Structural Genes And Transcription Factor Of The Phenylpropanoid Pathway In The Tea Plant (Camellia Sinensis)

The phenylpropanoid pathway of the tea plant(Camellia sinensis var.sinensis cv.Shuchazao)determines the transformation from primary metabolism to secondary metabolism.It can switch on the biosynthesis of compounds,such as catechins,flavonoids,anthocyanins,lignin and lignan,which play an important role in plant growth,development,and responses to various biotic and abiotic stresses.In addition,catechins and lignans have anti-tumor,antiviral and other pharmacological effects,which are of great significance to human health.The synthesis of these compounds depends on the catalysis of enzyme families and the regulation of transcription factors in the phenylpropane pathway.Currently,the functions of phenylalanine ammonia lyase(PAL)and pinoresinol/lariciresinol reductase(PLR)in tea plants still remain unclear.Besides,the function of transcription factor CsMYB 1 acting as a transcriptional repressor of the shikimate,phenylpropanoid,and lignin pathways has been reported previously,but we found that the transcription level of auxin signaling pathway of CsMYB1 transgenic tobacco also changed a lot.So,we conducted research on these problems in this paper.The main research results are as follows:1.Based on the sequencing data available from eight transcriptome projects,six PAL genes were screened out,cloned,and designated as CsPALa-CsPALf.Their transcript expression level displayed tissue-/induced-expression specificity in several tissues or under different exogenous treatments.The phylogenetic tree showed that CsPALs resulted from gene duplication.The subcellular localization result of CsPALs indicated that all six CsPALs exhibited indiscriminate cytosolic locations in epidermis cells and mesophyll cells.Furthermore,in vitro enzymatic assays showed that all six recombinant proteins were characterized by the strict substrate specificity toward L-Phe,but no activity toward L-Tyr,and they displayed subtle differences in kinetics and enzymatic properties.2.Two CsPLRs(CsPLR1 and CsPLR2)were identified in the tea plant by sequence homology searching using FiPLR.The phylogenetic tree showed that different clusters of PLRs were closely related to the catalytic function.The in vitro enzymatic assays showed that CsPLR1 could convert(+)-and(-)-pinoresinol into lariciresinol or secoisolariciresinol,whereas CsPLR2 catalyzed(+)-pinoresinol enantioselectively into(-)-secoisolariciresinol.Homology modeling and site-directed mutagenesis were used to examine the role of a variable loop(165-178 amino acids)in catalysis and substrate selectivity.The L174I mutant in CsPLR1 lost the capacity to reduce either(+)-or(-)-pinoresinol but retained the ability to catalyze the reduction of(-)-lariciresinol.3.Based on the previous transcriptome data analysis of the CsMYB1 transgenic tobacco,we found that CsMYB1 may regulate auxin signal pathway.The results of different concentrations of NAA treatments indicated that CsMYB1 transgenic tobacco was resistant to high concentration of NAA.The auxin inhibitors treatments and transcriptome analysis revealed that the target of CsMYB1 may be NtPIN2.Yeast one-hybrid assay,electrophoretic mobility shift assay and dual luciferase assay confirmed that CsMYB1 can bind the MYBCORE and AC-like elements of NtPIN2 promoter and activate the gene expression of NtPIN2.The NAA treatment of CsMYB1-truncated transgenic tobaccos proved that the C4 functional domain was an activation domain.These results revealed that CsMYB1 can regulate multiple targets and multiple pathways.