Arsenic Hyperaccumulator Fern Research Articles

Whole-Genome Sequence of an Arsenite-Oxidizing Bacterium, Pandoraea sp. Strain NE5, Isolated from the Rhizosphere of the Arsenic Hyperaccumulator Pteris vittata.

Pandoraea sp. strain NE5, an arsenite-oxidizing bacterium, was isolated from the rhizosphere of an arsenic hyperaccumulator fern (Pteris vittate). Here, the genome sequence of Pandoraea sp. strain NE5 is announced.

Soil texture and climate limit cultivation of the arsenic hyperaccumulator Pteris vittata for phytoextraction in a long-term field study

Long term field studies are required to bridge gaps between research and practical application of arsenic phytoextraction with the arsenic-hyperaccumulating fern Pteris vittata. In a 4-year field study, we investigated the effects of nutrient application (compost, inorganic or organic nitrogen, inorganic or organic phosphorus) and soil texture (13 % and 35 % clay) on arsenic phytoextraction with P. vittata in moderately contaminated soils (74–79 mg As/kg in the 0–15 cm depth interval). We found the highest phytoextraction rates, 5 ± 1 kg As/ha/y, in a coarse-textured compost-amended soil after 2 years of phytoextraction. Phytoextraction rates decreased over time, likely due to decreased root growth in mature stands, indicating plants should be replaced every 2–3 years to maintain phytoextraction efficiency. Across soil textures, nitrogen or phosphorus application led to a 60 % decrease in mean frond arsenic concentrations, leading to mean phytoextraction rates 54 % lower than in control ferns. In the fine-textured soil, frond arsenic concentrations were 54 % lower than in the coarse-textured soil, and fewer ferns survived from year 3 to 4. Across soil textures, compost application increased fern survival. We show that phytoextraction with P. vittata is limited to specific soil and climate conditions, narrower than those under which P. vittata grows in the wild.

Arsenic-induced response in roots of arsenic-hyperaccumulator fern and soil enzymatic activity changes

In a pot experiment, arsenic-hyperaccumulating Pteris cretica cv. Albo-lineata plant ferns were cultivated and exposed to low and high doses of arsenate (20 and 100 mg As/kg, respectively) for six months. Physiological and morphological changes of roots, as well as changes in soil quality of the root zone and bulk soil (water-soluble fraction of elements and activity of soil enzymes), were determined. The results showed that the accumulation of inorganic As, mainly in the form of As3+, did not significantly affect the yield of roots, but caused changes in root morphology (deformation of root cell walls due to lignification) and metabolism (decrease of auxin indole-3-acetic acid and 2-oxoindole-3-acetic acid contents). Although the soil quality results varied according to the As dose, there was a clear difference between the root zone and the bulk soil. The activities of enzymes in the root zone were greater that those in the bulk soil. The results showed a significant influence of the high dose of As (100 mg As/kg), which decreased the activity of arylsulfatase, nitrate reductase, and urease in the root zone, while a decrease in acid phosphatase and nitrate reductase was observed in the bulk soil. The water-soluble fractions of As, organic nitrogen, nitrate nitrogen and organic carbon were significantly affected by the high dose of As.

Characterization of a novel arsenite long-distance transporter from arsenic hyperaccumulator fern Pteris vittata.

Pteris vittata is an arsenic (As) hyperaccumulator that can accumulate several thousand mg As kg-1 DW in aboveground biomass. A key factor for its hyperaccumulation ability is its highly efficient As long-distance translocation system. However, the underlying molecular mechanisms remain unknown. We isolated PvAsE1 through the full-length cDNA over-expression library of P.vittata and characterized it through a yeast system, RNAi gametophytes and sporophytes, subcellular-location and insitu hybridization. Phylogenomic analysis was conducted to estimate the appearance time of PvAsE1. PvAsE1 was a plasma membrane-oriented arsenite (AsIII) effluxer. The silencing of PvAsE1 reduced AsIII long-distance translocation in P.vittata sporophytes. PvAsE1 was structurally similar to solute carrier (SLC)13 proteins. Its transcripts could be observed in parenchyma cells surrounding the xylem of roots. The appearance time was estimated at c. 52.7Ma. PvAsE1 was a previously uncharacterized SLC13-like AsIII effluxer, which may contribute to AsIII long-distance translocation via xylem loading. PvAsE1 appeared late in fern evolution and might be an adaptive subject to the selection pressure at the Cretaceaou-Paleogene boundary. The identification of PvAsE1 provides clues for revealing the special As hyperaccumulation characteristics of P.vittata.

