Oxidative stress, arising from an imbalance in the generation and removal of reactive oxygen species (ROS), is a challenge faced by all aerobic organisms. In plants, different pathways sense ROS from extracellular sources or organelles such as mitochondria, chloroplast or peroxisome. In our recent paper on Plant Molecular Biology1 we have studied the Arabidopsis thaliana early response to the generation of superoxide anion in chloroplasts during active photosynthesis. Transcript profile analysis revealed that the expression level of various genes encoding heat shock proteins (Hsps), increased after a short term of oxidative stress treatment. Furthermore, there was an induction of heat shock transcription factors HsfA2 and HsfA4A that were reported to be regulators of genes involved in stress response of Arabidopsis.1,2
In this addendum, we complement the expression analysis of two Hsp genes encoding Hsp70 and a 17.6 kDa class I small heat-shock protein (sHsp), and discuss their plausible role during oxidative stress, considering our data and other recently published papers.
heat shock factor; heat shock protein; chloroplast; oxidative stress; signalling
The heat stress (HS) response in eukaryotes is mainly regulated by heat shock factors (HSFs). Genetic disruption of the master HSF gene leads to dramatically reduced HS response and thermotolerance in several model organisms. However, it is not clear whether organisms devoid of the master regulator can still acclimate to heat. Previously, we showed that Arabidopsis HsfA1a, HsfA1b, and HsfA1d act as master regulators in the HS response. In this study, we examined the heat acclimation capacity of the Arabidopsis quadruple and triple T-DNA knockout mutants of HsfA1a, HsfA1b, HsfA1d, and HsfA1e. Our data showed that in the absence of the master regulators, a minimal but significant level of acquired thermotolerance could be attained in the Arabidopsis mutants after acclimation. The optimum acclimation temperature for the HsfA1 quadruple mutant was lower than that for the wild type plants, suggesting that plant cells have two HS-sensing mechanisms that can be distinguished genetically. The acquired thermotolerance of the quadruple mutant was likely due to the induction of a small number of HsfA1-independent HS response genes regulated by other transcription factors. Here, we discuss the possible candidates and propose a working model of the transcription network of the HS response by including the HsfA1-dependent and -independent pathways.
Arabidopis; heat shock factor; bZIP28; ER stress; acquired thermotolerance
So far little is known on the functional role of phosphorylation in the heat stress response of plants. Here we present evidence that heat stress activates the Arabidopsis mitogen-activated protein kinase MPK6. In vitro and in vivo evidence is provided that MPK6 specifically targets the major heat stress transcription factor HsfA2. Activation of MPK6 results in complex formation with HsfA2. MPK6 phosphorylates HsfA2 on T249 and changes its intracellular localisation. Protein kinase and phosphatase inhibitor studies indicate that HsfA2 protein stability is regulated in a phosphorylation-dependent manner, but this mechanism is independent of MPK6. Overall, our data show that heat stress-induced targeting of HsfA2 by MPK6 participates in the complex regulatory mechanism how plants respond to heat stress.
MAP kinase; Heat stress proteins; Heat stress transcription factors; Kinases; Arabidopsis
The class A heat shock factors HsfA1a and HsfA1b are highly conserved, interacting regulators, responsible for the immediate-early transcription of a subset of heat shock genes in Arabidopsis. In order to determine functional cooperation between them, we used a reporter assay based on transient over-expression in Arabidopsis protoplasts. Reporter plasmids containing promoters of Hsf target genes fused with the GFP coding region were co-transformed with Hsf effector plasmids. The GFP reporter gene activity was quantified using flow cytometry. Three of the tested target gene promoters (Hsp25.3, Hsp18.1-CI, Hsp26.5) resulted in a strong reporter gene activity, with HsfA1a or HsfA1b alone, and significantly enhanced GFP fluorescence when both effectors were co-transformed. A second set of heat shock promoters (HsfA2, Hsp17.6CII, Hsp17.6C-CI) was activated to much lower levels. These data suggest that HsfA1a/1b cooperate synergistically at a number of target gene promoters. These targets are also regulated via the late HsfA2, which is the most strongly heat-induced class A-Hsf in Arabidopsis. HsfA2 has also the capacity to interact with HsfA1a and HsfA1b as determined by bimolecular fluorescence complementation (BiFC) in Arabidopsis protoplasts and yeast-two-hybrid assay. However, there was no synergistic effect on Hsp18.1-CI promoter-GFP reporter gene expression when HsfA2 was co-expressed with either HsfA1a or HsfA1b. These data provide evidence that interaction between early and late HSF is possible, but only interaction between the early Hsfs results in a synergistic enhancement of expression of certain target genes. The interaction of HsfA1a/A1b with the major-late HsfA2 may possibly support recruitment of HsfA2 and replacement of HsfA1a/A1b at the same target gene promoters.
