Role In Thermotolerance Research Articles

The Transcriptional Co-activator MBF1c Is a Key Regulator of Thermotolerance in Arabidopsis thaliana

The ability of an organism to acclimate to its environment is a key determinant in its global distribution and capacity to compete with other organisms. The heat stress response, a highly conserved environmental and developmental program in eukaryotic and prokaryotic organisms, is an important component of the acclimation response of plants. Previous studies have shown that heat shock transcription factors play an important role in thermotolerance in plants and other organisms, controlling the expression of different heat shock proteins and detoxifying enzymes. In contrast, although several other pathways, involving ethylene, salicylic acid (SA), and trehalose, were recently shown to play a central role in thermotolerance in plants, a key regulator of these responses was not identified. Here we report that the highly conserved transcriptional co-activator, MBF1c (multiprotein bridging factor 1c), is a key regulator of thermotolerance in Arabidopsis thaliana. MBF1c protein accumulates rapidly and is localized to nuclei during heat stress. MBF1c is required for thermotolerance and functions upstream to SA, trehalose, ethylene, and pathogenesis-related protein 1 during heat stress. In contrast, MBF1c is not required for the expression of transcripts encoding HSFA2 and different heat shock proteins. Interestingly, MBF1c interacts with TPS5 (trehalose phosphate synthase 5), which is also heat-inducible, and mutants deficient in TPS5 are thermosensitive. Our results provide evidence for the existence of a tightly coordinated heat stress-response network, involving trehalose-, SA-, and ethylene-signaling pathways, that is under the control of MBF1c.

The C-terminal Extension of Saccharomyces cerevisiae Hsp104 Plays a Role in Oligomer Assembly

The Saccharomyces cerevisiae protein Hsp104, a member of the Hsp100/Clp AAA+ family of ATPases, and its orthologues in plants (Hsp101) and bacteria (ClpB) function to disaggregate and refold thermally denatured proteins following heat shock and play important roles in thermotolerance. The primary sequences of fungal Hsp104's contain a largely acidic C-terminal extension not present in bacterial ClpB's. In this work, deletion mutants were used to determine the role this extension plays in Hsp104 structure and function. Elimination of the C-terminal tetrapeptide DDLD diminishes binding of the tetratricopeptide repeat domain cochaperone Cpr7 but is dispensable for Hsp104-mediated thermotolerance. The acidic region of the extension is also dispensable for thermotolerance and for the stimulation of Hsp104 ATPase activity by poly-l-lysine, but its truncation results in an oligomerization defect and reduced ATPase activity in vitro. Finally, sequence alignments reveal that the C-terminal extension contains a sequence (VLPNH) that is conserved in fungal Hsp104's but not in other orthologues. Hsp104 lacking the entire C-terminal extension including the VLPNH region does not assemble and has very low ATPase activity. In the presence of a molecular crowding agent the ATPase activities of mutants with longer truncations are partially restored possibly through enhanced oligomer formation. However, elimination of the whole C-terminal extension results in an Hsp104 molecule which is unable to assemble and becomes aggregation prone at high temperature, highlighting a novel structural role for this region.

Atypical AAA+ Subunit Packing Creates an Expanded Cavity for Disaggregation by the Protein-Remodeling Factor Hsp104

SummaryHsp104, a yeast protein-remodeling factor of the AAA+ (ATPases associated with various cellular activities) superfamily, and its homologs in bacteria and plants mediate cell recovery after severe stress by disaggregating denatured proteins through a poorly understood mechanism. Here, we present cryo-electron microscopy maps and domain fitting of Hsp104 hexamers, revealing an unusual arrangement of AAA+ modules with the prominent coiled-coil domain intercalated between the AAA+ domains. This packing results in a greatly expanded cavity, which is capped at either end by N- and C-terminal domains. The fitted structures as well as mutation of conserved coiled-coil arginines suggest that the coiled-coil domain plays a major role in the extraction of proteins from aggregates, providing conserved residues for key functions in ATP hydrolysis and potentially for substrate interaction. The large cavity could enable the uptake of polypeptide loops without a requirement for exposed N or C termini.

