Overexpression of PbWRKY66 negatively regulate phosphate uptake and root-to-shoot distribution in Arabidopsis

Phoebe bournei is a valuable timber species endemic to southern China, renowned for its economic and ecological importance. However, the widespread low phosphorus level in the acidic soils of this region severely constrains the productivity of P. bournei plantations. Elucidating the molecular mechanisms governing phosphorus uptake, translocation, and homeostasis in P. bournei is therefore essential for breeding varieties with enhanced tolerance to low phosphorus conditions. The current study focuses on PbWRKY66, a group II WRKY transcription factor that was identified and characterized in Phoebe bournei. Under phosphorus deficient conditions, PbWRKY66 expression was markedly induced in leaves but strongly suppressed in roots during prolonged phosphorus deprivation. The PbWRKY66 protein was localized in the nucleus and exhibited transactivation activity. Expression analysis in different tissues revealed that PbWRKY66 transcripts accumulated predominantly in stems and leaves, while the assays of GUS reporter driven by promoter of PbWRKY66 demonstrated strong promoter activity in leaves vein, vascular tissues of stems and roots, flower stalk base, and lateral root tips. Transgenic Arabidopsis thaliana lines overexpressing PbWRKY66 displayed higher sensitivity to phosphorus deficiency, characterized by reduced root elongation, small aerial parts, and increased anthocyanin accumulation compared to the wild type. Phosphorus starvation also resulted in markedly lower total phosphorus in shoots and roots and reduced inorganic phosphate in leaves of the transgenic lines compared with those of the wild type plants. Moreover, the expression of AtPHT1;1, AtPHT1;4, and AtPHO1, which are central to phosphorus uptake and transport, was substantially repressed in PbWRKY66-OE roots, whereas AtSPX1, a negative regulator of the phosphate starvation response, was upregulated. Collectively, these findings point to PbWRKY66 functioning as a negative modulator in the control of phosphorus uptake, translocation from roots to shoots, and phosphate starvation signaling, thereby contributing to the modulation of phosphorus homeostasis under phosphorus deficiency stress in plants.