Electronic supplementary material
The online version of this article (doi:10.1007/s11103-010-9643-2) contains supplementary material, which is available to authorized users.
Heat shock transcription factor; Oligomerization domain; BiFC; Flow cytometry; Yeast-two-hybrid interaction; Protoplast transformation
The high sensitivity of male reproductive cells to high temperatures may be due to an inadequate heat stress response. The results of a comprehensive expression analysis of HsfA2 and Hsp17-CII, two important members of the heat stress system, in the developing anthers of a heat-tolerant tomato genotype are reported here. A transcriptional analysis at different developmental anther/pollen stages was performed using semi-quantitative and real-time PCR. The messengers were localized using in situ RNA hybridization, and protein accumulation was monitored using immunoblot analysis. Based on the analysis of the gene and protein expression profiles, HsfA2 and Hsp17-CII are finely regulated during anther development and are further induced under both short and prolonged heat stress conditions. These data suggest that HsfA2 may be directly involved in the activation of protection mechanisms in the tomato anther during heat stress and, thereby, may contribute to tomato fruit set under adverse temperatures.
Anther development; heat stress; HsfA2; Hsp17-CII; pollen; tomato
Heat-stressed crops suffer dehydration, depressed growth, and a consequent decline in water productivity, which is the yield of harvestable product as a function of lifetime water consumption and is a trait associated with plant growth and development. Heat shock transcription factor (HSF) genes have been implicated not only in thermotolerance but also in plant growth and development, and therefore could influence water productivity. Here it is demonstrated that Arabidopsis thaliana plants with increased HSFA1b expression showed increased water productivity and harvest index under water-replete and water-limiting conditions. In non-stressed HSFA1b-overexpressing (HSFA1bOx) plants, 509 genes showed altered expression, and these genes were not over-represented for development-associated genes but were for response to biotic stress. This confirmed an additional role for HSFA1b in maintaining basal disease resistance, which was stress hormone independent but involved H2O2 signalling. Fifty-five of the 509 genes harbour a variant of the heat shock element (HSE) in their promoters, here named HSE1b. Chromatin immunoprecipitation-PCR confirmed binding of HSFA1b to HSE1b in vivo, including in seven transcription factor genes. One of these is MULTIPROTEIN BRIDGING FACTOR1c (MBF1c). Plants overexpressing MBF1c showed enhanced basal resistance but not water productivity, thus partially phenocopying HSFA1bOx plants. A comparison of genes responsive to HSFA1b and MBF1c overexpression revealed a common group, none of which harbours a HSE1b motif. From this example, it is suggested that HSFA1b directly regulates 55 HSE1b-containing genes, which control the remaining 454 genes, collectively accounting for the stress defence and developmental phenotypes of HSFA1bOx.
Arabidopsis thaliana; basal resistance; biotic and abiotic stress; Brassica napus; drought stress; heat stress; Hyaloperonospora parasitica; hydrogen peroxide; Pseudomonas syringae; transcription factors; water productivity.