Role of Hsp104 in the propagation and inheritance of the [Het-s] prion.

The chaperones of the ClpB/HSP100 family play a central role in thermotolerance in bacteria, plants, and fungi by ensuring solubilization of heat-induced protein aggregates. In addition in yeast, Hsp104 was found to be required for prion propagation. Herein, we analyze the role of Podospora anserina Hsp104 (PaHsp104) in the formation and propagation of the [Het-s] prion. We show that DeltaPaHsp104 strains propagate [Het-s], making [Het-s] the first native fungal prion to be propagated in the absence of Hsp104. Nevertheless, we found that [Het-s]-propagon numbers, propagation rate, and spontaneous emergence are reduced in a DeltaPaHsp104 background. In addition, inactivation of PaHsp104 leads to severe meiotic instability of [Het-s] and abolishes its meiotic drive activity. Finally, we show that DeltaPaHSP104 strains are less susceptible than wild type to infection by exogenous recombinant HET-s(218-289) prion amyloids. Like [URE3] and [PIN(+)] in yeast but unlike [PSI(+)], [Het-s] is not cured by constitutive PaHsp104 overexpression. The observed effects of PaHsp104 inactivation are consistent with the described role of Hsp104 in prion aggregate shearing in yeast. However, Hsp104-dependency appears less stringent in P. anserina than in yeast; presumably because in Podospora prion propagation occurs in a syncitium.

Natural Variation in the Thermotolerance of Neural Function and Behavior due to a cGMP-Dependent Protein Kinase

Although it is acknowledged that genetic variation contributes to individual differences in thermotolerance, the specific genes and pathways involved and how they are modulated by the environment remain poorly understood. We link natural variation in the thermotolerance of neural function and behavior in Drosophila melanogaster to the foraging gene (for, which encodes a cGMP-dependent protein kinase (PKG)) as well as to its downstream target, protein phosphatase 2A (PP2A). Genetic and pharmacological manipulations revealed that reduced PKG (or PP2A) activity caused increased thermotolerance of synaptic transmission at the larval neuromuscular junction. Like synaptic transmission, feeding movements were preserved at higher temperatures in larvae with lower PKG levels. In a comparative assay, pharmacological manipulations altering thermotolerance in a central circuit of Locusta migratoria demonstrated conservation of this neuroprotective pathway. In this circuit, either the inhibition of PKG or PP2A induced robust thermotolerance of neural function. We suggest that PKG and therefore the polymorphism associated with the allelic variation in for may provide populations with natural variation in heat stress tolerance. for's function in behavior is conserved across most organisms, including ants, bees, nematodes, and mammals. PKG's role in thermotolerance may also apply to these and other species. Natural variation in thermotolerance arising from genes involved in the PKG pathway could impact the evolution of thermotolerance in natural populations.

Heat shock proteins in the skin

Heat shock proteins (hsp) are expressed in all cells and organisms. Their expression is induced by heat shock (temperatures above 42 degrees C) and other forms of pathophysiological stress. Elevated levels of hsp protect cells from further stress exposure. Hsp are expressed intracellularly. They are highly conserved throughout evolution indicating hsp being necessary for survival under potentially harmful environmental conditions. Hsp are divided into families according to their molecular weight. The majority of hsp function as molecular chaperones. Chaperone function is characterized by binding to other proteins and mediating their folding, transport and interaction with other molecules. In human epidermis hsp are abundantly expressed and have been linked with functions in cell differentiation and photobiology. Recent research has mainly focused on the 27 and 72 kD hsp that are constitutively expressed in human keratinocytes. ultraviolet radiation (UV)-induced cell death and sunburn cell formation can be inhibited by previous heat shock exposure and UV itself can induce hsp expression. The expression of the 27 kD hsp (hsp27) in epidermal keratinocytes in situ and in culture correlates with differentiation. Expression of hsp27 increases simultaneously with keratinocyte differentiation. For that reason, hsp27 is described as a marker of epidermal differentiation. Changes in the expression and inducibility of hsp have been linked with ageing. In the skin, recent data indicate that hsp72 expression remains remarkably stable with intrinsic ageing. In contrast, levels of hsp27 have been found to be elevated in sun-protected aged skin indicating a link between hsp27 expression and age-dependent epidermal alterations. Regulation of hsp can be modified by pharmacological intervention and the development of safe topical and systemic treatments for the prevention of skin damage and disorders of keratinocyte differentiation can be expected for the future.

