Identification of an arsenic tolerant double mutant with a thiol-mediated component and increased arsenic tolerance in phyA mutants

A genetic screen was performed to isolate mutants showing increased arsenic tolerance using an Arabidopsis thaliana population of activation tagged lines. The most arsenic-resistant mutant shows increased arsenate and arsenite tolerance. Genetic analyses of the mutant indicate that the mutant contains two loci that contribute to arsenic tolerance, designated ars4 and ars5. The ars4ars5 double mutant contains a single T-DNA insertion, ars4, which co-segregates with arsenic tolerance and is inserted in the Phytochrome A (PHYA) gene, strongly reducing the expression of PHYA. When grown under far-red light conditions ars4ars5 shows the same elongated hypocotyl phenotype as the previously described strong phyA-211 allele. Three independent phyA alleles, ars4, phyA-211 and a new T-DNA insertion allele (phyA-t) show increased tolerance to arsenate, although to a lesser degree than the ars4ars5 double mutant. Analyses of the ars5 single mutant show that ars5 exhibits stronger arsenic tolerance than ars4, and that ars5 is not linked to ars4. Arsenic tolerance assays with phyB-9 and phot1/phot2 mutants show that these photoreceptor mutants do not exhibit phyA-like arsenic tolerance. Fluorescence HPLC analyses show that elevated levels of phytochelatins were not detected in ars4, ars5 or ars4ars5, however increases in the thiols cysteine, gamma-glutamylcysteine and glutathione were observed. Compared with wild type, the total thiol levels in ars4, ars5 and ars4ars5 mutants were increased up to 80% with combined buthionine sulfoximine and arsenic treatments, suggesting the enhancement of mechanisms that mediate thiol synthesis in the mutants. The presented findings show that PHYA negatively regulates a pathway conferring arsenic tolerance, and that an enhanced thiol synthesis mechanism contributes to the arsenic tolerance of ars4ars5.

Mechanistic study of arsenic uptake, transformation and tolerance in arsenic hyperaccumulator Pteris vittata

Pteris vittata, the first reported arsenic hyperaccumulating plant, is potentially used in phytoremediation of arsenic, as it can accumulate up to 2.3% of arsenic in its fronds. In this study, the mechanisms of arsenic tolerance, uptake and transformation were studied in the plant. Arsenic species were analyzed by HPLC-AFS. Results showed that arsenic was mainly accumulated in leaflets, and inorganic arsenate and arsenite were only species in P. vittata. Arsenite was the predominant species in leaflets, whereas arsenate was the predominant species in roots. Arsenic induced the synthesis of thiol containing compounds in P. vittata. As-induced thiol was purified by a novel method: covalent chromatography following preparative HPLC. The purified thiol was characterized as a phytochelatin with two units (PC2). ^ In P. vittata, enhanced tolerance likely results from unusual intracellular detoxification mechanisms. Although PC-dependent sequestration of arsenic into vacuoles is essential for nonhyperaccumulators, this sequestration is not the major arsenic tolerance mechanisms in this arsenic hyperaccumulator. PC-independent sequestration of arsenic is likely the major arsenic tolerance mechanism. PC-dependent arsenic detoxification is probably a supplement to this major mechanism. ^ Interactions between arsenic and phosphate were studied. Under hydroponic condition, arsenic supply decreased the concentrations of phosphate in roots. In soil, arsenic increased the concentrations of phosphate in roots. Arsenic concentrations in rachises and leaflets were not affected by arsenic supply in either hydroponic or soil system. Phosphate decreased arsenic accumulation in roots, rachises and leaflets in the hydroponic system. ^ The uptake kinetics of arsenate, arsenite, monomethyl arsinic acid (MMA), dimethyl arsonic acid, and phosphate were studied in P. vittata. Phosphate uptake systems in Pteris vittata cannot distinguish phosphate and As(V), resulting in As hyperaccumulation. Arsenic hyperaccumulation in this plant is an inevitable consequence during phosphate acquisition. Arsenate, arsenite and MMA are transported via the phosphate uptake systems. The co-transport of arsenite/phosphate and MMA/phosphate is reported for the first time in plants. These unique phenomena are useful for understanding arsenic hyperaccumulation and the evolution of this capacity in P. vittata. ^

An arsenate tolerance gene on chromosome 6 of rice

The genetics of arsenic tolerance in plants has not been extensively studied and no arsenic tolerance gene has been genetically mapped. Screening 20 diverse genotypes of rice for reduced root growth in 13.3 μM arsenate identified marked differences in tolerance. The most sensitive variety, Dawn, is known to be highly susceptible to straighthead, a condition linked to arsenic contamination of soil. Screening 108 recombinant inbred lines of the Bala x Azucena mapping population revealed the presence of a major gene, AsTol, which mapped between markers RZ516 and RG213 on chromosome 6. This gene is a good target for further characterisation. It should prove valuable for investigations into the physiological and molecular mechanism behind arsenic tolerance in plants and may lead to strategies aimed at breeding for arsenic contaminated regions. © New Phytologist (2004).

