Study Of Molecular Mechanism Of Lignin Metabolism In Tea Plant(Camellia Sinensis)

Tea plant[Camellia sinensis(L.)O.Kuntze],belonging to theaceae,is a kind of perennial crops,and widely cultivated in China and the world.Bud and leaf are important nutrient organs of tea plant,which provide raw materials for the production of non-alcoholic beverage 'tea'.Tea is rich in beneficial to human body health of secondary metabolites,including flavonoids,theanine,carotenoids and tea polysaccharide,etc.The tea leaves are older or tender,is an important basis for judging tea quality.Lignin is the second largest class of molecular organic compounds in plants after cellulose,which play important roles in mechanical support,water transport and stress response.Lignin distributions were different among cultivar,organization,and developmental stage of tea plant.The lignification degree reflects the old or tender of tea fresh leaf,and determine the quality of tea.Lignin metabolism and flavonoids metabolism shared phenylpropanoid metabolic pathways.At present,researches on metabolites of tea plant mainly focus on flavonoids;while the lignin metabolism of tea plant has seldom been reported.Lignin metabolism related genes exist in multiple gene families;however,only a few genes play a major role in lignin synthesis.Therefore,screening and identification of the genes that involved in lignin synthesis are necessary,which provide effective target genes for lignin metabolism in future.Molecular methods(transgenic)and field practices(shading)were used to reduce lignin content,providing scientific basis for improving tea quality.In this study,family analysis and identification of CCR and CAD that involved in the synthesis of lignin in tea plant were carried out.Lignin accumulation and related genes in leaves and stems at different developmental stages of tea plant cultivars "Fudingdabai" and"Suchazao" were analyzed.The correlation between the accumulation of lignin and transcription of related genes in different shade levels were analyzed.By heterogenous expression,the function of CsWRKY13 in regulatory mechanism of lignin pathway and flavonoids pathway was studied.The specific research contents and conclusions are as follows:1.By using the bioinformatics method,a total of 43 CCR family members were identified based on whole genome sequences of tea plant.Evolution analysis shows that only CsCCR1 and CsCCR2 belong to bona fide CCR clade,namely,lignin synthesis related CCR clade.Selection pressure analysis showed that CCR genes of tea plant were mainly affected by purification selection during the evolution process,indicating that these genes were mainly functional genes and their functions were relatively conservative.The gene structure and motif composition were conserved in closely related CCR members of tea plant.Transcriptome data analysis showed that the transcription level of the same CCR gene was similar in different species of Section Thea,indicating that the function of these CCR genes in the evolution of Section Thea was relatively conservative.Transcriptome data from different tissues of tea plant showed that CCR genes existed widely in different tissues of tea plant,and CCR genes with close relatives often showed similar expression trend,indicating that tea plant CCR genes had some degree of functional redundancy.Quantitative analysis showed that multiple CCR genes were highly expressed in the stem tissues of tea plant,and the expression of CCR genes was different in different development stages of tea plant leaves.Based on comprehensive evolutionary analysis and gene expression,CsCCRl and CsCCR2 were recommended as CCR genes for the synthesis of lignin in tea plant.2.Based on tea plant genome data,30 CAD genes were screened and identified.Phylogenetic analysis showed that CAD proteins of tea plant and other species were mainly divided into four subfamilies,i.e.Classes(?-?).Among them,the tea plant CAD members are distributed in Classes(?-?).The study found that there were a large number of replication events in the evolution of tea plant CAD genes.The CAD members with close relationship showed similar motif and motif position.Transcriptome analysis showed that the transcription abundance of the same CAD gene was similar in 16 species of Section Thea,indicating that the function of CAD gene may be conservative in the evolutionary process of Section Thea.Transcriptional analysis of different tissues showed that CAD genes were widely expressed in different tissues of of tea plant,and their functions were diverse.Quantitative analysis showed that multiple CAD genes of tea tree were highly expressed in stem tissue,and different CAD genes had functions in the development of tea leaves,and their functions might be different.CsCAD24 is highly homologous to CAD genes responsible for lignin biosynthesis in other plants and highly expressed in stem tissues in tea plant,which suggest a priority as lignin metabolism CAD genes.3.In the present study,lignin accumulation was investigated in tea plant leaves and stems at three developmental stages.The lignin content continuously increased during leaf and stem development in both tea plant cultivars 'Fudingdabai' and 'Suchazao.' The lignin distribution and anatomical characteristics of the tea plant leaves coincided with lignin accumulation and showed that lignin is mainly distributed in the epidermis,xylem,and vascular bundle sheath.'Suchazao' exhibits a low lignin content and lacks a vascular bundle sheath.Twelve genes encoding the enzymes involved in the lignin biosynthesis of tea plant were identified and included CsPAL,CsC4H,Cs4CL,CsHCT,CsC3H,CsCCoAOMT,CsCCR,CsCAD,CsF5H,CsCOMT,CsPER,and CsLAC.The expression profiling of lignin biosynthesis-related genes and analysis of lignin accumulation may help elaborate the regulatory mechanisms of lignin biosynthesis in tea plant.4.To investigate the effects of shading on lignin accumulation of tea plant,tea plant cultivar 'Longjing 43' was treated with no shading,40%and 80%shading treatments.The leaf area and lignin content of tea plant leaves decreased under shading treatments(especially 80%shading treatment).The lignin distribution and anatomical characteristics of the tea plant leaves showed that lignin is mainly distributed in the xylem.Promoter analysis indicated that the genes involved in lignin pathway contain several light recognition elements.The transcript abundances of 12 lignin-associated genes were altered under shading treatments.Correlation analysis indicated that the most genes showed strong positive correlation with lignin content,and CsPAL,Cs4CL,CsF5H,and CsLAC exhibited significant positively correlation under 40%and 80%shading treatments.This work will potentially helpful to understand the regulation mechanism of lignin pathway under shading treatment.5.The Arabidopsis AtWRKY13 homologous gene,CsWRKY13,was cloned from tea plant 'Longjing 43'.The sequencing results showed that the open reading frame(ORF)of CsWRKY13 gene was 708 bp,encoding 235 amino acids.Sequence analysis showed that CsWRKY13 contained a conserved WRKYGQK amino acid sequence and a type zinc-finger-like motif CX4CX23HXH.Subcellular localization showed that CsWRKY13 was localized in the nucleus of onion epidermal cells.Cs WRKY13 gene was expressed in all tested tissues,especially in stem.Heterogeneously expressed CsWRKY13 gene reduced the content of lignin in transgenic A.thaliana by 5.2%,32%and 27%,respectively,and there was no significant phenotypic difference between the transgenic and wild type A.thaliana.Quantitative analysis showed that transcription levels of phenylpropanoid pathway and lignin-specific pathway related genes in transgenic A.thaliana were down-regulated,especially two CAD genes,AtCAD4 and AtCAD6;while,most flavonoid pathway related genes were significantly up-regulated.This study shows that CsWRKY13 negatively regulates the lignin pathway,while positively regulates the flavonoid pathway.