Research advances in mechanisms of arsenic hyperaccumulation of Pteris vittata: Perspectives from plant physiology, molecular biology, and phylogeny.

Pteris vittata, as the firstly discovered arsenic (As) hyperaccumulator, has great application value in As-contaminated soil remediation. Currently, the genes involved in As hyperaccumulation in P. vittata have been mined continuously, while they have not been used in practice to enhance phytoremediation efficiency. Aiming to better assist the practice of phytoremediation, this review collects 130 studies to clarify the progress in research into the As hyperaccumulation process in P. vittata from multiple perspectives. Antioxidant defense, rhizosphere activities, vacuolar sequestration, and As efflux are important physiological activities involved in As hyperaccumulation in P. vittata. Among related 19 genes, PHT, TIP, ACR3, ACR2 and HAC family genes play essential roles in arsenate (AsⅤ) transport, arsenite (AsⅢ) transport, vacuole sequestration of AsⅢ, and the reduction of AsⅤ to AsⅢ, respectively. Gene ontology enrichment analysis indicated it is necessary to further explore genes that can bind to related ions, with transport activity, or with function of transmembrane transport. Phylogeny analysis results implied ACR2, HAC and ACR3 family genes with rapid evolutionary rate may be the decisive factors for P. vittata as an As hyperaccumulator. A deeper understanding of the As hyperaccumulation network and key gene components could provide useful tools for further bio-engineered phytoremediation.

Copper enhanced arsenic-accumulation in As-hyperaccumulator Pteris vittata by upregulating its gene expression for As uptake, translocation, and sequestration.

In contaminated soils, arsenic (As) often co-exists with copper (Cu). However, its effects on As accumulation and the related mechanisms in As-hyperaccumulator Pteris vittata remain unclear. In this study, P. vittata plants were exposed to 50 µM As and/or 50 µM Cu under hydroponics to investigate the effects of Cu on plant growth and As accumulation, as well as gene expression related to arsenic uptake (P transporters), reduction (arsenate reductases), and translocation and sequestration (arsenite antiporters). After 14 d of growth and compared to the As treatment, the As concentration in P. vittata fronds increased by 1.4-times from 793 to 1131 mg·kg-1 and its biomass increased by 1.2-fold from 18.0 to 21.1 g·plant-1 in the As+Cu treatment. Copper-enhanced As accumulation was probably due to upregulated gene expressions related to As-metabolisms including As uptake (1.9-fold in P transporter PvPht1;3), translocation (2.1-2.4 fold in arsenite antiporters PvACR3/3;2) and sequestration (1.5-2.0 fold in arsenite antiporters PvACR3;1/3;3). Our results suggest that moderate amount of Cu can help to increase the As accumulation efficiency in P. vittata, which has implication in its application in phytoremedation in As and Cu co-contaminated soils.

Selenium Increased Arsenic Accumulation by Upregulating the Expression of Genes Responsible for Arsenic Reduction, Translocation, and Sequestration in Arsenic Hyperaccumulator Pteris vittata.

Selenate enhances arsenic (As) accumulation in As-hyperaccumulator Pteris vittata, but the associated molecular mechanisms are unclear. Here, we investigated the mechanisms of selenate-induced arsenic accumulation by exposing P. vittata to 50 µM arsenate (AsV50) and 1.25 (Se1.25) or 5 µM (Se5) selenate in hydroponics. After 2 weeks, plant biomass, plant As and Se contents, As speciation in plant and growth media, and important genes related to As detoxification in P. vittata were determined. These genes included P transporters PvPht1;3 and PvPht1;4 (AsV uptake), arsenate reductases PvHAC1 and PvHAC2 (AsV reduction), and arsenite (AsIII) antiporters PvACR3 and PvACR3;2 (AsIII translocation) in the roots, and AsIII antiporters PvACR3;1 and PvACR3;3 (AsIII sequestration) in the fronds. The results show that Se1.25 was more effective than Se5 in increasing As accumulation in both P. vittata roots and fronds, which increased by 27 and 153% to 353 and 506 mg kg-1. The As speciation analyses show that selenate increased the AsIII levels in P. vittata, with 124-282% more AsIII being translocated into the fronds. The qPCR analyses indicate that Se1.25 upregulated the gene expression of PvHAC1 by 1.2-fold, and PvACR3 and PvACR3;2 by 1.0- to 2.5-fold in the roots, and PvACR3;1 and PvACR3;3 by 0.6- to 1.1-fold in the fronds under AsV50 treatment. Though arsenate enhanced gene expression of P transporters PvPht1;3 and PvPht1;4, selenate had little effect. Our results indicate that selenate effectively increased As accumulation in P. vittata, mostly by increasing reduction of AsV to AsIII in the roots, AsIII translocation from the roots to fronds, and AsIII sequestration into the vacuoles in the fronds. The results suggest that selenate may be used to enhance phytoremediation of As-contaminated soils using P. vittata.

