PeHSF Activates PeWRKY1 To Confer Salt Tolerance In Populus Euphratica

Under salt stress, the salt-tolerant Populus euphratica has developed a complex pathway for balancing ion homeostasis and reactive oxygen species (ROS) homeostasis. In addition to the genes involved in balancing Na+/K+ and ROS homeostasis, some key transcription factors related to abiotic-stresses, such as WRKY, MYB and HSF, were also up-regulated by using Affymetrix microarray assay.Two transcription factor genes (PeHSF and PeWRKYl) were cloned and transformed into tobacoo seedlings to identify the phenotype of them subjected to salt stress, and then to figure out the interaction between PeHSF and PeWRKYl in Populus euphraticahas.PeHSF was induced to express by high-salinity (150 mM NaCl) in both leaves and callus cultures of P. euphratica and the expression level increased as the salt stress continued till peaked, then went down. The salt-stimulated PeHSF in P. euphratica callus cultures was markedly inhibited by DPI (an inhibitor of plasma membrane (PM) NADPH oxidase) and LaCl3 (an inhibitor of PM Ca2+ permeable channels), indicating that ROS and Ca2+ were involved in activation of HSF under 150 mM NaCl. Over-expression of PeHSF in tobacco significantly improved salt tolerance of transgenic plants, in term of seed germination and root growth. Compared to the wild-type, PeHSF-transgenic lines exhibited an increased capacity to control ROS homeostasis, but did not enhance the capacity to retain ionic homeostasis under salt stress. PeHSF expression in tobacco enhanced activities of ascorbate peroxidase, glutathione peroxidase, and glutathione reductase, due to the up-regulated transcriptional levels.We isolated another transcriptional factor PeWRKY1 from Populus euphratica, and there is high cDNA sequence identity between PeWRKY1 and PeWRKY1 from Populus x canescens. Transgenic tobacco plants overexpressing PeWRKYl showed improved salt tolerance in terms of survival rate, root growth, photosynthesis, and ion fluxes. The promoter of PeWRKY1 and PcWRKYl were further cloned by genomic walking. Promoter sequence analysis showed huge differences between two promoters. PeWRKY1 harbours four tandem repeats of heat shock element (HSE) in the upstream regulatory region. Yeast one-hybrid assay showed that PeHSF can directly binds the cis-acting HSE. To figure out whether the HSE cluster was important for salt-induced WRKY1 expression or not, the promoter-reporter construct PeWRKY1-pro::GUS and PcWRKYl-pro::GUS were transformed into tobacco plants. Compared to PcWRKY1-pro::GUS, β-glucuronidase activities increased in PeWRKYl-pro::GUS transgenic tabacco root, leaf, and stem tissues under salt stress. Furthermore, NaCl-treated P. euphratica exhibited a marked reduction of PeWRKYI when PeHSF was silenced via virus-induced gene silencing (VIGS) system in leaves. Therefore, we concluded that PeHSF plays a crucial role in ROS detoxification rather than in maintenance of the K+/Na+ under salt stress. It is likely that, the salt-induced H2O2 and cytosolic Ca2+ led to transcriptional activation of HSF, which initiated the transcription of genes encoding antioxidant enzymes in P. euphratica, and thus contributed to ROS homeostasis control under saline conditions. Besides that, PeHSF could binds the c/s-acting HSE of the PeWRKYI promoter, thus activating PeWRKY1 expression. The salt-elicited upregulation of PeWRKY1 helps salinized plants maintain K+/Na+ homeostasis under high-salinity conditions.