Stress tolerance in a yeast lipid mutant: membrane lipids influence tolerance to heat and ethanol independently of heat shock proteins and trehalose.

The response of a yeast unsaturated fatty acid auxotroph, defective in delta 9-desaturase activity, to heat and ethanol stresses was examined. The most heat- and ethanol-tolerant cells had membranes enriched with oleic acid (C18:1), followed in order by cells enriched with linoleic (C18:2) and linolenic (C18:3) acids. Cells subjected to a heat shock (25-37 degrees C for 30 min) accumulated trehalose and synthesized typical heat shock proteins. Although there were no obvious differences in protein profiles attributable to lipid supplementation of the mutant, relative protein synthesis as determined by densitometric analysis of autoradiograms suggested that hsp expression was different. However, there was no consistent relationship between the synthesis of heat shock proteins and the acquisition of thermotolerance in the lipid supplemented auxotroph or related wild type. Furthermore, trehalose accumulation was also not closely related to stress tolerance. On the other hand, the data presented indicated a more consistent role for membrane lipid composition in stress tolerance than trehalose, heat shock proteins, or ergosterol. We suggest that the sensitivity of C18:3-enriched cells to heat and ethanol may be attributable to membrane damage associated with increases in membrane fluidity and oxygen-derived free radical attack of membrane lipids.     

Heat-stable Proteins and Acquisition of Desiccation Tolerance in Peanut Seeds

The acquisition and induction of desiccation tolerance associated with the expression of heat-stable proteins in the developing peanut (Arachis hypogaea L. ) seeds were studied. Desiccation tolerance of peanut seeds was achieved during 45 to 65 DAP (days after pegging) embryogenesis, while a set of low molecular weight (9 to 15.5 kD) heat-stable polypeptides was preferentially expressed. Slow drying regime applied in vitro to 25 and 35 DAP peanut embryos induced desiccation tolerance and the expression of the same subset of polypeptides. Mature drying treatment enhanced the ability of 65 DAP peanut embryos to withstand fast drying, also increased the heat stability of arachins, the major peanut storage protein, which was heat labile during 45 to 65 DAP embryogenesis. It was concluded that the heat-stable proteins may contribute to desiccation tolerance of the peanut seeds, and the low molecular weight heat-stable polypeptides may confer nonspecifieally heat tolerance on peanut storage proteins which were normally heat labile.     

Analysis of abscisic acid responsive proteins in Brassica napus guard cells by multiplexed isobaric tagging

Guard cells, which form stomata on the leaf epidermis, play important roles in plant gas exchange and defense against pathogens. Abscisic acid (ABA) is a phytohormone that can be induced by drought and leads to stomatal closure. Guard cells have been a premier model system for studying ABA signal transduction. Despite significant progress on the identification of molecular components in the ABA signaling pathway, our knowledge of the protein components is very limited. Here, we employ a recently developed multiplexed isobaric tagging technology to identify ABA-responsive proteins in Brassica napus guard cells. A total of 431 unique proteins were identified with relative quantitative information in control and ABA-treated samples. Proteins involved in stress and defense constituted a major group among the 66 proteins with increased abundance. Thirty-eight proteins were decreased in abundance and fell into several functional groups including metabolism and protein synthesis. Many of the proteins have not been reported as being ABA responsive or involved in stomatal movement. A large percentage of the protein-coding genes contained ABA-responsive elements. This study not only established a comprehensive inventory of ABA-responsive proteins, but also identified new proteins for further investigation of their functions in guard cell ABA signaling.     

Repair of thermal damage to the Escherichia coli nucleoid.

The folded chromosome or nucleoid of Escherichia coli was analyzed by low-speed sedimentation in neutral sucrose gradients after heat treatment (30 min at 50 degrees C) and subsequent incubation of cells at 37 degrees C for various times. Heat treatment resulted in in vivo association of the nucleoids with cellular protein and in an increase in sedimentation coefficient. During incubation at 37 degrees C, a fraction of the nucleoids, from heated cells, because dissociated from cellular protein and regained their characteristic sedimentation coefficients. The percentage of nucleoids which returned to their control sedimentation position in the sucrose gradients corresponded to the percentage of cells able to repair thermal damage as assayed by enumeration on agar plates.     

