Enhancing effects of heat shock and gibberellic acid on the thermotolerance in etiolated Vigna radiata. I. Physiological aspects on thermotolerance

The effect of heat shock on the thermotolerance of etiolated mung bean seedlings ( Vigna radiata L. cv. Wilczek) and the effects of gibberellic acid (GA) were studied. The potentially lethal temperature of etiolated mung bean seedlings was 45°C. But, when seedlings were pretreated with a heat-shock period at 40°C for 1 h before incubation at 45°C, they become thermotolerant and survived the 45°C treatment. The addition of actinomycin D or cycloheximide during the heat-shock period decreased the subsequent thermotolerance of the seedlings. Depending upon the time of its application, GA appeared to have multiple effects: (1) when applied during the 40°C heat-shock period, GA enhanced the heat-shock effect; (2) when applied during the 45°C potentially lethal temperature period, GA enhanced the subsequent growth of hypocotyls. This suggests that GA makes the seedlings tolerant to the potentially lethal temperature; (3) when GA was applied during a following 25°C growth period to seedlings which had been exposed first to 40°C and then 45°C, it promoted growth, suggesting that GA enhanced the restoration of the seedlings from high temperature damage. The role of GA and heat shock in the acquisition of thermotolerance in etiolated mung bean seedlings are discussed.     

Net CO2 output by CAM plants in the light: the role of leaf conductance

Triadimefon [1-(4-chlorophenoxy)-3,3-dimethyl-l-(l,2,4-triazol-l-yl)-2-butanone] is a triazoie fungicide which protects bean ( Phaseolus vulgaris L. cv. Spring Green) plants from heat and chilling injury. When the plants were exposed to heat shock by dipping the shoots in hot (50°C) distilled water for 2 min or exposing the plants to cold (1°C) for 8 h the primary leaves showed visual symptoms of injury 2 days after treatment and thereafter there was a progressive decline in chlorophyll and an increase in electrolyte leakage indicative of a loss of membrane integrity. There was a loss of metabolic (respiratory) activity in the root meristems when the roots were dipped in hot (48°C) water. All these symptoms of heat and chilling injury in the controls were either delayed or prevented by root application of triadimefon.     

Imposition of water stress in wheat seedlings improves the efficacy of uniconazole-induced thermal resistance

Winter wheat ( Triticum aestivum L. cv. Fredrick) seedlings (seed) treated with different concentrations of uniconazole (S-3307) were water stressed by withholding water for 2, 4 and 6 days. Subsequently, the water-stressed seedlings were exposed to heat shock at 45°C for 3 h. Chlorophyll fluorescence, stress-ethylene and percentage survival were the parameters used to monitor the efficacy of protection against thermal stress by uniconazole. In the control seedlings, water stress for 2 days prior to heat shock provided minimal protection, whereas uniconazole protected both water-stressed and non-stressed seedlings from heat injury. However, the degree of protection by uniconazole was enhanced if the seedlings were subjected to water stress (2–4 days) prior to the heat shock. In addition to protection against heat injury, uniconazole also reduced stress-induced (water and heat) ethytene levels.     

Thermal tolerance and heat shock protein synthesis during development in the anuran Lepidobatrachus laevis

Development of the Paraguayan anuran Lepidobatrachus laevis is unusual in that the larvae are obligate carnivores, facultative cannibals and apparently exist at high environmental temperatures in their natural habitat. In the present study, the effect of environmental temperature on the rate of anuran development was investigated. The larvae have a thermotolerance range of 18°C for normal development between 19 and 37°C. The effect of temperature on the rate of development was dramatic; larvae that were incubated at 36.8°C develop to stage 24 (Gosner) in approximately 9 h compared with 24 h for larvae incubated at 19°C. The ability of larvae to survive heat shock was also examined; larvae did not survive a shock of 45°C for 15 min when it was administered at stages 3, 5, 9, 10 or 20. However, using the same heat shock conditions, 50% survival was observed when larvae were shocked at stage 16. To study protein synthesis during heat shock, larvae were pulsed with [³⁵S]-methionine during heat shock and labeled proteins were analyzed by electrophoresis under reducing and denaturing conditions. Larvae synthesized two sets of heat-shock proteins at doublet molecular weights of 83/78 and 62/59 kDa. These proteins were synthesized independently of the stage of development at which the shock was administered or the magnitude of the heat shock.     

