Effects of heat stress on yeast heat shock factor-promoter binding in vivo.

Heat shock factor-DNA interaction is critical for understanding the regulatory mechanisms of stress-induced gene expression in eukaryotes. In this study, we analyzed the in vivo binding of yeast heat shock factor (HSF) to the promoters of target genes ScSSA1, ScSSA4, HSP30 and HSP104, using chromatin immunoprecipitation. Previous work suggested that yeast HSF is constitutively bound to DNA at all temperatures. Expression of HSF target genes is regulated at the post-transcriptional level. However, our results indicated that HSF does not bind to the promoters of ScSSA4 and HSP30 at normal temperature (23 degrees C). Binding to these promoters is rapidly induced by heat stress at 39 degrees C. HSF binds to ScSSA1 and HSP104 promoters under non-stress conditions, but at a low level. Heat stress rapidly leads to a notable increase in the binding of HSF to these two genes. The kinetics of the level of HSF-promoter binding correlate well with the expression of target genes, suggesting that the expression of HSF target genes is at least partially the result of HSF-promoter binding stability and subsequent transcription stimulation.

Identification of novel heat shock factor-dependent genes and biochemical pathways in Arabidopsis thaliana.

In order to assess specific functional roles of plant heat shock transcription factors (HSF) we conducted a transcriptome analysis of Arabidopsis thaliana hsfA1a/hsfA1b double knock out mutants and wild-type plants. We used Affymetrix ATH1 microarrays (representing more than 24 000 genes) and conducted hybridizations for heat-treated or non-heat-treated leaf material of the respective lines. Heat stress had a severe impact on the transcriptome of mutant and wild-type plants. Approximately 11% of all monitored genes of the wild type showed a significant effect upon heat stress treatment. The difference in heat stress-induced gene expression between mutant and wild type revealed a number of HsfA1a/1b-regulated genes. Besides several heat shock protein and other stress-related genes, we found HSFA-1a/1b-regulated genes for other functions including protein biosynthesis and processing, signalling, metabolism and transport. By screening the profiling data for genes in biochemical pathways in which known HSF targets were involved, we discovered that at each step in the pathway leading to osmolytes, the expression of genes is regulated by heat stress and in several cases by HSF. Our results document that in the immediate early phase of the heat shock response HSF-dependent gene expression is not limited to known stress genes, which are involved in protection from proteotoxic effects. HsfA1a and HsfA1b-regulated gene expression also affects other pathways and mechanisms dealing with a broader range of physiological adaptations to stress.

Small heat shock proteins are differentially regulated during pollen development and following heat stress in tobacco.

In plants small heat shock proteins (sHsp) are abundantly expressed upon heat stress in vegetative tissue, however, sHsp expression is also developmentally induced in pollen. The developmental induction of sHsp has been related to the potential for stress-induced microspore embryogenesis. We investigated the polymorphism among sHsp and their expression during pollen development and after heat stress in tobacco. Real-time RT-PCR was used for quantification of mRNA of two known and nine newly isolated cDNAs representing cytosolic sHsp. At normal temperature most of these genes are not transcribed in vegetative tissues, however, all genes were expressed during pollen development. Low levels of mRNAs were found for sHsp-1A and -1B in early-unicellular stage, increasing four to sevenfold in mature pollen. Nine other genes are up-regulated in unicellular and down-regulated in bicellular pollen; three these genes show stage-specific expression. Western analysis revealed that cytosolic class I and II sHsp are developmentally expressed during all stages of pollen development. Different subsets of cytosolic sHsp genes are expressed in a stage-specific fashion suggesting that certain sHsp genes may play specific roles in early, others during later stages of pollen development. Heat stress results in a relatively weak and incomplete response in pollen: (i) the heat-induced levels of mRNA (excepting sHsp-2B, -3C and -6) are much lower than in leaves, (ii) several sHsp are not detected after heat stress in pollen, although, they are heat-inducibly expressed in leaves. Application of heat stress, cold, and starvation, which induce microspore embryogenesis, modify mRNA levels and the patterns of 2-D-separated sHsp, but only heat stress enhances the expression of sHsp in microspores. There is no correlation of the expression of specific sHsp with the potential for microspore embryogenesis.

Interaction between Arabidopsis heat shock transcription factor 1 and 70 kDa heat shock proteins.

The activity of the Arabidopsis heat shock transcription factor (HSF) is repressed at normal conditions but activated by cellular stresses. Circumstantial evidence suggests that HSP70 may function as a negative feedback regulator of HSF activity. Here the interaction between HSF and HSP70 is reported using electrophoretic mobility shift and yeast two-hybrid assays. Subdomain mapping indicates an interaction of the activation domain and DNA-binding domain of HSF1 with HSP70.