Pleurotus sajor-caju HSP100 complements a thermotolerance defect inhsp104 mutantSaccharomyces cerevisiae

A putative Hsp100 gene was cloned from the fungus Pleurotus sajor-caju. mRNA expression studies demonstrated that this gene (designated PsHsp100) is highly induced by high temperature,induced less strongly by exposure to ethanol, and not induced by drought or salinity. Heat shock induction is detectable at 37 degrees C and reaches a maximum level at 42 degrees C. PsHsp100 mRNA levels sharply increased within 15 min of exposure to high temperature, and reached a maximum expression level at 2 h that was maintained for several hours. These results indicate that PsHsp100 could work at an early step in thermotolerance. To examine its function, PsHsp100 was transformed into a temperature-sensitive hsp104 deletion mutant Saccharomycetes cerivisiae strain to test the hypothesis that PsHSP100 is an protein that functions in thermotolerance. Overexpression of PsHSP100 complemented the thermotolerance defect of the hsp104 mutant yeast, allowing them being survive even at 50 degree C for 4 h. These results indicate that PsHSP100 protein is functional as an HSP100 in yeast and could play and important role in thermotolerance in P. sajor-caju.

The Histidine Kinase Hik34 Is Involved in Thermotolerance by Regulating the Expression of Heat Shock Genes in Synechocystis

Histidine kinases (Hiks) in Synechocystis sp. PCC 6803 are involved in the transduction of signals associated with various kinds of environmental stress. To examine the potential role in thermotolerance of Hiks, we used genome microarray analysis to screen a Hik knockout library for mutations that affected the expression of genes for heat shock proteins. Mutation of the hik34 gene enhanced the levels of transcripts of a number of heat shock genes, including htpG and groESL1. Overexpression of the hik34 gene repressed the expression of these heat shock genes. In addition, the cells with a mutant gene for Hik34 (DeltaHik34 cells) survived incubation at 48 degrees C for 3 h, while wild-type cells and cells with mutations in other Hiks were killed. However, mutation of the hik34 gene had only an insignificant effect on the global expression of genes upon incubation of the mutant cells at 44 degrees C for 20 min. Quantitative two-dimensional gel electrophoresis revealed that levels of GroES and HspA were elevated in DeltaHik34 cells after incubation of cells at 42 degrees C for 60 min. We overexpressed recombinant Hik34 protein in Escherichia coli and purified it. We found that the protein was autophosphorylated in vitro at physiological temperatures, but not at elevated temperatures, such as 44 degrees C. These results suggest that Hik34 might negatively regulate the expression of certain heat shock genes that might be related to thermotolerance in Synechocystis.

Salicylic acid dependent signaling promotes basal thermotolerance but is not essential for acquired thermotolerance in Arabidopsis thaliana.

