Functional Characterization Of CsMYB4a Transcription Factor And Two Target Genes Cs4CLs In Tea Plant(Camellia Sinensis)

Green tea(Camellia sinensis,Cs)contains an abundant level of numerous phenylpropanoid compounds.How phenylpropanoid biosynthesis in tea plants is regulated,however,is largely unknown.The MYB4-subgroup is well-known transcriptional repressor of lignin production,and some genes such as C4 H is the target of its action.In addition,flavonoid accumulation is regulated by the MYB4-subgroup.In the present study,an R2R3-MYB transcription factor(CsMYB4a)was isolated from leaf tissues.Amino acid sequence alignment and phylogenetic analyses indicated that CsMYB4 a is a member of the MYB4-subgroup.Ectopic expression of CsMYB4 a in tobacco plants resulted in dwarf,shrinking and yellowish leaves,and early senescence phenotypes.Genome-wide transcriptomic analysis revealed significant downregulation of multiple genes involved in the shikimate and phenylpropanoid pathways which regulate the biosynthesis of phenylalanine(Phe),lignin,and other compounds.Multiple genes involved in the tricarboxylic acid(TCA)cycle,glycolysis,and sugar metabolism were also downregulated.Metabolic profiling revealed that there was a reduction of lignin in transgenic plants,as well as other phenylpropanoids,Phe and other amino acids,and sugars.The promoter sequences of 45 genes from tobacco and the coding region of 5 genes from tea plants were isolated.Four types of AC-elements were identified as binding sites.Both EMSA and Dual-luciferase analyses demonstrated that CsMYB4 a can bind to the promoters of the identified,down-regulated genes.The collective data indicate that CsMYB4 a is a repressor of the phenylpropanoid and shikimate pathways in tea and has pleiotropic effects on plant metabolism.Phenylpropanoid pathway is an important secondary metabolism pathway in plant,it connect the primary metabolism from shikimate pathway to other important secondary metablosims especially the lignin and flavonoid metablosims.The last strcture gene in phenylpropanoid is the 4CL(p-coumaric acid coenzyme A ligase).4CL is incolved in the lignin production and flavoionds production in plants.In this paper,two members of the 4CL gene(Cs4CL1 and Cs4CL2)in tea plant were identified and characterized.Amino acids anlignment and phylogenetic analyses indicated these two genes were all 4CL gene familiy members,Cs4CL1 clustered in Class I(lignin metabolism)and Cs4CL2 clustered in classII(flavoiond metabolism).The recombinant Cs4CL1 and Cs4CL2 proteins can catalyze the caffeic acid,p-coumaric acid and ferulic acid to caffeic acid-coa,p-coumaric acid-coa and ferulic acid-coa in vitro.Caffeic acid is the optimum substrate for Cs4CL1 and p-coumaric acid is the optimum substrate for Cs4CL2.Cs4CL1 and Cs4CL2 protein all localized in the cytoplasm and endoplasmic reticulum.Ectopic expression of Cs4CL1 and Cs4CL2 in tobacco plants results in more accumulation of lignin and flavonoid compounds.The promoters of Cs4CL1 and Cs4CL2 were cloned by the genome-walker.Abundant of the light sensitive elements were found in the promoters of Cs4CL1 and Cs4CL2.In addition,in Cs4CL1 promoter,more defense elements and meristem specific elemts were detected compared to the promoter of Cs4CL2.Our data verified the function of Cs4CL1 and Cs4CL2 in tea plant and the Cs4CL1 is probably involved in lignin metabolism and Cs4CL2 is probably involved in flavoiond metabolism in tea plants.