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。

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).

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. ^

植物对重金属砷和镉胁迫适应的分子机理初探

随着现代工业的发展，重金属污染日趋严重。重金属污染引发的环境和健康问题在许多国家都有报道，我国的重金属污染状况也不容乐观。土壤和水体中的重金属污染可以通过食物链进入人体，对人类健康造成很大的危害，如诱发癌症 和畸胎等。 植物修复是一种利用植物对重金属或有机污染物的超富集能力清除或减低污染的环境生物技术。植物修复的生物学机制的研究为这项技术走向实用化奠定了基础。植物修复近期的进展可能来自于可更有效地富集重金属的植物品种的选择、土壤条件的改善等;但长远看来，植物修复技术的巨大进步将取决于新的可更好地抵抗重金属或降解有机毒物的基因的鉴定和克隆，并通过转基因技术创造一批新的植物品种，如可迅速大量富集重金属的高生物量的用作环境净化的植物，以及可排拒重金属吸收的粮食、蔬菜和水果等作物。 本研究针对砷污染的植物修复机制，以超富集砷的凤尾蕨属植物——蜈蚣草为试材取得了如下进展： 1． 以从砷污染地区采集的蜈蚣草（Pteris vittataL．）为植物材料，利用抑制消减杂交(SSH)分离了经砷诱导处理与其对照间表达有差异的cDNA片段，以期得到与砷富集密切相关的基因。其中筛选到的一个cDNA片段与ABC transporter (ATP-binding cassette transporter)有较高的同源性。通过RACE方法对该基因进行了克隆，并进行了初步的结构和功能分析。结果表明所获得的PvABCTl (Accession No. AY496966)为一全长cDNA，长度为2165 bp，其中开读框架为1791 bp，编码597个氨基酸。该基因所编码的蛋白中含有2个ABC transporter特性结构域，1个ATP-binding cassette和2个ATP/GTP结合位点(P-loop)，没有明显的跨膜区。 2． 对蜈蚣草在砷胁迫下PvABCT1基因的表达模式进行了研究。转录水平分析表明PvABCT1的表达受砷的诱导。进一步通过PvABCTl-GFP融合基因在洋葱细胞中的表达进行亚细胞定位，结果显示该基因可能定位于细胞质中。 3． 为了研究所克隆的PvABCT1基因的功能，本研究构建了PvABCT1的酵母表达载体，把该基因转入因ACR3基因缺失而对砷敏感的酵母突变株。酵母功能互补实验表明PvABCT1不仅不能与ACR3基因功能互补，反而使酵母对砷的敏感性增加，同时酵母细胞中的砷含量较未转化的酵母细胞增加。即在转入PvABCT1后，酵母细胞吸收了更多的砷。这暗示该基因与蜈蚣草中砷的高吸收有关。 针对食品重金属污染问题，本研究探讨了减低蔬菜对重金属吸收的方法及其 作用机理，取得了如下进展： 1．研究了钙离子和镧离子对镉离子胁迫下生菜种子萌发和植株生长的影响，结果表明在种子萌发时外施4 mM CaCI2或0.04 mg/L La(N03)3均可提高生菜对重金属镉的抗性。 2．通过检测0.5 mM CdCl2胁迫下生菜植株中的镉含量以及外施钙离子或镧离子后相应的镉含量，发现4 mM CaCl2可以增加镉胁迫下生菜植株中镉的积累;而0.04 mg/L La(N03)3可以降低镉胁迫下生菜植株中镉的积累。 3．对生菜中植物络合素合酶基因进行了克隆，通过RT-PCR分析以及植物络合素( phytochelatins，PCs)的检测，探讨了外施钙离子或镧离子对镉胁迫下生菜植株中植物络合素合酶基因在转录水平的表达量、植物络合素含量以及镉的积累三者之间的关系。结果表明：4 mM CaCl2可以提高镉胁迫下生菜植株中植物络合素合酶基因在转录水平的表达以及植物络合素的含量，增加镉的积累;而0.04 mg/L La(N03)3虽然同样可以提高植物络合素合酶基因在转录水平的表达以及植物络合素的含量，却能降低镉胁迫下生菜植株中镉的积累。这暗示外施钙离子可以促进用于重金属污染环境修复的植物对重金属的吸收，而外施镧离子可以用于降低叶菜类蔬菜中重金属镉的积累。