In heat-stressed (HS) tomato (Lycopersicon peruvianum) cell cultures, the constitutively expressed HS transcription factor HsfA1 is complemented by two HS-inducible forms, HsfA2 and HsfB1. Because of its stability, HsfA2 accumulates to fairly high levels in the course of a prolonged HS and recovery regimen. Using immunofluorescence and cell fractionation experiments, we identified three states of HsfA2: (i) a soluble, cytoplasmic form in preinduced cultures maintained at 25°C, (ii) a salt-resistant, nuclear form found in HS cells, and (iii) a stored form of HsfA2 in cytoplasmic HS granules. The efficient nuclear transport of HsfA2 evidently requires interaction with HsfA1. When expressed in tobacco protoplasts by use of a transient-expression system, HsfA2 is mainly retained in the cytoplasm unless it is coexpressed with HsfA1. The essential parts for the interaction and nuclear cotransport of the two Hsfs are the homologous oligomerization domain (HR-A/B region of the A-type Hsfs) and functional nuclear localization signal motifs of both partners. Direct physical interaction of the two Hsfs with formation of relatively stabile hetero-oligomers was shown by a two-hybrid test in Saccharomyces cerevisiae as well as by coimmunoprecipitation using tomato and tobacco whole-cell lysates.
Based on the partial or complete sequences of 14 plant heat stress transcription factors (Hsfs) from tomato, soybean, Arabidopsis and maize we propose a general nomenclature with two basic classes, i.e. classes A and B each containing two or more types of Hsfs (HsfA1, HsfA2 etc.). Despite some plant-specific peculiarities, essential functional domains and modules of these proteins are conserved among plants, yeast, Drosophila and vertebrates. A revised terminology of these parts follows recommendations agreed upon among the authors and representatives from other laboratories working in this field (see legend to Fig.1). Similar to the situation with the small heat shock proteins (sHsps), the complexity of the hsf gene family in plants appears to be higher than in other eukaryotic organisms.
Tomato heat stress transcription factor HsfA2 is a shuttling protein with dominant cytoplasmic localization as a result of a nuclear import combined with an efficient export. Besides the nuclear localization signal (NLS) adjacent to the oligomerization domain, a C-terminal leucine-rich motif functions as a nuclear export signal (NES). Mutant forms of HsfA2 with a defective or an absent NES are nuclear proteins. The same is true for the wild-type HsfA2 if coexpressed with HsfA1 or in the presence of export inhibitor leptomycin B (LMB). Fusion of the NES domain of HsfA2 to HsfB1, which is a nuclear protein, caused export of the HsfB1-A2NES hybrid protein, and this effect was reversed by the addition of LMB. Due to the lack of background problems, Chinese hamster ovary (CHO) cells represent an excellent system for expression and functional analysis of tomato Hsfs. The results faithfully reflect the situation found in plant cells (tobacco protoplasts). The intriguing role of NLS and NES accessibility for the intracellular distribution of HsfA2 is underlined by the results of heat stress treatments of CHO cells (41°C). Despite the fact that nuclear import and export are not markedly affected, HsfA2 remains completely cytoplasmic at 41°C even in the presence of LMB. The temperature-dependent conformational transition of HsfA2 with shielding of the NLS evidently needs intramolecular interaction between the internal HR-A/B and the C-terminal HR-C regions. It is not observed with the HR oligomerization domain (HR-A/B region) deletion form of HsfA2 or in HsfA2-HsfA1 hetero-oligomers.
Background and Aims
Molecular-based studies of thermotolerance have rarely been performed on wild plants, although this trait is critical for summer survival. Here, we focused on thermotolerance and expression of heat shock transcription factor A2 (HSFA2) and its putative target gene (chloroplast-localized small heat shock protein, CP-sHSP) in two allied aquatic species of the genus Potamogeton (pondweeds) that differ in survival on land.
The degree of thermotolerance was examined using a chlorophyll bioassay to assess heat injury in plants cultivated under non- and heat-acclimation conditions. Potamogeton HSFA2 and CP-sHSP genes were identified and their heat-induction was quantified by real-time PCR.
The inhibition of chlorophyll accumulation after heat stress showed that Potamogeton malaianus had a higher basal thermotolerance and developed acquired thermotolerance, whereas Potamogeton perfoliatus was heat sensitive and unable to acquire thermotolerance. We found two duplicated HSFA2 and CP-sHSP genes in each species. These genes were induced by heat shock in P. malaianus, while one HSFA2a gene was not induced in P. perfoliatus. In non-heat-acclimated plants, transcript levels of HSFA2 and CP-sHSP were transiently elevated after heat shock. In heat-acclimated plants, transcripts were continuously induced during sublethal heat shock in P. malaianus, but not in P. perfoliatus. Instead, the minimum threshold temperature for heat induction of the CP-sHSP genes was elevated in P. perfoliatus.
