Molecular, genetic, and proteomic analysis of Arabidopsis glutathione S -transferases

Aaron P Smith, Purdue University

Abstract

Glutathione S-transferases (GSTs) are a ubiquitous collection of proteins that have diverse functions. GSTs in plants have been widely characterized as enzymes involved in xenobiotic detoxification, but little is known about the endogenous substrates of these enzymes. Deletion analyses identified two regions of the Arabidopsis AtGSTF2 promoter important for regulation of AtGSTF2 expression in response to several distinct stimuli. Previous studies have suggested that AtGSTF2 interacts with indole-3-acetic acid (IAA). Recombinant AtGSTF2 was shown to directly bind IAA and 1-N naphthylphthalamic acid, and interact with quercetin and kaempferol—flavonoids involved in the regulation of IAA transport in vivo. In addition, expression of AtGSTF2::GUS and localization of At GSTF2 protein were disrupted in flavonoid-deficient tt4 seedlings. These results indicate that AtGSTF2 is involved not only in stress responses but also in development under normal growth conditions. A genetic screen was used to identify mutants with altered expression of AtGSTF2::GUS to gain insight into the signaling mechanisms involved in AtGSTF2 expression. Three distinct mutant classes were identified: those with high induction of GSTF2 (hig) or low induction of GSTF2 (lig) in response to copper treatment, and a class that had relatively high constitutive expression of GSTF2 (ceg). Inheritance and complementation tests were used to characterize these mutants, and the genes responsible for two mutations, HIG3 and HIG5, were mapped using molecular markers as a prelude to identification and cloning of these genes based on their chromosomal position. Studies examining the regulation of GSTs in plants have depended heavily upon RNA expression analysis. In order to more critically evaluate the impact of RNA expression on the regulation of GSTs, the expression of GST proteins in Arabidopsis was evaluated using a proteomics approach. A total of eight Arabidopsis GST proteins were detected in Arabidopsis seedlings, and the expression of these GSTs were differentially induced by an herbicide safener (benoxacor) and copper. These results provide important information previously lacking in studies examining plant GST expression, and provide a framework for future proteomic analysis of this gene family in plants.

Degree

Ph.D.

Advisors

Goldsbrough, Purdue University.

Subject Area

Molecular biology|Botany|Biochemistry

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