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This project is aimed at functionally defining the expression patterns and substrate reactivities of the Arabidopsis thaliana cytochrome P450 monooxygenase (P450) gene family that represents approximately 0.6% of this plant's genome. The proteins encoded by these genes mediate oxidative transformations in a wide array of biosynthetic and detoxicative pathways essential for plant growth. Because of their roles in this wide diversity of metabolic processes and their relative lack of post-translational modification, they serve as downstream reporters for the direct activation of many different biochemical pathways responding to chemical, developmental and environmental cues. Extensive divergence of catalytic site as well as noncatalytic site residues has resulted in a high degree of primary structure variation in this very diverse P450 gene superfamily that presents unsurpassed complexity in assigning function to individual genomic DNA sequences. We will further the functional definition of Arabidopsis P450s by creating microarrays for analysis of the mRNA expression patterns in tissues at different developmental stages. Imaging of the responses of individual P450 genes with respect to a variety of internal and external chemical cues as well as changing environmental conditions (UV damage, pathogen attack, insect attack, cold stress, etc.) and collations of these response patterns with respect to plant biochemical pathways will provide the basis for assigning prospective functions to these enzymes. Over-expression of a subset of these P450s in yeast and baculovirus protein production systems and incorporation into membrane-scaffolding protein complexes suitable for high-throughput screening of substrate reactivities will provide the basis for assigning definitive function(s) to these enzymes. These results serve to elucidate this model plant's biochemical responses to a variety of stress conditions and provide genomic technology tools needed for assessment of the diverse P450 gene family as well as other membrane protein families. Data on the organization of the 273 P450 genes, alignments of available ESTs, patterns of mRNA expression defined by microarray analysis and P450 substrate preferences defined by functional protein expression will be made available as collected.