Isoprenyl diphosphate synthases of terpenoid biosynthesis in rose-scented geranium (Pelargonium graveolens).

The essential oil of Pelargonium graveolens (rose-scented geranium), an important aromatic plant, comprising mainly mono- and sesqui-terpenes, has applications in food and cosmetic industries. This study reports the characterization of isoprenyl disphosphate synthases (IDSs) involved in P. graveolens terpene biosynthesis. The six identified PgIDSs belonged to different classes of IDSs, comprising homomeric geranyl diphosphate synthases (GPPSs; PgGPPS1 and PgGPPS2), the large subunit of heteromeric GPPS or geranylgeranyl diphosphate synthases (GGPPSs; PgGGPPS), the small subunit of heteromeric GPPS (PgGPPS.SSUI and PgGPPS.SSUII), and farnesyl diphosphate synthases (FPPS; PgFPPS).All IDSs exhibited maximal expression in glandular trichomes (GTs), the site of aroma formation, and their expression except PgGPPS.SSUII was induced upon treatment with MeJA. Functional characterization of recombinant proteins revealed that PgGPPS1, PgGGPPS and PgFPPS were active enzymes producing GPP, GGPP/GPP, and FPP respectively, whereas both PgGPPS.SSUs and PgGPPS2 were inactive. Co-expression of PgGGPPS (that exhibited bifunctional G(G)PPS activity) with PgGPPS.SSUs in bacterial expression system showed lack of interaction between the two proteins, however, PgGGPPS interacted with a phylogenetically distant Antirrhinum majus GPPS.SSU. Further, transient expression of AmGPPS.SSU in P. graveolens leaf led to a significant increase in monoterpene levels. These findings provide insight into the types of IDSs and their role in providing precursors for different terpenoid components of P. graveolens essential oil.

Evidence for Reductions in Physical and Chemical Plant Defense Traits in Island Flora.

Reduced defense against large herbivores has been suggested to be part of the "island syndrome" in plants. However, empirical evidence for this pattern is mixed. In this paper, we present two studies that compare putative physical and chemical defense traits from plants on the California Channel Islands and nearby mainland based on sampling of both field and common garden plants. In the first study, we focus on five pairs of woody shrubs from three island and three mainland locations and find evidence for increased leaf area, decreased marginal leaf spines, and decreased concentrations of cyanogenic glycosides in island plants. We observed similar increases in leaf area and decreases in defense traits when comparing island and mainland genotypes grown together in botanic gardens, suggesting that trait differences are not solely driven by abiotic differences between island and mainland sites. In the second study, we conducted a common garden experiment with a perennial herb-Stachys bullata (Lamiaceae)-collected from two island and four mainland locations. Compared to their mainland relatives, island genotypes show highly reduced glandular trichomes and a nearly 100-fold reduction in mono- and sesquiterpene compounds from leaf surfaces. Island genotypes also had significantly higher specific leaf area, somewhat lower rates of gas exchange, and greater aboveground biomass than mainland genotypes across two years of study, potentially reflecting a broader shift in growth habit. Together, our results provide evidence for reduced expression of putative defense traits in island plants, though these results may reflect adaptation to both biotic (i.e., the historical absence of large herbivores) and climatic conditions on islands.

In Pursuit of Optimal Quality: Cultivar-Specific Drying Approaches for Medicinal Cannabis.

A limited number of studies have examined how drying conditions affect the cannabinoid and terpene content in cannabis inflorescences. In the present study, we evaluated the potential of controlled atmosphere drying chambers for drying medicinal cannabis inflorescence. Controlled atmosphere drying chambers were found to reduce the drying and curing time by at least 60% compared to traditional drying methods, while preserving the volatile terpene content. On the other hand, inflorescences subjected to traditional drying were highly infested by Alternaria alternata and also revealed low infestation of Botrytis cinerea. In the high-THC chemovar ("240"), controlled N2 and atm drying conditions preserved THCA concentration as compared to the initial time point (t0). On the other hand, in the hybrid chemovar ("Gen12") all of the employed drying conditions preserved THCA and CBDA content. The optimal drying conditions for preserving monoterpenes and sesquiterpenes in both chemovars were C5O5 (5% CO2, 5% O2, and 90% N2) and pure N2, respectively. The results of this study suggest that each chemovar may require tailored drying conditions in order to preserve specific terpenes and cannabinoids. Controlled atmosphere drying chambers could offer a cost-effective, fast, and efficient drying method for preserving cannabinoids and terpenes during the drying process while reducing the risk of mold growth.

