Arsenic (As) pollution is a global problem, and the plant-based cleanup of contaminated soils, called phytoremediation, is therefore of great interest. Recently, transgenic approaches have been designed to develop As phytoremediation technologies. Here, we used a one-gene transgenic approach for As tolerance and accumulation in Arabidopsis thaliana. PvACR3, a key arsenite [As(III)] antiporter in the As hyperaccumulator fern Pteris vittata, was expressed in Arabidopsis, driven by the CaMV 35S promoter. In response to As treatment, PvACR3 transgenic plants showed greatly enhanced tolerance. PvACR3 transgenic seeds could even germinate and grow in the presence of 80 μM As(III) or 1200 μM arsenate [As(V)] treatments that were lethal to wild-type seeds. PvACR3 localizes to the plasma membrane in Arabidopsis and increases arsenite efflux into external medium in short-term experiments. Arsenic determination showed that PvACR3 substantially reduced As concentrations in roots and simultaneously increased shoot As under 150 μM As(V). When cultivated in As(V)-containing soil (10 ppm As), transgenic plants accumulated approximately 7.5-fold more As in above-ground tissues than wild-type plants. This study provides important insights into the behavior of PvACR3 and the physiology of As metabolism in plants. Our work also provides a simple and practical PvACR3 transgenic approach for engineering As-tolerant and -hyperaccumulating plants for phytoremediation.

The mechanisms by which Pteris vittata (L.) hyperaccumulates arsenic (As) have not been fully elucidated. To investigate how P. vittata tolerates high concentrations of arsenite, we compared the toxicities of various As compounds to P. vittata and Arabidopsis thaliana (L.). The phytotoxicities of As species were found to be in the order of arsenite > arsenate > dimethylarsinic acid (DMAA) in A. thaliana, and in the order of DMAA > arsenate > arsenite in P. vittata. P. vittata calli displayed a weaker ability to absorb arsenite than arsenate. These results demonstrate that P. vittata possesses mechanisms of As accumulation and detoxification.

Nitric oxide (NO) is a bioactive gas and functions as a signaling molecule in plants exposed to diverse biotic and abiotic stresses including cadmium (Cd2+). Cd2+ is a non-essential and toxic heavy metal, which has been reported to induce programmed cell death (PCD) in plants. Here, we investigated the role of NO in Cd2+-induced PCD in tobacco BY-2 cells (Nicotiana tabacum L. cv. Bright Yellow 2). In this work, BY-2 cells exposed to 150 μM CdCl2 underwent PCD with TUNEL-positive nuclei, significant chromatin condensation and the increasing expression of a PCD-related gene Hsr203J. Accompanied with the occurring of PCD, the production of NO increased significantly. The supplement of NO by sodium nitroprusside (SNP) had accelerated the PCD, whereas the NO synthase inhibitor Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME) and NO-specific scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) alleviated this toxicity. To investigate the mechanism by which NO exerted its function, Cd2+ concentration was measured subsequently. SNP led more Cd2+ content than Cd2+ treatment alone. By contrast, the prevention of NO by l-NAME decreased Cd2+ accumulation. Using the scanning ion-selective electrode technique, we analyzed the pattern and rate of Cd2+ fluxes. This analysis revealed the promotion of Cd2+ influxes into cells by application of SNP, while l-NAME and cPTIO reduced the rate of Cd2+ uptake or even resulted in net Cd2+ efflux. Based on these founding, we concluded that NO played a positive role in CdCl2-induced PCD by modulating Cd2+ uptake and thus promoting Cd2+ accumulation in BY-2 cells.

