Low temperature acclimation mediated by ethanol production is essential for chilling tolerance in rice roots

Rice seedlings (Oryza sativa L.) were subjected to low temperature pretreatment (LT-PT; 10°C) for various length of time followed by a 48-h chilling temperature stress (2°C). Chilling tolerance of rice roots was improved with increasing duration of LT-PT, but HT-PT longer than 12 h gave no additional improvement. LT-PT did not change in fatty acid composition in rice roots under the present experimental condition. Alcohol dehydrogenase (ADH) activity and ethanol concentration in the roots were increased with increasing duration of LT-PT up to 12 h, which indicates that LT-PT increased ethanol fermentation in the roots. 4-Methylpyrazole, a potent inhibitor of ADH, reduced the ethanol concentration and the chilling tolerance in the roots. This reduction of the chilling tolerance recovered with exogenously applied ethanol. Ethanol also induced 21- and 33-kD protein synthesis in the roots and these proteins may contribute the improvement of the tolerance. The present research suggests that LT-PT may increase chilling tolerance in rice roots owing to ethanol production, and ethanol may trigger a signal transduction cascade, which might lead to a decrease in membrane damage and injury.Key words: acclimation, alcohol dehydrogenase, chilling tolerance, ethanol, heat shock protein, low temperature, Oryza sativaAlcohol dehydrogenase (ADH; EC.1.1.1.1) gene and protein were induced by low temperature in Arabidopsis, maize and rice seedlings.^1,2,3 ADH is an enzyme involved in ethanolic fermentation and essential for plants to survive under anaerobic conditions.^4,5 However, it is unlikely that the induction of ADH by low temperture is due to a switch from aerobic respiration to anaerobic respiration as reported with anaerobic conditions.^2,6 Therefore, it is not clear that biological meanings of the induction of ADH in low temperature conditions.Rice seedlings (Oryza sativa L. cv. Nipponbare) were subjected to low temperature pretreatment (LT-PT; 10°C) for various length of time (1, 2, 4, 6, 12, 18, 24 h) followed by a 48-h chilling temperature stress (2°C). Chilling tolerance of rice roots was improved with increasing duration of LT-PT, but HT-PT longer than 12 h gave no additional improvement. LT-PT did not change in any fatty acid compositions in rice roots under the present experimental condition. Several plant species, such as oat, rye and spinach increased freezing tolerance due to the increasing unsaturation of fatty acids in plasma membranes, but this cold acclimation process required exposure of these plants to subzero temperature for 2–3 weeks.^7,8LT-PT increased ADH activity and ethanol concentration in rice roots, and the activity and the concentration were increased with increasing duration of LT-PT up to 12 h. Thus, LT-PT induced ethanolic fermentation system and stimulated ethanol production in the roots. 4-Methylpyrazole, which is a potent inhibitor of ADH and prevents ethanol production,⁹ reduced rice root growth to 40% of LP-PT root growth (Fig. 1), and the ethanol accumulation in the roots. This growth inhibition by 4-methylpyrazole recovered with exogenously applied ethanol. These results suggest that ethanol produced by LT-PT may contribute the chilling tolerance in the roots of the rice seedlings. In addition, an ADH deficient mutant of maize seedlings, which can not produce ethanol, was more sensitive to chilling temperature than their wild types.