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This work was supported by the project VALEU (PTDC/EAM-AMB/30488/2017); by the strategic program UID/BIA/04050/2019 through the Fundação para a Ciência e a Tecnologia (FCT) I.P.; and by the European Regional Development Fund (ERDF) through the COMPETE2020-Programa Operacional Competitividade e Internacionalização (POCI). The work was also supported by a Ph.D grant (grant number PD/BD/146184/2019) to F.S.

Coniferous trees produce secondary or defense chemicals, such as terpenes, against pest insects. Terpenes could serve as constitutive or induced defensive mechanisms, defending the tree from invasive herbivores. The Mediterranean pine shoot beetle Tomicus destruens colonizes stems and branches of Pinus brutia trees and even can kill mature trees during periodic outbreaks. We investigated whether terpene profiles of needle and stem of P. brutia trees differ between health and those infested by T. destruens. We selected 20 healthy and T. destruens-infested trees and analyzed the monoterpenes and sesquiterpenes of their needles and phloem. We found higher concentrations of tricyclene, camphene and p-cymene in the phloem of infested trees. Similarly, the needles of infested trees had higher concentrations of α-pinene, β-pinene, myrcene, limonene, trans-β-caryophyllene and α-humulene than healthy trees. These results show that the monoterpene and sesquiterpene profiles of P. brutia trees differed between healthy and infested trees, suggesting that volatile terpenes may be an important part of plant-induced responses against T. destruens.

Background Zataria multiflora Boiss. is a medicinal and aromatic plant from the Lamiaceae family. It is extensively used in Iranian traditional medicine, mostly as a replacement for Thyme species. This study was focused on the analysis of chemical composition and the distribution and types of trichomes of Z. multiflora grown under different conditions. Equilibrium headspace analysis in combination with GC-FID-MS was used to identify volatile compounds released by aerial parts of Z. multiflora in development stages of 50 and 100% flowering under normal and drought-stress conditions. Results The main constituents were p-cymene (20.06–27.40%), γ-terpinene (12.44–16.93%), and α-pinene (6.91–16.58%) and thymol (8.52–9.99%). The highest content of p-cymene (27.40%) and thymol (9.99%) was observed in the 50% flowering stage at the 90% field capacity, while the maximum γ-terpinene (16.93%) content was recorded in the 100% flowering stage under normal conditions. Using the SEM method, it was found that peltate glandular and non-glandular trichomes are distributed on the surface of the leaf, stem, and outer side of the calyx. However, capitate trichomes only are detected on the stem and calyx in the 100% flowering and beginning of blooming stages, respectively. The type and structure of trichomes do not vary in different development stages, but they differ in density. The highest number of leaf peltate glandular trichomes was observed in the vegetative and beginning of blooming stages at 50% and 90% field capacity, respectively. Non-glandular trichomes of the stem were observed with high density in both normal and stress conditions, which are more densely in 90% field capacity. Conclusions Since this plant has strong potential to be used in the food and pharmacological industries, this study provides valuable information for its cultivation and harvesting at specific phenological stages, depending on desired compounds and their concentrations.

(−)-Carvone is a monoterpenoid with a spearmint flavor. A sustainable biotechnological production process for (−)-carvone is desirable. Although all enzymes in (−)-carvone biosynthesis have been functionally expressed in Escherichia coli independently, the yield was low in previous studies. When cytochrome P450 limonene-6-hydroxylase (P450)/cytochrome P450 reductase (CPR) and carveol dehydrogenase (CDH) were expressed in a single strain, by-product formation (dihydrocarveol and dihydrocarvone) was detected. We hypothesized that P450 and CDH expression levels differ in E. coli . Thus, two strains independently expressing P450/CPR and CDH were mixed with different ratios, confirming increased carvone production and decreased by-product formation when CDH input was reduced. The optimum ratio of enzyme expression to maximize (−)-carvone production was determined using the proteome analysis quantification concatamer (QconCAT) method. Thereafter, a single strain expressing both P450/CPR and CDH was constructed to imitate the optimum expression ratio. The upgraded strain showed a 15-fold improvement compared to the initial strain, showing a 44 ± 6.3 mg/L (−)-carvone production from 100 mg/L (−)-limonene. Our study showed the usefulness of the QconCAT proteome analysis method for strain development in the industrial biotechnology field.