Endoribonuclease of Vicia faba ssp. minor radicles, and enhancement of its activity by chilling stress or abscisic acid

Endoribonuclease in roots of 3-day-old seedlings of Vicia faba L. ssp, minor is a citrate activated glycoprotein of 35 kDa, with pH and temperature optima of 6.0 and 50°C. The same holds for endoribonuclease from seedlings treated with abscisic acid or exposed to a chilling stress of -3°C for 24 h, except that the temperature optimum was decreased to 40°C. The enzyme(s) preferentially hydrolysed poly (A) and poly (U). RNase activity in the radicles was enhanced by chilling stress or abscisic acid. ABA did not potentiate the effect brought about by chilling but slowed down the decrease in RNase activity in chill-stressed seedlings upon transfer to 25°C. Both factors modified the pattern of the isoelectric-points of the molecular forms of RNase.     

Oxygen sensitivity of heated cells of Escherichia coli O157:H7

Following defined heat treatments (55 °C for 100 min, 59 °C for 5 min, 61 °C for 1 min), a 6 decimal (6-D) reduction was obtained when cells of Escherichia coli O157:H7 were enumerated in aerobic growth medium. Part of this reduction (3-D) was due to thermal inactivation (as determined when cells were enumerated in anaerobic growth medium), and part (3-D) was due to the inability of sub-lethally heat-injured cells of E. coli O157:H7 to grow in the presence of oxygen. When held anaerobically, the injured cells regained their ability to grow in the presence of oxygen. Following heating at 59 °C for 5 min, repair took 4 h at 30 °C, 48 h at 20 °C, 95 h at 10 °C, but did not occur in 816 h at 5 °C. Recovery from sub-lethal heat injury was not influenced by heat shock. These findings are relevant to the safety of minimally-heated foods.     

Effect of pre- and post-heat shock temperature on the persistence of thermotolerance and heat shock-induced proteins in Listeria monocytogenes

The effect of incubation temperature, before and after a heat shock, on thermotolerance of Listeria monocytogenes at 58°C was investigated. Exposing cells grown at 10°C and 30°C to a heat shock resulted in similar rises in thermotolerance while the increase was significantly higher when cells were grown at 4°C prior to the heat shock. Cells held at 4°C and 10°C after heat shock maintained heat shock-induced thermotolerance for longer than cells held at 30°C. The growth temperature prior to inactivation had negligible effect on the persistence of heat shock-induced thermotolerance. Concurrent with measurements of thermotolerance were measurements of the levels of heat shock-induced proteins. Major proteins showing increased synthesis upon the heat shock had approximate molecular weights of 84, 74, 63, 25 and 19 kDa. There was little correlation between the loss of thermotolerance after the heat shock and the levels of these proteins. Thermotolerance of heat shocked and non-heat shocked cells was described by traditional log-linear kinetics and a model describing a sigmoidal death curve (logistic model). Employing log-linear kinetics resulted in a poor fit to a major part of the data whereas a good fit was achieved by the use of a logistic model.     

Effect of chilling, heat shock, and vigor on the growth of cucumber (Cucumis sativus) radicles

Chilling at 2.5°C reduced the subsequent growth of cucumber ( Cucumis sativus L.) radicles at 25°C. The reduction in radicle growth was linear for 1–3 days of chilling at ≈10% per day of treatment, but then it increased in a non-linear pattern until subsequent radicle growth was all but eliminated by 6 days of chilling. A heat shock of 40°C for 4–12 min increased chilling tolerance such that 4 days of chilling caused only a 36% decrease in radicle growth, compared to 66% for seedlings not heat shocked. Heat shocks were only able to protect that part of radicle growth that was in excess of the linear decrease in radicle growth projected from 0–3 days. There appear to be two effects of chilling on radicle growth. The first inhibition of subsequent growth was linear and was not affected by heat shocks. The second inhibition was much more severe; it appeared after 3 days of chilling and could be prevented by heat shock. Seeds classified with different levels of vigor (i.e., different initial rates of growth) did not respond significantly different to chilling stresses following heat-shock treatments.     

