Highly efficient xylem transport of arsenite in the arsenic hyperaccumulator Pteris vittata

The hyperaccumulator Pteris vittata translocates arsenic (As) from roots to fronds efficiently, but the form of As translocated in xylem and the main location of arsenate reduction have not been resolved. Here, P. vittata was exposed to 5 microM arsenate or arsenite for 1-24 h, with or without 100 microM phosphate. Arsenic speciation was determined in xylem sap, roots, fronds and nutrient solutions by high-performance liquid chromatography (HPLC) linked to inductively coupled plasma mass spectrometry (ICP-MS). The xylem sap As concentration was 18-73 times that in the nutrient solution. In both arsenate- and arsenite-treated plants, arsenite was the predominant species in the xylem sap, accounting for 93-98% of the total As. A portion of arsenate taken up by roots (30-40% of root As) was reduced to arsenite rapidly. The majority (c. 80%) of As in fronds was arsenite. Phosphate inhibited arsenate uptake, but not As translocation. More As was translocated to fronds in the arsenite-treated than in the arsenate-treated plants. There was little arsenite efflux from roots to the external solution. Roots are the main location of arsenate reduction in P. vittata. Arsenite is highly mobile in xylem transport, possibly because of efficient xylem loading, little complexation with thiols in roots, and little efflux to the external medium.     

Phytoremediation of arsenic contaminated soil by Pteris vittata L. II. Effect on arsenic uptake and rice yield

A greenhouse experiment evaluated the effect of phytoextraction of arsenic from a contaminated soil by Chinese Brake Fern (Pteris vittata L.) and its subsequent effects on growth and uptake of arsenic by rice (Oryza sativa L.) crop. Pteris vittata was grown for one or two growing cycles of four months each with two phosphate sources, using single super phosphate (SSP) and di-ammonium phosphate (DAP). Rice was grown on phytoextracted soils followed by measurements of biomass yield (grain, straw, and root), arsenic concentration and, uptake by individual plant parts. The biomass yield (grain, straw and rice) of rice was highest in soil phytoextracted with Pteris vittata grown for two cycles and fertilized with diammonium phosphate (DAP). Total arsenic uptake in contaminated soil ranged from 8.2 to 16.9 mg pot(-1) in first growing cycle and 5.5 to 12.0 mg pot(-1) in second growing cycle of Pteris vittata. There was thus a mean reduction of 52% in arsenic content of rice grain after two growing cycle of Pteris vittata and 29% after the one growing cycle. The phytoextraction of arsenic contaminated soil by Pteris vittata was beneficial for growing rice resulted in decreased arsenic content in rice grain of <1 ppm. There was a mean improvement in rice grain yield 14% after two growing cycle and 8% after the one growing cycle of brake fern.     

Antioxidant responses of hyper-accumulator and sensitive fern species to arsenic

Plant species capable of hyper-accumulating heavy metals are of considerable interest for phytoremediation, and differ in their ability to accumulate metals from the environment. This work aims to examine (i) arsenic accumulation in three fern species [Chinese brake fern (Pteris vittata L.), slender brake fern (Pteris ensiformis Burm. f.), and Boston fern (Nephrolepis exaltata L.)], which were exposed to 0, 150, or 300 muM of arsenic (Na(2)HAsO(4).7H(2)O), and (ii) the role of anti-oxidative metabolism in arsenic tolerance in these fern species. Arsenic accumulation increased with an increase in arsenic concentration in the growth medium, the most being found in P. vittata fronds showing no toxicity symptoms. In addition, accumulation was highest in the fronds, followed by the rhizome, and finally the roots, in all three fern species. Thiobarbituric acid-reacting substances, indicators of stress in plants, were found to be lowest in P. vittata, which corresponds with its observed tolerance to arsenic. All three ferns responded differentially to arsenic exposure in terms of anti-oxidative defence. Higher levels of superoxide dismutase, catalase, and ascorbate peroxidase were observed in P. vittata than in P. ensiformis and N. exaltata, showing their active involvement in the arsenic detoxification mechanism. However, no significant increase was observed in either guaiacol peroxides or glutathione reductase in arsenic-treated P. vittata. Higher activity of anti-oxidative enzymes and lower thiobarbituric acid-reacting substances in arsenic-treated P. vittata correspond with its arsenic hyper-accumulation and no symptoms of toxicity.     

