Inducible Thermotolerance Research Articles

Increased <i>Arabidopsis thaliana</i> cell culture resistance to sodium fluoride by constitutive expression of HSP101

Fluorine is one of the toxic elemental components of industrial emissions. Increased fluoride content in the atmosphere, soil or water negatively affects the growth and development of plants, as well as reducing resistance to various environmental stressors. An increase in ambient temperature causes a protective response in all organisms taking the form heat shock protein synthesis. The specific protein HSP101, which performs the function of protecting plant cells from heat damage, is also responsible for inducible thermotolerance, representing the ability of organisms to withstand the effects of severe heat shock that were previously exposed to mild heat stress, as a result of which heat shock proteins are induced. Heat shock proteins are involved in protecting not only against elevated temperatures, but also various other stress factors. In this work, the effect of sodium fluoride treatment on the viability of Arabidopsis thaliana cell culture, expression and synthesis of heat shock proteins was studied along with the role of heat shock protein HSP101 in providing resistance to fluoride. Sodium fluoride has been shown to significantly reduce the viability of A. thaliana cells by suppressing the activation of HSP101 gene expression with an increase in temperature. At the same time, the A. thaliana line, which has constitutive expression of the HSP101 gene, proved to be more resistant to the toxic effects of sodium fluoride.

Acclimation of photosynthetic tolerance to acute heat stress at elevated CO2 and N

Determining interactive effects of pre-heat-stress, CO2 and N on photosynthetic thermotolerance is necessary for predicting plant responses to global change. We grew Hordeum vulgare (barley, C3) and Zea mays (corn, C4) at current or elevated CO2 (370 and 700ppm) and limiting or optimal soil N (0.5 and 7.5mM). We assessed basal and inducible thermotolerance of net photosynthesis (Pn), photosystem II efficiency (F′v/F′m), photochemical quenching (qp), carboxylation efficiency (CE), and rubisco activase content. Inducible thermotolerance was measured on plants which were pre-heat-stressed (PHS) for 4h before heat stress. We also assayed content of several major heat-shock proteins (HSPs), as HSPs are primary adaptations to heat stress and affected by N. Acclimation of photosynthetic thermotolerance was dependent on species, CO2 and N treatment and the component in the photosynthetic processes. PHS had a positive effect on the production of HSP60 and sHSP in low-N barley and corn. These results indicate that stimulatory effects of elevated CO2 at normal temperatures on photosynthesis may be partly changed by the different interactive effects of CO2, heat stress and N for species with different photosynthetic pathways. Thus, PHS, CO2 and N effects on photosynthetic thermotolerance may contribute to changes in plant productivity, distribution, and diversity.

Thermotolerance and HSP70 expression in the Mediterranean fruit fly Ceratitis capitata

The relationship between Hsp70 expression and thermotolerance has been well documented in Drosophila melanogaster. However, there is limited information on this relationship in other insect species. In this report we describe the Hsp70-thermotolerance relationship in one of the major fruit fly pests, Ceratitis capitata (medfly). Hsp70 expression and thermotolerance were assayed at a range of temperatures in several stages of medfly development. The most thermotolerant stage was found to be the late larval stage (100% survival at 41 °C) followed by adult flies and late embryos (100% survival at 39 °C). These three stages showed a positive relationship between Hsp70 expression and thermotolerance. Mid-larval and mid-embryonic stages were found less thermotolerant and the Hsp70-thermotolerance relationship was not evident. Early embryos did not express Hsp70 at any temperature and exhibited the lowest thermotolerance. The relationship between Hsp70 and inducible thermotolerance was also studied in late larvae. A pretreatment at 37–39 °C increased thermotolerance at higher temperatures by approximately 1 °C. In parallel, the pretreatment increased Hsp70 expression suggesting a close link between Hsp70 expression and inducible thermotolerance. The increased Hsp70 levels after pretreatment were found to be due to the increased levels of the hsp70 RNA.

