The heat stress transcription factors HsfA3a/b enhance heat stress resistance by regulating HSP26.5-1/-2 in Sorbus pohuashanensis.

Heat stress impacts the growth and value of ornamental plants such as Sorbus pohuashanensis. Heat stress transcription factors (Hsfs) and heat shock proteins (HSPs) are critical in the response of plants to heat stress. However, the roles of HsfA3a/b and HSP26.5-1/-2 in the heat tolerance mechanisms of woody plants remain elusive. Here, we report the isolation of SpHsfA3a/b and SpHSP26.5-1/-2 from S. pohuashanensis and their molecular characterization. SpHsfA3a/b and SpHSP26.5-1/-2 were expressed at higher levels in leaves and fruits. Furthermore, their expression levels increased dramatically under heat stress. Overexpression of SpHsfA3a/b and SpHSP26.5-1/-2, as well as complementation of hsfa3 mutants with SpHsfA3a/b in Arabidopsis thaliana, significantly increased plant survival under heat stress. This enhanced survival was accompanied by reduced malondialdehyde (MDA) content, increased proline and soluble sugars, and elevated antioxidant enzyme activities. The positive regulatory genes related to the heat stress responses were upregulated, while the negative regulatory gene was downregulated in the transgenic SpHsfA3a/b-overexpressing Arabidopsis lines and the complemented hsfa3 mutant lines under high temperature stress. Yeast one-hybrid and dual-luciferase reporter assays showed that SpHsfA3a/b directly bound to and activated the promoters of SpHSP26.5-1/-2. In summary, SpHsfA3a/b directly regulate the expression of SpHSP26.5-1/-2 and play crucial, synergistic roles in enhancing thermotolerance. This study elucidates a key transcriptional regulatory module, SpHsfA3a/b-SpHSP26.5-1/-2, in the heat stress response of S. pohuashanensis and provides candidate genes for improving the heat resistance of forest trees.