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.

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.

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.

Nowadays, the combination of molecules influences their biological effects, and interesting outcomes can be obtained from different component interactions. Using a mixture design method, this research seeks to simulate the efficacy of essential oil combinations against various bacteria and forecast the ideal combination. The chemical compositions of Myrtus communis, Artemisia herba-alba and Thymus serpyllum essential oils were analyzed using CG/MS. Then, the combined antibacterial effects were evaluated by testing mixture design formulations using the microdilution bioassay. The main compounds detected for M. communis essential oil were myrtenyl acetate (33.67%), linalool (19.77%) and 1,8-cineole (10.65%). A. herba-alba had piperitone as a chemotype, representing 85%. By contrast, the T. serpyllum oil contained thymol (17.29%), γ-terpinene (18.31%) and p-cymene (36.15%). The antibacterial effect of the essential oils studied, and the optimum mixtures obtained were target strain-dependent. T. serpyllum alone ensured the optimal inhibition against S. aureus and E. coli, while a ternary mixture consisting of 17.1%, 39.6% and 43.1% of M. communis, A. herba-alba and T. serpyllum respectively, was associated with optimal inhibitory activity against B. subtilis. The outcome of this research supports the idea of the boosting effect of essential oil combinations toward better activities, giving better understanding of the usefulness of mixture designs for food, cosmetics, and pharmaceutical applications.

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.

Essential oils from Lavandula angustifolia Mill. and Lavandula x intermedia Emeric x Loisel composed of concentrated terpene mixtures, and are highly produced due to their economic value. In the Mediterranean area, the production is threatened by the increasing frequency and intensity of drought events, leading to the loss of crops. Thus, we aimed to evaluate the response to water stress and the tolerance of the main French cultivated varieties of L. angustifolia and L. x intermedia during flowering period, based on the response of their primary metabolic traits (e.g. growth, photosynthesis, stomatal conductance) and specialized metabolic traits (terpene storage and emissions related to the plant defense system). Two treatments were applied: a control where plants received 300 mL every day (approximate to 75% of substrate field capacity) and a stress where plants received 100 mL every two days (approximate to 25% of substrate field capacity). Our results showed that both species featured a drought-tolerant strategy to cope with water stress with a more competitive strategy in L. angustifolia. Water deficit modified the amounts of stored terpenes in both species which could be economically harmful for the sector related to essential oil production. Moreover, some compounds such as bornyl acetate were highlighted as potential defense compounds. The study also highlighted that the varieties 'Rapido' (L. angustifolia), arecent variety developed in France, and 'Sumian' (L. x intermedia), could be the best candidates to cultivate under intense drought.

Several studies have demonstrated the possible synergistic effect as an effective strategy to boost the bioactivity of essential oils. Using this framework, this study was conducted to effectively establish the ideal combination of six essential oils from different plants (Origanum compactum, Origanum majorana, Thymus serpyllum, Mentha spicata, Myrtus communis, and Artemisia herba-alba) that would express the best antioxidant activity. Each mixture was optimized using a mixture design approach to generate the most effective blend. The 2,2-diphenyl-1-picrylhydrazyl radical scavenging method was used as a reference method to assess the antioxidant activity. Each essential oil's composition was identified using the GC/MS method. The single essential oil activities demonstrated variable antioxidant effects, and following the mixture design approach, the optimal antioxidant blend was revealed, as two mixtures demonstrated the best antiradical activity with 79.46% obtained with the mixture of O. majorana (28%) and M. spicata (71%) and 78.8% obtained with the mixture O. compactum (64%), O. majorana (13%), and T. serpyllum (21%). This study proposes a practical way to elaborate mixtures in the search for a boosting effect that can be oriented for the food or pharmaceutical industry.

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 [...]

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.

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.

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.

Hydro-distilled essential oils from the aerial parts of Satureja briquetti, S. atlantica and S. alpina, which are growing wild in the middle Atlas Mountains of Morocco, were analyzed by GC-MS. Different compounds were identified in the three plant oils, representing 96.25 %, 98.19 % and 98.18 % of the total oil respectively. The major constituents of essential oil obtained from the plant material of S. briquetti, were borneol (27.64 %), beta-bisabolene (9.58 %), alpha-pinene (6.97 %) and linalool (6.77 %). The most abundant compounds in essential oil of S. atlantica were piperitenone oxide (24.27 %), limonene (20.57 %), pulegone (16.88 %) and cispiperitone oxide (15.55 %). Pulegone (87.74 %) was identified as the main component in the essential oil of S. alpina. The screening of antibacterial activity of essential oil samples was individually evaluated against twelve bacteria species using a disc diffusion method. In addition, the Minimal inhibitory concentration (MIC) was determined. The results showed that the essential oil of S. alpina has stronger and broader spectrum of antibacterial activity as compared to S. briquetti and S. atlantica.

