Mechanisms of arsenic hyperaccumulation in Pteris vittata. Uptake kinetics,interactions with phosphate,and arsenic speciation

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).     

Phytoremediation of arsenic contaminated soil by Pteris vittata L. II. Effect on arsenic uptake and rice yield

A greenhouse experiment evaluated the effect of phytoextraction of arsenic from a contaminated soil by Chinese Brake Fern (Pteris vittata L.) and its subsequent effects on growth and uptake of arsenic by rice (Oryza sativa L.) crop. Pteris vittata was grown for one or two growing cycles of four months each with two phosphate sources, using single super phosphate (SSP) and di-ammonium phosphate (DAP). Rice was grown on phytoextracted soils followed by measurements of biomass yield (grain, straw, and root), arsenic concentration and, uptake by individual plant parts. The biomass yield (grain, straw and rice) of rice was highest in soil phytoextracted with Pteris vittata grown for two cycles and fertilized with diammonium phosphate (DAP). Total arsenic uptake in contaminated soil ranged from 8.2 to 16.9 mg pot(-1) in first growing cycle and 5.5 to 12.0 mg pot(-1) in second growing cycle of Pteris vittata. There was thus a mean reduction of 52% in arsenic content of rice grain after two growing cycle of Pteris vittata and 29% after the one growing cycle. The phytoextraction of arsenic contaminated soil by Pteris vittata was beneficial for growing rice resulted in decreased arsenic content in rice grain of <1 ppm. There was a mean improvement in rice grain yield 14% after two growing cycle and 8% after the one growing cycle of brake fern.     

Arsenic-resistant bacteria solubilized arsenic in the growth media and increased growth of arsenic hyperaccumulator Pteris vittata L

The role of arsenic-resistant bacteria (ARB) in arsenic solubilization from growth media and growth enhancement of arsenic-hyperaccumulator Pteris vittata L. was examined. Seven ARB (tolerant to 10 mM arsenate) were isolated from the P. vittata rhizosphere and identified by 16S rRNA sequencing as Pseudomonas sp., Comamonas sp. and Stenotrophomonas sp. During 7-d hydroponic experiments, these bacteria effectively solubilized arsenic from the growth media spiked with insoluble FeAsO? and AlAsO? minerals (from < 5 μg L?1 to 5.04-7.37 mg L?1 As) and enhanced plant arsenic uptake (from 18.1-21.9 to 35.3-236 mg kg?1 As in the fronds). Production of (1) pyochelin-type siderophores by ARB (fluorescent under ultraviolet illumination and characterized with thin layer chromatography) and (2) root exudate (dissolved organic C) by P. vittata may be responsible for As solubilization. Increase in P. vittata root biomass from 1.5-2.2 to 3.4-4.2 g/plant dw by ARB and by arsenic was associated with arsenic-induced plant P uptake. Arsenic resistant bacteria may have potential to enhance phytoremediation of arsenic-contaminated soils by P. vittata.     

Comparison of root absorption, translocation and tolerance of arsenic in the hyperaccumulator Pteris vittata and the nonhyperaccumulator Pteris tremula

* Several fern species can hyperaccumulate arsenic, although the mechanisms are not fully understood. Here we investigate the roles of root absorption, translocation and tolerance in As hyperaccumulation by comparing the hyperaccumulator Pteris vittata and the nonhyperaccumulator Pteris tremula. * The two species were grown in a pot experiment with 0-500 mg As kg-1 added as arsenate, and in a short-term (8 h) uptake experiment with 5 microM arsenate under phosphorus-sufficient conditions. * In the pot experiment, P. vittata accumulated up to 2500 mg As kg-1 frond d. wt and suffered no phytotoxicity. P. tremula accumulated<100 mg As kg-1 frond d. wt and suffered severe phytotoxicity with additions of >or=25 mg As kg-1. In the short-term uptake experiment, P. vittata had a 2.2-fold higher rate of arsenate uptake than P. tremula, and distributed more As taken up to the fronds (76%) than did P. tremula (9%). * Our results show that enhanced root uptake, efficient root-to-shoot translocation, and a much elevated tolerance through internal detoxification all contribute to As hyperaccumulation in P. vittata.     