⁶Open in a separate windowFigure 1Effects of ethanol and 4-methylpyrazole on root growth of rice seedlings. Three-day-old rice seedlings were treated 12-h LT-PT (10°C) with or without 100 mM ethanol and/or 5 mM 4-methylpyrazole at 25°C for 24 h, and then subjected to chilling stress treatment (2°C, 48 h). Elongation of rice roots was determined over 48 h at 25°C after chilling stress treatment. Non-stressed seedlings and ethanol-treated seedlings were grown at 25°C. Chilling stressed seedlings were grown at 25°C for 24 h, and then subjected to chilling stress treatment. Means ± SE from five independent experiments with 20 plants for each determination are shown.When the seedlings were subjected to chilling temperature stress after ethanol treatment without LT-PT, the growth inhibition of rice roots by chilling temperature recovered from 22% to 71% of that of nonstressed roots (Fig. 1), which suggests that exogenously applied ethanol may improve chilling tolerance in the roots. It is also found that the ethanol treatment did not change in fatty acid composition in the roots at the temperature of this treatment (25°C).Chilling temperature induced lipid degradation in plant cells of cold-sensitive plants, such as cucumber, rice and soybean, as measured by an increase in malondialdehyde, which is a decomposition product of phospholipid peroxidation.¹⁰ Lipid peroxidation occurs when polyunsatured fatty acids are released from phospholipids by phospholipases and became substrates for lipoxygenases. Changes in the structural composition of the plasma membranes by lipid peroxidation cause the phase transition of the membrane from liquid to gel and the inactivation of membrane bound enzymes such as plasma membrane ATPase. Thus, the phase transition of the membranes was thought to be one of the primary causes of chilling injury.^11–13The addition of C₁ to C₆ alcohols including ethanol to model membranes increased fluidity of the membranes and lowered the phase transition temperature of the membranes.^14,15,16 Therefore, ethanol produced by LT-PT may prevent the phase transition of the membrane from liquid to gel, and lower the phase transition temperature of the membranes, which may contribute the acclimation to the chilling tolerance. In addition, ethanol induced an increase in ATPase activity in plasma membranes,⁶ and prevented chilling-induced ion leakage from plant tissues.¹⁷Ethanol is also known to stimulate the synthesis of heat shock protein (HSP) in yeast, bacteria and some other plants.^18,19 We thus determined the effect of ethanol on protein synthesis in rice roots by SDS-gel electrophoresis, and found that 21- and 33-kD protein synthesis were induced by ethanol. These proteins were also induced by heat shock treatment (45°C, 20 min). HSP was shown to be associated with the development of low temperature tolerance in spinach.^20,21 Thus, 21- and 33-kD proteins induced by ethanol may contribute the improvement of the chilling tolerance.The present research suggests that LT-PT-induced chilling tolerance may be owing to ethanol accumulation in rice roots. Accumulated ethanol may increase the fluidity of plasma membranes and lower the phase transition temperature of the membranes, and may also induce protein synthesis. This hypothesis is supported by exogenously applied ethanol which increased the chilling tolerance. Thus, ethanol might trigger a signal transduction cascade, which would lead to a decrease in membrane damage and injury. Further work needs to be done to test this possibility.     

