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Metabolic mapping of gene families : a new strategy for the discovery of overlooked pathways – METAMAP

Submission summary

Plants synthesize a vast array of diverse organic compounds, referred to as secondary metabolites or natural products that serve important adaptive functions in protection against pests, as structural components, allelochemicals and signalling molecules. Such plant natural products are also a major source of dyes, polymers, oils, perfumes, flavours and medicinal drugs. Exploitation of this resource however requires extensive description of plant metabolites and of their biosynthetic pathways. Recent knowledge acquired on plant genomes revealed that most plant metabolic networks remain completely unexplored : a vast majority of genes expected to be involved in plant metabolism have no associated function. We propose here a new strategy to assign a function to genes in plant metabolism and for highlighting so far overlooked pathways. This strategy is based on the observation that genes involved in the same pathway are usually co-regulated, and exploits the plethora of gene expression data presently available in databases for the model plant Arabidopsis thaliana. We used the largest multigene family involved in plant metabolism, encoding cytochromes P450, as a bait to identify sets of co-regulated genes and associate typical expression profiles. This results in a predictive mapping of plant metabolism with associated tissue-specific expression and potential biological functions. This approach is comforted by the correct prediction of the function of genes with demonstrated function. The aim of this proposal is to assess the predicted function of orphan genes and broad applicability of the strategy to the discovery of plant metabolism. Two groups of genes predicted with high scores to be involved in volatile monoterpene biosynthesis in flowers and triterpene metabolism in roots were selected for our research program. The first is formed of 2 co-expressed mono-terpene synthases and 3 P450 monooxygenases, the second of one tri-terpene synthase and two co-expressed P450 enzymes. All branches of the isoprenoid pathway have in common that they use universal precursors (such as geranyl-PP or 2,3-epoxysqualene) to synthesize a wide array of structurally very diverse compounds via terpene synthases (TPS). These products are then further modified, e.g. hydroxylated, to increase diversity of structures and biological functions. Oxygenation pattern of the final compounds usually plays a key role in their biological activities and organoleptic properties. Cytochromes P450 usually catalyze initial steps of terpene modification after synthesis by a TPS. In A. thaliana, most of the products of this isoprenoid metabolism were never described, nor their biological activities. We thus propose to express the appropriate TPS/P450 couples in yeast in order to identify the reaction products and determine their structure (GC-MS, NMR). To this end, yeast with optimal genetic backgrounds (accumulating GPP or 2,3-epoxysqualene) will be used or engineered. In vitro biological activity (antibacterial) and sensorial properties of the products will be determined. Simultaneously, gene function will be analysed in the plant. Predicted patterns of gene expression will be confirmed in promoter-GUS transformed plants and by qRT-PCR. Phenotypic analysis and metabolic profiling will be carried out in wild-type, constitutive overexpressors and TDNA-inactivated plants. Some of the relevant mutants are readily available and under investigation in the partner's labs. Mono- and tri-terpene metabolites will be analysed in flower and roots respectively, if relevant as volatiles or exudates, by GC-MS. Analysis of volatiles and flower isoprenoids will be carried out with the support of Firmenich (scent and flavour company) providing optimal analytical platform. Presence of gene orthologues and relevant metabolites will be investigated in Brassica napus and Vitis vinifera. Potential role of the candidate genes in plant ecophysiology will be first investigated by submitting promoter-GUS transformants to environmental stress (abiotic, insect/nematode infestation, bacterial/fungal infection). Impact of gene overexpression suppression will then be tested on growth of epiphytic microflora, bacterial/fungal pathogen attack and insect/nematode infestation. The latter step will be carried out as far as time allows and with the support of external collaborations. Confirmation of the efficiency of the metabolic mapping strategy will pave the way to the discovery of many overlooked aspects of plant secondary metabolism, in a first step in A. thalania and plants with sequenced genomes, many of which will be applicable to plant of major economic interest. Tools created by this project for optimal recombinant expression of enzymes in the mono- and tri-terpene pathways will provide support to further exploration of isoprenoid branch pathways in all plant species and to biotechnological applications of resulting active compounds.

Project coordination

Danièle WERCK-REICHHART (Organisme de recherche)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

Help of the ANR 380,000 euros
Beginning and duration of the scientific project: - 36 Months

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