INFLUENCE OF SOIL PROPERTIES AND PHOSPHATE ADDITION ON ARSENIC UPTAKE FROM POLLUTED SOILS BY VELVETGRASS (HOLCUS LANATUS)

Four kinds of soil material were used in a pot experiment with velvetgrass (Holcus lanatus). Two unpolluted soils: sand (S) and loam (L) were spiked with sodium arsenite (As III) and arsenate (As V), to obtain total arsenic (As) concentrations of 500 mg As kg^?1. Two other soils (ZS I, ZS III), containing 3320 and 5350 mg As kg^?1, were collected from Zloty Stok where gold and arsenic ores were mined and processed for several centuries. The effects of phosphate addition on plants growth and As uptake were investigated. Phosphate was applied to soils in the form of NH₄H₂PO₄ at the rate 0.2 g P/kg. Average concentrations of arsenic in the shoots of velvetgrass grown in spiked soils S and L without P amendment were in the range 18–210 mg As kg^?1 d.wt., whereas those in plants grown on ZS I and ZS II soils were considerably lower, and varied in the range 11–52 mg As kg^?1 d.wt. The addition of phosphate caused a significant increase in plant biomass and therefore the total amounts of As taken up by plants, however, the differences in As concentrations in the shoots of velvetgrass amended and non-amended with phosphate were not statistically significant.     

Arsenic uptake and accumulation in rice (Oryza sativa L.) irrigated with contaminated water

Long-term use of arsenic contaminated groundwater to irrigate crops, especially paddy rice (Oryza sativaL.) has resulted in elevated soil arsenic levels in Bangladesh. There is, therefore, concern regarding accumulation of arsenic in rice grown on these soils. A greenhouse pot experiment was conducted to evaluate the impact of arsenic-contaminated irrigation water on the growth and uptake of arsenic into rice grain, husk, straw and root. There were altogether 10 treatments which were a combination of five arsenate irrigation water concentrations (0–8 mg As l^–1) and two soil phosphate amendments. Use of arsenate containing irrigation water reduced plant height, decreased rice yield and affected development of root growth. Arsenic concentrations in all plant parts increased with increasing arsenate concentration in irrigation water. However, arsenic concentration in rice grain did not exceed the maximum permissible limit of 1.0 mg As kg^–1. Arsenic accumulation in rice straw at very high levels indicates that feeding cattle with such contaminated straw could be a direct threat for their health and also, indirectly, to human health via presumably contaminated bovine meat and milk. Phosphate application neither showed any significant difference in plant growth and development, nor in As concentrations in plant parts.     

Predicting arsenic bioavailability to hyperaccumulator Pteris vittata in arsenic-contaminated soils

Using chemical extraction to evaluate plant arsenic availability in contaminated soils is important to estimate the time frame for site cleanup during phytoremediation. It is also of great value to assess As mobility in soil and its risk in environmental contamination. In this study, four conventional chemical extraction methods (water, ammonium sulfate, ammonium phosphate, and Mehlich III) and a new root-exudate based method were used to evaluate As extractability and to correlate it with As accumulation in P. vittata growing in five As-contaminated soils under greenhouse condition. The relationship between different soil properties, and As extractability and plant As accumulation was also investigated. Arsenic extractability was 4.6%, 7.0%, 18%, 21%, and 46% for water, ammonium sulfate, organic acids, ammonium phosphate, and Mehlich III, respectively. Root exudate (organic acids) solution was suitable for assessing As bioavailability (81%) in the soils while Mehlich III (31%) overestimated the amount of As taken up by plants. Soil organic matter, P and Mg concentrations were positively correlated to plant As accumulation whereas Ca concentration was negatively correlated. Further investigation is needed on the effect of Ca and Mg on As uptake by P. vittata. Moreover, additional As contaminated soils with different properties should be tested.     

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.     

