Bouille, A. | Search bioRxiv

bioRxiv2025

Introducing furanocoumarin biosynthetic genes in tomato results in coumarins accumulation and impacted growth

Over the past three decades, eeorts to decipher plant metabolism have shed light on key enzymes driving specialized metabolite biosynthesis. Although only few pathways have been completely investigated to date, their characterization paves the way for exploring the potential eeects of specialized metabolites on plant physiology. Among them is the linear furanocoumarin pathway, which was recently completed to produce up to psoralen. In this study, we report the first metabolic engineering of the linear furanocoumarin pathway to enable artificial psoralen production in tomato, through the integration of four genes coding for the enzymes: Umbelliferone Synthase, Demethylsuberosin Synthase, Marmesin Synthase and Psoralen Synthase. Interestingly, coumarins were produced instead of furanocoumarins. Using morphophysiological, metabolomic, and transcriptomic analyses, we suggest how coumarins, particularly scopoletin, can impact growth and aeect plant physiology, even at low concentrations. As coumarins have increasingly attracted interest for agricultural applications due to their minimal environmental impact, this work both expands and challenges their potential by highlighting the physiological costs and benefits they may impose on tomato. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=164 SRC="FIGDIR/small/663522v2_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1df0bdeorg.highwire.dtl.DTLVardef@843d9borg.highwire.dtl.DTLVardef@1e898b6org.highwire.dtl.DTLVardef@18205eb_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

plant biology↗

bioRxiv2024

Using lineage-specific patterns to understand convergence of enzymatic functions led to the identification of Moraceae-specific P450s involved in furanocoumarin biosynthesis

O_LISpecialized metabolites are molecules involved in plants interaction with their environment. Elucidating their biosynthetic pathways is a challenging but rewarding task, leading to societal applications and ecological insights. Furanocoumarins emerged multiple times in Angiosperms, raising the question of how different enzymes evolved into catalyzing identical reactions. C_LIO_LITo identify enzymes producing lineage-specific metabolites, an evolutionary-based approach was developed and applied to furanocoumarin biosynthesis in Ficus carica (Moraceae). This led to the characterization of CYP71B129-131a, three P450 enzymes whose evolution of the function was investigated using phylogenetics, structural comparisons and site-directed mutagenesis. C_LIO_LICYP71B129 and CYP71B130,131a were found to hydroxylate umbelliferone (coumarin) and xanthotoxin (furanocoumarin), respectively. Results suggest that CYP71Bs xanthotoxin hydroxylase activity results from duplications and functional divergence of umbelliferone hydroxylase genes. Structural comparisons highlighted an amino acid affecting CYP71Bs substrate specificity, which may play a key role in allowing xanthotoxin hydroxylation in several P450 subfamilies. C_LIO_LICYP71B130-131a characterization validates the proposed enzyme-discovery approach, which can be applied to different pathways and help to avoid the classic bottlenecks of specialized metabolism elucidation. The CYP71Bs also exemplify how furanocoumarin-biosynthetic enzymes can stem from coumarin-biosynthetic ones and provides insights into the molecular mechanisms underlying the multiple emergences of xanthotoxin hydroxylation in distant P450 subfamilies. C_LI

plant biology↗