Arsenic accumulation in Pteris vittata: Time course, distribution, and arsenic-related gene expression in fronds and whole plantlets.

In this work, arsenic (As) accumulation and distribution over time in Pteris vittata young fronds from adult plants and in whole plantlets, grown on a highly contaminated As-soil, was determined by µ-XRF. A linear increase in As content up to 60 days was found in young fronds at different times, and a progressive distribution from the apex to the base of the fronds was observed. In whole plantlets, As signal was detectable from 9 to 20 days in the apex of a few fronds and fiddleheads. Later, up to 60 days, As was localized in all fronds, in the rhizome and in basal part of the roots. The dynamics of expression of As-related genes revealed a good correlation between As content and the level of the As (III)-antiporter PvACR3 transcript in plantlets roots and fronds and in young fronds. Moreover, the transcription of As (V)-related gametophytic genes PvGAPC1, PvOCT4 increases over time during As accumulation while PvGSTF1 is expressed only in roots. Here, we demonstrate the suitability of the µ-XRF technique to monitor As accumulation, which allowed us to propose that As is initially directly transported to fiddleheads and apex of fronds, is later distributed to the whole fronds and simultaneously accumulated in the rhizome and roots. We also provide indications on the expression of candidate genes possibly involved in As (hyper)accumulation.

Empirical Evidence of Arsenite Oxidase Gene as an Indicator Accounting for Arsenic Phytoextraction by Pteris vittata.

Arsenic (As) is a toxic semi-metallic element that is ubiquitous in the environment and poses serious human health risks. Phytoextraction by Pteris vittata is considered a low-cost and environmentally friendly approach to treat As-contaminated soil. P. vittata mainly absorbs arsenate thus the bioavailability of As to P. vittata depends on the chemical form of As. Microbial redox of As contributes to the biogeochemical cycling of As, and rhizobacterium-assisted phytoextraction by P. vittata was proposed. In this study, this microbe-assisted phytoextraction was applied to two fields, and the effectiveness of phytoextraction was evaluated. The results revealed that P. vittata was able to grow in temperate and subarctic climate zones. The biomass was influenced by the weather, and the As concentration in plants was dependent on the As content in the soil. The ratio of arsenite oxidase genes (aioA-like genes) to 16S rRNA genes was employed to evaluate the effect of As phytoextraction, and the results exhibited that the ratio was related to the As concentration in P. vittata. Our results showed that arsenite oxidation in the rhizosphere might not be achieved by single-strain inoculation, while this study provided empirical evidence that the rhizospheric aioA-like genes could be an indicator for evaluating the effectiveness of As phytoextraction.

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 in situ 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. 52.7 Ma. 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.

Novel phytase PvPHY1 from the As-hyperaccumulator Pteris vittata enhances P uptake and phytate hydrolysis, and inhibits As translocation in Plant.

Developing P-efficient plants helps improve P uptake from soils with low-available P and reduce environmental damage by P runoff. Here, we investigated a novel root-specific phytase PvPHY1 from As-hyperaccumulator Pteris vittata, which can efficiently utilize phytate, a recalcitrant organic phosphorus in soil. Unlike other plants, expression of PvPHY1 in P. vittata was greater in the roots than the fronds. A pure phytase with considerable activity was obtained via prokaryotic expression. Expressing PvPHY1 in tobacco (PvPHY1-Ex) enhanced its growth (2.8 to 3.5-3.9 g per plant) and increased its P accumulation by 10-50% under low- and adequate-P conditions. Further, PvPHY1-Ex tobacco showed 25-32% lower intracellular phytate and 30-56% higher inorganic P in the roots, likely due to phytase-mediated hydrolysis of phytate. Decrease of phytate levels up-regulated phosphate transporter genes (NbPht1;1, NbPht1;2 and NbPht1;6), leading to greater P and As uptake. However, As translocation to the shoots was low, probably due to competition from increased inorganic P via phytate hydrolysis. As such, PvPHY1 facilitated P uptake from soils and phytate hydrolysis in plants, thereby promoting tobacco growth. Overall, PvPHY1 from P. vittata helps better understand the novel phytase to increase soil P utilization efficiency, thereby reducing P fertilizer requirements for crop production.

