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1.  Promoter specificity and interactions between early and late Arabidopsis heat shock factors 
Plant Molecular Biology  2010;73(4-5):559-567.
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.
PMCID: PMC2882041  PMID: 20458611
Heat shock transcription factor; Oligomerization domain; BiFC; Flow cytometry; Yeast-two-hybrid interaction; Protoplast transformation
2.  The Balance of Nuclear Import and Export Determines the Intracellular Distribution and Function of Tomato Heat Stress Transcription Factor HsfA2 
Molecular and Cellular Biology  2001;21(5):1759-1768.
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.
PMCID: PMC86729  PMID: 11238913
3.  Modulation of heat shock factors accompanies salicylic acid-mediated potentiation of Hsp70 in tomato seedlings 
Journal of Experimental Botany  2008;59(8):2125-2132.
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.
PMCID: PMC2413278  PMID: 18468986
Heat shock; heat shock factors; HsfA1; HsfA2; HsfB1; Hsf–DNA binding; Hsp70; salicylic acid; tomato
4.  Acquired thermotolerance independent of heat shock factor A1 (HsfA1), the master regulator of the heat stress response 
Plant Signaling & Behavior  2012;7(5):547-550.
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.
PMCID: PMC3419016  PMID: 22516818
Arabidopis; heat shock factor; bZIP28; ER stress; acquired thermotolerance
5.  Comparative studies of thermotolerance: different modes of heat acclimation between tolerant and intolerant aquatic plants of the genus Potamogeton 
Annals of Botany  2011;109(2):443-452.
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.
Key Results
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.
PMCID: PMC3268545  PMID: 22147547
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
6.  A Seed Preferential Heat Shock Transcription Factor from Wheat Provides Abiotic Stress Tolerance and Yield Enhancement in Transgenic Arabidopsis under Heat Stress Environment 
PLoS ONE  2013;8(11):e79577.
Reduction in crop yield and quality due to various abiotic stresses is a worldwide phenomenon. In the present investigation, a heat shock factor (HSF) gene expressing preferentially in developing seed tissues of wheat grown under high temperatures was cloned. This newly identified heat shock factor possesses the characteristic domains of class A type plant HSFs and shows high similarity to rice OsHsfA2d, hence named as TaHsfA2d. The transcription factor activity of TaHsfA2d was confirmed through transactivation assay in yeast. Transgenic Arabidopsis plants overexpressing TaHsfA2d not only possess higher tolerance towards high temperature but also showed considerable tolerance to salinity and drought stresses, they also showed higher yield and biomass accumulation under constant heat stress conditions. Analysis of putative target genes of AtHSFA2 through quantitative RT-PCR showed higher and constitutive expression of several abiotic stress responsive genes in transgenic Arabidopsis plants over-expressing TaHsfA2d. Under stress conditions, TaHsfA2d can also functionally complement the T-DNA insertion mutants of AtHsfA2, although partially. These observations suggest that TaHsfA2d may be useful in molecular breeding of crop plants, especially wheat, to improve yield under abiotic stress conditions.
PMCID: PMC3827158  PMID: 24265778
7.  Investigating the role of plant heat shock proteins during oxidative stress 
Plant Signaling & Behavior  2008;3(10):856-857.
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.
PMCID: PMC2634396  PMID: 19704521
heat shock factor; heat shock protein; chloroplast; oxidative stress; signalling
8.  Heat shock factor HSFB2a involved in gametophyte development of Arabidopsis thaliana and its expression is controlled by a heat-inducible long non-coding antisense RNA 
Plant Molecular Biology  2014;85(6):541-550.
