Physiological And Molecular Mechanisms Of Plasma Membrane H~+-ATPase Mediated Uptake Of Fluoride By Tea Plant Roots

Tea plant(Camellia sinensis(L.)O.Kuntze)is a fluoride hyper-accumulator.Most of fluoride accumulates in the leaves of tea and enters the human body through tea consumption resulting in human health risks.Fluoride is usually absorbed by the roots of tea.However,the physiological and molecular mechanisms of transmembrane uptake fluoride by the roots of tea plant are still poorly documented.The plasma membrane H+-ATPase can provide the original power for the active transmembrane uptake of plants.Therefore,investigate the functional and role of plasma membrane H+-ATPase of active uptake fluoride by tea roots is great significance for revealing the physiological and molecular processes of excessive enrichment of tea.In this paper,the low concentration NaF and NaCl solution hydroponic "Shuchazao"tea seedlings were tested with deionized water as a blank control.To study the absorption and accumulation of fluoride under F-/Cl-conditions,and the effect of Cl-on the absorption of fluoride by tea roots.The effects of F-and Cl-on root activity and plasma membrane H+ATPase activity were analyzed.Screening the relevant suspected plasma membrane H+ATPase gene in RNA-Seq for expression pattern,cluster analysis and transgenic Arabidopsis heterologous system validation analysis were performed.The role of plasma membrane H+ATPase and the function of proton pump genes in the process of fluoride uptake in tea plants were preliminarily identified.The main results are as follows:(1)There was no significant change in the total length and total surface area of tea roots in low concentration F-/Cl-culture for 72 h,it indicating that the low concentration of F/Cl treatment had no apparent toxicity to the tea plant in short time.(2)The uptake of fluoride by the“Shuchazao”tea plant is not negatively affected by Cl-,which in turn promotes the absorption of fluorine by the tea tree.(3)F-increased the tea root activity and promoted the absorption of fluoride by tea roots.(4)F-increased the amount of plasma membrane H+-ATPase protein and activity in tea roots.Co-treatment of F-and Cl-makes the increase in protein amount and activity more significant.It is because F-/Cl-activate the root plasma membrane H+-ATPase,increases the amount of protein,promotes ATP hydrolysis H+efflux,and increases ΔH+,and the electrochemical potential outside the membrane drives F-into the cell through the secondary transport system Thereby promoting uptake of fluoride by tea plant.(5)Four plasma membrane H+-ATPase genes were identified from the transcriptome(RNA-Seq)KEGG database.namely CL2275,CL4876,Unigene12499 and Unigene24443.Further analysis by qPCR showed that F treatment up-regulated expression of CL4876,and all others were down-regulated.(6)According to the gene annotation in NCBI,it was found that CL22 75,CL4876,Unigene12499 and Unigene24443 are homologous genes with plasma membrane H+ATPase of Medicago truncatula(HA1),Oryza sativaJaponica Group(Os02t0797300),and Solanum lycopersicum(LHA2),respectively.However,the results of systematic cluster analysis showed that the three genes were not clustered in the same branch as the annotated genes.CL4876 founded a plasma membrane ATPase 8 of rice in NCBI,and is clustered in the same evolutionary branch with rice OsA9 and Arabidopsis thaliana AHA10(both belong to plasma membrane H+-ATPase subfamily Ⅲ),which deviated slightly from the RNA-Seq annotation results.We speculated that CL4876 and OsA8,OsA9,AHA10 may have similar expression patterns or similar biological functions(7)F-activated the CL4876 gene in the inorganic ion transport and metabolism pathway,which increased the expression level.Then,it activated plasma membrane H+-ATPase,increased the protein and activity,thereby increasing the absorption and accumulation of fluoride in tea plant.The addition of Cl-further enhances the mechanism of action of this physiological molecule(8)Arabidopsis thaliana with plasma membrane H+-ATPase CL4876 gene has significantly higher uptake of F than wild-type.It is further proved that the CL4876 gene may promote uptake of fluoride by tea plant.