砷超富集植物蜈蚣草(Pteris vittata L.)中砷解毒机制的研究

砷是一种具有致癌、致畸、致突变的有毒元素，在地表的含量本来很低。然而，随着现代社会的发展和工业活动的增加导致砷污染日趋严重。土壤和水体中的砷污染可以通过食物链进入人体，对人类的健康造成极大的危害。植物修复是一种利用植物对污染物的超富集能力来清除或减低污染的新型环境生物技术。植物修复的实际应用依赖于超富集植物的发现和超富集机制的阐明，特别是砷解毒过程(砷的吸收、还原和区域化)的研究及相关基因的克隆。砷超富集植物蜈蚣草(Pteris vittata L.)中砷解毒机制的阐明将为砷污染的植物修复及新型工程植物的研发提供理论基础。 本论文以蜈蚣草为试材，针对蜈蚣草的砷解毒机制取得了如下研究进展： 1.以砷超富集植物蜈蚣草为材料，建立了一个适于研究蜈蚣草砷吸收和解毒机制的新系统—愈伤组织悬浮培养体系。首次证明蜈蚣草愈伤组织与其孢子体及配子体一样具有对砷的抗性和砷超富集的能力。 2.以蜈蚣草愈伤组织为材料，通过比较亚砷酸盐、砷酸盐和二甲基胂酸盐对蜈蚣草和拟南芥植物毒性的差异，表明砷的还原可能是蜈蚣草对砷解毒的重要机制之一而砷的甲基化对蜈蚣草的砷解毒作用甚微。 3.以蜈蚣草愈伤组织为材料，通过对砷在蜈蚣草愈伤组织细胞中的亚细胞定位，首次直接证明植物液泡对砷具有非常明显的区隔化作用。暗示区隔化作用在蜈蚣草对砷的解毒过程中发挥着重要的作用。 4.通过测定蜈蚣草愈伤组织对不同化学态的砷处理下抗氧化物质的变化发现酸溶性巯基在蜈蚣草砷解毒中也发挥着重要作用。 5.以蜈蚣草愈伤组织为材料，发现磷和砷的吸收在高浓度范围下（﹥0.2 mM）存在明显的协同效应。对蜈蚣草高亲和磷酸盐转运蛋白基因－PvPHT基因功能的初步分析则表明PvPHT参与了蜈蚣草对磷和砷的吸收过程。

