Corentin Conart, Dikki Pedenla Bomzan, Xing-Qi Huang, +19, and Benoît Boachon

PNAS May 1, 2023; 120 (19) e2221440120

Significance

Geraniol, a volatile compound contributing to the unique smell of rose flowers, is synthesized through a noncanonical NUDX1-dependent pathway from cytosolic geranyl diphosphate (GPP), the origin of which remained unknown. We demonstrate that in contrast to the heterodimeric GPP synthase providing plastidic GPP in most plants, a cytosolic and bifunctional FPP synthase enzyme produces both GPP and farnesyl diphosphate (FPP) via the mevalonate pathway in rose flowers. This enzyme, conserved in Rosaceae species, evolved from ancestral FPPSs with two amino acids contributing to GPP/FPP product specificity. It plays a key role in the emission of geraniol and also germacrene D and dihydro-β-ionol, the latter is due to trafficking of RcG/FPPS1-dependent isoprenoid intermediates from the cytosol to plastids.

Abstract

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.

See https://www.pnas.org/doi/10.1073/pnas.2221440120

Fig. 2: The RcOB genome contains six IDS candidates of which two FPPS-like synthases localized in the cytosol. (A) Maximum likelihood tree of protein sequences of the six RcOB trans-short-chain IDSs (highlighted in pink) with characterized IDSs from Abies grandis (Ag), Antirrhinum majus (Am), Arabidopsis thaliana (At), Clarkia breweri (Cb), Catharanthus roseus (Cr), Humulus lupulus (Hl), Mentha piperita (Mp), Phalaenopsis bellina (Pb), Picea abies (Pa), Populus trichocarpa (Pt), Quercus robur (Qr), Rosa chinensis “Old Blush” (Rc), and Solanum lycopersicum (Sl). Numbers correspond to bootstrap values. Tree is rooted on RcGDS (Germacrene D synthase). Polyprenyl pyrophosphate synthase (PPPS). (B) Transcriptomic analysis of the six RcOB IDSs and RcNUDX1-1as compared to geraniol levels in R. chinensis “Old Blush” (OB) and 9 Rosa x hybrida cultivars including R. x hybrida “Akito” (AK), R. x hybrida “The Fairy” (FY), R. x damascena “Kazanlik” (KZ), R. x odorata “Lady Hillingdon” (LH), R. x hybrida “The Mc Cartney rose” (MC), R. x hybrida “Marius Ducher” (MD), R. x hybrida “Pariser Charme” (PC), R. x hybrida “Papa Meilland,” (PM) and R. x hybrida “Rouge Meilland” (RM). Heatmap on top panel shows the expression levels of the six IDS candidates and RcNUDX1-1as from RNA-Seq analysis on open flowers (Stage 5) from the 10 rose cultivars and expressed as transcripts per million (TPM). Data are means from three biological replicates. Expression level for RcNUDX1-1as represents the sum of TPMs of the five RcNUDX1-1as present in the RcOB genome (7). Heatmap on bottom panel shows the geraniol levels analyzed in the flowers (Stage 5) of these 10 cultivars. Data are means from two to seven biological replicates. Pearson’s r correlation coefficients comparing the transcript levels of each gene to geraniol content in the 10 rose cultivars are shown on the right of top panel. r values with significant P values (≤0.05) are highlighted in green. (C) Subcellular localization of RcOB IDSs in conical epidermal cells of RcOB petals. Schematic diagrams of the constructs used are shown on the left with corresponding transient expression in RcOB cells on the right. CDS of RcOB IDS candidates and RcNUDX1-1a were fused with eGFP or mCherry at their C terminus as indicated. mCherry was fused to different subcellular markers for cytosol (untargeted mCherry), plastids (CD3-999), and mitochondria (CD3-991). Merge channel shows both eGFP and mCherry signals with bright field (Scale bar, 10 µm.).