WRKY33 negatively regulates anthocyanin biosynthesis and cooperates with PHR1 to mediate acclimation to phosphate starvation

Anthocyanin accumulation is acknowledged as a phenotypic indicator under phosphate (Pi) starvation. However, the negative regulator of this process and the molecular mechanisms remain largely unexplored. In this study, our results revealed WRKY33 acts as a negative regulator of phosphorus-status-dependent anthocyanin biosynthesis. WRKY33 regulates the expression of dihydroflavonol 4-reductase (DFR), a rate-limiting enzyme in anthocyanin production, in a direct and indirect manner. WRKY33 directly binds to the DFR promoter to repress its expression or interacts with PAP1 by interfering with the MBW complex to influence its transcriptional activation indirectly. In -Pi conditions, PHR1 interacted with WRKY33, and the protein level of WRKY33 decreased, thereby the repression of WRKY33 on DFR expression attenuated, leading to anthocyanin accumulation in Arabidopsis. Further genetic and biochemical assays suggest that PHR1 is also involved in regulating factors that impact WRKY33 protein turnover. Taken together, our findings reveal that Pi starvation represses WRKY33, a repressor of anthocyanin biosynthesis, to finely tune anthocyanin biosynthesis. This 'double-negative logic' regulation of phosphorus-status-dependent anthocyanin biosynthesis is required for plants to maintain metabolic homeostasis in adaption to Pi starvation.