Thermotolerance of the photosynthetic light reactions in two <Emphasis Type="Italic">Phaseolus</Emphasis> species: a comparative study

The common bean (Phaseolus vulgaris L.) is sensitive to high temperature, while an ecologically contrasting species (Phaseolus acutifolius A. Gray) is cultivated successfully in hot environments. In this study, the two bean species were respectively acclimated to a control temperature of 25 °C and a moderately elevated temperature of 35 °C in order to compare the thermotolerance capabilities of their photosynthetic light reactions. Growth at 35 °C appeared to have no obvious adverse effect on the photosynthetic activities of the two beans, but changed their thermotolerance. After a short period of heat shock (40 °C for up to 4 h), the photosynthetic activities of 25 °C-grown P. vulgaris declined more severely than those of P. acutifolius grown at 25 °C, implying that the basal thermotolerance of P. vulgaris is inferior to that of P. acutifolius. But after acclimating to 35 °C, the thermotolerances of the two species were both greatly enhanced to about the same level, clearly demonstrating the induction of acquired thermotolerance in their chloroplasts, and P. vulgaris could be as good as P. acutifolius. Temperature acclimation also changed plants’ resistance to photoinhibition in a manner similar to those toward heat stress. In addition, acquisition of tolerance to heat and strong irradiance would reduce the dependency of the two beans on xanthophyll pigments to dissipate heat, and also seemed irrelevant to the agents with antioxidant activities such as SOD.     

Heat shock response in psychrophilic and psychrotrophic yeast from Antarctica

The response to heat stress in six yeast species isolated from Antarctica was examined. The yeast were classified into two groups: one psychrophilic, with a maximum growth temperature of 20°C, and the other psychrotrophic, capable of growth at temperatures above 20°C. In addition to species-specific heat shock protein (hsp) profiles, a heat shock (15°C–25°C for 3 h) induced the synthesis of a 110-kDa protein common to the psychrophiles, Mrakia stokesii, M. frigida, and M. gelida, but not evident in Leucosporidium antarcticum. Immunoblot analyses revealed heat shock inducible proteins (hsps) corresponding to hsps 70 and 90. Interestingly, no proteins corresponding to hsps 60 and 104 were observed in any of the psychrophilic species examined. In the psychrotrophic yeast, Leucosporidium fellii and L. scottii, in addition to the presence of hsps 70 and 90, a protein corresponding to hsp 104 was observed. In psychrotrophic yeast, as observed in psychrophilic yeast, the absence of a protein corresponding to hsp 60 was noted. Relatively high endogenous levels of trehalose which were elevated upon a heat shock were exhibited by all species. A 10 Celsius degree increase in temperature above the growth temperature (15°C) of psychrophiles and psychrotrophs was optimal for heat shock induced thermotolerance. On the other hand, in psychrotrophic yeast grown at 25°C, only a 5 Celsius degree increase in temperature was necessary for heat shock induced thermotolerance. Induced thermotolerance in all yeast species was coincident with hsp synthesis and trehalose accumulation. It was concluded that psychrophilic and psychrotrophic yeast, although exhibiting a stress response similar to mesophilic Saccharomyces cerevisiae, nevertheless had distinctive stress protein profiles. Received: August 7, 1997 / Accepted: October 22, 1997     

Heat shock and cold shock in Deinococcus radiodurans

On the basis of acquired thermotolerance and cryotolerance, the optimal heat shock and cold shock temperatures have been determined for Deinococcus radiodurans. A heat shock at 42°C maximized survival at the lethal temperature of 52°C and a cold shock at 20°C maximized survival after repeated freeze-thawing. Enhanced survival from heat shock was found to be strongly dependent on growth stage, with its greatest effect shortly after phase. Increased synthesis of a total of 67 proteins during heat shock and 42 proteins during cold shock were observed by two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and autoradiography. Eight of the most highly induced heat shock proteins shown by 2D PAGE were identified by MALDI-MS as Hsp20, GroEL, DnaK, SodA, Csp, Protease I and two proteins of unknown function.     

