超积累东南景天（Sedum alfredii Hance）对重金属（Zn/Cd/Pb）的解毒机制

【作者】 田生科；

【导师】 杨肖娥；

【作者基本信息】 浙江大学 ， 环境修复与资源再生， 2010， 博士

【摘要】 植物对重金属的超积累作用是当今国际研究热点和难点,深入研究超积累植物体内重金属稳态作用机制,无论对发展植物营养新理论,还是对超积累植物在污染环境修复中的实际应用均有重要意义。东南景天(Sedum alfredii Hance)是我国原生的锌镉超积累及铅富集植物,其对这三种重金属都具有很强的耐性和积累能力,是应用于污染土壤绿色植物修复技术的一种良好潜在材料。本文在综述超积累植物对重金属的解毒机理及其相关研究方法的基础上,以超积累植物体内重金属的区隔和螯合作用为研究中心,深入探讨了锌、镉和铅在东南景天体内的分布特征与赋存形态。综合运用同步辐射X射线荧光光谱(SR-XRF)、激光刻蚀-电感耦合等离子体质谱(LA-ICP-MS)等高通量金属组学研究技术,结合高分辨率的荧光探针显微技术,原位分析了锌镉铅在东南景天体内组织水平、细胞水平以及亚细胞水平上的分配特点,明确各类组织/细胞对重金属的区隔化在植物体耐受高浓度有毒重金属中所起的重要作用；同时,利用X射线吸收光谱(XAS)、微区X射线吸收光谱(μ-XAS)和高效液相色谱(HPLC)等技术,重点研究了重金属在植物组织中的赋存形态及其时空变化特点,探讨植物体内各类有机化合物对重金属的螯合作用及其对植物体耐受与积累重金属的可能影响,以期进一步揭示超积累特异植物对重金属的积累机理,为其在植物修复技术改良与优化中的应用提供科学依据,也为其在锌元素的生物强化中的潜在应用提供一定的理论参考。本研究取得的主要研究结果如下：1.采用水培实验,研究比较了镉对两种生态型东南景天根伸长量、活性氧自由基(ROS)、脂质过氧化、原生质膜完整性及谷胱甘肽代谢的影响。试验结果表明,镉对非超积累生态型东南景天(NHE)的根尖伸长表现出明显的抑制作用,而对超积累生态型(HE)无明显影响,低镉处理甚至在一定程度上促进了其根系的伸长。随着镉处理浓度的增加,NHE根尖的质膜受损与脂质过氧化程度也随之加深,根部也可检测到明显的活性氧自由基积累,但在HE的根部则无上述现象。镉处理诱导下,HE根尖中谷胱甘肽(GSH)和非蛋白巯基化合物(NPT)含量显著增加,氧化型谷胱甘肽(GSSH)含量却明显下降,但镉对NHE无类似影响。由此可见,与NHE相比,HE能通过一系列的耐性机制抵制介质中的高镉胁迫,从而维持其根系的正常生理功能。2.超积累植物东南景天中,锌(Zn)在茎中含量最高,叶次之,根最低。利用同步辐射X射线荧光(SR-XRF)分析技术结合荧光探针染色技术,对其茎叶中锌的分布特征进行原位分析,发现锌主要集中在茎叶的表皮层,维管组织次之。随着锌处理时间延长,茎叶表皮中锌含量迅速升高,且增幅较大,而叶肉细胞和维管束中的锌含量则趋向于饱和。由此可见,表皮层可能是超积累东南景天重要的锌贮存库之一,进一步研究表明茎表皮的蜡质层是锌大量积累的场所。然而,叶片、皮层等薄壁细胞,尤其是液泡,对锌的区隔作用亦不容小视,其一,薄壁细胞占植物体的总容量较大；其二,超积累生态型茎叶中这些组织的锌含量要远远高于非超积累生态型相应部位。3.镉(Cd)在超积累植物东南景天的分布特点与锌有所差异,其在叶中最高,茎次之,根最低。利用激光刻蚀-电感耦合等离子体质谱(LA-ICP-MS)分析技术,对茎叶中镉的分布特征进行原位分析。发现镉在叶片的主脉中最高,叶片边缘较低。