Development of suitable hydroponics system for phytoremediation of arsenic-contaminated water using an arsenic hyperaccumulator plant Pteris vittata.

In this study, we found that high-performance hydroponics of arsenic hyperaccumulator fern Pteris vittata is possible without any mechanical aeration system, if rhizomes of the ferns are kept over the water surface level. It was also found that very low-nutrition condition is better for root elongation of P. vittata that is an important factor of the arsenic removal from contaminated water. By the non-aeration and low-nutrition hydroponics for four months, roots of P. vittata were elongated more than 500 mm. The results of arsenate phytofiltration experiments showed that arsenic concentrations in water declined from the initial concentrations (50 μg/L, 500 μg/L, and 1000 μg/L) to lower than the detection limit (0.1 μg/L) and about 80% of arsenic removed was accumulated in the fern fronds. The improved hydroponics method for P. vittata developed in this study enables low-cost phytoremediation of arsenic-contaminated water and high-affinity removal of arsenic from water.

Arsenic Uptake and Translocation by Plants in Pot and Field Experiments

A work undertaken by pot and field experiments to assess the suitability of poplars and ferns for the in-situ, phytoextraction, of a dumping site with residues from the roasting process of arseno-pyrite is reported. The main characteristic of this site is the high content of both the As metalloid and heavy metals (e.g., Al, Fe, Cu, Co, Cr, Pb). Two poplar clones (Populus deltoides ‘Dvina’ and Populus x canadensis ‘Orion’) and Pteris vittata (Chinese brake fern) were planted in the contaminated soil both ex situ in pots and in situ. Plant survival, As accumulation in plant tissues, leaf content of pigments, soluble proteins, activity of catalase and SH-groups in both roots and leaves were evaluated during a 24-month study period. Both poplar and fern plants exhibited an increase in the activity of catalase and SH group contents when grown in the presence of pyrite ashes. The results showed that the co-planting system (arsenic-hyperaccumulator fern Pteris vittata and Populus clones) was suitable for phytoextraction of multi-contaminated dumping sites. Agronomic measures such as irrigation, soil tillage and amendments also seem to be necessary for the successful establishment of poplar trees and ferns in contaminated soils in order to enhance plant growth through the improvement of soil conditions.

Arsenic-induced morphogenic response in roots of arsenic hyperaccumulator fern Pteris vittata

On the assumption that arsenic induces stress morphogenetic responses involved in As tolerance and hyperaccumulation in the Pteris vittata fern, we analyzed the root system of young sporophytes grown in 250, 334, and 500μM As for five days and for 14 days. Anatomical and histological analyses were performed in plants grown for five days to evaluate the number, position, length and differentiation pattern of root hairs. AgNOR staining, employed to study nucleolus behavior in root apices, showed that arsenic influences nucleolar activity (evaluated by nucleolus size, number and absorbance) in the root meristem. In plants treated with 250 and 334μM As an acropetal shift of root hair development and an increase in hair length and density were observed, linked to an ectopic pattern of differentiation. The opposite trend was recorded in plants treated with 500μM As. It is worth noting the presence of living border-like cells, not yet observed in ferns, and their increase following As treatments. Analysis and vitality of border-like cells were surveyed after 14 days of treatments. In conclusion As treatments elicited a stress-induced morphogenic response which, by modifying the differentiation pattern, number and length of root hairs, modulating nucleolar activity and interacting with the rhizosphere by inducing border-like cell production, may adjust the rate of root uptake and its metabolic activity.

Assessment of Bioaccumulation of Heavy Metal by Pteris Vittata L. Growing in the Vicinity of Fly Ash

Pteris vittata L. subsp. vittata, a potential arsenic hyperaccumulator fern, growing naturally in the vicinity of fly ash was analyzed for the concentration of nine heavy metals (Fe, Cu, Zn Ni, Al, Cr, Pb, Si, and As) from five different sites around of Kanti Thermal Power Station at Muzaffarpur in Bihar State, India. Metal accumulation in P. vittata was correlated with the level of pollution at five selected sampling sites. The results revealed significantly more accumulation of these metals in the above ground parts of the plant than the parts below ground. Statistical parameters such as the coefficient of variation (CV%) showed a higher for As, Cu, Cr, and a lower one for Fe, Ni, Al. There was high spatial variability in the total metal concentration at different sites. The present study confirmed that P. vittata is a heavy metals accumulator and that it is a highly suitable candidate for phytoremediation of metal contaminated wastelands.