蜈蚣草(Pteris vittata L.)对砷和铜复合污染的响应及生理机制研究

随着现代工农业的发展，环境污染日益加剧。农用土壤作为我们赖以生存的基础，大量含砷（Arsenic, As）和铜（Copper, Cu）的化肥和农药的应用造成As、Cu及其他重金属的复合污染日趋加重。蜈蚣草作为一种天然的砷超富集植物，在清除土壤砷污染的应用中备受人们关注。然而，目前的研究多集中在蜈蚣草对单一砷污染的吸收、转运和超富集等方面，几乎没有对土壤复合污染尤其是As、Cu复合污染的的响应和生理机制的研究。本文针对As、Cu复合污染的现实，研究了不同浓度砷、铜复合污染胁迫对蜈蚣草生长和发育的影响，探讨了蜈蚣草对砷、铜复合污染抗性和富集的生理生化机制，以期为蜈蚣草在植物修复复合污染的应用提供理论基础。 主要研究结果如下： 1、以砷超富集植物蜈蚣草成熟孢子为外植体材料，建立了一套成熟的蜈蚣草孢子植株再生体系，包括配子体分化和愈伤组织分化两条途径。为蜈蚣草的生理生化机制以及将来的遗传转化奠定了基础。同时证明了蜈蚣草愈伤组织与其孢子体及配子体一样具有对砷的抗性和超富集特性以及铜抗性。 2、以组培的蜈蚣草孢子体为材料，通过比较高浓度砷、低浓度砷以及高浓度铜、低浓度铜的相互组合对孢子体毒性的差异，表明适度浓度砷可以增强蜈蚣草对铜的抗性，但高、低浓度铜都不能增强砷的抗性。同时测定了不同浓度砷、铜处理对蜈蚣草体内砷和铜的吸收分配情况，探讨了蜈蚣草对砷铜复合污染土壤的植物修复的潜在可能性 。 3、以蜈蚣草组培的配子体为材料，研究了蜈蚣草对不同浓度铜砷复合处理的生理生化响应。结果表明砷的加入可以缓解铜对蜈蚣草配子体的植物毒性，而铜的加入并没有缓解砷的植物毒性。而且随着砷浓度的提高可以显著降低铜在配子体中的积累，显著提高了配子体细胞生存能力，降低了配子体细胞膜透性，且可以改变铜砷在配子体中亚细胞定位，暗示砷对铜的积累具有拮抗作用。同时观察到适度的铜也可以降低砷在孢子体根中的积累，对砷在叶柄以及羽叶中的积累有一定的降低作用，但并不显著。 4、以蜈蚣草愈伤组织为材料，研究了蜈蚣草愈伤组织对砷和铜复合污染胁迫的生理生化响应，结果表明适度的砷可以显著提高蜈蚣草愈伤组织中抗氧化酶系统，进而提高抵御铜胁迫引起的ROS胁迫的能力而显著缓解铜对蜈蚣草愈伤组织的毒性。低浓度砷加入显著诱导POD、CAT活性的升高，提高了蜈蚣草愈伤组织抵抗ROS胁迫能力，尤其是POD与生长呈显著的正相关;SOD活性在0-1.0 mM Na3AsO4条件下并没有显著增加，暗示在相对较低的砷胁迫和铜胁迫条件下POD对蜈蚣草愈伤组织解毒起到重要作用。同时在高砷或者高铜胁迫条件下GR活性和GPx活性与之的相关系数可以达到显著水平，这说明高砷或者高铜胁迫条件下GR和GPx在蜈蚣草解毒中起到重要作用。 5、首次证明NO可能参与砷缓解铜对蜈蚣草的植物毒性的过程。发现加入0.2 μM NO加入并没有显著改善蜈蚣草砷的植物毒性，清除蜈蚣草愈伤组织内的NO后，显著增强了砷胁迫条件下对蜈蚣草愈伤组织生长的抑制作用。同时加入NO后却显著改善了蜈蚣草铜胁迫条件下愈伤组织的生长。测定了As、Cu处理后蜈蚣草愈伤组织内源NO含量的变化以及CAT抗ROS能力的变化，结果表明蜈蚣草愈伤组织NO合成显著受As诱导，但不受铜的诱导，同时内源NO浓度的升高，伴随着抵抗ROS胁迫的抗氧化的CAT活性升高。暗示砷可能是通过诱导内源NO浓度升高提高蜈蚣草愈伤组织抵抗ROS胁迫能力来缓解对铜的植物毒性。

Yellow earthworms:Distinctive pigmentation associated with arsenic- and copper-tolerance in Lumbricus rubellus

Lumbricus rubellus Hoffmeister, inhabiting soil at the 19th century Devon Great Consols mine at Tavistock, Devon, UK, show high tolerance to Cu- and As-toxicity and frequently have a striking yellow coloration. Specimens from this site (mature and immature) and from an uncontaminated site on Lancaster University campus (mature) were photographed, and the slide images digitized and analyzed. All L. rubellus showed reddish-purple pigmentation of the body wall that declined in intensity posteriorly. The metal- and metalloid-resistant earthworms, whether mature or immature, showed yellowing in the posterior half of the body. The source of the coloration was intense yellow pigmentation of the chloragogenous tissue surrounding the alimentary canal. The yellow pigmentation is masked by reddish-purple body wall pigmentation anteriorly. Total As concentrations in tissues were determined for the anterior, middle and posterior sections of resistant and non-resistant L. rubellus. Highest concentrations were in the middle sections of the mature and immature resistant L. rubellus (36.17 ± 19.77 and 27.77 ± 9.02 mg As kg^-1, respectively). Resistant immature L. rubellus lost condition over 28 d in soil treated with 750 mg As kg^-1, possibly due to a higher metabolism, whilst there was no loss in condition for resistant mature L. rubellus in the treated soil. As far as the authors are aware, this is the first report of yellow pigmentation of this kind in earthworms. The pigmentation may provide a useful indicator of exposure/resistance to soil contamination. © 2002 Elsevier Science Ltd. All rights reserved.