The role of salicylic acid in response of two rice cultivars to chilling stress

Two rice (Oryza sativa L.) cultivars differing in chilling sensitivity, Changbaijiu (chilling-tolerant) and Zhongjian (chilling-sensitive) were pre-treated with 0.5, 1.0 and 2.0 mM salicylic acid (SA) for 24 h before chilling at 5°C for 1 d. Chilling induced SA accumulation, particularly conjugated SA in both leaves and roots of the two rice cultivars. After SA administration, SA accumulated in the roots of both cultivars at a concentration-dependent manner, whereas only a slight increase was observed in their leaves. Conjugated SA accounted for most of the increase. The beneficial effect of SA treatment on protecting rice seedlings from chilling injury was not observed at any concentration in either cultivar. Pre-treatment with SA even decreased their chilling tolerance confirmed by increased electrolyte leakage and lipid peroxidation. Further, most of the activities of antioxidant enzymes decreased or remained unchanged in leaves and roots of SA pre-treated seedlings after chilling. These results implied that down-regulation of antioxidant defence might be involved in the reduction of chilling tolerance in SA-pre-treated plants.     

Glycinebetaine increases chilling tolerance and reduces chilling-induced lipid peroxidation in Zea mays L.

Chilling tolerance was increased in suspension‐cultured cells and seedlings of maize (Zea mays L. cv ‘Black Mexican Sweet’) grown in media containing glycinebetaine (GB). A triphenyl tetrazolium chloride (TTC) reduction test indicated that after a 7 d chilling period at 4 °C, cells treated with 1 mm GB at 26 °C for 1 d had a survival rate (30%) that was twice as high as that of untreated controls. The addition of 2·5 m M GB to the culture medium resulted in maximum chilling tolerance (40%). The results of a cell regrowth assay were consistent with viability determined by the TTC method. In suspension‐cultured cells supplemented with various concentrations of GB, accumulation of GB in the cells was proportional to the GB concentration in the medium and was saturated at a concentration of 240 μ mol (g DW) ^{? 1}. The degree of increased chilling tolerance was positively correlated with the level of GB accumulated in the cells. The increased chilling tolerance was time‐dependent; i.e. it was first observed 3 h after treatment and reached a plateau after 14 h. Feeding seedlings with 2·5 m M GB through the roots also improved their chilling tolerance, as evidenced by the prevention of chlorosis after chilling for 3 d at 4 °C/2 °C. Lipid peroxidation, as expressed by the production of malondialdehyde, was significantly reduced in GB‐treated cells compared with the untreated controls during chilling. These results suggest that increased chilling tolerance may be due, in part, to the reduction of lipid peroxidation of the cell membranes in the presence of GB.     

Effect of chilling on antioxidant enzymes and DPPH-radical scavenging activity of high- and low-vigour cucumber seedling radicles

The chilling tolerance of cucumber seedling radicles was influenced by their relative levels of vigour. Radicles of high‐vigour seedlings grew to 20 mm in length in 36 h at 25 °C, whereas it took 60 h for low‐vigour seedling radicles to reach that length. Chilling at 2·5 °C for 48 h inhibited the subsequent growth of high‐ and low‐vigour seedlings by 39 and 68%, respectively. The 2,3,5‐triphenyltetrazolium chloride (TTC) viability index, and α,α‐diphenyl‐β‐picrylhydrazyl (DPPH)‐radical scavenging activity were higher in high than low‐vigour radicles. Higher ascorbate peroxidase (APX) and catalase (CAT) enzyme activity, DPPH‐radical scavenging activity, and recovery of CAT activity after chilling in high‐vigour radicles corresponded with their higher level of chilling tolerance in comparison with low‐vigour radicles. In contrast, elevated levels of superoxide dismutase, glutathione reductase and guaiacol peroxidase appear to be correlated with chilling injury since they only showed substantial increases in activity in the more chilling‐­sensitive low‐vigour radicles after chilling. Manipulation of APX, CAT, and/or DPPH activity could produce plants with superior and persistent chilling tolerance.     

Anoxia tolerance in rice roots acclimated by several different periods of hypoxia

Rice (Oryza sativa L.) seedlings were subjected to hypoxic pretreatment (H-PT; incubated in 5% O2 atmosphere) for various lengths of time followed by an anoxic stress. Anoxia tolerance of rice roots was improved with increasing duration of H-PT, but longer H-PT than 12 h gave no additional improvement. Concentrations of ATP and ethanol, and activities of pyruvate decarboxylase (EC 4.1.1.1) and alcohol dehydrogenase (EC 1.1.1.1) in the roots were increased by H-PT, and the times and patterns of increasing in these concentrations and activities were similar to those of increasing in the anoxia tolerance. These results suggest that the H-PT may increase anoxia tolerance due to maintenance of ATP levels with rapid induction of ethanolic fermentation, and hypoxic acclimation may occur within 12 h.     

