ABSTRACT
Rice (Oryza sativa L.) grown on arsenic-containing soil and water become a primary dietary source of arsenic and pose a significant health risk. Gene modification is an important and practical approach to reduce arsenic accumulation in rice grains. Here, we reported a WaarsM gene of soil fungus Westerdykella aurantiaca, expressed in rice able to convert toxic inorganic arsenicals to methylated arsenic species, therefore, reduce arsenic accumulation in rice grains. In response to arsenic treatment in hydroponics, WaarsM expressing transgenic lines showed a marked increase in arsenic resistance and reduces its accumulation compared to NT. Also, WaarsM expressing transgenic Line 1 evolved ca. 157ng and ca. 43ng volatile arsenicals (mg-1 fresh weight) after 72h of exposure to 25µM AsIII and 250µM AsV, respectively. Transgenic Line 1, grown in soil irrigated with arsenic-containing water accumulates about 50% and 52% lower arsenic than NT in shoot and root, respectively; while arsenic concentration in polished seeds and husk of the transgenic line was reduced by 52% compared to NT. Thus, the present study demonstrates that the expression of WaarsM in rice induces arsenic methylation and volatilization, provides a potential strategy to reduce arsenic accumulation in rice grain.
Subject(s)
Arsenic/metabolism , Fungal Proteins/metabolism , Methyltransferases/metabolism , Oryza/metabolism , Soil Pollutants/metabolism , Edible Grain/metabolism , Food Contamination/prevention & control , Fungal Proteins/genetics , Methylation , Methyltransferases/genetics , Oryza/genetics , Plant Roots/metabolism , Plant Shoots/metabolism , Plants, Genetically Modified/metabolism , VolatilizationABSTRACT
Arsenic (As) is an acute poison and class I carcinogen, can cause a serious health risk. Staple crops like rice are the primary source of As contamination in human food. Rice grown on As contaminated areas accumulates higher As in their edible parts. Based on our previous transcriptome data, two rice glutaredoxins (OsGrx_C7 and OsGrx_C2.1) were identified that showed up-regulated expression during As stress. Here, we report OsGrx_C7 and OsGrx_C2.1 from rice involved in the regulation of intracellular arsenite (AsIII). To elucidate the mechanism of OsGrx mediated As tolerance, both OsGrxs were cloned and expressed in Escherichia coli (Δars) and Saccharomyces cerevisiae mutant strains (Δycf1, Δacr3). The expression of OsGrxs increased As tolerance in E. coli (Δars) mutant strain (up to 4 mM AsV and up to 0.6 mM AsIII). During AsIII exposure, S. cerevisiae (Δacr3) harboring OsGrx_C7 and OsGrx_C2.1 have lower intracellular AsIII accumulation (up to 30.43% and 24.90%, respectively), compared to vector control. Arsenic accumulation in As-sensitive S. cerevisiae mutant (Δycf1) also reduced significantly on exposure to inorganic As. The expression of OsGrxs in yeast maintained intracellular GSH pool and increased extracellular GSH concentration. Purified OsGrxs displays in vitro GSH-disulfide oxidoreductase, glutathione reductase and arsenate reductase activities. Also, both OsGrxs are involved in AsIII extrusion by altering the Fps1 transcripts in yeast and protect the cell by maintaining cellular GSH pool. Thus, our results strongly suggest that OsGrxs play a crucial role in the maintenance of the intracellular GSH pool and redox status of the cell during both AsV and AsIII stress and might be involved in regulating intracellular AsIII levels by modulation of aquaporin expression and functions.
Subject(s)
Aquaporins/metabolism , Arsenites/metabolism , Glutaredoxins/metabolism , Glutathione/metabolism , Oryza/metabolism , Saccharomyces cerevisiae/metabolism , Arsenate Reductases/metabolism , Biological Transport , Genes, Plant , Genetic Complementation Test , Glutathione Reductase/metabolism , Mutation/genetics , Oryza/genetics , Phenotype , Protein Disulfide Reductase (Glutathione)/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Elevated arsenic concentration in the environment and agricultural soil is a serious concern to crop production and human health. Among different detoxification mechanisms, the methylation of arsenic is a widespread phenomenon in nature. A number of microorganisms are able to methylate arsenic, but less is known about the arsenic metabolism in fungi. We identified a novel arsenic methyltransferase (WaarsM) gene from a soil fungus, Westerdykella aurantiaca. WaarsM showed sequence homology with all known arsenic methyltransferases having three conserved SAM binding motifs. The expression of WaarsM enhanced arsenic resistance in E. coli (Δars) and S. cerevisiae (Δacr2) strains by biomethylation and required endogenous reductants, preferably GSH, for methyltransferase activity. The purified WaarsM catalyzes the production of methylated arsenicals from both AsIII and AsV, and also displays AsV reductase activity. It displayed higher methyltransferase activity and lower KM 0.1945 ± 0.021 mM and KM 0.4034 ± 0.078 mM for AsIII and AsV, respectively. S. cerevisiae (Δacr2) cells expressing WaarsM produced 2.2 ppm volatile arsenic and 0.64 ppm DMA(v) with 0.58 ppm volatile arsenicals when exposed to 20 ppm AsV and 2 ppm AsIII, respectively. Arsenic tolerance in rice after co-culture with genetically engineered yeast suggested its potential role in arsenic bioremediation. Thus, characterization of WaarsM provides a potential strategy to reduce arsenic concentration in soil with reduced arsenic accumulation in crops grown in arsenic contaminated areas, and thereby alleviating human health risks.
