DRB1, DRB2 and DRB4 Are Required for Appropriate Regulation of the microRNA399/ Expression Module in .

Adequate phosphorous (P) is essential to plant cells to ensure normal plant growth and development. Therefore, plants employ elegant mechanisms to regulate P abundance across their developmentally distinct tissues. One such mechanism is PHOSPHATE2 (PHO2)-directed ubiquitin-mediated degradation of a cohort of phosphate (PO) transporters. is itself under tight regulation by the PO responsive microRNA (miRNA), miR399. The DOUBLE-STRANDED RNA BINDING (DRB) proteins, DRB1, DRB2 and DRB4, have each been assigned a specific functional role in the () miRNA pathway. Here, we assessed the requirement of DRB1, DRB2 and DRB4 to regulate the miR399/ expression module under PO starvations conditions. Via the phenotypic and molecular assessment of the knockout mutant plant lines, , and , we show here that; (1) DRB1 and DRB2 are required to maintain P homeostasis in shoot and root tissues; (2) DRB1 is the primary DRB required for miR399 production; (3) DRB2 and DRB4 play secondary roles in regulating miR399 production, and; (4) miR399 appears to direct expression regulation of the transcript via both an mRNA cleavage and translational repression mode of RNA silencing. Together, the hierarchical contribution of DRB1, DRB2 and DRB4 demonstrated here to be required for the appropriate regulation of the miR399/ expression module identifies the extreme importance of P homeostasis maintenance in to ensure that numerous vital cellular processes are maintained across tissues under a changing cellular environment.