Variation in Heat‐shock Proteins and Photosynthetic Thermotolerance among Natural Populations of Chenopodium album L. from Contrasting Thermal Environments: Implications for Plant Responses to Global Warming

Production of heat-shock proteins (Hsps) is a key adaptation to acute heat stress and will be important in determining plant responses to climate change. Further, intraspecifc variation in Hsps, which will influence species-level response to global warming, has rarely been examined in naturally occurring plants. To understand intraspecific variation in plant Hsps and its relevance to global warming, we examined Hsp content and thermotolerance in five naturally occurring populations of Chenopodium album L. from contrasting thermal environments grown at low and high temperatures. As expected, Hsp accumulation varied between populations, but this was related more to habitat variability than to mean temperature. Unexpectedly, Hsp accumulation decreased with increasing variability of habitat temperatures. Hsp accumulation also decreased with increased experimental growth temperatures. Physiological thermotolerance was partitioned into basal and induced components. As with Hsps, induced thermotolerance decreased with increasing temperature variability. Thus, populations native to the more stressful habitats, or grown at higher temperatures, had lower Hsp levels and induced thermotolerance, suggesting a greater reliance on basal mechanisms for thermotolerance. These results suggest that future global climate change will differentially impact ecotypes within species, possibly by selecting for increased basal versus inducible thermotolerance.

QTL for the thermotolerance effect of heat hardening, knockdown resistance to heat and chill‐coma recovery in an intercontinental set of recombinant inbred lines of Drosophila melanogaster

The thermotolerance effect of heat hardening (also called short-term acclimation), knockdown resistance to high temperature (KRHT) with and without heat hardening and chill-coma recovery (CCR) are important phenotypes of thermal adaptation in insects and other organisms. Drosophila melanogaster from Denmark and Australia were previously selected for low and high KRHT, respectively. These flies were crossed to construct recombinant inbred lines (RIL). KRHT was higher in heat-hardened than in nonhardened RIL. We quantify the heat-hardening effect (HHE) as the ratio in KRHT between heat-hardened and nonhardened RIL. Composite interval mapping revealed a more complex genetic architecture for KRHT without heat-hardening than for KRHT in heat-hardened insects. Five quantitative trait loci (QTL) were found for KRHT, but only two of them were significant after heat hardening. KRHT and CCR showed trade-off associations for QTL both in the middle of chromosome 2 and the right arm of chromosome 3, which should be the result of either pleiotropy or linkage. The major QTL on chromosome 2 explained 18% and 27-33% of the phenotypic variance in CCR and KRHT in nonhardened flies, respectively, but its KRHT effects decreased by heat hardening. We discuss candidate loci for each QTL. One HHE-QTL was found in the region of small heat-shock protein genes. However, HHE-QTL explained only a small fraction of the phenotypic variance. Most heat-resistance QTL did not colocalize with CCR-QTL. Large-effect QTL for CCR and KRHT without hardening (basal thermotolerance) were consistent across continents, with apparent transgressive segregation for CCR. HHE (inducible thermotolerance) was not regulated by large-effect QTL.

Inducible and constitutive heat shock gene expression responds to modification of Hsp70 copy number in Drosophila melanogaster but does not compensate for loss of thermotolerance in Hsp70null flies

BackgroundThe heat shock protein Hsp70 promotes inducible thermotolerance in nearly every organism examined to date. Hsp70 interacts with a network of other stress-response proteins, and dissecting the relative roles of these interactions in causing thermotolerance remains difficult. Here we examine the effect of Hsp70 gene copy number modification on thermotolerance and the expression of multiple stress-response genes in Drosophila melanogaster, to determine which genes may represent mechanisms of stress tolerance independent of Hsp70.ResultsHsp70 copy number in four strains is positively associated with Hsp70 expression and inducible thermotolerance of severe heat shock. When assayed at carefully chosen temperatures, Hsp70 null flies are almost entirely deficient in thermotolerance. In contrast to expectations, increasing Hsp70 expression levels induced by thermal pretreatment are associated with increasing levels of seven other inducible Hsps across strains. In addition, complete Hsp70 loss causes upregulation of the inducible Hsps and six constitutive stress-response genes following severe heat shocks.ConclusionModification of Hsp70 copy number quantitatively and qualitatively affects the expression of multiple other stress-response genes. A positive association between absolute expression levels of Hsp70 and other Hsps after thermal pretreatment suggests novel regulatory mechanisms. Severe heat shocks induce both novel gene expression patterns and almost total mortality in the Hsp70 null strain: alteration of gene expression in this strain does not compensate for Hsp70 loss but suggests candidates for overexpression studies.