Enhanced Thermotolerance of <Emphasis Type="Italic">E. coli</Emphasis> by Expressed OsHsp90 from Rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

A gene encoding the rice (Oryza sativa L.) 90-kDa heat shock protein (OsHsp90) was introduced into Escherichia coli using the pGEX-6p-3 expression vector with a glutathione-S-transferase (GST) tag to analyze the possible function of this protein under heat stress for the first time. We compared the survivability of E. coli (BL21) cells transformed with a recombinant plasmid containing GST-OsHsp90 fusion protein with control E. coli cells transformed with the plasmid containing GST and the wild type BL21 under heat shock after isopropyl β-d-thiogalactopyranoside induction. Cells expressing GST-OsHsp90 demonstrated thermotolerance at 42, 50, and 70°C, treatments that were more harmful to cells expressing GST and the wild type. Further studies were carried out to analyze the heat-induced characteristics of OsHsp90 at 42, 50, and 70°C in vitro. When cell lysates from E. coli transformants were heated at these heat stresses, expressed GST-OsHsp90 prevented the denaturation of bacterial proteins treated with 42°C heat shocks, and partially prevented that of proteins treated at 50 and 70°C; meanwhile, cells expressing GST-OsHsp90 withstood the duration at 50°C. These results indicate that OsHsp90 functioned as a chaperone, binding to a subset of substrates, and maintained E. coli growth well at high temperatures.     

Blood heat shock proteins evoked by some <Emphasis Type="Italic">Salmonella</Emphasis> strains infection in ducks

Bacterial heat-shock response is a global regulatory system required for effective adaptation to changes (stress) in the environment. An in vitro study was conducted to investigate the impact of a sublethal temperature (42°C) on heat shock protein (HSP) expression in 6 Salmonella strains (Salmonella Enteritidis, S. Typhimurium, S. Virchow, S. Shubra, S. Haifa and S. Eingedi). The 6 Salmonella strains were isolated from the tissues of ducklings that had died from avian salmonellosis. To determine the induction of HSP in the 6 Salmonella strains, they were exposed to the selected temperature level for 24 h and further kept for 48 h at culturing condition of 42°C. Growth under a sublethal temperature of 42°C increased the expression of several proteins of Salmonella, including a 63 kDa protein in addition to the generation and/or overexpression of 143 proteins which were specific to heat shock, concurrent to this acquired thermotolerance. The 6 Salmonella strains responded to 24 h of thermal stress at an elevated temperature 42°C by synthesizing different heat shock proteins (HSP) with molecular weights ranging between 13.62 and 96.61 kDa. At 48 h, the 6 Salmonella strains synthesized different HSPs with molecular weights ranging between 14.53 and 103.43 kDa. It follows that salmonellae would produce HSPs during the course of the infectious process. Salmonellosis produced several proteins after 24 and 48 h of infection. Seven of these proteins (100, 80, 60, 40, 30, 20 and 10 kDa) were recognized in the serum obtained from the ducklings infected with S. Enteritidis, S. Typhimurium, S. Virchow, S. Shubra, S. Haifa and S. Eingedi after 24 h of infection. After 48 h, the 1–7 kDa HSP became more evident and indicated their de novo generation.     

Effect of salicylic acid on the development of induced Thermotolerance and induction of heat shock protein synthesis in the <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> cell culture

Salicylic acid (SA) could be involved in the development of tolerance to abiotic stresses, to heat shock in particular. Under normal conditions (26°C), treatment with SA improved the tolerance of heterotrophic Arabidopsis thaliana (L.) Heynh culture to severe heat shock (50°C). Under mild heat shock (37°C) inducing the development of thermotolerance, the presence of SA, in contrast, reduced the capability of arabidopsis cells to tolerate high temperature (50°C) and simultaneously suppressed induction of HSP synthesis (Hsp101 and Hsp17.6) important for the development of induced thermotolerance. Since SA suppressed cell respiration and activated the alternative pathway of electron transport, SA is supposed, by modulating mitochondria functions, to be an endogenous regulator of plant stress gene expression.     

