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Functional mapping of the Arabidopsis Cytochrome P450 Gene superfamily based on Co-expression Analysis

Final Activity Report Summary - FUNCYP (Functional Mapping of the Arabidopsis Cytochrome P450 Gene Superfamily based on Co-Expression Analysis)

Cytochromes P450 are enzymes, more specifically monooxygenases, that constitute metabolic control points regulating the synthesis of the myriads of plant natural products found in plants. These play pivotal roles in plant development, adaptation, and acclimation to changing environments plant are poised to cope with, as they cannot avoid them by moving to a more suitable place. Recent plant genome initiatives have shown that large families of genes encode P450s, but that a surprisingly small fraction of them was characterised. This highlighted that our understanding of plant natural product metabolism is only at its infancy.

Our goal was to support identification of the biochemical functions of the orphan gene family members, not only to enhance our understanding of plant metabolism, but also to support the biotechnological exploitation of these compounds for human use, for example as medicinal drugs. A critical bottleneck in such functional characterisation was the generation of hypotheses regarding the actual biochemical reaction a given P450 performs, which can then be addressed experimentally using established techniques of functional genomics.

We used the wealth of published large scale gene expression data from the model plant Arabidopsis thaliana, quantifying the amounts of almost all gene transcripts present in this plant under more than 2 000 experimental conditions. These conditions cover most stages of plant development and numerous responses to environmental clues. We first showed that previously characterised genes acting in the same biochemical pathways are indeed co-expressed during development and in response to external clues. Thus, we compared the expression profile of each P450 gene with those from about 4 000 genes that have been implied to act in diverse metabolic pathways and identified computationally co-expressed genes. Based on the knowledge available in diverse databases for these co-expressed genes, we assigned likely metabolic pathways these uncharacterized P450s act in.

For many orphan P450 putative pathways were identified. The information resulting from this work has been organised as an open-access database. The largest group identified is related to chlorophyll / carotenoid biosynthesis and / or plastidial activity. Interesting candidates were selected for further biochemical characterization, which are predicted to be involved in terpenoid modulation (CYP76C and CYP71B families), oxylipin metabolism (CYP86B and CYP77A families), and pollen coat biosynthesis (CYP98A and CYP86B/C families). The functional characterisation by means of reverse genetics and reverse biochemistry is ongoing, but for two of these candidates novel biochemical functions have been identified that were consistent with the bioinformatic predictions.