The Molecular Basis for Heavy Metal Accumulation and Tolerance in the Hyperaccumulating Plant Species, Thlaspi Caerulescens

Leon V.Kochian有毒重金属污染土壤是一个严重的世界性问题，对人类健康和农业都是如此。据估计，仅在美国，目前使用的基于工程的技术清理危险废物就至少花费了4000亿美元。最近，人们对利用陆地植物作为一种替代的“绿色技术”修复受有毒重金属污染的表层土壤产生了相当大的兴趣。植物修复背后的原理是在受污染的地方种植植物，就像农作物在农业土壤上生长的方式一样。如果植物对土壤中的重金属有亲和力，它们可以从土壤中提取重金属，并将其积累到地上部分的生物量中。然后，这些含有重金属的嫩枝组织被收获并灰化，以减少它们的体积，然后储存在废物库中。这种植物生长和收获的过程被重复，直到土壤中的污染物水平降低到可接受的水平(通常是几年)。最近对金属污染土壤的植物修复感兴趣的一个主要因素是科学界越来越多地意识到，许多植物物种不仅可以容忍土壤中高水平的有毒重金属，而且实际上可以在容易收获的地上部分生物量中积累这些金属到非常高的水平。据报道，200多种陆生植物生长在高金属土壤上，能够耐受和积累高水平的重金属，如锌、镉、铜和镍。这些植物被称为超积累植物；这些有趣的金属超积累植物物种的存在本身表明，存在着植物修复成功的遗传潜力。然而，这些超积累植物中的大多数都很小，生长缓慢，而且由于它们生产的地上部生物量有限，它们大规模净化受污染土壤的潜力有限。将表达超积累表型的基因转移到地上部生物量较高的植物中，已被认为是提高植物修复作为一种可行的商业技术的潜力的一种途径。然而，由于缺乏对重金属超积累的基本分子、生化和生理机制的了解，这一目标的进展一直受到阻碍。最著名的金属超积累植物之一是结球甘蓝，它是卷心菜家族的成员，可以在地上部积累极高水平的重金属镉(Cd)和锌(Zn)。此外，据报道，某些生态型的菜青虫积累了高水平的其他重金属，包括镍和钴。菜青虫独特的重金属运输和耐受性使其成为一个非常有趣的基础研究实验系统，旨在阐明植物的重金属超积累机制和相关基因控制重金属超积累的相关基因。本研究的目的是识别植物中重金属超积累的基本机制，并分离和表征构成该超积累特性的一系列基因。Kochian博士的团队将利用植物分子生物学和基因组学的最新进展，确定与金属积累和耐受性有关的金属转运蛋白基因，以及参与产生低分子有机化合物(例如，肽、有机基因、氨基酸、金属硫蛋白、植物螯合素)的基因，这些基因可以结合和解毒植物细胞中的锌和镉。根据最近对拟南芥基因组的测序和分析，目前已知高等植物利用最近在酵母、细菌和哺乳动物中发现和表征的金属转运蛋白家族来实现金属积累和动态平衡。Kochian博士的团队已经从这些不同的金属转运蛋白基因家族中克隆出了球囊毛滴虫的基因，并将对这些转运蛋白进行表征，以确定它们在金属超积累中的作用。这一特征将包括确定在哪些植物组织和细胞类型中表达不同的基因，运输蛋白的膜定位以帮助分配每个转运蛋白的潜在作用，以及阐明单个金属转运蛋白的生理功能。他们还在酵母中表达菜青霉基因，通过产生金属结合的有机配体来寻找与金属耐受性有关的基因。这些方法应该可以让研究人员识别出一套允许菜青霉中重金属过度积累的基因，并阐明这一特征的分子机制(S)。这项研究的最终目标是利用这些超积累基因培育出既是金属超积累植物又能产生高生物量的转基因植物，从而非常适合于金属污染土壤的植物修复。该项目是由美国环保局、美国国家科学基金会、海军研究办公室和战略环境研究与发展计划共同发起的植物修复联合计划资助的。

0129844Leon V. KochianContamination of soils with toxic heavy metals is a serious worldwide problem both for human health and agriculture. Cleanup of hazardous wastes by the currently used engineering-based technologies has been estimated to cost at least $400 billion in the U.S. alone. Recently, there has been considerable interest in the use of terrestrial plants as an alternative, "green technology" for the remediation of surface soils contaminated with toxic heavy metals. The principal behind phytoremediation is to grow plants on a contaminated site in much the same way crop plants are grown on agricultural soils. If the plants have an affinity for the heavy metals in the soil, they can extract the metals from the soils and accumulate them in the above-ground shoot biomass. These heavy metal-containing shoot tissues are then harvested and ashed to reduce their volume prior to storage in a waste repository. This plant growth and harvesting process is repeated until the level of contaminant in the soil is reduced to acceptable levels (usually a number of years).A major factor behind the recent interest in phytoremediation of metal polluted soils has been the growing awareness by the scientific community of the existence of a number of plant species that not only can tolerate high levels of toxic heavy metals in the soil, but actually can accumulate these metals to very high levels in the easily harvested above-ground shoot biomass. Over 200 terrestrial species have been reported that grow on high metal soils and can tolerate and accumulate high levels of heavy metals such as Zn, Cd, Cu, and Ni in their shoots. These plants have been coined hyperaccumulators; the very existence of these interesting metal hyperaccumulator species suggests that the genetic potential exists for phytoremediation to be successful. Most of these hyperaccumulator