Efeito fitotóxico e potencial remediador de três espécies vegetais contaminadas com benzeno

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

苏铁叶绿体的超微结构和功能及对CO2浓度倍增的响应

本研究首次揭示了七种苏铁类植物叶绿体的超微结构。即苏铁科(Cycadaceae)的攀枝花苏铁(C. panzhihuaensis).苏铁(C. revoluta)、叉叶苏铁(C.micholitzii)、 刺叶苏铁(C.rumphii和多歧苏铁(C.multipinnata)，蕨铁科(Stangeriaceae)的蕨铁(Stangeria eriopus)和泽米科(Zamiaceae)的米德尔堡大苏铁(Encephalortos middelburgensis).根据它们叶绿体内膜结构的不同将其大体分为两种类型：1、阴生型叶绿体：多歧苏铁和刺叶苏铁为此类型，它们的叶绿体类囊体垛叠程度高，基粒垛较宽，单个基粒中类囊体的数量很多，有的甚至上百;2、阳生型叶绿体：攀枝花苏铁、米德尔堡大苏铁、苏铁、蕨铁和刺叶苏铁以及外类群的凤尾蕨(Pteris vittata)的叶绿体均有阳生型叶绿体的特征：类囊体膜垛叠程度低，基粒较小。根据它们叶绿体的结构特征，又将其分为两组： ( 1)攀枝花苏铁，米德尔堡大苏铁和叉叶苏铁叶绿体中均有类囊体膜膨大的现象： (2)苏铁和蕨铁在苏铁类中是比较原始的种类，它们的叶绿体与在系统进化上较苏铁类低等的凤尾蕨的叶绿体中都有几个基粒聚集成簇的现象，说明苏铁类在进化的同时一些较原始的性状仍保留了下来。这些苏铁都在温室相同的条件下生长了两年多，但它们的叶绿体结构仍然能够反应原产地生境特点，说明在长期进化中，叶绿体对环境的适应方式已在基因水平上稳定下来，苏铁类植物不同叶绿体结构的形成有其遗传基础。 多歧苏铁，攀枝花苏铁，叉叶苏铁的叶绿体膜垛叠程度依次降低，与此相应，它们的chla/b和F730/F684依次升高，反应了结构与功能的一致性。 选用具有阳生型叶绿体的攀枝花苏铁和有阴生型叶绿体的多歧苏铁用不同的C02浓度（350 umol mol-l和700umol mol-l）处理后观察其叶绿体结构的变化，结果发现C02浓度倍增对它们的叶绿体影响甚微，而作为对照的无论是C3植物小麦(Triticum italica)还是C4植物谷子(Setaria italica)在C02倍增的条件下叶绿体内均有大量淀粉粒积累，并有膜结构改变。这说明苏铁的叶绿体结构有保守性，这有可能是苏铁类能历经亿万年的沧桑而生存下来的结构基础之一。

Assessment of the Removal Capacity, Tolerance, and Anatomical Adaptation of Different Plant Species to Benzene Contamination

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

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

The Global Plankton Database: an inventory and data from the Former Soviet Union expeditions

At present time, there is a lack of knowledge on the interannual climate-related variability of zooplankton communities of the tropical Atlantic, central Mediterranean Sea, Caspian Sea, and Aral Sea, due to the absence of appropriate databases. In the mid latitudes, the North Atlantic Oscillation (NAO) is the dominant mode of atmospheric fluctuations over eastern North America, the northern Atlantic Ocean and Europe. Therefore, one of the issues that need to be addressed through data synthesis is the evaluation of interannual patterns in species abundance and species diversity over these regions in regard to the NAO. The database has been used to investigate the ecological role of the NAO in interannual variations of mesozooplankton abundance and biomass along the zonal array of the NAO influence. Basic approach to the proposed research involved: (1) development of co-operation between experts and data holders in Ukraine, Russia, Kazakhstan, Azerbaijan, UK, and USA to rescue and compile the oceanographic data sets and release them on CD-ROM, (2) organization and compilation of a database based on FSU cruises to the above regions, (3) analysis of the basin-scale interannual variability of the zooplankton species abundance, biomass, and species diversity.