Novel PvACR3;2 and PvACR3;3 genes from arsenic-hyperaccumulator Pteris vittata and their roles in manipulating plant arsenic accumulation.

Arsenite (AsIII) antiporter ACR3 is crucial for arsenic (As) translocation and sequestration in As-hyperaccumulator Pteris vittata, which has potential for phytoremediation of As-contaminated soils. In this study, two new ACR3 genes PvACR3;2 and PvACR3;3 were cloned from P. vittata and studied in model organism yeast (Saccharomyces cerevisiae) and model plant tobacco (Nicotiana tabacum). Both ACR3s mediated AsIII efflux in yeast, decreasing its As accumulation and enhancing its As tolerance. In addition, PvACR3;2 and PvACR3;3 were expressed in tobacco plant. Localized on the plasma membrane, PvACR3;2 mediated both AsIII translocation to the shoots and AsIII efflux from the roots in tobacco, resulting in 203 - 258% increase in shoot As after exposing to 5 µM AsIII under hydroponics. In comparison, localized to the vacuolar membrane, PvACR3;3 sequestrated AsIII in tobacco root vacuoles, leading to 18 - 20% higher As in the roots and 15 - 36% lower As in the shoots. Further, based on qRT-PCR, both genes were mainly expressed in P. vittata fronds, indicating PvACR3;2 and PvACR3;3 may play roles in AsIII translocation and sequestration in the fronds. This study provides not only new insights into the functions of new ACR3 genes in P. vittata, but also important gene resources for manipulating As accumulation in plants for phytoremediation and food safety.

Efficient arsenate reduction in As-hyperaccumulator Pteris vittata are mediated by novel arsenate reductases PvHAC1 and PvHAC2.

Arsenic-hyperaccumulator Pteris vittata is efficient in As absorption, reduction, and translocation. But the molecular mechanisms and locations of arsenate (AsV) reduction in P. vittata are still unclear. Here, we identified two new arsenate reductase genes from P. vittata, PvHAC1 and PvHAC2. Two PvHAC genes encoded a rhodanase-like protein, which were localized in the cytoplasm and nucleus. Both recombinant Escherichia coli strains and transgenic Arabidopsis thaliana lines showed arsenate reductase ability after expressing PvHAC genes. Further, expressing PvHAC2 enhanced As tolerance and reduced As accumulation in A. thaliana shoots under AsV exposure. Based on expression pattern analysis, PvHAC1 and PvHAC2 were predominantly expressed in the rhizomes and fronds of P. vittata. Different from those of HAC homologous genes in non-hyperaccumulators, little PvHAC was expressed in the roots. Besides, PvHAC1 expression was strongly upregulated under AsV exposure but not AsIII. The data suggest that arsenate reductase PvHAC1 in the rhizomes coupled with arsenate reductase PvHAC2 in the fronds of P. vittata played a critical role in As-hyperaccumulation by P. vittata, which helps to further improve its utility in phytoremediation of As-contaminated soils.

Stable expression of bacterial transporter ArsB attached to SNARE molecule enhances arsenic accumulation in Arabidopsis.

Acute and chronic arsenic (As) toxicity is a global health issue affecting millions of people, which leads to inactivation of over 200 enzymes, particularly those involved in cellular energy pathways and DNA synthesis and repair. The fern Pteris vittata acts as a hyperaccumulator of As and may be useful for phytoremediation to reduce disposal risks by utilizing metal-enriched plant biomass in energy and metal recovery. However, these ferns grow in limited environments and its transplantation and transport can be challenging. Therefore, we generated a transgenic Arabidopsis plant as a seed plant model, capable of accumulating As in their vacuole lumen. This was achieved by transforming the As-resistant bacterial As transporter, ArsB, via fusion with a organelle-targeting signal to the vacuolar membrane, N-ethyl-maleimide-sensitive factor attachment protein receptors (SNAREs) protein, VAMP711. In this study, we developed the iVenus assay as a method for detecting whether the N- or C-terminus of a membrane protein is located on the cytoplasmic or exoplasmic side, and from the result of the iVenus assay, we generated the transgenic plant introduced N-terminal end of ArsB with VAMP711, localized to the central vacuolar membrane to accumulate As in the shoot and differentiation zone of root.

