【作者】 傅晓萍；

【导师】 陈英旭；

【作者基本信息】 浙江大学 ， 环境工程， 2011， 博士

【摘要】 土壤重金属污染已经成为人类所面临的重大生态环境问题,引起了全世界人民的重视与关注。在所有重金属污染中,Cd污染最为严重,Cd也以其移动性大、毒性高成为最受关注的元素之一。我国农田土壤Cd污染不仅面积大,而且污染程度较重,已成为我国农产品安全生产的重大问题。近年来,我国学者发现报道了美洲商陆(Phytolacca ameircana L., pokeweed)、东南景天(Sedum alfredii Hance)等Cd耐性植物或超积累植物,为Cd污染土壤植物修复提供了良好的植物材料。然而,这些植物对Cd的吸收、累积、解毒和耐性机理还有待深入研究。本论文以美洲商陆为材料,通过温室水培实验,利用抑制剂检测了植物对Cd的吸收方式,用生化分析和透射电镜研究了Cd对植物生理生化和超微结构的影响,用超速离心及透射电镜+能谱技术考察了植物Cd的亚细胞分布规律,用化学试剂连续提取探讨了植物中Cd的化学形态及转化规律,用蛋白质SDS-PAGE和双向电泳2-DE分离了植物体内Cd胁迫表达差异蛋白。旨在阐明美洲商陆对Cd的吸收和耐性机理,为土壤重金属污染植物修复提供一定的理论和科学依据。通过研究取得了以下主要结果：美洲商陆具有很高的Cd积累能力,其根系中的Cd含量高于地上部。水培条件下,100μM Cd处理美洲商陆地上部和根系Cd含量分别可达966.9 mg/kg和8179.1 mg/kg,且受害症状较轻；即使在低浓度Cd处理下,美洲商陆仍具有很高的Cd吸收积累能力。解偶联剂DNP和钙离子通道抑制剂LaCl3对美洲商陆Cd吸收均有一定的抑制作用,说明美洲商陆对Cd的吸收可能存在主动吸收,同时与钙离子通道密切相关。不同浓度Cd处理对美洲商陆体内Cu、Fe、Mn、Zn和P、S等元素的积累影响程度不同。美洲商陆对Cd的吸收可能与Zn离子通道无关,同时植物体对Cd与Mn存在竞争性吸收作用,而P和S可能与美洲商陆对Cd的解毒和耐性过程相关。生理生化实验结果表明,当Cd处理浓度低于100μM时,美洲商陆叶绿素a、叶绿素b及膜脂过氧化产物丙二醛MDA含量无显著变化,说明一定浓度Cd处理对美洲商陆叶片光合作用和细胞膜没有明显影响。但是随着处理浓度继续增加,植物体内叶绿素a、叶绿素b和总叶绿素含量都呈现下降趋势,说明美洲商陆叶绿素的合成、光合作用进程和生长情况均可能受到了Cd不同程度的抑制；而MDA含量明显增加,则表明高浓度Cd处理使细胞膜受损加重。此外,美洲商陆叶片不同抗氧化酶活性的变化趋势不尽相同,抗氧化酶对植物Cd毒可能存在一定的缓解作用,但这种作用有一定限度。透射电镜结果也显示Cd胁迫下美洲商陆膜系统、叶绿体和线粒体等细胞结构受到不同程度的破坏,这种因结构破坏而呈现的植物衰老现象很可能是由于植物体内活性氧代谢失调、膜脂过氧化加剧而处于氧化状态所致。利用超速离心技术对美洲商陆亚细胞组分进行了分离,结果显示各亚细胞组分Cd的含量与处理浓度之间具有很好的相关性,且美洲商陆根和叶细胞中大部分Cd都存在于细胞可溶性组分(53.7%-68.3%)或者细胞壁(23.4%-29.1%)中,说明细胞可溶性组分(液泡)和细胞壁是美洲商陆体内Cd的主要分布位点。而不同化学提取剂对美洲商陆根、茎和叶的提取结果表明,美洲商陆不同组织中Cd的存在形态有所不同。在根中,大部分Cd以水溶态无机盐存在,而在地上部Cd主要与果胶酸或者蛋白质结合。各提取态Cd的含量与处理浓度之间具有很好的相关性。Cd形态分析同样证实液泡和细胞壁可能在美洲商陆对Cd的耐性和解毒中发挥了重要作用,从而使植物细胞免受或少受Cd的毒害。利用SDS-PAGE和2-DE技术研究了Cd胁迫对美洲商陆蛋白质表达的影响,SDS-PAGE结果显示美洲商陆根系和叶片蛋白的分子量均集中在10～100 kDa,且在～25 kDa和～50 kDa处出现蛋白主带。与对照相比,根系蛋白在Cd处理前后变化不明显,而美洲商陆叶片蛋白谱带强度在60～150 kDa范围内有所降低,在25～50 kDa则有所增加。2-DE结果表明Cd处理后美洲商陆根系有26个蛋白点在表达量上有显著变化,其中8个蛋白点下调表达,18个蛋白点上调表达；而植物叶蛋白2-DE图谱显示共有46个蛋白点的丰度发生变化,其中下调表达大于2倍的蛋白点有21个,上调表达大于2倍的蛋白点为25个。这些蛋白可能是植物对Cd胁迫的响应,参与Cd的螯合,对美洲商陆Cd吸收和耐性起着积极的作用。更多还原