An in vitro plant regeneration system was established from the spores of Pteris vittata and identification of its tolerance, and accumulation of gametophytes and callous, to arsenic (As) and copper (Cu) was investigated. The highest frequency (100%) of callus formation was achieved from gametophyte explants treated with 0.5 mg l−1 6-benzylaminopurine (6-BA) + 0.5 mg l−1 gibberellin acid (GA). Furthermore, sporophytes were differentiated from the callus tissue derived from gametophyte explants on MS medium supplemented with 0.5 mg l−1 6-BA, 0.5–1.0 mg l−1 GA and additional 300 mg l−1 lactalbumin hydrolysate (LH) for 4 weeks. The optimum combination of ½ MS + 1.0 mg l−1 GA + 0.5 mg l−1 6-BA + 300 mg l−1 LH promoted sporophyte formation on 75 ± 10% of the callus. Every callus derived from gametophyte explants could achieve 3–4 sporophytes. The in vitro growth of gametophyte and callus was accelerated in the medium containing Na3AsO4 lower than 0.5 mM, but this growth was inhibited with 2 mM Na3AsO4. And with the increase of Na3AsO4 in the culture medium from 0 to 2 mM, the As accumulation in gametophytes and callus increased and achieved a level of 763.3 and 315.4 mg kg−1, respectively. Gametophytes and calluses transplanted to culture medium, supplemented with different concentrations of CuSO4, are similar to those in Na3AsO4, and the Cu accumulation in gametophytes could achieve 7,940 mg kg−1 when gametophytes were subcultured in medium containing 3 mM CuSO4. These results suggested that the high efficiency propagation system could be a useful and rapid means to identify other heavy metal tolerance and accumulation. Further, the regeneration ability of callus made it possible for genetic transformation of this fern.

The callus of Pteris vittata was induced from gametophytes generated from spores in vitro, and grew rapidly with periodical medium change. Arsenic tolerance and accumulation of P. vittata callus were compared with those of Arabidopsis thaliana callus. Cell death was not detected in P. vittata callus even at arsenate concentrations up to 2 mM; however, A. thaliana callus died at low (0.2 mM) arsenate concentrations. Meanwhile, P. vittata callus accumulated almost three times more As than A. thaliana callus when exposed to 0.2 mM arsenate. About 60% of the total As was removed when 7.5 g of P. vittata callus was cultured on 150 ml of half-strength MS liquid medium containing 450 μg As for 2 days. Furthermore, P. vittata callus, sporophytes, and gametophytes all grew well under 1 mM of arsenate and accumulated 1,250; 1,150 and 2,180 mg kg−1 dry weight As when grown on 2 mM arsenate for 15 or 30 days. The characteristics of non-differentiated cells, large biomass, ease of culture, good synchronization, and excellent As sequestering, make the callus of P. vittata a new ideal system to study the mechanisms of As hyperaccumulation and phytoremediation in As-contaminated groundwater.

The goal of this study was to develop transgenic plants with increased tolerance for and accumulation of heavy metals and metalloids from soil by simultaneous overexpression of AsPCS1 and GSH1 (derived from garlic and baker’s yeast) in Arabidopsis thaliana. Phytochelatins (PCs) and glutathione (GSH) are the main binding peptides involved in chelating heavy metal ions in plants and other living organisms. Single-gene transgenic lines had higher tolerance to and accumulated more Cd and As than wild-type. Compared to single-gene transgenic lines, dual-gene transformants exhibited significantly higher tolerance to and accumulated more Cd and As. One of the dual-gene transgenic lines, PG1, accumulated twice the amount of Cd as single-gene transgenic lines. Simultaneous overexpression of AsPCS1 and GSH1 led to elevated total PC production in transgenic Arabidopsis. These results indicate that such a stacking of modified genes is capable of increasing Cd and As tolerance and accumulation in transgenic lines, and represents a highly promising new tool for use in phytoremediation efforts.