Proteomic analysis of Pteris vittata fronds: Two arbuscular mycorrhizal fungi differentially modulate protein expression under arsenic contamination

Arbuscular mycorrhizae (AM) are the most widespread mutualistic symbioses between the roots of most land plants and a phylum of soil fungi. AM are known to influence plant performance by improving mineral nutrition, protecting against pathogens and enhancing resistance or tolerance to biotic and abiotic stresses. The aim of this study was to investigate the frond proteome of the arsenic hyperaccumulator fern Pteris vittata in plants that had been inoculated with one of the two AM fungi (Glomus mosseae or Gigaspora margarita) with and without arsenic treatment. A protective role for AM fungi colonisation in the absence of arsenic was indicated by the down-regulation of oxidative damage-related proteins. Arsenic treatment of mycorrhizal ferns induced the differential expression of 130 leaf proteins with specific responses in G. mosseae- and Gi. margarita-colonised plants. Up-regulation of multiple forms of glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and enolase, primarily in G. mosseae-inoculated plants, suggests a central role for glycolytic enzymes in arsenic metabolism. Moreover, a putative arsenic transporter, PgPOR29, has been identified as an up-regulated protein by arsenic treatment.

Characterization of arsenic-resistant bacteria from the rhizosphere of arsenic hyperaccumulatorPteris vittata

Arsenic hyperaccumulator fern Pteris vittata L. produces large amounts of root exudates that are hypothesized to solubilize arsenic and maintain a unique rhizosphere microbial community. Total heterotrophic counts on rich or defined media supplemented with up to 400 mmol/L of arsenate showed a diverse arsenate-resistant microbial community from the rhizosphere of P. vittata growing in arsenic-contaminated sites. Twelve bacterial isolates tolerating 400 mmol/L of arsenate in liquid culture were identified. Selected bacterial isolates belonging to different genera were tested for their resistance to osmotic and oxidative stresses. Results showed that growth was generally better under osmotic stress generated by arsenic than under that generated by NaCl or PEG 6000, demonstrating that arsenic detoxification metabolism also cross-protected bacterial isolates from arsenic-induced osmotic stress. After 32 h of growth, all arsenate at 1 mmol/L was reduced to arsenite by strains Naxibacter sp. AH4, Mesorhizobium sp. AH5, and Pseudomonas sp. AH21, but arsenite at 1 mmol/L remained unchanged. Sensitivity to hydrogen peroxide was similar to that in broad-host pathogen Salmonella enterica sv. Typhimurium wild type, except strain AH4. The results suggested that these arsenic-resistant bacteria are metabolically adapted to arsenic-induced osmotic or oxidative stresses in addition to the specific bacterial system to exclude cellular arsenic. Both these adaptations contribute to the high arsenic resistance in the bacterial isolates.

Characterization of glutathione reductase and catalase in the fronds of two Pteris ferns upon arsenic exposure

To better understand the mechanisms of plant tolerance to high concentration of arsenic, we characterized two antioxidant enzymes, glutathione reductase (GR) and catalase (CAT), in the fronds of Pteris vittata, an arsenic-hyperaccumulating fern, and Pteris ensiformis, an arsenic-sensitive fern. The induction, activation and apparent kinetics of GR and CAT in the plants upon arsenic exposure were investigated. Under arsenic exposure (sodium arsenate), CAT activity in P. vittata was increased by 1.5-fold, but GR activity was unchanged. Further, GR was not inhibited or activated by the arsenic in assays. No significant differences in K(m) and V(max) values of GR or CAT were observed between the two ferns. However, CAT activity in P. vittata was activated by 200 microM arsenate up to 300% compared to the control. Similar but much smaller increases were observed for P. ensiformis and purified bovine liver catalase (133% and 120%, respectively). This research reports, for the first time, the activation of CAT by arsenic in P. vittata. The increased CAT activities may allow P. vittata to more efficiently mediate arsenic-induced stress by preparing the fern for the impeding production of reactive oxygen species resulting from arsenate reduction to arsenite in the fronds.

