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Fangjie Zhao - One of the best experts on this subject based on the ideXlab platform.
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oshac1 1 and oshac1 2 function as Arsenate reductases and regulate arsenic accumulation
Plant Physiology, 2016Co-Authors: Tao Wang, Ziru Chen, Zhong Tang, Zhongchang Wu, David E Salt, Daiyin Chao, Fangjie ZhaoAbstract:Rice is a major dietary source of the toxic metalloid arsenic (As). Reducing its accumulation in rice (Oryza sativa) grain is of critical importance to food safety. Rice roots take up Arsenate and arsenite depending on the prevailing soil conditions. The first step of Arsenate detoxification is its reduction to arsenite, but the enzyme(s) catalyzing this reaction in rice remains unknown. Here, we identify OsHAC1;1 and OsHAC1;2 as Arsenate reductases in rice. OsHAC1;1 and OsHAC1;2 are able to complement an Escherichia coli mutant lacking the endogenous Arsenate reductase and to reduce Arsenate to arsenite. OsHAC1:1 and OsHAC1;2 are predominantly expressed in roots, with OsHAC1;1 being abundant in the epidermis, root hairs, and pericycle cells while OsHAC1;2 is abundant in the epidermis, outer layers of cortex, and endodermis cells. Expression of the two genes was induced by Arsenate exposure. Knocking out OsHAC1;1 or OsHAC1;2 decreased the reduction of Arsenate to arsenite in roots, reducing arsenite efflux to the external medium. Loss of arsenite efflux was also associated with increased As accumulation in shoots. Greater effects were observed in a double mutant of the two genes. In contrast, overexpression of either OsHAC1;1 or OsHAC1;2 increased arsenite efflux, reduced As accumulation, and enhanced Arsenate tolerance. When grown under aerobic soil conditions, overexpression of either OsHAC1;1 or OsHAC1;2 also decreased As accumulation in rice grain, whereas grain As increased in the knockout mutants. We conclude that OsHAC1;1 and OsHAC1;2 are Arsenate reductases that play an important role in restricting As accumulation in rice shoots and grain.
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A novel pathway of Arsenate detoxification.
Molecular Microbiology, 2016Co-Authors: Fangjie ZhaoAbstract:Microorganisms have evolved various mechanisms to detoxify arsenic, an ubiquitous environmental toxin. Known mechanisms include arsenite efflux, Arsenate reduction followed by arsenite efflux and arsenite methylation. In this issue, Chen et al. describe a novel mechanism for Arsenate detoxification via synergistic interaction of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and a major facilitator superfamily protein (ArsJ). They propose that GAPDH catalyzes the formation of 1-arseno-3-phosphoglycerate, which is then extruded out of the cell by ArsJ. The significance of this pathway and questions for further research are discussed.
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genome wide association mapping identifies a new Arsenate reductase enzyme critical for limiting arsenic accumulation in plants
PLOS Biology, 2014Co-Authors: Daiyin Chao, Fangjie Zhao, Ziru Chen, Yi Chen, Jiugeng Chen, Chengcheng Wang, John Danku, David E SaltAbstract:Inorganic arsenic is a carcinogen, and its ingestion through foods such as rice presents a significant risk to human health. Plants chemically reduce Arsenate to arsenite. Using genome-wide association (GWA) mapping of loci controlling natural variation in arsenic accumulation in Arabidopsis thaliana allowed us to identify the Arsenate reductase required for this reduction, which we named High Arsenic Content 1 (HAC1). Complementation verified the identity of HAC1, and expression in Escherichia coli lacking a functional Arsenate reductase confirmed the Arsenate reductase activity of HAC1. The HAC1 protein accumulates in the epidermis, the outer cell layer of the root, and also in the pericycle cells surrounding the central vascular tissue. Plants lacking HAC1 lose their ability to efflux arsenite from roots, leading to both increased transport of arsenic into the central vascular tissue and on into the shoot. HAC1 therefore functions to reduce Arsenate to arsenite in the outer cell layer of the root, facilitating efflux of arsenic as arsenite back into the soil to limit both its accumulation in the root and transport to the shoot. Arsenate reduction by HAC1 in the pericycle may play a role in limiting arsenic loading into the xylem. Loss of HAC1-encoded arsenic reduction leads to a significant increase in arsenic accumulation in shoots, causing an increased sensitivity to Arsenate toxicity. We also confirmed the previous observation that the ACR2 Arsenate reductase in A. thaliana plays no detectable role in arsenic metabolism. Furthermore, ACR2 does not interact epistatically with HAC1, since arsenic metabolism in the acr2 hac1 double mutant is disrupted in an identical manner to that described for the hac1 single mutant. Our identification of HAC1 and its associated natural variation provides an important new resource for the development of low arsenic-containing food such as rice.