Polyamine content and chilling susceptibility are affected by seasonal changes in temperature and by conditioning temperature in cold-stored 'Fortune' mandarin fruit

‘Fortune’ mandarins are prone to develop pitting and necrosis upon exposure to low temperatures. We have examined the effect of field temperature during fruit maturation and the effect of conditioning temperatures (from 2 to 37°C) prior to cold storage on the content of polyamines (PAs) and on chilling susceptibility in order to understand the role of PAs in maturation and chilling tolerance of this citrus cultivar. Chilling susceptibility and the content of PAs were more affected by seasonal changes in field temperature than by the stage of fruit maturity. The highest putrescine (Put) and spermidine (Spd) content was found in fruits exposed to the lowest field temperatures. These fruits were in turn more susceptible to develop chilling injury (CI) after storage at 2°C. Spermine (Spm), however, decreased in attached fruit with time of exposure to temperatures below 12°C. Temperature pretreatments for 3 days above 20°C of fruits detached from the tree reduced CI, the more so the higher the conditioning temperature. Put and Spd increased with temperature conditioning in detached fruits, differing from the response of fruits attached to the tree. No direct relationship between induced levels of these PAs and the tolerance to CI was found. Levels of Put and Spd increased at temperatures (22, 30 and 37°C) which increased the tolerance and also at temperatures (6 and 12°C) which accelerated the appearance of chilling symptoms. In contrast, a significant increase in Spm levels was only found after conditioning at 30 or 37°C. After cold storage a general decline in PA levels occurred in all temperature‐conditioned mandarins. In most cases no significant differences among fruit exposed to effective and non‐effective pretreatments were observed. PA content increased again after transferring cold‐stored fruits to 20°C, whereas the CI index was barely affected. In conclusion, PA changes in the flavedo of ‘Fortune’ mandarins appear to be related to variations in temperature rather than to stage of maturity or tolerance to chilling.     

Effect of soil temperature on nitrogen fixation on roots of rice and reed

Summary The relation of nitrogenase activity (ethylene evolution) to soil temperature or incubation temperature of roots was determined on two genera of swamp plants, namely rice (Oryza sativa) cultivated in tropical climate and reed (Phragmites communis) grown in temperate regions. For both intact rice plants and excised rice roots the optimum temperature was 35°C. On excised roots nitrogenase activity responded more sensitivity to changes in temperature. In contrast to intact rice plants no ethylene evolution occurred on excised roots at 17 and 44°C. On reed roots temperature optimum was between 26 and 30°C which is clearly lower than on rice (35°C). The temperature range in which nitrogen fixation occurred was, however, similar to that of rice, although on a lower level. The results suggest a higher potential of the tropics for associative N₂ fixation, while in cooler climates the lower temperatures appear to be a major limiting factor.     

Prior temperature exposure affects subsequent chilling sensitivity

The chilling sensitivity of small discs or segments of tissue excised from chillingsensitive species was significantly altered by prior temperature exposure subsequent to holding the tissue at chilling temperatures as measured by a number of physiological processes sensitive to chilling. This temperature conditioning was reversible by an additional temperature exposure before chilling, and mature-green and red-ripe tomato tissue exhibit similar chilling sensitivities. Exposing pericarp discs excised from tomato fruit (Lycopersicon esculentum Mill. cv. Castelmart), a chilling-sensitive species, to temperatures from 0 to 37°C for 6 h before chilling the discs at 2.5°C for 4 days significantly altered the rate of ion leakage from the discs, but had no effect on the rate of ion leakage before chilling and only a minimal effect on discs held at a non-chilling temperature of 12°C. Exposing chillingsensitive tissue to temperatures below that required to induce heat-shock proteins but above 20°C significantly increased chilling sensitivity as compared to tissue exposed to temperatures between 10 and 20°C. Rates of ion leakage after 4 days of chilling at 2.5°C were higher from fruit and vegetative tissue of chilling-sensitive species (Cucumis sativus L. cv. Poinsett 76, and Cucurbita pepo L. cv. Young Beauty) that were previously exposed for 6 h to 32°C than from similar tissue exposed to 12°C. Exposure to 32 and 12°C had no effect on the rate of ion leakage from fruit tissue of chilling tolerant species (Malus domestica Borkh. cv. Golden Delicious, Pyrus communis L. cv. Bartlett). Ethylene and CO₂ production were higher and lycopene synthesis was lower in chilled tomato pericarp discs that were previously exposed for 6 h to 32°C than the values from tissue exposed to 12°C for 6 h before chilling. Increased chilling sensitivity induced by a 6 h exposure to 32°C could be reversed by subsequent exposure to 12°C for 6 h.     

