Heat Shock-induced Changes Research Articles

Eco-Physiological Screening of Different Tomato Genotypes in Response to High Temperatures: A Combined Field-to-Laboratory Approach.

High temperatures represent a limitation for growth and development of many crop species. Several studies have demonstrated that the yield reduction of tomato under high temperatures and drought is mainly due to a photosynthetic decline. In this paper, a set of 15 tomato genotypes were screened for tolerance to elevated temperatures by cultivating plants under plastic walk-in tunnels. To assess the potential tolerance of tomato genotypes to high temperatures, measurements of chlorophyll fluorescence, pigments content and leaf functional traits have been carried out together with the evaluation of the final yields. Based on the greenhouse trials, a group of eight putative heat-sensitive and heat-tolerant tomato genotypes was selected for laboratory experiments aimed at investigating the effects of short-term high temperatures treatments in controlled conditions. The chlorophyll fluorescence induction kinetics were recorded on detached leaves treated for 60 min at 35 °C or at 45 °C. The last treatment significantly affected the photosystem II (PSII) photochemical efficiency (namely maximum PSII quantum efficiency, Fv/Fm, and quantum yield of PSII electron transport, ΦPSII) and the non-photochemical quenching (NPQ) in the majority of genotypes. The short-term heat shock treatments also led to significant differences in the shape of the slow Kautsky kinetics and its significant time points (chlorophyll fluorescence levels minimum O, peak P, semi-steady state S, maximum M, terminal steady state T) compared to the control, demonstrating heat shock-induced changes in PSII functionality. Genotypes potentially tolerant to high temperatures have been identified. Our findings support the idea that chlorophyll fluorescence parameters (i.e., ΦPSII or NPQ) and some leaf functional traits may be used as a tool to detect high temperatures-tolerant tomato cultivars.

Heat Acclimation and Inhibition of Cytokinin Degradation Positively Affect Heat Stress Tolerance of Arabidopsis.

In order to pinpoint phytohormone changes associated with enhanced heat stress tolerance, the complex phytohormone profiles [cytokinins, auxin, abscisic acid (ABA), jasmonic acid (JA), salicylic acid and ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC)] were compared in Arabidopsis thaliana after direct heat shock (45°C, 3 h) and in heat-stressed pre-acclimated plants (1 h at 37°C followed by 2 h at optimal temperature 20°C). Organ-specific responses were followed in shoot apices, leaves, and roots immediately after heat shock and after 24-h recovery at 20°C. The stress strength was evaluated via membrane ion leakage and the activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOX) and antioxidant enzymes [superoxide dismutases, guaiacol peroxidases (POD), catalases, ascorbate peroxidases (APX)]. Heat acclimation diminished negative effects of heat stress, especially in apices and roots, no significant differences being observed in leaves. Low NOX1-3 activities indicated diminished production of reactive oxygen species. Higher activity of APX, POD1, and the occurrence of POD3-4 reflected acclimation-stimulated readiness of the antioxidant system. Acclimation diminished heat shock-induced changes of ABA, JA, cytokinin, and auxin levels in apices. Excess of ABA catabolites suggested an early stress response. The strong up-regulation of ABA and ACC in roots indicated defense boost in roots of acclimated plants compared to the non-acclimated ones. To evaluate the possibility to enhance stress tolerance by cytokinin pool modulation, INCYDE-F, an inhibitor of cytokinin oxidase/dehydrogenase, was applied. As cytokinin effects on stress tolerance may depend on timing of their regulation, INCYDE was applied at several time-points. In combination with acclimation, INCYDE treatment had a slight positive effect on heat stress tolerance, mainly when applied after 2-h period of the optimal temperature. INCYDE increased contents of cytokinins trans-zeatin and cis-zeatin in roots and auxin in all tissues after heat shock. INCYDE also helped to suppress the content of ABA in leaves, and ethylene in apices and roots. INCYDE application to non-acclimated plants (applied before or after heat shock) strengthened negative stress effects, probably by delaying of the repair processes. In conclusion, pre-treatment with moderately elevated temperature enhanced heat stress tolerance and accelerated recovery after stress. Inhibition of cytokinin degradation by INCYDE slightly improved recovery of acclimated plants.