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knocking out acr2 does not affect arsenic redox status in arabidopsis thaliana implications for as detoxification and accumulation in plants
PLOS ONE, 2012Co-Authors: Henk Schat, S P Mcgrath, David E Salt, Mathijs Bliek, Yi Chen, Graham N George, Fangjie ZhaoAbstract:Many plant species are able to reduce Arsenate to arsenite efficiently, which is an important step allowing detoxification of As through either efflux of arsenite or complexation with thiol compounds. It has been suggested that this reduction is catalyzed by ACR2, a plant homologue of the yeast Arsenate reductase ScACR2. Silencing of AtACR2 was reported to result in As hyperaccumulation in the shoots of Arabidopsis thaliana. However, no information of the in vivo As speciation has been reported. Here, we investigated the effect of AtACR2 knockout or overexpression on As speciation, arsenite efflux from roots and As accumulation in shoots. T-DNA insertion lines, overexpression lines and wild-type (WT) plants were exposed to different concentrations of Arsenate for different periods, and As speciation in plants and arsenite efflux were determined using HPLC-ICP-MS. There were no significant differences in As speciation between different lines, with arsenite accounting for >90% of the total extractable As in both roots and shoots. Arsenite efflux to the external medium represented on average 77% of the Arsenate taken up during 6 h exposure, but there were no significant differences between WT and mutants or overexpression lines. Accumulation of As in the shoots was also unaffected by AtACR2 knockout or overexpression. Additionally, after exposure to Arsenate, the yeast (Saccharomyces cerevisiae) strain with ScACR2 deleted showed similar As speciation as the WT with arsenite-thiol complexes being the predominant species. Our results suggest the existence of multiple pathways of Arsenate reduction in plants and yeast.
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arsenic accumulation by the aquatic fern azolla comparison of Arsenate uptake speciation and efflux by a caroliniana and a filiculoides
Environmental Pollution, 2008Co-Authors: Xin Zhang, Fangjie Zhao, Guozhong Xu, Gui-lan DuanAbstract:This study investigates As accumulation and tolerance of the aquatic fern Azolla. Fifty strains of Azolla showed a large variation in As accumulation. The highest- and lowest-accumulating ferns among the 50 strains were chosen for further investigations. Azolla caroliniana accumulated two times more As than Azolla filiculoides owing to a higher influx velocity for Arsenate. A. filiculoides was more resistant to external Arsenate due to a lower uptake. Both strains showed a similar degree of tolerance to internal As. Arsenate and arsenite were the dominant As species in both Azolla strains, with methlyated As species accounting for <5% of the total As. A. filiculoides had a higher proportion of arsenite than A. caroliniana. Both strains effluxed more Arsenate than arsenite, and the amount of As efflux was proportional to the amount of As accumulation. The potential of growing Azolla in paddy fields to reduce As transfer from soil and water to rice should be further evaluated.
Andrew A Meharg - One of the best experts on this subject based on the ideXlab platform.
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Arsenite transport into paddy rice (Oryza sativa) roots
New Phytologist, 2020Co-Authors: Andrew A Meharg, Louise JardineAbstract:Summary • Here the mechanism of arsenite transport into paddy rice ( Oryza sativa ) roots, uptake of which is described by Michaelis–Menten kinetics, is reported. A recent study on yeast ( Saccharomyces cerevisiae ) showed that undissociated arsenite (its pK a is 9.2) was transported across the plasma membrane via a glycerol transporting channel. To investigate whether the same mechanism of transport was involved for rice, competitive studies with glycerol, which is transported into cells via aquaporins, were performed. • Glycerol competed with arsenite for transport in a dose-dependent manner, indicating that arsenite and glycerol uptake mechanisms were the same. Arsenate transport was unaffected by glycerol, confirming that Arsenate and arsenite are taken up into cells by different mechanisms. • Antimonite, an arsenite analogue that is transported into S. cerevisiae cells by aquaporins, also competed with arsenite transport in a dose-dependent manner, providing further evidence that arsenite is transported into rice roots via glycerol transporting channels. Mercury (Hg 2+ ) inhibited both arsenite and Arsenate uptake, suggesting that inhibition of influx was due to general cellular stress rather than the specific action of Hg 2+ on aquaporins. • Arsenite uptake by pea ( Pisum sativum ) and wheat ( Triticum aestivum ) was also described by Michaelis–Menten kinetics.