An arsenic-accumulating, hypertolerant brassica, Isatis capadocica

Isatis capadocica, a brassica collected from Iranian arsenic-contaminated mine spoils and control populations, was examined to determine arsenate tolerance, metabolism and accumulation. I. cappadocica exhibited arsenate hypertolerance in both mine and nonmine populations, actively growing at concentrations of > 1 mm arsenate in hydroponic solution. I. cappadocica had an ability to accumulate high concentrations of arsenic in its shoots, in excess of 100 mg kg(-1) DW, with a shoot : root transfer ratio of > 1. The ability to accumulate arsenic was exhibited in both hydroponics and contaminated soils. Tolerance in this species was not achieved through suppression of high-affinity phosphate/arsenate root transport, in contrast to other monocotyledons and dicotyledons. A high percentage (> 50%) of arsenic in the tissues was phytochelatin complexed; however, it is argued that this is a constitutive, rather than an adaptive, mechanism of tolerance.

Rice-arsenate interactions in hydroponics: a three-gene model for tolerance

In this study, the genetic mapping of the tolerance of root growth to 13.3 muM arsenate [As(V)] using the BalaxAzucena population is improved, and candidate genes for further study are identified. A remarkable three-gene model of tolerance is advanced, which appears to involve epistatic interaction between three major genes, two on chromosome 6 and one on chromosome 10. Any combination of two of these genes inherited from the tolerant parent leads to the plant having tolerance. Lists of potential positional candidate genes are presented. These are then refined using whole genome transcriptomics data and bioinformatics. Physiological evidence is also provided that genes related to phosphate transport are unlikely to be behind the genetic loci conferring tolerance. These results offer testable hypotheses for genes related to As(V) tolerance that might offer strategies for mitigating arsenic (As) accumulation in consumed rice.

Effect of organic matter amendment, arsenic amendment and water management regime on rice grain arsenic species

Arsenic accumulation in rice grain has been identified as a major problem in some regions of Asia. A study was conducted to investigate the effect of increased organic matter in the soil on the release of arsenic into soil pore water and accumulation of arsenic species within rice grain. It was observed that high concentrations of soil arsenic and organic matter caused a reduction in plant growth and delayed flowering time. Total grain arsenic accumulation was higher in the plants grown in high soil arsenic in combination with high organic matter, with an increase in the percentage of organic arsenic species observed. The results indicate that the application of organic matter should be done with caution in paddy soils which have high soil arsenic, as this may lead to an increase in accumulation of arsenic within rice grains. Results also confirm that flooding conditions substantially increase grain arsenic. (c) 2013 Elsevier Ltd. All rights reserved.

Arsenic uptake and speciation in the rootless duckweed Wolffia globosa

Duckweeds are a common macrophyte in paddy and aquatic environments. Here, we investigated arsenic (As) accumulation, speciation and tolerance of the rootless duckweed Wolffia globosa and its potential for As phytofiltration.

When grown with 1 mu M arsenate, W. globosa accumulated two to 10 times more As than four other duckweed or Azolla species tested. W. globosa was able to accumulate > 1000 mg As kg(-1) in frond dry weight (DW), and tolerate up to 400 mg As kg-1 DW. At the low concentration range, uptake rate was similar for arsenate and arsenite, but at the high concentration range, arsenite was taken up at a faster rate.

Arsenite was the predominant As species (c. 90% of the total extractable As) in both arsenate-and arsenite-exposed duckweed. W. globosa was more resistant to external arsenate than arsenite, but showed a similar degree of tolerance internally. W. globosa decreased arsenate in solution rapidly, but also effluxed arsenite.

Wolffia globosa is a strong As accumulator and an interesting model plant to study As uptake and metabolism because of the lack of a root-to-frond translocation

A balanced polymorphism in biomass resource allocation controlled by phosphate in grasses screened through arsenate tolerance

The response of arsenate and non-tolerant Holcus lanatus L. phenotypes, where tolerance is achieved through suppression of high affinity phosphate/arsenate root uptake, was investigated under different growth regimes to investigate why there is a polymorphism in tolerance found in populations growing on uncontaminated soil. Tolerant plants screened from an arsenic uncontaminated population differed, when grown on the soil from the populations origin, from non-tolerants, in their biomass allocation under phosphate fertilization: non-tolerants put more resources into tiller production and down regulated investment in root production under phosphate fertilization while tolerants tillered less effectively and did not alter resource allocation to shoot biomass under phosphate fertilization. The two phenotypes also differed in their shoot mineral status having higher concentrations of copper, cadmium, lead and manganese, but phosphorus status differed little, suggesting tight homeostasis. The polymorphism was also widely present (40%) in other wild grass species suggesting an important ecological role for this gene that can be screened through plant root response to arsenate.