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.     

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.     

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.     

Effectiveness of applying arsenate reducing bacteria to enhance arsenic removal from polluted soils by Pteris vittata L

Arsenic is a common contaminant in soils and water. It is well established that the fern Pteris vittata L. is an As hyperaccumulator and therefore has potential to phyroremediate As-polluted soils. Also, it is accepted that rhizosphere microflora play an enhancing role in plant uptake of metallic elements from soils. Studies showed that hydroponiclly grown P. Vittata accumulated arsenite more than the arsenate form of As apparently because arsenate and phosphate are analogues and therefore its absorption is inhibited by phosphate. The objective of this study was to determine whether addition of five different arsenate-reducing bacteria would enhance arsenic uptake by P. vittata grown in arsenic polluted soils in afield experiment. Results showed that addition of the As reducing bacteria promoted the growth of P. vittata, increased As accumulation, activated soil insoluble As, and reduced As leaching compared to the untreated control. Plant biomass increased by 53% and As uptake by 44%. As leaching was reduced by 29% to 71% depending on the As reducing bacterium. The results in their entirety permitted some insight into the mechanisms by which the arsenate reducing bacteria enhanced the effectiveness of P. vittata to remove As from the polluted soil.     

Effects of arsenic species and concentrations on arsenic accumulation by different fern species in a hydroponic system

Two hydroponic experiments were conducted to evaluate factors affecting plant arsenic (As) hyperaccumulation. In the first experiment; two As hyperaccumulators (Pteris vittata and P. cretica mayii) were exposed to 1 and 10 mg L(-1) arsenite (AsIII) and monomethyl arsenic acid (MMA) for 4 wk. Total As concentrations in plants (fronds and roots) and solution were determined In the second experiment P. vittata and Nephrolepis exaltata (a non-As hyperaccumulator) were exposed to 5 mgL(-1) arsenate (AsV) and 20 mgL(-1) AsIIIfor 1 and 15 d. Total As and AsIII concentrations in plants were determined Compared to P. cretica mayii, P. vittata was more efficient in arsenic accumulation (1075-1666 vs. 249-627mg kg(-1) As in the fronds) partially because it is more efficient in As translocation. As translocation factor (As concentration ratio in fronds to roots) was 3.0-5.6 for P. vittata compared to 0.1 to 4.8 for P. cretica. Compared to N. exaltata, P. vittata was significantly more efficient in arsenic accumulation (38-542 vs. 4.8-71 mg kg(-1) As in thefronds) as well asAs translocation (1.3-5.6 vs. 0.2-0.5). In addition, P. vittata was much more efficient in As reduction from AsV to AsIII (83-84 vs. 13-24% AsIII in the fronds). Little As reduction occurred after 1-d exposure to AsV in both species indicates that As reduction was not instantaneous even in an As hyperaccumulator. Our data were consistent with the hypothesis that both As translocation and As reduction are important for plant As hyperaccumulation.     

Uptake kinetics of different arsenic species by Brassica carinata

The time- and concentration-dependent uptake kinetics for arsenate and arsenite were determined in 15-day-old excised roots. In both cases, arsenite showed a mono-phasic influx with the isotherm data fitting a linear model better than a non-linear one. The time- and the concentration-dependent uptake of arsenate displayed a hyperbolic kinetic. Greater uptake of arsenate, compared with arsenite, was found especially at lower external substrate concentrations. Competitive inhibition of uptake with phosphate showed that arsenite and arsenate were taken up by different uptake systems because arsenate uptake was strongly inhibited in the presence of phosphate, whereas arsenite uptake was not affected.     

Interaction of arsenic and phosphate on their uptake and accumulation in Chinese brake fern

Interactive effects of arsenate (As (V)) and phosphate (Pi) were investigated under hydroponic culture. Arsenic concentrations in fronds and roots of Chinese brake fern (Pteris vittata L.) significantly (p < 0.05) increased with increasing As (V), but decreased (p < 0.05) with increasing Pi in nutrient solution. Phosphate uptake was significantly (p < 0.05) inhibited by 1000 micromol L(-1) As (V). Under 100 micromol L(-1) As (V), frond phosphorus (P) increased at 100 and 1000 micromol L(-1) Pi, and root P increased at 250 micromol L(-1) Pi exposures. Arsenic and P concentrations in fronds and roots of Chinese brake fern were negatively correlated (p < 0.05). Arsenate treatments enhanced As and P transport to fronds, while increasing Pi inhibited their transportation, with highest frond P and As (%) obtained under 100 micromol L(-1) treatment. pH values in nutrient solution increased with increasing exposure time, but decreased with increasing Pi levels. Dissolved organic carbon (DOC) contents (dry weight) in nutrient solution decreased with increasing Pi levels, both for treatments with and without As (V). Arsenate at 1000 micromol L(-1) significantly (p < 0.05) increased DOC contents, especially for treatment without Pi. Six organic acids were detected in root exudates of Chinese brake fern, with oxalic and malic acids being most dominant.     

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.     

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.     

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.     