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arsenic as a food chain contaminant mechanisms of plant uptake and metabolism and mitigation strategies
Annual Review of Plant Biology, 2010Co-Authors: S P Mcgrath, Andrew A MehargAbstract:Arsenic (As) is an environmental and food chain contaminant. Excessive accumulation of As, particularly inorganic arsenic (Asi), in rice (Oryza sativa) poses a potential health risk to populations with high rice consumption. Rice is efficient at As accumulation owing to flooded paddy cultivation that leads to arsenite mobilization, and the inadvertent yet efficient uptake of arsenite through the silicon transport pathway. Iron, phosphorus, sulfur, and silicon interact strongly with As during its route from soil to plants. Plants take up Arsenate through the phosphate transporters, and arsenite and undissociated methylated As species through the nodulin 26-like intrinsic (NIP) aquaporin channels. Arsenate is readily reduced to arsenite in planta, which is detoxified by complexation with thiol-rich peptides such as phytochelatins and/or vacuolar sequestration. A range of mitigation methods, from agronomic measures and plant breeding to genetic modification, may be employed to reduce As uptake by food crops.
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direct evidence showing the effect of root surface iron plaque on arsenite and Arsenate uptake into rice oryza sativa roots
New Phytologist, 2004Co-Authors: Zheng Chen, Andrew A MehargAbstract:Summary • The present study aimed to investigate the effects of root surface iron plaque on the uptake kinetics of arsenite and Arsenate by excised roots of rice (Oryza sativa) seedlings. • The results demonstrated that the presence of iron plaque enhanced arsenite and decreased Arsenate uptake. • Arsenite and Arsenate uptake kinetics were adequately fitted by the Michaelis–Menten function in the absence of plaque, but produced poor fits to this function in the presence of plaque. • Phosphate in the uptake solution did not have a significant effect on arsenite uptake irrespective of the presence of iron plaque; however phosphate had a significant effect on Arsenate uptake. Without iron plaque, phosphate inhibited Arsenate uptake. The presence of iron plaque diminished the effect of phosphate on Arsenate uptake, possibly through a combined effect of Arsenate desorption from iron plaque.
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mechanisms of arsenic hyperaccumulation in pteris vittata uptake kinetics interactions with phosphate and arsenic speciation
Plant Physiology, 2002Co-Authors: Junru Wang, Andrew A Meharg, Fangjie Zhao, Andrea Raab, Joerg Feldmann, S P McgrathAbstract:The mechanisms of arsenic (As) hyperaccumulation in Pteris vittata , the first identified As hyperaccumulator, are unknown. We investigated the interactions of Arsenate and phosphate on the uptake and distribution of As and phosphorus (P), and As speciation in P. vittata . In an 18-d hydroponic experiment with varying concentrations of Arsenate and phosphate, P. vittata accumulated As in the fronds up to 27,000 mg As kg −1 dry weight, and the frond As to root As concentration ratio varied between 1.3 and 6.7. Increasing phosphate supply decreased As uptake markedly, with the effect being greater on root As concentration than on shoot As concentration. Increasing Arsenate supply decreased the P concentration in the roots, but not in the fronds. Presence of phosphate in the uptake solution decreased Arsenate influx markedly, whereas P starvation for 8 d increased the maximum net influx by 2.5-fold. The rate of arsenite uptake was 10% of that for Arsenate in the absence of phosphate. Neither P starvation nor the presence of phosphate affected arsenite uptake. Within 8 h, 50% to 78% of the As taken up was distributed to the fronds, with a higher translocation efficiency for arsenite than for Arsenate. In fronds, 49% to 94% of the As was extracted with a phosphate buffer (pH 5.6). Speciation analysis using high-performance liquid chromatography-inductively coupled plasma mass spectroscopy showed that >85% of the extracted As was in the form of arsenite, and the remaining mostly as Arsenate. We conclude that Arsenate is taken up by P. vittata via the phosphate transporters, reduced to arsenite, and sequestered in the fronds primarily as As(III).