species, however, are small and slow growing, and because they produce limited shoot biomass their potential for large-scale decontamination of polluted soils is limited. Transferring the genes expressing the hyperaccumulating phenotype to higher shoot biomass-producing plants has been suggested as an avenue for enhancing the potential of phytoremediation as a viable commercial technology. Progress towards this goal, however, has been hindered by a lack of understanding of the basic molecular, biochemical and physiological mechanisms involved in heavy metal hyperaccumulation.One of the best known metal hyperaccumulators is Thlaspi caerulescens, a member of the cabbage family that can accumulate the heavy metals cadmium (Cd) and zinc (Zn) to extremely high levels in the shoot. Additionally, certain ecotypes of T. caerulescens have been reported to accumulate high levels of other heavy metals, including Ni and Co. The unique physiology of heavy metal transport and tolerance in Thlaspi caerulescens makes it a very interesting experimental system for basic research aimed at elucidating plant mechanisms and the associated genes controlling heavy metal hyperaccumulation.The goals of this research are to identify the basic mechanisms of heavy metal hyperaccumulation in plants, and to isolate and characterize the suite of genes that underly this hyperaccumulation trait in Thlaspi caerulescens. Dr. Kochian's group will use recent advances in plant molecular biology and genomics to identify both metal transporter genes involved in metal accumulation and tolerance, as well as genes involved in the production of low molecular weight organic compounds (e.g., peptides, organic genes, amino acids, metallothioneins, phytochelatins) that can bind and detoxify Zn and Cd in plant cells. Based on the recent sequencing and analysis of the Arabidopsis genome, it is now known that higher plants employ the same families of metal transporters recently identified and characterized in yeast, bacteria and mammals for metal accumulation and homeostasis. Dr. Kochian's group has cloned genes in T. caerulescens from these different metal transporter gene families and will characterize these transporters to determine their role in metal hyperaccumulation. This characterization will include determining in which plant tissue and cell type different genes are expressed, the membrane localization of transport proteins to help assign a potential role for each transporter, and the elucidation of the physiological function of individual metal transporters. They also are expressing T. caerulescens genes in yeast to look for genes conferring metal tolerance through the production of metal binding organic ligands.These approaches should allow the investigators to identify the suite of genes that confer heavy metal hyperaccumulation in T. caerulescens and to elucidate the molecular mechanism(s) for this trait. The ultimate goal of this research is to use these hyperaccumulation genes to develop transgenic plants that both are metal hyperaccumulators and produce high shoot biomass , and thus will be well suited for the phytoremediation of metal contaminated soils.This project was funded through the Joint Program on Phytoremediation, co-sponsored by the Environmental Protection Agency, the National Science Foundation, the Office of Naval Research, and the Strategic Environmental Research and Development Program.