Effects of selenium on arsenic uptake in arsenic hyperaccumulator Pteris vittata L

Selenium (Se) is a non-metallic element, which has the capability to increase the antioxidative capacity and stress tolerance of plants to heavy metals. Plants vary considerably in their physiological response to Se. The reported research investigated the effects of Se on arsenic (As) uptake by As hyperaccumulator Pteris vittata L. and determined possible mechanisms of interaction. Pteris vittata plants were exposed hydroponically to 0, 150 or 300 microM of Na(2)HAsO(4) in the presence of 0, 5 or 10 microM of Na(2)SeO(4) for 5 or 10d. Application of 5 microM Se enhanced As concentration by P. vittata fronds by 7-45%. At 5 microM, Se acted as an antioxidant, inhibiting lipid peroxidation (reduced by 26-42% in the fronds) via increased levels of thiols and glutathione (increased by 24% in the fronds). The results suggest that Se is either an antioxidant or it activates plant protective mechanisms, thereby alleviating oxidative stress and improving arsenic uptake in P. vittata.     

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.     

Root exudates and arsenic accumulation in arsenic hyperaccumulating Pteris vittata and non-hyperaccumulating Nephrolepis exaltata

This study compared the roles of root exudates collected from two fern species, the As hyperaccumulating Chinese Brake fern (Pteris vittata L.) and the As-sensitive Boston fern (Nephrolepis exaltata L.), on As-mobilization of two As minerals (aluminum arsenate and iron arsenate) and a CCA (chromated copper arsenate)-contaminated soil as well as plant As accumulation. Chinese Brake fern exuded 2 times more dissolved organic carbon (DOC) than Boston fern and the difference was more pronounced under As stress. The composition of organic acids in the root exudates for both ferns consisted mainly of phytic acid and oxalic acid. However, Chinese Brake fern produced 0.46 to 1.06 times more phytic acid than Boston fern under As stress, and exuded 3–5 times more oxalic acid than Boston fern in all treatments. Consequently, root exudates from Chinese Brake fern mobilized more As from aluminum arsenate (3–4 times), iron arsenate (4–6 times) and CCA-contaminated soil (6–18 times) than Boston fern. Chinese Brake fern took up more As and translocated more As to the fronds than Boston fern. The molar ratio of P/As in the roots of Chinese Brake fern was greater than in the fronds whereas the reverse was observed in Boston fern. These results suggested that As-mobilization from the soil by the root exudates (enhancing plant uptake), coupled with efficient As translocation to the fronds (keeping a high molar ratio of P/As in the roots), are both important for As hyperaccumulation by Chinese Brake fern.     

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.     

Direct evidence showing the effect of root surface iron plaque on arsenite and arsenate uptake into rice (Oryza sativa) roots

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.     

Toxicity of arsenic (III) and (V) on plant growth, element uptake, and total amylolytic activity of mesquite (Prosopis juliflora x P. velutina)

The effects of arsenite [As(III)] and arsenate [As(V)] on the growth of roots, stems, and leaves and the uptake of arsenic (As), micro- and macronutrients, and total amylolytic activity were investigated to elucidate the phytotoxicity of As to the mesquite plant (Prosopis juliflora x P. velutina). The plant growth was evaluated by measuring the root and shoot length, and the element uptake was determined using inductively coupled plasma optical emission spectroscopy. The root and leaf elongation decreased significantly with increasing As(III) and As(V) concentrations; whereas, stem elongation remained unchanged. The As uptake increased with increasing As(III) or As(V) concentrations in the medium. Plants treated with 50 mg/L As(III) accumulated up to 920 mg/kg dry weight (d wt) in roots and 522 mg/kg d wt in leaves, while plants exposed to 50 mg/L As(V) accumulated 1980 and 210 mg/kg d wt in roots and leaves, respectively. Increasing the As(V) concentration up to 20 mg/L resulted in a decrease in the total amylolytic activity. On the contrary, total amylolytic activity in As(III)-treated plants increased with increasing As concentration up to 20 mg/L. The macro- and micronutrient concentrations changed in As-treated plants. In shoots, Mo and K were reduced but Ca was increased, while in roots Fe and Ca were increased but K was reduced. These changes reduced the size of the plants, mainly in the As(III)-treated plants; however, there were no visible sign of As toxicity.     

Mechanisms of arsenic hyperaccumulation in<Emphasis Type="Italic"> Pteris</Emphasis> species: root As influx and translocation

Several species of fern from the Pteris genus are able to accumulate extremely high concentrations of arsenic (As) in the fronds. We have conducted short-term unidirectional As influx and translocation experiments with ⁷³As-radiolabeled arsenate, and found that the concentration-dependent influx of arsenate into roots was significantly larger in two of these As-hyperaccumulating species, Pteris vittata (L.) and Pteris cretica cv. Mayii (L.), than in Nephrolepis exaltata (L.), a non-accumulating fern. The arsenate influx could be described by Michaelis-Menten kinetics and the kinetic parameter K _m was found to be lower in the Pteris species, indicating higher affinity of the transport protein for arsenate. Quantitative analysis of kinetic parameters showed that phosphate inhibited arsenate influx in a directly competitive manner, consistent with the hypothesis that arsenate enters plant roots on a phosphate-transport protein. The significantly augmented translocation of arsenic to the shoots that was seen in these As hyperaccumulator species is proposed to be due to a combination of the increased root influx and also decreased sequestration of As in the roots, as a larger fraction of As could be extracted from roots of the Pteris species than from roots of N. exaltata. This leaves a larger pool of mobile As available for translocation to the shoot, probably predominantly as arsenite.Abbreviations As ^V Arsenate - As ^III Arsenite - K _m Michaelis-Menten constant - P _i Phosphate - V _max Maximum rate of an enzyme-catalyzed reaction     

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.     

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.