Heat shock proteins in willow (Salix viminalis)

The heat shock response in three vegetatively propagated clones of Salix viminalis L. was studied. In the clone 78198, synthesis of a total of 58 proteins was induced or increased by heat shock. Of these proteins, 39 were found in both leaves and callus, 8 only in leaves, and 11 only in callus. The number of heat shock proteins differed between the three clones studied. The molecular weights of the heat shock proteins ranged from 18000 to over 94000. The optimal synthesis of heat shock proteins took place at 37–40°C, but several proteins could be induced at 25–30°C. The synthesis of the majority of the proteins present at a normal growth temperature (20°C) was not completely blocked by the heat shock. More than 12 h was needed for the reappearance of the normal protein synthesis pattern after heat shock.     

Proline accumulation is inhibitory to Arabidopsis seedlings during heat stress

The effect of proline (Pro) accumulation on heat sensitivity was investigated using transgenic Arabidopsis (Arabidopsis thaliana) plants ectopically expressing the Δ(1)-pyrroline-5-carboxylate synthetase 1 gene (AtP5CS1) under the control of a heat shock protein 17.6II gene promoter. During heat stress, the heat-inducible expression of the AtP5CS1 transgene was capable of enhancing Pro biosynthesis. Twelve-day-old seedlings were first treated with heat at 37 °C for 24 h to induce Pro and then were stressed at 50 °C for 4 h. After recovery at 22 °C for 96 h, the growth of Pro-overproducing plants was significantly more inhibited than that of control plants that do not accumulate Pro, manifested by lower survival rate, higher ion leakage, higher reactive oxygen species (ROS) and malondialdehyde levels, and increased activity of the Pro/P5C cycle. The activities of antioxidant enzymes superoxide dismutase, guaiacol peroxidase, and catalase, but not those of glutathione reductase and ascorbate peroxidase, increased in all lines after heat treatment, but the increase was more significant in Pro-overproducing seedlings. Staining with MitoSox-Red, reported for being able to specifically detect superoxide formed in mitochondria, showed that Pro accumulation during heat stress resulted in elevated levels of ROS in mitochondria. Interestingly, exogenous abscisic acid (ABA) and ethylene were found to partially rescue the heat-sensitive phenotype of Pro-overproducing seedlings. Measurement of ethylene and ABA levels further confirmed that these two hormones are negatively affected in Pro-overproducing seedlings during heat stress. Our results indicated that Pro accumulation under heat stress decreases the thermotolerance, probably by increased ROS production via the Pro/P5C cycle and inhibition of ABA and ethylene biosynthesis.     

Induction of heat shock-like proteins in Vigna sinensis seedlings growing under ultraviolet-B (280-320 nm) enhanced radiation

The effect of ultraviolet-B (UV-B) enhanced fluorescent radiation on protein profile and protein synthesis has been investigated in Vigna sinensis L. cv. Walp seedlings growing at various temperatures. In seedlings growing at 30°C, UV-B radiation decreased the level of several proteins as seen in Coomassie brilliant blue stained gel. However, fluorography of the same gel indicates induction of three sets of proteins in the range of 70. 53 and 16 k Da. Such induction under UV-B enhanced radiation resembled that found after heat shock treatments. In seedlings at 10 and 20°C, induction of such proteins varied both qualitatively and quantitatively. At 40°C. UV-B enhanced radiation caused a cumulative effect with temperature. Strong induction of specific proteins by UV-B radiation in seedlings growing under normal temperature indicates a possible protective role.     

Effects of temperature and moisture stress on the accumulation of abscisic acid in bean

Levels of abscisic acid (ABA) and several indicators of leaf-water status were measured in excised and intact primary leaves of bean ( Phaseolus vulgaris cv. Kinghorn) exposed to different temperature and moisture regimes. After 2 h at 5°, 25° or 45°C under moist conditions, the water status of detached leaves showed only minor changes, and there was no increase in ABA. Under conditions favoring water loss, ABA rose over 10-fold at 25°C, and trends towards higher ABA levels were observed at 5° and 45°C. When intact leaves on whole plants were exposed to the same temperatures for 10 h, there was still no evidence of a temperature-dependent rise in ABA that was not associated with a disturbance in the water balance of the plant. These data suggest that the rapid accumulation of ABA during temperature stress is a function of induced moisture deficits and does not result from high or low temperatures per se.     

