TaHsfA2-11-TaZAT8 module negatively regulates salt tolerance in wheat

Plant heat shock transcription factor (Hsf) can directly activate the expression of various genes related to abiotic stress responses, thereby enhancing plant stress resistance, making them to be important target genes in modern molecular breeding. The wheat Hsf family has numerous members with complex functions and mechanisms, and a single gene often participates in multiple stress responses. Our previous studies reported that overexpression of the core subfamily member TaHsfA2-11 (ACCESSION: KY774305) significantly improves plant thermotolerance, while its expression is significantly up-regulated by NaCl. Based on this, in this paper we generated transgenic plants of Arabidopsis thaliana and wheat and studied the salt resistance function of TaHsfA2-11. Phenotypic identification all revealed that both TaHsfA2-11-overexpressing Arabidopsis and wheat plants displayed more sensitive salt stress, with lower germination rate and cotyledons greening, and poorer growth vigor than the wild-type (WT). Moreover, TaHsfA2-11 overexpressing Arabidopsis mutant atHsfa2 (insensitive compared to the WT) also increased the sensitivity to salt stress. RNA-seq analysis results between the WT and overexpressed wheat revealed numerous differentially expressed genes, which were enriched in multiple biological processes, such as response to abiotic stimulus, reactive oxygen species, protein folding, protein complex oligomerization, etc, and pathways such as starch and sucrose metobolism, protein processing in endoplasmic reticulum, flavone and flavonol biosythesis, photosynthesis-antenna proteins, etc. RT-qPCR results indicated that majority of DEGs were upregulated by both salt and TaHsfA2-11. Among those, the Zinc finger protein 8 (TaZAT8), contains heat shock element (HSE) motif in the promotor, was prominently induced under NaCl treatment in TaHsfA2-11-OE wheat. Through experiments of electrophoretic mobility shift assay (EMSA) and dual luciferase reporter assay (DLR), we found that TaHsfA2-11 can bind to the HSE of TaZAT8 promoter and transcriptionally activate its expression. Overexpression of TaZAT8 in Arabidopsis thaliana WT and mutant athsfa2 markedly enhanced plant sensitivity to salt stress, displaying the similar salt resistance phenotypes with TaHsfA2-11-OE Arabidopsis and wheat. These findings indicated that TaHsfA2-11 perhaps regulates plant salt tolerance by modulating the expression of the negative regulator TaZAT8 by mediating pathways such as photosynthesis, ROS accumulation, and flavonoid synthesis, etc. The results not only enrich the theory of crop salt tolerance regulation, expand the functional scope of the wheat HsfA2 subfamily genes, but also provide a key target gene for editing breeding aimed at enhancing salt tolerance of wheat.