The liverwort Frullania tamarisci (L.) Dumort produces large amounts of terpenoids, among others the sesquiterpene alcohol tamariscol. Tamariscol has an earthy woody fragrance, and the use in perfurmes and production of it was patented in 1984. The microbial terpene synthase-like (MTPSL) enzyme FtMTPSL6 is shown to be responsible for the biosynthesis of tamariscol. FtMTPSL6 was obtained through RNA sequencing of wild growing F. tamarisci along with six other MTPSLs (FtMTPSL1-7). The biochemical activity was determined for three of them, and two others where metabolic active, but the product could not be identified. The three characterized enzymes are the tamariscol synthase (FtMTPSL6), copaene synthase (FtMTPSL1) and gurjunene synthase (FtMTPSL3). Thus, the biosynthesis of the economically attractive compound tamariscol is established, and this opens up for further exploitation of this molecule.

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 5β-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.

ABSTRACT Roses have been admired ever since antiquity. They have followed all of the human history for reasons other than just medicine and food, mostly because of their beauty and their fragrance. They have always been used in perfumes, in gardens, and yet again in the cut flower market. Humans have listed more than 25,000 cultivars to this day, while there are only a little over a hundred species in the wild. In this review, we will present the domestication history of roses used for perfumes, and the selection of garden roses and cut rose cultivars. We will also compare the scent of modern roses and wild roses and give the chemical analysis of representative volatile compounds that have been characterized in roses. We will then summarize the biochemical pathways that have been studied in roses at the gene level. We will conclude that scent is not a trait in itself but a multitude of traits driven by many genes. Their alleles could perhaps be used as markers for the selection of new cultivars.

Rosa is a complex taxon with more than 150 intertwined species. Only few have been domesticated by humans since Antiquity, reaching today more than 30,000 cultivars. One of the major traits that have been selected is scent. However, rose scent is a complex trait comprised of dozens of volatile molecules. Some of these molecules originate from a specific and uniquely evolved biosynthetic pathway in the genus Rosa, which arose from acquisition of the duplication and neofunctionalization of genes to become involved in the production of scent compounds. Examples include NUDX1, a gene involved in geraniol biosynthesis, specifically in roses (Magnard et al., 2015). We have shown that multiple trans- and cis-duplications of NUDX1 during the evolution of Rosaceae and Rosa, have led to the specialization of the paralog NUDX1-1a toward geraniol production (Conart et al., 2022). This paralog is not present in the more ancient wild roses making them unsuitable for crosses to obtain fragrant roses. Previously we showed that some hybrid cultivars with R. wichurana as one parent, have a different specialization: NUDX1-1a is inactive, while NUDX1-2c, another paralog, is active and involved in (E,E)-farnesol production (Sun et al., 2020). Furthermore, some genes well-known to be involved in scent production encode enzymes that are functional in vitro, but are not always highly expressed in planta. Examples include LIS and PAAS genes, respectively involved in linalool and 2-phenylethanol biosynthesis (Magnard et al., 2018; Roccia et al., 2019). Taken together, these results indicate different evolutionary scenarios in different rose species. A better understanding of the genes and alleles involved in the production of fragrant molecules is thus needed to help the selection of new, scented rose cultivars. This paper focuses on the NUDX1 gene evolution as an example of what knowledge of a gene family can bring to the breeding of roses.

