Metabolic adaptation of Pteris vittata L. gametophyte to arsenic induced oxidative stress

The sporophyte and gametophyte of Pteris vittata are arsenic hyperaccumulators, however, little is known about the mechanism by which the gametophyte deals with this toxic element. An in vitro system (spores grown in arsenic amended nutrient media) was used to investigate the impact of arsenic on growth of the gametophyte and the role of antioxidative systems in combating As-stress. When mature spores of P. vittata were grown in medium amended with 0-50 mg kg(-1) of arsenic (as arsenate), the arsenic concentration in the gametophyte increased, with increasing arsenate in the media, but did not inhibit the spore germination and biomass development. Increases in the level of antioxidant enzymes, superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase, and glutathione-Stransferase) and of ascorbic acid and glutathione probably enabled the gametophyte to withstand the oxidative stress caused by arsenate.     

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.     

Distributions of arsenic and essential elements in pinna of arsenic hyperaccumulator Pteris vittata L.

The distributions of arsenic and 6 essential elements in the pinna of As hyperaccumulator, Pteris vittata L., were studied using synchrotron radiation X-ray fluorescence (SRXRF). Significant correlation between the distribution and mobility of the elements revealed that SRXRF study on the elemental distribution was feasible to inspect the transportations of elements in plants. The distribution of As in the pinna showed that As had great abilities to be transported in xylem vessels and from xylem to mesophyll. The distribution of K, one of the most mobile elements in plants, was similar to that of As, whereas the distributions of Fe and Ca with less mobility in plants were almost opposite to that of As in the pinna.     

耐砷促生菌对超富集植物蜈蚣草砷吸收及根际微生物群落的调控作用

【目的】分析添加外源促植物生长微生物(plant growth-promoting bacteria,PGPB)对植物生长、砷富集和根际微生物的影响,为植物-微生物联合修复砷污染土壤提供参考。【方法】通过添加外源PGPB,研究蜈蚣草生物量和砷富集量与外源微生物促植物生长特性的关系,采用高通量测序技术分析蜈蚣草根际微生物群落在外源PGPB干预下的变化规律。【结果】2株根际菌(假单胞菌PG12、芽孢杆菌R19)和1株内生菌(恶臭假单胞菌S6)具备典型的促植物生长特性,对蜈蚣草的促生作用顺序为：PG12>S6>R19,与对照组相比,生物量分别提高了234%(P<0.01)、136%(P<0.01)和67%;添加外源PGPB后,蜈蚣草砷含量从对照的18.50 mg提高到了31.25-46.95 mg,增幅高达153%(PG12)和139%(S6),对应的蜈蚣草砷浓度从2616.34mg/kg降至1348.04-2 156.23 mg/kg,呈现出典型的砷“稀释效应”;α多样性指数Sobs、Chao和Ace显示,仅R19处理显著提高了根际微生物的群落多样性,而β多样性指数...     

Behavior of chloroplasts and chloroplast nuclei during spermatogenesis in the fern,Pteris vittata L.

Summary The fate of the chloroplasts and chloroplast nuclei (cp-nuclei) was followed during spermatogenesis in the fernPteris vittata L. by epifluorescence microscopy after staining with 4-6-diamidino-2-phenylindole (DAPI) and by quantitation of chloroplast DNA (cp-DNA) by fluorimetry using a video intensified microscope photon counting system (VIMPICS). The spores were grown on solid medium that contained antheridiogen (An_ptd), and formed an antheridium initial on the protonema cell. The antheridium initial divided and produced 16 spermatocytes and 3 surrounding cells. The chloroplasts in the spermatocytes decreased in volume as cell division was repeated, until finally the volume of each chloroplast was 1/15 of that of the primary chloroplasts. The DNA content of the chloroplasts was also reduced to 1/5 of the original value and when the sperm matured, the fluorescence of cp-DNA disappeared. In the 16-cell spermatocyte, the recognition of the fluorescence of chlorophyll in the chloroplasts with a green excitation filter became difficult. But, the plastids could be observed until the final stage of the sperm. From these observations, it appears that there are two steps in the metamorphosis of chloroplasts during spermatogenesis in the fern. The first step involves the decrease in the volume of chloroplasts, accompanied by reduction of the DNA content, and the second step involves the change of the physical state of chloroplasts to amyloplasts and the disappearance of the cp-DNA from the amyloplasts.     