通过微区分析发现,镉在叶脉和栅栏组织中较高,而下表皮中分布较少,运用荧光探针显微技术,发现其大量贮存于液泡中。在植物茎中,镉主要集中分布于髓和皮层等薄壁组织细胞中,在维管组织与表皮分布很少。可见,镉的主要区隔场所为以薄壁细胞组成的皮层、髓和叶肉组织中,且大部分储存于液泡内。此外,比较发现镉与锌的分布有较大区别,而与钙的分布特征极为类同,推测钙镉之间可能存在着相似的运输与存储机制。4.铅(Pb)的分配特点与锌镉大为不同,其大量滞留在根中,茎叶中含量较低,但在富集型东南景天中含量仍显著高于非富集生态型。利用同步辐射X射线荧光光谱(XRF)对其茎叶中的分布特征进行原位分析,发现在茎叶横切面中,铅主要积累在叶脉等维管组织中,其次则是表皮,而叶肉组织和皮层组织中铅含量很低。在植物茎部,90%以上的铅集中累积于维管组织(尤其是木质部)内。对维管束部位的精细扫描发现铅呈块状分散分布。木质部的主要组成成分为细胞壁,由此可推,细胞壁可能是铅在富集植物东南景天的重要区隔场所。5.利用同步辐射X射线吸收光谱(XAS)技术和高效液相色谱(HPLC)分别测定Zn胁迫下两种生态型东南景天不同组织和木质部汁液中有机酸组成与锌的形态特征。研究结果显示：两种生态型东南景天不同组织中Zn配位都是以Zn-O/N为主,但不同生态型之间以及同种生态型不同部位之间配位数和键长都有所差异。超积累生态型木质部汁液中锌的含量是非超积累生态型的6-9倍,而且在超积累生态型的木质部汁液中检测到大量的柠檬酸,且随锌处理增加而显著增加,但非超积累生态型木质部汁液中柠檬酸含量变化不明显。在超积累生态型东南景天的木质部汁液中存在着Zn-O/N配位,未发现Zn-S配位。对样品谱进行主成分分析发现,锌处理后,超积累生态型木质部汁液中Zn主要以Zn2+和锌-柠檬酸形态存在。上述结果表明,两种生态型东南景天体内锌可能与含氧的羧基或者羟基类配体相结合,而柠檬酸参与了东南景天锌的木质部运输过程,超积累生态型东南景天中锌-柠檬酸的运输可能是重要积累机理之一。6.采用高效液相色谱(HPLC)和同步辐射X射线吸收光谱(XAS)技术分别测定镉胁迫下超积累生态型东南景天中有机酸组成和镉的形态变化特征。研究结果显示：超积累生态型东南景天地上部检测到大量的苹果酸,其次为琥珀酸和柠檬酸,草酸的含量相对较低,而且地上部苹果酸含量随Cd处理浓度的增加而升高。在超积累生态型东南景天根的第一壳层中存在着Cd-S和Cd-O/N两种配位形式,第二壳层以Cd-C配位。而在叶片和茎则未发现Cd-S配位,第一配位层均以CdO/N和Cd/O为主,第二配位层则以Cd-C配位。上述结果结合镉的微区分布结果表明,在叶片中Cd可能主要存在于液泡,而液泡含有大量的苹果酸,通过与镉结合以达到解毒的目的,而并非是含硫的巯基化合物在起作用；但在根部,则有部分含巯基化合物参与镉的解毒过程。7.利用同步辐射X射线吸收光谱(XAS)对富集型东南景天体内铅的赋存形态进行研究。结果表明,铅在两种生态型东南景天中主要的化学形态与先前的研究结果一致,是一种铅-细胞壁复合物。但在富集型东南景天的茎和叶中,有相当比例的铅与巯基基团(GSH)相结合的。利用Micro-XANES法对组织切片进行研究的结果进一步证实,铅在表皮和髓中以铅-细胞壁及铅-胶质复合物的形式存在,而在维管束中有较高比例的Pb-GSH复合物存在,这表明铅可能是以Pb-GSH复合物的形式在植物体内运输的。然而在EDTA-Pb处理下,植物体内铅的形态有所改变,尽管细胞壁结合态的铅还是占主导地位,但Pb-EDTA结合态铅的比重明显增高,说明植物体内铅的形态并非一成不变,而可能随着外界环境中的变化而有所改变。更多还原