Geraniol derived from essential oils of various plant species is widely used in the cosmetic and perfume industries. It is also an essential trait of the pleasant smell of rose flowers. In contrast to other monoterpenes which are produced in plastids via the methyl erythritol phosphate pathway, geraniol biosynthesis in roses relies on cytosolic NUDX1 hydrolase which dephosphorylates geranyl diphosphate (GPP). However, the metabolic origin of cytosolic GPP remains unknown. By feeding Rosa chinensis “Old Blush” flowers with pathway-specific precursors and inhibitors, combined with metabolic profiling and functional characterization of enzymes in vitro and in planta, we show that geraniol is synthesized through the cytosolic mevalonate (MVA) pathway by a bifunctional geranyl/farnesyl diphosphate synthase, RcG/FPPS1, producing both GPP and farnesyl diphosphate (FPP). The downregulation and overexpression of RcG/FPPS1 in rose petals affected not only geraniol and germacrene D emissions but also dihydro-β-ionol, the latter due to metabolic cross talk of RcG/FPPS1-dependent isoprenoid intermediates trafficking from the cytosol to plastids. Phylogenetic analysis together with functional characterization of G/FPPS orthologs revealed that the G/FPPS activity is conserved among Rosaceae species. Site-directed mutagenesis and molecular dynamic simulations enabled to identify two conserved amino acids that evolved from ancestral FPPSs and contribute to GPP/FPP product specificity. Overall, this study elucidates the origin of the cytosolic GPP for NUDX1-dependent geraniol production, provides insights into the emergence of the RcG/FPPS1 GPPS activity from the ancestral FPPSs, and shows that RcG/FPPS1 plays a key role in the biosynthesis of volatile terpenoid compounds in rose flowers.

Nudix hydrolases are conserved enzymes ubiquitously present in all kingdoms of life. Recent research revealed that several Nudix hydrolases are involved in terpenoid metabolism in plants. In modern roses, RhNUDX1 is responsible for formation of geraniol, a major compound of rose scent. Nevertheless, this compound is produced by monoterpene synthases in many geraniol-producing plants. As a consequence, this raised the question about the origin of RhNUDX1 function and the NUDX1 gene evolution in Rosaceae, in wild roses or/and during the domestication process. Here, we showed that three distinct clades of NUDX1 emerged in the Rosoidae subfamily (Nudx1-1 to Nudx1-3 clades), and two subclades evolved in the Rosa genus (Nudx1-1a and Nudx1-1b subclades). We also showed that the Nudx1-1b subclade was more ancient than the Nudx1-1a subclade, and that the NUDX1-1a gene emerged by a trans-duplication of the more ancient NUDX1-1b gene. After the transposition, NUDX1-1a was cis-duplicated, leading to a gene dosage effect on the production of geraniol in different species. Furthermore, the NUDX1-1a appearance was accompanied by the evolution of its promoter, most likely from a Copia retrotransposon origin, leading to its petal-specific expression. Thus, our data strongly suggest that the unique function of NUDX1-1a in geraniol formation was evolved naturally in the genus Rosa before domestication.

SUMMARYRoses use a non‐canonical pathway involving a Nudix hydrolase, RhNUDX1, to synthesize their monoterpenes, especially geraniol. Here we report the characterization of another expressed NUDX1 gene from the rose cultivar Rosa x wichurana, RwNUDX1‐2. In order to study the function of the RwNUDX1‐2 protein, we analyzed the volatile profiles of an F1 progeny generated by crossing R. chinensis cv. ‘Old Blush’ with R. x wichurana. A correlation test of the volatilomes with gene expression data revealed that RwNUDX1‐2 is involved in the biosynthesis of a group of sesquiterpenoids, especially E,E‐farnesol, in addition to other sesquiterpenes. In vitro enzyme assays and heterologous in planta functional characterization of the RwNUDX1‐2 gene corroborated this result. A quantitative trait locus (QTL) analysis was performed using the data of E,E‐farnesol contents in the progeny and a genetic map was constructed based on gene markers. The RwNUDX1‐2 gene co‐localized with the QTL for E,E‐farnesol content, thereby confirming its function in sesquiterpenoid biosynthesis in R. x wichurana. Finally, in order to understand the structural bases for the substrate specificity of rose NUDX proteins, the RhNUDX1 protein was crystallized, and its structure was refined to 1.7 Å. By molecular modeling of different rose NUDX1 protein complexes with their respective substrates, a structural basis for substrate discrimination by rose NUDX1 proteins is proposed.