Chilling tolerance in <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> induced by seed priming with putrescine

Chilling stress is one of primary constraints to tobacco production in many parts of the world. The present study was conducted to induce chilling tolerance in tobacco by seed priming with putrescine (Put) in relation to physiological changes, using seeds from two tobacco varieties, MSk326 (chilling sensitive variety) and Honghuadajinyuan (HHDJY, chilling tolerant variety). Seed germination, seedling antioxidant enzyme activities and malondialdehyde (MDA) concentration, as well as polyamine concentration were determined under low temperature. During chilling stress at 11°C, seed priming with 0.01 mM Put for 48 h (Put0.01mM48 h) and seed priming with 0.1 mM Put for 48 h (Put0.1mM48 h) significantly increased germination percentage, germination index, seedling length and dry weight of both varieties compared to the controls without Put treatment. When seedlings of 4-leaf stage suffered a short chilling stress (5°C), Put 0.1 mM 48 h improved the activities of antioxidant enzymes including superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX), increased endogenous Put, Spd and Spm concentration and decreased the MDA concentration. The results showed that Put priming treatments were available to enhance the chilling tolerance of tobacco seedlings. The optimal treatment of Put was Put0.1 mM48 h.     

Association of polyamines in governing the chilling sensitivity of maize genotypes

Chilling stress is an important constraint of global production of maize. This study was undertaken to compare the chilling responses of different maize seedling tissues and to analyze changes in polyamines as a result of chilling stress. Reponses to chilling were characterized in two maize (Zea mays L.) inbred lines, ‘HuangC’ and ‘Mo17’, that putatively differ in chilling sensitivity. Seedlings were exposed to low temperature (5°C) and chilling injury was estimated by electrical conductivity (EC), malonaldehyde (MDA) concentration, and by changes in putrescine (Put), spermidine (Spd) and spermine (Spm) concentrations in root, mesocotyl, and coleoptile tissues. Membrane permeability (as measured by EC), MDA concentrations and Put concentrations in the three tissue of maize seedlings increased after chilling stress, except for the Put concentration in roots. Spd and Spm concentrations in the three tissues of seedlings decreased after chilling stress. The EC for cold stressed tissues were lower in HuangC than Mo17. Also, the EC of coleoptile tissues were lower than for mesocotyl in both inbred lines. We suggest that mesocotyl tissue can be used to evaluate cold tolerance in maize. Stepwise regression analyses showed that chilling injury in roots was generally correlated with Spd concentration while in the mesocotyl injury was mainly correlated with Put and Spd concentrations. Spermidine and Spm concentrations in the coleoptile were correlated with chilling injury. Characteristics changes of polyamines in chill-tolerant maize seedling combined with regression analysis are a reliable method for evaluating chill tolerance in maize lines.     

Nuclear Deoxyribonucleic Acid Metabolism and Membrane Fatty Acid Content Related to Chilling Resistance in Germinating Cotton (Gossypium barbadense)

Chilling damage was examined in the chilling-sensitive plant Gossypium barbadense. Between 30 and 36 h of germination at 34°C, the seedlings are extremely sensitive to temperatures below 10°C. The initiation of chilling damage by exposure to 2°C for 5 h during the sensitive period resulted in a large reduction in DNA synthesis. The reduction was correlated with a reduced efficiency of nuclear DNA polymerase activity. Comparing a more chilling resistant genotype to a more sensitive variety indicated that the resistant genotype nuclear DNA polymerase activity is more efficient when exposed to a chilling stress. Resistance was also correlated with a higher degree of unsaturated fatty acid content in the nuclear membranes of the resistant variety.     