Salicylic acid (SA) is reported to protect plants from heat shock (HS), but insufficient is known about its role in thermotolerance or how this relates to SA signaling in pathogen resistance. We tested thermotolerance and expression of pathogenesis-related (PR) and HS proteins (HSPs) in Arabidopsis thaliana genotypes with modified SA signaling: plants with the SA hydroxylase NahG transgene, the nonexpresser of PR proteins (npr1) mutant, and the constitutive expressers of PR proteins (cpr1 and cpr5) mutants. At all growth stages from seeds to 3-week-old plants, we found evidence for SA-dependent signaling in basal thermotolerance (i.e. tolerance of HS without prior heat acclimation). Endogenous SA correlated with basal thermotolerance, with the SA-deficient NahG and SA-accumulating cpr5 genotypes having lowest and highest thermotolerance, respectively. SA promoted thermotolerance during the HS itself and subsequent recovery. Recovery from HS apparently involved an NPR1-dependent pathway but thermotolerance during HS did not. SA reduced electrolyte leakage, indicating that it induced membrane thermoprotection. PR-1 and Hsp17.6 were induced by SA or HS, indicating common factors in pathogen and HS responses. SA-induced Hsp17.6 expression had a different dose-response to PR-1 expression. HS-induced Hsp17.6 protein appeared more slowly in NahG. However, SA only partially induced HSPs. Hsp17.6 induction by HS was more substantial than by SA, and we found no SA effect on Hsp101 expression. All genotypes, including NahG and npr1, were capable of expression of HSPs and acquisition of HS tolerance by prior heat acclimation. Although SA promotes basal thermotolerance, it is not essential for acquired thermotolerance.

Maize HSP101 plays important roles in both induced and basal thermotolerance and primary root growth.

HSP101 belongs to the ClpB protein subfamily whose members promote the renaturation of protein aggregates and are essential for the induction of thermotolerance. We found that maize HSP101 accumulated in mature kernels in the absence of heat stress. At optimal temperatures, HSP101 disappeared within the first 3 days after imbibition, although its levels increased in response to heat shock. In embryonic cells, HSP101 concentrated in the nucleus and in some nucleoli. Hsp101 maps near the umc132 and npi280 markers on chromosome 6. Five maize hsp101-m-::Mu1 alleles were isolated. Mutants were null for HSP101 and defective in both induced and basal thermotolerance. Moreover, during the first 3 days after imbibition, primary roots grew faster in the mutants at optimal temperature. Thus, HSP101 is a nucleus-localized protein that, in addition to its role in thermotolerance, negatively influences the growth rate of the primary root. HSP101 is dispensable for proper embryo and whole plant development in the absence of heat stress.

Evolution of the response to heat shock in genus Drosophila

Thermotolerance was studied in a wide spectrum of Drosophila species and strains originating from different climatic zones and considerably differing from one another in the ambient temperature of their habitats. The species that lived in hot climate have a higher thermotolerance. Most species of the virilis group exhibited positive correlation between the HSP70 accumulation after heat exposure and thermotolerance; however, this correlation was absent in some species and strains. For example, the D. melanogaster Oregon R strain, which had the highest sensitivity to heat shock (HS) among all strains and species studied, displayed the maximum level of HSP70 proteins after HS. The patterns of induction of various heat shock protein (HSP) families after heat exposure in a wide spectrum of Drosophila species were compared. The results obtained suggest that the HSP40 and low-molecular-weight HSPs (lmwHSPs) play a significant role in thermotolerance and adaptation to hot climate. Polymorphism in hsp70 gene clusters of Drosophila and variation in the numbers of gene copies and hsp70 isoforms in group virilis were found. The evolutionary role of the variation in the number of hsp70 gene copies observed in the strains and species of genus Drosophila is discussed.

