Molecular cloning and characterization of a phytochelatin synthase gene,PvPCS1, from Pteris vittata L.

Pteris vittata L. is a staggeringly efficient arsenic hyperaccumulator that has been shown to be capable of accumulating up to 23,000 μg arsenic g⁻¹, and thus represents a species that may fully exploit the adaptive potential of plants to toxic metals. However, the molecular mechanisms of adaptation to toxic metal tolerance and hyperaccumulation remain unknown, and P. vittata genes related to metal detoxification have not yet been identified. Here, we report the isolation of a full-length cDNA sequence encoding a phytochelatin synthase (PCS) from P. vittata. The cDNA, designated PvPCS1, predicts a protein of 512 amino acids with a molecular weight of 56.9 kDa. Homology analysis of the PvPCS1 nucleotide sequence revealed that it has low identity with most known plant PCS genes except AyPCS1, and the homology is largely confined to two highly conserved regions near the 5′-end, where the similarity is as high as 85–95%. The amino acid sequence of PvPCS1 contains two Cys-Cys motifs and 12 single Cys, only 4 of which (Cys-56, Cys-90/91, and Cys-109) in the N-terminal half of the protein are conserved in other known PCS polypeptides. When expressed in Saccharomyces cerevisae, PvPCS1 mediated increased Cd tolerance. Cloning of the PCS gene from an arsenic hyperaccumulator may provide information that will help further our understanding of the genetic basis underlying toxic metal tolerance and hyperaccumulation.

     

蜈蚣草中砷超富集的分子机制研究进展

砷是一种毒性很强的类金属元素,土壤砷污染可引发一系列食品安全问题,进而威胁人类健康。蜈蚣草具有极强的富集砷的能力,在砷污染土壤的植物修复中具有重要的应用价值。深入阐释蜈蚣草超富集砷的分子机制是植物修复技术的核心理论基础。文中综述了蜈蚣草超富集砷的组学研究进展,以及目前鉴定到的砷富集过程中的重要分子元件,并对未来的研究方向和趋势进行了展望。     

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.     

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.     

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.     

植物重金属超富集机理研究进展

植物超富集重金属机理主要涉及植物对金属离子高的吸收、运输能力,区域化作用及螯合作用等方面,其中跨膜运载蛋白的表达、调控对重金属超富集这一特性起了关键作用。金属阳离子运载蛋白家族主要包括CDF家族、NRAMP家族和ZIP家族等,在超富集植物中已克隆出多个家族的金属运载蛋白基因,这些基因的过量表达对重金属在细胞中的运输、分布和富集及提高植物的抗性方面发挥了重要作用。综述了近年来研究重金属超富集植物吸收、转运和贮存Zn、Ni、Cd等重金属的生理和分子机制所取得的主要进展。     

蜈蚣草砷超富集机制及其在砷污染修复中的应用

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

Arsenic hyperaccumulation in gametophytes of Pteris vittata. A new model system for analysis of arsenic hyperaccumulation

The sporophyte of the fern Pteris vittata is known to hyperaccumulate arsenic (As) in its fronds to >1% of its dry weight. Hyperaccumulation of As by plants has been identified as a valuable trait for the development of a practical phytoremediation processes for removal of this potentially toxic trace element from the environment. However, because the sporophyte of P. vittata is a slow growing perennial plant, with a large genome and no developed genetics tools, it is not ideal for investigations into the basic mechanisms underlying As hyperaccumulation in plants. However, like other homosporous ferns, P. vittata produces and releases abundant haploid spores from the parent sporophyte plant which upon germination develop as free-living, autotrophic haploid gametophyte consisting of a small (<1 mm) single-layered sheet of cells. Its small size, rapid growth rate, ease of culture, and haploid genome make the gametophyte a potentially ideal system for the application of both forward and reverse genetics for the study of As hyperaccumulation. Here we report that gametophytes of P. vittata hyperaccumulate As in a similar manner to that previously observed in the sporophyte. Gametophytes are able to grow normally in medium containing 20 mm arsenate and accumulate >2.5% of their dry weight as As. This contrasts with gametophytes of the related nonaccumulating fern Ceratopteris richardii, which die at even low (0.1 mm) As concentrations. Interestingly, gametophytes of the related As accumulator Pityrogramma calomelanos appear to tolerate and accumulate As to intermediate levels compared to P. vittata and C. richardii. Analysis of gametophyte populations from 40 different P. vittata sporophyte plants collected at different sites in Florida also revealed the existence of natural variability in As tolerance but not accumulation. Such observations should open the door to the application of new and powerful genetic tools for the dissection of the molecular mechanisms involved in As hyperaccumulation in P. vittata using gametophytes as an easily manipulated model system.     