Extracellular protectants produced by Clostridium perfringens cells at elevated temperatures

Aim:  The mechanisms of adaptation of Clostridium perfringens to high temperatures are not well understood. In this work, the involvement of extracellular compounds in protection to heat was determined.
Methods and Results:  Cells were grown in fluid thioglycollate medium or chicken broth. When mid-log phase was reached, they were heat-shocked at 50°C for 30 min. Then cultures were centrifuged and supernatants were transferred to nonshocked cells. Heat tolerance of these cells was performed at 55°C. Viable cells were determined. In some cases, supernatants were heated at 65°C or 100°C or treated with trypsin. Supernatants were fractionated and PAGE was made of fractions showing heat-protective activity. When C. perfringens was exposed to a heat shock at 50°C, extracellular factors were found in the culture supernatant that provided protection to cells not exposed to a heat shock. The extracellular factors were sensitive to heat and trypsin treatment suggesting a protein component. SDS-PAGE analysis of supernatant fractions from heat-treated cells revealed two induced proteins (56 and 125 kDa) that could be involved in heat tolerance.
Conclusion:  In this work, the presence and thermoprotective activity of extracellular factors produced by C. perfringens under a heat shock was demonstrated.
Significance and Impact of the Study:  The detection of thermoprotective extracellular factors of C . perfringens will aid in our understanding of the physiology of survival of C. perfringens in foods.     

The prevention of dehydration injury in celery seeds by polyethylene glycol, abscisic acid, dark and high temperature

Celery seeds ( Apium graveolens L.) given a germination induction period (3 days imbibition at 17°C in the light) could be prevented from germinating by up to 14 days subsequent exposure to high temperature (32°C), polyethylene glycol (PEG), abscisic acid (ABA) or dark (22°C). When the seeds were returned to 17°C in the light, germination occurred and, except for the high temperature treatment, was more rapid compared to seeds given a germination induction period only.
Celery seeds incubated for 3 days at 17°C in the light and then air-dried at 20°C germinated slowly when re-sown at 17°C in the light, and achieved only 19% germination after 21 days. Exposing the seeds to high temperature, PEG, ABA or dark for up to 14 days before drying maintained seed viability and subsequent germination was faster. The longer treatment periods gave increased benefit, and PEG was the most effective treatment. It is suggested that the effectiveness of the treatments in inducing dehydration tolerance relates to their ability to inhibit germination possibly via their prevention of cell expansion.     

Stabilization of Soluble Proteins in Vitro by Heat Shock Proteins-Enriched Ammonium Sulfate Fraction from Soybean Seedlings

The 70-100% ammonium sulfate fraction of postribosomal supernatantof heat shocked soybean seedlings contained a high percentageof all of the heat shock proteins. The proteins in this fractionwere resistant to heat denaturation, as judged by their unpelletabilityafter heat treatment. Moreover, this fraction, when added tothe postribosomal supernatant from control (non-heat shocked)seedlings, showed a significant ability to protect the controlproteins from heat denaturation. Heated at 55°C, some 50%of the control proteins, which were normally denatured afterheat treatment, were protected for at least 1 h when heat shockproteins-enriched fraction was added. The degree of protectionwas proportional to the amount of heat shock proteins-enrichedfraction added. However, when the ammonium sulfate fractionprepared from the seedlings with a heat treatment at 40°Cfor 3 h followed with a brief heat shock at 45°C which depletedmost of the 15–18 kDa and partial 68–70 kDa, 24kDa and 22 kDa heat shock proteins was added the effectivenessin preventing heat denaturation was lost. This suggests thatthe heat shock proteins of 15–18 kDa with those of 68–70kDa and perhaps 24 kDa and 22 kDa are important for providingthe protection from heat denaturation. (Received July 28, 1988; Accepted March 2, 1989)     

Breakage of Pseudotsuga menziesii seed dormancy by cold treatment as related to changes in seed ABA sensitivity and ABA levels

