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How to produce your furanocoumarins : the hidden pathway ~ From the characterisation of new P450s to the evolution of the furanocoumarin pathway and the development of tools allowing the study of furanocoumarins’ metabolic cost

Abstract : To cope with pests and pathogens, plants have evolved many defence mechanisms such as the production of various specialised metabolites. For instance, some species including the fig tree (Ficus carica) produce toxic molecules called furanocoumarins. As the use of pesticides is being restricted, these age-old adaptations represent an interesting source of inspiration to find crop protection alternatives. Therefore, improving our understanding of plant defences becomes essential to rethink our crop management strategies. The first objective of this project was to pursue the molecular elucidation of the furanocoumarin biosynthesis pathway and to gain evolutionary insights through the characterisation of new genes in F. carica. The second objective was to assess the metabolic cost of furanocoumarin production by inserting the associated pathway in the genome of tomato (Solanum lycopersicum), a plant that does not naturally produce these molecules. To identify new genes involved in the furanocoumarin biosynthesis, we combined transcriptomic and metabolomic approaches: we identified candidate genes in a F. carica differential RNAseq database, cloned their coding sequences, heterologously expressed them, and performed enzymatic assays. This led us to identify three enzymes with original activities. Among these, CYP76F112 plays a key role in the furanocoumarin biosynthesis by converting demethylsuberosin into marmesin with a very high affinity. A phylogenetic gene-family analysis strongly suggests that CYP76F112 evolved recently in a restricted taxon of the Moraceae family, through an expansion of the CYP76Fs. Modelling and site-directed mutagenesis experiments permitted to highlight four amino acids that impact CYP76F112 specificity and affinity. Moreover, the recent evolution of these amino acids has most certainly been critical for the emergence of the marmesin synthase activity. Therefore, CYP76F112 significantly improves our understanding of furanocoumarin production in higher plants, since its recent evolution supports the hypothesis that furanocoumarins have appeared by convergent evolution in distant plant families. In addition, as CYP76F112 completes the set of four enzymes that allow the conversion of coumaric acid (a common molecule) into psoralen (a toxic furanocoumarin), it opens new horizons for the use of furanocoumarins in the study of plant defence. For instance, by generating psoralen-producing tomatoes, it is now possible to evaluate the metabolic costs and defensive profits linked to psoralen production, which might lead us to better understand the trade-offs between growth and defence. Consequently, we used a multi-gene cloning technology called GoldenBraid to construct a plasmid harbouring the four genes of the psoralen biosynthesis pathway. Using transgenesis methods along with in vitro culture, we used this plasmid to initiate plant transformation, in order to generate psoralen-producing tomatoes. These transformations were not conclusive, but our study constitutes a pioneer work that will be continued. In particular, it allowed to the identification of some weaknesses in the initial strategy and the establishment of recommendations that will be essential to overcome these limits.
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https://hal.univ-lorraine.fr/tel-03184585
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Submitted on : Monday, March 29, 2021 - 4:09:46 PM
Last modification on : Tuesday, March 30, 2021 - 3:46:08 AM

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  • HAL Id : tel-03184585, version 1

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Cloé Villard. How to produce your furanocoumarins : the hidden pathway ~ From the characterisation of new P450s to the evolution of the furanocoumarin pathway and the development of tools allowing the study of furanocoumarins’ metabolic cost. Vegetal Biology. Université de Lorraine, 2020. English. ⟨NNT : 2020LORR0201⟩. ⟨tel-03184585⟩

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