The monooxygenase activity of engineered CYP102A1 on α-terpineol was investigated. CYP102A1 M850 mutant (F11Y/R47L/D68G/F81I/F87V/E143G/L188Q/E267V/H408R) showed the highest catalytic activity toward α-terpineol among the engineered mutants produced by random mutagenesis. The major product (P1) of α-terpineol, p -menth-1-ene-3,8-diol, was characterized by high-performance liquid chromatography, gas-chromatography mass spectrometry, and nuclear magnetic resonance spectroscopy. Three minor products (P2–P4) of α-terpineol were considered as 6-hydroxy-α,α,4-trimethyl-3-cyclohexene-1-methanol (P2), trans -sobrerol (P3), and carvone hydrate (P4). Optimal conditions for product formation were determined as pH 7.0 and 30 °C. Production of p -menth-1-ene-3,8-diol was 0.87 mM at 1 h. Structure modeling using PyMOL and CAVER Web 1.2 server indicated that several mutations of CYP102A1 M850 were involved in access tunnels and active sites, resulting in increased activity toward α-terpineol. The major product, p -menth-1-ene-3,8-diol, of α-terpineol was produced by engineered CYP102A1 M850 via regioselective carbon hydroxylation. The engineered CYP102A1 could be a suitable biocatalyst for producing α-terpineol derivatives.

Main conclusionThe transcription factor LiNAC100 has a novel function of regulating floral fragrance by directly regulating linalool synthase gene LiLiS.Lilium ‘Siberia’, an Oriental hybrid, is renowned as both a cut flower and garden plant, prized for its color and fragrance. The fragrance comprises volatile organic compounds (VOCs), primarily monoterpenes found in the plant. While the primary terpene synthases in Lilium ‘Siberia’ were identified, the transcriptional regulation of these terpene synthase (TPS) genes remains unclear. Thus, understanding the regulatory mechanisms of monoterpene biosynthesis is crucial for breeding flower fragrance, thereby improving ornamental and commercial values. In this study, we isolated a nuclear-localized LiNAC100 transcription factor from Lilium ‘Siberia’. The virus-induced gene silencing (VIGS) of LiNAC100 was found to down-regulate the expression of linalool synthase gene (LiLiS) and significantly inhibit linalool synthesis. Conversely, transient overexpression of LiNAC100 produced opposite effects. Additionally, yeast one-hybrid and dual-luciferase assays confirmed that LiNAC100 directly activates LiLiS expression. Our findings reveal that LiNAC100 plays a key role in monoterpene biosynthesis in Lilium ‘Siberia’, promoting linalool synthesis through the activation of LiLiS expression. These results offer insights into the molecular mechanisms of terpene biosynthesis in Lilium ‘Siberia’ and open avenues for biotechnological enhancement of floral scent.

Background Cinnamomum camphora is a valuable aromatic oil-producing species with significant economic and industrial importance. Linalool, a monoterpenoid compound and a major component of camphor leaf essential oil, is widely used in cosmetics, food, and pharmaceuticals. While microRNAs (miRNAs) are known to regulate terpenoid biosynthesis, their regulatory role in linalool biosynthesis remains largely unexplored. Results In this study, we performed small RNA and degradome sequencing on three C. camphora samples (H_MAR, H_MAY, and L_MAY) exhibiting significant differences in linalool content. A total of 199 known and 200 novel miRNAs were identified. Among them, 170 differentially expressed miRNAs (DEMs; 83 downregulated and 87 upregulated) were detected between H_MAY and H_MAR, whereas 77 DEMs (45 downregulated and 32 upregulated) were found between H_MAY and L_MAY. Degradome analysis predicted 223 target genes for 52 known miRNAs and 86 targets for 39 novel miRNAs. Network analysis revealed that the miRNA-SPL module may play a critical role in indirectly regulating linalool biosynthesis. Conversely, the miR167- Cca.gene21941 ( GPPS ) module may directly regulate monoterpene biosynthesis in linalool-type C. camphora . Additionally, miR5368 was found to target Cca.gene21642 (DHDDS) and Cca.gene34720 (GGDR), both of which may contribute directly to linalool biosynthesis. Conclusion These findings enhance the understanding of post-transcriptional regulation in linalool biosynthesis and provide insights for developing genetic improvement strategies for C. camphora .