Heat stress transcription factors (HSFs) are central regulators of the heat stress response. Plant HSFs of subgroup B lack a conserved sequence motif present in the transcriptional activation domain of class A-HSFs. Arabidopsis members were found to be involved in non-heat shock functions. In the present analysis we investigated the expression, regulation and function of HSFB2a. HSFB2a expression was counteracted by a natural long non-coding antisense RNA, asHSFB2a. In leaves, the antisense RNA gene is only expressed after heat stress and dependent on the activity of HSFA1a/HSFA1b. HSFB2a and asHSFB2a RNAs were also present in the absence of heat stress in the female gametophyte. Transgenic overexpression of HSFB2a resulted in a complete knock down of the asHSFB2a expression. Conversely, asHSFB2a overexpression leads to the absence of HSFB2a RNA. The knockdown of HSFB2a by asHSFB2a correlated with an improved, knockdown of asHSFB2a by HSFB2a overexpression with an impaired biomass production early in vegetative development. In both cases the development of female gametophytes was impaired. A T-DNA knock-out line did not segregate homozygous mutant plants, only heterozygots hsfB2a-tt1/+ were viable. Approximately 50 % of the female gametophytes were arrested in early development, before mitosis 3, resulting in 45 % of sterile ovules. Our analysis indicates that the “Yin–Yang” regulation of gene expression at the HSFB2a locus influences vegetative and gametophytic development in Arabidopsis.
Electronic supplementary material
The online version of this article (doi:10.1007/s11103-014-0202-0) contains supplementary material, which is available to authorized users.
PMCID: PMC4099531  PMID: 24874772
HSFB2a; Heat shock factor; Gametophyte development; Antisense RNA; Arabidopsis thaliana
9.  Arabidopsis HEAT SHOCK TRANSCRIPTION FACTORA1b overexpression enhances water productivity, resistance to drought, and infection 
Journal of Experimental Botany  2013;64(11):3467-3481.
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.
PMCID: PMC3733161  PMID: 23828547
Arabidopsis thaliana; basal resistance; biotic and abiotic stress; Brassica napus; drought stress; heat stress; Hyaloperonospora parasitica; hydrogen peroxide; Pseudomonas syringae; transcription factors; water productivity.
10.  Developmental and heat stress-regulated expression of HsfA2 and small heat shock proteins in tomato anthers 
Journal of Experimental Botany  2009;61(2):453-462.
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.
PMCID: PMC2803211  PMID: 19854799
Anther development; heat stress; HsfA2; Hsp17-CII; pollen; tomato
11.  Regulation of the heat stress response in Arabidopsis by MPK6-targeted phosphorylation of the heat stress factor HsfA2 
PeerJ  2013;1:e59.
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.
PMCID: PMC3628891  PMID: 23638397
MAP kinase; Heat stress proteins; Heat stress transcription factors; Kinases; Arabidopsis
12.  Cytosolic heat shock protein 90 regulates heat shock transcription factor in Arabidopsis thaliana 
Plant Signaling & Behavior  2008;3(9):660-662.
Plant survival requires the ability to acclimate to heat, which is involves the expression of heat-inducible genes. We found cytosolic heat shock protein (HSP) 90 serves as a negative regulator of heat shock transcription factor (HSF), which is responsible for the induction of heat-inducible genes in plant. Transient inhibition of HSP90 induces heat-inducible genes and heat acclimation in Arabidopsis thaliana seedlings. Most of upregulated genes by heat shock and HSP90 inhibitor treatments carry heat shock response element (HSE) in their promoter, which suggests that HSF participates in the response to HSP90 inhibition. A. thaliana HSP90.2 interacts with AtHsfA1d, which is one of the constitutively expressed HSFs in A. thaliana. Heat shock depleted cytosolic HSP90 activity, as shown by the activity of exogenously expressed glucocorticoid receptor (GR), which is a substrate of cytosolic HSP90. Thus, it appears that in the absence of heat shock, cytosolic HSP90 negatively regulates HsfA1. Upon heat shock, cytosolic HSP90 is transiently inactivated, and this may lead to the activation of HsfA1.
PMCID: PMC2634549  PMID: 19704818
HSP90; heat shock transcription factor; heat shock; heat shock response element; heat acclimation; geldanamycin; radicicol
13.  Heat Shock Factors HsfB1 and HsfB2b Are Involved in the Regulation of Pdf1.2 Expression and Pathogen Resistance in Arabidopsis 
Molecular Plant  2009;2(1):152-165.