Expression of a Pteris vittata glutaredoxin PvGRX5 in transgenic Arabidopsis thaliana increases plant arsenic tolerance and decreases arsenic accumulation in the leaves

Chinese brake fern Pteris vittata hyperaccumulates arsenic in its fronds. In a study to identify brake fern cDNAs in arsenic resistance, we implicated a glutaredoxin, PvGRX5, because when expressed in Escherichia coli, it improved arsenic tolerance in recombinant bacteria. Here, we asked whether PvGRX5 transgenic expression would alter plant arsenic tolerance and metabolism. Two lines of Arabidopsis thaliana constitutively expressing PvGrx5 cDNA were compared with vector control and wild-type lines. PvGRX5-expressors were significantly more tolerant to arsenic compared with control lines based on germination, root growth and whole plant growth under imposed arsenic stress. PvGRX5-expressors contained significantly lower total arsenic compared with control lines following treatment with arsenate. Additionally, PvGRX5-expressors were significantly more efficient in their arsenate reduction in vivo. Together, our results indicate that PvGRX5 has a role in arsenic tolerance via improving arsenate reduction and regulating cellular arsenic levels. Paradoxically, our results suggest that PvGRX5 from the arsenic hyperaccumulator fern can be used in a novel biotechnological solution to decrease arsenic in crops.

Herbivory induces a ROS burst and the release of volatile organic compounds in the fern Pteris vittata L.

The arsenic hyper-accumulating fern, Pteris vittata L., was studied to investigate the behavior of lower plants in response to both mechanical (MW) and herbivore wounding (HW). To this end, the cotton leaf worm Spodoptera littoralis was fed on P. vittata and the volatile organic compound (VOC) emission was detected from the fern's head space by using solid phase micro extraction (SPME) and analyzed by GC-MS. Controls were represented by unwounded and MW ferns. The fluorescent probe Amplex red was used to investigate the oxidative burst due to MW and HW by confocal laser scanning microscopy. Ferns responded to herbivore wounding events by emitting the sesquiterpenes (Z)-β-farnesene, (E)-β-farnesene, (Z,E)-α-farnesene, (E,E)-α-farnesene and (E)-nerolidol. An oxidative burst was found close to wounded areas in both MW and HW ferns, and young ferns reacted with a higher H2O2 production after HW. The reported data suggest that lower plants, such as ancient ferns, show two events commonly occurring and reported in many higher and more evolved plants after herbivore attack: an oxidative burst and the emission of volatile terpenoids.

An Arsenate-activated Glutaredoxin from the Arsenic Hyperaccumulator Fern Pteris vittata L. Regulates Intracellular Arsenite

To elucidate the mechanisms of arsenic resistance in the arsenic hyperaccumulator fern Pteris vittata L., a cDNA for a glutaredoxin (Grx) Pv5-6 was isolated from a frond expression cDNA library based on the ability of the cDNA to increase arsenic resistance in Escherichia coli. The deduced amino acid sequence of Pv5-6 showed high homology with an Arabidopsis chloroplastic Grx and contained two CXXS putative catalytic motifs. Purified recombinant Pv5-6 exhibited glutaredoxin activity that was increased 1.6-fold by 10 mm arsenate. Site-specific mutation of Cys(67) to Ala(67) resulted in the loss of both GRX activity and arsenic resistance. PvGrx5 was expressed in E. coli mutants in which the arsenic resistance genes of the ars operon were deleted (strain AW3110), a deletion of the gene for the ArsC arsenate reductase (strain WC3110), and a strain in which the ars operon was deleted and the gene for the GlpF aquaglyceroporin was disrupted (strain OSBR1). Expression of PvGrx5 increased arsenic tolerance in strains AW3110 and WC3110, but not in OSBR1, suggesting that PvGrx5 had a role in cellular arsenic resistance independent of the ars operon genes but dependent on GlpF. AW3110 cells expressing PvGrx5 had significantly lower levels of arsenite when compared with vector controls when cultured in medium containing 2.5 mm arsenate. Our results are consistent with PvGrx5 having a role in regulating intracellular arsenite levels, by either directly or indirectly modulating the aquaglyceroporin. To our knowledge, PvGrx5 is the first plant Grx implicated in arsenic metabolism.

Arsenic distribution and speciation in an arsenic hyperaccumulator fern by X-ray spectrometry utilizing a synchrotron radiation source

The arsenic distribution in the arsenic hyper-accumulating fern (Pteris vittata L.) was investigated using a synchrotron X-ray fluorescence microprobe. Fronds of various ages were subjected to XRF imaging analysis, and it was found that the arsenic distribution in the pinnae of fronds changed according to their stage of growth. The focused microbeam (3.5 × 5.5 μm2) produced by Kirkpatrick–Baez optics was applied to the fern in order to determine the elemental distribution in plant tissue and cell levels. The results indicated that high levels of arsenic accumulate at the base of sporangium with lamina of pinnae. Also, the fern was subjected to X-ray absorption near edge structure (XANES) analysis without any sample treatment to directly elucidate the arsenic oxidation state in fern. It was found that arsenic exists as the As(III) form in pinnae, and as a mixture of As(III) and As(V) in rachis, while As(V) is present in cultivated soil. These findings indicate that the fern uptakes arsenic as As(V) from soil and that the As(V) is then partially reduced to As(III) within the plant, with the arsenic finally accumulating as As(III) in a specific area of the pinna.