Our comparative study of thermotolerance showed that heat acclimation leads to species-specific changes in heat response. The development of acquired thermotolerance is beneficial for survival at extreme temperatures. However, the loss of acquired thermotolerance and plasticity in the minimum threshold temperature of heat response may be favourable for plants growing in moderate habitats with limited daily and seasonal temperature fluctuations.
Acquired thermotolerance; chloroplast-localized small heat shock protein (CP-sHSP); gene duplication; heat stress; heat acclimation; heterophylly; geographical distribution; heat shock transcription factor A2 (HSFA2); minimum threshold temperature; pondweed; Potamogeton
In plants, salicylic acid (SA) is a signalling molecule regulating disease resistance responses such as systemic acquired resistance (SAR) and the hypersensitive response (HR), and has been implicated in both basal and acquired thermotolerance. It has been shown that SA enhances heat-induced Hsp/Hsc70 accumulation in plants. To investigate the mechanism of how SA influences the heat shock response (HSR) in plants, tomato seedlings were treated with SA alone, heat shock, or a combination of both before analyses of hsp70 mRNA, heat shock factor (Hsf)–DNA binding, and gene expression of hsp70, hsfA1, hsfA2, and hsfB1. SA alone led to activation of Hsf–DNA binding, but not induction or transcription of hsp70 mRNA. SA had no significant effect on hsfA2 and hsfB1 gene expression, but potentiated the basal levels of hsfA1. In heat-shocked plants, Hsf–DNA binding was established, and increased hsfA1, hsfA2, and hsfB1 expression was followed by accumulation of Hsp70. SA plus heat shock showed enhanced Hsf–DNA binding, enhanced induction of hsp70 mRNA transcription, and gene expression of hsfA1, hsfA2, and hsfB1, resulting in potentiated levels of Hsp/Hsc70. Since increased hsp70 and hsf gene expression coincide with increased levels of Hsp70 accumulation, it is concluded that SA-mediated potentiation of Hsp70 is due to modulation of these Hsfs by SA. In our efforts to understand the role of Hsp70 in heat-related disease susceptibility, the degree of the complexity of the cross-talk between the pathways in which SA is involved, inter alia, the plant defence response, the HSR and thermotolerance, was further underscored.
Heat shock; heat shock factors; HsfA1; HsfA2; HsfB1; Hsf–DNA binding; Hsp70; salicylic acid; tomato
The heat-shock response, a fundamental defense mechanism against proteotoxic stress, is regulated by a family of heat-shock transcription factors (HSF). In humans HSF1 is considered the central regulator of heat-induced transcriptional responses. The main targets for HSF1 are specific promoter elements (HSE) located upstream of heat-shock genes encoding cytoprotective heat-shock proteins (HSP) with chaperone function. In addition to its cytoprotective function, HSF1 was recently hypothesized to play a more complex role, regulating the expression of non-HSP genes; however, the non-canonical role of HSF1 is still poorly understood. Herein we report that heat-stress promotes the expression of cyclooxygenase-2 (COX-2), a key regulator of inflammation controlling prostanoid and thromboxane synthesis, resulting in the production of high levels of prostaglandin-E2 in human cells. We show that heat-induced COX-2 expression is regulated at the transcriptional level via HSF1-mediated signaling and identify, by in-vitro reporter gene activity assay and deletion-mutant constructs analysis, the COX-2 heat-responsive promoter region and a new distal cis-acting HSE located at position −2495 from the transcription start site. As shown by ChIP analysis, HSF1 is recruited to the COX-2 promoter rapidly after heat treatment; by using shRNA-mediated HSF1 suppression and HSE-deletion from the COX-2 promoter, we demonstrate that HSF1 plays a central role in the transcriptional control of COX-2 by heat. Finally, COX-2 transcription is also induced at febrile temperatures in endothelial cells, suggesting that HSF1-dependent COX-2 expression could contribute to increasing blood prostaglandin levels during fever. The results identify COX-2 as a human non-classical heat-responsive gene, unveiling a new aspect of HSF1 function.