In order to assess the functional roles of heat stress-induced class B-heat shock factors in Arabidopsis, we investigated T-DNA knockout mutants of AtHsfB1 and AtHsfB2b. Micorarray analysis of double knockout hsfB1/hsfB2b plants revealed as strong an up-regulation of the basal mRNA-levels of the defensin genes Pdf1.2a/b in mutant plants. The Pdf expression was further enhanced by jasmonic acid treatment or infection with the necrotrophic fungus Alternaria brassicicola. The single mutant hsfB2b and the double mutant hsfB1/B2b were significantly improved in disease resistance after A. brassicicola infection. There was no indication for a direct interaction of Hsf with the promoter of Pdf1.2, which is devoid of perfect HSE consensus Hsf-binding sequences. However, changes in the formation of late HsfA2-dependent HSE binding were detected in hsfB1/B2b plants. This suggests that HsfB1/B2b may interact with class A-Hsf in regulating the shut-off of the heat shock response. The identification of Pdf genes as targets of Hsf-dependent negative regulation is the first evidence for an interconnection of Hsf in the regulation of biotic and abiotic responses.
PMCID: PMC2639743  PMID: 19529832
Abiotic/environmental stress; transcriptional control and transcription factors; transcriptome analysis; defense responses; disease resistance; Arabidopsis
14.  Transcription factor OsHsfC1b regulates salt tolerance and development in Oryza sativa ssp. japonica 
AoB Plants  2012;2012:pls011.
The paper describes the functional analysis of a class C heat shock transcription factor from rice (Oryza sativa). OsHsfC1b is shown to play a role in ABA-mediated salt stress tolerance and is required for plant growth under non-stress conditions.
Background and aims
Salt stress leads to attenuated growth and productivity in rice. Transcription factors like heat shock factors (HSFs) represent central regulators of stress adaptation. Heat shock factors of the classes A and B are well established as regulators of thermal and non-thermal stress responses in plants; however, the role of class C HSFs is unknown. Here we characterized the function of the OsHsfC1b (Os01g53220) transcription factor from rice.
We analysed the expression of OsHsfC1b in the rice japonica cultivars Dongjin and Nipponbare exposed to salt stress as well as after mannitol, abscisic acid (ABA) and H2O2 treatment. For functional characterization of OsHsfC1b, we analysed the physiological response of a T-DNA insertion line (hsfc1b) and two artificial micro-RNA (amiRNA) knock-down lines to salt, mannitol and ABA treatment. In addition, we quantified the expression of small Heat Shock Protein (sHSP) genes and those related to signalling and ion homeostasis by quantitative real-time polymerase chain reaction in roots exposed to salt. The subcellular localization of OsHsfC1b protein fused to green fluorescent protein (GFP) was determined in Arabidopsis mesophyll cell protoplasts.
Principal results
Expression of OsHsfC1b was induced by salt, mannitol and ABA, but not by H2O2. Impaired function of OsHsfC1b in the hsfc1b mutant and the amiRNA lines led to decreased salt and osmotic stress tolerance, increased sensitivity to ABA, and temporal misregulation of salt-responsive genes involved in signalling and ion homeostasis. Furthermore, sHSP genes showed enhanced expression in knock-down plants under salt stress. We observed retarded growth of hsfc1b and knock-down lines in comparison with control plants under non-stress conditions. Transient expression of OsHsfC1b fused to GFP in protoplasts revealed nuclear localization of the transcription factor.
OsHsfC1b plays a role in ABA-mediated salt stress tolerance in rice. Furthermore, OsHsfC1b is involved in the response to osmotic stress and is required for plant growth under non-stress conditions.
PMCID: PMC3357053  PMID: 22616023
15.  Co-overexpression of two Heat Shock Factors results in enhanced seed longevity and in synergistic effects on seedling tolerance to severe dehydration and oxidative stress 
BMC Plant Biology  2014;14:56.
