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ItemThe roles of a novel group of plastid transporters in glutathione metabolism in arabidopsis(University of Melbourne, 2010)The tripeptide glutathione (GSH) is an important compound with a broad spectrum of functions in plants, including stress responses and is a key element of the intracellular redox buffer that protects cells from oxidation. In Arabidopsis GSH is synthesized in two enzymatic steps by GSH1, found exclusively in the plastids, forming the pathway intermediate ?-glutamylcysteine (?-EC) and then by GSH2 which is located in both plastids and cytosol. This suggests a mechanism for ?-EC export from the plastids and, because the majority of GSH2 transcripts (90%) encode the cytosolic isoform, it is believed that the cytosol may be the main compartment for GSH biosynthesis. In addition, GSH may be efficiently transported between the two compartments to maintain proper homeostasis. However, comparatively little is understood about the mechanisms that control and interconnect these two subcellular GSH pools. The identification and characterization of a novel family of transporters, the CRT Like Transporters (CLTs), has provided further insights into glutathione metabolism in Arabidopsis. The CLT1 gene was originally identified in a screen for mutants resistant to L-Buthionine-SR-sulfoximine (BSO), a synthetic compound used to specifically inhibit GSH1 and GSH biosynthesis. Two paralogues in Arabidopsis, CLT2 and CLT3, were identified and all three CLTs are localized to the plastids. Previous studies have shown the clt1clt2clt3 triple mutant is GSH-deficient in roots and cadmium-sensitive suggesting a role for the CLTs in transporting ?-EC and/or GSH between the plastid and cytosol compartments. In this study redirecting GSH1 activity or GSH biosynthesis exclusively to the cytosol by complementing the Arabidopsis gsh1 and gsh2 mutants with E. coli GSHA and GSHB, respectively, has no significant impact on phenotypes or stress resistance suggesting efficient exchange of ?-EC and GSH between the plastid and cytosol compartments. The use of redox-sensitive roGFP2 reporters in combination with confocal imaging with monochlorobimane and measurements of cytosolic thiols has demonstrated that the clt1clt2clt3 mutant is GSH-deficient in the cytosol but not the plastid in both roots and shoots consistent with a predicted role for CLTs in ?-EC and/or GSH transport. The Arabidopsis gsh2 mutant accumulates high levels of ?-EC in the cytosol. However, the combination of the clt1clt2clt3 mutations with gsh2 did not decrease ?-EC levels suggesting CLTs may not contribute to ?-EC transport in vivo. To further study the roles of the CLTs separately on ?-EC and GSH transport in planta different clt mutations were introduced into transgenic lines expressing plastid- or cytosol-targeted GSH2. These observations suggest that de novo GSH biosynthesis in wildtype Arabidopsis occurs largely in the plastids and that CLT2 and CLT1/CLT3 are mainly involved in ?-EC and GSH export, respectively, in planta.