Fritz Schöffl

HSF3, a new heat shock factor from Arabidopsis thaliana, derepresses the heat shock response and confers thermotolerance when overexpressed in transgenic plants

Abstract Organisms synthesize heat shock proteins (HSPs) in response to sublethal heat stress and concomitantly acquire increased tolerance against a subsequent, otherwise lethal, heat shock. Heat shock factor (HSF) is essential for the transcription of many HSP genes. We report the isolation of two HSF genes, HSF3 and HSF4, from an Arabidopsis cDNA library. Transgenic Arabidopsis plants were generated containing constructs that allow expression of HSF3 and HSF4 or the respective translational β-glucuronidase (GUS) fusions. Overexpression of HSF3 or HSF3-GUS, but not of HSF4 or HSF4-GUS, causes HSP synthesis at the non-heat-shock temperature of 25° C in transgenic Arabidopsis. In transgenic plants bearing HSF3/HSF3-GUS, transcription of several heat shock genes is derepressed. Electrophoretic mobility shift assays suggest that derepression of the heat shock response is mediated by HSF3/HSF3-GUS functioning as transcription factor. HSF3/HSF3-GUS-overexpressing Arabidopsis plants show an increase in basal thermotolerance, indicating the importance of HSFs and HSF-regulated genes as determinants of thermoprotective processes. Plants transgenic for HSF3/HSF3-GUS exhibit no other obvious phenotypic alterations. Derepression of HSF activity upon overexpression suggests the titration of a negative regulator of HSF3 or an intrinsic constitutive activity of HSF3. We assume that stable overexpression of HSFs may be applied to other organisms as a means of derepressing the heat shock response.

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::β-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.

Two different heat shock transcription factors regulate immediate early expression of stress genes in Arabidopsis

In order to assess the specific functional roles of different plant heat shock transcription factors (HSFs) we have isolated T-DNA insertion mutants in the AtHsf1 and AtHsf3 genes of Arabidopsis thaliana. Complete and selective loss of the promoter binding activities of AtHSF1 or AtHSF3, verified by immunoprecipitation assays, had no obvious effects on the heat shock (HS) response in the individual mutant lines. Only hsf1--/hsf3--double mutants were significantly impaired in HS gene expression. In these plants the inability to form high-molecular-weight HSE-binding complexes correlates with a dramatic change in the kinetics of mRNA accumulation from all HSF target genes tested, including members of the Hsp100, Hsp90, Hsp70 and small Hsp families, and genes for two heat-inducible class B-HSFs. After prolonged HS, the amounts of most heat shock mRNAs expressed, except transcripts of Hsp18.2, reached approximately the same levels as in wild type plants. Our data indicate that AtHSF1 and AtHSF3 are key regulators of the immediate stress-induced activation of HS gene transcription, and consequently determine the kinetics of the negative feed back loop that is responsible for the transience of HS gene expression in wild type.

An Hsp70 antisense gene affects the expression of HSP70/HSC70, the regulation of HSF, and the acquisition of thermotolerance in transgenic Arabidopsis thaliana

Abstract The genes and proteins of the HSP70 family, are involved in important processes in cells and organelles at normal temperature and after heat stress. Constitutive Hsc70 and heat-inducible Hsp70 genes are known in all organisms including plants. The goal of our present investigation was to generate an Hsp70 mutation in Arabidopsis thaliana. In a transgenic approach a heat-inducible antisense Hsp70 gene was constructed, plants were transformed and screened for lack of heat-inducible HSP70 mRNA; two such lines were further investigated. In these plants the Hsp70 gene was not induced by heat shock, and the level of HSC70 RNA was also greatly reduced. This negative antisense effect was specific for genes of the HSP70 family and the induction of mRNAs encoding the small HSP18 class of heat shock protein (HSP) was not affected. The level of HSP70/HSC70 proteins was significantly reduced in transgenic plants, but HSP18 was induced to the same level in different transgenic lines and in untransformed plants. The acquisition of thermotolerance was negatively affected in artisense plants, the survival temperature being 2°C below the survival temperature of the wild type and other transgenic lines. Another major effect concerning the regulation of the endogenous heat shock transcription factor HSF was detected by testing the ability to form heterotrimers between authentic HSF and recombinant HSF-GUS (β-glucuronidase) proteins. The shut-off time, required to turn off HSF activity during recovery from heat stress, was significantly prolonged in antisense plants compared with wild-type and other transgenic lines. Our results imply a dual role of HSP70 in plants, a protective role in thermotolerance and a regulatory effect on HSF activity and hence the autoregulation of the heat shock response.

