Regulatory mechanisms controlling expression of cytochrome P450 genes from mammals have been well deliniated, whereas the analysis of plant P450 gene regulation has just recently begun. The description of plant P459 inducers is documented in the literature, yet no single plant P450 gene's controlling element(s) has been defined. This grant request proposed studies which will further understanding of the mechanism by which an avacado cytochrome P450 gene (cyp71A1) is regulated during developmental processes, and analyzes specific upstream segments which respond to ethylene evolved either as a result of wounding or ripening. This will further the understanding of the mechanism by which this plant gene whose product is involved in oxidative metabolism responds to different stimuli via a hormone mediated signal transduction apparatus.
Aim 1 To determine if ethylene evolved either during ripening or wounding is required and/or sufficient to activate cyp71A1. Initial studies will define temporal expression of P450 antigen and cyp71a1 mRNA following mechanical wounding of avocado leaves (1A). Additionally the role of ethylene during either ripening or wounding will be assayed (1B,1C,1D).
Aim 2 To identify where the ethylene-responsive elements of cyp71A1 are located. The upstream region of cyp71A1 has been studied in the context of ri[pe avocado fruit mesocarp protoplasts through a transient expression assay. Proposed studies will further narrow the previously identified broad segments critical for ethylene regulation, and will differentiate between wounding and ripening-specific elements, and those which directly respond to ethylene. The proposed studies include both functional assays assessing activity of various promoter fragments in ripe avocado fruit protoplasts (2A), transgenic tobacco either wounded (2B) or gassed with ethylene (2C) and gel mobility shifts to reveal whether these identify the same element(s) (2D).
Aim 3 To train students in contemporary molecular biology techniques (analyzing hormone-mediated gene regulation) that are used in research conducted at biotechnology companies, hospitals and universities. Understanding the regulation of this first fully-analyzed plant (P450 will serve as a basis for comparison with other plant and animal P450 regulatory mechanisms.
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