We have previously reported that the seed-specific overexpression of sunflower (Helianthus annuus L.) Heat Shock Factor A9 (HaHSFA9) enhanced seed longevity in transgenic tobacco (Nicotiana tabacum L.). In addition, the overexpression of HaHSFA9 in vegetative organs conferred tolerance to drastic levels of dehydration and oxidative stress.
Here we found that the combined overexpression of sunflower Heat Shock Factor A4a (HaHSFA4a) and HaHSFA9 enhanced all the previously reported phenotypes described for the overexpression of HaHSFA9 alone. The improved phenotypes occurred in coincidence with only subtle changes in the accumulation of small Heat Shock Proteins (sHSP) that are encoded by genes activated by HaHSFA9. The single overexpression of HaHSFA4a in vegetative organs (which lack endogenous HSFA9 proteins) did not induce sHSP accumulation under control growth conditions; neither it conferred thermotolerance. The overexpression of HaHSFA4a alone also failed to induce tolerance to severe abiotic stress. Thus, a synergistic functional effect of both factors was evident in seedlings.
Our study revealed that HaHSFA4a requires HaHSFA9 for in planta function. Our results strongly support the involvement of HaHSFA4a and HaHSFA9 in transcriptional co-activation of a genetic program of longevity and desiccation tolerance in sunflower seeds. These results would also have potential application for improving seed longevity and tolerance to severe stress in vegetative organs.
PMCID: PMC4081658  PMID: 24593798
Combined overexpression; Drastic oxidative stress; Enhanced seed longevity; Heat Shock Factors; Severe dehydration; Stress tolerance; Transgenic tobacco
16.  The heat shock factor family from Triticum aestivum in response to heat and other major abiotic stresses and their role in regulation of heat shock protein genes 
Journal of Experimental Botany  2013;65(2):539-557.
Heat shock factors (Hsfs) play a central regulatory role in acquired thermotolerance. To understand the role of the major molecular players in wheat adaptation to heat stress, the Hsf family was investigated in Triticum aestivum. Bioinformatic and phylogenetic analyses identified 56 TaHsf members, which are classified into A, B, and C classes. Many TaHsfs were constitutively expressed. Subclass A6 members were predominantly expressed in the endosperm under non-stress conditions. Upon heat stress, the transcript levels of A2 and A6 members became the dominant Hsfs, suggesting an important regulatory role during heat stress. Many TaHsfA members as well as B1, C1, and C2 members were also up-regulated during drought and salt stresses. The heat-induced expression profiles of many heat shock protein (Hsp) genes were paralleled by those of A2 and A6 members. Transactivation analysis revealed that in addition to TaHsfA members (A2b and A4e), overexpression of TaHsfC2a activated expression of TaHsp promoter-driven reporter genes under non-stress conditions, while TaHsfB1b and TaHsfC1b did not. Functional heat shock elements (HSEs) interacting with TaHsfA2b were identified in four TaHsp promoters. Promoter mutagenesis analysis demonstrated that an atypical HSE (GAACATTTTGGAA) in the TaHsp17 promoter is functional for heat-inducible expression and transactivation by Hsf proteins. The transactivation of Hsp promoter-driven reporter genes by TaHsfC2a also relied on the presence of HSE. An activation motif in the C-terminal domain of TaHsfC2a was identified by amino residue substitution analysis. These data demonstrate the role of HsfA and HsfC2 in regulation of Hsp genes in wheat.
PMCID: PMC3904712  PMID: 24323502
Gene expression; gene regulation; heat shock factors; heat shock proteins; heat stress; transcription factors; wheat.
17.  Genome-wide analysis of the Hsf family in soybean and functional identification of GmHsf-34 involvement in drought and heat stresses 
BMC Genomics  2014;15(1):1009.