S P Mcgrath - One of the best experts on this subject based on the ideXlab platform.
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knocking out acr2 does not affect arsenic redox status in arabidopsis thaliana implications for as detoxification and accumulation in plants
PLOS ONE, 2012Co-Authors: Henk Schat, S P Mcgrath, David E Salt, Mathijs Bliek, Yi Chen, Graham N George, Fangjie ZhaoAbstract:Many plant species are able to reduce Arsenate to arsenite efficiently, which is an important step allowing detoxification of As through either efflux of arsenite or complexation with thiol compounds. It has been suggested that this reduction is catalyzed by ACR2, a plant homologue of the yeast Arsenate reductase ScACR2. Silencing of AtACR2 was reported to result in As hyperaccumulation in the shoots of Arabidopsis thaliana. However, no information of the in vivo As speciation has been reported. Here, we investigated the effect of AtACR2 knockout or overexpression on As speciation, arsenite efflux from roots and As accumulation in shoots. T-DNA insertion lines, overexpression lines and wild-type (WT) plants were exposed to different concentrations of Arsenate for different periods, and As speciation in plants and arsenite efflux were determined using HPLC-ICP-MS. There were no significant differences in As speciation between different lines, with arsenite accounting for >90% of the total extractable As in both roots and shoots. Arsenite efflux to the external medium represented on average 77% of the Arsenate taken up during 6 h exposure, but there were no significant differences between WT and mutants or overexpression lines. Accumulation of As in the shoots was also unaffected by AtACR2 knockout or overexpression. Additionally, after exposure to Arsenate, the yeast (Saccharomyces cerevisiae) strain with ScACR2 deleted showed similar As speciation as the WT with arsenite-thiol complexes being the predominant species. Our results suggest the existence of multiple pathways of Arsenate reduction in plants and yeast.
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arsenic as a food chain contaminant mechanisms of plant uptake and metabolism and mitigation strategies
Annual Review of Plant Biology, 2010Co-Authors: S P Mcgrath, Andrew A MehargAbstract:Arsenic (As) is an environmental and food chain contaminant. Excessive accumulation of As, particularly inorganic arsenic (Asi), in rice (Oryza sativa) poses a potential health risk to populations with high rice consumption. Rice is efficient at As accumulation owing to flooded paddy cultivation that leads to arsenite mobilization, and the inadvertent yet efficient uptake of arsenite through the silicon transport pathway. Iron, phosphorus, sulfur, and silicon interact strongly with As during its route from soil to plants. Plants take up Arsenate through the phosphate transporters, and arsenite and undissociated methylated As species through the nodulin 26-like intrinsic (NIP) aquaporin channels. Arsenate is readily reduced to arsenite in planta, which is detoxified by complexation with thiol-rich peptides such as phytochelatins and/or vacuolar sequestration. A range of mitigation methods, from agronomic measures and plant breeding to genetic modification, may be employed to reduce As uptake by food crops.
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highly efficient xylem transport of arsenite in the arsenic hyperaccumulator pteris vittata
New Phytologist, 2008Co-Authors: S P Mcgrath, Y H Su, Fangjie ZhaoAbstract:Summary • The hyperaccumulator Pteris vittata translocates arsenic (As) from roots to fronds efficiently, but the form of As translocated in xylem and the main location of Arsenate reduction have not been resolved. • Here, P. vittata was exposed to 5 µM Arsenate or arsenite for 1–24 h, with or without 100 µM phosphate. Arsenic speciation was determined in xylem sap, roots, fronds and nutrient solutions by high-performance liquid chromatography (HPLC) linked to inductively coupled plasma mass spectrometry (ICP-MS). • The xylem sap As concentration was 18–73 times that in the nutrient solution. In both Arsenate- and arsenite-treated plants, arsenite was the predominant species in the xylem sap, accounting for 93–98% of the total As. A portion of Arsenate taken up by roots (30–40% of root As) was reduced to arsenite rapidly. The majority (c. 80%) of As in fronds was arsenite. Phosphate inhibited Arsenate uptake, but not As translocation. More As was translocated to fronds in the arsenite-treated than in the Arsenate-treated plants. There was little arsenite efflux from roots to the external solution. • Roots are the main location of Arsenate reduction in P. vittata. Arsenite is highly mobile in xylem transport, possibly because of efficient xylem loading, little complexation with thiols in roots, and little efflux to the external medium.