Phytofiltration of arsenic from drinking water using arsenic-hyperaccumulating ferns.

Arsenic contamination of drinking water poses serious health risks to millions of people worldwide. Current technologies used to clean arsenic-contaminated water have significant drawbacks, such as high cost and generation of large volumes of toxic waste. In this study, we investigated the potential of using recently identified arsenic-hyperaccumulating ferns to remove arsenic from drinking water. Hydroponically cultivated, two arsenic-hyperaccumulating fern species (Pteris vittata and Pteris cretica cv. Mayii) and a nonaccumulating fern species (Nephrolepis exaltata) were suspended in water containing 73As-labeled arsenic with initial arsenic concentrations ranging from 20 to 500 microg L(-1). The efficiency of arsenic phytofiltration by these fern species was determined by continuously monitoring the depletion of 73As-labeled arsenic concentration in the water. With an initial water arsenic concentration of 200 microg L(-1), P. vittata reduced the arsenic concentration by 98.6% to 2.8 microg L(-1) in 24 h. When the initial water arsenic was 20 microg L(-1), P. vittata reduced the arsenic concentration to 7.2 microg L(-1) in 6 h and to 0.4 microg L(-1) in 24 h. At similar plant ages, both P. vittata and P. cretica had similar arsenic phytofiltration efficiency and were able to rapidly remove arsenic from water to achieve arsenic levels below the new drinking water limit of 10 microg L(-1). However, N. exaltata failed to reduce water arsenic to achieve the limit under the same experimental conditions. The significantly higher efficiency of arsenic phytofiltration by arsenic-hyperaccumulating fern species is associated with their ability to rapidly translocate absorbed arsenic from roots to shoots. The nonaccumulating fern N. exaltata was unable to translocate the absorbed arsenic to the shoots. Our results demonstrate that the arsenic-phytofiltration technique may provide the basis for a solar-powered hydroponic technique that enables small-scale cleanup of arsenic-contaminated drinking water.

Phytoremediation of Arsenic-Contaminated Groundwater by the Arsenic Hyperaccumulating Fern Pteris vittata L.

ABSTRACT Arsenic concentrations in a much larger fraction of U.S. groundwater sources will exceed the maximum contaminant limit when the new 10 μg L−1 EPA standard for drinking water takes effect in 2006. Thus, it is important to develop remediation technologies that can meet this new standard. Phytoremediation of arsenic-contaminated groundwater is a relatively new idea. In this research, an arsenic-hyperaccumulating fern, commonly known as Chinese Brake fern (Pteris vittata L.), was grown hydroponically to examine its effectiveness in arsenic removal from what is believed to be herbicide-contaminated groundwater. One plant grown in 600 mL of groundwater effectively reduced the arsenic concentration from 46 to less than 10 μg L−1 in 3 days. Re-used plants continued to take up arsenic from the groundwater, albeit at a slower rate (from 46 to 20 μg L− 1 during the same time). Young fern plants were more efficient in removing arsenic than were older fern plants of similar size. The addition of a supplement of phosphate-free Hoagland nutrition to the groundwater had little effect on arsenic removal, but the addition of phosphate nutrition significantly reduced its arsenic affinity and, thus, inhibited the arsenic removal. This study suggested that Chinese Brake has some potential to remove arsenic from groundwater.

XAS speciation of arsenic in a hyper-accumulating fern.

The coordination environment and the redox speciation of arsenic in a newly discovered arsenic hyper-accumulating fern (Pteris vittata L) were investigated by X-ray absorption spectroscopy. This method allowed us to probe arsenic directly, i.e., with minimal sample preparation. The results indicate that arsenic is predominantly accumulated as As(III) in the leaves. XANES and EXAFS results show that As(III) in the leaves is primarily present as aqueous arsenite species. The plant actively maintains arsenic in this reduced oxidation state, because after sample collection and subsequent aging and drying of the plant material, As(III) is gradually oxidized to As(V). We think that these arsenite species are sequestered in vacuoles. At extremely high As concentrations (ca. 1% As per dry weight) arsenic in the fern leaves is coordinated to a significant degree by sulfur in addition to oxygen. This spectral signature indicates that thiol-rich compounds are implicated in the biochemical transformations of arsenic within the plant.