Interactions of Opuntia ficus-indica with Dactylopius coccus and D. opuntiae (Hemiptera: Dactylopiidae) through the Study of Their Volatile Compounds.

Opuntia ficus-indica has always interacted with many phytophagous insects; two of them are Dactylopius coccus and D. opuntiae. Fine cochineal (D. coccus) is produced to extract carminic acid, and D. opuntiae, or wild cochineal, is an invasive pest of O. ficus-indica in more than 20 countries around the world. Despite the economic and environmental relevance of this cactus, D. opuntiae, and D. coccus, there are few studies that have explored volatile organic compounds (VOCs) derived from the plant-insect interaction. The aim of this work was to determine the VOCs produced by D. coccus and D. opuntiae and to identify different VOCs in cladodes infested by each Dactylopius species. The VOCs (essential oils) were obtained by hydrodistillation and identified by GC-MS. A total of 66 VOCs from both Dactylopius species were identified, and 125 from the Esmeralda and Rojo Pelón cultivars infested by D. coccus and D. opuntiae, respectively, were determined. Differential VOC production due to infestation by each Dactylopius species was also found. Some changes in methyl salicylate, terpenes such as linalool, or the alcohol p-vinylguaiacol were related to Dactylopius feeding on the cladodes of their respective cultivars. Changes in these VOCs and their probable role in plant defense mechanisms should receive more attention because this knowledge could improve D. coccus rearing or its inclusion in breeding programs for D. opuntiae control in regions where it is a key pest of O. ficus-indica.

Improved Long-Term Preservation of Cannabis Inflorescence by Utilizing Integrated Pre-Harvest Hexanoic Acid Treatment and Optimal Post-Harvest Storage Conditions.

The effort to maintain cannabinoid and terpene levels in harvested medicinal cannabis inflorescence is crucial, as many studies demonstrated a significant concentration decrease in these compounds during the drying, curing, and storage steps. These stages are critical for the preparation and preservation of medicinal cannabis for end-use, and any decline in cannabinoid and terpene content could potentially reduce the therapeutic efficacy of the product. Consequently, in the present study, we determined the efficacy of pre-harvest hexanoic acid treatment alongside four months of post-harvest vacuum storage in prolonging the shelf life of high THCA cannabis inflorescence. Our findings indicate that hexanoic acid treatment led to elevated concentrations of certain cannabinoids and terpenes on the day of harvest and subsequent to the drying and curing processes. Furthermore, the combination of hexanoic acid treatment and vacuum storage yielded the longest shelf life and the highest cannabinoid and mono-terpene content as compared to all other groups studied. Specifically, the major cannabinoid's-(-)-Δ9-trans-tetrahydrocannabinolic acid (THCA)-concentration decreased by 4-23% during the four months of storage with the lowest reduction observed following hexanoic acid pre-harvest treatment and post-harvest vacuum storage. Hexanoic acid spray application displayed a more pronounced impact on mono-terpene preservation than storage under vacuum without hexanoic acid treatment. Conversely, sesqui-terpenes were observed to be less prone to degradation than mono-terpenes over an extended storage duration. In summation, appropriate pre-harvest treatment coupled with optimized storage conditions can significantly extend the shelf life of cannabis inflorescence and preserve high active compound concentration over an extended time period.

Eucalypt Extracts Prepared by a No-Waste Method and Their 3D-Printed Dosage Forms Show Antimicrobial and Anti-Inflammatory Activity.