Organisms respond to circumstances threatening the cellular protein homeostasis by activation of heat-shock transcription factors (HSFs), which play important roles in stress resistance, development, and longevity. Of the four HSFs in vertebrates (HSF1-4), HSF1 is activated by stress, whereas HSF2 lacks intrinsic stress responsiveness. The mechanism by which HSF2 is recruited to stress-inducible promoters and how HSF2 is activated is not known. However, changes in the HSF2 expression occur, coinciding with the functions of HSF2 in development. Here, we demonstrate that HSF1 and HSF2 form heterotrimers when bound to satellite III DNA in nuclear stress bodies, subnuclear structures in which HSF1 induces transcription. By depleting HSF2, we show that HSF1-HSF2 heterotrimerization is a mechanism regulating transcription. Upon stress, HSF2 DNA binding is HSF1 dependent. Intriguingly, when the elevated expression of HSF2 during development is mimicked, HSF2 binds to DNA and becomes transcriptionally competent. HSF2 activation leads to activation of also HSF1, revealing a functional interdependency that is mediated through the conserved trimerization domains of these factors. We propose that heterotrimerization of HSF1 and HSF2 integrates transcriptional activation in response to distinct stress and developmental stimuli.
The heat shock transcription factors (Hsfs) activate the stress-inducible expression of heat shock proteins (Hsps) and other molecular chaperones in response to stress and, therefore, play an essential role in protein disaggregation and protein folding. In humans, missense mutation in the hsf4 gene causes cataract, and mice bearing a targeted disruption of the hsf4 gene exhibit defects in lens fiber cell differentiation and early cataract formation. Here, we show that Hsf4b is a direct target of the mitogen-activated protein (MAP) kinase extracellular signal-related kinase (ERK) and that phosphorylation of Hsf4b by ERK leads to increased ability of Hsf4b to bind DNA. Surprisingly, Hsf4b also interacts with an ERK-specific dual-specificity tyrosine phosphatase named DUSP26 identified from a yeast two-hybrid screen. While activated ERK phosphorylates Hsf4b, DUSP26 controls the activity of ERK, leading to phosphorylation/dephosphorylation of Hsf4b, altering its ability to bind DNA. Therefore, DUSP26 interaction with Hsf4b places this transcription factor within a regulatory circuit in the MAP kinase signaling pathway.
A genetic program that in sunflower seeds is activated by Heat Shock transcription Factor A9 (HaHSFA9) has been analyzed in transgenic tobacco seedlings. The ectopic overexpression of the HSFA9 program protected photosynthetic membranes, which resisted extreme dehydration and oxidative stress conditions. In contrast, heat acclimation of seedlings induced thermotolerance but not resistance to the harsh stress conditions employed. The HSFA9 program was found to include the expression of plastidial small Heat Shock Proteins that accumulate only at lower abundance in heat-stressed vegetative organs. Photosystem II (PSII) maximum quantum yield was higher for transgenic seedlings than for non-transgenic seedlings, after either stress treatment. Furthermore, protection of both PSII and Photosystem I (PSI) membrane protein complexes was observed in the transgenic seedlings, leading to their survival after the stress treatments. It was also shown that the plastidial D1 protein, a labile component of the PSII reaction center, and the PSI core protein PsaB were shielded from oxidative damage and degradation. We infer that natural expression of the HSFA9 program during embryogenesis may protect seed pro-plastids from developmental desiccation.
Heat stress transcription factors (Hsfs) are the central regulators of defense response to heat stress. We identified a total of 25 rice Hsf genes by genome-wide analysis of rice (Oryza sativa L.) genome, including the subspecies of O. japonica and O. indica. Proteins encoded by OsHsfs were divided into three classes according to their structures. Digital Northern analysis showed that OsHsfs were expressed constitutively. The expressions of these OsHsfs in response to heat stress and oxidative stress differed among the members of the gene family. Promoter analysis identified a number of stress-related cis-elements in the promoter regions of these OsHsfs. No significant correlation, however, was found between the heat-shock responses of genes and their cis-elements. Overall, our results provide a foundation for future research of OsHsfs function.
