Characterization of the function and ethylene-regulation of a senescence-related glutathione S-transferase gene from carnation
Abstract
The senescence of carnation flower petals represents a genetic program of events which serve to strategically eliminate this unnecessary organ following its transient function to attract pollinators. While the mechanism of this programmed cell death is unknown, it is clearly enhanced by the plant hormone, ethylene. Treatment of flowers with exogenous ethylene promotes premature petal senescence, while inhibitors of ethylene action or biosynthesis delay the onset of senescence. As a regulator of carnation petal senescence, ethylene directs the expression of several senescence-related (SR) genes. I have analyzed the expression of the pSR8 gene which has a predicted amino acid sequence previously shown to share homology with glutathione s-transferases (GSTs). GSTs are members of a large multigene family present in all aerobic organisms. Detoxification of xenobiotic compounds through their conjugation to glutathione and intracellular ligand binding are two demonstrated functions in plants. Western blot analysis confirms that the pSR8 gene product is specifically expressed in floral organs during the peak of ethylene production that is characteristic of senescence. Unlike other plant or mammalian GSTs, the putative carnation GST is not active against the trademark substrates used to classify this large family of isozymes. Its overexpression in transgenic petunia does not result in any significant alteration in phenotype. While its endogenous substrate has not been identified, expression of the senescence-related GST appears to be under strict regulation by ethylene. Promoter analysis of a genomic clone (GST1) led to the identification of a 197 bp ethylene-responsive element (ERE) that was both necessary and sufficient for conferring ethylene regulation to a heterologous promoter. I further defined a 20 bp region that was capable of binding carnation petal nuclear proteins. Using Southwestern blot analysis of a cDNA expression library, a cDNA clone encoding a DNA binding protein was identified (ERE-BP1). The predicted peptide sequence revealed a 32 kD protein containing a putative RNA binding motif (RNP). Both eukaryotic and prokaryotic transcription factors have been identified as RNP-containing proteins. Speculation on the dual function of ERE-BP1 and its role in ethylene signal transduction will be presented.
Degree
Ph.D.
Advisors
Woodson, Purdue University.
Subject Area
Molecular biology|Botany
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