The pharmaceutical industry usually utilizes either hydrophobic or hydrophilic substances extracted from raw plant materials to prepare a final product. However, the waste products from the plant material still contain biologically active components with the opposite solubility. The aim of this study was to enhance the comprehensive usability of plant materials by developing a new no-waste extraction method for eucalypt leaves and by investigating the phytochemical and pharmacological properties of eucalypt extracts and their 3D-printed dosage forms. The present extraction method enabled us to prepare both hydrophobic soft extracts and hydrophilic (aqueous) dry extracts. We identified a total of 28 terpenes in the hydrophobic soft extract. In the hydrophilic dry extract, a total of 57 substances were identified, and 26 of them were successfully isolated. The eucalypt extracts studied showed significant antimicrobial activity against Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans, Corynebacterium diphtheriae gravis, and Corynebacterium diphtheriae mitis. The anti-inflammatory activity of the dry extract was studied using a formalin-induced-edema model in mice. The maximum anti-exudative effect of the dry extract was 61.5% at a dose of 20 mg/kg. Composite gels of polyethylene oxide (PEO) and eucalypt extract were developed, and the key process parameters for semi-solid extrusion (SSE) 3D printing of such gels were verified. The SSE 3D-printed preparations of novel synergistically acting eucalypt extracts could have uses in antimicrobial and anti-inflammatory medicinal applications.

Evaluation of the Antimicrobial Activity of Geraniol and Selected Geraniol Transformation Products against Gram-Positive Bacteria.

Both geraniol and the products of its transformation, thanks to their beneficial properties, find a variety of applications in cosmetics. Due to their antioxidant and moisturizing properties, these compounds can be added to skin care products such as face creams, lotions, oils, and masks. In addition, these compounds show some antibacterial and antifungal properties, making them suitable for application in skin care products to help fight against bacteria or fungi. This study determined the antimicrobial activity of geraniol and the compounds which were formed during its transformation in relation to selected Gram-positive bacteria, and the preliminary assessment was made whether these compounds can act as ingredients of preparations with potential antimicrobial activity in the treatment of various human diseases (for example diseases of the skin, digestive system, or urinary tract). In addition, this work presents studies on the microbiological purity of cream samples obtained with different contents of geraniol and its transformation products (contents of the tested compounds: 0.5%, 1.5%, 2.5%, 4%, 8%, and 12%). Antibacterial activity tests were performed using the disc diffusion method against Gram-positive cocci, including the reference strains Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212, and against the clinical strains Staphylococcus aureus MRSA, Staphylococcus epidermidis, Enterococcus faecalis VRE VanB, Enterococcus faecium VRE VanA, and Enterococcus faecium VRE VanB. The most active ingredient against bacteria of the Staphylococcus genus was citral, followed by linalool and then geraniol. During our tests, in the case of bacteria of the Enterococcus genus, citral also showed the highest activity, but linalool, ocimenes, and geraniol showed a slightly lower activity. Moreover, this study examined the microbiological purity of cream samples obtained with various contents of geraniol and its transformation products. In the tests of the microbiological purity of cream samples, no growth of aerobic bacteria and fungi was found, which proves the lack of microbiological contamination of the obtained cosmetic preparations. On this basis, it was assessed that these compounds have preservative properties in the prepared creams. The addition of the analyzed compounds also had influence on the durability of the creams and had no effect on the change in their consistency, did not negatively affect the separation of phases during storage, and even had a positive effect on organoleptic sensations by enriching the smell of the tested samples.

Two-Phase Fermentation Systems for Microbial Production of Plant-Derived Terpenes.