Effects of arsenate and phosphate on their accumulation by an arsenic-hyperaccumulator Pteris vittata L.

Arsenate and phosphate interactions are important for better understanding their uptake and accumulation by plant due to their similarities in chemical behaviors. The present study examined the effects of arsenate and phosphate on plant biomass and uptake of arsenate and phosphate by Chinese brake (Pteris vittata L.), a newly-discovered arsenic hyperaccumulator. The plants were grown for 20 weeks in a soil, which received the combinations of 670, 2670, or 5340 mol kg^–1 arsenate and 800, 1600, or 3200 mol kg^–1 phosphate, respectively. Interactions between arsenate and phosphate influenced their availability in the soil, and thus plant growth and uptake of arsenate and phosphate. At low and medium arsenate levels (670 and 2670 mol kg^–1), phosphate had slight effects on arsenate uptake by and growth of Chinese brake. However, phosphate substantially increased plant biomass and arsenate accumulation by alleviating arsenate phytotoxicity at high arsenate levels (5340 mol kg^–1). Moderate doses of arsenate increased plant phosphate uptake, but decreased phosphate concentrations at high doses because of its phytotoxicity. Based on our results, the minimum P/As molar ratios should be at least 1.2 in soil solution or 1.0 in fern fronds for the growth of Chinese brake. Our findings suggest that phosphate application may be an important strategy for efficient use of Chinese brake to phytoremediate arsenic contaminated soils. Further study is needed on the mechanisms of interactive effects of arsenate and phosphate on Chinese brake in hydroponic systems.     

Production of aposporous gametophytes and calli from Pteris vittata L. pinnae strips cultured in vitro

Murashige and Skoog's modified medium in 1% Difco Bacto-agar supplemented with sugar alcohols (sorbsitol, mannitol), growth regulators (1-naphthalenacetic acid, 2,4-dichlorophenoxyacetic acid, benzyladenine, kinetin) and sugars (fructose, glucose, sucrose) induced aposporous gametophytes from pinnae of Pteris vittata cultured in vitro at lower concentrations of all the mentioned components. Aposporous gametophytes and vegetative calli were produced at higher concentrations. The calli regenerated sporophytes when cultured on MS medium without growth regulators. The gametophytes grew vegetatively on MS medium but produced sporophytes when transferred into 0.1 strength MS medium. This is the first report of simultaneous production of calli and gametophytes from fern explants.     

Role of trichome ofPteris vittata L. in arsenic hyperaccumulation

Environmental scanning electron microscope (ESEM) fitted with an energy dispersive X-ray microanalyzer (EDX) was used to investigate the surface micromorphology and arsenic (As) micro-distribution in Chinese brake (Pteris vittata L.). It was found that amounts of trichome, which possessed multicellular structure with the average length of 160 μm and with an average diameter of 28 μm, existed in the frond ofP. vittata, and the density of trichome on the pinnate axial surface was higher than that on the petiole. Visible X-ray peak of As was recorded in the epidermal cell and trichome. The relative weight of As in the pinnate trichome, which contained the highest concentration of As among all tissues of the plant, was 2.4 and 3.9 times as much as that in the epidermal and mesophyllous cells, respectively. The As concentrations in the basal and stalk cells of the same trichome were higher than that in its cap cell. This is the first time to report that the trichome ofP. vittata plays an important role in arsenic hyperaccumulation. The finding from the present study implies that much attention should be paid to the role of the trichome in understanding the hyperaccumulation and detoxicity of As in the hyperaccumulator and improving the ability of As accumulation.     

Arsenate reductase activity in roots from the arsenic hyperaccumulator Pteris vittata utilizes both glutathione and dithiothreitol as reductants

Abstract

The role of glutathione and dithiothreitol as reductants supporting arsenate reductase activity in root extract from the arsenic hyperaccumulator Pteris vittata was examined. The two reductants in combination enhanced arsenate reduction in vitro more than glutathione alone. The implications of these results for in vivo arsenate reduction are discussed.     

