CUP-SHAPED COTYLEDON2 activatesMIR319Ctranscription and promotes cell proliferation in Arabidopsis leaf primordia

Abstract

AbstractThe 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 ofMIR319C, 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 maintainMIR319Cexpression in leaf primordia are yet unknown. Here, we report the CUP-SHAPED COTYLEDON2 (CUC2) transcription factor as a direct activator ofMIR319Ctranscription. In a yeast one-hybrid (Y1H) screen, we identified several NAC domain TFs as potential regulators ofMIR319C. Subsequent ex vivo binding and transactivation assays suggested that CUC2 may bind to distal promoter region ofMIR319C. Mutants with compromisedCUC2andMIR319Cactivities exhibited smaller leaf areas and reduced pavement cell numbers due to early cell proliferation-to-differentiation transition. Morphometric analysis of higher orderCUC2andMIR319loss-of-function mutants highlighted the crucial role of theCUC2-MIR319module in promoting leaf cell proliferation and indicated functional redundancy among the threeMIR319genes in regulating JAW-TCP levels during leaf growth. Additionally, the phenotypes of mutants with alteredCUC2andMIR319/JAW-TCPactivities demonstrated thatCUC2enhances leaf size through theMIR319C-JAW-TCPpathway. Overall, our findings uncovered a novel role for CUC2 in promoting cell proliferation by activatingMIR319Ctranscription in the leaf primordia.