Identification And Functional Analysis Of Caffeoyl-coenzyme A O-Methyltransferase Family Members In Tea Plant

Tea plant [Camellia sinensis L.(O.)Kuntze],a perennial woody plant of Theaceae family,is one of the economically important crops,which is widely cultivated in China and the world.Buds and leaves of tea plants are the raw materials for tea production and processing.The tenderness of tea plant leaves plays a vital role for the quality and production of the tea industry.The accumulation of lignin is closely related to the tenderness of tea plant leaves and affects the taste,quality and economic value of tea.Until now,there are relatively few researches on genes involved in lignin biosynthesis pathway of tea plant.Although genes related to lignin biosynthesis usually exist in multiple gene families,only a minority of genes play a critical role in lignin metabolism.Therefore,it is necessary to screen and identify the genes in lignin biosynthesis,which provide a reference for further study of regulation mechanism of lignin biosynthesis in tea plant.In this study,lignin accumulation and expression patterns of genes related to lignin biosynthesis in leaves and stems at two different developmental stages of Camellia sinensis cv.‘Longjing 43’,‘Baiyeyihao’ and ‘Fudingdabaicha’ were analyzed.Identification of CCo AOMT family members that involved in the biosynthesis of lignin in tea plant were carried out by bioinformatics,and Cs CCo AOMT6 gene was screened out.By transgenic technology,the function of Cs CCo AOMT6 in lignin biosynthesis pathway was verified.The main results of this article are as follows:1.We collected leaves and stems of three tea cultivars(‘Longjing 43’,‘Baiyeyihao’and ‘Fudingdabaicha’)at two developmental stages,analyzed the lignin contents and characterized the distribution of lignin in tea leaves and stems by using histochemical staining and autofluorescence microscopy.The expression profiles of genes involved in lignin biosynthesis were determined by RT-q PCR.Histochemical staining and autofluorescence microscopy results showed that lignin in tea leaves and stems was mostly deposited on the cell walls of xylem vessels.During the development of tea leaves and stems,lignin contents continuously increased.‘Baiyeyihao’ exhibited the lowest lignin content in the leaves and stems at different developmental stages among the three cultivars.The expression of Cs CCo AOMT gene displayed significantly positively correlated with lignin content at the two developmental stages of leaves and stems.2.A total of 10 Cs CCo AOMTs were identified based on tea plant genome.Systematic analysis of CCo AOMTs was conducted for its physicochemical properties,phylogenetic relationships,conserved motifs,gene structure,and promoter cis-element prediction.Phylogenetic analysis suggested that all the Cs CCo AOMT proteins can be categorized into three clades.The promoters of six Cs CCo AOMT genes possessed lignin-specific ciselements,indicating that they are possibly essential for lignin biosynthesis.According to the distinct tempo-spatial expression profiles,five genes were substantially expressed in eight tested tissues.Most Cs CCo AOMT genes were expressed in stems and leaves in three tea plant cultivars ‘Longjing 43’,‘Baiyeyihao’,and ‘Fudingdabaicha’ by RT-q PCR detection and analysis.The expression levels of two genes(Cs CCo AOMT5 and Cs CCo AOMT6)were higher than those of the other genes.The expression levels of most Cs CCo AOMT genes in ‘Longjing 43’ were significantly higher than that those in‘Baiyeyihao’ and ‘Fudingdabaicha’.Correlation analysis revealed that only the expression levels of Cs CCo AOMT6 were positively correlated with lignin content in the leaves and stems.3.Cs CCo AOMT6 was cloned from C.sinensis cv.‘Longjing 43’.Sequence analysis shows that the open reading frame length of Cs CCo AOMT6 was 738 bp,encoding 245 amino acids.Cs CCo AOMT6 belonged to Ado Met＿MTases super-family containing a highly conserved region called SAM domain.The results of subcellular localization indicated that Cs CCo AOMT6 was a cytoplasmic protein.RT-q PCR analysis revealed that the Cs CCo AOMT6 gene was expressed in all tested tissues,and the expression level was highest in stems,followed by old leaves.In order to further understand the function of this gene,the overexpression of Cs CCo AOMT6 in transgenic Arabidopsis thaliana were obtained through the floral dip method.Compared with wild-type Arabidopsis,the lignin contents in the leaves and stems of transgenic Arabidopsis were significantly increased by35.5 % and 19.2 %,respectively.The expression levels of most genes involved in lignin biosynthesis were significantly enhanced in transgenic plants,indicating that Cs CCo AOMT6 gene played an important role in lignin biosynthesis.