Heat shock; Transcription factors; Rice; Protein structure; Expression analysis
Diverse cellular and environmental stresses can activate the heat shock response, an evolutionarily conserved mechanism to protect proteins from denaturation. Stressors activate heat shock transcription factor 1 (HSF1), which binds to heat shock elements in the genes for heat shock proteins, leading to rapid induction of these important molecular chaperones. Both heat and noise stress are known to activate the heat shock response in the cochlea and protect it from subsequent noise trauma. However, the contribution of HSF1 to induction of heat shock proteins following noise trauma has not been investigated at the molecular level. We evaluated the role of HSF1 in the cochlea following noise stress by examining induction of heat shock proteins in Hsf1+/− control and Hsf1−/− mice. Heat stress rapidly induced expression of Hsp25, Hsp47, Hsp70.1, Hsp70.3, Hsp84, Hsp86, and Hsp110 in the cochleae of wild-type and Hsf1+/− mice, but not in Hsf1−/− mice, confirming the essential role of HSF1 in mediating the heat shock response. Exposure to broadband noise (2–20 kHz) at 106 dB SPL for 2 h produced partial hearing loss. Maximal induction of heat shock proteins occurred 4 h after the noise. In comparison to heat stress, noise stress resulted in lower induced levels of Hsp25, Hsp70.1, Hsp70.3, Hsp86, and Hsp110 in Hsf1+/− mice. Induction of these heat shock proteins was attenuated, but not completely eliminated, in Hsf1−/− mice. These same noise exposure conditions induced genes for several immediate early transcription factors and maximum induction occurred earlier than for heat shock proteins. Thus, additional signaling pathways and transcriptional regulators that are activated by noise probably contribute to induction of heat shock proteins in the cochlea.
quantitative RT-PCR; heat shock proteins; noise-induced hearing loss; ABR
Heat shock transcription factor (Hsf)–1 and Hsf2 are members of the heat shock factor (HSF) protein family involved in heat shock protein (hsp) gene regulation, a regulation that is critical for the ability of cells to survive exposure to stress conditions. Although the role of Hsf1 in binding and activating transcription of hsp gene promoters in response to cell stress is well established, how Hsf2 enhances stress-induced hsp expression is not understood. To gain an insight into the critical conserved features of the regulation and function of Hsf2, we have identified and characterized the Hsf2 protein from Xenopus laevis. We found that, similar to its human counterpart, Xenopus Hsf2 is sumoylated at lysine 82 and that, as it does in human Hsf2, the modification event of the small ubiquitin–related modifier 1 functions to increase the deoxyribonucleic acid–binding activity of this transcription factor in Xenopus. These results indicate that sumoylation is an evolutionarily conserved modification of Hsf2 proteins, supporting the position of this modification as a critical regulator of Hsf2 function.
Inhibition of proteasome-mediated protein degradation machinery is a potent stress stimulus that causes accumulation of ubiquitinated proteins and increased expression of heat shock proteins (Hsps). Hsps play pivotal roles in homeostasis and protection in a cell, through their well-recognized properties as molecular chaperones. The inducible Hsp expression is regulated by the heat shock transcription factors (HSFs). Among mammalian HSFs, HSF1 has been shown to be important for regulation of the heat-induced stress gene expression, whereas the function of HSF2 in stress response is unclear. Recent reports have suggested that both HSF1 and HSF2 are affected during down-regulation of ubiquitin-proteasome pathway (Y. Kawazoe et al., Eur. J. Biochem. 255:356–362, 1998; A. Mathew et al., Mol. Cell. Biol. 18:5091–5098, 1998; D. Kim et al., Biochem. Biophys. Res. Commun. 254:264–268, 1999). To date, however, no unambiguous evidence has been presented as to whether a single specific HSF or multiple members of the HSF family are required for transcriptional induction of heat shock genes when proteasome activity is down-regulated. Therefore, by using loss-of-function and gain-of-function strategies, we investigated the specific roles of mammalian HSFs in regulation of the ubiquitin-proteasome-mediated stress response. Here we demonstrate that HSF1, but not HSF2, is essential and sufficient for up-regulation of Hsp70 expression during down-regulation of the ubiquitin proteolytic pathway. We propose that specificity of HSF1 could be an important therapeutic target during disease pathogenesis associated with abnormal ubiquitin-dependent proteasome function.