Involvement of Stat3 phosphorylation in mild heat shock-induced thermotolerance

Thermotolerance is a phenomenon in which cells become resistant to stress by prior exposure to heat shock, and its development is associated with the induction of heat shock proteins (Hsps), including Hsp70. We previously showed that the expression of Hsp70 is regulated by the cytokine signaling transcription factor Stat3, but the role of Stat3 in thermotolerance is not known. In this study, we examined the possible involvement of Stat3 in the acquisition of thermotolerance. We found that severe heat shock-induced morphological changes and decreases in cell viability, which were suppressed by exposure to non-lethal mild heat shock prior to severe heat shock. This thermotolerance development was accompanied by Stat3 phosphorylation and the induction of Hsps such as Hsp105, Hsp70, and Hsp27. Stat3 phosphorylation and Hsp induction were inhibited by AG490, an inhibitor of JAK tyrosine kinase. Consistent with this, we found that mild heat shock-induced thermotolerance was partially suppressed by AG490 or knockdown of Hsp105. We also found that the Stat3 inhibitor Stattic suppresses the acquisition of thermotolerance by inhibiting the mild heat shock-induced Stat3 phosphorylation and Hsp105 expression. These results suggest that the mild heat shock-dependent stimulation of the JAK-Stat signaling pathway contributes to the development of thermotolerance via the induction of Hsps including Hsp105. This signaling pathway may be a useful target for hyperthermia cancer therapy.

Heat shock exerts anticancer effects on liver cancer via autophagic degradation of aquaporin 5.

Previous studies described that the expression of aquaporin 5 (AQP5) was altered in tumors of various organs. AQP5 is attracting attention as a new cancer therapeutic target. In the present study, heat shock-induced changes in AQP5 expression were evaluated by immunofluorescent staining(IF) and western blotting(WB) of liver cancer cells. AQP5 knockdown experiments or a heat shock treatment were conducted, and their effects on cell volume, proliferation, cell cycle, the activity of apoptosis and migration/invasion were compared. Cycloheximide(CHX) chase experiments and double IF of AQP5 and light chain 3B(LC3B) were performed to investigate the mechanisms underlying changes in AQP5 expression. The results showed that IF and WB revealed decrease in AQP5 expression on cellular membranes and in the cytoplasm of heated cells. AQP5 knockdown and heat shock similarly decreased cell volume, suppressed migration/invasion and proliferation, and induced early apoptosis and partial G0/G1 arrest. CHX chase experiments revealed that heat shock accelerated the degradation of AQP5, which was rescued under CHX and the autophagy inhibitor, bafilomycinA1 (BafA1). Double IF showed the co-localization of AQP5 and LC3B on BafA1-treated heated cells. In conclusion, we demonstrated that heat shock decreased AQP5 on cellular membranes and in the cytoplasm by activating autophagic degradation, and heat shock and AQP5 knockdown exerted similar anticancer effects, suggesting that heat shock exerts anticancer effects via the autophagic degradation of AQP5.

Bacterial transcriptome reorganization in thermal adaptive evolution.

BackgroundEvolution optimizes a living system at both the genome and transcriptome levels. Few studies have investigated transcriptome evolution, whereas many studies have explored genome evolution in experimentally evolved cells. However, a comprehensive understanding of evolutionary mechanisms requires knowledge of how evolution shapes gene expression. Here, we analyzed Escherichia coli strains acquired during long-term thermal adaptive evolution.ResultsEvolved and ancestor Escherichia coli cells were exponentially grown under normal and high temperatures for subsequent transcriptome analysis. We found that both the ancestor and evolved cells had comparable magnitudes of transcriptional change in response to heat shock, although the evolutionary progression of their expression patterns during exponential growth was different at either normal or high temperatures. We also identified inverse transcriptional changes that were mediated by differences in growth temperatures and genotypes, as well as negative epistasis between genotype—and heat shock-induced transcriptional changes. Principal component analysis revealed that transcriptome evolution neither approached the responsive state at the high temperature nor returned to the steady state at the regular temperature. We propose that the molecular mechanisms of thermal adaptive evolution involve the optimization of steady-state transcriptomes at high temperatures without disturbing the heat shock response.ConclusionsOur results suggest that transcriptome evolution works to maintain steady-state gene expression during constrained differentiation at various evolutionary stages, while also maintaining responsiveness to environmental stimuli and transcriptome homeostasis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1999-x) contains supplementary material, which is available to authorized users.