Effect of low temperature on ethanolic fermentation in rice seedlings

Rice seedlings (Oryza sativa L.) were incubated at 5-30 degrees C for 48 h and the effect of temperature on ethanolic fermentation in the seedlings was investigated in terms of low-temperature adaptation. Activities of alcohol dehydrogenase (ADH, EC 1.1.1.1) and pyruvate decarboxylase (PDC, EC 4.1.1.1) in roots and shoots of the seedlings were low at temperatures of 20-30 degrees C, whereas temperatures of 5, 7.5 and 10 degrees C significantly increased ADH and PDC activities in the roots and shoots. Temperatures of 5-10 degrees C also increased ethanol concentrations in the roots and shoots. The ethanol concentrations in the roots and shoots at 7.5 degrees C were 16- and 12-times greater than those in the roots and shoots at 25 degrees C, respectively. These results indicate that low temperatures (5-10 degrees C) induced ethanolic fermentation in the roots and shoots of the seedlings. Ethanol is known to prevent lipid degradation in plant membrane, and increased membrane-lipid fluidization. In addition, an ADH inhibitor, 4-methylpyrazole, decreased low-temperature tolerance in roots and shoots of rice seedlings and this reduction in the tolerance was recovered by exogenous applied ethanol. Therefore, production of ethanol by ethanolic fermentation may lead to low-temperature adaptation in rice plants by altering the physical properties of membrane lipids.     

Pre-treating paclobutrazol enhanced chilling tolerance of sweetpotato

The objective of this work was to study changes in low molecular weight antioxidants and antioxidative enzymes in chilling-stressed sweetpotato, as affected by paclobutrazol (PBZ) pre-treatment 24 h prior to exposure to chilling conditions. Sweetpotato ‘TN71’ and ‘TN65’ were treated with 300 mg PBZ/5 ml/plant, after which plants were subjected to 7°C/7°C (day/night) for periods of 1, 3 and 5 days, followed by a 3-day recovery period at 24°C/20°C (day/night). A factorial experiment in completely randomized design with four replications was used in this study. Young fully expanded leaves at each temperature and period of time were clipped for antioxidative system measurement. We concluded that different varieties displayed variations in their oxidative system, and the differential expressions of each genotype were associated with chilling stress response. Plants with various antioxidative systems responded differently to chilling stress according to the duration of the chilling period and subsequent re-warming period. ASA, GSH and GSSG contents were enhanced in TN71 prior to chilling stress. Increased APX, GR, ASA and MDA activities accounted for chilling tolerance in TN65. Furthermore, our results indicate that the elevated levels of the antioxidative system observed after PBZ pre-treatments afforded the sweetpotato leaf improved chilling-stress tolerance. The levels of ASA and GSSG of both TN71 and TN65 under chilling were significantly raised by pre-treating with PBZ. PBZ pre-treatment exhibited the important function of enhancing the restoration of leaf oxidative damage under chilling stress and increasing the chilling tolerance of plants to mitigate chilling stress effects.     

The importance of fluctuating thermal regimes for repairing chill injuries in the tropical beetle Alphitobius diaperinus (Coleoptera: Tenebrionidae) during exposure to low temperature

Abstract. In this study, the impact of acclimation (1 month at 15 °C vs. breeding at 30 °C) and fluctuating thermal regimes (daily transfers from low temperatures to various higher temperatures for 2 h) on the cold tolerance of the tropical beetle, Alphitobius diaperinus Panzer (Coleoptera: Tenebrionidae) was examined. Acclimation increased significantly the duration of survival (Lt₅₀) at a constant 5 °C (7.7 ± 0.3 days to 9.7 ± 0.5 days). Survival of acclimated and nonacclimated beetles increased slightly at alternating temperatures of 5 °C/10 °C or 5 °C/15 °C. When daily transfer to 20 °C was applied, survival (Lt₅₀) was improved markedly (nonacclimated: 15.5 ± 0.7 days, acclimated: 19.6 ± 0.6 days). The higher temperatures may allow progressive repair of injuries, and the effects of chilling may be repaired completely at 25 and 30 °C, a phenomenon recorded here for the first time. It is estimated that the theoretical upper threshold of chill injury (Th) of nonacclimated beetles is 15.1 °C whereas it is shifted down to 11.2 °C in acclimated beetles, which might enable this temperature to allow effective repair of injury.     