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rapid reduction of Arsenate in the medium mediated by plant roots
New Phytologist, 2007Co-Authors: S P Mcgrath, X Y Xu, Fangjie ZhaoAbstract:Summary • Microbes detoxify Arsenate by reduction and efflux of arsenite. Plants have a high capacity to reduce Arsenate, but arsenic efflux has not been reported. • Tomato (Lycopersicon esculentum) and rice (Oryza sativa) were grown hydroponically and supplied with 10 µm Arsenate or arsenite, with or without phosphate, for 1–3 d. The chemical species of As in nutrient solutions, roots and xylem sap were monitored, roles of microbes and root exudates in As transformation were investigated and efflux of As species from tomato roots was determined. • Arsenite remained stable in the nutrient solution, whereas Arsenate was rapidly reduced to arsenite. Microbes and root exudates contributed little to the reduction of external Arsenate. Arsenite was the predominant species in roots and xylem sap. Phosphate inhibited Arsenate uptake and the appearance of arsenite in the nutrient solution, but the reduction was near complete in 24 h in both –P- and +P-treated tomato. Phosphate had a greater effect in rice than tomato. Efflux of both arsenite and Arsenate was observed; the former was inhibited and the latter enhanced by the metabolic inhibitor carbonylcyanide m-chlorophenylhydrazone. • Tomato and rice roots rapidly reduce Arsenate to arsenite, some of which is actively effluxed to the medium. The study reveals a new aspect of As metabolism in plants.
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mechanisms of arsenic hyperaccumulation in pteris vittata uptake kinetics interactions with phosphate and arsenic speciation
Plant Physiology, 2002Co-Authors: Junru Wang, Andrew A Meharg, Fangjie Zhao, Andrea Raab, Joerg Feldmann, S P McgrathAbstract:The mechanisms of arsenic (As) hyperaccumulation in Pteris vittata , the first identified As hyperaccumulator, are unknown. We investigated the interactions of Arsenate and phosphate on the uptake and distribution of As and phosphorus (P), and As speciation in P. vittata . In an 18-d hydroponic experiment with varying concentrations of Arsenate and phosphate, P. vittata accumulated As in the fronds up to 27,000 mg As kg −1 dry weight, and the frond As to root As concentration ratio varied between 1.3 and 6.7. Increasing phosphate supply decreased As uptake markedly, with the effect being greater on root As concentration than on shoot As concentration. Increasing Arsenate supply decreased the P concentration in the roots, but not in the fronds. Presence of phosphate in the uptake solution decreased Arsenate influx markedly, whereas P starvation for 8 d increased the maximum net influx by 2.5-fold. The rate of arsenite uptake was 10% of that for Arsenate in the absence of phosphate. Neither P starvation nor the presence of phosphate affected arsenite uptake. Within 8 h, 50% to 78% of the As taken up was distributed to the fronds, with a higher translocation efficiency for arsenite than for Arsenate. In fronds, 49% to 94% of the As was extracted with a phosphate buffer (pH 5.6). Speciation analysis using high-performance liquid chromatography-inductively coupled plasma mass spectroscopy showed that >85% of the extracted As was in the form of arsenite, and the remaining mostly as Arsenate. We conclude that Arsenate is taken up by P. vittata via the phosphate transporters, reduced to arsenite, and sequestered in the fronds primarily as As(III).
Gui-lan Duan - One of the best experts on this subject based on the ideXlab platform.
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Characterization of Arsenate transformation and identification of Arsenate reductase in a green alga Chlamydomonas reinhardtii.
