Isolation and characterization of six heat shock transcription factor cDNA clones from soybean (original) (raw)
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Biological Chemistry, 2003
Using UV laser cross-linking and immunoprecipitation we measured the in vivo binding of Arabidopsis heat shock transcription factor HSF1 to the promoters of target genes, Hsp18.2 and Hsp70. The amplification of promoter sequences, co-precipitated with HSF1-specific antibodies, indicated that HSF1 is not bound in the absence of heat stress. Binding to promoter sequences of target genes is rapidly induced by heat stress, continues throughout the heat treatment, and declines during subsequent recovery at room temperature. The molecular mechanisms underlying the differences between Hsp18.2 and Hsp70 in the kinetics of HSF1/promoter binding and corresponding mRNA expression profiles are discussed.
DNA Sequence and Transcript Mapping of a Soybean Gene Encoding a Small Heat Shock Protein
Proceedings of The National Academy of Sciences, 1985
The DNA sequence of a gene (Gmhspl7.5-E) encoding a small heat shock protein of soybean, Glycine max, has been determined. Nuclease Si mapping of the 5' terminus of the corresponding RNA indicates that the start site for transcription is located 82 bases upstream from the coding region and 24 bases downstream from a "TATA"-like region (-T-T-T-A-A-A-T-A-). The 5' flanking region of Gmhspl7.5-E contains two imperfect dyads that closely resemble regulatory elements present in the promoters of heat-inducible genes of Drosophia. One, positioned 18 bases upstream from the TATAlike region, shows 90% homology to the Drosophila heat shock consensus sequence. The other overlaps an upstream TATA sequence and is located at position -213. Analysis of the derived amino acid sequence indicates that the protein encoded by Gmhspl7.5-E is related structurally to the four small heat shock proteins of Drosophila. This relationship is most evident by comparison of hydropathy profiles; they show conservation of several major hydrophilic and hydrophobic regions, which suggests that these proteins have common structural features.
Upstream sequences required for efficient expression of a soybean heat shock gene
Molecular and cellular biology, 1986
A soybean gene (Gmhsp17.5-E) encoding a small heat shock protein was introduced into primary sunflower tumors via T-DNA-mediated transformation. RNA blot hybridizations and S1-nuclease hybrid protection studies indicated that the heat shock gene containing 3.25 kilobases of 5'-flanking sequences was strongly transcribed in a thermoinducible (40 degrees C) manner. Transcriptional induction also occurred to a lesser extent upon treatment of whole tumors with sodium arsenite and CdCl2. Basal (26 degrees C) transcription was not detected in soybean seedlings, but it was quite evident in transformed tumor tissue. A 5' deletion to -1,175 base pairs with respect to the CAP site had no effect on the levels of thermoinducible transcription, but it resulted in a large increase in basal transcription. Further removal of DNA sequences (including the TATA-distal heat shock consensus element) to -95 base pairs reduced thermoinducible transcription by 95% and also greatly decreased basal t...
In Vitro Interaction of Nuclear Proteins with the Promoter of Soybean Heat Shock Gene Gmhsp17.5E
PLANT PHYSIOLOGY, 1990
Proteins present in crude nuclear extracts of soybean (Glycine max) plumules were shown to bind in vitro to the 5' flanking sequences of the soybean heat shock gene Gmhspl7.5E. The specificity of binding activity present in extracts from both control (280C) and heat shocked (400C) tissues was demonstrated by reciprocal competition experiments using gel mobility retardation assays. Footprinting experiments using DNase I with crude nuclear extracts indicated that a continuous stretch of 5' flanking sequences extending from -40 to -153 was protected from digestion in vitro. Nuclear proteins that were partially purified by heparin agarose chromatography were shown to bind specific TATA-proximal sequences containing the heat shock consensus elements (HSEs) (-73 to -49; -107 to -84) and AT-rich motifs (-119 to -153). Other binding sites within AT-rich sequences (-906 to -888, -868 to 863, -859 to 853, and -841 to -830), distal HSE elements (-568 to -532) and a TATA/dyad (-234 to -207) were also identified by DNase I footprinting of TATA-distal probes. DNA binding activities specific for the HSE and AT-rich sequences were present in nuclear extracts from both control and heat shocked tissues. Both types of binding activity were increased after heat shock treatment; HSE binding increased from 1.8to 2.7-fold, and binding to AT-rich sequences showed an increase from 1.3to 1.7-fold.
