Expressing ScACR3 in rice enhanced arsenite efflux and reduced arsenic accumulation in rice grains

Arsenic (As) accumulation in rice grain poses a serious health risk to populations with high rice consumption. Extrusion of arsenite [As(III)] by ScAcr3p is the major arsenic detoxification mechanism in Saccharomyces cerevisiae. However, ScAcr3p homolog is absent in higher plants, including rice. In this study, ScACR3 was introduced into rice and expressed under the control of the Cauliflower mosaic virus (CaMV) 35S promoter. In the transgenic lines, As concentrations in shoots and roots were about 30% lower than in the wild type, while the As translocation factors were similar between transgenic lines and the wild type. The roots of transgenic plants exhibited significantly higher As efflux activities than those of the wild type. Within 24 h exposure to 10 μM arsenate [As(V)], roots of ScACR3-expressing plants extruded 80% of absorbed As(V) to the external solution as As(III), while roots of the wild type extruded 50% of absorbed As(V). Additionally, by exposing the As-containing rice plants to an As-lacking solution for 24 h, about 30% of the total As derived from pre-treatment was extruded to the external solution by ScACR3-expressing plants, while about 15% of As was extruded by wild-type plants. Importantly, ScACR3 expression significantly reduced As accumulation in rice straws and grains. When grown in flooded soil irrigated with As(III)-containing water, the As concentration in husk and brown rice of the transgenic lines was reduced by 30 and 20%, respectively, compared with the wild type. This study reports a potential strategy to reduce As accumulation in the food chain by expressing heterologous genes in crops.     

Heterologous expression of the yeast arsenite efflux system ACR3 improves Arabidopsis thaliana tolerance to arsenic stress

? Arsenic contamination has a negative impact on crop cultivation and on human health. As yet, no proteins have been identified in plants that mediate the extrusion of arsenic. Here, we heterologously expressed the yeast (Saccharomyces cerevisiae) arsenite efflux transporter ACR3 into Arabidopsis to evaluate how this affects plant tolerance and tissue arsenic contents. ? ACR3 was cloned from yeast and transformed into wild-type and nip7;1 Arabidopsis. Arsenic tolerance was determined at the cellular level using vitality stains in protoplasts, in intact seedlings grown on agar plates and in mature plants grown hydroponically. Arsenic efflux was measured from protoplasts and from intact plants, and arsenic levels were measured in roots and shoots of plants exposed to arsenate. ? At the cellular level, all transgenic lines showed increased tolerance to arsenite and arsenate and a greater capacity for arsenate efflux. With intact plants, three of four stably transformed lines showed improved growth, whereas only transgenic lines in the wild-type background showed increased efflux of arsenite into the external medium. The presence of ACR3 hardly affected tissue arsenic levels, but increased arsenic translocation to the shoot. ? Heterologous expression of yeast ACR3 endows plants with greater arsenic resistance, but does not lower significantly arsenic tissue levels.     

Rice is more efficient in arsenite uptake and translocation than wheat and barley

Rice is efficient at arsenic (As) accumulation, thus posing a potential health risk to humans and animals. Arsenic bioavailability in submerged paddy soil is enhanced due to mobilisation of arsenite, but rice may also have an inherently greater ability to take up and translocate arsenite than other cereal crops. To test this hypothesis, rice, wheat and barley were exposed to 5 µM arsenate or arsenite for 24 h. Arsenic uptake and distribution, and As speciation in the xylem sap and nutrient solution were determined. Regardless of the As form supplied to plants, rice accumulated more As in the shoots than wheat or barley. Arsenite uptake by rice was double of that by wheat or barley, whereas arsenate uptake was similar between rice and wheat and approximately a third smaller in barley. The efficiency of As translocation from roots to shoots was greater when plants were supplied with arsenite than with arsenate, and in both treatments rice showed the highest translocation efficiency. Arsenite was the main species of As (86–97%) in the xylem sap from arsenite-treated plants of all three species. In the arsenate-treated plants, 84%, 45% and 63% of As in the xylem sap of rice, wheat and barley, respectively, was arsenite. Arsenite efflux to the external medium was also observed in all three plant species exposed to arsenate. The results show that rice is more efficient than wheat or barley in arsenite uptake and translocation, probably through the highly efficient pathway for silicon.     

Highly efficient xylem transport of arsenite in the arsenic hyperaccumulator Pteris vittata

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 microM arsenate or arsenite for 1-24 h, with or without 100 microM 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.     

Characterization of arsenate reductase in the extract of roots and fronds of Chinese brake fern, an arsenic hyperaccumulator

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.     

