Transcriptome Analysis Of Camellia Sinensis Response To Fluoride Treatment And Functional Characterization Of Fluoride Export Protein CsFEX Gene

Tea plant(Camellia sinensis(L.)O.Kuntze)is one of the main economic crops in China.The tea made from its fresh leaves contribute to human health.Studies have shown that 40%-90%of the F in C.sinensis leaves can be easily released into the tea soup by brewing,and then absorbed by human body through drinking tea,so drinking tea is the main pathway for human body to absorb F.Ingesting proper amount of F by drinking tea is beneficial to maintain the stability of human bones and prevent caries and bones,however,excessive absorption for long time will increase the risk of ’tea drinking fluorosis’,so the F content in tea is related to human health.A large number of studies have found that C.sinensis can hyperaccumulated F,especially in its leaves,which can accumulate a large amount of F without showing any toxic symptoms.The mechanism of analyzing F accumulation/detoxification in C.sinensis is to provide a theoretical basis for regulating the F content in C.sinensis leaves and guide the breeding of low-F tea varieties.The ’Longjing Changye’ tea variety was used as experimental material in this study,and the high-throughput sequencing and molecular biology techniques was used to explore the response mechanism of C.sinensis leaves at the transcriptome level under F treatment,and the potential molecular mechanism of F treatment on the accumulation of major bioactive components in C.sinensis leaves,and the functions of fluoride export protein gene CsFEX,the results and conclusions are as follows:1.The transcriptome of C.sinensis under F treatment was analyzed through RNA-Seq technology based on Illumina HiSeqTM 2000 platform.The results indicated that numerous differentially expressed genes involved in ion absorption and transport exists in the process of F absorption in C.sinensis leaves.The up-regulated expression of genes associated with Ca2+and K+uptake and transport(CNGC,TPC1,CAX,and VHA)increased the intracellular and vacuole cation concentration during F accumulation in C.sinensis leaves,increasing the capacity of C.sinensis to absorb F.We also found that some of AQP(PIPs and TIPs)genes may promote F into the intracellular spaces and vacuoles in C.sinensis leaves,respectively.This provides new candidate genes for exploring the molecular mechanism of hyperaccumulation of F in C.sinensis.In addition,genes associated with plant hormone metabolism and signaling pathways(ABA,auxin,and GA),Transcription factors(WRKY,MYB,NAC,bHLH and AP2/ERF),and the ubiquitin system(E2s and E3s)were identified as differentially expressed genes,indicating that they are responsible for the response of C.sinensis leaves to F.Overall,these findings provide a comprehensive understanding of the mechanism of F accumulation/detoxification in C.sinensis.2.The effects of F treatment on the accumulation of F and major bioactive compounds and gene expression were investigated,revealing the molecular mechanisms affecting the accumulation of bioactive compounds by F treatment.The results showed that exogenous F treatment induced an increase in F content and total flavonoid content,but the content of tea polyphenols and free amino acids decreased significantly.Some of the special amino acids,such as sulfur-containing amino acids and proline,had the opposite trend of free amino acids.Caffeine was obviously induced by exogenous F,while the theanine content peaked after two day-treatment.These results suggest that the F accumulation and content of bioactive compounds were dramatically affected by F treatment.Furthermore,differentially expressed genes(DEGs)related to the metabolism of main bioactive compounds and amino acids,especially the pivotal regulatory genes of catechins,caffeine,and theanine biosynthesis pathways,were identified and analyzed using high-throughput Illumina RNA-Seq technology and qRT-PCR.The expression of pivotal regulatory genes is consistent with the changes of the main bioactive compounds in C.sinensis leaves,indicating a complicated molecular mechanism for the above findings.Overall,these data provide a reference for exploring the possible molecular mechanism of the accumulation of major bioactive components in tea leaves under fluoride treatment,which involves changes in the content of flavonoid,catechins,caffeine,theanine,and other amino acids.3.A fluoride exporter protein gene(CsFEX)was newly found and isolated from C.sinensis and its functions in detoxifying F were investigated in transgenic Escherichia coli and Arabidopsis thaliana.CsFEX contains two crcB domains,which is the typical structure in plants.The expression of CsFEXin C.sinensis is tissue specific and related to maturity of leaves,and its expression is significantly induced by F treatments in different tissues of C.sinensis,particularly in leaves.Additionally,the growth of C.sinensis,E.coli,and A.thaliana can all be inhibited by F treatment.However,the growth of CsFEX-overexpression E.coli was increased with lower F content under F treatment compared to the control.Similarly,the germination and growth of CsFEX-overexpression A.thaliana were enhanced with lower F content under F treatment compared to wild type.CsFEX relieves F toxicity in the transgenic E.coli and A.thaliana by alleviating F accumulation.