Physiological and biochemical changes related to methyl jasmonate-induced chilling tolerance of rice (Oryza sativa L.) seedlings

Physiological and biochemical changes related to methyl jasmonate (MeJA)-induced chilling tolerance of rice (Oryza sativa L. cv. Taichung Native 1) seedlings were investigated. Treatment of whole plants with 10 mmol m^?3 MeJA for 48 h before chilling (5 °C) was optimal for the induction of chilling tolerance. MeJA greatly improved the survival ratio of chilled seedlings and ameliorated chilling injury such as demolition of membrane structure (estimated by electrolyte leakage). MeJA also prevented water loss in chilled seedlings by reducing the opening of stomata and decreasing the root bleeding rate. Putrescine and spermine levels in shoots increased but spermidine levels decreased on exposure to MeJA. In roots, putrescine levels also increased and spermidine levels increased transiently on exposure to MeJA. Activities of arginine decarboxylase (ADC; EC 4.1.1.19) and S-adenosylmethionine decarboxylase (SAMDC; EC 4.1.1.50) in both shoots and roots increased on exposure to MeJA, while the activity of ornithine decarboxylase (ODC; EC 4.1.1.17) remained unchanged. The MeJA-induced putrescine increase was inhibited by 50 mmol m^?3α-difluoromethylarginine (DFMA), an irreversible inhibitor of ADC, but not by 50 mmol m^?3α-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC. The effect of MeJA on the induction of chilling tolerance was also reduced by 50 mmol m^?3 DFMA. The effects of DFMA were partly prevented by 1 mol m^?3 putrescine. This indicates that putrescine accumulation is required for the induction of chilling tolerance of rice seedlings by MeJA.     

Polypeptide markers for low temperature stress during seed germination in sunflower

Sunflower seeds behaved as chilling and freezing sensitive and also exhibited acclimation under low seed moisture content (< 1 %). At high seed moisture content (approx. 22 %) they tolerated chilling stress but failed to acclimate under freezing temperatures. Pre-imbibitional chilling (5 °C) or freezing (−5 or −10 °C) stress significantly enhanced total soluble protein (TSP) content. Chilling treatment after imbibition (in contrast to pre-imbibition) enhanced germination and this was accompanied by increase in 30, 24 and 21.9 kDa TSPs content (3 d after germination). Freezing at −5 and −10 °C suppressed seed germination and increased content of 78 and 56.2 kDa wall bound proteins. Chilling acclimation decreased 35.4, 33.9, 29.5, 23.4 and 21.4 kDa TSPs.     

Heat shock increases chilling tolerance of mung bean hypocotyl tissue

The effects of heat shock on the chilling tolerance of mung bean [Vigna radiata (L.) Wilczek] seedling tissue were studied by using two measurements of chilling injury: increased 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase activity and solute leakage. ACC oxidase activity (measured as ACC-induced ethylene production) of freshly excised mung bean hypocotyl segments was highly dependent on the temperature at which the seedlings were grown. However, this highly temperature-dependent level of ACC oxidase activity was probably a wound response since it was almost entirely eliminated by incubating the excised segments at 20°C for 3 h. In contrast, heating of excised segments to 40°C for up to 4 h resulted in a time-dependent increase in ACC oxidase activity which was sensitive to cycloheximide, indicating rapid protein synthesis during the heat treatment. ACC oxidase activity fell sharply during subsequent chilling at 2. 5°C. After 3 days of chilling, all treated segments, regardless of their initial ACC oxidase activity, showed a decline to the same low activity level and ACC oxidase activity continued to fall slowly for up to 9 days at 2. 5°C. Hypocotyl segments excised from seedlings held at 15°C showed no change in solute leakage, but leakage increased rapidly when seedlings were either chilled at 2. 5°C or heated to 32°C (just below the heat shock temperature). Chill-induced leakage from non-heat-shocked segments increased steadily with chilling duration and was unaffected by cycloheximide concentration up to day 6. Within the elevated rate of leakage on day 9, however, leakage was lower from segments exposed to 10 and 50 μM cycloheximide. Solute leakage was markedly reduced for up to 9 days when segments were heat shocked at 40°C for 3 or 4 h with or without 10 M cycloheximide, but the presence of 50 μM cycloheximide caused an initial doubling of solute leakage and a 3-fold increase after 3 days of chilling. Cycloheximide prevented formation of heat shock protection against chilling from the start at 50 μM and after 9 days at 10 μM. These results indicate that the protection afforded by heat shock against chilling damage is quantitative and probably involves protein synthesis.     