Investigation of heavy metal hyperaccumulation at the cellular level: development and characterization of Thlaspi caerulescens suspension cell lines

The ability of Thlaspi caerulescens, a zinc (Zn)/cadmium (Cd) hyperaccumulator, to accumulate extremely high foliar concentrations of toxic heavy metals requires coordination of uptake, transport, and sequestration to avoid damage to the photosynthetic machinery. The study of these metal hyperaccumulation processes at the cellular level in T. caerulescens has been hampered by the lack of a cellular system that mimics the whole plant, is easily transformable, and competent for longer term studies. Therefore, to better understand the contribution of the cellular physiology and molecular biology to Zn/Cd hyperaccumulation in the intact plant, T. caerulescens suspension cell lines were developed. Differences in cellular metal tolerance and accumulation between the cell lines of T. caerulescens and the related nonhyperaccumulator, Arabidopsis (Arabidopsis thaliana), were examined. A number of Zn/Cd transport-related differences between T. caerulescens and Arabidopsis cell lines were identified that also are seen in the whole plant. T. caerulescens suspension cell lines exhibited: (1) higher growth requirements for Zn; (2) much greater Zn and Cd tolerance; (3) enhanced expression of specific metal transport-related genes; and (4) significant differences in metal fluxes compared with Arabidopsis. One interesting feature exhibited by the T. caerulescens cell lines was that they accumulated less Zn and Cd than the Arabidopsis cell lines, most likely due to a greater metal efflux. This finding suggests that the T. caerulescens suspension cells represent cells of the Zn/Cd transport pathway between the root epidermis and leaf. We also show it is possible to stably transform T. caerulescens suspension cells, which will allow us to alter the expression of candidate hyperaccumulation genes and thus dissect the molecular and physiological processes underlying metal hyperaccumulation in T. caerulescens.     

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.     

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.     

The Genetic Basis of Metal Hyperaccumulation in Plants

Referee: Professor Alan J.M. Baker, School of Botany, The University of Melbourne, VIC 3010, Australia A relatively small yet diverse group of plants are capable of sequestering metals in their shoot tissues at remarkably high concentrations that would be toxic to most organisms. This process, known as metal hyperaccumulation, is of interest for several reasons, including its relevance to the phytoremediation of metalpolluted soils. Most research on hyperaccumulators has focused on the physiological mechanisms of metal uptake, transport, and sequestration, but relatively little is known regarding the genetic basis of hyperaccumulation. There are no known cases of major genetic polymorphisms in which some members of a species are capable of hyperaccumulation and others are not. This is in contrast to the related phenomenon of metal tolerance, in which most species that possess any metal tolerance are polymorphic, evolving tolerance only in local populations on metalliferous soil. However, although some degree of hyperaccumulation occurs in all members of the species that can hyperaccumulate, there is evidence of quantitative genetic variation in ability to hyperaccumulate, both between and within populations. Such variation does not appear to correlate positively with either the metal concentration in the soil or the degree of metal tolerance in the plant. Studies using controlled crosses, interspecific hybrids, and molecular markers are beginning to shed light on the genetic control of this variation. As molecular physiology provides greater insights into the specific genes that control metal accumulation, we may learn more about the genetic and regulatory factors that influence variable expression of the hyperaccumulation phenotype.     

Constitutively elevated salicylic acid signals glutathione-mediated nickel tolerance in Thlaspi nickel hyperaccumulators

Progress is being made in understanding the biochemical and molecular basis of nickel (Ni)/zinc (Zn) hyperaccumulation in Thlaspi; however, the molecular signaling pathways that control these mechanisms are not understood. We observed that elevated concentrations of salicylic acid (SA), a molecule known to be involved in signaling induced pathogen defense responses in plants, is a strong predictor of Ni hyperaccumulation in the six diverse Thlaspi species investigated, including the hyperaccumulators Thlaspi goesingense, Thlaspi rosulare, Thlaspi oxyceras, and Thlaspi caerulescens and the nonaccumulators Thlaspi arvense and Thlaspi perfoliatum. Furthermore, the SA metabolites phenylalanine, cinnamic acid, salicyloyl-glucose, and catechol are also elevated in the hyperaccumulator T. goesingense when compared to the nonaccumulators Arabidopsis (Arabidopsis thaliana) and T. arvense. Elevation of free SA levels in Arabidopsis, both genetically and by exogenous feeding, enhances the specific activity of serine acetyltransferase, leading to elevated glutathione and increased Ni resistance. Such SA-mediated Ni resistance in Arabidopsis phenocopies the glutathione-based Ni tolerance previously observed in Thlaspi, suggesting a biochemical linkage between SA and Ni tolerance in this genus. Intriguingly, the hyperaccumulator T. goesingense also shows enhanced sensitivity to the pathogen powdery mildew (Erysiphe cruciferarum) and fails to induce SA biosynthesis after infection. Nickel hyperaccumulation reverses this pathogen hypersensitivity, suggesting that the interaction between pathogen resistance and Ni tolerance and hyperaccumulation may have played a critical role in the evolution of metal hyperaccumulation in the Thlaspi genus.     

