SUMMARY (See instructions): Soils and waters with high levels of toxic heavy metal(loid)s such as arsenic, cadmium and mercury are detrimental to human and environmental health. These three metal(loid)s are among the Superfund's top 7 priority hazardous substances. 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 important genes and pathways that function in heavy metal over-accumulation in plants remain to be identified. In recent research we have made major advances at understanding key mechanisms that function in heavy metal detoxification, transport and accumulation in plants. We will combine powerful genomic, genetic, biochemical and physiological approaches to test new central hypotheses by pursuing the following Specific Aims: L Characterize newly identified vacuolar membrane transporters that function in uptake and accumulation of phytochelatin-heavy metal(loid) complexes into plant vacuoles and analyze their bioremediation potential. \ l Understanding the control of heavy metal accumulation and distribution in roots and shoots is critical for engineering of plants for bioremediation. Determine the mechanisms by which a new peptide transporter mutant, opt3, causes hyper-accumulation in roots and under-accumulation of cadmium in plant leaves. iii Our recent research has shown that heavy metal-chelating and detoxifying thiols undergo long distance transport in plants. However, the plasma membrane transporters for uptake of glutathione (GSH) and phytochelatins (PCs) remain unknown. Using a high-throughput screen we have now identified OPT4 as a putative GSH & PC-Cd transporter. We will characterize 0PT4 and additional transporters to determine their underlying GSH/PC-Cd/As transport mechanisms and their functions in heavy metal distribution in plants. IV. The mechanisms and transcription factors that mediate rapid heavy metal-induced gene expression in plants remain largely unknown, but are important for heavy metal resistance and accumulation. The genes of newly isolated mutants impaired in cadmium-induced gene expression will be identified and their functions characterized. Furthermore, a potent genome-wide approach has been developed and will be pursued to identify and characterize key transcription factors and repressors that control cadmium- and arsenic-induced gene expression. We will work closely with the RTC and CEC in sharing our advances and foremost in translational analyses of our findings for their potential in phytoremediafion of contaminated soils and waters.