Regulation of a LON-like protease and its influence on developmental expression of the CMS-associated peptide, ORF239, in common bean mitochondria
Abstract
Cytoplasmic male sterility in the common bean plant is associated with a dominant mitochondrial mutation designated pvs-orf239. The sequence is transcribed in both vegetative and reproductive tissues, but the translation product, ORF239, is present only in reproductive tissues. I present evidence to support a model of post-translational regulation of ORF239 expression based on the following observations. In organello translation experiments using purified mitochondria from young seedlings demonstrated accumulation of ORF239 only when a protease inhibitor was included. Proteolytic activity against ORF239 was observed in mitochondrial extracts, fractionating with the mitochondrial inner membrane. A serine type protease, similar to the LON protease of E. coli, was cloned from the Arabidopsis genome (AtLON) and demonstrated proteolytic activity against ORF239 in vitro, with features resembling the activity detected in mitochondrial inner membrane preparations. Antibodies generated against the cloned, overexpressed LON homologue reduced proteolytic activity against ORF239 when added to mitochondrial extracts. These data suggest that ORF239 is undetected in vegetative tissue due to rapid turnover by at least one mitochondrial protease that acts against ORF239. Additionally, I have isolated the AtLON promoter and performed preliminary studies of its expression pattern and stress response. I have also studied a second means of pvs-orf239 regulation in common bean lines that have undergone spontaneous reversion to fertility. This reversion to fertility is associated with the shifting to substoichiometric levels of the DNA molecule containing pvs-orf239. In vitro mitochondrial incubation results demonstrated that the genomic shift of the pvs-orf239 -containing molecule to substoichiometric levels upon spontaneous reversion was a reversible phenomenon. Moreover, I have demonstrated that substoichiometric forms, apparently silent with regard to gene expression, are transcriptionally and translationally active once amplified.
Degree
Ph.D.
Advisors
Mackenzie, Purdue University.
Subject Area
Molecular biology|Biochemistry|Cellular biology
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