Functional Identification Of Phosphate Transporter PvPht1;4 In Peteris Vittata And Its Application Of Controlling Arsenic Accumulation In Plants

Arsenic(As)-hyperaccumulator Pteris vittata is efficient in As uptake and translocation.Arsenate [As(V)] is the main arsenic species and the main fern-available form of arsenic in the natural environment.Arsenate was absorbed via the phosphate transporters in soils for the similar chemical structure between phosphate(Pi)and As V.High affinity phosphate transporter(Pht1)plays an important role in transferring phosphate as well as As(V)from soil solution to plant root cells.Here,all PvPht1 genes were screened and analyzed from the transcriptome database of Peteris vittata,and for the first time,we cloned a new PvPht1;4 gene from Pteris vittata and investigated its role in As(V)uptake and transport in yeast and transgenic tobacco plants.In this study,a total of five PvPht1 genes,PvPht1;1-5,were obtained from the transcriptome database of Peteris vittata,among which PvPht1;4-5 were the genes discovered for the first time.The results of bioinformatics analysis showed that the sequences of genes in PvPht1 family were conserved.Among them,PvPht1;3 and PvPht1;4 showed high sequence identity and a relatively closer evolutionary distance.They may have similar As(V)/Pi absorption functions.Based on q RT-PCR,PvPht1;4was mainly expressed in the roots of Peteris vittata,with its transcripts in the roots being induced by both Pi deficiency and As(V)exposure,which may participate in the process of the efficient As(V)/Pi absorption in Peteris vittata.Based on the prediction of the protein tertiary structure and the transmembrane structure,PvPht1;4 encoded a typical phosphate transporter.Further identication of the subcellular localization showed that PvPht1;4 localized to the plasma membrane which was responsible for the transmembrane transport of Pi from extracellular to intracellular.To clarify the kinetic characteristics of PvPht1;4 in transporting As(V)and Pi,we heterogeneous expressed PvPht1;4 in the yeast-mutant strain which is defective in Pi uptake.PvPht1;4 showed higher Pi transport capacity than PvPht1;3.Under As(V)exposure,PvPht1;4 yeast transformants showed comparable tolerance as PvPht1;3,but higher As accumulation than PvPht1;2 transformants,indicating that PvPht1;4 had considerable As(V)and Pi transport activity.However,in soil and hydroponic experiments,PvPht1;4 expressing tobacco lines accumulated 26-44% and37-55% lower As in the shoots than WT plants,with lower root-to-shoot As translocation.In the roots of PvPht1;4 lines,higher glutathione(GSH)contents and expression levels of GSH synthetase gene Nt GSH2 corroborated with better As(III)complexation and detoxification.In addition,the transcripts of As-GSH transporter Nt ABCC1 in PvPht1;4 lines were up-regulated.The data suggested that PvPht1;4lines probably detoxified As by complexing As with GSH,which was stored in root vacuoles,thereby reducing As translocation in transgenic tobacco.Given its strong As(V)transport capacity,expression of PvPht1;4 provides a new molecular approach to reduce As-accumulation in plant shoots.