Populus euphratica WRKY1 binds the promoter of PeHA1 to enhance gene expression and salt tolerance

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 promoter region was
verified by a yeast one-hybrid assay, electrophoretic mobility shift
assay, 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 H+
pumping activity in both in vivo and in vitro assays. We conclude that
salt stress upregulated 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.