The Molecular Mechanism Of Arginine Methyltransferse SKB1 Controlling Flowering Time In Arabidopsis

Plant flowering is a crucial developmental transition from the vegetative to reproductive phase and is properly timed by a number of intrinsic and environmental cues. Investigations in Arabidopsis have identified four major pathways (photoperiod, vernalization, gibberellin (GA), and autonomous) involved in regulating flowering time. Plant flowering is influenced by epigenetic factors. Epigenetic regulations mainly involve the mechanism of histone modification and chromatin remodeling regulating gene transcription. Chromatin covalent modification included acetylation and methylation of lysine and arginine. The recent characterization of FLC repressors and activators has shown that some of these regulatory proteins are involved in the covalent modification of FLC chromatin and controlling the flowering time.Mammalian SKB1(Shk1 kinase-bingding protein 1),also called PRMT5( Protein Arginine methyltransferases 5)has been shown to regulate chromatin remodelling, gene transcription, RNA splicing, and cell proliferation and differentiation. The Arabidopsis genome contains a single-copy SKB1 gene (At4g31120) shows high homology with the human PRMT5. However, little is known about the functions of histone arginine methylation in plants. In this dissertation, we focused on the genetic and biochemical mechanism of SKB1 controlling flowering time in Arabidopsis and obtained the following results:1. By comparison of Arabidopsis SKB1 amino acid sequence with homologous proteins of diverse organisms, we found that SKB1 contains five conserved motifs of protein arginine methyltransferases. The sequence of SKB1 is highly aligned with PRMT5, which is the homologue of mammalian. To analyze biochemical characterization of SKB1, the His-SKB1 recombinannt protein was purified in E.coli cells. The antibody to SKB1 was produced by using of the method of cutting and reclaiming gels. We assayed in vitro a GST-SKB1 fusion protein for methylation activity with 3H-SAM. Only H4 was methylated.2. We identified two T-DNA insertional mutants. SKB1 lesion results in late flowering. The skb1 mutant plants also formed leaves slightly more curled and darker than wild-type plants. We introduced SKB1 cDNA into skb1mutant plants. 35S::SKB1 could rescue the skb1-1 mutant, resulting in transgenic plants with a wild-type phenotype. Plants overexpressing SKB1 had early flowering feature and SKB1-promoted flowering time showed a stoichiometric relation with SKB1 protein level. So SKB1 is a positive regulator of floral initiation and prevents late flowering.3. We sought to identify the pathway that SKB1 was involved in by treating skb1 mutant plants with the different conditions. Under long-day or short-day photoperiods, skb1 mutant displayed later flowering than wild-type plants, which indicates that skb1 mutants were sensitive to photoperiod. Vernalization and GA treatment reversed in part the delayed flowering of skb1 mutant plants. Taken together, these results suggest that SKB1 is a new member of the autonomous pathway.4. RT-PCR result indicated that the expression of the flowering repressor gene FLC was significantly up-regulated in skb1 mutant. We introduced skb1 into FLC-null mutant. The late-flowering phenotype of skb1 was suppressed by flc mutant. Thus, the flowering time control by SKB1 is targeted towards FLC expression.5. The temporal and spatial expression pattern of SKB1 was analyzed with use of a SKB1 promoter driving GUS, in situ hybridization and Western with a specific antibody against SKB1. SKB1 was more abundant in shoot apex and young leaves in seedlings, which was similar to that of genes in autonomous pathway.6. We used specific antibodies modificated histone of specific site to examine whether SKB1 methylates H4R3. The results suggested that SKB1 can methylate H4R3 symmetrically in vitro and in vivo.7. Chromatin immunoprecipitation (ChIP) assay revealed that SKB1 binds to the FLC promoter and mediates histone H4R3 symmetric dimethylation (H4R3sme2) to control flowering time.Thus, SKB1 affects flowering development by alterating FLC expression via symmetric dimethylation of H4R3 in its promoter.SKB1-mediated H4R3sme2 is a novel histone mark required for repression of FLC expression and flowering time control. Base on these results of this dissertation, we not only clarified the molecular mechanism of aginine methyltransferse SKB1 controlling flowering time in Arabidopsis, but the new histone code of epigenetic inheritance to regulate plant development.