The main aims of the present work were to investigate whether a chilling treatment which breaks dormancy of Douglas fir ( Pseudotsuga menziesii (Mirb.) Franco) seeds induces changes in the sensitivity of seeds to exogenous ABA or in ABA levels in the embryo and the megagametophyte, and whether these changes are related to the breaking of dormancy. Dormant seeds germinated very slowly within a narrow range of temperatures (20–30°C), the thermal optimum being approximately 25°C. The seeds were also very sensitive to oxygen deprivation. Treatment of dormant seeds at 5°C improved further germination, and resulted in a widening of the temperature range within which germination occurred and in better germination in low oxygen concentrations. In dry dormant seeds the embryo contained about one-third of the ABA in the megagametophyte. ABA content of both organs increased during the first 4 weeks of chilling. It then decreased sharply in the megagametophyte to the level in the embryo after 7–15 weeks of chilling. At 15°C, a temperature at which dormancy was expressed, the ABA level increased in the embryo and the megagametophyte of dormant unchilled seeds whereas it decreased in the organs of chilled seeds. The longer the chilling treatment, the faster the decrease in ABA after the transfer of seeds from 5°C to higher temperatures, and the decrease was faster at 25 than at 15°C. These results suggest that the breaking of dormancy by cold was associated with a lower capacity of ABA biosynthesis and/or a higher ABA catabolism in the seeds subsequently placed at 15 or 25°C. Moreover, the chilling treatment resulted in a progressive decrease in the sensitivity of seeds to exogenous ABA. However, seeds remained more sensitive to ABA at 15 than at 25°C. The possible involvement of ABA synthesis and of responsiveness of seeds to ABA in the breaking of dormancy by cold treatment is discussed.     

Effects of heat shock upon the expression of developmentally regulated genes in Myxococcus xanthus

Abstract The effects of heat shock upon the expression of several developmentally regulated genes of Myxococcus xanthus were examined. No effects were observed on levels or timing of developmentally regulated β-galactosidase expression in eight randomly selected Tn5lac insertion mutants. However, heat shock significantly affected the fruiting behavior of temperature-sensitive aggregation ( tag ) mutants of M. xanthus . The tag mutant phenotype exhibits the normal aggregation of cells to form fruiting bodies at temperatures < 34°C, but cells fail to aggregate at temperatures ⩾ 34°C. Heat shock administered to tag mutant strains prior to starvation prohibited fruiting body formation at permissive temperatures. Additionally, tag mutant strains were found to be extremely sensitive to killing at 40°C. Heat shock was also found to increase tagA and tagE expression by 22 and 47%, respectively. Mutations in tagA blocked heat shock induced expression of tagE .     

The effect of prior heat shock on the thermoresistance of Salmonella thompson in foods

The resistance of Salmonella thompson to heating at 54° or 60°C in tryptone soya broth, liquid whole egg, 10% or 40% reconstituted dried milk or minced beef was increased if cells were held at 48°C for 30 min before heating at the higher temperatures. Induction of thermotolerance by mild heat shock is thus not confined to cells grown and heated in broth systems. The heat shock phenomenon may therefore have implications for the safety of foods given marginal heat treatment.     

The effects of heat shock and acclimation temperature on hsp70 and hsp30 mRNA expression in rainbow trout: in vivo and in vitro comparisons

Heat shock (25° C) of 10° C-acclimated rainbow trout Oncorhynchus mykiss led to increases in heat shock protein 70 (hsp70) mRNA in blood, brain, heart, liver, red and white muscle, with levels in blood being amongst the highest. Hsp30 mRNA also increased with heat shock in all tissues with the exception of blood. When rainbow trout blood was heat shocked in vitro , both hsp70 and hsp30 mRNA increased significantly. In addition, these in vitro experiments demonstrated that blood from fish acclimated to 17° C water had a lower hsp70 mRNA heat shock induction temperature than did 5° C acclimated fish (20 v. 25° C). The hsp30 mRNA induction temperature (25° C), however, was unaffected by thermal acclimation. While increases in hsp70 mRNA levels in blood may serve as an early indicator of temperature stress in fish, tissue type, thermal history and the particular family of hsp must be considered when evaluating stress by these molecular means.     

Effect of Whole Plant and Local Heating on the ABA Content in Cucumber Seedling Leaves and Roots and on Their Heat Tolerance

The effects of heating at 38°C of whole cucumber (Cucumis sativus L.) seedlings or local heating of their shoots and roots on ABA content and heat tolerance of leaves and roots were investigated. During the initial period of the high-temperature treatment of whole seedlings, the ABA concentration in leaves and roots increased considerably. Local heating of the shoot or root resulted in an increase in the ABA concentration not only in the heated organ, but also in unheated seedling parts. A high-temperature treatment of the whole seedlings and the local treatment of shoots or roots caused an increase in the heat tolerance of leaf cells. The heat tolerance of root cells virtually did not change after heating of the whole seedlings or shoots, but decreased after heating of roots. The possible role of ABA in changing the heat tolerance of leaf and root cells by local heating of the seedling is discussed.