Transcriptomic and metabolic responses of Staphylococcus aureus exposed to supra-physiological temperatures

BackgroundPrevious evaluation by different molecular and physiological assays of Staphylococcus aureus (S. aureus) responses to heat shock exposure yielded a still fragmentary view of the mechanisms determining bacterial survival or death at supra-physiological temperatures. This study analyzed diverse facets of S. aureus heat-shock adjustment by recording global transcriptomic and metabolic responses of bacterial cultures shifted for 10 min from 37°C to a sub-lethal (43°C) or eventually lethal (48°C) temperature. A relevant metabolic model of the combined action of specific stress response mechanisms with more general, energy-regulating metabolic pathways in heat-shocked S. aureus is presented.ResultsWhile S. aureus cultures shifted to 43°C or left at 37°C showed marginal differences in growth and survival rates, bacterial cultures exposed to 48°C showed a rapid growth arrest followed by a subsequent decline in viable counts. The most substantial heat shock-induced changes at both 43°C and 48°C occurred in transcript levels of HrcA- and CtsR-regulated genes, encoding classical chaperones DnaK and GroESL, and some Hsp100/Clp ATPases components, respectively. Other metabolic pathways up-regulated by S. aureus exposure at 48°C included genes encoding several enzymes coping with oxidative stress, and DNA damage, or/and impaired osmotic balance. Some major components of the pentose phosphate cycle and gluconeogenesis were also up-regulated, which reflected depletion of free glucose by bacterial cultures grown in Mueller-Hinton broth prior to heat shock. In contrast, most purine- and pyrimidine-synthesis pathway components and amino acyl-tRNA synthetases were down-regulated at 48°C, as well as arginine deiminase and major fermentative pathway components, such as alcohol, lactate and formate dehydrogenases. Despite the heat-induced, increased requirements for ATP-dependent macromolecular repair mechanisms combined with declining energy sources, intracellular ATP levels remained remarkably constant during heat shock.ConclusionThe sequential loss of replication and viability at 48°C cannot be explained by significant reductions in intracellular ATP levels, but may reflect ATP rerouting for macromolecular repair mechanisms and cell survival. Our metabolic model also suggests that heat-stressed S. aureus should down-regulate the production of potential, DNA-damaging reactive oxygen species that might result from electron transport-generated ATP, involving excessive levels of free heavy metals, in particular iron.

Cell-specific association of heat shock-induced proton flux with actin ring formation in Chenopodium cells: comparison of auto- and heterotroph cultures

A comparison of the responses of extracellular pH, buffering capacity and actin cytoskeleton in autotroph and heterotroph Chenopodium rubrum cells to heat shock revealed cell-specific reactions: alkalinization caused by the heat shock at 25-35 degrees C was higher in heterotroph cells and characterized by heat shock-induced changes in the actin cytoskeleton and ring formation at 35-37 degrees C. Rings (diameter up to 3 mum) disappeared and extracellular pH recovered after the heat-shocked cells were transferred into control medium. At 41 degrees C, no rings but a network of coarse actin filaments were induced; at higher temperatures, fragmentation of the actin cytoskeleton and release of buffering compounds occurred, indicating sudden membrane leakage at 45-47 degrees C. The calcium chelator EGTA [ethylene-glycol-bis(beta-aminoethyl-ether)-N,N,N',N'-tetraacetic-acid] increased the frequency of heat shock-induced rings. Ionophore (10 microM nigericin) and the sodium/proton antiport blocker [100 microM 5-(N-ethyl-N-isopropyl)-amiloride] mimicked the effect of the 37 degrees C heat shock. The cytoskeleton inhibitors latrunculin B, cytochalasin D and 2,3-butanedione monoxime inhibited ring formation but not alkalinization. In autotroph cells, the treatment with nigericin (10 microM) produced rings, although the actin cytoskeleton was not affected by temperatures up to 45 degrees C. We conclude that Chenopodium cells express a specific temperature sensor that has ascendancy over the organization of the actin cytoskeleton; this is probably a temperature- and potential-sensitive proton-transporting mechanism that is dependent on the culture conditions of the heterotroph cells.

