Abstract
Anthocyanin accumulation is acknowledged as a phenotypic indicator of phosphate (Pi) starvation. However, negative regulators of this process and their molecular mechanisms remain largely unexplored. In this study, we demonstrate that WRKY33 acts as a negative regulator of phosphorus-status-dependent anthocyanin biosynthesis. WRKY33 regulates the expression of the gene encoding dihydroflavonol 4-reductase (DFR), a rate-limiting enzyme in anthocyanin production, both directly and indirectly. WRKY33 binds directly to the DFR promoter to repress its expression and also interferes with the MBW complex through interacting with PAP1 to indirectly influence DFR transcriptional activation. Under -Pi conditions, PHR1 interacts with WRKY33, and the protein level of WRKY33 decreases; the repression of DFR expression by WRKY33 is thus attenuated, leading to anthocyanin accumulation in Arabidopsis. Further genetic and biochemical assays suggest that PHR1 is also involved in regulating factors that affect 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 the maintenance of plant metabolic homeostasis during acclimation to Pi starvation.