Arsenic Resistant Bacteria And Root Organic Acid Promoted Plant Growth And Arsenic Uptake In As-hyperaccumulator Pteris Vittata

Arsenic（As）is toxic to plants,and in soils it primarily exists in its oxidized form,arsenate（AsV）.Arsenate is a phosphate（P）analog sharing similar chemical properties and behavior.Iron（Fe）is an essential element for living organism and plays a major role in metabolism of higher plants and bacteria.In soils,As and P often bind with metals including Ca,A1 and Fe,making them insoluble and unavailable for plant uptake.Thus they need to be solubilized before being taken up by plants or bacteria.It is known that plants and microorganisms produce organic ligands to solubilize Fe from poorly available sources.Microorganisms produce siderophores under Fe-deficiency conditions,serving as their main strategy to acquire Fe from poorly available forms.Plant roots release exudates into the rhizosphere to enhance their uptake of nutrients（e.g.,Fe and P）from poorly available sources.Among root exudates,LMW organic acids（LMWOAs）are of particular importance due to their ubiquitous presence in soils and their ability to mobilize insoluble nutrients.Pteris vittata（Chinese Brake fern）,the first known As-hyperaccumulator.P.vittata prefers to grow in calcareous soils,which is characterized by low available Fe and As contents.This suggests that P.vittata and the associated rhizobacteria may have evolved efficient strategies to acquire Fe and mobilize As from insoluble minerals in soils.However,limited information is available about As/Fe/P solubilization and the interactions in rhizosphere of P.vittata.Therefore,the overall goal of this study was to examine the characteristic and ability of Pseudomonas PG12 in solubilizing FeAsO4 mineral,the response of root organic acids to external As and interactions between rhizobacteria and plant in mineral solubilization as well as mechanisms in enhancing plant growth.The main results are as the following:（1）Arsenic-hyperaccumulator Pteris vittata was grown for 7 d in Fe-free Hoagland solution containing FeAsO4 mineral and PG12-siderophore or fungal-siderophore desferrioxamine B（DFOB）.Standard siderophore assays indicated that PG12-siderophore was catecholate-type.PG12-siderophore was more effective in promoting FeAsO4 dissolution,and Fe and As plant uptake than DFOB.Media soluble Fe and As in PG12 treatment were 34.6 and 3.07 μM,1.6-and 1.4-fold of that in DFOB.Plant Fe content increased from 2.93 to 6.24 g kg^-1 in the roots and As content increased from 14.3 to 78.5 mg kg^-1 in the fronds.Besides,P.vittata in PG12 treatment showed 2.6-times greater biomass than DFOB.While P.vittata fronds in PG12 treatment were dominated by AsIII,those in DFOB treatment was dominated by AsV（61 to 77%）.This study showed that siderophore-producing arsenic-resistant rhizobacteria may have potential in enhancing phytoremediation of arsenic-contaminated soils.（2）We evaluated the mechanisms used by Pteris vittata（PV）to mobilize As/Fe by examining the impact of As on root exudates in four plants:As-hyperaccumulators PV and Pteris multifida（PM）,non-hyperaccumulator Pteris ensiformis（PE）and angiosperm plant tomato（Solanum lycopersicum）.Phytate and oxalate were dominant in fern plants（>93%）,which were 50-83,15-42 and 0-32 mg kg^-1 phytate and 10^-15,7-26 and 4^-12 mg kg^-1 oxalate for PV,PM and PE respectively,with higher As inducing greater phytate exudation and no phytate being detected in tomato exudates.PV treated with phytate+FeAsO4 had higher As/Fe contents and larger biomass than phytate or FeAsO4,which were 340 vs.20 and 130 mg kg^-1 As in fronds and 7,900 vs.1,600 and 4,100 mg kg^-1 Fe in roots.We hypothesized that As-induced phytate exudation helped PV to take up Fe and As from insoluble FeAsO4 and promoted PV growth.Our study suggests that phytate exudation may be special to fern plants,which may play an important role in enhancing As/Fe solubilization and plant uptake,thereby increasing its efficiency in phytoremediation of As-contaminated soils.（3）Arsenic（As）in soils is often adsorbed on Fe-（hydro）oxides surface,rendering them more resistant to dissolution,which is undesirable for phytoremediation of As-contaminated soils.Arsenic hyperaccumulator Pteris vittata prefers to grow in calcareous soils where available Fe and As are low.To elucidate its mechanisms of acquiring Fe and As from insoluble sources in soils,we investigated dissolution of goethite with pre-adsorbed arsenate（AsV;As-goethite）in presence of four organic ligands,including two root exudates（oxalate and phytate,dominant in P.vittata）and two microbial siderophores（PG12-siderophore and desferrioxamine B）.Their presence increased As solubilization from As-goethite from 0.03 to 0.27-5.33 mg L^-1 compared to the control.The siderophore/phytate bi-ligand treatment released 7.42 mg L^-1 soluble Fe,which was 1.2-fold that of the sum of siderophore and phytate,showing a synergy in promoting As-goethite dissolution.In ligand-mineral-plant system,siderophore/phytate was most effective in releasing As and Fe from As-goethite.Moreover,the continuous plant uptake induced more As-goethite dissolution.The continued release of As and Fe significantly enhanced their plant uptake(from 0.01 to 0.43 mg plant^-1 As and 2.7 to 14.8 mg plant^-1 Fe)and plant growth(from 1.2 to 3.1 g plant^-1 fw)in P.vittata.Since microbial siderophores and root exudates often coexist in soil rhizosphere,their synergy in enhancing dissolution of insoluble As-Fe minerals may play an important role in efficient phytoremediation of As-contaminated soils.（4）Phytate is abundant organic P in soils,which is stable and unavailable for plant uptake.Its origin is poorly understood,but it occurs in root exudates of As-hyperaccumulator Pteris vittata（PV）.To elucidate its effect on As uptake and growth,P.vittata was examined on agar media（63 μM P）containing 50μM As and/or 50 or 500 μM phytate with non As-hyperaccumulator Pteris ensiformis（PE）as a congeneric control.Phytate induced efficient As and P uptake,and enhanced growth in PV,but had little effects on PE.The As concentrations in PV fronds and roots were 157 and 31 mg kg^-1 in Asso+phytateso,2.2-and 3.1-fold that of As50 treatment.Phosphorus uptake by PV was reduced by 27%in As treatment than the control（P vs.P+As）but increased by 73%comparing to phytate500 and phytate500+As,indicating that PV effectively take up P from phytate.Neither As nor phytate affected Fe accumulation in PV,but phytate reduced root Fe concentration in PE（46-56%）.As such,the increased As and P and the unsuppressed Fe uptake in PV probably promoted its growth.Thus,supplying phytate to As-contaminated soils may promote As uptake and growth in PV and its phytoremediation ability.