BH4 Peptide Derived from BCL-xL and BAX-Inhibitor Peptide Suppresses Heat Shock-Induced Apoptopic Changes in Bovine Oocyte Mitochondria.

BH4 Peptide Derived from BCL-xL and BAX-Inhibitor Peptide Suppresses Heat Shock-Induced Apoptopic Changes in Bovine Oocyte Mitochondria.

Chromosomal Protein HMGN1 Enhances the Heat Shock-induced Remodeling of Hsp70 Chromatin

The nucleosome-binding protein HMGN1 affects the structure and function of chromatin; however, its role in regulating specific gene expression in living cells is not fully understood. Here we use embryonic fibroblasts from Hmgn1(+/+) and Hmgn1(-/-) mice to examine the effect of HMGN1 on the heat shock-induced transcriptional activation of Hsp70, a well characterized gene known to undergo a rapid chromatin re-structuring during transcriptional activation. We find that loss of HMGN1 decreases the levels of Hsp70 transcripts at the early stages of heat shock. HMGN1 enhances the rate of heat shockinduced changes in the Hsp70 chromatin but does not affect the chromatin structure before induction, an indication that it does not predispose the gene to rapid activation. Heat shock elevates the levels of H3K14 acetylation in the Hsp70 chromatin of wild type cells more efficiently than in the chromatin of Hmgn1(-/-) cells, whereas treatment with histone deacetylase inhibitors abrogates the effects of HMGN1 on the heat shock response. We suggest that HMGN1 enhances the rate of heat shock-induced H3K14 acetylation in the Hsp70 promoter, thereby enhancing the rate of chromatin remodeling and the subsequent transcription during the early rounds of Hsp70 activation when the gene is still associated with histones in a nucleosomal conformation.

Heat Shock-Induced Changes in Lipid and Protein Metabolism in the Endoplasmic Reticulum of Barley Aleurone Layers

Heat shock in barley aleurone layers induces heat shock protein synthesis and suppresses secretory protein synthesis by selectively destabilizing their mRNAs. In addition, the endoplasmic reticulum (ER) membranes upon which secretory protein mRNAs are translated become vesiculated during heat shock, leading to the hypothesis that ER dissociation and targeted mRNA destabilization are linked mechanistically. Supporting this, ER can be heat adapted, and heat-adapted ER has higher levels of fatty acid saturation in membrane phospholipids which do not vesiculate upon heat shock. Secretory protein mRNAs are also more stable in heat-adapted cells. To understand better heat shock-induced changes in ER membranes, we examined ER membrane proteins and enzymes involved in phosphatidylcholine biosynthesis and phospholipid turnover in heat-shocked aleurone cells. Heat shock significantly increased the activity of phospholipases A2 and D, and shortly thereafter significant but gradual increases in choline kinase and phosphocholine glyceride transferase activities and a sharp increase in phosphorylcholine citidyl transferase activity were observed. Only minor changes were observed in SDS-PAGE analyses of proteins from sonicated ER membranes fractionated on continuous sucrose gradients. Overall, heat shock reduced total lipid in ER membranes relative to protein, and in intact, ultracentrifuged aleurone cells examined by light and electron microscopy the ER band appeared to increase in density. The changes in phospholipid metabolism coupled with the suppression of secretory protein synthesis indicate that in addition to inducing a classic heat shock response, high temperature also induces a classic unfolded protein response in the ER of this secretory cell.

Heat Shock-Induced Apoptosis in Preimplantation Bovine Embryos Is a Developmentally Regulated Phenomenon1

