| dc.description.abstract | The evolutionarily conserved microRNA miR319 and its target transcription factors encoded by five CIN-TCPs (TCP2, 3, 4, 10 & 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 & 3. In addition to the ability of the CUC proteins to bind to the MIR319C promoter region in yeast, the expression domain of CUC2 overlaps with that of MIR319C in early leaf primordia, suggesting a role for CUCs in the activation of MIR319C during leaf development. Loss of CUC2 activity significantly reduced the MIR319C expression domain, whereas increased CUC2 level led to a distal expansion of MIR319C expression. Elevated CUC2 level partly rescued the TCP4-mediated suppression of MIR319C expression suggesting that CUC2 and TCP4 interact to establish the domain of MIR319C expression in leaf primordia. Thus, we have identified CUCs as the activators of MIR319C in the leaf primordia.
In conclusion, we propose a model where the CUC proteins initially activate MIR319C throughout early leaf primordia. As development progresses, the CIN-TCP genes are expressed towards the distal end of the primordia by the action of yet unidentified factors, and the onset of CIN-TCP activity results in the downregulation of MIR319C transcription in the distal primordia, possibly by recruiting chromatin modifiers. Strong CUC activity at the base sustains MIR319C expression in the proximal region, where CIN-TCP transcripts are degraded by mature miR319. Thus, our study provides evidence that a CUC-MIR319C-CIN-TCP module patterns a uniformly growing leaf primordium into the proximal and the distal growth domains, where the cells in the basal region continue to divide and grow, whereas cells in the distal region stop dividing and start differentiating. | en_US |
