High NaCl (200 mM) increases the transcription of phospholipase Dδ (PLDδ) in roots and leaves of the salt-resistant woody species Populus euphratica. We isolated a 1138 bp promoter fragment upstream of the translation initiation codon of PePLDδ. A promoter-reporter construct, PePLDδ-pro::GUS, was introduced into Arabidopsis plants (Arabidopsis thaliana) to demonstrate the NaCl-induced PePLDδ promoter activity in root and leaf tissues. Mass spectrometry analysis of DNA pull-down-enriched proteins in P. euphratica revealed that PeGLABRA3, a basic helix-loop-helix transcription factor, was the target transcription factor for binding the promoter region of PePLDδ. The PeGLABRA3 binding to PePLDδ-pro was further verified by virus-induced gene silencing, luciferase reporter assay (LRA), yeast one-hybrid assay, and electrophoretic mobility shift assay (EMSA). In addition, the PeGLABRA3 gene was cloned and overexpressed in Arabidopsis to determine the function of PeGLABRA3 in salt tolerance. PeGLABRA3-overexpressed Arabidopsis lines (OE1 and OE2) had a greater capacity to scavenge reactive oxygen species (ROS) and to extrude Na+ under salinity stress. Furthermore, the EMSA and LRA results confirmed that PeGLABRA3 interacted with the promoter of AtPLDδ in transgenic plants. The upregulated AtPLDδ in PeGLABRA3-transgenic lines resulted in an increase in phosphatidic acid species under no-salt and saline conditions. We conclude that PeGLABRA3 activated AtPLDδ transcription under salt stress by binding to the AtPLDδ promoter region, conferring Na+ and ROS homeostasis control via signaling pathways mediated by PLDδ and phosphatidic acid.
Keywords: DNA pull-down; Na+/H+ anti-transporter; NaCl; PeGLABRA3; Populus euphratica; ROS; electrophoretic mobility shift assay; luciferase reporter assay; noninvasive micro-test technique; phosphatidic acid; phospholipase D; virus-induced gene silencing.