Microbial cell factories, renowned for their economic and environmental benefits, have emerged as a key trend in academic and industrial areas, particularly in the fermentation of natural compounds. Among these, plant-derived terpenes stand out as a significant class of bioactive natural products. The large-scale production of such terpenes, exemplified by artemisinic acid-a crucial precursor to artemisinin-is now feasible through microbial cell factories. In the fermentation of terpenes, two-phase fermentation technology has been widely applied due to its unique advantages. It facilitates in situ product extraction or adsorption, effectively mitigating the detrimental impact of product accumulation on microbial cells, thereby significantly bolstering the efficiency of microbial production of plant-derived terpenes. This paper reviews the latest developments in two-phase fermentation system applications, focusing on microbial fermentation of plant-derived terpenes. It also discusses the mechanisms influencing microbial biosynthesis of terpenes. Moreover, we introduce some new two-phase fermentation techniques, currently unexplored in terpene fermentation, with the aim of providing more thoughts and explorations on the future applications of two-phase fermentation technology. Lastly, we discuss several challenges in the industrial application of two-phase fermentation systems, especially in downstream processing.

Therapeutic Potential of Fungal Terpenes and Terpenoids: Application in Skin Diseases.

Terpenes and their derivatives comprise a diverse group of natural compounds with versatile medicinal properties. This article elucidates the general characteristics of fungal terpenes and terpenoids, encompassing their structure and biogenesis. The focal point of this work involves a comprehensive overview of these compounds, highlighting their therapeutic properties, mechanisms of action, and potential applications in treating specific skin conditions. Numerous isolated terpenes and terpenoids have demonstrated noteworthy anti-inflammatory and anti-microbial effects, rivalling or surpassing the efficacy of currently employed treatments for inflammation or skin infections. Due to their well-documented antioxidant and anti-cancer attributes, these compounds exhibit promise in both preventing and treating skin cancer. Terpenes and terpenoids sourced from fungi display the capability to inhibit tyrosinase, suggesting potential applications in addressing skin pigmentation disorders and cancers linked to melanogenesis dysfunctions. This paper further disseminates the findings of clinical and in vivo research on fungal terpenes and terpenoids conducted thus far.

Drought induces moderate, diverse changes in the odour of grassland species.

Plants react to drought stress with numerous changes including altered emissions of volatile organic compounds (VOC) from leaves, which provide protection against oxidative tissue damage and mediate numerous biotic interactions. Despite the share of grasslands in the terrestrial biosphere, their importance as carbon sinks and their contribution to global biodiversity, little is known about the influence of drought on VOC profiles of grassland species. Using coupled gas chromatography-mass spectrometry, we analysed the odorants emitted by 22 European grassland species exposed to an eight-week-lasting drought treatment (DT; 30% water holding capacity, WHC). We focused on the odorants emitted during the light phase from whole plant shoots in their vegetative stage. Emission rates were standardised to the dry weight of each shoot. Well-watered (WW) plants (70% WHC) served as control. Drought-induced significant changes included an increase in total emission rates of plant VOC in six and a decrease in three species. Diverging effects on the number of emitted VOC (chemical richness) or on the Shannon diversity of the VOC profiles were detected in 13 species. Biosynthetic pathways-targeted analyses revealed 13 species showing drought-induced higher emission rates of VOC from one, two, three, or four major biosynthetic pathways (lipoxygenase, shikimate, mevalonate and methylerythritol phosphate pathway), while six species exhibited reduced emission rates from one or two of these pathways. Similarity trees of odorant profiles and their drought-induced changes based on a biosynthetically informed distance metric did not match species phylogeny. However, a phylogenetic signal was detected for the amount of terpenoids released by the studied species under WW and DT conditions. A comparative analysis of emission rates of single compounds released by WW and DT plants revealed significant VOC profile dissimilarities in four species only. The moderate drought-induced changes in the odorant emissions of grassland species are discussed with respect to their impact on trophic interactions across the food web. (294 words).

Organotransition Metal Chemistry of Terpenes: Syntheses, Structures, Reactivity and Molecular Rearrangements.

