Essential oils (EOs) are a potential source of biologically active molecules. Knowing exactly the constituents of an EO is fundamental to verifying its quality and predicting its biological potential and toxicity. Thus, the objective of this work is to determine the chemical constituents and the chemotype of the EO of Otanthus maritimus (L.) Hoffmanns. & Link growing wild in Zemmouri (Boumerdes, Algeria). EOs from the aerial flowering parts of two harvests, December and February, of O. maritimus, were isolated by hydrodistillation in a 0.19% and 0.14% yield, respectively. Gas chromatography-mass spectrometry analysis allows the identification of both EOs 36 constituents representing 87.35%-92.7% of the total oil composition dominated by irregular sesquiterpenes 35.05% and 29.34%, respectively. In the EO of the December harvest, the major compounds were α-isocomene (15.75%), β-isocomene (7.92%), artemisyl acetate (6.63%) and α-epi-bisabolol (6.16%). Whereas, α-isocomene (18.73%), artemisyl acetate (9.65%), α-epi-bisfabolol (8.96%), germacrene-D (7.13%) and β-isocomene (6.63%) were dominant in the EO of February harvest. This is the first report of the α-isocomene present as the first main component in the EO of this plant in parallel with an exceptional richness in α-epi-bisabolol, which has never been detected previously, which justifies that it is another different chemotype of O. maritimus from Algeria.

Sesquiterpenes are key molecules nurturing the complex and subtle base notes of pelargonium fragrances. Yet, their contribution to the essential oil olfactory profile and their biosynthesis are far from being well understood. We therefore explored sesquiterpene composition in 10 pelargonium accessions and revealed that, although lowly accumulated, their wide diversity participates to the biochemical uniqueness of each fragrance. We further investigated sesquiterpene biosynthesis thanks to a multiomic approach, integrating transcriptomic together with metabolomic data. A phylogenetic analysis of pelargonium TPS-a subfamily first revealed a total of 21 groups of orthology and denoted a strong transcriptional regulation of related enzymes. Seven sesquiterpene synthases were then wisely selected for functional characterization, both in vitro and in planta. Among them, two 6,9-guaidiene synthases were characterized for the first time in plants. Finally, we explored the role of amino acids located in the active site of sesquiterpene synthases in the enzymatic mechanism using site-directed mutagenesis. Altogether, this work highlights the great potential of the multiomic approach to predict TPS functions, but also exposes its limitations, inherent to terpene synthase's plasticity.

Proton Transfer Reaction Time-of-Flight Mass Spectrometry (PTR-TOF-MS) provides high-resolution, real-time tracking of volatile organic compounds (VOCs), yet its analytical workflows for complex mixtures are often underexplored. This study presents a flexible data analysis workflow tailored for PTR-TOF-MS, designed to manage datasets with multiple replicates and experimental factors efficiently. Using lavender plants (Lavandula angustifolia Mill. var. diva) in a controlled greenhouse environment, we analyzed BVOC emissions over a 24-hour cycle. The workflow integrates data importation and optimization via the provoc R package, utilizing Multivariate Curve Resolution (MCR) to decompose spectra into biologically meaningful components and Redundancy Analysis (RDA) to assess component relevance. For this specific model experiment, we determined that normalization using the RUVr method and using and setting the MCR on a number of five components gave the most accurate results. However, this configuration is optimized for the current dataset; other experimental designs may require different normalization methods or numbers of components to achieve optimal results. The workflow enables effective differentiation of VOC emission patterns depending on experimental factors as well as an effective normalization method selection. It provides a systematic approach to PTR-TOF-MS data interpretation, adaptable to various experimental designs and scientific questions, making it valuable for studying complex VOC mixtures in chemical ecology and metabolomics. This method supports long-term, non-destructive sampling while delivering precise VOC profiling.

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 Citronellol is a monoterpene alcohol biosynthesized by various plant species belonging to different families of Angiosperm. Bioinspired by the metabolism of Rosa sp., able to produce (–)-cis-rose oxide from citronellol, we have studied and optimized a laccase-catalyzed oxidation of (±)-, (R)-, and (S)-citronellol into rose oxide diastereomers in the presence of mediators. The reaction was found to be diastereomerically cis-selective but completely non-enantioselective. The laccase-mediator system was then applied on citronellol-containing essential oils such as lemongrass (Cymbopogon citratus) and geranium (Pelargonium graveolens) essential oils in order to modify their composition beyond the plant metabolism and increase their rose oxides content, thereby tuning their olfactory properties.