Selection on knockdown performance inDrosophila melanogasterimpacts thermotolerance and heat-shock response differently in females and males

We studied adaptive thermotolerance in replicate populations of Drosophila melanogaster artificially selected for high and low knockdown temperature (T(KD)), the upper temperature at which flies can no longer remain upright or locomote effectively. Responses to selection have generated High T(KD) populations capable of maintaining locomotor function at approximately 40 degrees C, and Low T(KD) populations with T(KD) of approximately 35 degrees C. We examined inducible knockdown thermotolerance, as well as inducible thermal survivorship, following a pretreatment heat-shock (known to induce heat-shock proteins) for males and females from the T(KD) selected lines. Both selection for knockdown and sex influenced inducible knockdown thermotolerance, whereas inducible thermal survivorship was influenced only by sex, and not by selection. Overall, our findings suggest that the relationships between basal and inducible thermotolerance are contingent upon the methods used to gauge thermotolerance, as well as the sex of the flies. Finally, we compared temporal profiles of the combined expression of two major heat-shock proteins, HSC70 and HSP70, during heat stress among the females and males from the selected T(KD) lines. The temporal profiles of the proteins differed between High and Low T(KD) females, suggesting divergence of the heat-shock response. We discuss a possible mechanism that may lead to the heat-shock protein patterns observed in the selected females.

Remarkable Site Specificity of Local Transposition Into the Hsp70 Promoter of Drosophila melanogaster

Heat-shock genes have numerous features that ought to predispose them to insertional mutagenesis via transposition. To elucidate the evolvability of heat-shock genes via transposition, we have exploited a local transposition technique and Drosophila melanogaster strains with EPgy2 insertions near the Hsp70 gene cluster at 87A7 to produce numerous novel EPgy2 insertions into these Hsp70 genes. More than 50% of 45 independent insertions were made into two adjacent nucleotides in the proximal promoter at positions -96 and -97, and no insertions were into a coding or 3'-flanking sequence. All inserted transposons were in inverse orientation to the starting transposon. The frequent insertion into nucleotides -96 and -97 is consistent with the DNase hypersensitivity, absence of nucleosomes, flanking GAGA-factor-binding sites, and nucleotide sequence of this region. These experimental insertions recapitulated many of the phenotypes of natural transposition into Hsp70: reduced mRNA expression, less Hsp70 protein, and decreased inducible thermotolerance. The results suggest that the distinctive features of heat-shock promoters, which underlie the massive and rapid expression of heat-shock genes upon heat shock, also are a source of evolutionary variation on which natural selection can act.

Adaptive differentiation of thermotolerance in Drosophila along a microclimatic gradient

We examined whether a remarkable occurrence - the physiological evolution of two Drosophila melanogaster populations, despite a spatial separation of only 100-400 m, was idiosyncratic and temporary, or persisted over multiple years. We ascertained the high-temperature tolerance of Drosophila descended from populations on the north-facing slope (NFS) and south-facing slope (SFS) of 'Evolution Canyon' (Lower Nahal Oren, Mt Carmel, Israel), which were collected in 1997, 1999, and 2000. Results for these Drosophila uniformly resembled other studies in many respects: an inverse relationship between survival and heat-shock temperature, male-female differences in thermotolerance, and inducible thermotolerance. Importantly, for all years of collection, SFS flies consistently exceeded NFS flies in basal and inducible thermotolerance after diverse heat shocks, with and without thermal pretreatment, and whether isofemale lines, synthetic populations, or inbred lines were compared. Inbred lines, however, had lower thermotolerance than outbred lines. Several nonexclusive processes may explain the evolution of such physiological differentiation.