Synergistic effect of upstream sequences, CCAAT box elements, and HSE sequences for enhanced expression of chimaeric heat shock genes in transgenic tobacco

SummaryThe thermoregulated expression of the soybean heat shock (hs) gene Gmhsp17.3-B is regulated via the heat shock promoter elements (HSEs), but full promoter activity requires additional sequences located upstream of the HSE-containing region. Structural features within this putative enhancer region include a run of simple sequences which are also present upstream of HSE-like sequences of other soybean hs genes, and three perfect CCAAT box sequences located immediately upstream from the most distal HSE of the promoter. A series of heterologous and homologous promoter fusions linked to the chloramphenicol acetyl transferase (CAT) gene was constructed and examined in transgenic tobacco plants. The region containing the AT-rich domain of the 5′ flanking region was unable to direct transcription from the TATA box of a truncated ΔCaMV35S promoter. Heat-inducible CAT activity was detectable when additional sequences from the native promoter containing three CCAAT boxes and a single HSE were present in the constructions. Complete reconstitution of the native hs promoter/enhancer region increased hs specific CAT activities only very little, but deletion of CCAAT box sequences reduced CAT expression fivefold. Our results suggest that AT-rich sequences have a moderate effect on thermoinducible expression levels of the soybean heat shock gene and that CCAAT box sequences act cooporatively with HSEs to increase the hs promoter activity.

Arabidopsis heat shock factor: isolation and characterization of the gene and the recombinant protein

We have isolated the Arabidopsis heat shock factor gene Athsf1 as genomic and corresponding cDNA sequences via cross-hybridization with tomato clones. Sequence analysis indicates only a partial homology with the HSFs from tomato and other organisms which is confined to the DNA-binding and the oligomerization domains. The gene is constitutively expressed but the level of mRNA for Athsf1 increases two-fold upon heat shock. However, the putative promoter region lacks the canonical heat shock elements. After expression in Escherichia coli the recombinant Athsf1 protein binds specifically to a synthetic oligonucleotide containing five heat shock elements. The native size of recombinant ATHSF1 in vitro is consistent with a trimer as demonstrated by chemical cross-linking and pore exclusion limit analysis.

The DNA sequence analysis of soybean heat-shock genes and identification of possible regulatory promoter elements.

The soybean possesses a gene family encoding the major low mol. wt. heat‐shock proteins of 15–18 kd. We have determined the primary DNA sequences of two of the genes, both located on the same subgenomic DNA fragment. The protein coding regions are characterized by long uninterrupted open reading frames and by sequence homology of 92% and 100% with a heat‐shock specific cDNA. One protein sequence deduced from the completely cloned gene hs6871 is composed of 153 amino acids with a total mol. wt. of 17.3 kd; the other protein is a truncated polypeptide containing 73 amino acids at the carboxy‐terminal end of an incompletely cloned heat‐shock gene designated hs6834. Investigations of the hydrophilic/hydrophobic characteristics of the polypeptides revealed a conservation of structural features between heat‐shock proteins from soybean, Caenorhabditis and Drosophila and mammalian lens α‐crystallin. The 5′ end of the soybean heat‐shock gene hs6871 was mapped by S1 nuclease at a position which is ˜100 nucleotides upstream from the translation start codon and 25 nucleotides downstream from a TATA‐box sequence. Six other potential promoter elements which are homologous to the Drosophila heat‐shock consensus sequence CT‐GAA‐TTC‐AG‐, are present within ˜150 nucleotides upstream from the TATA‐box. The possible functions of these promoter elements in transcriptional regulation of expression of soybean heat‐shock gene are discussed.

An SAR sequence containing 395 bp DNA fragment mediates enhanced, gene-dosage-correlated expression of a chimaeric heat shock gene in transgenic tobacco plants

A 395 bp fragment located downstream from the soybean heat shock geneGmhsp 17.6-L exhibits several characteristics of scaffold attachment region (SAR) sequences. It contains matrix consensus elements, a topoisomerase II binding sequence and it associates with the isolated nuclear scaffold of soybeanin vitro. Chimaeric genes containing the SARL fragment either at one side (5′ or 3′) or at both sides of a heat shock promoter-regulated β-glucuronidase reporter gene were constructed. A five-to nine-fold increase of heat-inducible β-glucuronidase activity was observed in transgenic tobacco plants containing constructs with SARL fragments either at both sides or with at least one SARL copy located upstream from the reporter gene. The gene copy number is positively correlated with the level of heat-inducible reporter gene activity in these. plants but positional effects are not entirely eliminated. Thus, SAR sequences may potentially be used to increase gene expression, via as yet unknown mechanisms, and to reduce adverse effects on the expression of multiple gene copies in transgenic plants.

Functional analysis of sequences required for transcriptional activation of a soybean heat shock gene in transgenic tobacco plants.

The 5′ DNA sequences involved in the thermal inducibility of the soybean heat shock gene hs6871 were analysed in transgenic tobacco plants. The transcriptional activity of various in vitro generated deletion mutants was examined by Northern blot analysis, S1 nuclease mapping and dot‐blot hybridization. At least 181 bp upstream from the translational start site are sufficient for thermal induction at 40°C and correct initiation of transcripts. Full promoter activity with the induction of wild‐type levels of transcripts requires additional upstream sequences contained within 439 bp 5′ to the coding sequence. Our results suggest that faithful regulation and the generation of high levels of hs6871‐specific mRNA depend on the presence of sequences which show homology to the 14‐bp heat shock consensus element of Drosophila and, in addition, on as yet unidentified enhancer‐like upstream sequences.

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, −3Cand -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.