Ethanol treatment triggers a heat shock-like response but no thermotolerance in soybean (Glycine max cv. Kaohsiung No.8) seedlings

Non‐lethal heat‐shock (HS) treatment has previously been shown to induce thermotolerance in soybean (Glycine max cv. Kaohsiung No.8) seedlings. This acquired thermotolerance correlates with the de novo synthesis of heat‐shock proteins (HSPs). Interestingly, we found that ethanol treatments also elicited HS‐like responses in aetiolated soybean seedlings at their normal growth temperature of 28 °C. Northern blot analyses revealed that the expression of HS genes hsp17.5, hsp70 and hsc 70 was induced by ethanol. Radioactive amino acids were preferentially incorporated into high molecular weight (HMW) HSPs rather than class I low molecular weight (LMW) HSPs during non‐lethal ethanol treatments. Immunoblot analysis confirmed that no accumulation of class I LMW HSPs occurred after non‐lethal ethanol treatment. Pre‐treatment with a non‐lethal dose of ethanol did not provide thermotolerance, as the aetiolated soybean seedlings could not survive a subsequent heat shock of 45 °C for 2 h. In contrast, non‐lethal HS pre‐treatment, 40 °C for 2 h, conferred tolerance on aetiolated soybean seedlings to otherwise lethal treatments of 7·5% ethanol for 8 h or 10% ethanol for 4 h. These results suggest that plant class I LMW HSPs may play important roles in providing both thermotolerance and ethanol tolerance.     

Stress proteins and stress tolerance in an Antarctic, psychrophilic yeast, Candida psychrophila

Conditions are described for the heat shock acquisition of thermotolerance, peroxide tolerance and synthesis of heat shock proteins (hsps) in the Antarctic, psychrophilic yeast Candida psychrophila. Cells grown at 15°C and heat shocked at 25°C (3 h) acquired tolerance to heat (35°C) and hydrogen peroxide (100 mM). Novel heat shock inducible proteins at 80 and 110 kDa were observed as well as the presence of hsp 90, 70 and 60. The latter hsps were not significantly heat shock inducible. The absence of hsp 104 was intriguing and it was speculated that the 110 kDa protein may play a role in stress tolerance in psychrophilic yeasts, similar to that of hsp 104 in mesophilic species.     

Impact of heat shock on heat shock proteins expression, biological and commercial traits of Bombyx mori

We report the thermotolerance of new bivoltine silkworm, Bombyx mori strains NB₄D₂, KSO₁, NP₂, CSR₂ and CSR₄and differential expression of heat shock proteins at different instars. Different instars of silkworm larva were subjected to heat shock at 35°C, 40°C and 45°C for 2 hours followed by 2 hours recovery. Heat shock proteins were analyzed by SDS‐PAGE. The impact of heat shock on commercial traits of cocoons was analyzed by following different strategies in terms of acquired thermotolerance over control. Comparatively NP₂ exhibited better survivability than other strains. Resistance to heat shock was increased as larval development proceeds in the order of first instar > second instar > third instar > fourth instar > fifth instar in all silkworm strains. Expression of heat shock proteins varies in different instars. 90 kDa in the first, second and third instars, 84 kDa in the fourth instar and 84, 62, 60, 47 and 33 kDa heat shock proteins in fifth instar was observed in response to heat shock. Relative influence of heat shock on commercial traits that correspond to different stages was significant in all strains. In NB₄D₂, cocoon and shell weight significantly increased to 17.52% and 19.44% over control respectively. Heat shock proteins as molecular markers for evaluation and evolution of thermotolerant silkworm strains for tropics was discussed.     

CHROMOSOMAL ANALYSIS OF HEAT-SHOCK TOLERANCE IN DROSOPHILA MELANOGASTER EVOLVING AT DIFFERENT TEMPERATURES IN THE LABORATORY