Naturally Occurring Transposable Elements Disrupt hsp70 Promoter Function in Drosophila melanogaster

Naturally occurring transposable element (TE) insertions that disrupt Drosophila promoters are correlated with modified promoter function and are posited to play a significant role in regulatory evolution, but their phenotypes have not been established directly. To establish the functional consequences of these TE insertions, we created constructs with either TE-bearing or TE-lacking hsp70 promoters fused to a luciferase reporter gene and assayed luciferase luminescence in transiently transfected Drosophila cells. Each of the four TEs reduces luciferase signal after heat shock and heat inducibility of the hsp70 promoter. To test if the differences in hsp70 promoter activity are TE-sequence dependent, we replaced each of the TEs with multiple intergenic sequences of equal length. These replacement insertions similarly reduced luciferase signal, suggesting that the TEs affect hsp70 promoter function by altering promoter architecture. These results are consistent with differences in Hsp70 expression levels, inducible thermotolerance, and fecundity previously associated with the TEs. That two different varieties of TEs in two different hsp70 genes have common effects suggests that TE insertion represents a general mechanism through which selection manipulates hsp70 gene expression.

EVOLUTION OF HSP90 EXPRESSION IN TETRAHYMENA THERMOPHILA (PROTOZOA, CILIATA) POPULATIONS EXPOSED TO THERMALLY VARIABLE ENVIRONMENTS

Evolutionary consequences of thermally varying environments were studied in the ciliated protozoan Tetrahymena thermophila. Replicated lines were propagated for 60 days, a maximum of 500 generations, in stable, slowly fluctuating (red spectrum), and rapidly fluctuating (blue spectrum) temperatures. The red and blue fluctuations had a dominant period length of 15 days and two hours, respectively. The mean temperature of all time series was 25°C and the fluctuating temperatures had the same minimum (10°C), maximum (40°C), and variance. During the experiment, population sizes and biomasses were monitored at three-day intervals. After the experiment, carrying capacity and maximum growth rate were measured at low (15°C), intermediate (25°C), and high (35°C) temperatures for each experimental line. Physiological changes in the lines were assessed by measuring the expression of stress-induced heat shock protein Hsp90 at 25°C, 35°C, and 39°C. Population sizes and biomasses showed no differences between stable, blue, or red temperature treatments during the experiment. Also, after the experiment, mean carrying capacities and maximum growth rates were comparable in the stable, blue, and red temperature treatments. The expression of Hsp90 was higher in lines from the blue environment than in lines from the stable environment. Lines from the red environment had an intermediate level of Hsp90 expression. This supports the hypothesis that inducible thermotolerance and expression of canalizing genes can evolve in response to rapidly varying environments. Furthermore, we found correlative evidence of benefits and disadvantages of high Hsp90 expression. Lines with high expression of Hsp90 had an increased growth rate at the highest temperature when food resources were not limiting growth. At low and intermediate temperatures the same lines had the lowest carrying capacities.

Evolutionary changes in heat-inducible gene expression in lines of Escherichia coli adapted to high temperature.

The involvement of heat-inducible genes, including the heat-shock genes, in the acute response to temperature stress is well established. However, their importance in genetic adaptation to long-term temperature stress is less clear. Here we use high-density arrays to examine changes in expression for 35 heat-inducible genes in three independent lines of Escherichia coli that evolved at high temperature (41.5 degrees C) for 2,000 generations. These lines exhibited significant changes in heat-inducible gene expression relative to their ancestor, including parallel changes in fkpA, gapA, and hslT. As a group, the heat-inducible genes were significantly more likely than noncandidate genes to have evolved changes in expression. Genes encoding molecular chaperones and ATP-dependent proteases, key components of the cytoplasmic stress response, exhibit relatively little expression change; whereas genes with periplasmic functions exhibit significant expression changes suggesting a key role for the extracytoplasmic stress response in the adaptation to high temperature. Following acclimation at 41.5 degrees C, two of the three lines exhibited significantly improved survival at 50 degrees C, indicating changes in inducible thermotolerance. Thus evolution at high temperature led to significant changes at the molecular level in heat-inducible gene expression and at the organismal level in inducible thermotolerance and fitness.

Modification of heat-shock gene expression in Drosophila melanogaster populations via transposable elements.

