Transcriptional regulation of a microRNA encoding gene MIR319C during leaf development in Arabidopsis thaliana

Abstract

The evolutionarily conserved microRNA miR319 and its target transcription factors encoded by five CIN-TCPs (TCP2, 3, 4, 10 and 24) regulate leaf morphogenesis in Arabidopsis by triggering the division to differentiation switch of the leaf cells. In a young leaf, the expression of the miR319 encoding gene MIR319C is restricted at the basal region coinciding with the cell proliferation zone, whereas the CIN-TCP transcripts are detected in the more distal region where differentiation is initiated. How the complementary expression patterns of MIR319C and CIN-TCPs are established in leaf primordia is unknown. Moreover, the factors that activate and maintain MIR319C expression in the leaf primordia are yet to be uncovered. Here, a detailed spatiotemporal analysis of the predominantly expressed TCP4 and MIR319C genes suggested the possibility of CIN-TCP mediated downregulation of MIR319C promoter activity in the leaf primordia. Loss of multiple CIN-TCPs resulted in the distal extension of the MIR319C expression domain, whereas ectopic TCP4 activity restricted the MIR319C domain more proximally. TCP4 was enriched at the MIR319C promoter, and increased TCP4 activity enhanced the deposition of H3K27me3 repressive marks on the MIR319C. Additionally, transgenic lines carrying mutations in TCP binding sites on MIR319C promoter exhibited miR319 overexpression phenotypes. Together with the previous knowledge that miR319 degrades CIN-TCP transcripts, our study suggests the existence of a double-negative feedback loop involving the miR319-CIN-TCP module in regulating leaf morphogenesis in Arabidopsis. To uncover the activators of MIR319C in leaf primordia, we screened a leaf-specific Arabidopsis transcription factor (TF) library using a yeast one-hybrid assay to isolate proteins that bind to the 2.7 kb promoter of MIR319C. The screen yielded 57 positives including the six NAM/ATAF1/ATAF2/CUC (NAC) domain-containing TFs with DNA-binding preferences similar to that of the CUC sub-group of NAC TFs, i.e., CUC1, 2 and 3. In addition to...