Potential use of the Pteris vittata arsenic hyperaccumulation-regulation network for phytoremediation.

Arsenic accumulation in soil is a global problem typically addressed using phytoremediation methods. Pteris vittata, a model arsenic hyperaccumulator, has great potential as a genetically engineered plant for phytoremediation. However, the lack of omic information on this species has severely limited the identification and application of its arsenic hyperaccumulation and regulation components. In this study, we used an optimized single-molecular real-time (SMRT) strategy to create a de novo full-length transcriptomic-tonoplast proteomic database for this unsequenced fern and to determine the genetic components underlying its arsenic hyperaccumulation-regulation mechanisms. We established a comprehensive network consisting of six major transporter families, two novel resistance pathways, and a regulatory system by examining alternative splicing (AS) and long non-coding RNA (lncRNA) in different tissues following As(III) and As(V) treatment. The database and network established in this study will deepen our understanding of the unique hyperaccumulation and regulation mechanisms of P. vittata, ultimately providing a valuable resource for futher research on phytoremediation of arsenic-contaminated soil.

Root transcripts associated with arsenic accumulation in hyperaccumulator Pteris vittata.

Hyperaccumulation of arsenic (As) by brake fern Pteris vittata has been described as an important genetic trait that provides an option for development of a sustainable phytoremediation process for As mitigation. Accumulation of very high concentration of arsenic in above-ground tissues may be the result of arsenic vacuole compartmentalization, but the mechanism(s) of arsenic uptake and transport by underground tissues are largely unknown. In this study, we made an attempt towards understanding the molecular mechanism of As hyperaccumulation in this plant. A time-dependent As accumulation study indicates an exponential accumulation of As from 7 to 30 days of arsenic exposure in fronds, and day 3-7 in roots. Root transcriptome analysis identified 554,973 transcripts. Further, subsets of 824 transcripts were differentially expressed between treated and control samples. Many of the genes of critical As-stress response, transcription factors and metal transporters, biosynthesis of chelating compounds involved in uptake and accumulation mechanisms were identified. The genes that were highly expressed such as cysteine-rich RLK, and ABC transporter G family member 26 needs further studies along with arsenite transmembrane transporter. The analysis of generated transcriptome dataset has provided valuable information and platform for further functional studies.

Dissecting the components controlling root-to-shoot arsenic translocation in Arabidopsis thaliana.

Arsenic (As) is an important environmental and food-chain toxin. We investigated the key components controlling As accumulation and tolerance in Arabidopsis thaliana. We tested the effects of different combinations of gene knockout, including arsenate reductase (HAC1), γ-glutamyl-cysteine synthetase (γ-ECS), phytochelatin synthase (PCS1) and phosphate effluxer (PHO1), and the heterologous expression of the As-hyperaccumulator Pteris vittata arsenite efflux (PvACR3), on As tolerance, accumulation, translocation and speciation in A. thaliana. Heterologous expression of PvACR3 markedly increased As tolerance and root-to-shoot As translocation in A. thaliana, with PvACR3 being localized to the plasma membrane. Combining PvACR3 expression with HAC1 mutation led to As hyperaccumulation in the shoots, whereas combining HAC1 and PHO1 mutation decreased As accumulation. Mutants of γ-ECS and PCS1 were hypersensitive to As and had higher root-to-shoot As translocation. Combining γ-ECS or PCS1 with HAC1 mutation did not alter As tolerance or accumulation beyond the levels observed in the single mutants. PvACR3 and HAC1 have large effects on root-to-shoot As translocation. Arsenic hyperaccumulation can be engineered in A. thaliana by knocking out the HAC1 gene and expressing PvACR3. PvACR3 and HAC1 also affect As tolerance, but not to the extent of γ-ECS and PCS1.

Effects of indole-3-acetic acid on arsenic uptake and antioxidative enzymes in Pteris cretica var. nervosa and Pteris ensiformis.

