Study On Mechanism Of Fluorine Accumulation In Cell Wall Of Tea Plant Leaf

Tea is one of the three largest non-alcoholic beverages in the world,which is widely favored by consumers.Tea plant is a fluorine(F)accumulator,which can enrich certain amount of F in its mature leaves.Chronically and largely drinking some types of tea processed from older leaves(such as brick tea)may cause excessive absorption of F by the human body,posing potential risks to human health.Therefore,conducting researchs on F enrichment mechanism of tea plant can provide a theoretical basis for fundamentally solving the problem of F safety by drinking tea.The cell wall is the main part for F accumulation in tea plant leaf,whereas the specific mechanism of F accumulation in tea leaf cell wall is currently unclear.In this study,tea seedlings were treated with different concentrations of F or Ca-F interactions to further analyze the relationship between the metabolic profiling,pectin biosynthesis,cell wall structure and compositions and the F accumulation in tea leaf cell wall,aiming to provide a theoretical reference for regulating the level of F in tea plants.The main research contents and results are as follows:1.F treatments significantly affect the carbohydrate metabolism in leaves of tea plant.Metabolomics studies on tea plant in response to F stress indicated that F made significant effect on the metabolism of tea leaves.Univariate and multivariate statistical analysis of GC-TOF-MS data from different F treatments identified differential metabolites including malic acid,galacturonic acid,glucaric acid,fructose,sorbose,maltose,lactose,inositol,dihydrogen quercetin,etc.and pathway analysis identified key metabolic pathways including galactose metabolism,fructose and mannose metabolism,starch and sucrose metabolism,TCA cycle and pectin biosynthesis.F promoted the biosynthesis of pectin materials in tea leaf.Further study was focused on the changes of pectin biosynthesis pathway in tea plant leaf under F treatments.It was found that F could decrease the relative content of the precursors for pectin biosynthesis,increase pectin content,induce the over-expression of key genes involved in pectin biosynthesis and raise the activity of corresponding enzymes.All ofthese results indicated that F made a promotion effect on pectin biosynthesis.2.Binding F in pectin fraction is an important way for the enrichment of F in tea plant.It was found that 67%-92% of leaf F were distributed in the cell wall part,of which56%-71% of cell wall F were assigned to the pectin component,followed by hemicellulose and cellulose components,accounting for 20%-28% and 8%-15%,respectively,indicating that pectin was the main binding site for the accumulation of F in tea leaf.The proportion of F in pectin was raised with the increase of exogenous F concentration,indicating that pectin combining with F was crucial for tea plant to accumulate exogenous F.3.The change of tea plant leaf cell wall structure and composition is closely related to its enrichment of F.The content of metal ions in tea leaf cell wall is closely related to its F enrichment.F significantly increased the content of Cu,Mg,Zn,Al,Ca,Ba,and B in the cell wall of tea leaf.Moreover,within different F treatments,the changes in the cell wall F content and Cu,Mg,Zn content exhibited significantly positive correlation,indicating that tea leaf cell wall accumulating F was closely related to the accumulation of metals and B ion.Tea leaf cell wall accumulating F is closely related to the pectin structure.F induced an increase in the content of total cell wall,total pectin and cellulose but an decrease in the content of xyloglucan and RGI type pectin in the cell wall of tea plant leaf.Moreover,the results of FT-IR analysis showed that F caused a decrease in the number of esterified carboxyl functional groups but an increase in the number of ionized carboxyl functional groups in tea leaf cell wall.The results of immol/Lunohistochemical staining revealed that F could induce an increase in the fluorescence intensity of low methylesterified pectin and a decrease in the fluorescence intensity of high methylesterified pectin.These two results indicated that F could promote the demethylesterification of tea leaf pectin.F-induced increase of pectin content and pectin demethylesterification will cause an increase in the number of free carboxyl groups in the cell wall,which provides more binding sites for cell wall metals.It is speculated that F may be chelated in the cell wall by forming a certain complex with pectin and metal ions.4.The Ca-regulated F enrichment in tea plant is related to the tea plant physiological status and cell wall structure.An appropriate exogenous Ca concentration(0.5 or 5 mmol/L)protected the tea plant from F damage,however,both Ca deficiency and excess induced excessive accumulation of exogenous F and caused double stress of Ca and F in tea plant,which indicated that an extreme calcium concentration can induce excessive absorption of F by tea plants.Ca significantly reduced the accumulation of exogenous F in the cell wall of tea plant leaves,of which high Ca treatments could cause a decrease in the proportion of cell wall F to total leaf F.Analysis on the F content in each cell wall component showed that Ca inhibited the F adsorption in pectin and hemicellulose components,while promoted the accumulation of F in cellulose.Moreover,the assignment of cell wall F to both pectin and hemicellulose fractions was reduced.These results indicated that the reason why Ca could decrease the F level in tea leaf was that Ca inhibited the F accumulation in cell wall pectin and hemicellulose.Ca-regulated cell wall metals level is related to its F-binding ability.Under the F-present condition,in different Ca concentrations,the F content in tea leaf cell wall and its pectin and hemicellulose fractions was significantly positively correlated with the content of cell wall Al,Mg,Cu,Zn,Mn,K and B.This is consistent with the results of different F treatment experiments in this study,which showed that the enrichment of F was closely related to the accumulation of metal ions and B ions in the cell wall of tea leaf.Ca-regulated cell wall pectin structure is related to its F-binding ability.The Ca-induced changes in cell wall materials and xyloglucan content were significantly positively correlated with the F content.Although the change of pectin content showed an opposite trend with F content under different calcium concentrations,immol/Lunohistochemical analysis on pectin structure observed that F induced an increase in the content of high-methylesterified pectin,while a decrease in the content of LM19-labeled low-methylesterified pectin,which would cause a decrease of binding sitesfor cell wall metals.The above results indicate that the inhibition effect of Ca on the F enrichment in tea leaf cell wall is related to the decreased cell wall and xyloglucan content,increased pectin esterification and decreased metals contents.Ca may cause the reduction of cell wall metal ions level by affecting the methyl esterification structure of tea leaf cell wall pectin,which leads to a decrease in the cell wall's ability to chelate F ions.