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.     

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。     

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.     

Phytoremediation of an arsenic-contaminated site using Pteris vittata L.: a two-year study

A field study was conducted to determine the efficiency of Chinese brake fern (Pteris vittata L.), an arsenic hyperaccumulator, on removal of arsenic from soil at an arsenic-contaminated site. Chinese brake ferns were planted on a site previously used to treat wood with chromated copper arsenate (CCA). Arsenic concentrations in surface and profile soil samples were determined for 2000, 2001, and 2002. In both 2001 and 2002, senesced and senescing fronds only, as well as all fronds, were harvested. Frond arsenic concentrations were not significantly different between the three harvests. Compared to senesced fronds, live fronds resulted in the greatest amount of arsenic removal. There were no significant differences in soil arsenic concentrations between 2000, 2001, and 2002, primarily due to the extreme variability in soil arsenic concentrations. However, the mean surface soil arsenic was reduced from 190 to 140 mg kg(-1). Approximately 19.3 g of arsenic were removed from the soil by Chinese brake fern. Therefore, this fern is capable of accumulating arsenic from the CCA -contaminated site and may be competitive, in terms of cost, to conventional remediation systems. However, better agronomic practices are needed to enhance plant growth and arsenic uptake to obtain maximum soil arsenic removal and to minimize remediation time.     

Calcium in Xylem Sap and the Regulation of its Delivery to the Shoot

Amounts of total and free calcium in root and shoot xylem sapwere quantified for a number of species grown in comparableenvironments and in a rooting medium not deficient in calcium.The potential for the shoot to sequester calcium was also examined,along with the ability for ABA to regulate calcium flux to theleaf. Xylem sap calcium showed considerable interspecific and diurnalvariation, even though the plants were grown with similar rhizosphericcalcium concentrations. The potential for the shoot to sequesterxylem sap calcium was also highly variable between species andimplied an ability, at least in some species, to regulate thecalcium reaching the shoot in the transpiration stream. Long distance transport of calcium in the xylem was not primarilyby mass flow, because neither calcium uptake nor distributionwere closely related to water uptake or transpiration. The diurnalchanges in xylem sap total ion concentration appeared to benegatively correlated with transpiration while, in contrast,the calcium ion concentration showed two peaks, one occurringin the dark and the other in the light period. The application of ABA to roots caused an increase in the rateof exudation from the xylem of detopped well-watered plants.These experiments suggest that changes in root water relationsdriven by ionic fluxes were the likely cause for enhanced sapexudation from ABA-treated roots. The steady-state concentrationof calcium in the xylem sap was unaffected by ABA when exudationrate increased and, consequently, the flux of calcium must alsohave increased. Key words: Abscisic acid, calcium, xylem sap, ionic fluxes     

Effectiveness of applying arsenate reducing bacteria to enhance arsenic removal from polluted soils by Pteris vittata L

Arsenic is a common contaminant in soils and water. It is well established that the fern Pteris vittata L. is an As hyperaccumulator and therefore has potential to phyroremediate As-polluted soils. Also, it is accepted that rhizosphere microflora play an enhancing role in plant uptake of metallic elements from soils. Studies showed that hydroponiclly grown P. Vittata accumulated arsenite more than the arsenate form of As apparently because arsenate and phosphate are analogues and therefore its absorption is inhibited by phosphate. The objective of this study was to determine whether addition of five different arsenate-reducing bacteria would enhance arsenic uptake by P. vittata grown in arsenic polluted soils in afield experiment. Results showed that addition of the As reducing bacteria promoted the growth of P. vittata, increased As accumulation, activated soil insoluble As, and reduced As leaching compared to the untreated control. Plant biomass increased by 53% and As uptake by 44%. As leaching was reduced by 29% to 71% depending on the As reducing bacterium. The results in their entirety permitted some insight into the mechanisms by which the arsenate reducing bacteria enhanced the effectiveness of P. vittata to remove As from the polluted soil.     