Evolution and function of phytochelatin synthases

Both essential and non-essential transition metal ions can easily be toxic to cells. The physiological range for essential metals between deficiency and toxicity is therefore extremely narrow and a tightly controlled metal homeostasis network to adjust to fluctuations in micronutrient availability is a necessity for all organisms. One protective strategy against metal excess is the expression of high-affinity binding sites to suppress uncontrolled binding of metal ions to physiologically important functional groups. The synthesis of phytochelatins, glutathione-derived metal binding peptides, represents the major detoxification mechanism for cadmium and arsenic in plants and an unknown range of other organisms. A few years ago genes encoding phytochelatin synthases (PCS) were cloned from plants, fungi and nematodes. Since then it has become apparent that PCS genes are far more widespread than ever anticipated. Searches in sequence databases indicate PCS expression in representatives of all eukaryotic kingdoms and the presence of PCS-like proteins in several prokaryotes. The almost ubiquitous presence in the plant kingdom and beyond as well as the constitutive expression of PCS genes and PCS activity in all major plant tissues are still mysterious. It is unclear, how the extremely rare need to cope with an excess of cadmium or arsenic ions could explain the evolution and distribution of PCS genes. Possible answers to this question are discussed. Also, the molecular characterization of phytochelatin synthases and our current knowledge about the enzymology of phytochelatin synthesis are reviewed.     

Expression of phytochelatin synthase from aquatic macrophyte Ceratophyllum demersum L. enhances cadmium and arsenic accumulation in tobacco

Phytochelatin synthase (PCS), the key enzyme involved in heavy metal detoxification and accumulation has been used from various sources to develop transgenic plants for the purpose of phytoremediation. However, some of the earlier studies provided contradictory results. Most of the PCS genes were isolated from plants that are not potential metal accumulators. In this study, we have isolated PCS gene from Ceratophyllum demersum cv. L. (CdPCS1), a submerged rootless aquatic macrophyte, which is considered as potential accumulator of heavy metals. The CdPCS1 cDNA of 1,757?bp encodes a polypeptide of 501 amino acid residues and differs from other known PCS with respect to the presence of a number of cysteine residues known for their interaction with heavy metals. Complementation of cad1-3 mutant of Arabidopsis deficient in PC (phytochelatin) biosynthesis by CdPCS1 suggests its role in the synthesis of PCs. Transgenic tobacco plants expressing CdPCS1 showed several-fold increased PC content and precursor non-protein thiols with enhanced accumulation of cadmium (Cd) and arsenic (As) without significant decrease in plant growth. We conclude that CdPCS1 encodes functional PCS and may be part of metal detoxification mechanism of the heavy metal accumulating plant C. demersum. KEY MESSAGE: Heterologous expression of PCS gene from C. demersum complements Arabidopsis cad1-3 mutant and leads to enhanced accumulation of Cd and As in transgenic tobacco.     

Proteomic analysis as a tool for investigating arsenic stress in Pteris vittata roots colonized or not by arbuscular mycorrhizal symbiosis

Pteris vittata can tolerate very high soil arsenic concentration and rapidly accumulates the metalloid in its fronds. However, its tolerance to arsenic has not been completely explored. Arbuscular mycorrhizal (AM) fungi colonize the root of most terrestrial plants, including ferns. Mycorrhizae are known to affect plant responses in many ways: improving plant nutrition, promoting plant tolerance or resistance to pathogens, drought, salinity and heavy metal stresses. It has been observed that plants growing on arsenic polluted soils are usually mycorrhizal and that AM fungi enhance arsenic tolerance in a number of plant species. The aim of the present work was to study the effects of the AM fungus Glomus mosseae on P. vittata plants treated with arsenic using a proteomic approach. Image analysis showed that 37 spots were differently affected (21 identified). Arsenic treatment affected the expression of 14 spots (12 up-regulated and 2 down-regulated), while in presence of G. mosseae modulated 3 spots (1 up-regulated and 2 down-regulated). G. mosseae, in absence of arsenic, modulated 17 spots (13 up-regulated and 4 down-regulated). Arsenic stress was observed even in an arsenic tolerant plant as P. vittata and a protective effect of AM symbiosis toward arsenic stress was observed.     

砷超富集植物蜈蚣草原生质体的分离及其抗砷性分析

蜈蚣草(Pteris vittata)是一种砷超富集植物, 能够通过根从土壤中吸收砷, 并将其输送至羽叶中富集。为了探索蜈蚣草单个细胞在砷积累和砷抗性中的特性, 本文首次通过酶解方法获得了这一砷超富集蕨类植物的原生质体, 并研究了原生质体在不同浓度砷胁迫下的生活力。结果显示, 蜈蚣草原生质体的抗砷性远高于烟草原生质体的抗砷性, 与其整体植株的抗性一致。这为探索砷抗性和超富集机理提供了一个新的研究体系。     

砷超富集植物蜈蚣草原生质体的分离及其抗砷性分析

蜈蚣草（Pteris vittata）是一种砷超富集植物,能够通过根从土壤中吸收砷,并将其输送至羽叶中富集.为了探索蜈蚣草单个细胞在砷积累和砷抗性中的特性,本文首次通过酶解方法获得了这一砷超富集蕨类植物的原生质体,并研究了原生质体在不同浓度砷胁迫下的生活力.结果显示,蜈蚣草原生质体的抗砷性远高于烟草原生质体的抗砷性,与其整体植株的抗性一致.这为探索砷抗性和超富集机理提供了一个新的研究体系.