蜈蚣草砷超富集机制及其在砷污染修复中的应用

蕨类植物蜈蚣草能够从土壤中吸收砷,并储存于地上部分羽叶的液泡中。蜈蚣草具有高效的抗氧化系统,以降低砷的毒害;其砷酸还原系统和液泡区隔化是蜈蚣草进行砷解毒和砷超富集的重要机制。本文综述了目前蜈蚣草砷超富集机制研究的主要进展,并对其在修复砷污染环境的应用中进行了讨论。     

Arsenic-resistant bacteria isolated from agricultural soils of Bangladesh and characterization of arsenate-reducing strains

Aims:  To analyse the arsenic-resistant bacterial communities of two agricultural soils of Bangladesh, to isolate arsenic-resistant bacteria, to study their potential role in arsenic transformation and to investigate the genetic determinants for arsenic resistance among the isolates.
Methods and Results:  Enrichment cultures were performed in a minimal medium in the presence of As(III) and As(V) to isolate resistant bacteria. Twenty-one arsenic-resistant bacteria belonging to different genera of Gram-positive and Gram-negative bacteria were isolated. The isolates, with the exception of Oceanimonas doudoroffii Dhal Rw, reduced 2 mmol l⁻¹ As(V) completely to As(III) in aerobic conditions. Putative gene fragments for arsenite efflux pumps were amplified in isolates from Dhal soil and a putative arsenate reductase gene fragment was amplified from a Bacillus sp. from Rice soil.
Conclusions:  Phylogenetically diverse arsenic-resistant bacteria present in agricultural soils of Bangladesh are capable of reducing arsenate to arsenite under aerobic conditions apparently for detoxification purpose.
Significance and Impact of the Study:  This study provides results on identification, levels of arsenic resistance and reduction of arsenate by the bacterial isolates which could play an important role in arsenic cycling in the two arsenic-contaminated soils in Bangladesh.     

Phytoremediation of arsenic contaminated soil by Pteris vittata L. I. Influence of phosphatic fertilizers and repeated harvests

A greenhouse experiment was conducted to evaluate the effectiveness of diammonium phosphate (DAP), single superphosphate (SSP) and two growing cycles on arsenic removal by Chinese Brake Fern (Pteris vittata L.) from an arsenic contaminated Typic Haplustept of the Indian state of West Bengal. After harvest of Pteris vittata the total, Olsen's extractable and other five soil arsenic fractions were determined. The total biomass yield of P. vittata ranged from 10.7 to 16.2 g pot(-1) in first growing cycle and from 7.53 to 11.57 g pot(-1) in second growing cycle. The frond arsenic concentrations ranged from 990 to 1374 mg kg(-1) in first growing cycle and from 875 to 1371 mg kg(-1) in second growing cycle. DAP was most efficient in enhancing biomass yield, frond and root arsenic concentrations and total arsenic removal from soil. After first growing cycle, P. vittata reduced soil arsenic by 10 to 20%, while after two growing cycles Pteris reduced it by 18 to 34%. Among the different arsenic fractions, Fe-bound arsenic dominated over other fractions. Two successive harvests with DAP as the phosphate fertilizer emerged as the promising management strategy for amelioration of arsenic contaminated soil of West Bengal through phyotoextraction by P. vittata.     

Effects of arsenate and phosphate on their accumulation by an arsenic-hyperaccumulator Pteris vittata L.

Arsenate and phosphate interactions are important for better understanding their uptake and accumulation by plant due to their similarities in chemical behaviors. The present study examined the effects of arsenate and phosphate on plant biomass and uptake of arsenate and phosphate by Chinese brake (Pteris vittata L.), a newly-discovered arsenic hyperaccumulator. The plants were grown for 20 weeks in a soil, which received the combinations of 670, 2670, or 5340 mol kg^–1 arsenate and 800, 1600, or 3200 mol kg^–1 phosphate, respectively. Interactions between arsenate and phosphate influenced their availability in the soil, and thus plant growth and uptake of arsenate and phosphate. At low and medium arsenate levels (670 and 2670 mol kg^–1), phosphate had slight effects on arsenate uptake by and growth of Chinese brake. However, phosphate substantially increased plant biomass and arsenate accumulation by alleviating arsenate phytotoxicity at high arsenate levels (5340 mol kg^–1). Moderate doses of arsenate increased plant phosphate uptake, but decreased phosphate concentrations at high doses because of its phytotoxicity. Based on our results, the minimum P/As molar ratios should be at least 1.2 in soil solution or 1.0 in fern fronds for the growth of Chinese brake. Our findings suggest that phosphate application may be an important strategy for efficient use of Chinese brake to phytoremediate arsenic contaminated soils. Further study is needed on the mechanisms of interactive effects of arsenate and phosphate on Chinese brake in hydroponic systems.     