Journal of Environmental Sciences-china, 2011Co-Authors: Lihong Wang, Gui-lan DuanAbstract:Arsenic (As) is a pervasive and ubiquitous environmental toxin that has created catastrophic human health problems world-wide. Chlamydomonas reinhardtii is a unicellular green alga, which exists ubiquitously in freshwater aquatic systems. Arsenic metabolism processes of this alga through Arsenate reduction and sequent store and efflux were investigated. When supplied with 10 μmol/L Arsenate, arsenic speciation analysis showed that arsenite concentration increased from 5.7 to 15.7 mg/kg dry weight during a 7-day period, accounting for 18%–24% of the total As in alga. When treated with different levels of Arsenate (10, 20, 30, 40, 50 μmol/L) for 7 days, the arsenite concentration increased with increasing external Arsenate concentrations, the proportion of arsenite was up to 23%–28% of the total As in alga. In efflux experiments, both Arsenate and arsenite could be found in the efflux solutions. Additionally, the efflux of Arsenate was more than that of arsenite. Furthermore, two Arsenate reductase genes of C. reinhardtii (CrACR2s) were cloned and expressed in Escherichia coli strain WC3110 (ΔarsC) for the first time. The abilities of both CrACR2s genes to complement the Arsenate-sensitive strain were examined. CrACRIA restored Arsenate resistance at 0.8 mmol/L. However, CrACR2.2 showed much less ability to complement. The gene products were demonstrated to reduce Arsenate to arsenite in vivo. In agreement with the complementation results, CrACR2.1 showed higher reduction ability than CrACR2.2, when treated with 0.4 mmol/L Arsenate for 16 hr incubation.
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arsenic accumulation by the aquatic fern azolla comparison of Arsenate uptake speciation and efflux by a caroliniana and a filiculoides
Environmental Pollution, 2008Co-Authors: Xin Zhang, Fangjie Zhao, Guozhong Xu, Gui-lan DuanAbstract:This study investigates As accumulation and tolerance of the aquatic fern Azolla. Fifty strains of Azolla showed a large variation in As accumulation. The highest- and lowest-accumulating ferns among the 50 strains were chosen for further investigations. Azolla caroliniana accumulated two times more As than Azolla filiculoides owing to a higher influx velocity for Arsenate. A. filiculoides was more resistant to external Arsenate due to a lower uptake. Both strains showed a similar degree of tolerance to internal As. Arsenate and arsenite were the dominant As species in both Azolla strains, with methlyated As species accounting for <5% of the total As. A. filiculoides had a higher proportion of arsenite than A. caroliniana. Both strains effluxed more Arsenate than arsenite, and the amount of As efflux was proportional to the amount of As accumulation. The potential of growing Azolla in paddy fields to reduce As transfer from soil and water to rice should be further evaluated.
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a cdc25 homologue from rice functions as an Arsenate reductase
New Phytologist, 2007Co-Authors: Gui-lan Duan, Yao Zhou, Yiping Tong, Rita Mukhopadhyay, Barry P. RosenAbstract:Summary • Enzymatic reduction of Arsenate to arsenite is the first step in Arsenate metabolism in all organisms studied. The rice genome contains two ACR2-like genes, OsACR2.1 and OsACR2.2, which may be involved in regulating arsenic metabolism in rice. • Here, we cloned both OsACR2 genes and expressed them in an Escherichia coli strain in which the arsC gene was deleted and in a yeast (Saccharomyces cerevisiae) strain with a disrupted ACR2 gene. OsACR2.1 complemented the Arsenate hypersensitive phenotype of E. coli and yeast. OsACR2.2 showed much less ability to complement. • The gene products were purified and demonstrated to reduce Arsenate to arsenite in vitro, and both exhibited phosphatase activity. In agreement with the complementation results, OsACR2.1 exhibited higher reductase activity than OsACR2.2. Mutagenesis of cysteine residues in the putative active site HC(X)5R motif led to nearly complete loss of both phosphatase and Arsenate reductase activities. • In planta expression of OsACR2.1 increased dramatically after exposure to Arsenate. OsACR2.2 was observed only in roots following Arsenate exposure, and its expression was less than OsACR2.1.