High temperature affects organism growth and metabolic activity. Heat shock transcription factors (Hsfs) are key regulators in heat shock response in eukaryotes and prokaryotes. Under high temperature conditions, Hsfs activate heat shock proteins (Hsps) by combining with heat stress elements (HSEs) in their promoters, leading to defense of heat stress. Since the first plant Hsf gene was identified in tomato, several plant Hsf family genes have been thoroughly characterized. Although soybean (Glycine max), an important oilseed crops, genome sequences have been available, the Hsf family genes in soybean have not been characterized accurately.
We analyzed the Hsf genetic structures and protein function domains using the GSDS, Pfam, SMART, PredictNLS, and NetNES online tools. The genome scanning of dicots (soybean and Arabidopsis) and monocots (rice and maize) revealed that the whole-genome replication occurred twice in soybean evolution. The plant Hsfs were classified into 3 classes and 16 subclasses according to protein structure domains. The A8 and B3 subclasses existed only in dicots and the A9 and C2 occurred only in monocots. Thirty eight soybean Hsfs were systematically identified and grouped into 3 classes and 12 subclasses, and located on 15 soybean chromosomes. The promoter regions of the soybean Hsfs contained cis-elements that likely participate in drought, low temperature, and ABA stress responses. There were large differences among Hsfs based on transcriptional levels under the stress conditions. The transcriptional levels of the A1 and A2 subclass genes were extraordinarily high. In addition, differences in the expression levels occurred for each gene in the different organs and at the different developmental stages. Several genes were chosen to determine their subcellular localizations and functions. The subcellular localization results revealed that GmHsf-04, GmHsf-33, and GmHsf-34 were located in the nucleus. Overexpression of the GmHsf-34 gene improved the tolerances to drought and heat stresses in Arabidopsis plants.
This present investigation of the quantity, structural features, expression characteristics, subcellular localizations, and functional roles provides a scientific basis for further research on soybean Hsf functions.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-1009) contains supplementary material, which is available to authorized users.
PMCID: PMC4253008  PMID: 25416131
Hsfs; Genome-wide identification; Expression pattern; Subcellular localization; Functional identification; Soybean
18.  Transcriptional profiling of Arabidopsis heat shock proteins and transcription factors reveals extensive overlap between heat and non-heat stress response pathways 
BMC Genomics  2007;8:125.
The heat shock response of Arabidopsis thaliana is dependent upon a complex regulatory network involving twenty-one known transcription factors and four heat shock protein families. It is known that heat shock proteins (Hsps) and transcription factors (Hsfs) are involved in cellular response to various forms of stress besides heat. However, the role of Hsps and Hsfs under cold and non-thermal stress conditions is not well understood, and it is unclear which types of stress interact least and most strongly with Hsp and Hsf response pathways. To address this issue, we have analyzed transcriptional response profiles of Arabidopsis Hsfs and Hsps to a range of abiotic and biotic stress treatments (heat, cold, osmotic stress, salt, drought, genotoxic stress, ultraviolet light, oxidative stress, wounding, and pathogen infection) in both above and below-ground plant tissues.
All stress treatments interact with Hsf and Hsp response pathways to varying extents, suggesting considerable cross-talk between heat and non-heat stress regulatory networks. In general, Hsf and Hsp expression was strongly induced by heat, cold, salt, and osmotic stress, while other types of stress exhibited family or tissue-specific response patterns. With respect to the Hsp20 protein family, for instance, large expression responses occurred under all types of stress, with striking similarity among expression response profiles. Several genes belonging to the Hsp20, Hsp70 and Hsp100 families were specifically upregulated twelve hours after wounding in root tissue, and exhibited a parallel expression response pattern during recovery from heat stress. Among all Hsf and Hsp families, large expression responses occurred under ultraviolet-B light stress in aerial tissue (shoots) but not subterranean tissue (roots).