We report multiple cases in which disruption of hsp70 regulatory regions by transposable element (TE) insertions underlies natural variation in expression of the stress-inducible molecular chaperone Hsp70 in Drosophila melanogaster. Three D. melanogaster populations from different continents are polymorphic for jockey or P element insertions in the promoter of the hsp70Ba gene. All three TE insertions are within the same 87-bp region of hsp70Ba promoter, and we suggest that the distinctive promoter architecture of hsp genes may make them vulnerable to TE insertions. Each of the TE insertions reduces Hsp70 levels, and RNase protection assays demonstrate that such insertions can reduce transcription of the hsp70Ba gene. In addition, the TEs alter two measures of organismal fitness, inducible thermotolerance and female reproductive success. Thus, transposition can create quantitative genetic variation in gene expression within populations, on which natural selection can act.

Evolvability of Hsp70 expression under artificial election for inducible thermotolerance in independent populations of Drosophila melanogaster.

To test whether expression of the inducible heat-shock protein Hsp70 increases under selection for inducible thermotolerance in Drosophila melanogaster, we performed artificial selection on replicate sets of Drosophila lines founded from two independent populations. Selection entailed pretreatment at 36 degrees C to induce thermotolerance and Hsp70 expression, followed by a more severe heat shock, whose temperature varied between sexes and among generations to achieve 50% mortality. Inducible thermotolerance increased slowly and continuously in selected lines and was 37%-50% greater than in controls after 10-11 generations. Lines founded from the two populations differed in their coevolution of Hsp70 expression. In lines founded from Evolution Canyon, Israel, Hsp70 level initially increased and thereafter was unchanged; replicate lines exhibited two temporal patterns of response to selection. In lines founded from Australia, Hsp70 levels increased throughout selection. In both cases, however, the increase in Hsp70 level averaged only 15%, suggesting that pleiotropy in Hsp70 function constrains evolutionary increase in its expression.

Hsp 27 is better associated with the expression of inducible thermotolerance in human pancreatic tumor cell lines than hsp 70, p53 or p21/waf1/cip1

We evaluated the levels of p53, p21/waf1/cip1, hsp 27 and hsp 70 proteins in confluent cultures of human pancreatic tumor cell lines during the expression of inducible thermotolerance (IT). Two of these cell lines have wild type, whereas the other two have mutant, p53 status. Three cell lines expressed a significant amount of IT ( p<0.05 and better, one-tailed Student's t-test); the fourth manifested little IT. Hsp 70 was upregulated in all cell lines. In contrast, hsp 27 was upregulated only in the cell lines that expressed IT. There was no correlation between either p53, or p21, protein levels and propensity to express IT. We conclude that hsp 27 is better associated with the expression of IT than hsp 70 in these cell lines.

A Drosophila melanogaster strain from sub-equatorial Africa has exceptional thermotolerance but decreased Hsp70 expression.

Drosophila melanogaster collected in sub-equatorial Africa in the 1970s are remarkably tolerant of sustained laboratory culture above 30 degrees C and of acute exposure to much warmer temperatures. Inducible thermotolerance of high temperatures, which in Drosophila melanogaster is due in part to the inducible molecular chaperone Hsp70, is only modest in this strain. Expression of Hsp70 protein and hsp70 mRNA is likewise reduced and has slower kinetics in this strain (T) than in a standard wild-type strain (Oregon R). These strains also differed in constitutive and heat-inducible levels of other molecular chaperones. The lower Hsp70 expression in the T strain apparently has no basis in the activation of the heat-shock transcription factor HSF, which is similar in T and Oregon R flies. Rather, the reduced expression may stem from insertion of two transposable elements, H.M.S. Beagle in the intergenic region of the 87A7 hsp70 gene cluster and Jockey in the hsp70Ba gene promoter. We hypothesize that the reduced Hsp70 expression in a Drosophila melanogaster strain living chronically at intermediate temperatures may represent an evolved suppression of the deleterious phenotypes of Hsp70.