We investigated the heat tolerance of adults of three replicated lines of Drosophila melanogaster that have been evolving independently by laboratory natural selection for 15 yr at “nonextreme” temperatures (18°C, 25°C, or 28°C). These lines are known to have diverged in body size and in the thermal dependence of several life-history traits. Here we show that they differ also in tolerance of extreme high temperature as well as in induced thermotolerance (“heat hardening”). For example, the 28°C flies had the highest probability of surviving a heat shock, whereas the 18°C flies generally had the lowest probability. A short heat pretreatment increased the heat tolerance of the 18°C and 25°C lines, and the threshold temperature necessary to induce thermotolerance was lower for the 18°C line than for the 25°C line. However, neither heat pretreatment nor acclimation to different temperatures influenced heat tolerance of the 28°C line, suggesting the loss of capacity for induced thermotolerance and for acclimation. Thus, patterns of tolerance of extreme heat, of acclimation, and of induced thermotolerance have evolved as correlated responses to natural selection at nonextreme temperatures. A genetic analysis of heat tolerance of a representative replicate population each from the 18°C and 28°C lines indicates that chromosomes 1, 2, and 3 have significant effects on heat tolerance. However, the cytoplasm has little influence, contrary to findings in an earlier study of other stocks that had been evolving for 7 yr at 14°C versus 25°C. Because genes for heat stress proteins (hsps) are concentrated on chromosome 3, the potential role of hsps in the heat tolerance and of induced thermotolerance in these naturally selected lines is currently unclear. In any case, species of Drosophila possess considerable genetic variation in thermal sensitivity and thus have the potential to evolve rapidly in response to climate change; but predicting that response may be difficult.     

Expression of heat shock proteins in response to high and low temperature extremes in diapausing pharate larvae of the gypsy moth,Lymantria dispar

Diapausing pharate first instars of the gypsy moth, Lymantria dispar, respond to high temperature (37–41°C) by suppressing normal protein synthesis and synthesizing a set of seven heat shock proteins with M_rs of 90,000, 75,000, 73,000, 60,000, 42,000, 29,000, and 22,000 as determined by SDS-PAGE. During recovery at 25°C from heat shock, synthesis of the heat shock proteins gradually decreases over a period of 6 h, while normal protein synthesis is restored. A subset of these same heat shock proteins is also expressed during recovery at 4°C or 25°C from brief exposures to low temperature (-10 to 20°C), and its expression is more intense with increased severity of cold exposure. During recovery at 4°C after 24 h at ?20°C, both 90,000 and 75,000 M_r heat shock proteins are expressed for more than 96 h. While normal protein synthesis is suppressed during heat shock and recovery from heat shock, normal protein synthesis coincides with synthesis of the heat shock proteins during recovery from low temperatures, thus implying that expression of the heat shock proteins is not invariably linked to suppression of normal protein synthesis. Western transfer, using a monoclonal antibody that recognizes the inducible form of the human 70,000 M_r heat shock protein, demonstrates that immunologically related proteins in the gypsy moth are expressed at 4°C and during recovery from cold and heat shock.     

Adaptation of the yeast <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> to heat shock

The adaptive response of the yeast Yarrowia lipolytica to heat shock has been studied. Experiments showed that, after 10 min of incubation at 45°C, the survival rate of Yarrowia lipolytica cells was less than 0.1%. Stationary-phase yeast cells were found to be more thermotolerant than exponential-phase cells. A 60-min preincubation of cells at 37°C or pretreatment with low concentrations of H₂O₂ (0.5 mM) or menadione (0.05 mM) made them more tolerant to heat and to oxidative stress (120 mM hydrogen peroxide). The pH dependence of yeast thermotolerance has also been studied. The adaptation of yeast cells to heat shock and oxidative stress was found to be associated with a decrease in the intracellular level of cAMP and an increase in the activity of antioxidant enzymes (catalase, superoxide dismutase, glucose-6-phosphate dehydrogenase, and glutathione reductase).     

Thermotolerance of IVM‐derived bovine oocytes and embryos after short‐term heat shock

