Molecular Mechanisms of Heavy Metal Detoxification

描述(申请人提供)：土壤和水体含有高水平的有毒重金属，如镉、砷、铅和汞，对人类和环境健康有害。这4种金属(固体)S是超级基金的7大重点危险物质之一。最近的研究和应用表明，通过植物根系吸收重金属和在植物地上部分积累重金属可以为重金属污染土壤和水体的去除和修复提供一种经济有效的途径。然而，许多在植物体内重金属过度积累中起作用的基因、机制和途径仍有待于识别和表征。植物络合素是植物体内主要的重金属和类金属螯合解毒硫代多肽。在最近的研究中，我们在了解植物中重金属解毒和运输的机制方面取得了进展，包括植物络合素合成酶基因的分离，镉从根到地转移机制的表征，重金属积累拟南芥突变体的分离，基于微阵列的快速突变克隆方法的发展，以及基于微阵列的可能与重金属运输有关的转运蛋白基因的鉴定。研究人员将验证以下假设：植物螯合素影响有毒金属从根到叶的长距离维管运输；新有毒金属积累的特征 突变体将导致鉴定在植物重金属积累中起作用的限速步骤；植物中的重金属感应和信号转导机制对于植物对重金属的抗性和积累是重要的。为了验证这些假说，拟议的项目将在特定的目标1和2中，利用生理、基因组、生化和膜运输分析来表征重金属和植物螯合素从根到地被运输的新的生理和分子机制。通过在合作研究中寻求一种新的高通量筛选方法，研究人员已经确定了影响叶片中有毒金属积累的拟南芥突变体。在具体目标3中，将使用一种新开发的基于基因组微阵列的快速突变定位和克隆方法来分离选定的重金属积累突变基因，并对其潜在机制进行表征。具体目标4将是描述重金属生物传感和转导的特征 利用荧光素酶报告筛查植物中的机制。将与Edenspace公司合作(具体目标5)，对来自超级基金地点的受污染土壤进行试验，以评估利用本研究产生的转基因和突变植物监测生物可利用重金属以及利用转基因植物和突变植物将重金属和类金属过度积累到植物根部和地上部的可行性。

DESCRIPTION (provided by applicant): Soils and waters with high levels of toxic heavy metals such as cadmium, arsenic, lead and mercury are detrimental to human and environmental health. These 4 metal(loid)s are among the Superfund's top 7 priority hazardous substances. Recent research and applications indicate that uptake of heavy metals into plants via the root system and accumulation of heavy metals in plant shoots could provide a cost effective approach for toxic metal removal and remediation of heavy metal-laden soils and waters. However many genes, mechanisms and pathways that function in heavy metal over-accumulation in plants remain to be identified and characterized. Phytochelatins are major heavy metal and metalloid chelating and detoxifying thiolate peptides in plants. In recent research we have made advances at understanding mechanisms that contribute to heavy metal detoxification and transport in plants, including isolation of phytochelatin synthase genes, characterization of mechanisms for root to shoot transfer of cadmium, isolation of heavy metal accumulation Arabidopsis mutants, development of a novel microarray-based rapid mutant cloning approach and microarray-based identification of putative transporter genes that may contribute to heavy metal transport. The investigators will test the hypotheses that, phytochelatins affect long distance root to leaf vascular transport of toxic metals; characterization of new toxic metal accumulation mutants will lead to identification of rate-limiting steps that function in plant heavy metal accumulation; and heavy metal sensing and signal transduction mechanisms in plants are important for plant heavy metal resistance and accumulation. To test these hypotheses, the proposed project will, in Specific Aims 1 and 2, characterize novel physiological and molecular mechanisms of root to shoot transport of heavy metals and phytochelatins using physiological, genomic, biochemical and membrane transport analyses. By pursuing a new high-throughput screening approach in collaborative research, the investigators have identified Arabidopsis mutants that affect the accumulation of toxic metals in leaves. In Specific Aim 3, a newly developed genomic microarray-based rapid mutant mapping and cloning approach will be used to isolate selected heavy metal accumulation mutant genes and characterize the underlying mechanisms. Specific Aim 4 will be to characterize heavy metal biosensing and transduction mechanisms in plants using a luciferase reporter screen. Trials with contaminated soils from Superfund sites will be pursued in collaboration with Edenspace Corp (in Specific Aim 5), to assess the feasibility of monitoring bioavailable heavy metals and of hyperaccumulating heavy metals and metalloids into plant roots and shoots using transgenic and mutant plants generated in this research.