CUP-SHAPED COTYLEDON2 activates MIR319C transcription and promotes cell proliferation in Arabidopsis leaf primordia

The microRNA miR319 regulates leaf shape and size in diverse plant species by reducing the level of the target transcripts that encode JAW-TCP proteins, the transcription factors (TF) which commit the proliferating pavement cells to differentiation. Expression of MIR319C, one of the three miR319-producing genes in Arabidopsis, is expressed throughout the incipient leaf primordia, and the expression domain gets restricted to the base at later stages, partly due to the direct action of JAW-TCPs. However, the factors that activate and maintain MIR319C expression in leaf primordia are yet unknown. Here, we report the CUP-SHAPED COTYLEDON2 (CUC2) transcription factor as a direct activator of MIR319C transcription. In a yeast one-hybrid (Y1H) screen, we identified several NAC domain TFs as potential regulators of MIR319C. Subsequent ex vivo binding and transactivation assays suggested that CUC2 may bind to distal promoter region of MIR319C. Mutants with compromised CUC2 and MIR319C activities exhibited smaller leaf areas and reduced pavement cell numbers due to early cell proliferation-to-differentiation transition. Morphometric analysis of higher order CUC2 and MIR319 loss-of-function mutants highlighted the crucial role of the CUC2-MIR319 module in promoting leaf cell proliferation and indicated functional redundancy among the three MIR319 genes in regulating JAW-TCP levels during leaf growth. Additionally, the phenotypes of mutants with altered CUC2 and MIR319/JAW-TCP activities demonstrated that CUC2 enhances leaf size through the MIR319C-JAW-TCP pathway. Overall, our findings uncovered a novel role for CUC2 in promoting cell proliferation by activating MIR319C transcription in the leaf primordia.