Inactivation of Geobacillus stearothermophilus spores by high-pressure carbon dioxide treatment

High-pressure CO2 treatment has been studied as a promising method for inactivating bacterial spores. In the present study, we compared this method with other sterilization techniques, including heat and pressure treatment. Spores of Bacillus coagulans, Bacillus subtilis, Bacillus cereus, Bacillus licheniformis, and Geobacillus stearothermophilus were subjected to CO2 treatment at 30 MPa and 35 degrees C, to high-hydrostatic-pressure treatment at 200 MPa and 65 degrees C, or to heat treatment at 0.1 MPa and 85 degrees C. All of the bacterial spores except the G. stearothermophilus spores were easily inactivated by the heat treatment. The highly heat- and pressure-resistant spores of G. stearothermophilus were not the most resistant to CO2 treatment. We also investigated the influence of temperature on CO2 inactivation of G. stearothermophilus. Treatment with CO2 and 30 MPa of pressure at 95 degrees C for 120 min resulted in 5-log-order spore inactivation, whereas heat treatment at 95 degrees C for 120 min and high-hydrostatic-pressure treatment at 30 MPa and 95 degrees C for 120 min had little effect. The activation energy required for CO2 treatment of G. stearothermophilus spores was lower than the activation energy for heat or pressure treatment. Although heat was not necessary for inactivationby CO2 treatment of G. stearothermophilus spores, CO2 treatment at 95 degrees C was more effective than treatment at 95 degrees C alone.     

Effect of heat shock on the intracellular distribution of proteins

Exposure of cells to heat induces thermotolerance, a transient resistance to subsequent heat challenges. It has been shown that thermotolerance is correlated in time with the enhanced synthesis of heat shock proteins. In this study, the association of induced heat shock proteins with various cellular fractions was investigated and the heat-induced changes in skeletal protein composition in thermotolerant and control cells was compared. All three major heat shock proteins induced in Chinese hamster fibroblasts after a 46 degrees C, 4-min heat treatment (70, 87, and 110 kDa) were purified with the cytoplasmic fraction, whereas only the 70-kDa protein was also found in other cell fractions, including that containing the cellular skeleton. Immediately after a second heat treatment at 45 degrees C for 45 min, the 110-kDa protein from thermotolerant cells also purified extensively with the cellular skeletal fraction. In this regard, the 110-kDa protein behaved similarly to many other cellular proteins, since we observed an overall temperature-dependent increase in the total labeled protein content of the high-salt-resistant cellular skeletal fraction after heat shock. Pulse-chase studies demonstrated that this increased protein content gradually returned to normal levels after a 3-hr incubation at 37 degrees C. The alteration or recovery kinetics of the total labeled protein content of the cellular skeletal fraction after heat shock did not correlate with the dramatic increase in survival observed in thermotolerant cells. The relationship between heat shock proteins and thermotolerance, therefore, does not correlate directly with changes in the heat-induced cellular alterations leading to differences in protein fractionation.     

Abscisic acid-induced freezing tolerance in the moss Physcomitrella patens is accompanied by increased expression of stress-related genes

Abscisic acid (ABA)-induced genes are implicated in the development of freezing tolerance during cold acclimation in higher plants, but their roles in lower land plants have not been determined. We examined ABA- and cold-induced changes in freezing tolerance and gene expression in the moss Physcomitrella patens. Slow equilibrium freezing to -4 degrees C of P. patens protonemata grown under normal growth conditions killed more than 90% of the cells, indicating that the protonema cells are freezing-sensitive. ABA treatment for 24 h dramatically increased the freezing tolerance of the protonemata, while cold treatment only slightly increased the freezing tolerance within the same period. We examined the expressions of fourteen Physcomitrella patens ABA-responsive genes (PPARs), isolated from ABA-treated protonemata. ABA treatment resulted in a remarkable increase in the expression of all the PPAR genes within 24 h. Several of the PPAR genes (PPAR 1 to 8, and 14) were also responsive to cold, but the response was much slower than that to ABA. Treatment with hyperosmotic concentrations of NaCl and mannitol increased freezing tolerance of protonemata and also increased the expression levels of eleven PPAR genes (PPAR2, 3, 5 to 8, and 10 to 14). These results suggest that ABA and environmental stresses positively affect the expression of common genes that participate in protection of protonema cells leading to the development of freezing tolerance.     