Uptake kinetics of arsenic species in rice plants

Arsenic (As) finds its way into soils used for rice (Oryza sativa) cultivation through polluted irrigation water, and through historic contamination with As-based pesticides. As is known to be present as a number of chemical species in such soils, so we wished to investigate how these species were accumulated by rice. As species found in soil solution from a greenhouse experiment where rice was irrigated with arsenate contaminated water were arsenite, arsenate, dimethylarsinic acid, and monomethylarsonic acid. The short-term uptake kinetics for these four As species were determined in 7-d-old excised rice roots. High-affinity uptake (0-0.0532 mM) for arsenite and arsenate with eight rice varieties, covering two growing seasons, rice var. Boro (dry season) and rice var. Aman (wet season), showed that uptake of both arsenite and arsenate by Boro varieties was less than that of Aman varieties. Arsenite uptake was active, and was taken up at approximately the same rate as arsenate. Greater uptake of arsenite, compared with arsenate, was found at higher substrate concentration (low-affinity uptake system). Competitive inhibition of uptake with phosphate showed that arsenite and arsenate were taken up by different uptake systems because arsenate uptake was strongly suppressed in the presence of phosphate, whereas arsenite transport was not affected by phosphate. At a slow rate, there was a hyperbolic uptake of monomethylarsonic acid, and limited uptake of dimethylarsinic acid.     

砷对土壤-蜈蚣草系统中磷生物有效性的影响

磷是植物必需的大量营养元素,而其同族元素砷却不是植物生长发育所必需的。通过等温吸附平衡实验发现土壤中存在的砷可以降低褐土对磷的吸附,褐土对砷的吸附率大于等于褐土对磷的吸附率。对砷超富集植物蜈蚣草而言,土壤中砷的添加量不超过800mg／kg时,蜈蚣草地上部和地下部磷含量显著提高,结果初步表明,砷可以提高土壤中磷的生物有效性。     

Metabolic adaptation of Pteris vittata L. gametophyte to arsenic induced oxidative stress

The sporophyte and gametophyte of Pteris vittata are arsenic hyperaccumulators, however, little is known about the mechanism by which the gametophyte deals with this toxic element. An in vitro system (spores grown in arsenic amended nutrient media) was used to investigate the impact of arsenic on growth of the gametophyte and the role of antioxidative systems in combating As-stress. When mature spores of P. vittata were grown in medium amended with 0-50 mg kg(-1) of arsenic (as arsenate), the arsenic concentration in the gametophyte increased, with increasing arsenate in the media, but did not inhibit the spore germination and biomass development. Increases in the level of antioxidant enzymes, superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase, and glutathione-Stransferase) and of ascorbic acid and glutathione probably enabled the gametophyte to withstand the oxidative stress caused by arsenate.     

Effects of Phosphate on Arsenate Uptake and Translocation in Nonmetallicolous and Metallicolous Populations of Pteris Vittata L. Under Solution Culture

An arsenic hyperaccumulator, Pteris vittata L., is common in nature and could occur either on As-contaminated soils or on uncontaminated soils. However, it is not clear whether phosphate transporter play similar roles in As uptake and translocation in nonmetallicolous and metallicolous populations of P. vittata. Five populations were used to investigate effects of phosphate on arsenate uptake and translocation in the plants growing in 1.2 L 20% modified Hoagland's nutrient solution containing either 100 μM phosphate or no phosphate and 10 μM arsenate for 1, 2, 6, 12, 24 h, respectively. The results showed that the nonmetallicolous populations accumulated apparently more As in their fronds and roots than the metallicolous populations at both P supply levels. Phosphate significantly (P < 0.01) decreased frond and root concentrations of As during short time solution culture. In addition, the effects of phosphate on As translocation in P. vittata varied among different time-points during time-course hydroponics (1–24 h). The present results indicated that the inhibitory effect of phosphate on arsenate uptake was larger in the three nonmetallicolous populations than those in the two metallicolous populations of P. vittata.     