Appropriate responses of organisms to heat stress are essential for their survival. In eukaryotes, adaptation to high temperatures is mediated by heat shock transcription factors (HSFs). HSFs regulate the expression of heat shock proteins, which function as molecular chaperones assisting in protein folding and stability. In many model organisms a great deal is known about the products of hsf genes. An important exception is the filamentous fungus and model eukaryote Neurospora crassa. Here we show that two Neurospora crassa genes whose protein products share similarity to known HSFs play different biological roles. We report that heat shock factor 1 (hsf1) is an essential gene and that hsf2 is required for asexual development. Conidiation may be blocked in the hsf2 knockout (hsf2KO) strain because HSF2 is an integral element of the conidiation pathway or because it affects the availability of protein chaperones. We report that genes expressed during conidiation, for example fluffy, conidiation-10, and repressor of conidiation-1 show wild-type levels of expression in a hsf2KO strain. However, consistent with the lack of macroconidium development, levels of eas are much reduced. Cultures of the hsf2KO strain along with two other aconidial strains, the fluffy and aconidial-2 strains, took longer than the wild type to recover from heat shock. Altered expression profiles of hsp90 and a putative hsp90-associated protein in the hsf2KO strain after exposure to heat shock may in part account for its reduced ability to cope with heat stress.
Heat shock transcription factor (HSF) and the promoter heat shock element (HSE) are among the most highly conserved transcriptional regulatory elements in nature. HSF mediates the transcriptional response of eukaryotic cells to heat, infection and inflammation, pharmacological agents, and other stresses. While HSF is essential for cell viability in Saccharomyces cerevisiae, oogenesis and early development in Drosophila melanogaster, extended life span in Caenorhabditis elegans, and extraembryonic development and stress resistance in mammals, little is known about its full range of biological target genes. We used whole-genome analyses to identify virtually all of the direct transcriptional targets of yeast HSF, representing nearly 3% of the genomic loci. The majority of the identified loci are heat-inducibly bound by yeast HSF, and the target genes encode proteins that have a broad range of biological functions including protein folding and degradation, energy generation, protein trafficking, maintenance of cell integrity, small molecule transport, cell signaling, and transcription. This genome-wide identification of HSF target genes provides novel insights into the role of HSF in growth, development, disease, and aging and in the complex metabolic reprogramming that occurs in all cells in response to stress.
The heat shock transcription factor (HSF) is a conserved regulator of heat shock-inducible gene expression. Organismal roles for HSF in physiological processes such as development, aging, and immunity have been defined largely through studies of the single C. elegans HSF homolog, hsf-1. However, the molecular and cell biological properties of hsf-1 in C. elegans are incompletely understood. We generated animals expressing physiological levels of an HSF-1::GFP fusion protein and examined its function, localization, and regulation in vivo. HSF-1::GFP was functional as measured by its ability to rescue phenotypes associated with two hsf-1 mutant alleles. Rescue of hsf-1 stress, aging, and development phenotypes was abolished in a DNA-binding-deficient mutant, demonstrating that the transcriptional targets of hsf-1 are critical to its function even in the absence of stress. Under non-stress conditions, HSF-1::GFP was found primarily in the nucleus. Following heat shock, HSF-1::GFP rapidly and reversibly redistributed into dynamic, sub-nuclear structures that share many properties with human nuclear stress granules, including colocalization with markers of active transcription. Rapid formation of HSF-1 stress granules required HSF-1 DNA binding activity and the threshold for stress granule formation was altered by growth temperature. HSF-1 stress granule formation was not induced by inhibition of IGF signaling, a pathway previously suggested to function upstream of hsf-1. Our findings suggest that development, stress, and aging pathways may regulate HSF-1 function in distinct ways, and that HSF-1 nuclear stress granule formation is an evolutionarily conserved aspect of HSF-1 regulation in vivo.