Rapid degradation of starch in chloroplasts and concomitant accumulation of soluble sugars associated with ABA-induced freezing tolerance in the moss Physcomitrella patens

Abscisic acid (ABA) has been postulated to play a role in the development of freezing tolerance during the cold acclimation process in higher plants, but its role in cold tolerance in tower land plants has not been elucidated. The moss Physcomitrella patens rapidly developed freezing tolerance when its protonemata were grown in a medium containing ABA, with dramatic changes in the LT50 value from -2 degrees C to over -10 degrees C. We examined physiological and morphological alterations in protonema cells caused by ABA treatment to elucidate early cellular events responsible for rapid enhancement of freezing tolerance. Microscopic observations revealed that ABA treatment for 1 day resulted in a dramatic alteration in the appearance of intracellular organelles. ABA-treated cells had slender chloroplasts, with a reduced amount of starch grains, in comparison with those of non-treated cells. The ABA-treated cells also had several segmented vacuoles while many of non-treated cells had one central vacuole. When frozen to -4 degrees C, freezing injury-associated ultrastructural changes such as formation of aparticulate domains and fracture-jump lesions were frequently observed in the plasma membrane of non-treated protonema cells but not in that of ABA-treated cells. The ABA treatment increased the osmotic concentration of the protonema cells, in correlation with accumulation of free soluble sugars. These results suggest that ABA-induced accumulation of soluble sugars, associated with morphological changes in organelles, mitigated freezing-induced structural damage in the plasma membrane, eventually leading to enhancement of freezing tolerance in the protonema cells.     

Effect of prior heat shock on heat resistance of Listeria monocytogenes in meat.

The effect of prior heat shock on the thermal resistance of Listeria monocytogenes in meat was investigated. A sausage mix inoculated with approximately 10(7) L. monocytogenes per g was initially subjected to a heat shock temperature of 48 degrees C before being heated at a final test temperature of 62 or 64 degrees C. Although cells heat shocked at 48 degrees C for 30 or 60 min did not show a significant increase in thermotolerance as compared with control cells (non-heat shocked), bacteria heat shocked for 120 min did, showing an average 2.4-fold increase in the D64 degrees C value. Heat-shocked cells shifted to 4 degrees C appeared to maintain their thermotolerance for at least 24 h after heat shock.     

Relationship between Proline and Abscisic Acid in the Induction of Chilling Tolerance in Maize Suspension-Cultured Cells

Both proline and abscisic acid (ABA) induce chilling tolerance in chilling-sensitive plants. However, the relationship between proline and ABA in the induction of chilling tolerance is unclear. We compared the time course of the increase in chilling tolerance induced by proline and ABA, and the time course of the uptake of both into the cultured cells of maize (Zea mays L. cv Black Mexican Sweet) at 28[deg]C. The plateau of proline-induced chilling tolerance preceded by 12 h the plateau of ABA-induced chilling tolerance. The uptake of exogenous ABA into the cells reached a plateau in 1 h, whereas the uptake of exogenous proline gradually increased throughout the 24-h culture period. Although the proline content in ABA-treated cells was 2-fold higher than in untreated cells at the end of the 24-h ABA treatment at 28[deg]C, the correlation between the endogenous free proline content and the chilling tolerance in the ABA-treated cells was insignificant. Isobutyric acid treatment, which resulted in a larger accumulation of proline in the cells than ABA treatment, did not increase chilling tolerance. The induction of chilling tolerance by proline and ABA appeared to be additive. Cycloheximide inhibited ABA-induced chilling tolerance, but it did not inhibit proline-induced chilling tolerance. Newly synthesized proteins accumulate in ABA-treated cells at 28[deg]C while the chilling tolerance is developing (Z. Xin and P.H. Li [1993] Plant Physiol 101: 277-284), but none of these proteins were observed in the proline-treated cells. Results suggest that proline and ABA induce chilling tolerance in maize cultured cells by different mechanisms.     