Arsenic Pollution of a Loam Soil: Retention Form and Decontamination

A study was conducted to assess the retention form of arsenic in soil and to evaluate the use of phosphate for releasing it from the soil. In this study, a loam soil was artificially polluted with arsenate at pH 5.5, which is one of the pH values at which maximum arsenic adsorption occurred. The soil was kept for 2.5 months under wet conditions to allow for stabilization. The soil was maintained under aerobic condition and losses of arsenic by volatilization were determined to be minimal. The soil was then sequentially extracted with a series of chemicals to identify the soil fractions in which the arsenic was bound. The percentage of arsenic found in the Fe bound-exchangeable, reducible-residual, Al bound exchangeable, residual, calcium bound exchangeable, and easily exchangeable forms was 31.6, 27.3, 25.2, 5.5, 4.9, and 4.7%, respectively. A batch experiment showed that at 20°C, 80% of the bound arsenic was removed by phosphate in the pH range of 5 to 7. A power function model was found to fit the data with a desorption rate constant of 402?mg/kg As h^?1.     

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.     

Selecting appropriate forms of nitrogen fertilizer to enhance soil arsenic removal by Pteris vittata: a new approach in phytoremediation

Certain plant species have been shown to vigorously accumulate some metals from soil, and thus represent promising and effective remediation alternatives. In order to select the optimum forms of nitrogen (N) fertilizers for the arsenic (As) hyperaccumulator, Pteris vittata L., to maximize As extraction, five forms of N were added individually to different treatments to study the effect of N forms on As uptake of the plants under soil culture in a greenhouse. Although shoot As concentration tended to decrease and As translocation from root to shoot was inhibited, overall As accumulation was greater due to higher biomass when N fertilizer was added. Arsenic accumulation in plants with N fertilization was 100-300% more than in the plants without N fertilization. There were obvious differences in plant biomass and As accumulation among the N forms, i.e., NH4HCO3, (NH4)2S04, Ca(NO3)2, KNO3, urea. The total As accumulation in the plants grown in As-supplied soil, under different forms of N fertilizer, decreased as NH4HCO3>(NH4)2S04 > urea > Ca(NO3)2 >KNO3>CK. The plants treated with N and As accumulated up to 5.3-7.97 mg As/pot and removed 3.7-5.5% As from the soils, compared to approximately 2.3% of As removal in the control. NH4+ -N was apparently more effective than other N fertilizers in stimulating As removal when soil was supplied with As at initiation. No significant differences in available As were found among different forms of N fertilizer after phytoremediation. It is concluded that NH4+ -N was the preferable fertilizer for P. vittata to maximize As removal.     

Inhibition of microbial arsenate reduction by phosphate

The ratio of arsenite (As(III)) to arsenate (As(V)) in soils and natural waters is often controlled by the activity of As-transforming microorganisms. Phosphate is a chemical analog to As(V) and, consequently, may competitively inhibit microbial uptake and enzymatic binding of As(V), thus preventing its reduction to the more toxic, mobile, and bioavailable form - As(III). Five As-transforming bacteria isolated either from As-treated soil columns or from As-impacted soils were used to evaluate the effects of phosphate on As(V) reduction and As(III) oxidation. Cultures were initially spiked with various P:As ratios, incubated for approximately 48 h, and analyzed periodically for As(V) and As(III) concentration. Arsenate reduction was inhibited at high P:As ratios and completely suppressed at elevated levels of phosphate (500 and 1,000 μM; P inhibition constant (K(i))～20-100 μM). While high P:As ratios effectively shut down microbial As(V) reduction, the expression of the arsenate reductase gene (arsC) was not inhibited under these conditions in the As(V)-reducing isolate, Agrobacterium tumefaciens str. 5B. Further, high phosphate ameliorated As(V)-induced cell growth inhibition caused by high (1mM) As pressure. These results indicate that phosphate may inhibit As(V) reduction by impeding As(V) uptake by the cell via phosphate transport systems or by competitively binding to the active site of ArsC.