Molecular characterization of glutathione S-transferase genes from Arabidopsis thaliana
Abstract
Gluthathione S-transferases (GSTs) are a ubiquitous group of enzymes that are involved in cellular detoxification. A cDNA encoding a protein (AtGST2) with significant homology to both plant and animal GSTs was previously isolated from Arabidopsis. When expressed in E. coli, this protein could be purified by GSH-affinity chromatography and displayed activity with CDNB. AtGST2 mRNA was found in all three organs tested: roots, leaves and stems. The highest level of expression was found in roots and this expression was shown to be controlled by ethylene. The developmental expression of AtGST2 mRNA was examined in leaves of wild type and etr1 plants. AtGST2 mRNA reached similar levels in aging leaves of both genotypes, suggesting that factors other than ethylene regulate the expression of this gene. Several other stimuli, including salicylic acid, NAA, GSH, H$\sb2$O$\sb2$, paraquat, heavy metals (Cu, Zn, Cd) and cold treatment, increased the level of AtGST2 mRNA in both wild type and etr1 seedlings. These results suggest that AtGST2 is a general stress protein induced by both internal and external stimuli that lead to oxidative stress. This is consistent with the Se-independent peroxidase activity reported for this enzyme. Two highly related DNA fragments were isolated from the Arabidopsis genome; AtGST2A encoded the AtGST2 mRNA while the other contained a pseudogene, AtGST2B. DNA sequence comparisons suggest that AtGST2B arose by gene duplication followed by gene conversion. The transcriptional start site of AtGST2 mRNA was determined by primer extension analysis. Several 5$\sp\prime$ deletions of the promoter region from AtGST2A were fused to the $\beta$-glucuronidase (GUS) reporter gene and introduced into Arabidopsis. Analysis of GUS activity in transgenic plants indicated that, first, accumulation of AtGST2 mRNA in response to GSH, DTT, copper, and paraquat is at least partially due to the transcriptional activation and, second, 220 bp of the 5$\sp\prime$ flanking region contain regulatory sequences that confer induction in response to these inducers.
Degree
Ph.D.
Advisors
Goldsbrough, Purdue University.
Subject Area
Botany|Molecular biology|Genetics
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