Responses of Saccharomyces cerevisiae to thermal stress

We studied the mechanisms involved in heat gradient-induced thermotolerance of Saccharomyces cerevisiae. Yeasts were slowly heated in a nutrient medium from 25 to 50 degrees C at 0.5 degrees C/min or immediately heat shocked at 50 degrees C, and both sets of cultures were maintained at this temperature for 1 h. Cells that had been slowly heated showed a 50-fold higher survival rate than the rapidly heated cells. Such thermotolerance was found not to be related to protein synthesis. Indeed Hsp104 a known protein involved in yeast thermal resistance induced by a preconditioning mild heat treatment, was not synthesized and cycloheximide addition, a protein synthesis inhibitor, did not affect the thermoprotective effect. Moreover, a rapid cooling from 50 to 25 degrees C applied immediately after the heat slope treatment inhibited the mechanisms involved in thermotolerance. Such observations lead us to conclude that heat gradient-induced thermal resistance is not directly linked to mechanisms involving intracellular molecules synthesis or activity such as proteins (Hsps, enzymes) or osmolytes (trehalose). Other factors such as plasma membrane phospholipid denaturation could be involved in this phenomenon.     

An abscisic acid analog inhibits abscisic acid-induced freezing tolerance and protein accumulation, but not abscisic acid-induced sucrose uptake in a bromegrass (Bromus inermis Leyss) cell culture

The application of abscisic acid (ABA), either as a racemic mixture or as optically resolved isomers, increases freezing tolerance in a bromegrass (Bromus inermis Leyss) cell culture and induces the accumulation of several heat-stable proteins. Two stereoisomers of an ABA analog, 23 dihydroacetylenic abscisyl alcohol (DHA), were used to study the role of ABA-induced processes in the acquisition of freezing tolerance in these cells. Freezing tolerance was unchanged in the presence of (–) DHA (LT₅₀ -9°C), and no increase in heat-stable protein accumulation was detected; however, the (+) enantiomer increased the freezing tolerance (LT₅₀ -13°C) and induced the accumulation of these polypeptides. All three forms of ABA increased freezing tolerance in the bromegrass cells, although (–) ABA was less effective than either (+) or (±) ABA when added at equal concentrations. Cells pretreated with 20 or 50 M (–) DHA displayed lower levels of freezing tolerance following the addition of 2.5, 7.5 or 25 M (±) ABA. Full freezing tolerance could be restored by increasing the concentration of (±) ABA to > 25 M. Pretreatment of cells with (–) DHA (20 or 50 M) had no effect on freezing tolerance when 25 M (+) ABA was added. The induction of freezing tolerance by 25 M (–) ABA was completely inhibited by the presence of 20 M (–) DHA. The accumulation of ABA-responsive heat-stable proteins was inhibited by pretreatment with 20 M (–) DHA in cells treated with 2.5 or 7.5M (+) ABA, and in cells treated with 25 M (–) ABA. The accumulation of these polypeptides was restored when (±) or (+) ABA was added at a concentration of 25 M. The analysis of proteins which cross-reacted with a dehydrin antibody revealed a similar inhibitory pattern as seen with the other ABA-responsive proteins. The effects of the various isomers of ABA and DHA on cell osmolarity and sucrose uptake was also investigated. In both cases, (±) and (+) ABA had pronounced effects on the parameters measured, whereas (–) ABA treated cells gave substantially different results. In both sucrose uptake and cell osmolarity, DHA had no significant effect on the results obtained following (±) or (+) ABA treatment. Maximum freezing tolerance was only observed in cells when both heat-stable protein accumulation and sucrose uptake were observed.Abbreviations ABA abscisic acid - DHA 2,3 dihydroacetylenicabscisyl alcohols - DMSO dimethyl sulfoxide - LT₅₀ temperature at which 50% of cells are killed The authors would like to acknowledge the technical assistance of Angela Bollman, Bruce Ewan and Angela Shaw. This work was supported by grants from the Natural Science and Engineering Research Council of Canada to L.V.G. and N.H.L., and a grant from the University of Saskatchewan to R.W.W.     