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characterization of Arsenate reductase in the extract of roots and fronds of chinese brake fern an arsenic hyperaccumulator
Plant Physiology, 2005Co-Authors: Gui-lan Duan, Yiping Tong, Ralf KneerAbstract:Root extracts from the arsenic (As) hyperaccumulating Chinese brake fern (Pteris vittata) were shown to be able to reduce Arsenate to arsenite. An Arsenate reductase (AR) in the fern showed a reaction mechanism similar to the previously reported Acr2p, an AR from yeast (Saccharomyces cerevisiae), using glutathione as the electron donor. Substrate specificity as well as sensitivity toward inhibitors for the fern AR (phosphate as a competitive inhibitor, arsenite as a noncompetitive inhibitor) was also similar to Acr2p. Kinetic analysis showed that the fern AR had a Michaelis constant value of 2.33 mm for Arsenate, 15-fold lower than the purified Acr2p. The AR-specific activity of the fern roots treated with 2 mm Arsenate for 9 d was at least 7 times higher than those of roots and shoots of plant species that are known not to tolerate Arsenate. A T-DNA knockout mutant of Arabidopsis (Arabidopsis thaliana) with disruption in the putative Acr2 gene had no AR activity. We could not detect AR activity in shoots of the fern. These results indicate that (1) arsenite, the previously reported main storage form of As in the fern fronds, may come mainly from the reduction of Arsenate in roots; and (2) AR plays an important role in the detoxification of As in the As hyperaccumulating fern.
David E Salt - One of the best experts on this subject based on the ideXlab platform.
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oshac1 1 and oshac1 2 function as Arsenate reductases and regulate arsenic accumulation
Plant Physiology, 2016Co-Authors: Tao Wang, Ziru Chen, Zhong Tang, Zhongchang Wu, David E Salt, Daiyin Chao, Fangjie ZhaoAbstract:Rice is a major dietary source of the toxic metalloid arsenic (As). Reducing its accumulation in rice (Oryza sativa) grain is of critical importance to food safety. Rice roots take up Arsenate and arsenite depending on the prevailing soil conditions. The first step of Arsenate detoxification is its reduction to arsenite, but the enzyme(s) catalyzing this reaction in rice remains unknown. Here, we identify OsHAC1;1 and OsHAC1;2 as Arsenate reductases in rice. OsHAC1;1 and OsHAC1;2 are able to complement an Escherichia coli mutant lacking the endogenous Arsenate reductase and to reduce Arsenate to arsenite. OsHAC1:1 and OsHAC1;2 are predominantly expressed in roots, with OsHAC1;1 being abundant in the epidermis, root hairs, and pericycle cells while OsHAC1;2 is abundant in the epidermis, outer layers of cortex, and endodermis cells. Expression of the two genes was induced by Arsenate exposure. Knocking out OsHAC1;1 or OsHAC1;2 decreased the reduction of Arsenate to arsenite in roots, reducing arsenite efflux to the external medium. Loss of arsenite efflux was also associated with increased As accumulation in shoots. Greater effects were observed in a double mutant of the two genes. In contrast, overexpression of either OsHAC1;1 or OsHAC1;2 increased arsenite efflux, reduced As accumulation, and enhanced Arsenate tolerance. When grown under aerobic soil conditions, overexpression of either OsHAC1;1 or OsHAC1;2 also decreased As accumulation in rice grain, whereas grain As increased in the knockout mutants. We conclude that OsHAC1;1 and OsHAC1;2 are Arsenate reductases that play an important role in restricting As accumulation in rice shoots and grain.
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genome wide association mapping identifies a new Arsenate reductase enzyme critical for limiting arsenic accumulation in plants
PLOS Biology, 2014Co-Authors: Daiyin Chao, Fangjie Zhao, Ziru Chen, Yi Chen, Jiugeng Chen, Chengcheng Wang, John Danku, David E SaltAbstract:Inorganic arsenic is a carcinogen, and its ingestion through foods such as rice presents a significant risk to human health. Plants chemically reduce Arsenate to arsenite. Using genome-wide association (GWA) mapping of loci controlling natural variation in arsenic accumulation in Arabidopsis thaliana allowed us to identify the Arsenate reductase required for this reduction, which we named High Arsenic Content 1 (HAC1). Complementation verified the identity of HAC1, and expression in Escherichia coli lacking a functional Arsenate reductase confirmed the Arsenate reductase activity of HAC1. The HAC1 protein accumulates in the epidermis, the outer cell layer of the root, and also in the pericycle cells surrounding the central vascular tissue. Plants lacking HAC1 lose their ability to efflux arsenite from roots, leading to both increased transport of arsenic into the central vascular tissue and on into the shoot. HAC1 therefore functions to reduce Arsenate to arsenite in the outer cell layer of the root, facilitating efflux of arsenic as arsenite back into the soil to limit both its accumulation in the root and transport to the shoot. Arsenate reduction by HAC1 in the pericycle may play a role in limiting arsenic loading into the xylem. Loss of HAC1-encoded arsenic reduction leads to a significant increase in arsenic accumulation in shoots, causing an increased sensitivity to Arsenate toxicity. We also confirmed the previous observation that the ACR2 Arsenate reductase in A. thaliana plays no detectable role in arsenic metabolism. Furthermore, ACR2 does not interact epistatically with HAC1, since arsenic metabolism in the acr2 hac1 double mutant is disrupted in an identical manner to that described for the hac1 single mutant. Our identification of HAC1 and its associated natural variation provides an important new resource for the development of low arsenic-containing food such as rice.