Lavandula angustifolia Mill. or true lavender is an emblematic plant from dry and calcareous hills of the south of France. Lavender essential oils obtained from inflorescences harvested at bloom stage are mainly composed of monoterpenes and few sesquiterpenes. The characteristic lavender smell is due to linalool and linalyl acetate that represent more than half of the emitted volatile organic compound (VOC) quantity. These monoterpenoids are derived from plastidial pathway precursors. The regulation of this pathway in lavender is mainly managed by the successive expression of two DXS during flower development. In the light of 10 years of published data on lavender, it appears that biochemical pathways of main terpenes are mostly depicted. Several transcriptomic libraries are available and a genome at chromosome scale has been assembled and molecular markers developed. The diversity of lavender terpenes and their biosynthesis are reviewed with additional original data regarding the anatomical glandular structures and expression of genes involved in IPP biosynthesis.

As requested by the Editorial Office, the authors remove the scientific consortium “Camille Nous” from the author list and the Author Contributions section in the published paper [...]

Abstract Ammodaucus leucotrichus is one of the desert aromatic plants most commonly used in Algerian and Moroccan traditional medicine. The plant is a rich source of bioactive compounds especially essential oils (EOs). In this work, we reported for the first time the characterization of the chemical composition of EOs from vegetative and reproductive organs of this species. Root, stem/leaf, flower and fruit EOs were obtained by Clevenger-hydrodistillation and analyzed by gas chromatography-mass spectrometry (GC-MS). The percentage yield of EOs varied between 0.52 and 3.04 % (w/w, based on dry weight). ATotal of 89 (90.0-97.7 %) components were identified. The EO chemical profiles of plant organs were different from each other exhibited a significant chemo-diversity. (Z)-falcarinol (24.3 %), β-bisabolene (19.6 %) and myristicin (14.0 %) were the major constituents in root oil; methyl eugenol (33.5 %), α-pinene (26.3 %) and muurola-4,10(14)-dien-1-β-ol (10.5 %) in stem/leaf oil; methyl eugenol (24.9 %), β-bisabolene (16.0 %) and geranyl acetate (14.5 %) in flower oil; perillaldehyde (53.7 %) and limonene (25.3 %) in fruit oil.

The true lavender Lavandula angustifolia Miller is a Mediterranean aromatic shrub widely cultivated for its high quality essential oil used in perfumery and phytotherapy. Despite its economic importance, the intra-specific diversity among wild, non-cultivated plants remains poorly understood. We analyzed the structure of the chemical and genetic diversity of plants from 14 sites sampled over the entire native range of the true lavender. Volatile organic compounds of inflorescences were analyzed using gas chromatography coupled to mass spectrometry. Genotyping was performed with fingerprinting genetic markers. To limit the influence of environmental variability on chemical composition, plants were grown in the same conditions in a common garden. Without prior knowledge, discriminant analysis of principal component identified unambiguously four distinct chemotypes among three genetic populations. Co-inertia analysis and supervised analysis which integrated multiple datasets indicated a strong congruency between chemical and genetic patterns. Two distinct genetic units were located at the edge of the distribution area in the south of Italy and in the northeast of Spain, and were associated with two distinct chemotypes. Our results confirmed the existence of three genetically distinct entities, suggesting speciation. All French populations and the Italian Piedmontese population were genetically homogeneous but separated in two distinct chemotypes. The dominant chemotype was present in the center of the native range in southeastern France and was at the origin of the current most cultivated French varieties. Its main compounds were linalyl acetate, linalool, and caryophyllene oxide. The second French chemotype was found in south of Massif Central and presented high abundance of valuable linalyl and lavandulyl acetates. Linalool, eucalyptol, β-caryophyllene, borneol, camphor, and cis-sabinene-hydrate were significantly associated with southern latitudes and their role would be worth exploring.

Background Myrtus communis L. is an aromatic evergreen plant common in Morocco. In addition to its culinary uses, it has been used medicinally as a disinfectant, an antiseptic or as a hypoglycemic agent. However, its cytotoxic activity has not been well investigated so far. The current study describes the chemical composition, cytotoxic and antioxidant activities of Myrtus communis L essential oil obtained from different regions of Morocco. Methods Myrtus communis essential oils were obtained by hydrodistillation, and analyzed by gas chromatography coupled with mass spectrometry. Cytotoxic activity was evaluated in murine mastocytoma P815 and MCF-7 breast cancer cells, using the MTT assay. In addition, DNA fragmentation was assessed by gel electrophoresis. The antioxidant effect was determined by measuring bleaching of β-carotene with the linoleic acid and the DPPH radical scavenging methods. Results GC-MS analysis showed high amounts of methyl eugenol (18.7%), α-terpineol (15.5%) and geranyl acetate (11.64%) in essential oil from the Benslimane region. In contrast, essential oil from Ouazzane was particularly rich in 1,8-cineole (36.3%). The cytotoxicity results showed that MCF-7 cells were more sensitive than P815 cells to the essential oils from Ouazzane and Benslimane regions with IC50 values of 4 and 6.25 µg/mL, respectively. Moreover, this cytotoxicity was partly associated with DNA fragmentation, which is one of the characteristics of apoptosis. The tested essential oils did not show strong antioxidant activity. Conclusions Myrtus communis L. essential oil exhibits a weak antioxidant effect, but induced remarkable cytotoxic activity by a mechanism related to apoptosis, suggesting a possible application of the bioactive compounds as natural anticancer compounds.

