The cytosolic class I small heat shock proteins (sHSP-CI) represent the most abundant sHSP in plants. Here, we report the characterization and the expression profile of nine members of the sHSP-CI gene family in rice (Oryza sativa Tainung No.67), of which Oshsp16.9A, Oshsp16.9B, Oshsp16.9C, Oshsp16.9D and Oshsp17.9B are clustered on chromosome 1, and Oshsp17.3, Oshsp17.7, Oshsp17.9A and Oshsp18.0 are clustered on chromosome 3. Oshsp17.3 and Oshsp18.0 are linked by a 356-bp putative bi-directional promoter. Individual gene products were identified from the protein subunits of a heat shock complex (HSC) and from in vitro transcription/ translation products by two-dimensional gel electrophoreses (2-DE). All sHSP-CI genes except Oshsp17.9B were induced strongly after a 2-h heat shock treatment. The genes on chromosome 3 were induced rapidly at 32 and 41 degrees C, whereas those on chromosome 1 were induced slowly by similar conditions. Seven of these genes, except Oshsp16.9D and Oshsp17.9B, were induced by arsenite (As), but only genes on chromosome 3 were strongly induced by azetidine-2-carboxylic acid (Aze, a proline analog) and cadmium (Cd). A similar expression profile of all sHSP-CI genes at a lower level was evoked by ethanol, H2O2 and CuCl2 treatments. Transient expression assays of the promoter activity by linking to GUS reporter gene also supported the in vivo selective expression of the sHSP-CI genes by Aze treatment indicating the differential induction of rice sHSP-CI genes is most likely regulated at the transcriptional level. Only Oshsp16.9A abundantly accumulated in mature dry seed also suggested additionally prominent roles played by this HSP in development.
In rice, the class I small heat shock protein (sHSP-CI) genes were found to be selectively induced by L-azetidine-2-carboxylic acid (AZC) on chromosome 3 but not chromosome 1. Here it is shown that a novel cis-responsive element contributed to the differential regulation. By serial deletion and computational analysis, a 9 bp putative AZC-responsive element (AZRE), GTCCTGGAC, located between nucleotides –186 and –178 relative to the transcription initiation site of Oshsp17.3 was revealed. Deletion of this putative AZRE from the promoter abolished its ability to be induced by AZC. Moreover, electrophoretic mobility shift assay (EMSA) revealed that the AZRE interacted specifically with nuclear proteins from AZC-treated rice seedlings. Two AZRE–protein complexes were detected by EMSA, one of which could be competed out by a canonical heat shock element (HSE). Deletion of the AZRE also affected the HS response. Furthermore, transient co-expression of the heat shock factor OsHsfA4b with the AZRE in the promoter of Oshsp17.3 was effective. The requirement for the putative AZRE for AZC and HS responses in transgenic Arabidopsis was also shown. Thus, AZRE represents an alternative form of heat HSE, and its interaction with canonical HSEs through heat shock factors may be required to respond to HS and AZC.
Non‐lethal heat‐shock (HS) treatment has previously been shown to induce thermotolerance in soybean (Glycine max cv. Kaohsiung No.8) seedlings. This acquired thermotolerance correlates with the de novo synthesis of heat‐shock proteins (HSPs). Interestingly, we found that ethanol treatments also elicited HS‐like responses in aetiolated soybean seedlings at their normal growth temperature of 28 °C. Northern blot analyses revealed that the expression of HS genes hsp17.5, hsp70 and hsc 70 was induced by ethanol. Radioactive amino acids were preferentially incorporated into high molecular weight (HMW) HSPs rather than class I low molecular weight (LMW) HSPs during non‐lethal ethanol treatments. Immunoblot analysis confirmed that no accumulation of class I LMW HSPs occurred after non‐lethal ethanol treatment. Pre‐treatment with a non‐lethal dose of ethanol did not provide thermotolerance, as the aetiolated soybean seedlings could not survive a subsequent heat shock of 45 °C for 2 h. In contrast, non‐lethal HS pre‐treatment, 40 °C for 2 h, conferred tolerance on aetiolated soybean seedlings to otherwise lethal treatments of 7·5% ethanol for 8 h or 10% ethanol for 4 h. These results suggest that plant class I LMW HSPs may play important roles in providing both thermotolerance and ethanol tolerance.
A monospecific polyclonal antibody was used to study the tissuetype specificity and intracellular localization of class I lowmolecular-weight (LMW) heat-shock proteins (HSPs) in soybean (Glycine max) under different heat-shock regimes. In etiolated soybean seedlings, the root meristematic regions contained the highest levels of LMW HSP. No tissue-type-specific expression of class I LMW HSP was detected using the tissue-printing method. In immunolocalization studies of seedlings treated with HS (40°C for 2 h) the class I LMW HSPs were found in the aggregated granular structures, which were distributed randomly in the cytoplasm and in the nucleus. When the heat shock was released, the granular structures disappeared and the class I LMW HSPs became distributed homogeneously in the cytoplasm. When the seedlings were then given a more severe heat shock following the initial 40°C + 28°C treatment, a large proportion of the class I LMW HSPs that originally localized in the cytoplasm were translocated into the nucleus and nucleolus. Class I LMW HSPs may assist in the resolubilization of proteins denatured or aggregated by heat and may also participate in the restoration of organellar function after heat shock.
A rice class I low-molecular-mass heat shock protein (LMM HSP) Oshsp 16.9 was overexpressed in Escherichia coli. Oligomerized complexes of Oshsp16.9 were harvested and electron microscopic observations of purified complexes revealed globular structures of 10-20 nm in diameter (with majority of 15-18 nm) and calculated to comprise approx. 12 monomers per complex. In comparison, complexes from native rice class I LMM HSPs were observed as larger ellipsoid- or globular-like random aggregated hetero-oligomers. To characterize the biochemical functions of the hydrophobic N-terminal region of Oshsp16.9, a truncation in the N-terminal region was constructed and introduced into E. coli. Results showed that the N-terminal truncated Oshsp16.9 mutant was capable of forming complexes similar to the full-length Oshsp16.9; however, the deletion protein failed to confer in vitro protein thermostability under elevated temperatures. Protein assays from in vivo treatments at higher temperatures exhibited that non-specific interactions of E. coli cellular proteins only occurred with full-length Oshsp16.9 complexes but not with the mutant complex. In vitro immunoprecipitation of cellular proteins from E. coli overexpressing full-length Oshsp16.9 showed that interactions between plant LMM HSP and E. coli cellular proteins are temperature-dependent. Taken together, the hydrophobic N-terminal region of rice class I LMM HSP is critical in the ability of the protein to interact/bind with its potential substrates.
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