A hydroponic experiment was conducted to investigate the effects of indole-3-acetic acid (IAA) on arsenic (As) uptake and antioxidative enzymes in fronds of Pteris cretica var. nervosa (As hyperaccumulator) and Pteris ensiformis (non-hyperaccumulator). Plants were exposed to 2 mg L-1 As(III), As(V) or dimethylarsinic acid (DMA) and IAA concentrations for 14 d. The biomass and total As in the plants significantly increased at 30 mg L-1 IAA. Superoxide dismutase (SOD) activities significantly increased with IAA addition. Catalase (CAT) activities showed a significant increase in P. ensiformis exposed to three As species at 30 or 50 mg L-1 IAA but varied in P. cretica var. nervosa. Peroxidase (POD) activities were unchanged in P. ensiformis except for a significant decrease at 50 mg L-1 IAA under As(III) treatment. However, a significant increase was observed in P. cretica var. nervosa at 10 mg L-1 IAA under As(III) or DMA treatment and at 50 mg L-1 IAA under As(V) treatment. Under DMA stress, malondialdehyde contents in fronds of P. cretica var. nervosa showed a significant decrease at 10 mg L-1 IAA but remained unchanged in P. ensiformis. Therefore, IAA enhanced As uptake and frond POD activity in P. cretica var. nervosa under As stress.

Photosynthetic electron-transfer reactions in the gametophyte of Pteris multifida reveal the presence of allelopathic interference from the invasive plant species Bidens pilosa.

To date, the response of the fern gametophyte to its environment has received considerable attention. However, studies on the influence of plant invasion on the fern gametophyte are fewer. Allelopathy has been hypothesized to play an important role in biological invasion. Hence, it is necessary to study the allelopathy of invasive plant species to the fern gametophyte and elucidate the mechanisms by which invasive plants cause phytotoxicity. As one of the main invasive plants in China, Bidens pilosa exhibits allelopathic effects on the gametophytic growth of Pteris multifida. The root exudate plays an important role among various allelochemical delivery mechanisms in B. pilosa. The effect invasive plant species has on photosynthesis in native species is poorly understood. To elucidate this effect, the changes in photosynthesis in the gametophytes of P. multifida are analyzed to examine the mechanisms of the root exudates of B. pilosa. Meanwhile, a non-invasive plant, Coreopsis basalis, was also applied to investigate the effects on fluorescence and pigments in P. multifida gametophytes. We found that gametophytes exposed to both B. pilosa and C. basalis had decreased fluorescence parameters in comparison with the control, except for non-photochemical quenching. Furthermore, it was found that these parameters were markedly affected from day 2 to day 10 in the presence of both exudates at a concentration of 25% or above. B. pilosa exudate had a negative dose-dependent effect on chlorophyll a, chlorophyll b, carotenoid, and the total chlorophyll in the gametophyte. The inhibitory effects increased with increasing exudate concentrations of both species, exhibiting the greatest inhibition at day 10. In conclusion, B. pilosa irreversibly affected the photosynthesis of P. multifida on both PS I and PS II. Root exudates caused the primary damage with respect to the decrease of the acceptors and donors of photon and electron in photosynthetic units and the production and the relative yield of photochemical quantum in PS II. With the effects of exudates, part of the energy is released as heat in chloroplasts. The comparison of invasive and non-invasive plants in allelopathic experiments demonstrated that invasive plants were responsible for the critical damage to the photosynthetic process in local species.

An aquaporin PvTIP4;1 from Pteris vittata may mediate arsenite uptake.

The fern Pteris vittata is an arsenic hyperaccumulator. The genes involved in arsenite (As(III)) transport are not yet clear. Here, we describe the isolation and characterization of a new P. vittata aquaporin gene, PvTIP4;1, which may mediate As(III) uptake. PvTIP4;1 was identified from yeast functional complement cDNA library of P. vittata. Arsenic toxicity and accumulating activities of PvTIP4;1 were analyzed in Saccharomyces cerevisiae and Arabidopsis. Subcellular localization of PvTIP4;1-GFP fusion protein in P. vittata protoplast and callus was conducted. The tissue expression of PvTIP4;1 was investigated by quantitative real-time PCR. Site-directed mutagenesis of the PvTIP4;1 aromatic/arginine (Ar/R) domain was studied. Heterologous expression in yeast demonstrates that PvTIP4;1 was able to facilitate As(III) diffusion. Transgenic Arabidopsis showed that PvTIP4;1 increases arsenic accumulation and induces arsenic sensitivity. Images and FM4-64 staining suggest that PvTIP4;1 localizes to the plasma membrane in P. vittata cells. A tissue location study shows that PvTIP4;1 transcripts are mainly expressed in roots. Site-directed mutation in yeast further proved that the cysteine at the LE1 position of PvTIP4;1 Ar/R domain is a functional site. PvTIP4;1 is a new represented tonoplast intrinsic protein (TIP) aquaporin from P. vittata and the function and location results imply that PvTIP4;1 may be involved in As(III) uptake.

