Plant systems utilize a diverse array of cytochrome monooxygenase (P450s) in their biosynthetic and detoxification pathways. The first plant P450 characterized at the nucleotide level was a ripening induced avocado (Persea americana) P450 designated cyp71A1. As cyp71A1 was initially characterized as a ripening-induced gene, it was thought the encoded protein (CYP71) had a specific, yet undefined role in ripening perhaps involving accumulation of chemicals involved in flavor or aroma. More recent studies have revealed the induction of the CYP71A1 protein in wounded non-fruit tissues, as well as the activation of the upstream region of cyp71A1 protein in wounded non-fruit tissues, as well as the activation of the upstream region of cyp71A1 by the phytohormone ethylene. Therefore this proposal furthers characterization of biotic and abiotic stress-response of both the cyp71A1 gene in avocado cells, as well as activation patterns of the cyp71A1 upstream region in a transgene Arabidopsis system. Toward the end of predicting the role of CYP71A1 in the avocado plant through analysis of its activation patterns this proposal's first specific aim is to characterize CYP71A1 induction in sterile avocado root suspension cell cultures with respect to ethylene treatment as well as both chemical and fungal elicitors. Once the induction pattern of cyp71A1 is known in avocado root cells the second aim is to identify activators of the cyp71A1 promoter in transgenic Arabidopsis plants which will be determined through use of the reporter gene GUS. To confirm the ethylene-induction of the cyp71A1 promoter in transgenic Arabidopsis plants, the hybrids plants resulting from the cross between a dominant ethylene-insensitive mutant and the cyp7pro/GUS chimera will be monitored for loss of GUS from the cross between a dominant ethylene-insensitive mutant and the cyp71pro/GUS chimera will be monitored for loss of GUS staining after ethylene- treatment (third aim).
The fourth aim of this proposal is to identify cyp71A1 upstream regions critical for biotic and abiotic stress-induction in transgenic plants, focusing on the -400 through-332 region previously identified as important for expression of the reporter gene GUS in a transient expression assay in avocado fruit cells. The complete characterization of ripening-specific promoters is becoming increasingly important to the production of transgenic plants which are engineered to produce pharmaceutically important chemicals in specific tissues (such as edible ripe fruit) instead of constitutively throughout the plant. The creation of """"""""edible vaccines"""""""" will be optimized if the gene encoding the antigen protein is placed under control of a ripening-specific promoter, if it is intended that the plant is to be used to produce antigen proteins in the ripe fruit which can be ingested instead of vaccine immunization.
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