Our findings show that Hsf and Hsp family member genes represent an interaction point between multiple stress response pathways, and therefore warrant functional analysis under conditions apart from heat shock treatment. In addition, our analysis revealed several family and tissue-specific heat shock gene expression patterns that have not been previously described. These results have implications regarding the molecular basis of cross-tolerance in plant species, and raise new questions to be pursued in future experimental studies of the Arabidopsis heat shock response network.
PMCID: PMC1887538  PMID: 17519032
19.  The HaDREB2 transcription factor enhances basal thermotolerance and longevity of seeds through functional interaction with HaHSFA9 
BMC Plant Biology  2009;9:75.
Transcription factor HaDREB2 was identified in sunflower (Helianthus annuus L.) as a drought-responsive element-binding factor 2 (DREB2) with unique properties. HaDREB2 and the sunflower Heat Shock Factor A9 (HaHSFA9) co-activated the Hahsp17.6G1 promoter in sunflower embryos. Both factors could be involved in transcriptional co-activation of additional small heat stress protein (sHSP) promoters, and thus contribute to the HaHSFA9-mediated enhancement of longevity and basal thermotolerance of seeds.
We found that overexpression of HaDREB2 in seeds did not enhance longevity. This was deduced from assays of basal thermotolerance and controlled seed-deterioration, which were performed with transgenic tobacco. Furthermore, the constitutive overexpression of HaDREB2 did not increase thermotolerance in seedlings or result in the accumulation of HSPs at normal growth temperatures. In contrast, when HaDREB2 and HaHSFA9 were conjointly overexpressed in seeds, we observed positive effects on seed longevity, beyond those observed with overexpression of HaHSFA9 alone. Such additional effects are accompanied by a subtle enhancement of the accumulation of subsets of sHSPs belonging to the CI and CII cytosolic classes.
Our results reveal the functional interdependency of HaDREB2 and HaHSFA9 in seeds. HaDREB2 differs from other previously characterized DREB2 factors in plants in terms of its unique functional interaction with the seed-specific HaHSFA9 factor. No functional interaction between HaDREB2 and HaHSFA9 was observed when both factors were conjointly overexpressed in vegetative tissues. We therefore suggest that additional, seed-specific factors, or protein modifications, could be required for the functional interaction between HaDREB2 and HaHSFA9.
PMCID: PMC2706249  PMID: 19545370
20.  OsHsfA2c and OsHsfB4b are involved in the transcriptional regulation of cytoplasmic OsClpB (Hsp100) gene in rice (Oryza sativa L.) 
Cell Stress & Chaperones  2011;17(2):243-254.
ClpB-cytoplasmic (ClpB-cyt)/Hsp100 is an important chaperone protein in rice. Cellular expression of OsClpB-cyt transcript is governed by heat stress, metal stress, and developmental cues. Transgenic rice plants produced with 2 kb OsClpB-cyt promoter driving Gus reporter gene showed heat- and metal-regulated Gus expression in vegetative tissues and constitutive Gus expression in calli, flowering tissues, and embryonal half of seeds. Rice seedlings regenerated with OsClpB-cyt promoter fragment with deletion of its canonical heat shock element sequence (HSE−273 to −280) showed not only heat shock inducibility of Gus transcript/protein but also constitutive expression of Gus in vegetative tissues. It thus emerges that the only classical HSE present in OsClpB-cyt promoter is involved in repressing expression of OsClpB-cyt transcript under unstressed control conditions. Yeast one-hybrid assays suggested that OsHsfA2c specifically interacts with OsClpB-cyt promoter. OsHsfA2c also showed binding with OsClpB-cyt and OsHsfB4b showed binding with OsClpB-cyt; notably, interaction of OsHsfB4b was seen for all three OsClpB/Hsp100 protein isoforms (i.e., ClpB-cytoplasmic, ClpB-mitochondrial, and ClpB-chloroplastic). Furthermore, OsHsfB4b showed interaction with OsHsfA2c. This study suggests that OsHsfA2c may play a role as transcriptional activator and that OsHsfB4b is an important part of this heat shock responsive circuitry.