Apoptosis is a form of cell death that can function to eliminate cells damaged by environmental stress. One stress that can compromise embryonic development is elevated temperature (i.e., heat shock). For the current studies, we hypothesized that heat shock induces apoptosis in bovine embryos in a developmentally regulated manner. Studies were performed to 1) determine whether heat shock can induce apoptosis in preimplantation embryos, 2) test whether heat-induced apoptosis is developmentally regulated, 3) evaluate whether heat shock-induced changes in caspase activity parallel patterns of apoptosis, and 4) ascertain whether exposure to a mild heat shock can protect embryos from heat-induced apoptosis. As determined by TUNEL reaction, exposure of bovine embryos > or =16 cells on Day 5 after insemination to 41 or 42 degrees C for 9 h increased the percentage of cells undergoing apoptosis. In addition, there was a duration-dependent increase in the proportion of blastomeres that were apoptotic when embryos were exposed to temperatures of 40 or 41 degrees C, which are more characteristic of temperatures experienced by heat-stressed cows. Heat shock also increased caspase activity in Day 5 embryos. However, heat shock did not induce apoptosis in 2- or 4-cell embryos, nor did it increase caspase activity in 2-cell embryos. The apoptotic response of 8- to 16-cell-stage bovine embryos to heat shock depended upon the day after insemination that heat shock occurred. When 8- to 16-cell embryos were collected on Day 3 after insemination, heat shock of 41 degrees C for 9 h did not induce apoptosis. In contrast, when 8- to 16-cell embryos were collected on Day 4 after insemination and exposed to heat shock, there was an increase in the percentage of cells undergoing apoptosis. Exposure of 8- to 16-cell embryos at Day 4 to a mild heat shock of 40 degrees C for 80 min blocked the apoptotic response to a subsequent, more-severe heat shock of 41 degrees C for 9 h. In conclusion, apoptosis is a developmentally acquired phenomenon that occurs in embryos exposed to elevated temperature, and it can be prevented by induced thermotolerance.

Heat shock-induced changes in the respiration of the yeast Saccharomyces cerevisiae

The incubation of Saccharomyces cerevisiae at elevated temperature (45 degrees C) stimulated the respiration of yeast cells and decreased their survival rate. The respiration-deficient mutant of this yeast was found to be more tolerant to the elevated temperature than the wild-type strain. At the same time, the cultivation of the wild-type strain in an ethanol-containing medium enhanced the respiration, catalase activity, and thermotolerance of yeast cells, as compared with their growth in a glucose-containing medium. It is suggested that the enhanced respiration of yeast cells at 45 degrees C leads to an intense accumulation of reactive oxygen species, which may be one of the reasons for the heat shock-induced cell death.

The synthesis of HSPs in sugar beet suspension culture cells under hyperthermia exhibits differential sensitivity to calcium.

Experiments aimed at testing the effect of Ca2+ on heat shock-induced changes in protein synthesis of cultured sugar beet cells were performed. Heat shock inhibits the synthesis of non-heat shock proteins (non-HSPs) and promotes the synthesis of a set of HSPs. Extracellular Ca2+ appeared to be strictly required for the synthesis of non-HSPs. Calcium was found to differentially exert its effect on the HSP synthesis: calcium induced (96 and 76 kDa), stimulated (94, 67, 58, 52, 32, 30, 26 and 22 kDa) or did not influence (82, 17 kDa) the de novo production of various HSPs. Cell injury increased if the cells were exposed to high temperature in a Ca(2+)-deficient medium. Calcium supplement to Ca(2+)-depleted cells resulted in the recovery of HSP synthesis and reduced cell injury by heat shock.

Metabolite changes associated with heat shocked avian fibroblast mitochondria.

A previous report from our laboratory (Collier et al 1993) showed that the elongated tubules of mitochondria in the cytoplasm of cultured chicken embryo fibroblasts collapsed to irregularly shaped structures surrounding the nuclear membrane after a 1 h heat shock treatment. The normal mitochondrial morphology reappeared upon removal of the thermal stress. We have now determined that several changes occurred in mitochondrial-related metabolites under these same heat shock and recovery conditions. Among these were significant decreases in the levels of fumarate and malate and increases in the amounts of aspartate and glutamate. In contrast, other intermediates of the tri-carboxylic acid cycle were unaltered as were levels of ATP and phosphocreatine. The changes observed might result from heat shock-induced changes in enzyme activities of the mitochondria, from alterations in the membrane-embedded specialized carrier proteins that transport metabolites between cytosol and mitochondria or from a disorganization of the electron-transport system normally coupled to oxidative metabolism. The rapid recovery, however, suggested that these changes were transient and readily reversible.