Changing fitness consequences of hsp70 copy number in transgenic Drosophila larvae undergoing natural thermal stress

Abstract1. Transgenic manipulation of the gene copy number of hsp70, which encodes the major inducible heat‐shock protein of Drosophila melanogaster (Hsp70), affects both Hsp70 levels and inducible thermotolerance in the laboratory; here parallel effects in transgenic Drosophila larvae undergoing natural or simulated natural thermal stress are demonstrated.2. Necrotic fruit was infested with larvae of either of two transgenic strains, one transformed with 12 extra copies of the hsp70 gene (extra‐copy strain) and a sister strain possessing only the wild‐type number (10) of hsp70 genes (excision strain), and then allowed to heat to variable extents.3. As the intensity of thermal stress increased, the consequences of extra hsp70 copies reversed. After no or moderate thermal stress, excision larvae survived better than did extra copy larvae. By contrast, extra copy larvae tolerated intense hyperthermia better than did excision larvae.4. These results establish that the Hsp70‐mediated enhancement of stress tolerance, previously demonstrated only for artificial stress regimes in the laboratory, extends to natural stress regimes.5. Mortality due to overexpression of Hsp70, however, also increases under mild natural stress regimes, buttressing the ecological relevance of a hypothesized evolutionary trade‐off of the benefits and adverse consequences of Hsp70 expression.

Evolution of Senescence: Longevity and the Expression of Heat Shock Proteins

Senescence is the progressive deterioration of organismal function leading to accelerating rates of mortality. Cumulative extrinsic and intrinsic stresses are thought to contribute to senescence. Molecular chaperones, such as heat shock proteins, are hypothesized to modulate senescence through their ability to mitigate protein damage. Recent discoveries made with the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster lend strong support to this theory. Longevity extending mutants of the nematode also increase intrinsic and inducible thermotolerance, and they overexpress heat shock proteins upon thermal shock. Intriguingly, these genes regulate dauer (diapause) formation, and are associated with an insulin-like dependent signal transduction pathway. Direct evidence for a casual role of hsp70 in aging is provided by analysis of transgenic fruit flies. When hsp70 is induced by mild heat shock, flies that overexpress the protein have greatly reduced mortality rates during subsequent weeks of aging at normal temperatures. Current work with fruit flies focuses on the relationship between insulin-like receptors, ovarian diapause, heat shock and aging.

Engineering Candidate Genes in Studies of Adaptation: The Heat-Shock Protein Hsp70 in Drosophila melanogaster.

One approach to elucidating the relationships among genes, traits, performance, and fitness is to choose genes of candidate evolutionary interest whose significance is well understood from in vitro and cellular studies and to explore the consequences of manipulating these genes in whole organisms in an evolutionary context. Ongoing research on Hsp70 in Drosophila exemplifies this candidate gene approach. The heat-shock protein Hsp70 was chosen for analysis because its biochemical phenotype (i.e., molecular chaperone activity), regulation, and encoding genes are well understood. Investigation of the thermal environment of Drosophila larvae in the wild and natural Hsp70 expression establishes an ecological context for the candidate gene. Engineering of the hsp70 genes via site-specific homologous recombination, expression off of a heterologous promoter, and expression of an innocuous protein off of the hsp70 promoter show that Hsp70 can be sufficient for a significant component of inducible thermotolerance but can have deleterious consequences. Study of nonengineered variation in Hsp70 expression reveals that the genetically engineered effects have counterparts in nature. Thus, in complementary fashion, both genetic engineering and the more classical approaches of evolutionary biology each contribute essential insights to adaptation.

Engineering Candidate Genes in Studies of Adaptation: The Heat-Shock Protein Hsp70 in Drosophila melanogaster

One approach to elucidating the relationships among genes, traits, performance, and fitness is to choose genes of candidate evolutionary interest whose significance is well understood from in vitro and cellular studies and to explore the consequences of manipulating these genes in whole organisms in an evolutionary context. Ongoing research on Hsp70 in Drosophila exemplifies this candidate gene approach. The heat‐shock protein Hsp70 was chosen for analysis because its biochemical phenotype (i.e., molecular chaperone activity), regulation, and encoding genes are well understood. Investigation of the thermal environment of Drosophila larvae in the wild and natural Hsp70 expression establishes an ecological context for the candidate gene. Engineering of the hsp70 genes via site‐specific homologous recombination, expression off of a heterologous promoter, and expression of an innocuous protein off of the hsp70 promoter show that Hsp70 can be sufficient for a significant component of inducible thermotolerance but can have deleterious consequences. Study of nonengineered variation in Hsp70 expression reveals that the genetically engineered effects have counterparts in nature. Thus, in complementary fashion, both genetic engineering and the more classical approaches of evolutionary biology each contribute essential insights to adaptation.