Simultaneous exposure of Xenopus A6 kidney epithelial cells to concurrent mild sodium arsenite and heat stress results in enhanced hsp30 and hsp70 gene expression and the acquisition of thermotolerance

In this study, we examined the effect of concurrent low concentrations of sodium arsenite and mild heat shock temperatures on hsp30 and hsp70 gene expression in Xenopus A6 kidney epithelial cells. RNA blot hybridization and immunoblot analysis revealed that exposure of A6 cells to 1–10 µM sodium arsenite at a mild heat shock temperature of 30 °C enhanced hsp30 and hsp70 gene expression to a much greater extent than found with either stress individually. In cells treated simultaneously with 10 µM sodium arsenite and different heat shock temperatures, enhanced accumulation of HSP30 and HSP70 protein was first detected at 26 °C with larger responses at 28 and 30 °C. HSF1 activity was involved in combined stress-induced hsp gene expression since the HSF1 activation inhibitor, KNK437, inhibited HSP30 and HSP70 accumulation. Immunocytochemical analysis revealed that HSP30 was present in a granular pattern primarily in the cytoplasm in cells treated simultaneously with both stresses. Finally, prior exposure of A6 cells to concurrent sodium arsenite (10 µM) and heat shock (30 °C) treatment conferred thermotolerance since it protected them against a subsequent thermal challenge (37 °C). Acquired thermotolerance was not observed with cells treated with the two mild stresses individually.     

Heat stress-induced H2O2 is required for effective expression of heat shock genes in Arabidopsis

The mechanisms of sensing and signalling of heat and oxidative stresses are not well understood. The central question of this paper is whether in plant cells oxidative stress, in particular H₂O₂, is required for heat stress- and heat shock factor (HSF)-dependent expression of genes. Heat stress increases intracellular accumulation of H₂O₂ in Arabidopsis cell culture. The accumulation was greatly diminished using ascorbate as a scavenger or respectively diphenyleneiodonium chloride (DPI) as an inhibitor of reactive oxygen species production. The mRNA of heat shock protein (HSP) genes, exemplified by Hsp17.6, Hsp18.2, and the two cytosolic ascorbate peroxidase genes Apx1, Apx2, reached similar levels by moderate heat stress (37°C) or by treatment with H₂O₂, butylperoxide and diamide at room temperature. The heat-induced expression levels were significantly reduced in the presence of ascorbate or DPI indicating that H₂O₂ is an essential component in the heat stress signalling pathway. Rapid (15 min) formation of heat shock promoter element (HSE) protein-binding complex of high molecular weight in extracts of heat-stressed or H₂O₂-treated cells and the inability to form this complex after ascorbate treatment suggests that oxidative stress affects gene expression via HSF activation and conversely, that H₂O₂ is involved in HSF activation during the early phase of heat stress. The heat stress induction of a high mobility HSE-binding complex, characteristic for later phase of heat shock response, was blocked by ascorbate and DPI. H₂O₂ was unable to induce this complex suggesting that H₂O₂ is involved only in the early stages of HSF activation. Significant induction of the genes tested after diamid treatment and moderate expression of the sHSP genes in the presence of 50 mM ascorbate at 37°C occurred without activation of HSF, indicating that other mechanisms may be involved in stress signalling. Electronic Supplementary Material Supplementary material is available for this article at http//dx.doi.org/10.1007/s11103-006-0045-4 Roman A. Volkov and Irina I. Panchuk contributed equally