Hot plants, hot yeast

Members of the heat-shock protein 70 (Hsp70) family act as central players when vertebrates and flies acquire tolerance to otherwise-lethal temperatures. Bacteria and yeast, by contrast, use the Hsp100 family in this role. The Vierling 1 Hong S-W. Vierling E. Mutants of Arabidopsis thaliana defective in the acquisition of tolerance to high temperature stress. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4392-4397 Crossref Scopus (321) Google Scholar , 2 Queitsch C. et al. Heatshock protein 101 plays a crucial role in thermotolerance in Arabidopsis. Plant Cell. 2000; 12: 479-492 Google Scholar and Lindquist 2 Queitsch C. et al. Heatshock protein 101 plays a crucial role in thermotolerance in Arabidopsis. Plant Cell. 2000; 12: 479-492 Google Scholar labs now place plants squarely in the microbial camp. In a direct assault on the problem, Queitch et al.2 Queitsch C. et al. Heatshock protein 101 plays a crucial role in thermotolerance in Arabidopsis. Plant Cell. 2000; 12: 479-492 Google Scholar generated transgenic Arabidopsis plants that underexpressed (by antisense and co-suppression methods) or constitutively accumulated Hsp101. Low-expressing plants lost most of the capacity to acquire thermotolerance, whereas the constitutive lines exhibited high levels of basal, uninduced resistance to elevated temperatures. Furthermore, the antisense plants produced seeds lacking both developmentally regulated Hsp101 expression and the high level of basal thermotolerance characteristic of seeds and early germination. These changes in thermotolerance were the only detectable phenotypic consquences of the modulated Hsp101 levels.

Taking the Heat

Heat shock proteins (HSPs) are well-known mediators of the stress response in many organisms. Although heat shock proteins have been implicated in plant response to thermal stress, the role of specific heat shock proteins in mediating the response to thermotolerance is poorly defined. Basal thermotolerance is the ability to withstand a nonlethal heat stress; acquired thermotolerance is the ability to withstand a normally lethal heat stress by preconditioning with a milder heat stress. Queitsch et al. demonstrate that HSP101 is essential to mediating the response to both acquired and basal thermotolerance. Arabidopsis plants with decreased levels of HSP101 due to antisense inhibition or cosuppression did not exhibit acquired thermotolerance in whole-plant recovery assays or hypocotyl elongation assays. Furthermore, constitutive expression of HSP101 resulted in a growth advantage for plants subjected to heat shock such that plants overexpressing HSP101 recovered from 45-minute heat shock, but control plants did not. Germinating Arabidopsis seeds have a high basal thermotolerance that is lost if HSP101 expression is inhibited. Thus, HSP101 plays an important role in plant thermotolerance. W. Gurley highlights this work in a commentary. Queitsch, C., Hong, S.-W., Vierling, E., and Lindquist, S. (2000) Heat shock protein 101 plays a crucial role in thermotolerance in Arabidopsis. Plant Cell 12 : 479-492. [Abstract] [Full Text] Gurley, W.B. (2000) HSP101: A key component for the acquisition of thermotolerance in plants. Plant Cell 12 : 457-460. [Full Text]

Taking the Heat

Heat shock proteins (HSPs) are well-known mediators of the stress response in many organisms. Although heat shock proteins have been implicated in plant response to thermal stress, the role of specific heat shock proteins in mediating the response to thermotolerance is poorly defined. Basal thermotolerance is the ability to withstand a nonlethal heat stress; acquired thermotolerance is the ability to withstand a normally lethal heat stress by preconditioning with a milder heat stress. Queitsch et al. demonstrate that HSP101 is essential to mediating the response to both acquired and basal thermotolerance. Arabidopsis plants with decreased levels of HSP101 due to antisense inhibition or cosuppression did not exhibit acquired thermotolerance in whole-plant recovery assays or hypocotyl elongation assays. Furthermore, constitutive expression of HSP101 resulted in a growth advantage for plants subjected to heat shock such that plants overexpressing HSP101 recovered from 45-minute heat shock, but control plants did not. Germinating Arabidopsis seeds have a high basal thermotolerance that is lost if HSP101 expression is inhibited. Thus, HSP101 plays an important role in plant thermotolerance. W. Gurley highlights this work in a commentary.Queitsch, C., Hong, S.-W., Vierling, E., and Lindquist, S. (2000) Heat shock protein 101 plays a crucial role in thermotolerance in Arabidopsis. Plant Cell 12: 479-492. [Abstract] [Full Text]Gurley, W.B. (2000) HSP101: A key component for the acquisition of thermotolerance in plants. Plant Cell 12: 457-460. [Full Text]

