Identification And Characterization Of Genes Related To Growth And Secondary Metabolism In Tea Plant

Tea（Camellia sinensis）is the most frequently consumed natural beverage across the world produced with the young leaves and shoots of the evergreen perennial plant Camellia sinensis（L.）O.Kuntze due to high content of secondary metabolites especially catechin and anthocyanins which collectively determine the rich flavors and health benefits of the infusion.Light is an important source of energy as well as external factor for the regulation of biosynthesis and accumulation of this multiple secondary metabolites in tea plants.Light emitting diodes（LEDs）have recently been considered as an efficient artificial light source in plant factories for enhancing plant growth and secondary metabolism accumulation.The pathways of secondary metabolites（catechin and anthocyaninss）has been generally established.However,studies about the gene expression for growth and secondary metabolism in tea（C.sinensis）under blue and red light have not been clearly reported.The purpose of this study is to evaluate the gene expression that correlated with secondary metabolites accumulation and growth of tea plant cultivar under various types of LED lights.In this research,different cultivars of tea plants,Zhong Ming 6（ZM6）and Longjing 43（LJ43）and overexpression of A.thaliana were used as materials to study the plant growth and secondary metabolism and the function of over expression BAHD acyltransferase gene（TEA031065）respectively,which is correlated with plant growth and secondary metabolites accumulation,in tea plant.High performance liquid chromatography（HPLC）,real-time quantitive PCR（q RT-PCR）and 60CRT dual-wavelength with double-beam Prove UV-2550 spectrophotometer were applied to explore the gene function and secondary metabolites accumulation.The main conclusions are as follows:ZM6 is a green tea（C.sinensis）cultivar that highly accumulates TGGP（1,2,6-tri-O-galloyl-β-D-glucopyranose）were used to investigate the variation in phenotypic characteristics with secondary metabolites and related gene expression under blue light(BL,200μmol m^–2 s^–1),red light(RL,200μmol m^–2 s^–1)and white light(CK/WL,200μmol m^–2 s^–1).The correlated analysis of secondary metabolites analysis and the related differentially expression genes（DEGs）with TFs（transcription factors）was conducted to identify the regulatory networks.Chlorophyll content,Caffeine,Gallic acid and TGGP accumulations were also identified.Moreover,these data revealed DEGs for regulation of secondary metabolites in ZM6 tea cultivar especially for catechin and anthocyaninss biosynthesis pathways.In this research,BL showed high accumulation of secondary metabolites with small rosettes but RL displayed high amount of chlorophyll with specious phenotypes.Due to the upregulation of the CsGSTF12 gene in the flavonoid biosynthesis pathway,anthocyanins production under BL was much higher than those under RL and WL.However,CsMYB75,Cs3GGT,b HLH-MYC,and other R2R3-MYBs were also highly upregulated under BL,which increased the accumulation of TC and anthocyaninss in tea plants.While CsMYB4 gene showed the highest expression that positively correlated with TC and anthocyanins accumulation under RL.Results showed that BL increased the malondialdehyde（MDA）content as well as the activity of superoxide dismutase（SOD）and peroxidase（POD）due to enhancing the secondary metabolites（catechin and anthocyaninss）as compared to RL and WL.To explore the molecular mechanism of secondary metabolites with growth and development of tea plants,TEA031065（BAHD acyltransferase）gene was selected in LJ43 following the RNA sequencing data from the previous study for further study.Twenty one BAHD acyltransferase genes were screened based on the tea plant（C.sinensis var.sinensis）genome database.The result showed that among the BAHD acyltransferase genes in tea cultivars,the expression of TEA031065 was highest in buds and young leaves following the RNA sequencing data,which was coincident with the tissue rich in catechin and other flavonoids.The expression mode of TEA031065 in young leaves were further investigated.Then these gene was transformed into wild-type Arabidopsis as an over-expression（OX）line 1 and line 2 in?MS media to verify its function.In the wild types（WT）,the primary root length,number of secondary roots,and total root weight were significantly higher at24%,15%,and 53.92%,respectively,compared to the transgenic lines（OX1 and OX2）.By contrast,the leaves displayed larger rosettes（21.58%）,with higher total leaf weight（32.64%）in the transgenic lines than in the wild type（WT）.These result is consistent with dcr mutant At5g23940 gene in A.thaliana.While,anthocyanins content in transgenic lines was also increased（21.65%）as compared to WT.Accroding to RNA sequencing data,IAA（Auxin）and GA（Gibberellin）biosynthesis were downregulated in the transgenic lines,which might be correlated with the phenotype changes in roots.Moreover,the upregulation of plant growth regulation genes,such as UGT73C4（zeatin）,ARR15,GH3.5,ETR2,ERS2,APH4 and SAG113 might be responsible for massive leaf growth.Transgenic lines shown high anthocyanins accumulation due to the upregulation of the AT and 3 GSTF particularly,GSTF12 genes in the flavonoid biosynthesis pathway.However,the TFs such as,CCo AMT,b HLH,WRKY,CYP and other MYBs were also significantly upregulated in transgenic lines,which increased the content of anthocyaninss in A.thaliana seedlings.Based on the above results,it suggested that BL increased the secondary metabolites specially catechin and anthocyanins and antioxident.Meanwhile,RL facilitated the plant growth and development with chlorophyll content.In addition,TEA031065（a member of BAHD acyltransferase）gene can be facilitated the plant growth and secondary metabolites accumulations in tea plant（C.sinensis）.