The Study Of XTH Gene Family Response To Different Concentrations Of Aluminum And Fluoride Treatments On Camellia Sinensis

Tea plant(Camellia sinensis)is a typical plant that accumulates aluminum(Al)and fluoride(F),and the mechanism of accumulating A1 and F is closely related to the construction of cell wall,but the detailed mechanism remains unclear.The main role of xyloglucan endoglycosylase/hydrolases(XTH)is to disrupt and reconnect the xyloglucan chains,modify the cellulose-xyloglucan complex structure in the cell wall,and make the cell wall reconstruct.At the same time,XTH is able to catalyze the newly synthesized xyloglucan molecules and transfer them to the original cell wall network structure,achieving the purpose of maintaining the thickness and integrity during cell wall reconstruction.Studies have shown that XTH plays a key role in the aluminum tolerance of tea plants,but its role in fluoride resistance in tea plants has not been reported.Therefore,this study screened 45 XTH genes in our existing transcriptome database.The expression of each gene was analyzed with different aluminum concentrations(0,0.1,0.4,2.0,and 4.0 mmol/L)and fluorine concentrations(0,8,16 mg/L).By measuring the activity of XET(Xyloglucan endotransglucosylase)and labeling xyloglucans with different degrees of substitution by immunofluorescence,the mechanism of XTH involved in aluminum-rich and quasi-fluorescein in tea plants was explored.Fourteen XTH genes were identified based on 45 XTH candidate genes from the existing tea plant transcriptome database.These genes were named as CsXTH1-CsXTH14.Thirty-three AtXTH proteins from Arabidopsis and the fourteen CsXTH proteins from tea plants were used for constructing an unrooted phylogenetic tree.Tea plant CsXTH6,-7,-11 and-14 belong to subgroup I;CsXTH2,-4,-5,-13 belong to subgroup II;CsXTH1,-3,-9 belong to subgroup IIIA;CsXTH8,-10,-12 belong to subgroup IIIB.Phylogenetic tree and sequence alignment showed that the N(aspartate)-glycosylation site is an essential structure for XTH to perform XET activity.In order to study the function of the CsXTH gene family,their relative expression in different tissues of Longjing 43 tea plant was analyzed by qRT-PCR.The results showed that the expression of 14 CsXTH genes in tea plants was significantly different in different tissues.Among them,CsXTH2,-3,-8 and-13 were highly expressed in roots;CsXTH2,-3,-7,-9 and-11 were higher in stems;CsXTH3,-6,-10 and-11 expression levels were higher in young leaves;CsXTH1,-4,-5,-6,-7,-11,-12 and-14 have higher expression levels in flowers;CsXTH12 has a higher expression level in pollen;CsXTH1,-4,-5 and-7 showed higher expression in fruits;In the old leaves,all of the CsXTH expression levels were relatively low,we speculate that the possible reason is that CsXTH is related to the growth of plants,while the old leaves grow slowly,so the CsXTH expression level is lower than other tissues.In order to investigate the role of CsXTH gene in tea-resistant aluminum fluoride,tea plants were treated with different concentrations of aluminum and fluoride to quantitatively analyze the gene expression.The results showed that under different aluminum stress treatments,CsXTH7,-9,-13 and-14 were all increased in the 0.4 mM aluminum concentration,CsXTH7 expression showed the most significant change.CsXTH1,-4,-6,-7,-8,-12,-13 and-14 showed the highest expression levels when the fluorine concentration was 8 mg/L.The content of xyloglucan in different degrees of substitution in tea plant roots was determined by immunofluorescence labeling.The results showed that when the aluminum concentration was 0.4 mM and the fluorine concentration was 8 mg/L,the brightness of the LM25(It can bind to xyloglucan polymers)was the brightest,indicating that the concentration at this time promoted the growth of the tea plant roots.The double-antibody sandwich method was used to determine the XET activity in tea roots under different treatments.The results showed that when the aluminum concentration was 0.4 mM and the fluorine concentration was 8 mg/L,the XET activity was significantly higher than that of the control group.When the concentration continued to rise,the activity also decreased.This result guides tea planting and further research.This trend is consistent with the results of fluorescent labeling.