The Function Analysis Of R2R3-MYB Transcription Factor CsMYB4a In The Tea Plants (Camellia Sinensis)

The R2R3-MYB subgroup 4(SG4)transcription factor is a well-known transcriptional repressor of the phenylpropanoid pathway and the lignin pathway.This thesis demonstrated that Cs MYB4 a not only inhibits the synthesis of phenylpropanoid and lignin,but also regulates plant growth and leaf polarity in tea plants.The function of the Cs MYB4 a transcriptional repressors was regulated by its phosphorylation modifications.The principal findings are summarized as follows:1.Transcriptome analysis revealed that Cs MYB4 a inhibits genes related to the auxin signaling pathway in transgenic tobacco earlier than the repression of phenylpropanoid pathway.Yeast two-hybrid assay showed Cs IAA4 is a target protein of Cs MYB4 a,which shares similar expression patterns with Cs MYB4 a in flowers and roots.Further gene silencing assay demonstrated that both Cs MYB4 a and Cs IAA4 repress flower filament elongation in tea plants.These findings suggest that Cs MYB4 a regulate filament elongation by regulating the Cs IAA4-mediated growth hormone signaling pathway.2.This thesis confirmed the interaction between Cs MYB4 a and Cs IAA4 through protein modeling and protein-protein interaction technology.The site of interaction located at the N-terminus of Cs IAA4.Protein degradation assay also revealed that Cs MYB4 a enhances the stability of Cs IAA4.Knocking out Nt IAA4,the homologous gene of Cs IAA4 in Cs MYB4 a transgenic tobacco,resulted in alleviated inhibition on plant growth and filament elongation by Cs MYB4 a.In conclusion,our findings suggest that Cs MYB4 a regulates the auxin signaling pathway by increasing the stability of Cs IAA4.3.Phosphorylation modifications regulate the repress function of Cs MYB4 a.In this thesis,sequence alignment analysis showed that R2R3-MYB subgroup IV transcription factors have varying phosphorylation sites in different plants.Based on differences of amino acid sequences in the C2 domain,members of this subgroup can be classified into four categories:SL type,SP type,MYB3 type,and Fa type.Among them,only SP type has a mitogen-activated protein kinase(MAPK)phosphorylation site located within its C2 domain.Cs MYB4 a belongs to the SP type.4.In this thesis,Cs MYB4 a was found that interact with and be phosphorylated by Cs MPK3-2 through protein-protein interaction.Further experiments revealed that phosphorylation of Cs MYB4 a weakened its ability to inhibit the expression of phenylpropanoid pathway genes PAL,C4 H,and 4CL.Notably,when the C2 domain was either truncated or phosphorylated,the activation ability of the C1 domain in Cs MYB4 a was displayed.Additionally,after phosphorylation by Cs MPK3-2,expression of transcription factor YABBY5 which maintains leaf adaxial-abaxial polarity could be activated by Cs MYB4 a.Knocking out Nt YAB5 in Cs MYB4 a transgenic tobacco resulted in reduced leaf shrinkage phenotype caused by Nt YAB5.Taken together,these results indicate that phosphorylation modification weakens inhibitory effect of Cs MYB4 a on phenylpropanoid pathway genes.Additionally,phosphorylated Cs MYB4 a activates transcriptional activation via its C1 domain and induces YAB5 gene expression.This ultimately affects leaf polarity,leading to shrinkage and curling.