Photoperiod and root-zone temperature: Interacting effects on growth and mineral nutrients of maize

Factorial effects of photoperiod (6, 12 and 18 h) and root-zone temperatures (9, 15 and 21°C) on the growth and mineral nutrient concentration and partitioning in maize (Zea mays L.) were investigated. Strong interactions were observed between photoperiod and root-zone temperature on the growth and concentration of numerous mineral elements in the plant tops and roots. For example, a threefold increase in photoperiod (from 6 to 18 h) did not affect the growth of tops or roots if the root-zone temperature was 9°C but increased them each by eightfold if the root-zone temperature was 21°C. On the other hand, raising the root-zone temperature from 9 to 21°C increased the growth of tops and root each by ca. threefold when plants were grown with 6 h of light. At 18 h photoperiod, however, plant growth was increased 20- to 30-fold by the same rise in the root-zone temperature. The concentrations of different mineral elements in the roots and tops were affected quite differently by the interacting effects of photoperiod and root-zone temperature. In general, increasing the photoperiod at a given root-zone temperature decreased the concentrations of elements while increasing the root-zone temperature at a given photoperiod increased the concentrations of most elements in both roots and tops. The exceptions were K and B which reacted opposite to each other: K concentration in both tops and roots was relatively insensitive to photoperiod but very sensitive to root-zone temperature and the reverse was true for boron. The relative insensitivity of plant growth to increased day length as long as the roots are subjected to suboptimal (low) soil temperatures may have survival significance and point to the predominant role of root temperature over that of day length in the early growth of maize. A possible mechanism by which photoperiod and root-zone temperature might interactively alter the nutrient uptake by the roots is discussed.     

Transcript profiling in Vitis riparia during chilling requirement fulfillment reveals coordination of gene expression patterns with optimized bud break

Endodormant grapevine buds require a period of chilling before they break and begin to grow. Custom Vitis bud cDNA microarrays (9,216 features) were used to examine gene expression patterns in overwintering Vitis riparia buds during 2,000 h of 4°C chilling. Three-node cuttings collected concurrently with buds were monitored to determine dormancy status. Chilling requirement was fulfilled after 1,500 h of chilling; however, 2,000 h of chilling significantly increased the rate of bud break. Microarray analysis identified 1,469 significantly differentially expressed (p value < 0.05) array features when 1,000, 1,500, and 2,000 h of chilling were compared to 500 h of chilling. Functional classification revealed that the majority of genes were involved in metabolism, cell defense/stress response, and genetic information processing. The number of significantly differentially expressed genes increased with chilling hour accumulation. The expression of a group of 130 genes constantly decreased during the chilling period. Up-regulated genes were not detected until the later stages of chilling accumulation. Hierarchical clustering of non-redundant expressed sequence tags revealed inhibition of genes involved in carbohydrate and energy metabolism and activation of genes involved in signaling and cell growth. Clusters with expression patterns associated with increased chilling and bud break were identified, indicating several candidate genes that may serve as indicators of bud chilling requirement fulfillment. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.