Will heat-stable proteins nonspecifically protect cells from thermal stress?

In cell-free systems, stress-resistant proteins nonspecifically stabilize stress-susceptible proteins. This mechanism has been suggested to contribute to thermotolerance in cells (Minton et al.: Proc. Natl. Acad. Sci. USA, 79: 7107-7117, 1982). To test this hypothesis, red-blood-cell-mediated microinjection was used to transfer macromolecules into monolayers of CHO cells. We introduced the heat-stable proteins fetuin and ovomucoid into RBCs during hypotonic hemolysis and then fused the RBCs to CHO cells with polyethylene glycol as fusogen. Fetuin and ovomucoid were successfully transferred into 36-55% of the CHO cells as demonstrated by fluorescence of FITC-conjugated proteins. The plating efficiency of these CHO cells after fusion ranged from 35% to 60%. Three hours after fusion, CHO cells microinjected with fetuin or ovomucoid were exposed to 43 degrees C for 0-180 min or 45 degrees C for 0-40 min, and thermal survival was determined. There was no difference in cell survival between control untreated cells, control cells fused with nonloaded RBCs, and cells fused with RBCs loaded with fetuin or ovomucoid. While our results do not support the hypothesis that heat-stable proteins nonspecifically protect cells from thermal stress, several possible explanations are provided for this observation.     

Drying rate and dehydrin synthesis associated with abscisic acid-induced dehydration tolerance in Spathoglottis plicata orchidaceae protocorms

Dehydration tolerance of in vitro orchid protocorms was investigated under controlled drying conditions and after abscisic acid (ABA) pretreatment. Protocorms were obtained by germinating seeds on Murashige and Skoog (MS) medium containing 10% (v/v) coconut water, 2% (w/v) sucrose and 0.8% (w/v) agar, and were dehydrated in relative humidities (RH) ranging from 7% to 93% at 25 degrees C. The critical water content of dehydration tolerance was determined, using the electrolyte leakage method. Drying rate affected the critical water content. Slow drying under high RH conditions achieved the greatest tolerance to dehydration. ABA pretreatment decreased the drying rate of protocorms, and increased dehydration tolerance. Improved tolerance to dehydration after ABA treatment was correlated with the effect of ABA on drying rate of protocorms. When critical water content of protocorms dried under different RH was plotted as a function of actual drying rate, no significant difference in tolerance to dehydration was observed between ABA-treated and control protocorms. ABA pretreatment and dehydration of orchid protocorms induced the synthesis of dehydrin, especially under the slow drying conditions. ABA pretreatment also promoted dry matter accumulation such as carbohydrates and soluble proteins and increased the concentration of K(+) and Na(+) ions in protocorms. The ABA-induced decrease in drying rate was correlated with lower osmotic potential, the enhanced maturity of protocorms and the accumulation of dehydrin in protocorms during pretreatment.     

The effects of low temperatures on intracellular transport of newly synthesized albumin and haptoglobin in rat hepatocytes.

Isolated rat hepatocytes were pulse-labelled with [35S]methionine at 37 degrees C and subsequently incubated (chased) for different periods of time at different temperatures (37-16 degrees C). The time courses for the secretion of [35S]methionine-labelled albumin and haptoglobin were determined by quantitative immunoprecipitation of the detergent-solubilized cells and of the chase media. Both proteins appeared in the chase medium only after a lag period, the length of which increased markedly with decreasing chase temperature: from about 10 and 20 min at 37 degrees C to about 60 and 120 min at 20 degrees C for albumin and haptoglobin respectively. The rates at which the proteins were externalized after the lag period were also strongly affected by temperature, the half-time for secretion being 20 min at 37 degrees C and 200 min at 20 degrees C for albumin; at 16 degrees C no secretion could be detected after incubation for 270 min. Analysis by subcellular fractionation showed that part of the lag occurred in the endoplasmic reticulum and that the rate of transfer to the Golgi complex was very temperature-dependent. The maximum amount of the two pulse-labelled proteins in Golgi fractions prepared from cells after different times of chase decreased with decreasing incubation temperatures, indicating that the transport from the Golgi complex to the cell surface was less affected by low temperatures than was the transport from the endoplasmic reticulum to the Golgi complex.     