Stress-induced transcriptional activation

Living cells, both prokaryotic and eukaryotic, employ specific sensory and signalling systems to obtain and transmit information from their environment in order to adjust cellular metabolism, growth, and development to environmental alterations. Among external factors that trigger such molecular communications are nutrients, ions, drugs and other compounds, and physical parameters such as temperature and pressure. One could consider stress imposed on cells as any disturbance of the normal growth condition and even as any deviation from optimal growth circumstances. It may be worthwhile to distinguish specific and general stress circumstances. Reasoning from this angle, the extensively studied response to heat stress on the one hand is a specific response of cells challenged with supra-optimal temperatures. This response makes use of the sophisticated chaperoning mechanisms playing a role during normal protein folding and turnover. The response is aimed primarily at protection and repair of cellular components and partly at acquisition of heat tolerance. In addition, heat stress conditions induce a general response, in common with other metabolically adverse circumstances leading to physiological perturbations, such as oxidative stress or osmostress. Furthermore, it is obvious that limitation of essential nutrients, such as glucose or amino acids for yeasts, leads to such a metabolic response. The purpose of the general response may be to promote rapid recovery from the stressful condition and resumption of normal growth. This review focuses on the changes in gene expression that occur when cells are challenged by stress, with major emphasis on the transcription factors involved, their cognate promoter elements, and the modulation of their activity upon stress signal transduction. With respect to heat shock-induced changes, a wealth of information on both prokaryotic and eukaryotic organisms, including yeasts, is available. As far as the concept of the general (metabolic) stress response is concerned, major attention will be paid to Saccharomyces cerevisiae.

Stress-induced transcriptional activation.

Living cells, both prokaryotic and eukaryotic, employ specific sensory and signalling systems to obtain and transmit information from their environment in order to adjust cellular metabolism, growth, and development to environmental alterations. Among external factors that trigger such molecular communications are nutrients, ions, drugs and other compounds, and physical parameters such as temperature and pressure. One could consider stress imposed on cells as any disturbance of the normal growth condition and even as any deviation from optimal growth circumstances. It may be worthwhile to distinguish specific and general stress circumstances. Reasoning from this angle, the extensively studied response to heat stress on the one hand is a specific response of cells challenged with supra-optimal temperatures. This response makes use of the sophisticated chaperoning mechanisms playing a role during normal protein folding and turnover. The response is aimed primarily at protection and repair of cellular components and partly at acquisition of heat tolerance. In addition, heat stress conditions induce a general response, in common with other metabolically adverse circumstances leading to physiological perturbations, such as oxidative stress or osmostress. Furthermore, it is obvious that limitation of essential nutrients, such as glucose or amino acids for yeasts, leads to such a metabolic response. The purpose of the general response may be to promote rapid recovery from the stressful condition and resumption of normal growth. This review focuses on the changes in gene expression that occur when cells are challenged by stress, with major emphasis on the transcription factors involved, their cognate promoter elements, and the modulation of their activity upon stress signal transduction. With respect to heat shock-induced changes, a wealth of information on both prokaryotic and eukaryotic organisms, including yeasts, is available. As far as the concept of the general (metabolic) stress response is concerned, major attention will be paid to Saccharomyces cerevisiae.

Heat Shock Proteins Are Not Required for the Degradation of α-Amylase mRNA and the Delamellation of Endoplasmic Reticulum in Heat-Stressed Barley Aleurone Cells

When barley (Hordeum vulgare) aleurone layers are heat shocked, the synthesis and secretion of alpha-amylase and other secretory proteins is arrested and the synthesis of heat shock proteins (hsps) is induced. alpha-Amylase mRNA, normally a very stable mRNA, is actively degraded during heat shock. In addition, endoplasmic reticulum (ER) is delamellated during heat shock, possibly causing the destabilization of the mRNA for the secreted alpha-amylase. To ascertain whether or not hsps play any role in the destabilization of alpha-amylase mRNA or in the delamellation process of ER, heat shocked cells were treated with the transcription inhibitor cordycepin, which effectively inhibits the synthesis of hsps yet does not affect alpha-amylase synthesis after this enzyme has been fully induced by gibberellic acid (12 hours). In the absence of hsp expression, heat shock still causes the destabilization of alpha-amylase mRNA and the delamellation of ER. Alternatively, the synthesis of hsps may be induced in the absence of temperature increase by incubating cells in the presence of arsenite. Arsenite-induced expression of some hsps in the absence of increased temperature does not result in the destabilization of alpha-amylase mRNA or in the delamellation of ER. If cordycepin or cycloheximide are used to inhibit hsp synthesis during heat shock, the tissue recovers from heat shock with normal recovery kinetics. Although hsps have been implicated in the establishment of thermotolerance, our observations indicate that hsps do not play a role in the other heat shock-induced changes observable in aleurone cells. Furthermore, if the synthesis of hsp mRNA is inhibited during heat shock (by cordycepin) hsp mRNAs are synthesized later, during recovery, indicating that there is a stable inducer of hsp synthesis in aleurone tissues.

