| Tea plant[Camellia sinensis(L.)O.Kuntze]is a super accumulator of fluoride,which can absorb fluoride from the environment continuously through the roots and accumulate a large quantity of fluoride in leaves.There are 40%to 90%of the fluorides in tea leaves can be released into the tea soup.Then people will take in fluoride by drinking tea.However,long-term consumption of tea or tea products with high fluoride content will increase the risk of“tea drinking fluorosis”,which may lead to the emergence of dental fluorosis,osteofluorosis and other diseases.In addition,the absorption and accumulation of fluoride in Camellia sinensis are mainly affected by external pH.And the soil environment which is suitable for the growth of C.sinensis is acidic.Therefore,it is more practical to study the characteristics of absorbing and accumulating fluoride and the mechanism of fluoride tolerance in C.sinensis under treatments of different pH(4.5-6.5).And the study on the mechanism of fluoride accumulation and fluoride tolerance of C.sinensis can provide theoretical basis for regulating fluoride accumulation and reducing fluoride content of tea.In this paper,the accumulation of fluoride in different tissues and the changes of antioxidant capacity of C.sinensis under fluoride and different pH treatments were determined.And the expression of CsFEX gene under different treatments was investigated.The function of CsFEX gene under different pH was further verified by heterologous expression.At the same time,the proton dynamic flow and membrane potential of the roots of C.sinensis and Arabidopsis thaliana under different treatments were monitored by NMT(Non-invasive Micro-test Technology),in order to understand the microscopic process of fluoride absorption and transport.The main results are as follows.1.The accumulation and transport of fluoride in the roots,stems and leaves of C.sinensis,the expression levels of CsFEX gene and the changes of antioxidant activities in C.sinensis were investigated by treating C.sinensis with different concentrations of fluoride and different pH.The results showed that the fluoride accumulations of C.sinensis were different under different pH conditions.The accumulations of fluoride in roots,stems and leaves were higher at pH 6.5.And the fluoride transport efficiency of C.sinensis was the highest under pH 4.5.The content of fluoride in C.sinensis was related to the expression levels of CsFEX gene and the change of antioxidant properties.SOD activities decreased significantly after 2 days of fluoride treatments;POD and CAT activities increased significantly after fluoride treatments;MDA contents also increased significantly after treatments.And the degree of the increase of activities or contents were all the most under pH 6.5.The CsFEX gene in roots,stems and leaves responded stronger to pH 4.5 and 6.5after fluoride treatments.2.The fluoride tolerance and fluoride accumulation of transgenic Escherichia coli and A.thaliana were compared to the wild types under different concentrations of fluoride and different pH.The results showed that the higher the fluoride concentration or the lower the pH,the more sensitive wild type E.coli and A.thaliana to fluoride.The transgenic E.coli and A.thaliana could enhance the fluoride tolerance by reducing the accumulation of fluoride,indicating that CsFEX gene played a vital role in the process of fluoride transport,especially at low pH.3.The changes of proton flow and membrane potential in the roots of C.sinensis and A.thaliana under different pH and fluoride treatments were measured by NMT.The results showed that the influx trend of proton and the decrease of membrane potential increased with the addition of fluoride or the decrease of pH,which indicated that the treatment of fluoride and pH affected the absorption and accumulation of F~-by changing the electrochemical gradient of the cells.Under the same treatments,the influx of proton and the decrease of membrane potential of transgenic A.thaliana were lower than that of the wild type,indicating that CsFEX protein helped the root cells stabilize the membrane potential to a certain extent by alleviating fluoride accumulation.In conclusion,different pH affected the absorption and accumulation of fluoride in C.sinensis by changing the electrochemical gradient of the cells.CsFEX gene played an important role in the process of F~-transport,and its expressions were also affected by different pH. |
