Citronellol biosynthesis in pelargonium is a multistep pathway involving progesterone 5ß-reductase and/or iridoid synthase-like enzymes.

Citronellol is a pleasant-smelling compound produced in rose (Rosa spp.) flowers and in the leaves of many aromatic plants, including pelargoniums (Pelargonium spp.). Although geraniol production has been well studied in several plants, citronellol biosynthesis has been documented only in crab-lipped spider orchid (Caladenia plicata) and its mechanism remains open to question in other species. We therefore profiled 10 pelargonium accessions using RNA sequencing and gas chromatography-MS analysis. Three enzymes from the progesterone 5ß-reductase and/or iridoid synthase-like enzymes (PRISE) family were characterized in vitroand subsequently identified as citral reductases (named PhCIRs). Transgenic RNAi lines supported a role for PhCIRs in the biosynthesis of citronellol as well as in the production of mint-scented terpenes. Despite their high amino acid sequence identity, the 3 enzymes showed contrasting stereoselectivity, either producing mainly (S)-citronellal or a racemate of both (R)- and (S)-citronellal. Using site-directed mutagenesis, we identified a single amino acid substitution as being primarily responsible for the enzyme's enantioselectivity. Phylogenetic analysis of pelargonium PRISEs revealed 3 clades and 7 groups of orthologs. PRISEs from different groups exhibited differential affinities toward substrates (citral and progesterone) and cofactors (NADH/NADPH), but most were able to reduce both substrates, prompting hypotheses regarding the evolutionary history of PhCIRs. Our results demonstrate that pelargoniums evolved citronellol biosynthesis independently through a 3-step pathway involving PRISE homologs and both citral and citronellal as intermediates. In addition, these enzymes control the enantiomeric ratio of citronellol thanks to small alterations of the catalytic site.

Laccase-Catalyzed Oxidation of Allylbenzene Derivatives: Towards a Green Equivalent of Ozonolysis.

Laccase-based biocatalytic reactions have been tested with and without mediators and optimized in the oxidation of allylbenzene derivatives, such as methyl eugenol taken as a model substrate. The reaction primarily consisted in the hydroxylation of the propenyl side chain, either upon isomerization of the double bond or not. Two pathways were then observed; oxidation of both allylic alcohol intermediates could either lead to the corresponding α,ß-unsaturated carbonyl compound, or the corresponding benzaldehyde derivative by oxidative cleavage. Such a process constitutes a green equivalent of ozonolysis or other dangerous or waste-generating oxidation reactions. The conversion rate was sensitive to the substitution patterns of the benzenic ring and subsequent electronic effects.

Functional Analysis of Four Terpene Synthases in Rose-Scented Pelargonium Cultivars (Pelargonium × hybridum) and Evolution of Scent in the Pelargonium Genus.

Pelargonium genus contains about 280 species among which at least 30 species are odorant. Aromas produced by scented species are remarkably diverse such as rose, mint, lemon, nutmeg, ginger and many others scents. Amongst odorant species, rose-scented pelargoniums, also named pelargonium rosat, are the most famous hybrids for their production of essential oil (EO), widely used by perfume and cosmetic industries. Although EO composition has been extensively studied, the underlying biosynthetic pathways and their regulation, most notably of terpenes, are largely unknown. To gain a better understanding of the terpene metabolic pathways in pelargonium rosat, we generated a transcriptome dataset of pelargonium leaf and used a candidate gene approach to functionally characterise four terpene synthases (TPSs), including a geraniol synthase, a key enzyme responsible for the biosynthesis of the main rose-scented terpenes. We also report for the first time the characterisation of a novel sesquiterpene synthase catalysing the biosynthesis of 10-epi-γ-eudesmol. We found a strong correlation between expression of the four genes encoding the respective TPSs and accumulation of the corresponding products in several pelargonium cultivars and species. Finally, using publically available RNA-Seq data and de novo transcriptome assemblies, we inferred a maximum likelihood phylogeny from 270 pelargonium TPSs, including the four newly discovered enzymes, providing clues about TPS evolution in the Pelargonium genus. Notably, we show that, by contrast to other TPSs, geraniol synthases from the TPS-g subfamily conserved their molecular function throughout evolution.

Linalool and linalool nerolidol synthases in roses, several genes for little scent.

Roses are widely appreciated for the appearance of their flowers and for their fragrance. This latter character results from the combination of different odorant molecules among which monoterpenes are often prevalent constituents. In this study, we report the cloning and characterization of three rose monoterpene synthases. In vitro functional characterization of these enzymes showed that one is a (-)-(3R)-linalool synthase whereas the others have a dual (+)-(3S)-linalool nerolidol synthase activity. However, given that the characterized rose cultivars were only able to produce the (-)-(3R)-linalool stereoisomer, the linalool nerolidol synthases are probably not active in planta. Furthermore, these three enzymes were also characterized by a weak expression level as assessed by RT-qPCR and by the low abundance of the corresponding sequences in an EST library. This characteristic is likely to explain why linalool is generally a minor constituent in rose flowers' scents. On this basis, we propose that in roses the monoterpene biosynthesis effort is focused on the production of acyclic monoterpenes derived from geraniol through the recently characterized Nudix biosynthesis pathway, at the expense of conventional monoterpene biosynthesis via terpene synthases such as linalool or linalool nerolidol synthases.