摘要
我国是茶叶生产和贸易大国,茶产业在国民经济和国际贸易中一直占有重要地位。近年来,我国茶产业发展虽然非常稳健,但茶叶的安全质量问题仍然是其发展的瓶颈之一,其中茶叶铅污染问题一直颇受关注。铅是众所周知的生理性和神经性毒物,几乎对人体所有重要的器官和系统均会产生不良影响。因此,开展茶叶铅污染研究对于我国茶产业可持续发展和消费者身体健康具有十分重要的意义。
茶多酚(Tea polyphenols, TP)作为茶树(Camellia sinensis L.)体内天然的金属有机配体,其调控重金属有效性的环境功能值得深入研究。因此,本研究首先对不同品种茶树的铅积累特性及其与茶多酚的关系进行调查评价,然后以实验室土培和水培模拟试验为主要手段,系统探讨了茶多酚对茶树铅生物有效性的调控作用及其机制。取得的主要研究结果如下：
(1)明确了不同品种茶树的铅积累特性及其影响因素,发现茶树组织和土壤中的茶多酚含量与茶树铅积累量具有密切关系。茶树不同品种、不同组织间的铅含量存在明显差异。土壤pH值、有机质含量、铅的化学形态和叶片铅吸附能力是影响不同品种茶树铅积累特性差异的重要因子。不同品种茶树老叶、老根和根区土壤之间的茶多酚含量存在明显差异。根区土壤多酚含量与土壤pH值、有机质含量、铅的化学形态和叶片铅吸附能力的相关关系显著。茶树组织茶多酚含量与叶片铅吸附能力、茶树体内铅含量存在显著的相关关系。
(2)茶多酚处理对茶树的铅毒害和吸收积累具有明显的调控作用。低浓度铅对茶树的生长发育、光合作用和生物量积累有一定的促进作用,高浓度则产生抑制作用。随着铅污染浓度的增加,茶树体内的铅含量升高,但不同器官的分布规律变化不大。茶多酚处理后,铅污染茶树的生长发育状况明显改善,生物量积累增加。低浓度下,茶多酚能降低铅在茶树体内各组织中的积累,高浓度下,茶多酚提高了根系对铅的吸收积累,而减少了叶片的铅含量。
(3)铅污染下,细胞整体结构发生了显著变化,结构不完整,大量细胞器消失,叶绿体、细胞核、线粒体等细胞器结构受到明显损伤。胞间连丝结构发生明显改变,大量内质网消失。细胞壁有大量云雾状黑色沉淀,沉淀颗粒细密均匀,在细胞壁角隅地带密度较大。茶多酚处理后,茶树叶片、根尖细胞的整体结构受损状况明显好转,叶绿体、线粒体等细胞器整体结构接近正常状态,特别值得关注的是,在细胞壁、胞间连丝、细胞核、内质网等处发现了大量块状或颗粒状黑色沉淀,与单一铅污染处理沉淀形态明显不同。
(4)茶多酚对土壤理化性质具有明显的调控作用,从而对土壤铅的生物有效性产生影响。茶多酚添加到土壤后,土壤pH值降低,多酚和有机质含量提高,土壤矿物物相有所改变,表现为晶型更为完整,特别是大量铁锰氧化物与茶多酚发生氧化还原反应,土壤铅的化学形态发生了明显转化,低浓度下有效态比例提高,而高浓度下有效态比例降低。土壤铅的化学结合形态由占较大比例的磷酸铅向茶多酚-铅结合形态转化。茶树干物质积累、体内铅积累与茶多酚导致的土壤理化性质和铅生物有效性变化关系密切。
(5)茶多酚对茶树体内铅的微区分布和形态转化具有明显的影响。铅处理下,茶树根尖细胞的铅主要沉积在细胞壁,组织水平上主要分布于叶片的海绵组织和下表皮,根尖的表皮、内皮层和中柱鞘等处。茶多酚处理后,铅则主要沉积在根尖细胞的液泡中,组织水平上铅在叶片上下表皮的分布明显高于叶肉组织,铅在叶脉纵向和根尖不同组织的分布也发生了明显变化。微区分布的重新分配避免了铅对重要细胞和组织的直接伤害,这可能是茶多酚降低铅毒害的重要机制之一
傅立叶变换红外光谱研究表明,茶树根、叶组织的特征官能团具有多样性,主要包括羟基、羧基、氨基、酰胺基、甲基、羰基和醚基等。在铅的吸收积累与解毒中,茶树组织中羟基、酰胺基、羰基等起到了重要作用,特别是添加茶多酚之后,明显提高了这些官能团的含量,从而提高了茶树对铅的解毒能力。
Pb处理下,茶树根中Pb的主要结合形态为Pb-茶多酚、氯化铅、Pb-茶氨酸、Pb-富啡酸和磷酸铅等。茶多酚处理后,茶多酚与Pb的结合形态比例大大提高,说明茶多酚在调控茶树体内Pb的分子形态方面具有重要作用。
China is a tea production and trading power and tea industry has played an important role in the national economy and international trade. In recent years, the issue of tea quality safety has become one of the bottlenecks of tea industry development in China, in which lead contamination of tea has been attracted attentions widely. Lead (Pb) is a physiological and neurological toxin affecting almost every important organ and system in the human body. Therefore, it is very important to study on lead pollution in tea for sustainable development of Chinese tea industry and health of consumers.
