Epigenetic regulation of miR396 expression by SWR1-C and the effect of miR396 on leaf growth and developmental phase transition in Arabidopsis.

In this study, we investigated the regulatory function of miR396 in the phase transition in Arabidopsis thaliana. Using AtMIR396a/b knockout mutants generated through clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-directed genome editing, we showed that miR396 negatively regulates the leaf size and vegetative phase transition, and the first leaf with abaxial trichomes appeared earlier in the mir396ab double mutant than in the wild type (WT) and was significantly delayed in miR396 overexpression lines. Moreover, mir396ab exhibited early flowering, whereas 35S:MIR396a/b and cib4-1 delayed flowering, and the flowering time was negatively correlated with FT gene expression. Furthermore, in arp6 and pie1 mutants, which are deficient in the ATP-dependent chromatin remodeling complex (SWR1-C), miR396 expression was significantly repressed. Compared with the WT, reduced H2A.Z deposit and stronger relative nucleosome occupancy in the promoter region of MIR396a was found in the arp6 mutant, indicating that SWR1-C contributes to the transcriptional activation of MIR396a via nucleosome dynamics. In addition, miR396 displayed specific spatio-temporal expression patterns in the leaf, which was altered in arp6 and pie1, and therefore affected the transcript levels of CIB4 and FT in these mutants. We propose that miR396 is not only a marker of cell differentiation, but also an age signal for leaf development and phase change. Meanwhile, SWR1-C-mediated epigenetic regulation contributes to the age-dependent enhancement of miR396 expression and differential miR396 accumulation among leaves.