Study On The Function Of Arsenate Reductase Genes In Arsenic Hyperaccumulator Pteris Vittata

Pteris vittata is the first known As(As)-hyperaccumulator in the world,and it is efficient in As adsorption,reduction,and translocation.In aerobic soils where P.vittata usually grows,As mainly exists as arsenate(As(?)).After being taken up by plants,As(?)is first reduced to As(?),then it can be either extruded out of the roots or complexed with GSH or phytochelatins(PCs).As(?)reduction is an important part of the plant's As detoxification.There have been many studies on the As(?)reduction process of common plants,but the characteristics of that in As hyperaccumulator are not clear yet.Therefore,studying the As reduction process in P.vittata is of great significance to the investigation of the fact that how P.vittata hyperaccumulate As.In addition,most research on the molecular mechanism of plant As metabolism only considers a single As pollution condition,most studies did not consider the situation of multi-metal compound pollution.In this study,we conducted a functional study on the arsenate reductase gene of the As hyperaccumulator P.vittata,including the reported PvACR2 and the newly cloned PvHAC1 and PvHAC2.The bioinformatics characteristics and expression patterns of different arsenate reductase genes in P.vittata were analyzed,the functions of PvHAC1 and PvHAC2 were identified,and the expression patterns of arsenate reductase genes under compound pollution conditions were integrated to clarify the different roles of reductase gene in the process of As(?)reduction in P.vittata.First,according to sequence homology,we identified two new arsenate reductase genes from P.vittata,PvHAC1 and PvHAC2.Bioinformatic analysis showed that PvHAC1 and PvHAC2 had a relatively distant relationship with PvACR2,and their amino acid sequences were quite different;and PvHAC1 and PvHAC2 had similar conserved regions with At HAC1,with the typical catalytic site and structural features of arsenate reductase.This indicated that PvHAC1 and PvHAC2 were very likely to encode arsenate reductase but different in function from other arsenate reductase genes.In the expression pattern analysis of P.vittata arsenate reductase genes,PvHAC1 was mainly expressed in the rhizomes,and was strongly induced by As(?);PvHAC2 and PvACR2 were mainly expressed in the roots and fronds.Differently,other arsenate reductase genes like PvACR2,At HAC1,OsHAC1;1/2/4 were all expressed only in the roots.These completely different expression patterns suggested PvHAC1 and PvHAC2 are likely to play a special role in the As(?)reduction process in P.vittata.To identify the function of PvHAC1 and PvHAC2,subcellular localization analysis showed that two PvHAC genes were localized in the cytoplasm and nucleus which was consistent with their possible function.To further verify their As(?)-reducing ability,PvHAC1 and PvHAC2 were expressed in the mutant Escherichia coli ?ars C who lacks its arsenate reductase.We found that both PvHAC genes can complement the As(?)-reducing ability of the mutant E.coli,noticeability enhanced the As(?)tolerance of the strains.At the same time,the concentration of As(?)in the culture medium was also significantly higher than that of the control,indicating that both PvHAC1 and PvHAC2 have arsenate reductase activity.Additionally,we also expressed the PvHAC genes in Arabidopsis.Under 10?M As(?),the As(?)concentration in the roots of PvHAC1 and PvHAC2 transgenic Arabidopsis was significantly lower than that of the wild-type,indicating that PvHAC1 and PvHAC2 have arsenate reductase activity in both recombinant E.coli and transgenic Arabidopsis.Interestingly,we also found that expressing PvHAC2 decreased As accumulation in Arabidopsis shoots under long-term As exposure.Combining function investigation with the tissue-specific expression pattern of PvHAC genes and physiological analysis,we proposed a possible schematic of As(?)reduction in P.vittata: As reduction is completed by PvACR2 in the root,PvHAC1 in the rhizome,and PvHAC2 in the frond together.And most of the As(?)is reduced by PvHAC1 in the rhizome.Finally,we investigated how the arsenate reductase genes in P.vittata responded to multiple heavy metal stress.After exposing to As(?)combined with lead(Pb),cadmium(Cd),chromium(Cr)or zinc(Zn),the As accumulation and the expression level of arsenate reductase genes in P.vittata roots were determined.It was found that under +As+Cr exposure,most As(?)was reduced to As(?)in the root of P.vittata,as the expression level of PvACR2 was up-regulated.Which destroyed the original As(?)reduction pattern of P.vittata,therefore inhibiting its ability to hyperaccumulate As.The As speciation of P.vittata roots was analyzed,it was found that the root As(?)in P.vittata of the +As+Cr treatment was 1.3 times that of the +As treatment,which was consistent with the change in the expression pattern of PvACR2.Although cadmium(Cd)could promote the hyperaccumulation of As by P.vittata,this had nothing to do with the arsenate reductase genes.lt may cause by the upregulation of the expression level of the As-absorption gene PvPht1;4.While lead(Pb)and zinc(Zn)had no significant effect on the As hyperaccumulation ability of P.vittata and the expression level of the arsenate reductase gene.In this study,the functions of the new arsenate reductase genes PvHAC1 and PvHAC2 in P.vittata were identified for the first time.And the possible As(?)reduction mechanism and its response to multiple heavy metal stress were inferred and explored.This study helps to understand the molecular mechanisms of As hyperaccumulation by P.vittata and improve its efficiency in phytoremediation of As-contaminated soils.