A series of experiments were designed to study the effect of elevated temperatures on developmental competence of bovine oocytes and embryos produced in vitro. In experiment 1, the effect of heat shock (HS) by a mild elevated temperature (40.5°C) for 0, 30, or 60 min on the viability of in vitro matured (IVM) oocytes was tested following in vitro fertilization (IVF) and culture. No significant difference was observed between the control (39°C) and the heat‐treated groups in cleavage, blastocyst formation, or hatching (P > 0.05). In experiment 2, when the HS temperature was increased to 41.5°C, neither the cleavage rate nor blastocyst development was affected by treatment. However, the rate of blastocyst hatching appeared lower in the HS groups (13% in control group vs. 3.9% and 5.6% in 30 min and 60 min, respectively; P < 0.05). When IVM oocytes were treated at 43°C prior to IVF (experiment 3), no difference was detected in blastocyst and expanded blastocyst development following heat treatment for 0, 15, or 30 min, but heat treatment of oocytes for 45 or 60 min significantly reduced blastocyst and expanded blastocyst formation (P < 0.05). In experiment 4, the thermotolerance of day 3 and day 4 bovine IVF embryos were compared. When embryos were pre‐treated with a mild elevated temperature (40.5°C) for 1 hr, and then with a higher temperature (43°C) for 1 hr, no improvement in thermotolerance of the embryos was observed as compared to those treated at 43°C alone. However, a higher thermotolerance was observed in day 4 than day 3 embryos. In conclusion, treatment at 43°C, but not 40.5°C or 41.5°C significantly reduced oocyte developmental competence. An increase in thermotolerance was observed from day 3 to day 4 of in vitro embryonic development, which corresponds to the maternal to zygotic transition of gene expression in bovine embryos. Mol. Reprod. Dev. 53:336–340, 1999. © 1999 Wiley‐Liss, Inc.     

Effects of circadian rhythms of fluctuating temperature on growth and biochemical composition of <Emphasis Type="Italic">Ulva pertusa</Emphasis>

The marcoalga Ulva pertusa was cultured under (20 ± 2)°C, (20 ± 4)°C, (20 ± 6)°C, (20 ± 8)°C and (20 ± 10)°C circadian rhythms of fluctuating temperature conditions, and constant temperature of 20°C was used as the control. The growth rate of macroalga at (20 ± 2)°C, (20 ± 4)°C and (20 ± 6)°C were significantly higher than that at constant temperature of 20°C, while growth rate at (20 ± 8)°C and (20 ± 10)°C were significantly lower than that at constant temperature of 20°C. The growth rate of macroalga was a quadratic function of the thermal amplitude. Such a growth model can be described by G = β ₀ + β ₁(TA) + β ₂(TA)², where G represents the relative growth rate, TA is thermal amplitude in degree Celsius, β ₀ is the intercept on the G axis, and β ₁ and β ₂ are the regression coefficients. The optimal thermal amplitude for the growth of thallus at mean temperature of 20°C was estimated to be ± 3.69°C. Analysis of biochemical composition at the final stages of thaulls growth revealed that diel fluctuating temperature caused various influences (P < 0.05). The content of chlorophyll, protein and total solute carbohydrate at (20 ± 2)°C and (20 ± 4)°C were slightly higher than those at constant temperature of 20°C, however no statistically significant differences were found among them (P > 0.05). While osmolytes (total solute carbohydrate and free proline) at (20 ± 10)°C were significantly higher than that at 20°C (P < 0.05). Therefore, more chlorophyll and carbohydrate production might account for the enhancement in the growth of macroalga at the diel fluctuating temperatures in the present study. Handling editor: S. M. Thomaz     

Developmental study of thermotolerance and the heat shock response inLucilia cuprina (Weidemann)

The ability to withstand thermal stress in a laboratory population of the blowflyLucilia cuprina (measured as per cent adult survival following varying periods of exposure to elevated temperature up to a maximum of 48°C) was in the order pupa > larva > adult. Pre-exposure to a mild heat shock (37°C) induced tolerance to temperatures which were otherwise lethal. An analysis of heat shock-induced protein synthesis during development at similar elevated temperatures presented patterns corresponding to the above observations on thermotolerance. The induced level of synthesis of major heat shock proteins (viz., 79, 69, 28, 20 and 19 kDa) were greater in larval tissues than in most of the adult tissues except gonads. The response varied between young (2 days) and old (30 days) adults in a tissue-specific manner. In general, heat shock protein 69 kDa was most abundant in all the tissues studied. Control as well as heat shocked Malpighian tubules of adults uniquely showed two major [³⁵S]methionine labelled bands corresponding to approximately 62 and 64 kDa. Immunoblots showed the 62 kDa protein to cross react with an antibody againstHelioihis HSP60. Although the synthesis of the 62 kDa polypeptide was prominent only in Malpighian tubules of adult blowflies, nearly equal levels of this HSP60 family polypeptide were present in all tissues (control as well heat shocked) except the larval salivary glands.     

