【作者】 徐劼；

【导师】 陈英旭；

【作者基本信息】 浙江大学 ， 环境工程， 2011， 博士

【摘要】 人类的大量开采和使用导致Pb成为环境中广泛存在的重金属污染物,对自然环境和人体健康构成严重威胁。茶作为消耗量仅次于水的无酒精型饮料,由于具有较高的营养价值及保健功效颇受世界各国人民的喜爱。Pb因其独特的特性已经作为茶叶中主要的重金属污染物而成为研究热点。因此开展茶树Pb的吸收、累积及耐性机制研究,对控制成品茶Pb污染加剧的趋势、促进我国茶产业良性发展和保障消费者身体健康都具有十分重要的意义。本研究通过茶园实际调查和试验室模拟水培试验深入探讨茶树对Pb的吸收和耐性机制,揭示茶树Pb毒害效应的品种差异,根系对Pb的吸收、累积、分布机理,Pb在茶树体内的化学形态及亚细胞分布的品种差异,根细胞壁在Pb累积中的作用,Pb及其他元素在茶树不同器官组织的微区分布特征,主要研究结果如下：(1)明确了不同品种茶树铅吸收累积的品种差异及影响因素。同一生长环境中不同品种茶树对Pb的累积、分布表现出差异,其分布存在三种规律。茶树根际土中的Pb主要以残渣态和铁/锰氧化物结合态为主,碳酸盐结合态次之,根际土中可交换态Pb与碳酸盐结合态Pb是茶树可利用Pb的主要形态。不同品种茶树根际土Pb形态、pH值、有机质含量存在显著差异,是影响茶树Pb吸收、累积的重要因子。不同品种茶树成熟叶片对Pb的吸附能力存在一定差异。(2)两个品种茶树在Pb的耐性方面存在强弱之分。在同一铅胁迫条件下,龙井43(LJ)和迎霜(YS)根系形貌、植株生长,叶片净光合速率大小,根系活力、根系质膜透性均表现出一定的差异。Pb胁迫导致根细胞细胞核溶解消失,线粒体脊突部分膨胀,细胞膜内陷,细胞壁增厚；导致茎细胞内叶绿体变形,基粒片层消失,类囊体紊乱,出现大的脂质颗粒；导致叶细胞叶绿体结构发生明显劣变,出现叶绿体双层膜破裂,基粒片层结构排列紊乱、基质类囊体肿胀等现象。但Pb胁迫对两个品种茶树叶细胞结构的影响存在差异,Pb不溶沉积物在根组织细胞中的形态也存在差异。YS品种植株对Pb毒害的耐受能力要强于LJ品种。(3)两个品种茶树根细胞壁对Pb的吸附具有不同特征。傅里叶红外光谱研究表明Pb主要与细胞壁纤维素、半纤维素和木质素的羧基官能团发生结合作用,同时根细胞壁上的蛋白质和果胶质也可能参与了Pb的结合。LJ根细胞壁中羧基在Pb吸附中的贡献作用较大,YS根细胞壁中氨基在Pb吸附中的贡献作用较大,果胶质在两个品种茶树根细胞壁Pb吸附中的作用相当。两个品种茶树根细胞壁上羧基(-COOH)、氨基基团(-NH2)和蛋白质百分含量存在差异。(4)两个品种茶树对Pb的吸收及定位分布存在一定差异。LJ和YS不同器官组织中Pb含量的分布表现出相同规律：根>茎>新叶>老叶,但两个品种新叶Pb含量的变化存在差异,新根和主根对Pb的吸收能力也存在差异。根系对Pb不存在主动吸收过程,但根系对Pb的吸收与Ca离子通道密切相关,Ca离子的介入对LJ和YS根系Pb的吸收产生了抑制作用。两个品种茶树根组织中的表皮层和内皮层区域Pb浓度较高,而皮层与维管束区域Pb含量较低,但在内皮层和表皮层区域Pb含量的高低分布上两个品种存在差异。两个茶树品种根组织Pb的亚细胞百分含量分布表现出相同规律：细胞壁组分>细胞器组分>可溶物质组分>细胞膜组分,但不同亚细胞组分Pb的百分含量在品种间存在差异。根组织中Pb的化学结合形态主要以FNaCl提取态、FHAc提取态为主,但各形态Pb的百分含量水平同样在两个品种间存在差异。透射电镜加能谱分析结果表明在LJ和YS根细胞壁、细胞间隙上的大量黑色物质除含Pb外还含有较多的P、O,推测这些沉积物可能是磷酸盐沉淀,如Pb5(PO4)3OH, Pb3(PO4)2, Pb(H2PO4)2等。(5)Pb胁迫影响了根组织对其它元素的吸收。与对照处理相比Pb胁迫显著促进了LJ和YS根系组织对Cu和Fe元素的吸收,但却显著抑制了Mn和Mg元素的吸收,对Ca、Zn元素的吸收两个品种则表现出差异。Fe在YS根表皮层的含量相对明显高于根部的其他部位,而Ca在内皮层和维管柱的交界处含量最高。LJ根横切面上Ca、Fe的分布则无明显规律。正常水培条件下,LJ和YS新叶中Fe、K、Mn元素含量最高的区域为靠近近轴面表皮的叶肉组织(栅栏组织),而Pb胁迫下,这种分布现象遭到弱化。更多还原