Heat shock factor; aging; longevity
Heat shock (HS) leads to the activation of molecular mechanisms, known as HS-response, that prevent damage and enhance survival under stress. Plants have a flexible and specialized network of Heat Shock Factors (HSFs), which are transcription factors that induce the expression of heat shock proteins. The present work aimed to identify and characterize the Glycine max HSF repertory in the Soybean Genome Project (GENOSOJA platform), comparing them with other legumes (Medicago truncatula and Lotus japonicus) in view of current knowledge of Arabidopsis thaliana. The HSF characterization in leguminous plants led to the identification of 25, 19 and 21 candidate ESTs in soybean, Lotus and Medicago, respectively. A search in the SuperSAGE libraries revealed 68 tags distributed in seven HSF gene types. From the total number of obtained tags, more than 70% were related to root tissues (water deficit stress libraries vs. controls), indicating their role in abiotic stress responses, since the root is the first tissue to sense and respond to abiotic stress. Moreover, as heat stress is related to the pressure of dryness, a higher HSF expression was expected at the water deficit libraries. On the other hand, expressive HSF candidates were obtained from the library inoculated with Asian Soybean Rust, inferring crosstalk among genes associated with abiotic and biotic stresses. Evolutionary relationships among sequences were consistent with different HSF classes and subclasses. Expression profiling indicated that regulation of specific genes is associated with the stage of plant development and also with stimuli from other abiotic stresses pointing to the maintenance of HSF expression at a basal level in soybean, favoring its activation under heat-stress conditions.
HSF; Fabaceae; bioinformatics; abiotic stress; transcription factor
The cellular heat stress response is well studied in Drosophila in respect to the role of heat shock proteins (Hsp). Hsps are molecular chaperones, highly expressed during and after exposure to numerous stress types. Hsps are all regulated by a common transcription factor, the heat shock factor (HSF), and it is known that HSF is controlling other, so far uncharacterised, heat-responsive genes. In this study, we investigate whether novel candidate genes for heat resistance, identified by microarray experiments, are regulated by HSF. The microarray experiments recently identified several strongly upregulated genes in response to a short, non-lethal heat treatment in Drosophila melanogaster. To test whether or not a subset of these genes are HSF-induced, we studied 11 currently unannotated genes using quantitative polymerase chain reaction on HSF mutant flies with a non-functional HSF at elevated temperatures. We found indication of HSF regulation in most of the studied genes, suggesting a role of these unknown genes in heat tolerance. Surprisingly, some of the genes seemed to be upregulated independent of HSF function. The high induction in response to heat, which mimics the expression profile of Hsps, implies a role in the cellular heat response of these genes as well.
Above-optimal temperatures reduce yield in tomato largely because of the high heat stress (HS) sensitivity of the developing pollen grains. The high temperature response, especially at this most HS-sensitive stage of the plant, is poorly understood. To obtain an overview of molecular mechanisms underlying the HS response (HSR) of microspores, a detailed transcriptomic analysis of heat-stressed maturing tomato microspores was carried out using a combination of Affymetrix Tomato Genome Array and cDNA-amplified fragment length polymorphism (AFLP) techniques. The results were corroborated by reverse transcription-PCR (RT-PCR) and immunoblot analyses. The data obtained reveal the involvement of specific members of the small heat shock protein (HSP) gene family, HSP70 and HSP90, in addition to the HS transcription factors A2 (HSFA2) and HSFA3, as well as factors other than the classical HS-responsive genes. The results also indicate HS regulation of reactive oxygen species (ROS) scavengers, sugars, plant hormones, and regulatory genes that were previously implicated in other types of stress. The use of cDNA-AFLP enabled the detection of genes representing pollen-specific functions that are missing from the tomato Affymetrix chip, such as those involved in vesicle-mediated transport and a pollen-specific, calcium-dependent protein kinase (CDPK2). For several genes, including LeHSFA2, LeHSP17.4-CII, as well as homologues of LeHSP90 and AtVAMP725, higher basal expression levels were detected in microspores of cv. Hazera 3042 (a heat-tolerant cultivar) compared with microspores of cv. Hazera 3017 (a heat-sensitive cultivar), marking these genes as candidates for taking part in microspore thermotolerance. This work provides a comprehensive analysis of the molecular events underlying the HSR of maturing microspores of a crop plant, tomato.
cDNA-AFLP; gene expression; heat stress response; microarray; microspore maturation; tomato