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。     

The potential of Thelypteris palustris and Asparagus sprengeri in phytoremediation of arsenic contamination

The potential of two plants, Thelypteris palustris (marsh fern) and Asparagus sprengeri (asparagus fern), for phytoremediation of arsenic contamination was evaluated. The plants were chosen for this study because of the discovery of the arsenic hyperaccumulating fern, Pteris vittata (Ma et al., 2001) and previous research indicating asparagus fern's ability to tolerate > 1200 ppm soil arsenic. Objectives were (1) to assess if selected plants are arsenic hyperaccumulators; and (2) to assess changes in the species of arsenic upon accumulation in selected plants. Greenhouse hydroponic experiments arsenic treatment levels were established by adding potassium arsenate to solution. All plants were placed into the hydroponic experiments while still potted in their growth media. Marsh fern and Asparagus fern can both accumulate arsenic. Marsh fern bioaccumulation factors (> 10) are in the range of known hyperaccumulator, Pteris vittata Therefore, Thelypteris palustris is may be a good candidate for remediation of arsenic soil contamination levels of < or = 500 microg/L arsenic. Total oxidation of As (III) to As (V) does not occur in asparagus fern. The asparagus fern is arsenic tolerant (bioaccumulation factors < 10), but is not considered a good potential phytoremediation candidate.     

Cadmium uptake and xylem loading are active processes in the hyperaccumulator Sedum alfredii

Sedum alfredii is a well known cadmium (Cd) hyperaccumulator native to China; however, the mechanism behind its hyperaccumulation of Cd is not fully understood. Through several hydroponic experiments, characteristics of Cd uptake and translocation were investigated in the hyperaccumulating ecotype (HE) of S. alfredii in comparison with its non-hyperaccumulating ecotype (NHE). The results showed that at Cd level of 10 microM measured Cd uptake in HE was 3-4 times higher than the implied Cd uptake calculated from transpiration rate. Furthermore, inhibition of transpiration rate in the HE has no essential effect on Cd accumulation in shoots of the plants. Low temperature treatment (4 degrees C) significantly inhibited Cd uptake and reduced upward translocation of Cd to shoots for 9 times in HE plants, whereas no such effect was observed in NHE. Cadmium concentration was 3-4-fold higher in xylem sap of HE, as compared with that in external uptake solution, whereas opposite results were obtained for NHE. Cadmium concentration in xylem sap of HE was significantly reduced by the addition of metabolic inhibitors, carbonyl cyanide m-chlorophenylhydrazone (CCCP) and 2,4-dinitrophenol (DNP), in the uptake solutions, whereas no such effect was noted in NHE. These results suggest that Cd uptake and translocation is an active process in plants of HE S. alfredii, symplastic pathway rather than apoplastic bypass contributes greatly to root uptake, xylem loading and translocation of Cd to the shoots of HE, in comparison with the NHE plants.     

Interaction of arsenic and phosphate on their uptake and accumulation in Chinese brake fern

Interactive effects of arsenate (As (V)) and phosphate (Pi) were investigated under hydroponic culture. Arsenic concentrations in fronds and roots of Chinese brake fern (Pteris vittata L.) significantly (p < 0.05) increased with increasing As (V), but decreased (p < 0.05) with increasing Pi in nutrient solution. Phosphate uptake was significantly (p < 0.05) inhibited by 1000 micromol L(-1) As (V). Under 100 micromol L(-1) As (V), frond phosphorus (P) increased at 100 and 1000 micromol L(-1) Pi, and root P increased at 250 micromol L(-1) Pi exposures. Arsenic and P concentrations in fronds and roots of Chinese brake fern were negatively correlated (p < 0.05). Arsenate treatments enhanced As and P transport to fronds, while increasing Pi inhibited their transportation, with highest frond P and As (%) obtained under 100 micromol L(-1) treatment. pH values in nutrient solution increased with increasing exposure time, but decreased with increasing Pi levels. Dissolved organic carbon (DOC) contents (dry weight) in nutrient solution decreased with increasing Pi levels, both for treatments with and without As (V). Arsenate at 1000 micromol L(-1) significantly (p < 0.05) increased DOC contents, especially for treatment without Pi. Six organic acids were detected in root exudates of Chinese brake fern, with oxalic and malic acids being most dominant.     