The fern Pteris vittata is an arsenic (As) hyperaccumulator and can take up very high concentrations of arsenic from the soil. However, little is known about its response to co-contamination with arsenic and copper (Cu). In this study, we used an in vitro model system of P. vittata gametophytes to investigate the impact of changes in As and Cu status on growth, chlorophyll (chl) concentration, metal accumulation, and subcellular localization. A remarkable inhibition of growth occurred when gametophytes were exposed to concentrations ⩾1.0 mM Na3AsO4 or ⩾0.5 mM CuSO4. chl concentration decreased significantly when gametophytes were exposed to >0.25 mM of CuSO4, but increased steadily with concentration to ⩽2 mM Na3AsO4. Interestingly, the inhibitory effect caused by Cu was reduced in the presence of 0.25 mM Na3AsO4. However, the inhibition caused by exposure to 1.0 mM Na3AsO4 was not alleviated by 0.25 mM CuSO4. Further studies showed that 0.25 mM Na3AsO4 increased cell viability (CV) and chl concentration, while decreasing cell membrane permeability (CMP) of gametophytes with 1.0 mM CuSO4 stress. In contrast, 0.25 mM CuSO4 decreased CV and chl concentration, while increasing CMP when gametophytes were treated with 1.0 mM Na3AsO4. In addition, the subcellular distribution of As and Cu in P. vittata gametophytes differed. As was found primarily in the cytoplasm, while Cu was mainly localized in the cell wall. These results suggest that As can reduce Cu phytotoxicity in the As hyperaccumulator P. vittata, and that this may serve as a biological mechanism for the fern to adapt to soils co-contaminated with As and Cu.

In Arabidopsis, the nodulin 26-like intrinsic protein (NIP) subfamily of aquaporin proteins consists of nine members, five of which (NIP1;1, NIP1;2, NIP5;1, NIP6;1, and NIP7;1) were previously identified to be permeable to arsenite. However, the roles of NIPs in the root-to-shoot translocation of arsenite in plants remain poorly understood. In this study, using reverse genetic strategies, Arabidopsis NIP3;1 was identified to play an important role in both the arsenic uptake and root-to-shoot distribution under arsenite stress conditions. The nip3;1 loss-of-function mutants displayed obvious improvements in arsenite tolerance for aboveground growth and accumulated less arsenic in shoots than those of the wild-type plants, whereas the nip3;1 nip1;1 double mutant showed strong arsenite tolerance and improved growth of both roots and shoots under arsenite stress conditions. A promoter-β-glucuronidase analysis revealed that NIP3;1 was expressed almost exclusively in roots (with the exception of the root tips), and heterologous expression in the yeast Saccharomyces cerevisiae demonstrated that NIP3;1 was able to mediate arsenite transport. Taken together, our results suggest that NIP3;1 is involved in arsenite uptake and root-to-shoot translocation in Arabidopsis, probably as a passive and bidirectional arsenite transporter.

Cadmium (Cd) is very toxic to plant cells and Cd2+ stress induces programmed cell death (PCD) in Nicotiana tabacum L. cv. bright yellow-2 (BY-2) cells. In plants, PCD can be regulated through the endoplasmic reticulum (ER) stress–cell death signaling pathway. However, the mechanism of Cd2+-induced PCD remains unclear. In this study, we found that Cd2+ treatment induced ER stress in tobacco BY-2 cells. The expression of two ER stress markers NtBLP4 and NtPDI and an unfolded protein response related transcription factor NtbZIP60 were upregulated with Cd2+ stress. Meanwhile, the PCD triggered by prolonged Cd2+ stress could be relieved by two ER chemical chaperones, 4-phenylbutyric acid and tauroursodeoxycholic acid. These results demonstrate that the ER stress–cell death signaling pathway participates in the mediation of Cd2+-induced PCD. Furthermore, the ER chaperone AtBiP2 protein alleviated Cd2+-induced ER stress and PCD in BY-2 cells based on the fact that heterologous expression of AtBiP2 in tobacco BY-2 cells reduced the expression of NtBLP4 and a PCD-related gene NtHsr203J under Cd2+ stress conditions. In summary, these results suggest that the ER stress–cell death signaling pathway regulates Cd2+-induced PCD in tobacco BY-2 cells, and that the AtBiP2 protein act as a negative regulator in this process.