A Pi-responsive GRAS transcription factor regulates PmSPX1 promoter activity in Pinus massoniana

Abstract

Phosphate (Pi) deficiency is a major environmental constraint limiting plant growth and forest productivity. Although SPX proteins are central regulators of phosphate homeostasis, the upstream transcriptional mechanisms controlling SPX1 expression in conifers remain poorly understood. Here, yeast one-hybrid (Y1H) screening using the PmSPX1 promoter as bait identified the Pinus massoniana GRAS transcription factor PmGRAS8 as a candidate promoter-interacting protein, and a dual-luciferase assay showed that PmGRAS8 enhanced PmSPX1 promoter activity in Nicotiana benthamiana . PmGRAS8 encoded a typical GRAS-family protein and showed nuclear-enriched, but not exclusively nuclear, localization. Its expression under Pi deficiency was strongly tissue- and time-dependent, with marked induction in roots at intermediate treatment stages and variable responses in shoots. Heterologous overexpression of PmGRAS8 in Arabidopsis thaliana was associated with increased ACP, SOD, and POD activities under low Pi. Root phenotypes observed in the initial plate assay are treated as qualitative because WT and transgenic seedlings were grown on separate plates. AtSPX1 expression also differed between WT and overexpression lines under low Pi, but the present data do not establish direct regulation of the Arabidopsis promoter by PmGRAS8. Collectively, the results identify PmGRAS8 as a candidate upstream regulator associated with activation of the PmSPX1 promoter and support a possible role in Pi-deficiency responses. Identification of the PmGRAS8-binding motif and direct biochemical or chromatin-based validation will be required to establish direct DNA binding and native function in P. massoniana .