Persian cumin ( Bunium persicum ) is a widely used plant in Iranian cuisine, valued for its aromatic and therapeutic compounds, primarily monoterpenoids such as γ-terpinene, cuminaldehyde, and p-cymene. Many ambiguities surround the exact process by which monoterpenoids are synthesized in plants. This study investigates the key genes and biosynthetic pathways involved in monoterpenoid production during fruit development. To achieve this, RNA sequencing and GC-MS analysis were performed on two tissues (inflorescence and stem) to identify differentially expressed genes associated with volatile compound biosynthesis. Our findings revealed four highly expressed genes, terpinene synthetase (TPS1, and TPS2), and two geraniol hydroxylases—correlated with γ-terpinene and cuminaldehyde production. We also propose that cuminaldehyde biosynthesis from p-cymene likely involves two hydroxylation steps mediated by cytochrome P450 enzymes (CYP76B) family genes. Additionally, we identified other candidate genes, including those from the P450 family, alcohol dehydrogenases, and hydroxylases, that may contribute to the pathway of cuminaldehyde biosynthesis. Ultimately, this study presents a comprehensive analysis of Bunium persicum ’s transcriptome and proposes potential key genes involved in the biosynthesis of cuminaldehyde and γ-terpinene.

The citral-type is the most common chemotype in Cinnamomum bodinieri Levl ( C. bodinieri ), which has been widely used in the daily necessities, cosmetics, biomedicine, and aromatic areas due to their high citral content. Despite of this economic prospect, the possible gene-regulatory roles of citral biosynthesis in the same geographic environment remains unknown. In this study, the essential oils (EOs) of three citral type (B1, B2, B3) and one non-citral type (B0) varieties of C. bodinieri were identified by GC-MS after hydrodistillation extraction in July. 43 components more than 0.10% were identified in the EOs, mainly composed of monoterpenes (75.8–91.84%), and high content citral (80.63–86.33%) were identified in citral-type. Combined transcriptome and metabolite profiling analysis, plant-pathogen interaction(ko04626), MAPK signaling pathway-plant(ko04016), starch and sucrose metabolism(ko00500), plant hormone signal transduction(ko04075), terpenoid backbone biosynthesis (ko00900) and monoterpenoid biosynthesis (ko00902) pathways were enriched significantly. The gene expression of differential genes were linked to the monoterpene content, and the geraniol synthase ( CbGES ), alcohol dehydrogenase ( CbADH ), geraniol 8-hydroxylase-like ( CbCYP76B6-like ) and 8-hydroxygeraniol dehydrogenase ( Cb10HGO ) were upregulated in the citral-type, indicating that they were associated with high content of geraniol and citral. The activities of CbGES and CbADH in citral type were higher than in non-citral type, which was corroborated by enzyme-linked immunosorbent assay ( ELISA ). This study on the accumulation mechanism of citral provides a theoretical basis for the development of essential oil of C. bodinieri .

Mint oil is a key source of natural flavors with wide industrial applications. Two unbalanced polyploid cultivars named Native ( Mentha Spicata L) and Scotch ( M.  ×  gracilis Sole) are the main producers of spearmint type oil, which is characterized by high levels of the monoterpenes (−)-carvone and (−)-limonene. These cultivars have been the backbone of spearmint oil production for decades, while breeding and improvement remained largely unexplored, in part, due to sterility in cultivated lines. Here we show that sexual breeding at the diploid level can be leveraged to develop new varieties that produce spearmint type oil, along with the improvement of other important traits. Using field trials and GC-FID oil analysis we characterized plant materials from a public germplasm repository and identified a diploid accession that exhibited 89.5% increase in oil yield, compared to the industry standard, and another that produces spearmint type oil. Spearmint-type oil was present at high frequency in a segregating F 2 population (32/160) produced from these two accessions. Field-testing of ten of these F 2 lines showed segregation for oil yield and confirmed the production of spearmint-type oil profiles. Two of these lines combined high yield and spearmint-type oil with acceptable analytic and sensory profiles. These results demonstrate that spearmint-type oil can be produced in a diploid background with high yield potential, providing a simpler genetic system for the development of improved spearmint varieties.