Tea polyphenols (TP) are the main compounds and natural metal organic ligands in tea plant. It is worthwhile to make a further research on tea polyphenols'environment function to regulate the availability of heavy metals. Therefore, in this work the relationship between TP and accumulation characteristics of lead in different varieties of tea plant was investigated and assessed firstly, and then the pot soil experiment and solution culture experiment were carried out to study regulation mechanisms of TP on the bioavailability of lead in tea plant. The main results obtained are as follows:
(1) It was made clear for characteristics of lead accumulation in different varieties of tea plant and its influencing factors and found that TP content in the soil and tea plant was closely related to lead accumulation amount in tea plant. There were significantly differences among the lead content in different varieties, the same as lead content in different organs. Soil pH, organic matter content, chemical species of lead and lead adsorption capacity of leaf were important factors affecting lead accumulation difference in different varieties of tea plant. There was significant difference between TP content in old leaves/old roots and that in the soil of root zone for different varieties of tea plant. Polyphenol content in the soil of root zone had significant correlation with soil pH, organic matter content, chemical species of lead, and lead adsorption capacity of leaf. The content of TP in tea plant was also significantly related to lead adsorption capacity of tea leaf and lead content in tea plants.
(2) TP had obviously regulative effect on the uptake, accumulation, and detoxification of lead in tea plant. Under low concentration of lead, growth and development, photosynthesis, and biomass production of tea plant were promoted in a certain level, but inhibited under high concentrations. With the increase of the concentration of lead, the lead content in tea plant increased, but there was little change of lead distribution in the different organs of tea plant. With TP treatment, the growth and development condition of tea plant was significantly improved and biomass production increased although under lead pollution. Under low concentration of TP, the accumulation amount of lead in tea plant was reduced, but uptake amount of lead by roots was increased and the lead content of leaves was reduced with high concentrations of TP.
(3) Under lead stress, some disturbances in ultrastructure of drganelles were found. The overall structures of cells were significantly changed and not complete, a large number of organelles even disappeared. The structures of chloroplast, nucleus, mitochondria and other organelles were markedly damaged. Plasmodesmata structure was changed significantly, a mass of endoplasmic reticulum disappeared. Abundant cloudy black depositions were found in cell wall. It was worth mentioning that the deposition particles were fine and well-distributed, mainly in the cell wall corners. After TP treatment, the overall structure of tea leaf, root tip cells damaged by lead stress had been taken a significant turn for the better. The overall-structure-condition of the chloroplast, mitochondrion was nearly same as normal state. And it was a remarkable phenomenon that block or granular black depositions were found in the cell wall, plasmodesmata, nucleus, endoplasmic reticulum, which were distinctly different from depositions under only lead stress treatment. (4) TP played an obviously regulative role on soil physical and chemical properties, and thus it had an effect on the lead bioavailability of soil. After TP was added to the soil, the soil pH values decreased, the contents of polyphenols and organic matter increased. The soil mineral phase was changed which showed a more complete crystal, and particularly redox reaction occurred between Fe-Mn oxides and polyphenols. The chemical species of lead in soil had been significantly transformed, the proportion of availability species of lead increased under low concentration of lead, but decreased under high concentrations. Chemical bound species of lead in soil had been transformed from the large proportion of lead phosphate to the combined species of TP-lead. Dry matter production and lead accumulation of tea plants was closely correlative to the changes of soil physical and chemical properties, and bioavailability of lead caused by TP treatment. (5) TP had a significant effect on the micro-area distribution and transformation of lead in tea plant. Under only lead treatment, lead was deposited mainly in the cell wall of root tip cells, while on the tissue level lead was mainly deposited in the spongy tissue and lower epidermis of leaves, and epidermis, endodermis and pericycle of root tips etc. After TP treatment, lead was deposited mainly in the vacuole of root tips, and on the tissue level the content of lead in the upper and lower epidermis of leaf was significantly higher than that in mesophyll tissue. The distribution of lead in the vertical vein of leaf and different tissues of the root tips was changed obviously. Direct damage of the important cells and tissues were avoided by the re-distribution of micro-area distribution of lead in tea plant, which may be one of the important mechanisms of TP to reduce lead toxicity for tea plant.
Fourier transform infrared spectroscopy studies showed that tea roots and leaves had diverse functional groups, including hydroxyl, carboxyl, amino, amide, methyl, carbonyl, and ether group, etc. Functional groups in tea plant tissues like hydroxyl, amide, carbonyl, etc. played an important role in the absorption, accumulation, and detoxification of lead in tea plants. Especially after adding TP the content of these functional groups significantly increased, which accordingly inhibited the toxicity of lead.
Under only lead treatment, combined species of the lead in root of tea plant were mostly Pb-TP, chloride, lead, Pb-theanine, Pb-fulvic acid and phosphoric acid lead, etc. After TP treatment, the ratio of binding species of Pb-TP greatly increased, which showed that TP had an important role in the regulation of the lead molecular species in tea plant.
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