Glutathione is one of the most abundant organic compounds found in many biological systems. It is a non-protein tripeptide of Glutamic acid, Cysteine and Glycine. Due to its stability, high cellular concentrations and structural features like gammaglutamyl linkage and sulfhydryl group, glutathione involves with many biological pathways including: cellular defense against xenobiotics and naturally occurring deleterious compounds, like free radicals, metal sequestering, and maintaining cellular sulfhydryl status. Glutathione has been broadly studied however the literature associated with Zn2+ coordination is not clear. This study is focused on collecting thermodynamic data of glutathione binding to Zn2+ metal ions using calorimetric technique. Isothermal titration calorimetry has been recommended as an excellent method to determine the association constant (K), enthalpy change (delta H), and binding stoichiometry (n) of a binding process. These parameters associated with Zn2+ binding glutathione deconvoluted from a series of complex equilibria provide an insight into what drives these reactions forward.

Plant mechanisms regulating environmental heavy metal interactions are vital for plant survival. Plants must maintain adequate metal levels while preventing excesses. Several mechanisms involved in heavy metal uptake and sequestration have been identified and studied in hyperaccumulating plants such as Thlaspi caerulescens. These plants accumulate large quantities of metals, but their use in remediation is limited by their small size. On the other hand, mechanisms in high-biomass, non-hyperaccumulating perennial species such as poplar (Populus spp.) are unknown. The central goal of this project is to delineate specific mechanisms in poplar regulating the heavy metal zinc (Zn) for potential use in bioremediation and real-time monitoring. Specifically, project aims are: 1) Determine the role of HMA4 and PCS1 genes in poplar; 2) Delineate the ZIP gene family including ZIP1 and ZNT1 activity; and 3) Harness fluorescent energy transfer to engineer a poplar tree that monitors Zn-soil contamination. These are addressed using current technologies including phylogenetic analysis, gene transformation, expression assays, promoter-GUS assays, fluorescent-gene imaging, and metal assays. Through these experiments, mechanisms controlling heavy metal interaction are identified and characterized in poplar. Poplar contains a large number of genes in both the ZIP and HMA4 families, but only two members in the PCS family. Poplar also contains several genes that share close sequence and structural homology to those in hyperaccumulators. However, there is an overall divergence from hyperaccumulators in regards to expression across an environmental Zn gradient. Poplar tightly regulates Zn intake by suppressing absorption avenues under Zn excess. Over-expression of HMA4 and PCS1 resulted in more tolerance and more accumulation, respectively, in poplar lines. These findings support a regulatory system used in poplar to limit Zn under excess and promote Zn under deficiency. Using ZNT1 and its natural expression gradient, a chimeric protein was created that served as a biosensor in both poplar and Arabidopsis thaliana host plants and was able to discriminate between 1μM and 10mM Zn concentrations. These findings add to current knowledge of heavy metal regulation and help fill the gap of knowledge currently existing on the regulatory mechanisms that perennial trees use to control heavy metals.

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