Thermotolerance induced by heat shock in Chlorella

Thermotolerance in cultures of Chlorella zofingiensis was induced by heat shock treatment at supraoptimal temperatures (40and 45 °C for 30 min). Thermotolerance was assayed by two methods: the survival of the cells at 70 °C and the growth of diluted cultures at 35 and 45 °C. A culture without heat shock treatment was unable to grow at 45 °C. According to eletrophoretic analyses, the synthesis of proteins of 95, 73, 60, 43 and 27 kDa was induced by heat shock treatment. The large molecular weight proteins (95, 73, 60 and43 kDa) were present in non-heat treated cells, but the heat shock treatment increased their quantity in cells. The synthesis of a low molecular weight protein (27 kDa) was induced by heat shock treatment. The induced thermotolerance could be inhibited by the presence of an 80S ribosomal translation inhibitor, cycloheximide(CHI). The first 12 amino acid residues from the N-terminus of the27 kDa heat shock induced protein are Val-Glu-Trp-Try-Gly-Pro-Asn-Arg-Ala-Lys-Phe-Leu. This revised version was published online in August 2006 with corrections to the Cover Date.     

MTH1745, a protein disulfide isomerase-like protein from thermophilic archaea, <Emphasis Type="Italic">Methanothermobacter thermoautotrophicum</Emphasis> involving in stress response

MTH1745 is a putative protein disulfide isomerase characterized with 151 amino acid residues and a CPAC active-site from the anaerobic archaea Methanothermobacter thermoautotrophicum. The potential functions of MTH1745 are not clear. In the present study, we show a crucial role of MTH1745 in protecting cells against stress which may be related to its functions as a disulfide isomerase and its chaperone properties. Using real-time polymerase chain reaction analyses, the level of MTH1745 messenger RNA (mRNA) in the thermophilic archaea M. thermoautotrophicum was found to be stress-induced in that it was significantly higher under low (50°C) and high (70°C) growth temperatures than under the optimal growth temperature for the organism (65°C). Additionally, the expression of MTH1745 mRNA was up-regulated by cold shock (4°C). Furthermore, the survival of MTH1745 expressing Escherichia coli cells was markedly higher than that of control cells in response to heat shock (51.0°C). These results indicated that MTH1745 plays an important role in the resistance of stress. By assay of enzyme activities in vitro, MTH1745 also exhibited a chaperone function by promoting the functional folding of citrate synthase after thermodenaturation. On the other hand, MTH1745 was also shown to function as a disulfide isomerase on the refolding of denatured and reduced ribonuclease A. On the basis of its single thioredoxin domain, function as a disulfide isomerase, and its chaperone activity, we suggest that MTH1745 may be an ancient protein disulfide isomerase. These studies may provide clues to the understanding of the function of protein disulfide isomerase in archaea.     

Heat shock response ofChlorella protothecoides during greening

Heterotrophically grown cells ofChlorella protothecoides were transferred to autotrophic medium and allowed to green at 25°C. The protein synthetic activity of the greening cells measured in terms of incorporation of [³5S]-methionine showed a maximum around 20 h of greening and thereafter started declining. Similarly, an analysis of densitometric tracings of the fluorographic profile of the polypeptides associated with both total cellular fraction and membrane fractions during different hours of greening revealed that maximum number of polypeptides were getting labelled around 20 h of greening. At 20 h of greening, the cells were shifted to 40°C and the effect of heat shock on protein synthesis was studied. The heat shock treatment caused a definite decrease in the incorporation of [³⁵S]-methionine into proteins. Due to heat shock, the synthesis of total soluble proteins was affected much more than that of the thylakoid membrane bound proteins. When the cells were transferred back to 25°C after a brief period of heat shock at 40°C, there was a considerable recovery in the protein synthesis and this recovery was found to be significant in the case of soluble proteins, while there was no such definite recovery in the synthesis of thylakoid membrane bound proteins.