Roses have high cultural and economic importance as ornamental plants and in the perfume industry. We report the rose whole-genome sequencing and assembly and resequencing of major genotypes that contributed to rose domestication. We generated a homozygous genotype from a heterozygous diploid modern rose progenitor, Rosa chinensis 'Old Blush'. Using single-molecule real-time sequencing and a meta-assembly approach, we obtained one of the most comprehensive plant genomes to date. Diversity analyses highlighted the mosaic origin of 'La France', one of the first hybrids combining the growth vigor of European species and the recurrent blooming of Chinese species. Genomic segments of Chinese ancestry identified new candidate genes for recurrent blooming. Reconstructing regulatory and secondary metabolism pathways allowed us to propose a model of interconnected regulation of scent and flower color. This genome provides a foundation for understanding the mechanisms governing rose traits and should accelerate improvement in roses, Rosaceae and ornamentals.

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.

In this work, the chemical composition, the antioxidant, and the antibacterial activities of two Moroccan essential oils less studied, extracted from Pelargonium asperum and Ormenis mixta, were investigated. According to the gas chromatography coupled to mass spectrometry analysis, citronellol (25.07%), citronellyl ester (10.52%), geraniol (10.46%), and buthyl anthranilate (5.93%) were found to be the major components of P. asperum, while O. mixta was mainly composed of D-germacrene (11.46%), 1,8-cineole (10.28%), and cis-methyl isoeugenol (9.04%). Moreover, O. mixta essential oil exhibited an important antioxidant activity being significantly higher than that exhibited by P. asperum oil (P < 0.001). As regards the antimicrobial activity of both essential oils, the zones of growth inhibition and the minimum inhibitory concentration values showed that P. asperum essential oil was more active than that of O. mixta. Thereafter, the impact of the binary combination of essential oils on their antimicrobial effect was investigated against Staphylococcus aureus using the fractional inhibitory concentration index calculation. The results showed a promising synergistic antibacterial interaction between essential oils studied.

The combination of essential oils (EOs) is a novel alternative to improve their preservative effects and to reduce their organoleptic impact in food. In this context, this work aims to investigate the antibacterial combined effect of two EOs combinations through the calculation of the fractional inhibitory concentration index. The combinations tested consists of Lavandula dentata/Origanum majorana EOs and Thymus serpyllum/Origanum majorana EOs. Their chemical compositions were identified by CG/MS analyses. The main compounds of O. majorana EO were (-) - terpinene-4-ol and trans-4-thujanol. Those of L. dentata EO were -pinene and 1,8-cineole, and those of T. serpyllum EO were p-cymene and -terpinene. Regarding the outcomes, results highlighted partial synergistic and additive interactions. Two combinations of marjoram and thyme EOs had antibacterial activities against S. aureus. The first one corresponded to the quarter of the minimum inhibitory concentration of marjoram and half that of thyme. The second one was the mix of half and quarter of the minimum inhibitory concentration of respectively marjoram and thyme EOs. This last combination also showed an antibacterial effect against E. coli. The quarter and the half of their minimum inhibitory concentration of marjoram and lavender combination, respectively, gave a partial synergy against both strains. Henceforth, these findings could be largely exploited in food preservation through the use of minimal doses of these plant products without affecting the antibacterial and the organoleptic properties in foods.

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.

Several studies have revealed the efficiency of essential oils as natural antimicrobial agents in foods. However, limited reports describe the synergistic or antagonistic effects of their combinations. The aim of this study was to investigate the combined antibacterial effect of three Moroccan essential oils (Origanum compactum, Origanum majorana and Thymus serpyllum) and to predict the optimal combination using the mixture design approach coupled to microdilution assay, for the first time to our knowledge. The chemical composition of the oils under study was also explored.The experimental antibacterial activity exhibited by the essential oils mixtures depended on the proportion of each oil in the combination and on the target strain. Moreover, the response surface analysis showed significant synergistic effects in some binary and ternary mixtures. The optimal mixture predicted against Bacillus subtilis and Staphylococcus aureus corresponded to 28%, 30% and 42% of O. compactum, O. majorana and T. serpyllum, respectively. While the optimal mixture predicted against Escherichia coli was composed by O. compactum and O. majorana essential oils at 75% and 25%, respectively. Our findings demonstrated the usefulness of mixture design in the estimation of antibacterial interaction of essential oils. The synergistic effect showed for some combinations may contribute to their successful application as natural preservatives in foods.