Terpenes and the thermotolerance of photosynthesis

A walk through the forest is a good way to become aquainted with the variety of terpenes emitted by plants. The most fragrant of these are the monoterpenes, which lend a distinctive smell to trees (e.g. α‐ and β‐pinene from pines), mints (e.g. menthol from peppermint), fruits (e.g. limonene from citrus) and flowers (e.g. geraniol from roses). In addition to adding fragrance, phytogenic hydrocarbons play an important role in atmospheric chemistry. This was recognized as early as 1960 by Fritz Went who noted that blue hazes often formed above forested areas (Went, 1960). This idea was picked up by US President Ronald Reagan who accused trees of polluting the atmosphere, although in truth phytogenic hydrocarbons only contribute to pollution in air that is already contaminated by industry or automobile exhaust (Feshenfeld et al., 1992).Terpenes serve a variety of functions in plants including deterring herbivores and attracting pollinators. Until recently, the function of terpene emission from some groups of plants, which do not store the terpenes in their tissues, has been a mystery. Following the discovery that isoprene, a hemiterpene, increases the thermotolerance of photosynthesis in some species (Sharkey & Singsaas, 1995; Singsaas et al., 1997), Loreto et al. (1998) discovered that thermotolerance of the monoterpene‐emitting oak, Quercus ilex, increases when leaves are fumigated with monoterpenes. Now, a report by Delfine et al. in this issue (pp. 27–36) provides further support for the hypothesis that terpenes play a role in thermotolerance of leaves and suggests, intriguingly, that the mechanism by which this occurs is not limited to plants that make these compounds.

The RheA repressor is the thermosensor of the HSP18 heat shock response in Streptomyces albus

Microorganisms have mechanisms to sense their environment and rapidly adapt to survive changes in conditions. In Streptomyces albus, various transcriptional repressors mediate the induction of heat shock genes. The RheA repressor regulates the synthesis of HSP18, a small heat shock protein, which plays a role in thermotolerance. The RheA protein was purified to determine how it responds rapidly to temperature. Gel retardation assays and footprinting experiments identified the specific target of RheA as an inverted repeat (TGTCATC 5N GATGACA) located in Phsp18, PrheA which is the common promoter region of the divergon. Gel retardation assays detected RheA-complexes formed with the hsp18-rheA promoters. The complexes did not form at higher temperature. In vitro transcription experiments showed that RheA is an autoregulatory protein and that its activity is inhibited by high temperature. The temperature-induced derepression by RheA is reversible. Dichroism circular spectroscopy revealed a reversible change of RheA conformation in relation with the temperature that could represent a transition between an active and an inactive form. Our experiments demonstrate that RheA acts as a cellular thermometer in hsp18 regulation.

The RheA repressor is the thermosensor of the HSP18 heat shock response in Streptomyces albus.

Microorganisms have mechanisms to sense their environment and rapidly adapt to survive changes in conditions. In Streptomyces albus, various transcriptional repressors mediate the induction of heat shock genes. The RheA repressor regulates the synthesis of HSP18, a small heat shock protein, which plays a role in thermotolerance. The RheA protein was purified to determine how it responds rapidly to temperature. Gel retardation assays and footprinting experiments identified the specific target of RheA as an inverted repeat (TGTCATC 5N GATGACA) located in Phsp18, PrheA which is the common promoter region of the divergon. Gel retardation assays detected RheA-complexes formed with the hsp18-rheA promoters. The complexes did not form at higher temperature. In vitro transcription experiments showed that RheA is an autoregulatory protein and that its activity is inhibited by high temperature. The temperature-induced derepression by RheA is reversible. Dichroism circular spectroscopy revealed a reversible change of RheA conformation in relation with the temperature that could represent a transition between an active and an inactive form. Our experiments demonstrate that RheA acts as a cellular thermometer in hsp18 regulation.