Heat shock-induced changes in the structural stability of proteinaceous karyoskeletal elements in vitro and morphological effects in situ.

Karyoskeletal protein fractions prepared from Drosophila melanogaster embryos contain morphologically identifiable remnants of nuclear pore complexes and peripheral lamina as well as what appears to be an internal nuclear "matrix" (Fisher, P. A., M. Berrios, and G. Blobel, 1982, J. Cell Biol., 92: 674-686). Structural stability of these proteinaceous assemblies is dependent on thermal incubation in vitro (37 degrees C, 15 min) before subfractionation of nuclei. In the absence of such incubation, greater than 90% of the total karyoskeletal protein including major polypeptide components of internal "matrix," pore complexes, and the peripheral lamina, is solubilized by 1 M NaCl. In vivo heat shock induces karyoskeletal stabilization resembling that resulting from thermal incubation in vitro. Immunocytochemical studies have been used to establish the effects of heat shock on the organization and distribution of major karyoskeletal marker proteins in situ. Taken together, these results are consistent with the notion that in vivo, regulation of karyoskeletal plasticity (and perhaps form) may be a functionally significant component of the Drosophila heat shock response. They also have broad practical implications for studies pertaining to the structure and function of karyoskeletal protein (nuclear "matrix") fractions isolated from higher eukaryotic cells.

Changes in chromatin and the phosphorylation of nuclear proteins during heat shock of Achlya ambisexualis.

Heat shock led to marked changes in the apparent levels of phosphorylation of nuclear proteins in the fungus Achlya ambisexualis. We characterized these heat shock-induced changes in nuclear proteins on two types of two-dimensional polyacrylamide gel systems. We report here that one of two Achlya H3 histones (H3.1) and also the oomycete histone alpha appear to be highly phosphorylated with heat shock. Additional changes observed in acid-soluble nuclear proteins included an apparent increase in the 32P labeling of a 43,000-molecular-weight protein and the dephosphorylation of a major group of Achlya phosphoproteins in the 30,000-to-32,000-molecular-weight range. The changes in protein phosphorylation were accompanied by striking changes in the morphology of Achlya nuclei. Nuclei in the heat-shocked cells, but not in control cells, exhibited marked chromatin condensation and contained bundles of filaments which were approximately 4 nm in diameter. Concomitantly, the bulk of chromatin from heat-shocked nuclei showed a decreased sensitivity to digestion with the enzyme DNase I relative to chromatin from control cells.

Changes in chromatin and the phosphorylation of nuclear proteins during heat shock of Achlya ambisexualis.

Heat shock led to marked changes in the apparent levels of phosphorylation of nuclear proteins in the fungus Achlya ambisexualis. We characterized these heat shock-induced changes in nuclear proteins on two types of two-dimensional polyacrylamide gel systems. We report here that one of two Achlya H3 histones (H3.1) and also the oomycete histone alpha appear to be highly phosphorylated with heat shock. Additional changes observed in acid-soluble nuclear proteins included an apparent increase in the 32P labeling of a 43,000-molecular-weight protein and the dephosphorylation of a major group of Achlya phosphoproteins in the 30,000-to-32,000-molecular-weight range. The changes in protein phosphorylation were accompanied by striking changes in the morphology of Achlya nuclei. Nuclei in the heat-shocked cells, but not in control cells, exhibited marked chromatin condensation and contained bundles of filaments which were approximately 4 nm in diameter. Concomitantly, the bulk of chromatin from heat-shocked nuclei showed a decreased sensitivity to digestion with the enzyme DNase I relative to chromatin from control cells.