Rapid reduction of arsenate in the medium mediated by plant roots

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 microm 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.     

A novel arsenate reductase from the arsenic hyperaccumulating fern Pteris vittata

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 HCX5R 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.     

Influence of sulphur on arsenic accumulation and metabolism in rice seedlings

The influence of sulphur on the accumulation and metabolism of arsenic in rice was investigated. Rice seedlings were grown in nutrient solutions with low sulphate (1.8 μM SO₄²⁻) or high sulphate (0.7 mM SO₄²⁻) for 12 or 14 d, before being exposed to 10 μM arsenite or arsenate for 2 or 1 d, respectively. In the arsenite exposure treatment, low sulphate-pretreated rice accumulated less arsenite than high sulphate pretreated plants, but the arsenite concentrations in shoots of low sulphate pretreated rice were higher than those of high sulphate pretreated. In the arsenate exposure treatment, the low sulphate pre-treatments also resulted in less arsenite accumulation in rice roots. Sulphur deprivation in nutrient solution decreased the concentrations of non-protein thiols in rice roots exposed to either arsenite or arsenate. The low sulphate-pretreated plants had a higher arsenic transfer factor than the high sulphate-pretreated plants. The results suggest that rice sulphate nutrition plays an important role in regulating arsenic translocation from roots to shoots, possibly through the complexation of arsenite-phytochelatins.     

Characterization of As efflux from the roots of As hyperaccumulator <Emphasis Type="Italic">Pteris vittata</Emphasis> L.

In some plant species, various arsenic (As) species have been reported to efflux from the roots. However, the details of As efflux by the As hyperaccumulator Pteris vittata remain unknown. In this study, root As efflux was investigated for different phosphorus (P) supply conditions during or after a 24-h arsenate uptake experiment under hydroponic growth conditions. During an 8-h arsenate uptake experiment, P-supplied (P+) P. vittata exhibited much greater arsenite efflux relative to arsenate uptake when compared with P-deprived (P–) P. vittata, indicating that arsenite efflux was not proportional to arsenate uptake. In the As efflux experiment following 24 h of arsenate uptake, arsenate efflux was also observed with arsenite efflux in the external solution. All the results showed relatively low rates of arsenate efflux, ranging from 5.4 to 16.1% of the previously absorbed As, indicating that a low rate of arsenate efflux to the external solution is also a characteristic of P. vittata, as was reported with arsenite efflux. In conclusion, after 24 h of arsenate uptake, both P+ and P– P. vittata loaded/effluxed similar amounts of arsenite to the fronds and the external solution, indicating a similar process of xylem loading and efflux for arsenite, with the order of the arsenite concentrations being solution ≪ roots ≪ fronds.     

Visualisation of gradients in arsenic concentrations around individual roots of Zea mays L. using agar-immobilized bioreporter bacteria

The classical concept of arsenic transfer into plants through arsenate uptake via phosphate transporters, reduction to arsenite, complexation and compartmentation within vacuoles is challenged by recent identification of bidirectional transporters for arsenite and their potential role in plant As status regulation. Soil-based studies with chemical analysis of soil solution require root mat formation amplifying root effects on their surroundings and additionally denying investigations along individual roots differing in age and function. We tried to overcome these shortcomings by using bioreporter bacteria to visualise the spatial distribution of inorganic arsenic along roots and to characterize inorganic arsenic gradients in the rhizosphere concurrent with root age and branching. Therefore we developed an agar-based carrier element ensuring intimate contact between bioreporters and root-soil system and enabling fast and easy reporter output analysis. We show that inorganic arsenic distribution is related to root development with the highest bioreporter signal induction around lateral roots, which are known to show the highest expression of transporters responsible for bidirectional arsenite flux. Since there is so far no evidence for an arsenate efflux mechanism this is a strong indicator that we observed rather arsenite than arsenate efflux. No signal was detected along the distal region of young adventitious roots, i.e. the region of extension growth and root hair formation. The novel bioreporter assay may thus complement conventional measurements by providing information on the spatial distribution of inorganic arsenic on mm to cm-scale.     