Binding activity of glucocorticoid receptors after heat shock

The response of glucocorticoid receptors (GR) to heat was measured by the change in ligand binding activity both in control cells and in cells made tolerant to heat by a prior mild heat exposure. The study was prompted by earlier data showing that one of the heat shock proteins (HSP90) is an essential component of the GR complex and that treatment of mammalian cells with hydrocortisone induces resistance to heat damage. The GR rapidly loses binding activity after commencement of heating. There is a 50% loss of activity after 4 min at 45 degrees C, 8 min at 44 degrees C, or 17 min at 43 degrees C. The reduction in binding is due mainly to a reduction in affinity of binding to the ligand. The ability to bind glucocorticoid recovers quickly after heat treatment. Activity returns to levels 60-80% of normal by 2 h after a heat treatment that initially reduces binding to less than 20% of normal. However, complete restoration of binding activity takes approximately 3 days. The recovery of binding activity does not require protein synthesis. Pretreatment of cells with hydrocortisone, using conditions that induce heat resistance, reduces the activity to 10-20% of control, but residual receptors display a heat sensitivity similar to that of control cells. There was evidence for a limited degree of protection of GR from heat damage in thermotolerant cells.     

Loss of heat-shock acquisition of thermotolerance in yeast is not correlated with loss of heat-shock proteins

Yeast cells when subjected to a primary heat shock, defined as a temperature shift from 23 to 37 degrees C for 30 min, acquired tolerance to heat stress (52 degrees C/5 min). Primary heat shocked cells incubated at 23 degrees C for up to 3 h, progressively lost thermotolerance but retained high levels of the major heat-shock proteins as observed on polyacrylamide gels. On the other hand, a temperature shift back up to 37 degrees C for 30 min fully restored thermotolerance. The major high-molecular-mass heat-shock proteins (hsp) identified were of approximate molecular mass 100 kDa (hsp 100), 80 kDa (hsp 80) and 70 kDa (hsp 70). The results indicate that loss of heat-shock acquisition of thermotolerance is not correlated with loss of heat-shock proteins.     

Effect of prior heat shock on heat resistance of Listeria monocytogenes in meat

The effect of prior heat shock on the thermal resistance of Listeria monocytogenes in meat was investigated. A sausage mix inoculated with approximately 10(7) L. monocytogenes per g was initially subjected to a heat shock temperature of 48 degrees C before being heated at a final test temperature of 62 or 64 degrees C. Although cells heat shocked at 48 degrees C for 30 or 60 min did not show a significant increase in thermotolerance as compared with control cells (non-heat shocked), bacteria heat shocked for 120 min did, showing an average 2.4-fold increase in the D64 degrees C value. Heat-shocked cells shifted to 4 degrees C appeared to maintain their thermotolerance for at least 24 h after heat shock.     

Abscisic Acid-induced chilling tolerance in maize suspension-cultured cells

The induction of chilling tolerance by abscisic acid (ABA) in maize (Zea mays L. cv Black Mexican Sweet) suspension cultured cells was examined. Cell viability during exposure to chilling was estimated by triphenyl tetrazolium chloride reduction immediately after chilling and a filter paper growth assay. Both methods yielded comparable results. Chilling tolerance was induced by transferring 5-day-old cultures (late log phase) to a fresh medium containing ABA (10 to 100 micromolar). The greatest chilling tolerance was achieved with ABA at 100 micromolar. Growth of cells was inhibited at this concentration. After a 7-day exposure to 4°C in the dark, the survival of ABA-treated cells (100 micromolar ABA, 28°C for 24 h in the dark) was sevenfold greater than untreated cells. Effective induction of chilling tolerance was first observed when cells were held at 28°C for 6 hours after adding ABA. No tolerance was induced if the culture was chilled at the inception of ABA treatment. Induction of chilling tolerance was inhibited by cycloheximide. These results indicate that ABA is capable of inducing chilling tolerance when ABA-treated cells are incubated at a warm temperature before exposure to chilling, and this induction requires de novo synthesis of proteins.