【Abstract】 Heavy metal pollution is a widespread and important environmental concern. Various in situ and ex situ cleanup technologies have been employed, of these methods, phytoremediation is promising due to its low implementation costs and significant environmental benefits. Hyperaccumulator and accumulator species, which efficiently tolerate and accumulate heavy metals from the soil into shoots, have great promise in phytoremediation of contaminated environments. Understanding the mechanisms of metal tolerance and accumulation will provide insight into the identification and management of these hyperaccumulating species. Sedum alfredii Hance (Crassulaceae) is the recently identified Zn/Cd co-hyperaccumulator native to Pb/Zn rich regions of China. This species exhibits a strong ability to both tolerate and accumulate considerable amounts of Pb. A better understanding of the biological mechanism involved in heavy metal accumulation in this species would help to improve its practical application. The aim of this study was to obtain fundamental information on heavy metals accumulation and tolerance mechanisms in S. alfredii, by investigation of the metal localization and ligand abundance using several techniques. The main results include:1. Short-term responses of roots of the Cd hyperaccumulating ecotype (HE) S. alfredii to Cd exposure were compared with its non-hyperaccumulating ecotype (NHE). Marked root elongation inhibition was observed in roots of NHE in response to Cd exposure, while such inhibition was much slighter in HE, and stimulation of root elongation was observed with 10μM Cd treatment. The loss of plasma membrane integrity and lipid peroxidation in roots tips of NHE increased significantly with Cd treatments, whereas these effects of Cd was only pronounced at 400μM in HE root tips. A strong dose-dependent accumulation of reactive oxygen species (ROS) with increasing Cd was noted in the NHE root tips, but not in roots of the HE plants. In response to Cd exposure, GSH increased in the HE root tips but tended to decrease in those of NHE. A dose-dependent decrease in GSSG and an increase in NPT caused by Cd were marked in root tips of HE, but were not seen in the NHE plants. These results suggested that in contrast to the NHE, the HE S. alfredii tolerates high Cd in the environment and maintains root function through the differential expression in a number of hypertolerance mechanisms including altered glutathione metabolism, reduced ROS accumulation and hence prevent lipid peroxidation, loss of plasma integrity, and inhibition of root elongation.2. The characteristics of in vivo Zn distribution in stems and leaves of the HE and NHE S. alfredii were investigated by synchrotron radiation X-ray fluorescence analysis, together with a Zn probe. Preferential Zn accumulation in leaf and stem epidermis was observed in both ecotypes, but to a much greater extent for HE. Epidermal Zn increased largely in leaf and stem of HE as exposure time prolonged, while Zn saturation occurred relatively early in its leaf mesophyll cells and stem vascular bundles. A second peak of Zn enrichment in stem and leaf vascular systems was shown in both ecotypes. However, the proportion of Zn accumulated in stem vascular bundles relative to other tissues was much greater for HE than for NHE. Leaf and stem distribution patterns of P and S in the HE were very like that for Zn, while the Ca distribution pattern was the reverse of that for Zn. No such relationship was observed in NHE. Our study mainly, suggested that epidermal layers serve as large storage sites for the excess Zn in the hyperaccumulator HE S. alfredii:3. The spatial information of Cd distribution within the leaf of HE S. alfredii was obtained by LA-ICP-MS together with XRF. The results showed that Cd was preferentially distributed in the veins of the whole leaf, whereas the content of the element in the leaf edge is relatively low. The localization of Zn appears to be the reverse of that for Cd. By analysis of the leaf cross-section using XRF, it is found that Cd was largely localized in the vascular bundles and upper epidermis, whereas its content in the lower epidermis is lower. Zinc was extremely concentrated in the epidermis, indicating a very different sequestration of the two elements within the leaf of HE S. alfredii.4. To investigate the spatial distribution of Pb in the accumulator plant, micro scanning XRF mapping was performed on both Accumulating ecotype (AE) and Non-accumulating ecotype (NAE) S. alfredii. The leaf cross-section image of AE revealed that Pb was accumulated mainly in the leaf veins, with a second peak in the epidermis. Much lower content of Pb was found in either spongy or palisade mesophyll cells. An almost exclusive localization and accumulation of Pb within vascular bundles was noted in the AE stem. Distribution patterns of S in both leaf and stsem cross-sections were quite similar to that of Pb, and a significant positive correlation between the XRF intensities of Pb and S was observed, whereas no such correlation were observed NAE plants. Besides, addition of EDTA in the culture solution did not change the preferential distribution of Pb in the vascular bundles, although the intensity of Pb was much lower, as compared with those with Pb alone.5. Zinc speciation in the different tissue and xylem sap of HE and NHE S. alfredii were determined by EXAFS and HPLC. The results showed that the majority of Zn in all tissues was coordinated with oxygen or nitrogen ligands both two S. alfredii ecotypes. After 24 h exposure to 100μM Zn, Zn speciation in the xylem sap of HE occurred in three forms, including Zn2+, Zn-Ctrate and Zn-Malate, of 55.87%,42.31% and 6.07%,respectively. Analysis of organic acids in the xylem sap of the plants with Zn treatments supported the possible role of citrate in Zn speciation. Abundant Citrate occurred constitutively, and increased by Zn exposure in the xylem sap of both ecotypes of S. alfredii. These results suggested that Zn-Citrate complex might play an important role in Zn hyperaccumulation of HE S. alfredii.6. Cadmium speciation and organic acid concentration in the different tissue of HE S. alfredii were determined by using EXAFS and HPLC, respectively. The results showed that the majority of organic acids in the leaf was malate, and malate concentration in the shoot was affected by increasing Cd concentration in the solution, suggesting that malate synthesis is induced by Cd. In root, Cd was coordinated by O/N and S ligands in the first shell, which indicates strong ligands are required for metal detoxification. In young leaf and mature leaf, Cd was coordinated by O/N and O in the fisrt shell and by carbon ligands in the seconds shell.7. In vivo speciation of Pb in tissues of the accumulator plant, was investigated using powder-and micro-X-ray absorption near edge structure (XANES) spectroscopy. The dominant chemical form of Pb in tissues of both accumulating (AE) and non-accumulating ecotype (NAE) S. alfredii was similar to previously defined Pb-cell wall compounds, though a much higher percentage of the Pb in the stem and leaf of AE appeared to be associated with SH-groups (GSH). Micro-XANES analysis of tissue cross sections further verified that Pb in the epidermis and pith were indicative of Pb-cell wall and Pb-pectin complexes, whereas Pb within vascular bundles was predominantly Pb-GSH complex, suggesting that this species may transport Pb via a Pb-GSH complex. By application of EDTA, the complexation of Pb-EDTA was detected in the plant samples, indicating the speciation of Pb within the plants may also change due to the environment circumstance.更多还原