance. Cucumber plants were exposed to 100 micromol m(-2) s(-1) at 9/7 degrees C (day/night) for 10 d and were then returned to optimal conditions for 2 d. Chilling resulted in more significant reductions in rbcL and rbcS transcripts, ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) content and initial Rubisco activity, leading to higher electron flux to O2 in the chilling-sensitive genotype than in the chilling-tolerant genotype. The chilling-tolerant genotype showed lower H2O2 contents in the chloroplasts by maintaining higher H2O2-scavenging activity in the chloroplasts than in the chilling-sensitive genotype. H2O2 accumulation in chloroplast was negatively correlated with the initial Rubisco activity and photosynthetic rate.

Antioxidants↗

Rapid postmortem pH decline and delayed chilling reduce quality of turkey breast meat.

Effects of rapid postmortem metabolism and delayed chilling on turkey breast meat quality were assessed using color measurements, protein extractability, and gelation characteristics. Based on 15 min postmortem breast muscle pH, tom turkey carcasses were classified as rapid glycolyzing (RG), pH < 5.80, or normal glycolyzing (NG), pH > 6.00. Two carcasses per group with similar ultimate pH values were selected on four occasions for a total of 16 carcasses. One half of each carcass was immersion-chilled at 20 min postmortem, the other half was maintained at body temperature for 110 min and then chilled. Breast meat from RG carcasses had higher carcass temperature (15 min) and lower protein (sarcoplasmic and myofibrillar) extractability than breast meat from NG carcasses. Delayed chilling increased all breast meat color values (L*, a*, b*), and decreased protein extractability and cook yield compared to breast meat from immediately chilled carcass halves. The true strain of cooked gels was reduced for RG carcasses. Delayed chilling reduced both true strain and stress of meat gels. There were no interactions between the rate of postmortem pH decline and initiation of chilling, indicating that reductions in meat quality due to delayed chilling were independent of the carcass classification for rate of postmortem glycolysis. Results indicate that factors that affect both rate of postmortem glycolysis and carcass temperature decline are important to turkey breast meat quality.

Animals↗

Net CO(2) assimilation of cacao seedlings following dark chilling.

Overnight exposure of cacao (Theobroma cacao L.) seedlings to chilling temperatures between 4.7 and 15.8 degrees C reduced net CO(2) assimilation rate (A) and stomatal conductance to water vapor (g(s)), with temperatures below 10 degrees C causing severe inhibition. Net CO(2) assimilation rates of chilled seedlings recovered to those of nonchilled plants within 7 days. No differences in daytime intercellular CO(2) concentration (c(i)) with overnight temperature were observed on the first day after the chilling treatment, which indicates that the reduction in photosynthesis was not caused by the reduction in stomatal conductance. However, c(i) of chilled plants was much less than that of nonchilled plants on the second day after treatment, which suggests that chilling caused a change in stomatal response to CO(2) concentration. Even 7 days after treatment, when A had recovered to control values, g(s) of chilled leaves was only approximately 70% that of controls. Chilling did not inhibit A through an effect on leaf water potential, which was higher in chilled plants than in unchilled plants.

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