The rose is the queen of flowers and is widely used as a garden plant and for the cut flower market. Roses are also used for the production of essential oil for the cosmetic and perfume industries. A lot of botanical roses are scented and use their volatiles to attract pollinators. Fragrances in garden roses are very diverse and scent has always been an important character in the selection process. Breeders have recently tried to introduce new fragrances, for instance reminiscent of fruit or spice odors. But despite their efforts, some roses on the market are not very fragrant, especially the ones selected for the cut flower market. The cause of this lack of scent is not known. In modern roses or Hybrid Tea roses, scent is mainly produced by petals, although stamens can also contribute to the emission of volatiles. Scent compounds are concentrated in rose petal epidermis, probably in small lipid droplets. In spite of numerous chemical studies, the biosynthetic pathways of many rose scent compounds are unknown. We are studying several genes involved in the biosynthesis of scent in rose. For example, we recently characterized the enzymes responsible for the socalled "tea scent" emitted by Chinese roses. Apart from "tea scent", terpenoids, especially monoterpenes, are also major constituents of rose flowers, mostly responsible for the "typical rose scent". Generally, terpenoid biosynthesis in plants is achieved by various terpene synthases. However, with a combination of transcriptomic and genetic approaches, our research group recently discovered a terpene synthase-independent pathway. A key enzyme of this pathway is RhNUDX1, belonging to the Nudix protein family. It has geranyl diphosphate diphosphohydrolase activity in vitro. A positive correlation was found between the expression levels of RhNUDX1 gene and the production of the monoterpene geraniol, indicating its essential role in scent production in roses.

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.

The genus Lavandula L. (Lamiaceae) comprises 40 species, of which only a few are used to extract essential oils for perfume and detergent industries. To date, the compound diversity of Lavandula essential oils, and most notably terpene variation amongst species, is poorly documented. In this work, we report the diversity of terpene content in 33 accessions from the subgenera Lavandula and Fabricia. We used these terpenes to distinguish subgenera and sections and to find autapomorphic and synapomorphic traits as taxonomic markers. We demonstrate that terpene composition is not appropriate for phylogenetic reconstruction and chemotaxonomy in Lavandula, but we show nevertheless that terpene diversity allow for distinguishing the two subgenera, Lavandula and Fabricia and for separating the sections, respectively i/Dentatae, Stoechas and Lavandula, and ii/Pterostoechas, Chaetostachys, Subnudae and Hasikenses. Furthermore, amongst terpenes with putative ecological roles, we found that camphor, 1,8-cineole and carvacrol are synapomorphic traits, or at least markers respective of each subgenus, that trans-α-necrodol is an autapomorphic trait of L. stoechas subsp. luisieri and that linalool is either a symplesiomorphic trait or a homoplasic trait. Based on these results, we propose evolutionary hypotheses and future work directions relative to the putative ecological role of these terpenes in other species and to terpene biosynthesis studies in Lavandula.

The purpose of this study was to elucidate the chemical composition of essential oil from Lavandula stoechas and its antibacterial activity against two phytopathogenic bacteria, Erwinia amylovora and Pectobacterium carotovorum subsp. carotovorum. These bacteria are respectively isolated from Algerian apples and potatoes. The Clevenger-hydrodistilled essential oil from dried flowers showed a good yield (1.13 ± 0.11 % w/w). Chemical composition of the oil was determined by capillary gas chromatography-mass spectrometry (GC-MS) from which 75 compounds were identified corresponding to 95.21% of the oil. The main constituents were fenchone (40.78%), camphor (9.76%), myrtenyl acetate (8.94%) and bornyl acetate (5.10%). The antibacterial effect evaluated by disk diffusion method revealed that the essential oil of flowers of Lavandula stoechas exhibited a good inhibitory activity with MIC values between 1.25 and 20 μg/ml.

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.