Plant Molecular Biology, 2000
Based on phylogeny of DNA-binding domains and the organization of hydrophobic repeats, two families of heat shock transcription factors (HSFs) exist in plants. Class A HSFs are involved in the activation of the heat shock response, but the role of class B HSFs is not clear. When transcriptional activities of full-length HSFs were monitored in tobacco protoplasts, no class B HSFs from soybean or Arabidopsis showed activity under control or heat stress conditions. Additional assays confirmed the finding that the class B HSFs lacked the capacity to activate transcription. Fusion of a heterologous activation domain from human HSF1 (AD2) to the C-terminus of GmHSFB1-34 gave no evidence of synergistic enhancement of AD2 activity, which would be expected if weak activation domains were present. Furthermore, activity of AtHSFB1-4 (class B) was not rescued by coexpression with AtHSFA4-21 (class A) indicating that the class A HSF was not able to provide a missing function required for class B activity. The transcriptional activation potential of Arabidopsis AtHSFA4-21 was mapped primarily to a 39 amino acid fragment in the C-terminus enriched in bulky hydrophobic and acidic residues. Deletion mutagenesis of the C-terminal activator regions of tomato and Arabidopsis HSFs indicated that these plant HSFs lack heat-inducible regulatory regions analogous to those of mammalian HSF1. These findings suggest that heat shock regulation in plants may differ from metazoans by partitioning negative and positive functional domains onto separate HSF proteins. Class A HSFs are primarily responsible for stress-inducible activation of heat shock genes whereas some of the inert class B HSFs may be specialized for repression, or down-regulation, of the heat shock response.
Structural and Functional Diversity of Plant Heat Shock Factors
Eukaryotic response towards abiotic and biotic stress is mediated by production of molecular chaperons like heat shock proteins (HSPs), which protect cellular proteins from damage. These HSPs are under tight regulation of transcription factors known as Heat Shock Factors (HSFs). They bind to the palindromic repeat motif, Heat Shock Element (HSE), present in promoter of stress responsive genes and modulate their expression. Plants have multi member HSF family as compared to other eukaryotes. HSFs have conserved domains of specialised functions, which have been characterised as DNA binding domain, oligomerization domain, nuclear localisation and export signal and C-terminal activation domain. Based on structural peculiarities, plant HSFs have been grouped in three different classes: Class A, B and C. Although plant HSFs are structurally conserved family of DNA binding proteins, they are functionally diverse. Functional diversity and redundancy within HSF members has evolutionary significance in combating variety of stress conditions, which usually occurs in combinations during plant life cycle. HSFs play significant role not only in stress tolerance but also in various aspects of plant development.
Molecular and Cellular Biology
Soybeans, Glycine max, synthesize a family of low-molecular-weight heat shock (HS) proteins in response to HS. The DNA sequences of two genes encoding 17.5and 17.6-kilodalton HS proteins were determined. Nuclease Si mapping of the corresponding mRNA indicated multiple start termini at the 5' end and multiple stop termini at the 3' end. These two genes were compared with two other soybean HS genes of similar size. A comparison among the 5' flanking regions encompassing the presumptive HS promoter of the soybean HS-protein genes demonstrated this region to be extremely homologous. Analysis of the DNA sequences in the 5' flanking regions of the soybean genes with the corresponding regions of Drosophila melanogaster HS-protein genes revealed striking similarity between plants and animals in the presumptive promoter structure of thermoinducible genes. Sequences related to the Drosophila HS consensus regulatory element were found 57 to 62 base pairs 5' to the start of transcription in addition to secondary HS consensus elements located further upstream. Comparative analysis of the deduced amino acid sequences of four soybean HS proteins illustrated that these proteins were greater than 90% homologous. Comparison of the amino acid sequence for soybean HS proteins with other organisms showed much lower homology (less than 20%). Hydropathy profiles for Drosophila, Xenopus, Caenorhabditis elegans, and G. max HS proteins showed a similarity of major hydrophilic and hydrophobic regions, which suggests conservation of functional domains for these proteins among widely dispersed organisms.
Genome-wide analysis of heat shock transcription factor families in rice and Arabidopsis
Journal of Genetics and Genomics, 2008
Plant heat shock transcription factors (Hsfs) play a significant role in adoption under abiotic stress conditions by modulating the expression of several stress-responsive genes. Analysis of the Hsf gene family will serve to understand the molecular mechanism which is involved in response to abiotic stress. The Ziziphus species grows in warm and dry regions and is inherently tolerant to abiotic stress conditions; thus, Ziziphus is a highly enriched source of genes conferring abiotic stress tolerance. Therefore, the present study provides a comprehensive genome-wide analysis of the Hsf gene family in Z. jujuba. Identified 21 non-redundant Hsf genes were grouped into three major classes (classes A, B, and C) based on the phylogenetic analysis. Promoter and gene ontology analysis suggested that ZjHsfs perform diverse functions in response to abiotic stress conditions. Two paralogous pairs resulting from tandem gene duplication events were identified. Also, physio-chemical properties of chromosomal locations, gene structure, motifs, and protein domain organization of Hsfs were analyzed. Real-time PCR expression analyses revealed that most of the Z. jujuba Hsf genes are differentially expressed in response to heat stress. The analysis suggested ZjHsf-2, ZjHsf-3, ZjHsf-5, ZjHsf-7, ZjHsf-8, ZjHsf-10, ZjHsf-12, ZjHsf-17, and ZjHsf-18 were the outstanding candidate genes for imparting heat stress tolerance and for future functional analysis. The present analysis laid the foundation for understanding the molecular mechanism of the Hsf gene family regulating Z. jujuba development and tolerance to abiotic stress conditions.