万顺铅锌矿区蜈蚣草内生细菌砷氧化基因(aoxB)的多样性分析

【背景】近年来,由于金属矿的开采和冶炼、砷产品的加工与使用、煤的燃烧等各种因素,导致土壤环境中的砷污染越来越严重,导致许多人暴露于极度危险的砷毒毒害之下。【目的】研究四川的万顺铅锌矿区蜈蚣草根组织内生细菌aoxB基因的多样性,为提高土壤重金属污染生态修复效率提供理论依据。【方法】利用实时荧光定量PCR (Real-time quantitative PCR,qPCR)和限制性片段长度多态性(Restriction fragment length polymorphism,RFLP)技术,对四川省汉源县万顺铅锌矿区蜈蚣草根组织内生细菌aoxB基因表达量及多样性进行研究。【结果】qPCR结果表明,不同采样点间的蜈蚣草根组织内生细菌aoxB基因表达量存在明显差异,表现为选矿区进山口弃渣场尾矿区矿口。酶切图谱结果表明,不同采样点蜈蚣草根组织内生细菌aoxB基因多样性存在明显差异,多样性指数表现为尾矿矿口弃渣场进山口选矿区。Pearson相关分析显示,aoxB基因的表达量与重金属As之间呈显著负相关(P0.05),多样性指数则与重金属Pb和As之间呈极显著正相关(P0.01)。系统发育分析显示,aoxB基因的优势菌群为α-变形菌门(Alphaproteobacteria)。【结论】蜈蚣草根组织中存在丰富的含aoxB基因内生细菌种群,这些内生细菌表现出潜在的应用价值。     

砷、钙对蜈蚣草中金属元素吸收和转运的影响

蜈蚣草是砷的超富集植物和钙质土壤的指示植物。本试验在砂培条件下,研究砷、钙对蜈蚣草吸收和转运必需金属元素K、Mg、Mn、Fe、Zn和Cu的影响。结果表明。提高营养液中的砷浓度显著降低根部Mg和Zn的吸收。但对根部其它元素的浓度没有明显影响;叶柄中的Mn和地上部的Fe浓度因介质中添加砷而显著减少。其它元素在地上部的分布不受抑制。添加砷限制Fe从地下部向地上部转运,但促进其从叶柄向羽叶中运输;另外,还显著促进Mn由叶柄向羽叶和Zn由根向羽叶的转运。提高钙处理浓度对蜈蚣草吸收Fe、Zn、Cu无显著影响,但显著限制K、Mg和Mn的吸收。Mn是研究的6种金属元素中惟一一种明显向地上部转运富集的元素。从根部到羽叶中。金属元素间的相关性增强,在根部Ca与各种金属元素都无相关性;叶柄中Ca和Fe浓度呈极显著正相关;在羽叶中,Ca与K、Mg、Mn和Zn浓度呈显著负相关。     

Arsenic resistance in Pteris vittata L.: identification of a cytosolic triosephosphate isomerase based on cDNA expression cloning in Escherichia coli

Arsenic hyperaccumulator Pteris vittata L. (Chinese brake fern) grows well in arsenic-contaminated media, with an extraordinary ability to tolerate high levels of arsenic. An expression cloning strategy was employed to identify cDNAs for the genes involved in arsenic resistance in P. vittata. Excised plasmids from the cDNA library of P. vittata fronds were introduced into Escherichia coli XL-1 Blue and plated on medium containing 4 mM of arsenate, a common form of arsenic in the environment. The deduced amino acid sequence of an arsenate-resistant clone, PV4-8, had cDNA highly homologous to plant cytosolic triosephosphate isomerases (cTPI). Cell-free extracts of PV4-8 had 3-fold higher level of triosephosphate isomerase (TPI) specific activities than that found in E. coli XL-1 Blue and had a 42 kD fusion protein immunoreactive to polyclonal antibodies raised against recombinant Solanum chacoense cTPI. The PV4-8 cDNA complemented a TPI-deficient E. coli mutant. PV4-8 expression improved arsenate resistance in E. coli WC3110, a strain deficient in arsenate reductase but not in AW3110 deficient for the whole ars operon. This is consistent with the hypothesis that PV4-8 TPI increased arsenate resistance in E. coli by directly or indirectly functioning as an arsenate reductase. When E. coli tpi gene was expressed in the same vector, bacterial arsenate resistance was not altered, indicating that arsenate tolerance was specific to P. vittata TPI. Paradoxically, P. vittata TPI activity was not more resistant to inhibition by arsenate in vitro than its bacterial counterpart suggesting that arsenate resistance of conventional TPI reaction was not the basis for the cellular arsenate resistance. P. vittata TPI activity was inhibited by incubation with reduced glutathione while bacterial TPI was unaffected. Consistent with cTPI’s role in arsenate reduction, bacterial cells expressing fern TPI had significantly greater per cent of cellular arsenic as arsenite compared to cells expressing E. coli TPI. Excised frond tissue infiltrated with arsenate reduced arsenate significantly more under light than dark. This research highlights a novel role for P. vittata cTPI in arsenate reduction.     

