Studies On The Mechanisms Of Leaf Adaxial-Abaxial Polarity Formation Regualted By MiR396 And 26S Proteasome Pathway In Arabidopsis

During leaf development, the adaxial-abaxial formation is critical for subsequent leaf morphogenesis and functions. In recent years, it has been demonstrated that the processes of leaf adaxial-abaxial formation are very complex and involve a complicated gene regulatory network, including the regulations at different levels mediated by a number of factors such as transcriptional regulation by transcription factors, post-transcriptional regulation by non-coding small RNA and protein translational and posttranslational regulation mediated by ribosome proteins and the 26S proteasome. Within the network, two types of small RNAs, miR165/166 and tasiR-ARF, play critical roles in leaf adaxial-abaxial formation through breaking the transcripts of targeted transcription factors to repressing gene expression posttranscriptionally. However, it is not clear if there are other small RNAs involved in the leaf adaxial-abaxial polarity formation. Previous studies have demonstrated that the proteolytic function of 26S proteasome is required for leaf polarity formation, but the target genes of 26S proteasome pathway and their action mechanisms remain unknown.In this study, we first investigated the functions of miR396 in leaf morphogenesis of Arabidopsis. We found that miR396 function as a negative regulator of cell proliferation by repressing the expression of multiple GROWTH-REGULATING FACTORs (AtGRFs) and play important roles in the leaf adaxial-abaxial polarity formation during leaf morphogenesis. We made the constructs to overexpress miR396 in wild type plants, and found that transgenic miR396 overexpression resulted in plants with narrower leaves with decreased cell number and enlarged cell size. By qRT-PCR, the expressions of multiple AtGRFs genes targeted by miR396 were markedly decreased in transgenic plants overexpressing miR396. ASYMMETRIC LEAVES1 and 2 (AS1 and AS2) are two critical regulators of leaf adaxial identity in Arabidopsis. To probe if miR396 has functions in leaf polarity formation, miR396 was further overexpressed in the as1/2 mutants. We found that cell proliferation defects by transgenic miR396 overexpression could enhance the leaf polarity defects of asymmetric leaves2 (as2), a mutant with mild abaxialized leaves, resulting in plants with the needle- or lotus-like leaves with severe leaf polarity defects, which could be rescued by the introduction of a miR396-resistant version of AtGRF9 gene. The cell proliferation in leaves largely depends on the proper progression of mitotic cell cycle. MiR396 is mainly expressed in the cells that arrested for cell division, and transgenic miR396 overexpression markedly decreased the expression of several marker genes of different phases of cell cycle, indicating that the miR396-AtGRFs pathway likely regulates the entry of mitotic cell cycle. Additionally, we found that miR396 is highly expressed in the leaf trichomes and miR396-targeted AtGRFs are required for trichome cell differentiation by regulating trichome-specific gene expression. Taken together, our results demonstrated that leaf morphogenesis requires a coordination process of cell proliferation and differentiation regulated by miR396-AtGRFs module.To dissect the mechanisms of the leaf polarity formation regulated by the 26S proteasome pathway, we performed an EMS mutagenesis using the seeds of ae3-2 as1-1double mutant and seeked for the suppressor mutants that may rescue the leaf polarity defects of double mutant. In this study, we analyzed one of the suppressor mutants of ae3-2 as1-1—107. The suppressor 107 could rescue the leaf polarity defects ae3-2 as1-1, such as the needle- and lotus-like leaves. Analyzing of F2 progeny of 107 crossed with wild type indicated that the 107 mutation segregated according to Mendel'law of recombination. The 107 single mutants were obtained by genetic analysis and exhibited pleitrophic phenotypes including defects in floral organ formation and deep leaf serrations. The 107 gene was mapped to the upper arm of chromosome 5 by using a set of molecular markers. Within this region, we found that a mutant of protein kinase showed the phenotype similar to 107. Sequencing this protein kinase gene in 107 mutant indicates that 107 gene likely encode this protein kinase. We are studying the underlying mechanisms of leaf polarity formation regulated by 107 gene and the relationship between 107 and 26S proteasome.