【Abstract】 Lead has been regarded as one of the widespread heavy metal pollutants in the environment because of its large-scale exploitation and use, which poses a serious threat to both the natural environment and human health. High in nutritional value and beneficial to human health, tea has won the great popularity all over the world. As a non-alcoholic beverage, tea consumption is only second to water. Pb, with its unique characteristics, has become a research hotspot as the main heavy metal pollutant in the tea. Therefore, the research about the mechanisms of lead uptake/accumulation and tolerance in tea plants is of great significance for controlling the tea Pb pollution, promoting the healthy development of the tea industry as well as guaranteeing the consumers’health.In this study, we made a further probe into the mechanisms of Pb uptake and tolerance in tea plants, with the aim to reveal the variety differences in the Pb toxic effect and Pb chemical forms and subcellular distribution in tea plant, the mechanism of Pb absorption, distribution and accumulation in different tea plant organs, and the role of root cell walls in Pb accumulation and the micro-distribution of both Pb and other elements in different tea plant organs. The main results are as follows:(1) It was made clear for characteristics of lead accumulation in differernt tea plant varieties and the factors influencing lead absorption. Different varieties of tea plants growing in the same environment showed some differences in Pb accumulation and distribution. There were three patterns of Pb distribution in different organs among the eight varieties. The rhizosphere speciation of Pb was similar for all tea plant varieties, with Pb being predominantly in the residual and oxide-bound forms, followed by carbonate-bound forms. Analyses indicate that exchangeable and carbonate-bound Pb fractions appeared to be readily bioavilable for tea plants. There were significant differences in lead chemical speciation, soil pH, organica matter content between different tea plant varieties, which were important factors influencing lead uptake and accumulaton by tea plant. Some certain differences were also found in Pb absorption capacitiy of mature leaves in different tea plant varieties.(2) Under the same treatment, there appeared to be differences between LJ and YS in root morphology, plant growth, net photosynthetic rate, root acivity and membrane permeability. We found the following Pb toxic effects on the ultrastructure of root cells including disappearance of nuclear, mitochondrial swelling or disappearance of cristae, membrane invagination and cell wall thickening; the Pb toxic effects on the ultrastructure of shoot cells including chloroplast deformation, disappearance of grana lamellae, thylakoid disorder and appearance of large lipid particles; the Pb toxic effects on the ultrastructure of leaf cells including chloroplast damage, mitochondrial swelling and so on. Our comparative study indicated that there were some differences in the leaf cell structure of the two varieties under Pb stress. Besides, the forms of Pb sediments in root cells were also different. The Pb tolerance ability of YS was relatively stronger than LJ.(3) Fourier transform infrared spectroscopy studies show that Pb was found to be mainly bound with the carboxyl group in the root cell walls. At the same time, the protein and pectin might also be involved in binding Pb. Carboxyl group played a greater role in binding Pb for LJ, while amino group played a greater role for YS. And pectin in root cell walls of the two varieties showed the same effect on binding Pb. Some differences existsed in the carboxyl group, amino group and protein percentage between the two tea plant varieties.(4) Roots played a significant role in fixing the Pb by tea plants. Pb content in different tea plant organs showed the same distribution order:roots> stems> young leaves> mature leaves. But the analysis found that there was a difference in Pb content variation in the leaves of two tea plant vareities and also Pb absorption capacity between fine roots and coarse roots. Compared with the control, Pb stress promoted the absorption of Cu and Fe, but inhibited the absorption of Mn and Mg by roots of two tea plant varieties. Besides, the two varieties showed different characteristics in the absorption of Ca and Zn. The results proved that the absorption of Pb in tea plants might not be an active process. LaCl3, an ion(Ca) channel inhibitor, significantly inhibited the absorption of Pb by roots, which indicated that the Pb absorption had a close relationship with ion(Ca) channels. Synchrotron radiation X-ray fluorescence scan results also showed that, to some extent, the Ca ions inhibited the absorption of Pb by roots.(5) Pb stress affected the uptake of other elements by tea plants. Compared with the control, Pb stress promoted the absorption of Cu and Fe, but inhibited the absorption of Mn and Mg by roots of two tea plant varieties. Besides, the two tea plant varieties showed different characteristics in the absorption of Ca and Zn. The Fe content in the YS root epidermis was relatively higher than other parts of the root, but Ca content was higher in the interface of YS root endodermis and vascular cylinder. No remarkable distribution rules were found about Ca and Fe in LJ root cross section under Pb stress. Under normal conditions of hydroponic culture, the highest content of elements such as Fe, K and Mn was found in the region close to the adaxial epidermis of LJ and YS leaf (palisade tissue). While under the Pb stress, the distribution rule, i.e., the element content appears higer in leaf edges (upper and lower epidermis) was weakened.更多还原