Rhizosphere bacteria affect growth and metal uptake of heavy metal accumulating willows

A variety of plants growing on metalliferous soils accumulate metals in their harvestable parts and have the potential to be used for phytoremediation of heavy metal polluted land. There is increasing evidence that rhizosphere bacteria contribute to the metal extraction process, but the mechanisms of this plant–microbe interaction are not yet understood. In this study ten rhizosphere isolates obtained from heavy metal accumulating willows affiliating with Pseudomonas, Janthinobacterium, Serratia, Flavobacterium, Streptomyces and Agromyces were analysed for their effect on plant growth, Zn and Cd uptake. In plate assays Zn, Cd and Pb resistances and the ability of the bacteria to produce indole-3-acetic acid (IAA), 1-amino-cyclopropane-1-carboxylic acid deaminase (ACC deaminase) and siderophores were determined. The isolates showed resistance to high Zn concentrations, indicating an adaptation to high concentrations of mobile Zn in the rhizosphere of Salix caprea. Four siderophore producers, two IAA producers and one strain producing both siderophores and IAA were identified. None of the analysed strains produced ACC deaminase. Metal mobilization by bacterial metabolites was assessed by extracting Zn and Cd from soil with supernatants of liquid cultures. Strain Agromyces AR33 almost doubled Zn and Cd extractability, probably by the relase of Zn and Cd specific ligands. The remaining strains, immobilized both metals. When Salix caprea plantlets were grown in γ-sterilized, Zn/Cd/Pb contaminated soil and inoculated with the Zn resistant isolates, Streptomyces AR17 enhanced Zn and Cd uptake. Agromyces AR33 tendentiously promoted plant growth and thereby increased the total amount of Zn and Cd extracted from soil. The IAA producing strains did not affect plant growth, and the siderophore producers did not enhance Zn and Cd accumulation. Apparently other mechanisms than the production of IAA, ACC deaminase and siderophores were involved in the observed plant–microbe interactions.     

蜈蚣草孢壁发育及其系统学意义

利用光镜、扫描电镜和透射电镜对凤尾蕨科(Pteridaceae)蜈蚣草(Pteris vittata L.)孢壁的形成和发育进行研究。结果表明:蜈蚣草孢子四面体型,极面观钝三角圆形,赤道面观半圆形或超半圆形,近极面具瘤状纹饰和近极脊,远极面具脊并连成网状,具赤道环;孢子具乌毛蕨型外壁,由外壁外层构成纹饰的轮廓;实心型周壁由2层构成,且内层薄、外层具小球体。结合孢子外壁和周壁的发育特征,认为凤尾蕨科与裸子蕨科和水蕨科的亲缘关系较近,支持将裸子蕨科和水蕨科置于凤尾蕨科。     

Plant regeneration of the arsenic hyperaccumulator <Emphasis Type="Italic">Pteris vittata</Emphasis> L<Emphasis Type="Italic">.</Emphasis> from spores and identification of its tolerance and accumulation of arsenic and copper

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.     

