Functional Characterization Of Arsenic Methylation In As(?)S-Adenosylmethytransferase (arsM) Transgenic Arabidopsis Thaliana

Arsenic(As)is a ubiquitous and toxic metalloid.Inorganic As has been classified as a class one carcinogen by the International Agency for Research on Cancer.As is introduced into the environment through both geological and anthropogenic sources,such as mining activities,utilization of arsenic-based pesticides or herbicides,burning of fossil fuels.Excessive exposure to arsenic can cause a number of damages to humans,plants and microbes.Many researches have been done to find the ways to reduce the inorganic arsenic in soil and plants.Arsenic methylation has been considered as a detoxification mechanism in microorganisms and humans.Because of the lack of genes responsible for As methylation,higher plants appear to be unable to methylate inorganic As to organic As species.One potential strategy is to genetically engineer plants to enable them to methylate and volatilize As.This study hypothesized that eukaryotic arsM gene could produce a high efficiency of As methylation and genetically engineered two ecotypes of Arabidopsis thaliana(Col-0 and Kr-0)with the arsM from the eukaryotic alga Chlamydomonas reinhardtii to identify this gene's ability of arsenic methylation in plants.In addition,a new strain from arsenic contaminated paddy was isolated,named SM-1,with a srong ability of As methylation and volatilization.So ArarsM from SM-1 was also expressed in A.thaliana to test its function of arsenic methylation.CrarsM and ArarsM genes were transformed into A.thaliana by the floral dip method,and transgenic lines were screened on 1/2 MS medium containing 50 ?g/mL hygromycin and were further tested by PCR and RT-PCR.CrarsM and ArarsM were well expressed in both shoots and roots.Two lines each in the background of Col-0 and Kr-0 and their wide-type(WT)plants were used for further experiments.WT and transgenic plants were treated with 10,25?M As(?)for 24 or 72 hours to investigate if transgenic plants expressing CrarsM and ArarsM can methylate As.Arsenic speciation was analysed by HPLC-ICP-MS.Dimethylarsenate(DMA)was found to be the predominant As species in the shoots of transgenic plants in both the Col-0 and Kr-0 backgrounds.In contrast,As(?)was the only speciation in both shoots and roots in WT plants.These results demonstrated that the transgenic plants expressing CrarsM or ArarsM can methylate As(?)to DMA efficiently.Arsenic tolerance experiments were designed to test if the expression of CrarsM or ArarsM affects the As tolerance in A.thaliana.The seeds of WT and transgenic plants were sown on 1/2 strength MS medium with As(?)concentration of 10,20 and 30 pM in Petri plates.After 20 days,root length and shoot biomass were measured and found that transgenic plants were more sensitive to As(?)in medium than WT plants.WT and transgenic plants were grown in hydroponic culture amended with 10 ?M As(?),and volatile arsenicals were trapped in a hermetical box by using silica gel impregnated with 10%AgNO3 solution for 7 days.Nutrient solution was changed every 3 days.Small amounts of trimethylarsine oxid(TMAO)was detected from transgenic plants in both backgrounds of A.thaliana,whereas no volatile As was detected in WT plants.A grafting experiment was designed to clarify the plants tissue where the arsenic methylation occurred.Shoots and roots of WT and transgenic plants were grafted reciprocally and exposed to 10 ?M As(?).By analyzing the As species of shoots and roots respectively,we found that arsenic methylation occured in both shoots and roots of transgenic plants,but the methylation product DMA was transported from roots to shoots quickly.Finally,a pot experiment was conducted in which WT and transgenic A.thaliana plants were treated with 10 ?M As(?)once at the seeding stage and twice at the bolting stage.Arsenic species and the amounts of total arsenic in the seeds were determined.As(?)was the main arsenic species in WT plants,while DMA and MMA were also detected in the seeds of CrarysM transgenic A.thaliana.In conclusion,CrarsM and ArarsM transgenic plants of both Col-0 and Kr-0 backgrounds showed strong abilities to methylate As(?)to DMA.Arsenic methylation occurs in both shoots and roots of the transgenic plants.Small amounts of volatile arsenicals were released from transgenic plants.However,transgenic plants became more sensitive to As(?)than WT in mediums,suggesting that DMA is more toxic to plants than As(?).