【作者】 施积炎；

【导师】 陈英旭；

【作者基本信息】 浙江大学 ， 环境工程， 2004， 博士

【摘要】 铜是一种用途广泛而又对人体危害较大的重金属元素。我国重金属污染土壤中，铜是一种主要污染元素。在江西、浙江等地均存在大面积的铜污染土壤。近年来，我国学者发现报道了海州香薷（Elsholtzia splendens）、鸭跖草（Commelina communis）等铜富集植物，为铜污染土壤植物修复提供了很好的植物材料。然而，这些富集植物对铜的吸收累积机理还有待深入研究。本论文以海州香薷和鸭跖草为材料，对比研究两种植物对铜的吸收方式及其与根质膜Fe（Ⅲ）—螯合物还原酶（FCR）活性的关系；用同步辐射X—射线荧光（SRXRF）研究植物中元素的微区分布规律；用X射线吸收光谱（XAS）探明植物中Cu的分子形态及转化规律；并分离植物中铜结合蛋白。旨在阐明海州香薷和鸭跖草对铜的吸收累积机理，为土壤重金属污染植物修复及调控重金属在食物链中的迁移提供理论依据。通过研究取得了以下主要结果： 海州香薷和鸭跖草根分泌物对污染土壤中Cu有一定的活化能力，但缺铁，缺锌及铜处理间差异不大，鸭跖草根泌物对Cu的活化能力大于海州香薷。不同处理根泌物中有机酸种类或含量有所差异，但主要以草酸和出峰时间为6.5min左右的未知有机酸为主。假单胞菌（P.aeruginosa ZD4-3）对污染土壤Cu，Pb和Zn有很强的活化作用；白腐真菌（P.Chrysosprim）对Cu，Zn有一定活化作用，对Pb则表现为固定作用；假单胞菌和白腐真菌混合接种提高土壤Cu，Pb和Zn的活性，但比单独接种假单胞菌时效果差。海州香薷根泌物降低了其假单胞菌对土壤Cu，Zn和Pb的活化效果，但促进了白腐真菌对土壤Cu，Pb的活化，对Zn活性的影响不大。 海州香薷和鸭跖草对Cu都具有较强的富集能力，但海州香薷根部和地上部Cu的累积量均显著高于鸭跖草。鸭跖草地上部／根Cu比值大于海州香薷，说明鸭跖草对Cu的运输能力大于海州香薷。解偶联剂2,4—二硝基苯酚（DNP）和P—型ATP酶抑制剂Na₃VO₄对海州香薷和鸭跖草Cu吸收均有一定的抑制作用，两种植物对Cu可能存在主动吸收。蛋白合成抑制剂放线菌酮（CHD）对海州香薷Cu吸收没有抑制作用，而显著抑制了鸭跖草对Cu的吸收。钙离子通道抑制剂LaCl₃对海州香薷Cu吸收有抑制作用，而对鸭跖草Cu吸收没有抑制作用。说明海州香薷Cu吸收与钙离子通道密切相关，而鸭跖草Cu吸收则不通过钙离子通道进行。 缺铁可以显著促进海州香蕾和鸭拓草对Cu的吸收，也可以提高对不同形态CuEDTA一Cu，NTA一Cu，草酸一Cu和柠檬酸一Cu的植物利用性。随着缺铁诱导时间的延长，海州香蕾和鸭踌草对Cu的吸收累积量呈先上升后下降的趋势。缺铁会导致草植物根际酸化效应，这与根质膜H十一ATPase的激活有密切关系。但在P型一ATP酶抑制剂处理下，缺铁处理鸭而草对铜的吸收量仍显著高于完全培养液生长的植株，说明缺铁对Cu吸收的促进作用可能与P一型ATP酶活性无关。缺铁没有显著提高海州香蕾根质膜FCR活性，但缺铜可以提高海州香蕾根质膜FCR活性。缺铁和缺铜均可以提高鸭拓草根质膜FCR活性。根质膜FCR还原Fe（m）鳌合物反应的最适pH为5.5左右。随着缺铁和缺铜处理时间的延长，FCR活性均总体呈先上升后下降的趋势。蛋白合成抑制剂放线菌酮可以同时抑制缺铁处理鸭环草对铜的吸收和根质膜FCR活性。另外，缺铁处理9天的鸭拓草同时出现铜吸收和FCR活性峰。以上结果说明鸭踌草Cu吸收可能与根质膜FCR活性密切相关。 用SRXRf点扫描和面扫描两种方式都能很好地检测海州香蕾和鸭踌草根茎叶切片上p，S，CI，K，Ca，Mn，Fe，eu和zn等元素的分布情况，并发现sRXRf所测定的元素相对含量和ICP^AES所测的元素浓度呈极显著线性相关，说明SRXRf是一种灵敏的微量元素检测技术。海州香莆和鸭拓草根表皮Cu含量最高，从外皮层至内皮层Cu逐渐降低，透过内皮层后到达维管柱含量又增高，进一步说明根可以主动地吸收Cu。Cu在茎和叶柄的维管组织中最高，而在叶肉细胞比叶表皮中更高。大部分元素在表皮毛中的含量并不比其它部位高，因此重金属区隔化分布在叶表皮或表皮毛细胞并不是所有植物的共有特征。P，S，Mn，Fe和zn的分布情况与Cu相似。K则集中分布在根皮层，茎的维管组织以及叶栅栏组织中，而Ca则集中分布在根、茎的表皮以及叶栅栏组织中。Cu与P、S、Ca的分布呈显著相关，表明P，S，Ca在海州香蕾和鸭踌草累积Cu的过程中起着重要作用。Cu与Mn，Fe及Zn的分布也呈显著相关，进一步说明Cu，Mn，Fe和Zn可能借助一些有较宽底物范围的转运蛋白进行吸收转运。 海州香蕾CuK边EXAFS研究结果表明，Cu处理初期海州香蕾根茎叶中Cu配位以Cu-O键为主，但根茎叶中Cu一O键的配位数和配位距离有所差异。随着Cu处理时间的延长，植物中Cu一O键有向Cu-S键转化的趋势，而且茎和叶比根表现更为明显。另外，海州香蕾根茎叶细胞壁的Cu配位主要以Cu一O键为主。鸭踌草中Cu主要与O配位，同时存在一定的Cu一S键，而且从根一茎一叶Cu-S键的比例有增加的趋势。从海州香蕾和鸭踌草cuK边XANEs发现，Cu处理下两种植物都会发生c矛+的还原现象，其中茎和叶中Cu价态比根中Cu价态降低更多。而且随着处理时间的延长，植物c了十的还原现象加更加明显。EXAFs和XANEs的结更多还原