Influence of glutathione chemical effectors in the response of maize to arsenic exposure

To support the key role of glutathione (GSH) in the mechanisms of tolerance and accumulation of arsenic in plants, this work examines the impact of several effectors of GSH synthesis or action in the response of maize (Zea mays L.) to arsenic. Maize was exposed in hydroponics to iso-toxic rates of 150 μM arsenate or 75 μM arsenite for 9 days and GSH effectors, flurazole (an herbicide safener), l-buthionine-sulfoximine (BSO, a known inhibitor of GSH biosynthesis), and dimercaptosuccinate (DMS) and dimercaptopropanesulfonate (DMPS) (two thiols able to displace GSH from arsenite-GSH complexes) were assayed. The main responses of plants to arsenic exposure consisted of a biomass reduction (fresh weight basis) of about 50%, an increase of non-protein thiol (NPTs) levels (especially in the GSH precursor γ-glutamylcysteine and the phytochelatins PC? and PC?) in roots, with little effect in shoots, and an accumulation of between 600 and 1000 ppm of As (dry weight basis) in roots with very little translocation to shoots. Growth inhibition caused by arsenic was partially or completely reversed in plants co-treated with flurazole and arsenate or arsenite, respectively, highly exacerbated in plants co-treated with BSO, and not modified in plants co-treated with DMS or DMPS. These responses correlated well with an increase of both NPTs levels in roots and glutathione transferase activity in roots and shoots due to flurazole treatment, the decrease of NPTs levels in roots caused by BSO and the lack of effect on NPT levels caused by both DMS and DMPS. Regarding to arsenic accumulation in roots, it was not modified by flurazole, highly reduced by BSO, and increased between 2.5- and 4.0-fold by DMS and DMPS. Therefore, tolerance and accumulation of arsenic by maize could be manipulated pharmacologically by chemical effectors of GSH.     

The distribution of arsenate and arsenite in shoots and roots of Holcus lanatus is influenced by arsenic tolerance and arsenate and phosphate supply

The recent discovery that phytochelatins are important for arsenic (As) detoxification in terrestrial plants results in the necessity to understand As speciation and metabolism in plant material. A hydroponic study was therefore conducted to examine the effects of different levels of phosphate and arsenate [As(V)] on As speciation and distribution in tolerant and non-tolerant clones of Holcus lanatus. Speciation of As in tissue (using high-performance liquid chromatography-inductively coupled plasma mass spectrometry) revealed that the predominant species present were the inorganic As species (As(V) and arsenite [As(III)]), although small levels (<1%) of organic As species (dimethylarsinic acid and monomethylarsonic acid) were detected in shoot material. In roots, the proportion of total As present as As(III) generally increased with increasing levels of As(V) in the nutrient solution, whereas in shoots, the proportion of total As present as As(III) generally decreased with increasing levels of As(V). H. lanatus plants growing in the high-phosphorus (P) (100 micro M) solution contained a higher proportion of As(V) (with regard to total As) in both roots and shoots than plants supplied with low P (10 micro M); in addition, tolerant clones generally contained a higher proportion of As(V) with regard to total As than non-tolerant clones. The study further revealed that As(V) can be reduced to As(III) in both roots and shoots. Although the reduction capacity was limited, the reduction was closely regulated by As influx for all treatments. The results therefore provide a new understanding about As metabolism in H. lanatus.     

Arsenate, arsenite and dimethyl arsinic acid (DMA) uptake and tolerance in maize (Zea mays L.)

The influx of arsenate, arsenite and dimethyl arsinic acid (DMA) were studied in 7-day-old excised maize roots (Zea mays L.), and then related to arsenate, arsenite and DMA toxicity. Arsenate, arsenite and DMA influx was all found concentration dependent with significant genotypic differences for arsenite and DMA. Arsenate influx in phosphate starved plants best fitted the four-parameter Michaelis–Menten model corresponding to an additive high and low affinity uptake system, while the uptake of phosphate replete plants followed the two parameter model of Michaelis–Menten kinetics. Arsenite influx was well described by the two parameter model of ‘Michaelis–Menten’ kinetics. DMA influx was comprised of linear phase and a hyperbolic phase. DMA influx was much lower than that for arsenite and arsenate. Arsenate and DMA influx decreased when phosphate was given as a pre-treatment as opposed to phosphate starved plants. The +P treatment tended to decrease influx by 50% for arsenate while this figure was 90% for DMA. Arsenite influx increasing slightly at higher arsenite concentrations in P starved plants but at lower arsenite concentrations, there was little or no difference in arsenite uptake. Low toxicity was found for DMA on maize compared with arsenate and arsenite and the relative toxicity of arsenic species was As(V) > As(III) >> DMA.     

Do radial oxygen loss and external aeration affect iron plaque formation and arsenic accumulation and speciation in rice?

