Populus euphratica WRKY1 binds the promoter of H+-ATPase gene to enhance gene expression and salt tolerance

Abstract

Plasma membrane proton pumps play a crucial role in maintaining ionic homeostasis in salt-resistant Populus euphratica under saline conditions. High levels of NaCl (200 mM) induced PeHA1 expression in P. euphratica roots and leaves. We isolated a 2022 bp promoter fragment upstream of the translational start of PeHA1 from P. euphratica. The promoter-reporter construct PeHA1-pro::GUS was transferred to tobacco plants, demonstrating that β-glucuronidase activities increased in root, leaf, and stem tissues under salt stress. DNA affinity purification sequencing revealed that PeWRKY1 protein targeted the PeHA1 gene. We assessed the salt-induced transcriptional response of PeWRKY1 and its interaction with PeHA1 in P. euphratica. PeWRKY1 binding to the PeHA1 W-box in the promoter region was verified by a yeast one-hybrid assay, EMSA, luciferase reporter assay, and virus-induced gene silencing. Transgenic tobacco plants overexpressing PeWRKY1 had improved expression of NtHA4, which has a cis-acting W-box in the regulatory region, and improved H+ pumping activity in both in vivo and in vitro assays. We conclude that salt stress up-regulated PeHA1 transcription due to the binding of PeWRKY1 to the W-box in the promoter region of PeHA1. Thus, we conclude that enhanced H+ pumping activity enabled salt-stressed plants to retain Na+ homeostasis.

Keywords: DNA affinity purification sequencing; H+-ATPase; NaCl; PeWRKY1; W-box; electrophoretic mobility shift assay; luciferase reporter assay; non-invasive microtest technique; virus-induced gene silencing.