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knocking out acr2 does not affect arsenic redox status in arabidopsis thaliana implications for as detoxification and accumulation in plants
PLOS ONE, 2012Co-Authors: Henk Schat, S P Mcgrath, David E Salt, Mathijs Bliek, Yi Chen, Graham N George, Fangjie ZhaoAbstract:Many plant species are able to reduce Arsenate to arsenite efficiently, which is an important step allowing detoxification of As through either efflux of arsenite or complexation with thiol compounds. It has been suggested that this reduction is catalyzed by ACR2, a plant homologue of the yeast Arsenate reductase ScACR2. Silencing of AtACR2 was reported to result in As hyperaccumulation in the shoots of Arabidopsis thaliana. However, no information of the in vivo As speciation has been reported. Here, we investigated the effect of AtACR2 knockout or overexpression on As speciation, arsenite efflux from roots and As accumulation in shoots. T-DNA insertion lines, overexpression lines and wild-type (WT) plants were exposed to different concentrations of Arsenate for different periods, and As speciation in plants and arsenite efflux were determined using HPLC-ICP-MS. There were no significant differences in As speciation between different lines, with arsenite accounting for >90% of the total extractable As in both roots and shoots. Arsenite efflux to the external medium represented on average 77% of the Arsenate taken up during 6 h exposure, but there were no significant differences between WT and mutants or overexpression lines. Accumulation of As in the shoots was also unaffected by AtACR2 knockout or overexpression. Additionally, after exposure to Arsenate, the yeast (Saccharomyces cerevisiae) strain with ScACR2 deleted showed similar As speciation as the WT with arsenite-thiol complexes being the predominant species. Our results suggest the existence of multiple pathways of Arsenate reduction in plants and yeast.
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a novel Arsenate reductase from the arsenic hyperaccumulating fern pteris vittata
Plant Physiology, 2006Co-Authors: Danielle R Ellis, Luke Gumaelius, Emily Indriolo, Ingrid J Pickering, Jo Ann Banks, David E SaltAbstract:Pteris vittata sporophytes hyperaccumulate arsenic to 1% to 2% of their dry weight. Like the sporophyte, the gametophyte was found to reduce Arsenate [As(V)] to arsenite [As(III)] and store arsenic as free As(III). Here, we report the isolation of an Arsenate reductase gene (PvACR2) from gametophytes that can suppress the Arsenate sensitivity and arsenic hyperaccumulation phenotypes of yeast (Saccharomyces cerevisiae) lacking the Arsenate reductase gene ScACR2. Recombinant PvACR2 protein has in vitro Arsenate reductase activity similar to ScACR2. While PvACR2 and ScACR2 have sequence similarities to the CDC25 protein tyrosine phosphatases, they lack phosphatase activity. In contrast, Arath;CDC25, an Arabidopsis (Arabidopsis thaliana) homolog of PvACR2 was found to have both Arsenate reductase and phosphatase activities. To our knowledge, PvACR2 is the first reported plant Arsenate reductase that lacks phosphatase activity. CDC25 protein tyrosine phosphatases and Arsenate reductases have a conserved HCX 5 R motif that defines the active site. PvACR2 is unique in that the arginine of this motif, previously shown to be essential for phosphatase and reductase activity, is replaced with a serine. Steady-state levels of PvACR2 expression in gametophytes were found to be similar in the absence and presence of Arsenate, while total Arsenate reductase activity in P. vittata gametophytes was found to be constitutive and unaffected by Arsenate, consistent with other known metal hyperaccumulation mechanisms in plants. The unusual active site of PvACR2 and the Arsenate reductase activities of cell-free extracts correlate with the ability of P. vittata to hyperaccumulate arsenite, suggesting that PvACR2 may play an important role in this process.