Modified expression of a carrot small heat shock protein gene, hsp17. 7, results in increased or decreased thermotolerancedouble dagger

We have determined that one small heat shock protein gene, encoding Hsp17.7, plays an important role in the ability of carrot cells and plants to survive thermal stress. Transgenic cells and regenerated plants were generated in which the carrot Hsp17.7 gene was either constitutively expressed (denoted CaS lines) or expressed as a heat inducible antisense RNA (denoted AH lines). Thermotolerance measurements demonstrated that CaS lines were more thermotolerant than vector controls and AH antisense lines were less thermo- tolerant than vector controls. RNA analysis demonstrated that Hsp17. 7 mRNA was detectable, but not abundant, prior to heat shock in CaS cells, but not in vector control cells. Conversely, RNA analysis of antisense cells showed that, after heat shock, the amounts of mRNA for Hsp17.7 was moderately less abundant in AH cells than in vector controls. Analysis of protein synthesis in CaS cells did not indicate substantial synthesis or accumulation of Hsp17.7, or any small Hsp, at 23 degrees C. However, in the most thermotolerant line, protein synthesis was maintained at a higher rate than in other cell lines at a more extreme heat shock (42 degrees C). In contrast, antisense AH cells showed reduced synthesis of many Hsp, large and small. These results suggest that the Hsp17.7 gene plays a critical, although as yet not understood, role in thermotolerance in carrot. This represents the first demonstration of the ability to both increase and decrease thermotolerance by the manipulation of expression of a single gene.

PREHARVEST FIELD HEAT AND POSTHARVEST FRUIT RESPONSE

Postharvest heat treatments of fruit are being used for disinfestation of quarantine pests, and for reducing postharvest chilling injury in cold-sensitive fruits. Preharvest high temperatures of the fruit may influence tolerance to high and low postharvest temperatures. To assess the effect of field temperature history of fruit, we monitored temperatures in the flesh of apple and avocado fruit in the field over diurnal cycles, and followed the postharvest response of these fruit. In both fruits, flesh temperatures exceeded 35°C, and sometimes reached 40°C and above, for 2 h or more in direct sunlight, even though air temperatures did not exceed 30°C. With night temperatures dropping to 10°C or lower, the diurnal range sometimes exceeded 25°C. A ubiquitous response to high temperatures in all organisms is the induction of heat-shock proteins (hsp) genes and associated protein synthesis. Hsp play a role in thermotolerance. In both fruits, mRNA for hsp were upregulated at temperatures above about 33°C, and the transcripts remained at an elevated level over the cooler night period. With warming up of fruit, hsp's mRNA levels rapidly declined until temperatures reached inducible levels again. This induction of hsp in fruit in the field indicates that fruit, even under temperate conditions, respond daily to temperatures commonly regarded as being in the stress range. Postharvest responses associated with prior high temperature or exposure history of the fruit included exposed sides of apple fruit having higher flesh firmness, and previously exposed avocado fruit having greater resistance to low temperature injury, slower ripening rates, and higher thermotolerance. Both long-term exposure of fruit and immediate temperature history close to harvest, are likely to influence fruit responses to high temperature postharvest treatments and low temperature storage.

UspB, a new sigmaS-regulated gene in Escherichia coli which is required for stationary-phase resistance to ethanol.

The open reading frame immediately upstream of uspA is demonstrated to encode a 14-kDa protein which we named UspB (universal stress protein B) because of its general responsiveness to different starvation and stress conditions. UspB is predicted to be an integral membrane protein with at least one and perhaps two membrane-spanning domains. Overexpression of UspB causes cell death in stationary phase, whereas mutants of uspB are sensitive to exposure to ethanol but not heat in stationary phase. In contrast to uspA, stationary-phase induction of uspB requires the sigma factor sigmaS. The expression of uspB is modulated by H-NS, consistent with the role of H-NS in altering sigmaS levels. Our results demonstrate that a gene of the RpoS regulon is involved in the development of stationary-phase resistance to ethanol, in addition to the regulon's previously known role in thermotolerance, osmotolerance, and oxidative stress resistance.