Transcriptome Analysis Of Tea Shoot Tenderness And Functional Study Of CsLAC3 Involved In Lignin Synthesis

Tea plant [Camellia sinensis(L.)O.Kuntze] is the most economically valuable plant in Theaceae,whose fresh leaves are processed into one of the world’s top three non-alcoholic beverages.As the basic raw material of tea,the tenderness of shoots is of great significance to the formation of tea quality.In this study,based on the results of RNA-Seq analysis,the contents and biosynthesis of cell wall components such as lignin,cellulose,hemicelluloses and pectin were comprehensively analyzed,and the function and regulatory mechanism of CsLAC33 gene was further discussed.The main research results and conclusions are as follows:(1)Based on RNA-Seq analysis,8548 and 6246 DEGs were identified in leaves and stems,respectively.GO and KEGG enrichment results showed that DEGs were mainly enriched in photosynthesis,DNA assembly,cell wall biosynthesis and metabolism.The comprehensive analysis of phenylpropane metabolic and its downstream pathway showed that with the decrease of shoot tenderness,catechin and anthocyanin contents and biosynthesis-related Cs CHS,Cs ANR,Cs LAR and Cs ANS genes were down-regulated.The expression levels of Cs HCT,Cs CAD and Cs PER genes in lignin synthesis branches are up-regulated,and Cs CHS and Cs HCT may be the key node genes controlling the flow direction of the two branches.At the same time,this process may be regulated by MYB,WRKY and other transcription factors,among which MYB may be a key transcription factor regulating lignin biosynthesis and flavonoid synthesis.On the other hand,cellulose content increased with the decrease of shoot tenderness,and the expressions of synthetase Cs Ces A and Cs COBL were up-regulated.Similarly,the decrease of shoot tenderness was accompanied by the increase of hemicellulose content and the increase of Cs IRX,Cs CSL and other synthase gene expression levels.In addition,the total pectin content was positively correlated with shoot tenderness,which might be regulated by the increase of Cs GAUT gene expression level.Cs PME,as a key enzyme gene of pectin morphological transformation,is mainly involved in the accumulation of soluble pectin.(2)Among the differentially expressed CsLAC3 genes in stems and leaves,the expression levels of 10 CsLAC3 genes increased with the decrease of shoot tenderness,indicating that they played an important role in the lignin synthesis process of shoots.The full-length c DNA sequence of CsLAC32 ～ 6 genes were cloned,and the protein sequences encoded by these genes were highly conserved and localized in cell membrane or cell wall.In particular,CsLAC33 was linked to LAC,which is known to regulate lignin synthesis in Arabidopsis thaliana.The lignin content in Arabidopsis thaliana stems with overexpression of CsLAC33 was significantly higher than that in control lines,indicating its key role in mediating lignin synthesis.In addition,in vitro experiments confirmed that the expression of CsLAC33 could be inhibited by Csmi RNA397,and the expression of CsLAC33 homologous gene At LAC2 in Arabidopsis thaliana that overexpressed Csmi RNA397 was also significantly inhibited.In addition,overexpressed Csmi RNA397 strains showed the characteristic phenotypes related to lignin content,such as root length increasing,leaf area decreasing,secondary xylem thinning and mechanical support decreasing,which indicated that Csmi RNA397 might affect lignin biosynthesis by inhibiting the expression of CsLAC33.This study helps to elucidate the molecular mechanism of CsLAC3-mediated lignin synthesis involved in the regulation of the new tenderness of tea plant,provides a theoretical reference for the study of tea shoot tenderness and is expected to serve the improvement of tea shoot tenderness and tea quality.