【Abstract】 Cu is a heavy metal, fully used but harmful for people’s health. It is one of major element in our country’s contaminated soil, especially in Jiangxi and Zhejiang province. E. splendens and C. communis were reported as copper-accumulated plants, which were benefit for phytoremediation of Cu-contaminated soil. However, the mechanism of Cu accumulation needed for further research. In this paper, we compared the Cu uptake way with these two plants and the relationship between Cu uptake and the root Fe(III)-chelate reductase (FCR) activity, studied the characters of Cu and other elements distribution using synchrotron radiation X-ray fluorescence spectroscopy (SRXRF), investigated Cu molecular speciation and its transformation with X-ray absorption spectroscopy (XAS), and then separated the Cu-binding proteins. The purpose of our study was to clarify the mechanism of Cu uptake and accumulation in E. splendens and C. communis, which could provid theory basis for phytoremediation. The main results were as follows:The results showed heavy metals in contaminated soil could be activated by root exudates of E. splendens and C. communis, and there was no significant difference among the root exudates with control, high Cu Fe deficiency and Zn deficiency treatment. There organic acid in the root exudates was different among different treatments, but the oxalic acid and an unknown organic acid were dominants. The mobility of Cu and Zn in contaminated soil was improved obviously by P. aeruginosa ZD4-3 and P. Chrysosprim, and the mobility of Pb was improved by P. aeruginosa ZD4-3 while inhibited by P. Chrysosprim. Root exudates of E. splendens inhibited the activation of P. aeruginosa ZD4-3 to heavy metals, while promoted the activation of P. Chrysosprim.The ability of Cu accumulation in E. splendens was higher than in C. communis, but the ability of Cu transport was lower. Both the uncoupler of oxidative phosphorylation DNP and P-type ATPase inhibitor Na3VO4 could inhibit Cu uptake, which showed properly the two plants could uptake Cu initiatively. Ca2+ ion channel inhibitor LaCl3 inhibited Cu uptake of E. splendens while not inhibit Cu uptake of C. communis, which demonstrated Cu uptake of E. splendens was related to Ca2+ ion channel, while Cu uptake of C. communis was not. On the contrary, Cu uptake of C. communis was inhibited by the protein synthesis inhibitor cycloheximide, while Cu uptake of E. splendens was not. Cu uptake was hardly affected byZn deficiency at low Cu, while Cu uptake was promoted at high Cu.The results showed that Cu uptake and accumulation in C. communis and E. splendens was significantly enhanced by Fe deficiency, and the Cu concentration in roots and shoots was increased, then decreased with the time of Fe deficiency treatment. The bioavailability of different Cu speciation, such as EDTA-Cu, NTA-Cu, oxalate-Cu, and citrate-Cu, could be promoted by Fe deficiency. It suggested that there was an inherent relationship between Cu uptake and element Fe. Fe deficiency could acidify the rhizosphere of C. communis. Cu uptake in Fe-deficient plants was still higher than that of Fe-sufficient plants when P-type ATPase inhibitor Na3VO4 was added in the solution, which demonstrated that Fe deficiency induced stimulation of the plasma membrane H+-ATPase did not play a role in the enhanced Cu uptake.Both Fe deficiency and Cu deficiency could induce root FCR activity in C. communis. Cu deficiency could induce root FCR activity in E. splendens, While Fe deficiency not. Both FCR activity and Cu concentration in roots of C. communis inhibited distinctly by cycloheximide, a kind of protein synthesis inhibitor. During the course of Fe deficiency, there simultaneously appeared Cu concentration and FCR activity peaks in roots at day 9. All above evidences demonstrated Cu uptake and accumulation in C. communis were probably related to the root FCR activity.Synchrotron radiation X-ray fluorescence spectroscopy (SRXRF) microprobe was used to study Cu and other elements distribution in E. splendens and C. communis. P更多还原