Arsenic hyperaccumulation in the Chinese brake fern (Pteris vittata) deters grasshopper (Schistocerca americana) herbivory

Brake fern, Pteris vittata, not only tolerates arsenic but also hyperaccumulates it in the frond. The hypothesis that arsenic hyperaccumulation in this fern could function as a defense against insect herbivory was tested. Fronds from control and arsenic-treated ferns were presented to nymphs of the grasshopper Schistocerca americana. Feeding damage was recorded by visual observation and quantification of the fresh weight of frond left uneaten and number of fecal pellets produced over a 2-d period. Grasshopper weight was determined before and after 5 d of feeding. Grasshoppers consumed significantly greater amounts of the frond tissue, produced more fecal pellets and had increased body weight on control plants compared with grasshoppers fed arsenic-treated ferns. Very little or none of the arsenic-treated ferns were consumed indicating feeding deterrence. In a feeding deterrent experiment with lettuce, sodium arsenite at 1.0 mm deterred grasshoppers from feeding whereas 0.1 mm did not. In a choice experiment, grasshoppers preferred to feed on lettuce dipped in water compared with lettuce dipped in 1.0 mm sodium arsenite. Our results show that arsenic hyperaccumulation in brake fern is an elemental defense against grasshopper herbivory.     

Mechanisms of arsenic hyperaccumulation in Pteris vittata. Uptake kinetics,interactions with phosphate,and arsenic speciation

The mechanisms of arsenic (As) hyperaccumulation in Pteris vittata, the first identified As hyperaccumulator, are unknown. We investigated the interactions of arsenate and phosphate on the uptake and distribution of As and phosphorus (P), and As speciation in P. vittata. In an 18-d hydroponic experiment with varying concentrations of arsenate and phosphate, P. vittata accumulated As in the fronds up to 27,000 mg As kg(-1) dry weight, and the frond As to root As concentration ratio varied between 1.3 and 6.7. Increasing phosphate supply decreased As uptake markedly, with the effect being greater on root As concentration than on shoot As concentration. Increasing arsenate supply decreased the P concentration in the roots, but not in the fronds. Presence of phosphate in the uptake solution decreased arsenate influx markedly, whereas P starvation for 8 d increased the maximum net influx by 2.5-fold. The rate of arsenite uptake was 10% of that for arsenate in the absence of phosphate. Neither P starvation nor the presence of phosphate affected arsenite uptake. Within 8 h, 50% to 78% of the As taken up was distributed to the fronds, with a higher translocation efficiency for arsenite than for arsenate. In fronds, 49% to 94% of the As was extracted with a phosphate buffer (pH 5.6). Speciation analysis using high-performance liquid chromatography-inductively coupled plasma mass spectroscopy showed that >85% of the extracted As was in the form of arsenite, and the remaining mostly as arsenate. We conclude that arsenate is taken up by P. vittata via the phosphate transporters, reduced to arsenite, and sequestered in the fronds primarily as As(III).     

Measurement of xylem sap amino acid concentrations in conjunction with whole tree transpiration estimates spring N remobilization by cherry (Prunus avium L.) trees

Prunus avium trees were grown in sand culture for one vegetative season with contrasting N supplies, in order to precondition their N storage capacities. During the spring of the second year a constant amount of ¹⁵N was supplied to all the trees, and the recovery of unlabelled N in the new biomass production was used as a direct measure of N remobilization. Destructive harvests were taken during spring to determine the pattern of N remobilization and uptake. Measurements of both xylem sap amino acid profiles and whole tree transpiration rates were taken, to determine whether specific amino acids are translocated as a consequence of N remobilization and if remobilization can be quantified by calculating the flux of these amino acids in the xylem. Whereas remobilization started immediately after bud burst, N derived from uptake by root appeared in the leaves only 3 weeks later. The tree internal N status affected both the amount of N remobilization and its dynamics. The concentration of xylem sap amino acids peaked shortly after bud burst, concurrently with the period of fastest remobilization. Few amino acids and amides (Gln, Asn and Asp) were responsible for most of N translocated through the xylem; however, their relative concentration varied over spring, demonstrating that the transport of remobilized N occurred mainly with Gln whereas transport of N taken up from roots occurred mainly with Asn. Coupling measurements of amino acid N in the xylem sap with transpiration values was well correlated with the recovery of unlabelled N in the new biomass production. These results are discussed in relation to the possibility of measuring the spring remobilization of N in field‐grown trees by calculating the flux of N translocation in the xylem.     