MAIN CONCLUSION:The use of a VIGS approach to silence the newly characterized apple tree SQS isoforms points out the biological function of phytosterols in plastid pigmentation and leaf development. Triterpenoids are beneficial health compounds highly accumulated in apple; however, their metabolic regulation is poorly understood. Squalene synthase (SQS) is a key branch point enzyme involved in both phytosterol and triterpene biosynthesis. In this study, two SQS isoforms were identified in apple tree genome. Both isoforms are located at the endoplasmic reticulum surface and were demonstrated to be functional SQS enzymes using an in vitro activity assay. MdSQS1 and MdSQS2 display specificities in their expression profiles with respect to plant organs and environmental constraints. This indicates a possible preferential involvement of each isoform in phytosterol and/or triterpene metabolic pathways as further argued using RNAseq meta-transcriptomic analyses. Finally, a virus-induced gene silencing (VIGS) approach was used to silence MdSQS1 and MdSQS2. The concomitant down-regulation of both MdSQS isoforms strongly affected phytosterol synthesis without alteration in triterpene accumulation, since triterpene-specific oxidosqualene synthases were found to be up-regulated to compensate metabolic flux reduction. Phytosterol deficiencies in silenced plants clearly disturbed chloroplast pigmentation and led to abnormal development impacting leaf division rather than elongation or differentiation. In conclusion, beyond the characterization of two SQS isoforms in apple tree, this work brings clues for a specific involvement of each isoform in phytosterol and triterpene pathways and emphasizes the biological function of phytosterols in development and chloroplast integrity. Our report also opens the door to metabolism studies in Malus domestica using the apple latent spherical virus-based VIGS method.

Lavender essential oils (EOs) of higher quality are produced by a few Lavandula angustifolia cultivars and mainly used in the perfume industry. Undesirable compounds such as camphor and borneol are also synthesized by lavender leading to a depreciated EO. Here, we report the cloning of bornyl diphosphate synthase of lavender (LaBPPS), an enzyme that catalyzes the production of bornyl diphosphate (BPP) and then by-products such as borneol or camphor, from an EST library. Compared to the BPPS of Salvia officinalis, the functional characterization of LaBPPS showed several differences in amino acid sequence, and the distribution of catalyzed products. Molecular modeling of the enzyme's active site suggests that the carbocation intermediates are more stable in LaBPPS than in SoBPPS leading probably to a lower efficiency of LaBPPS to convert GPP into BPP. Quantitative RT-PCR performed from leaves and flowers at different development stages of L. angustifolia samples show a clear correlation between transcript level of LaBPPS and accumulation of borneol/camphor, suggesting that LaBPPS is mainly responsible of in vivo biosynthesis of borneol/camphor in fine lavender. A phylogenetic analysis of terpene synthases (TPS) pointed out the basal position of LaBPPS in the TPSb clade, suggesting that LaBPPS could be an ancestor of others lavender TPSb. Finally, borneol could be one of the first monoterpenes to be synthesized in the Lavandula subgenus. Knowledge gained from these experiments will facilitate future studies to improve the lavender oils through metabolic engineering or plant breeding. Accession numbers: LaBPPS: KM015221.

Les graminées constituent un vaste ensemble de plantes, pour la plupart herbacées, comprenant en France deux groupes d’inégale importance pour le nombre de représentants. Le premier groupe comporte deux genres avec chacun une espèce : Zea maïs (le maïs, originaire d’Amérique du Sud qui est cultivé) et Coix lacrymajobi (la larme de Job, originaire des régions tempérées-chaudes d'Asie qui est naturalisée aux environs de Nice). Ce sont des plantes monoïques ne présentant que des fleurs unisexuées, formant deux types d’inflorescences séparées. Le deuxième groupe (qui va nous intéresser) comporte tous les genres et toutes les espèces qui sont caractérisés par des fleurs complètes (= hermaphrodites), parfois accompagnées de fleurs mâles stériles.

The genus Lavandula L. consists of 39 species distributed from the North Atlantic Islands, across the Mediterranean Basin to India. We analysed 36 taxa of the genus Lavandula representing two of the three subgenera and six of the eight sections according to the most recent classification (Upson & Andrews 2004). We achieved a phylogenetic reconstruction from partial sequences from plastid trnK and matK genes; the genome size was estimated by flow cytometer measurements. The primary aim was to track phylogenetic patterns through the maternal inherited marker at the sectional level and identify possible genome duplications. The cpDNA tree shows the phylogenetic relationships between subgenus, sections and also elucidates for the first time the relationships between the endemic species of Macaronesia, Morocco and Arabia. The ancestral split between the two subgenera could be explained by dispersal followed by an early vicariance event. The C-value shows genome up-sizing within several phylogenetic clades and geographical areas. An ancestral genome-up sizing is characterized at the node of section Dentatae and Lavandula. The cpDNA tree suggests that the taxa L. angustifolia subsp. pyrenaica (DC.) Guinea and L. stoechas subsp. luiseiri are best treated as a distinct species.