Electronic supplementary material
The online version of this article (doi:10.1007/s12192-011-0303-5) contains supplementary material, which is available to authorized users.
PMCID: PMC3273560  PMID: 22147560
cis-Acting sequences; Heat shock factor (Hsf); Heat shock protein100 (Hsp100); High temperature; Rice; trans-Acting factors
21.  C. elegans HSF-1 is an essential nuclear protein that forms stress granule-like structures following heat shock 
Aging cell  2012;12(1):112-120.
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.
PMCID: PMC3552056  PMID: 23107491
Heat shock factor; aging; longevity
22.  Heat shock factor 2 is required for maintaining proteostasis against febrile-range thermal stress and polyglutamine aggregation 
Molecular Biology of the Cell  2011;22(19):3571-3583.
HSF2 regulates proteostasis capacity against febrile-range thermal stress, which provides temperature-dependent mechanisms of cellular adaptation to thermal stress. Furthermore, HSF2 has a strong impact on disease progression of Huntington's disease R6/2 mice, suggesting that it could be a promising therapeutic target for protein misfolding diseases.
Heat shock response is characterized by the induction of heat shock proteins (HSPs), which facilitate protein folding, and non-HSP proteins with diverse functions, including protein degradation, and is regulated by heat shock factors (HSFs). HSF1 is a master regulator of HSP expression during heat shock in mammals, as is HSF3 in avians. HSF2 plays roles in development of the brain and reproductive organs. However, the fundamental roles of HSF2 in vertebrate cells have not been identified. Here we find that vertebrate HSF2 is activated during heat shock in the physiological range. HSF2 deficiency reduces threshold for chicken HSF3 or mouse HSF1 activation, resulting in increased HSP expression during mild heat shock. HSF2-null cells are more sensitive to sustained mild heat shock than wild-type cells, associated with the accumulation of ubiquitylated misfolded proteins. Furthermore, loss of HSF2 function increases the accumulation of aggregated polyglutamine protein and shortens the lifespan of R6/2 Huntington's disease mice, partly through αB-crystallin expression. These results identify HSF2 as a major regulator of proteostasis capacity against febrile-range thermal stress and suggest that HSF2 could be a promising therapeutic target for protein-misfolding diseases.
PMCID: PMC3183013  PMID: 21813737
23.  Regulation of Cyclooxygenase-2 Expression by Heat: A Novel Aspect of Heat Shock Factor 1 Function in Human Cells 
PLoS ONE  2012;7(2):e31304.
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.
PMCID: PMC3275557  PMID: 22347460
24.  New candidate genes for heat resistance in Drosophila melanogaster are regulated by HSF 
Cell Stress & Chaperones  2008;13(2):177-182.
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.
PMCID: PMC2673890  PMID: 18759003
25.  Overexpression of Arabidopsis Dehydration-Responsive Element-Binding Protein 2C Confers Tolerance to Oxidative Stress 
Molecules and Cells  2012;33(2):135-140.
Dehydration-responsive element-binding proteins (DREBs) regulate plant responses to environmental stresses. In the current study, transcription of DREB2C, a class 2 Arabidopsis DREB, was induced by a superoxide anion propagator, methyl viologen (MV). The oxidative stress tolerance of DREB2C-overexpressing transgenic plants was significantly greater than that of wild-type plants, as measured by ion leakage and chlorophyll fluorescence under light conditions. The transcriptional activity of several ascorbate peroxidase (APX) genes as well as APX protein activity was induced in DREB2C overexpressors. Additionally, the level of H2O2 in the overexpressors was lower than in wt plants under similar oxidative stress conditions. An electrophoretic mobility shift assay and transient activator-reporter assay showed that APX2 expression was regulated by heat shock factor A3 (HsfA3) and that HsfA3 is regulated at the transcriptional level by DREB2C. These results suggest that DREB2C plays an important role in promoting oxidative stress tolerance in Arabidopsis.
PMCID: PMC3887724  PMID: 22286229
gene expression; signaling; transcription factor; transgenic plant

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