A member of the Phosphate transporter 1 (Pht1) family from the arsenic-hyperaccumulating fern Pteris vittata is a high-affinity arsenate transporter.

Pteris vittata exhibits enhanced arsenic uptake, but the corresponding mechanisms are not well known. The prevalent form of arsenic in most soils is arsenate, which is a phosphate analog and a substrate for Phosphate transporter 1 (Pht1) transporters. Herein we identify and characterize three P. vittata Pht1 transporters. Pteris vittata Pht1 cDNAs were isolated and characterized via heterologous expression in Saccharomyces cerevisiae (yeast) and Nicotiana benthamiana leaves. Expression of the PvPht1 loci in P. vittata gametophytes was also examined in response to phosphate deficiency and arsenate exposure. Expression of each of the PvPht1 cDNAs complemented the phosphate uptake defect of a yeast mutant. Compared with yeast cells expressing Arabidopsis thaliana Pht1;5, cells expressing PvPht1;3 were more sensitive to arsenate, and accumulated more arsenic. Uptake assays with yeast cells and radiolabeled (32)P revealed that PvPht1;3 and AtPht1;5 have similar affinities for phosphate, but the affinity of PvPht1;3 for arsenate is much greater. In P. vittata gametophytes, PvPht1;3 transcript levels increased in response to phosphate (Pi) deficiency and arsenate exposure. PvPht1;3 is induced by Pi deficiency and arsenate, and encodes a phosphate transporter that has a high affinity for arsenate. PvPht1;3 probably contributes to the enhanced arsenate uptake capacity and affinity exhibited by P. vittata.

The arsenic hyperaccumulating Pteris vittata expresses two arsenate reductases.

Enzymatic reduction of arsenate to arsenite is the first known step in arsenate metabolism in all organisms. Although the presence of one mRNA arsenate reductase (PvACR2) has been characterized in gametophytes of P. vittata, no arsenate reductase protein has been directly observed in this arsenic hyperaccumulating fern, yet. In order to assess the possible presence of arsenate reductase in P. vittata, two recombinant proteins, ACR2-His6 and Trx-His6-S-Pv2.5-8 were prepared in Escherichia coli, purified and used to produce polyclonal antibodies. The presence of these two enzymes was evaluated by qRT-PCR, immunoblotting and direct MS analysis. Enzymatic activity was detected in crude extracts. For the first time we detected and identified two arsenate reductase proteins (PvACR2 and Pv2.5-8) in sporophytes and gametophytes of P. vittata. Despite an increase of the mRNA levels for both proteins in roots, no difference was observed at the protein level after arsenic treatment. Overall, our data demonstrate the constitutive protein expression of PvACR2 and Pv2.5-8 in P. vittata tissues and propose their specific role in the complex metabolic network of arsenic reduction.

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.

Morphology and ploidy level determination of Pteris vittata callus during induction and regeneration.

BACKGROUND: Morphological and ploidy changes of the arsenic hyperaccumulator, Chinese brake fern (Pteris vittata) callus tissue are described here to provide insight into fern life cycle biology and for possible biotechnology applications. Pteris vittata callus was studied using transmission and scanning electron microscopy, and flow cytometry. RESULTS: Callus induction occurred both in light and dark culture conditions from prothallus tissues, whereas rhizoid formation occurred only in dark culture conditions. Callus tissues contained two types of cells: one actively dividing and the other containing a single large vacuole undergoing exocytosis. Sporophytes regenerated from callus asynchronously form clusters of cells in a manner apparently analogous to direct organogenesis. Extracellular matrices were observed in actively-growing callus and at the base of regenerating sporophytes. Callus tissue nuclei were found to be primarily diploid at induction and throughout maintenance of cultures indicating that callus cell fate is determined at induction, which closely follows apogamous sporophyte development. Presence of a dense extracellular matrix in conjunction with sporophyte development suggests a link between the suspensor-like activity of the embryonic foot during normal fern embryo development and the suspected functions of extracellular matrices in angiosperms. CONCLUSIONS: Further investigation could lead to a better understanding of genes involved in P. vittata embryo development and apogamous sporophyte development. The methodology could be useful for in vitro propagation of rare and valuable fern germplasm.