Role of dissimilatory fermentative iron-reducing bacteria in Fe uptake by common bean (Phaseolus vulgaris L.) plants grown in alkaline soil

Iron (Fe) is an essential element for plant growth and development. Some plant growth-promoting rhizobacteria can increase Fe uptake by plants through reduction of Fe(III) to Fe(II) at the root surface. The aim of this work was to identify novel bacterial strains with high Fe(III) reduction ability and to evaluate their role in plant Fe uptake. Four bacterial strains (UMCV1 to UMCV4) showing dissimilatory Fe-reducing activity were isolated from the rhizosphere of bean and maize plants and further identified by 16S rDNA amplification and sequence analysis. From these analyses, UMCV1 and UMCV2 isolates were identified as Bacillus megaterium and Arthrobacter spp., respectively, whereas UMCV3 and UMCV4 were identified as Stenotrophomonas maltophilia. All four isolates showed Fe reduction in a nonflooded soil and when associated with roots of bean plants grown in alkaline soil or in mineral medium. In addition, the bacterial isolates were able to stimulate plant growth in vitro and on a broad level, plants grown in inoculated soil were generally bigger and with higher Fe content than those grown in sterilized soil. These results indicate that bacterial species isolated from the rhizosphere of bean and maize plants contribute significantly to Fe uptake by plants likely through increased Fe(III) reduction in the rhizosphere.     

A comparison of arsenic mobility in Phaseolus vulgaris, Mentha aquatica, and Pteris cretica rhizosphere

The ability of Phaseolus vulgaris, Mentha aquatica, and Pteris cretica to release arsenic (As) species from contaminated soil was tested in rhizobox experiments in three soils differing in their physicochemical parameters and total and mobile As concentration. Relatively low uptake of arsenic by P. vulgaris and M. aquatica resulted in very low and ambiguous changes in rhizosphere soil compared to bulk soil. However, there were observed differences in the distribution of the mobile As portion in soil to individual As species as affected by plant species and/or plantation conditions of these plants. Higher percentage of mobile arsenite in mint rhizosphere seems to be related to more reducing conditions during cultivation of these wetland plants. P. cretica planted in the soils containing between 36 and 1436 mg As kg⁻¹ was able to accumulate between 80 and 500 mg As kg⁻¹ in aboveground biomass. The extractable concentrations of As compounds in rhizosphere soil of P. cretica showed a clear depletion of arsenate (representing more than 90% of extractable arsenic) with the distance from plant roots. However, the As uptake mechanisms, as well as As transformation within hyperaccumulating fern plants, differ substantially from those in higher plants. Therefore the finding of suitable higher plant tolerant to the As soil contamination with good ability to accumulate As in aboveground biomass remains for the further research.     

Acinetobacter strains IH9 and OCI1, two rhizospheric phosphate solubilizing isolates able to promote plant growth,constitute a new genomovar of Acinetobacter calcoaceticus

During a screening of phosphate solubilizing bacteria (PSB) in agricultural soils, two strains, IH9 and OCI1, were isolated from the rhizosphere of grasses in Spain, and they showed a high ability to solubilize phosphate in vitro. Inoculation experiments in chickpea and barley were conducted with both strains and the results demonstrated their ability to promote plant growth. The 16S rRNA gene sequences of these strains were nearly identical to each other and to those of Acinetobacter calcoaceticus DSM 30006^T, as well as the strain CIP 70.29 representing genomospecies 3. Their phenotypic characteristics also coincided with those of strains forming the A. calcoaceticus–baumannii complex. They differed from A. calcoaceticus in the utilization of l-tartrate as a carbon source and from genomospecies 3 in the use of d-asparagine as a carbon source. The 16S–23S intergenic spacer (ITS) sequences of the two isolates showed nearly 98% identities to those of A. calcoaceticus, confirming that they belong to this phylogenetic group. However, the isolates appeared as a separate branch from the A. calcoaceticus sequences, indicating their molecular separation from other A. calcoaceticus strains. The analysis of three housekeeping genes, recA, rpoD and gyrB, confirmed that IH9 and OCI1 form a distinct lineage within A. calcoaceticus. These results were congruent with those from DNA–DNA hybridization, indicating that strains IH9 and OCI1 constitute a new genomovar for which we propose the name A. calcoaceticus genomovar rhizosphaerae.