【Abstract】 Heavy metals are toxic at excessive concentrations and may pollute the natural and man-made environment ecosystem. In recent years, heavy metal pollution of soil has received increasing attentions worldwide mainly because of the public awareness of environmental issues. Among all of the heavy metals, cadmium (Cd) is of most concern, for its great mobility and high toxicity in the soil environment. Meanwhile, large area of farmland is deeply contaminated by Cd, which leads to one of the major issues of agricultural product safety in China. Phytolacca americana L. (pokeweed) and Sedum alfredii Hance were reported to be Cd-tolerant plants or hyperaccumulators, which were benefit for phytoremediation of Cd polluted soil. However, the mechanisms of Cd uptake, accumulation, detoxification and tolerance were needed for further research. In the present work, we used hydroponically grown seedlings of pokeweed to find out the mechanisms in countering Cd stress in the plant cultures. We used inhibitors to study the Cd uptake way, investigated the impacts of Cd on plant physiology and biology characters and ultrastructure changes through biochemical analysis and transmission electron microscope (TEM) observation, examined cellular distribution of Cd with ultracentrifugation, TEM and energy disperse spectroscopy (EDX) analysis, isolated differential expression proteins from plant using SDS-PAGE and two dimensional electrophoresis (2-DE) technologies. The purpose of our study was to clarify the mechanisms of Cd uptake and tolerance in pokeweed, which could provide theoretical and scientific basis for phytoremediation of heavy metal contaminated soil. The main results were summarized as follows:Pokeweed is of high ability for accumulating Cd and this metal is mainly accumulated in plant root. With the hydraulic cultivation of 100μM Cd, the Cd contents were as high as 966.9 mg/kg in shoots and 8179.1 mg/kg in roots, respectively, and the plant showed a little toxicity symptoms. Meanwhile, pokeweed is still of high accumulating ability even under relatively low Cd concentration treatments. Both uncoupling agents 2,4-dinitrophenol (DNP) and Ca2+ channel inhibitor LaCl3 may inhibit the absorption of Cd into plant, suggesting that the uptake of Cd in pokeweed may be an active absorption process and it is closely related to Ca2+ channels. Furthermore, different Cd treatments have different effects on the accumulation of elements in plant. Pokeweed may have competitive absorption towards Cd and Mn. The uptake of Cd may have no/little relationship with Zn2+ channels while P and S may take part in the process of Cd tolerance and detoxification in pokeweed.Physiological and biochemical analysises showed that under 100μM Cd treatments, both chlorophyll and MDA (the peroxidating substance of plasma membrane) in pokeweed leaves were not significantly changed, indicating that the photosynthesis was not inhibited and plasma membrane was not injured. However, as the concentrations of Cd treatments continue to increase, all chlorophyll a, chlorophyll b and the total chlorophyll contents were declined while the MDA contents increased dramatically, suggesting chlorophyll synthesis, photosynthesis process and plant growth may all likely be inhibited by Cd. Besides, differences were noted in different antioxidase activities under Cd stress and antioxidase system may have certain extent to help relieve the toxicity of Cd in pokeweed. TEM results also illustrated varying degrees of damages in membrane system, chloroplast, mitochondria and other cellular organelles and these phenomenons may be due to the loss of cellular redox homeostasis and an increase of lipid peroxidation.Cd analysis at the subcellular level of plant tissue demonstrated that large proportion of Cd was stored in the soluble fraction (53.7%～68.3%) or bound to the cell wall fraction (23.4%～29.1%). Besides, Cd must be existed in different chemical forms among different tissues and Cd concentrations bound to each form in the plant increased in a concentration-dependent manner following Cd exposure. In roots, the majority of Cd was in inorganic form, while in stems and leaves most of the Cd was integrated with pectates and protein. Both the vacuoles and the cell walls might be involved in the Cd tolerance mechanisms to protect metabolically active cellular compartments from toxic Cd concentrationsDifferential expression proteins in pokeweed tissues under Cd stress were studied by SDS-PAGE and 2-DE techniques. SDS-PAGE results showed that the molecular weight of proteins isolated from roots and leaves of pokeweed is concentrated between 10～100 kDa, while in the 25 kDa and 50 kDa position scales there were two main protein bands. Compared with the control, root proteins did not change significantly after Cd treatment, while in leaves the intensity of protein bands decreased in the 60～150 kDa position scales and increased in the 25～50 kDa position scales, respectively. Moreover,2-DE results indicated that 26 proteins’ expression changed in pokeweed roots after Cd treatment. Among them,8 proteins were disappeared or down-regulated, while 18 proteins were induced or up-regulated. In plant leaves,2-DE results indicated that a total of 46 proteins’abundance were altered, among them,21 proteins were two fold down-regulated and 25 proteins were two fold up-regulated These proteins may be related to the response of pokeweed to Cd stress, involved in the chelation of Cd and play an active role in the uptake and tolerance processes of Cd in pokeweed. Therefore, further studies with respect to protein identification and their functions should be carried out.更多还原