Molecular mechanism of microRNA396 mediating pistil development in Arabidopsis

The precise control of gene regulation, and hence, correct spatiotemporal tissue patterning, is crucial for plant development. Plant microRNAs can constrain the expression of their target genes at posttranscriptional levels. Recently, microRNA396 (miR396) has been characterized to regulate leaf development by mediating cleavage of its GROWTH-REGULATING FACTOR (GRF) targets. miR396 is also preferentially expressed in flowers. However, its function in flower development is unclear. In addition to narrow leaves, pistils with a single carpel were also observed in miR396 overexpression plants. The dramatically reduced expression levels of miR396 targets (GRF1, GRF2, GRF3, GRF4, GRF7, GRF8, and GRF9) caused pistil abnormalities, because the miR396-resistant version of GRF was able to rescue miR396-overexpressing plants. Both GRF and GRF-INTERACTING FACTOR (GIF) genes are highly expressed in developing pistils, and their expression patterns are negatively correlated with that of miR396. GRF interacted with GIF to form the GRF/GIF complex in plant cell nucleus. miR396 suppressed the expression of GRF genes, resulting in reduction of GRF/GIF complex. gif single mutant displayed normal pistils, whereas gif triple mutant gif1/gif2/gif3 produced abnormal pistils, which was a phenocopy of 35S:MIR396a/grf5 plants. GRF and GIF function as cotranscription factors, and both are required for pistil development. Our analyses reveal an important role for miR396 in controlling carpel number and pistil development via regulation of the GRF/GIF complex.