Heat-stress-dependency and developmental modulation of gene expression: the potential of house-keeping genes as internal standards in mRNA expression profiling using real-time RT-PCR.

The potential of different house-keeping genes for their use as internal standards of gene expression under changing environmental conditions and in different organs of plants was assessed. Using real-time PCR mRNA levels were precisely quantified for preselected actin and ribosomal protein genes in Arabidopsis thaliana (L.) Heinh. and Nicotiana tabacum L. grown at normal temperature and following heat stress. In tobacco leaves the mRNA levels of the constitutively expressed ribosomal protein gene Nt-L25 and the actin genes Nt-ACT9 and At-ACT66 were strongly reduced (to approximately 10%) during heat stress. Heat stress applied at the temperature optimum (37 degrees C) for elicitation of a heat stress response to Arabidopsis leaves resulted in a strong induction (several thousand-fold) of the mRNA heat shock protein genes, At-HSP17.6 and At-HSP18.2. Concomitantly, the mRNA levels of constitutively expressed actin 2 (At-ACT2) and ribosomal protein L23 (At-L23a) genes were reduced to approximately 50% of the levels in leaves incubated at room temperature. Conversely, under severe heat stress conditions (44 degrees C), the induction of At-HSP17.6 and At-HSP18.2 mRNAs was insignificant, the mRNA levels of At-ACT2 remained at approximately the same levels as in leaves incubated at room temperature, whereas the mRNA level of At-L23 declined. The mRNA levels of At-ACT2 and At-L23a examined in stem, flower and siliques of Arabidopsis plants grown under non-stress condition showed differential alterations; the mRNA level of ribosomal protein L23 correlates with the metabolic activity of tissues. The potential use of house-keeping gene expression as standards in expression profiling and the mechanisms modulating the mRNA levels are discussed.

Detecting DNA-binding of proteins in vivo by UV-crosslinking and immunoprecipitation.

The temporal and spatial binding of proteins on DNA is important to the regulation of genome expression and maintenance. However, examining how the protein-DNA complexes assemble in living cells is challenging. The development of UV-crosslinking/immunoprecipitation (UV-X-ChIP) technique and the progress of its applications show the powerful potential of this method in detecting such binding behavior in vivo. UV light is a zero length crosslinker and is believed to produce less perturbation of the complex than chemical crosslinker. The use of UV laser as UV light source allows the number of photons required for crosslinking to be delivered in nano- or pico- or femtosecond intervals, extremely shortening the irradiation time and achieving higher crosslinking efficiency than conventional UV lamp, thus being well suitable for kinetic studies. UV-X-ChIP technique has been successfully applied on the study of DNA replication, transcription, chromatin structure, and genome-wide location of DNA-binding proteins.

Galactinol synthase1. A novel heat shock factor target gene responsible for heat-induced synthesis of raffinose family oligosaccharides in Arabidopsis.

Heat shock factors (HSFs) are transcriptional regulators of the heat shock response. The major target of HSFs are the genes encoding heat shock proteins (HSPs), which are known to have a protective function that counteracts cytotoxic effects. To identify other HSF target genes, which may be important determinants for the generation of stress tolerance in Arabidopsis, we screened a library enriched for genes that are up-regulated in HSF3 (AtHsfA1b)-overexpressing transgenic plants (TPs). Galactinol synthase1 (GolS1) is one of the genes that is heat-inducible in wild type, but shows constitutive mRNA levels in HSF3 TPs. The generation and analysis of TPs containing GolS1-promoter::beta-glucuronidase-reporter gene constructs showed that, upon heat stress, the expression is transcriptionally controlled and occurs in all vegetative tissues. Functional consequences of GolS1 expression were investigated by the quantification of raffinose, stachyose, and galactinol contents in wild type, HSF3 TPs, and two different GolS1 knockout mutants (gols1-1 and gols1-2). This analysis demonstrates that (1) raffinose content in leaves increases upon heat stress in wild-type but not in the GolS1 mutant plants; and (2) the level of raffinose is enhanced and stachyose is present at normal temperature in HSF3 TPs. These data provide evidence that GolS1 is a novel HSF target gene, which is responsible for heat stress-dependent synthesis of raffinose, a member of the raffinose family oligosaccharides. The biological function of this osmoprotective substance and the role of HSF-dependent genes in this biochemical pathway are discussed.