Hydroponic experiments were conducted to investigate the effect of radial oxygen loss (ROL) and external aeration on iron (Fe) plaque formation, and arsenic (As) accumulation and speciation in rice (Oryza sativa L.). The data showed that there were significant correlations between ROL and Fe concentrations in Fe plaque produced on different genotypes of rice. There were also significant differences in the amounts of Fe plaque formed between different genotypes in different positions of roots and under different aeration conditions (aerated, normal, and stagnant treatments). In aerated treatments, rice tended to have a higher Fe plaque formation than in a stagnant solution, with the greatest formation at the root tip decreasing with increasing distances away, in accordance with a trend of spatial ROL. Genotypes with higher rates of ROL induced higher degrees of Fe plaque formation. Plaques sequestered As on rice roots, with arsenate almost double that with arsenite, leading to decreased As accumulation in both roots and shoots. The major As species detected in roots and shoots was arsenite, ranging from 34 to 78% of the total As in the different treatments and genotypes. These results contribute to our understanding of genotypic differences in As uptake by rice and the mechanisms causing rice genotypes with higher ROL to show lower overall As accumulation.     

Effects of Iron and Manganese Plaques on Arsenic Uptake by Rice Seedlings (Oryza sativa L.) Grown in Solution Culture Supplied with Arsenate and Arsenite

We have shown previously that phosphorus nutrition and iron plaque on the surface of rice roots influence arsenate uptake and translocation by rice in hydroponic culture. We have now investigated the role of iron (Fe) and manganese (Mn) plaque on arsenate and arsenite uptake and translocation in rice seedlings grown hydroponically. Fe and Mn plaques were clearly visible as reddish or brown coatings on the root surface after 12 h induction, and Fe plaque was much more apparent than Mn plaque. Arsenite or arsenate supply did not decrease plant dry weights significantly. There were significant differences in shoot dry weights but little difference in root dry weights between some plaque treatments. Arsenic (As) concentrations in Fe plaque when arsenate was supplied were significantly higher than those in no plaque (control) and Mn plaque treatments, and much higher than those supplied with arsenite. This showed that Fe plaque on the rice root had higher affinity to arsenate than to arsenite. In Fe plaque treatment, the results indicated that most As was sequestered in roots when arsenite was supplied and most As concentrated in Fe plaque when arsenate was supplied. Most As was accumulated in rice roots in Mn plaque and no plaque treatments for both As species.     

Response to Arsenate Treatment in Schizosaccharomyces pombe and the Role of Its Arsenate Reductase Activity

Arsenic toxicity has been studied for a long time due to its effects in humans. Although epidemiological studies have demonstrated multiple effects in human physiology, there are many open questions about the cellular targets and the mechanisms of response to arsenic. Using the fission yeast Schizosaccharomyces pombe as model system, we have been able to demonstrate a strong activation of the MAPK Spc1/Sty1 in response to arsenate. This activation is dependent on Wis1 activation and Pyp2 phosphatase inactivation. Using arsenic speciation analysis we have also demonstrated the previously unknown capacity of S. pombe cells to reduce As (V) to As (III). Genetic analysis of several fission yeast mutants point towards the cell cycle phosphatase Cdc25 as a possible candidate to carry out this arsenate reductase activity. We propose that arsenate reduction and intracellular accumulation of arsenite are the key mechanisms of arsenate tolerance in fission yeast.     

A CDC25 homologue from rice functions as an arsenate reductase

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)(5)R 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.     

High-resolution secondary ion mass spectrometry reveals the contrasting subcellular distribution of arsenic and silicon in rice roots

Rice (Oryza sativa) takes up arsenite mainly through the silicic acid transport pathway. Understanding the uptake and sequestration of arsenic (As) into the rice plant is important for developing strategies to reduce As concentration in rice grain. In this study, the cellular and subcellular distributions of As and silicon (Si) in rice roots were investigated using high-pressure freezing, high-resolution secondary ion mass spectrometry, and transmission electron microscopy. Rice plants, both the lsi2 mutant lacking the Si/arsenite efflux transporter Lsi2 and its wild-type cultivar, with or without an iron plaque, were treated with arsenate or arsenite. The formation of iron plaque on the root surface resulted in strong accumulation of As and phosphorous on the epidermis. The lsi2 mutant showed stronger As accumulation in the endodermal vacuoles, where the Lsi2 transporter is located in the plasma membranes, than the wild-type line. As also accumulated in the vacuoles of some xylem parenchyma cells and in some pericycle cells, particularly in the wild-type mature root zone. Vacuolar accumulation of As is associated with sulfur, suggesting that As may be stored as arsenite-phytochelatin complexes. Si was localized in the cell walls of the endodermal cells with little apparent effect of the Lsi2 mutation on its distribution. This study reveals the vacuolar sequestration of As in rice roots and contrasting patterns of As and Si subcellular localization, despite both being transported across the plasma membranes by the same transporters.