改良FIASCO方法筛选砷超富集植物蜈蚣草SSR分子标记

蜈蚣草（Pteris vittata L.）是目前用于砷污染土壤修复最好的超富集植物,但其分子水平上的研究数据较少。为了开发蜈蚣草特异性SSR遗传标记,本文采用改良的FIASCO方法从蜈蚣草AG和AC微卫星富集文库中随机挑选100个克隆,分离得到51个微卫星位点,其中60%为完美型（Perfect）SSR。根据这些位点设计、合成了25对引物,并对江西庐山及湖北恩施两地蜈蚣草种群各20个个体进行了遗传多样性检测,结果发现：其中8个完美型及1个间断型（intermittent）SSR位点的引物能够扩增出清晰、稳定且具有多态性的条带。9对引物共扩增出41个等位基因,各位点等位基因数在2~7之间,平均等位基因数为4.56个;期望杂合度在0.0494~0.8169之间;没有连锁不平衡现象发生。采用大叶井栏边草（Pteris multifida Poir.）进行跨种扩增,结果发现其中6对引物能够进行种间扩增。这些SSR分子标记的开发有助于蜈蚣草生态适应性进化分析、揭示蜈蚣草地理分布格局以及探讨蜈蚣草遗传多样性,还可用于品种鉴定及选育等。     

Effects of Se on the uptake of essential elements in Pteris vittata L.

Selenium has been proven to be an antioxidant in plants at low dosages. To understand better the mechanisms of Se toxicity and benefit to plants, more investigations about effects of Se on the uptake of essential elements in plants would be desirable. In this study, hydroponic (nutrient solution culture) and pot (soil culture) experiments were simultaneously conducted to investigate the effects of Se on the uptake and distribution of essential elements in Pteris vittata. L (Chinese brake fern), an arsenic (As)-hyperaccumulator and a selenium (Se)-accumulator. Chinese brake fern took up much more Se in nutrient solution culture than in soil culture, with the highest Se content recorded as 1,573 mg kg^?1 in the roots, demonstrating remarkable tolerance to Se. In soil culture, Chinese brake fern also accumulated high content of Se, with the highest content measured as 81 mg kg^?1 and 233 mg kg^?1, in the fronds and roots, respectively. In soil culture, the addition of Se suppressed the uptake of most measured elements, including magnesium (Mg), potassium (K), phosphorus (P), iron (Fe), copper (Cu) and zinc (Zn). In nutrient solution culture, when the Se content in the tissues of Chinese brake fern was relatively low, the supplementation of Se suppressed the uptake of most essential elements; however, with the increase of Se content, stimulation effects of Se on the uptake of Ca, Mg, K were observed. An initial decrease followed by a rapid increase of Fe content in the fronds of Chinese brake fern was found with Se addition and tissue Se content increasing in nutrient solution culture, suggesting antagonistic and synergic roles of Se on these elements under low and high Se exposure, respectively. We suggest that Ca, Mg, K may be involved in the tolerance mechanism of Se, and that the regulation of Fe accumulation by Se in the fronds might be partially due to the dual effects of Se on Chinese brake fern.     

Interactive effects of selenium and arsenic on their uptake by Pteris vittata L. under hydroponic conditions

The interactive effects of selenium (Se) and arsenic (As) on plant uptake of Se and As have rarely been documented. In this study, the interactive effects of As and Se on their uptake by Chinese brake fern (Pteris vittata), an As-hyperaccumulator and Se-accumulator, were explored in two hydroponic experiments based on a two-factor, five-level central composite design. At Se levels of less than 2.5 mg L^?1, increasing amounts of As stimulated the uptake of Se in Chinese brake fern roots, possibly because of the beneficial effects of Se. In contrast, at Se concentrations greater than 2.5 mg L^?1, As suppressed the uptake of Se in Chinese brake fern roots. Uptake of As by both fronds and roots of Chinese brake fern was suppressed by the addition of Se, indicating the antagonistic effects of Se on As. In addition, at Se concentrations of less than 2.5 mg L^?1, As stimulated the translocation of Se from roots to fronds; meanwhile, the addition of Se resulted in reduced translocation of As from roots to fronds. These findings demonstrate the interactive effects of As and Se on their uptake by Chinese brake fern.