The phloem protein 2 (PP2) is positively regulated by ThNAC13 that enhances salt tolerance of Tamarix

NAC transcription factors play a significant role in regulating plant responses to biological processes and environmental stresses. Our previous study revealed 21 ThNAC proteins, and ThNAC13 is a nuclear protein that responds to saline stress in Tamarix hispida. To determine whether ThNAC13 regulates downstream genes, RNA sequencing (RNA-seq) and DNA affinity purification sequencing (DAP-seq) were used to identify the binding sites of ThNAC13 at the whole-genome level. In RNAi-silenced ThNAC13 transgenic Tamarix, a total of 4 downregulated differentially expressed genes (DEGs) and 114 peaks strongly associated with four different motifs were identified compared with those in empty vector transgenic Tamarix under salt stress. The c48149.graph_c0 gene contains the key domain of the phloem protein 2 (PP2) protein and is a candidate gene downstream of ThNAC13. Yeast one-hybrid (Y1H), chromatin immunoprecipitation (ChIP) and dual-luciferase reporter (LUC) assays were used to identify ThNAC13 as an upstream regulator of ThPP2. Transgenic Tamarix plants in which ThPP2 was transiently expressed or silenced via RNAi were used for functional gain and loss experiments. The ThPP2-overexpressing plants exhibited decreased hydrogen peroxide (H2O2), superoxide (O-2(center dot-)), and electrolyte leakage (EL) and malondialdehyde (MDA) levels and increased superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activity. In contrast, transient RNAi-mediated silencing of ThPP2 in Tamarix had the opposite effect. ThNAC13 binds to the promoter region of the ThPP2 gene and reduces the reactive oxygen species (ROS) accumulation and enhances antioxidant enzyme activity, positively regulating salt stress tolerance in Tamarix.