The impact of organometallic chemistry on the terpene field only really blossomed in the 1960s and 1970s with the realisation that carbon-carbon bond formation under mild conditions could be achieved by using nickel or iron carbonyls as synthetic reagents. Concomitantly, the development of palladium derivatives capable of the controlled coupling of isoprene units attracted the attention of numerous highly talented researchers, including future Nobel laureates. We discuss briefly how early work on the syntheses of simple monoterpenes soon progressed to sesquiterpenes and diterpenes of increasing complexity, such as humulene, flexibilene, vitamin A, or pheromones of commercial value, in particular those used in perfumery (muscone, lavandulol), or grandisol and red scale pheromone as replacements for harmful pesticides. As the field progressed, there has been more emphasis on developing organometallic routes to enantiopure rather than racemic products, as well as gaining precise mechanistic data on the transformations, notably the course of metal-promoted molecular rearrangements that have long been a feature of terpene chemistry. We note the impact of the enormously enhanced analytical techniques, high-field NMR spectroscopy and X-ray crystallography, and their use to re-examine the originally proposed structures of terpenes and their organometallic derivatives. Finally, we highlight the very recent ground-breaking use of the crystalline sponge method to acquire structural data on low-melting or volatile terpenes. The literature cited herein covers the period 1959 to 2023.

Engineering Terpene Production Pathways in Methylobacterium extorquens AM1.

Terpenes are diverse specialized metabolites naturally found within plants and have important roles in inter-species communication, adaptation and interaction with the environment. Their industrial applications span a broad range, including fragrances, flavors, cosmetics, natural colorants to agrochemicals and therapeutics, yet formal chemical synthesis is economically challenging due to structural complexities. Engineering terpene biosynthesis could represent an alternative in microbial biotechnological workhorses, such as Saccharomyces cerevisiae or Escherichi coli, utilizing sugars or complex media as feedstocks. Host species that metabolize renewable and affordable carbon sources may offer unique sustainable biotechnological alternatives. Methylotrophs are bacteria with the capacity to utilize one-carbon feedstocks, such as methanol or formate. They colonize the phyllosphere (above-ground area) of plants, and many accumulate abundant carotenoid pigments. Methylotrophs have the capacity to take up and use a subset of the rare earth elements known as lanthanides. These metals can enhance one-carbon (methylotrophic) metabolism. Here, we investigated whether manipulating the metabolism enables and enhances terpene production. A carotenoid-deficient mutant potentially liberates carbon, which may contribute to bioproduct accumulation. To test this hypothesis, terpene-producing bacterial strains regulated by two distinct promoters were generated. Wildtype Methylobacterium extorquens, ∆Meta1_3665, a methylotrophic mutant lacking the carotenoid pathway, and an E. coli strain were transformed with an exogenous terpene pathway and grown both in the presence and absence of lanthanides. The extraction, and the comparison of analytical profiles, provided evidence that engineered cultured M. extorquens under control of a native, inducible methylotrophic promoter can yield the sesquiterpene patchoulol when supplemented with lanthanide. In contrast, using a moderate-strength constitutive promoter failed to give production. We demonstrated colonization of the phyllosphere with the engineered strains, supporting the future engineering of selected species of the plant microbiome and with promising implications for the synthetic biology of small molecules.

Simultaneous analysis of cannabinoids and terpenes in Cannabis sativa inflorescence using full comprehensive two-dimensional liquid chromatography coupled to smart active modulation.

Nowadays, the higher peak capacity achievable by comprehensive two-dimensional liquid chromatography (LC×LC) for the analysis of vegetal samples is well-recognized. In addition, numerous compounds may be present in very different amounts. Cannabinoids and terpenes represent the main components of Cannabis sativa inflorescence samples, whose quantities are relevant for many application purposes. The analyses of both families are performed by different methods, at least two different separation methodologies, mainly according to their chemical characteristics and concentration levels. In this work, concentration differences and sample complexity issues were addressed using an LC×LC method that incorporates an optimized modulation strategy, namely smart active modulation, for the simultaneous analysis of cannabinoids and terpenes. The system was built by interposing an active flow splitter pump between both dimensions. This set up aimed to exploit the known advantages of LC×LC. In addition, here we proposed to use the splitter pump for online control over the splitting ratio to facilitate the selective dilution of different eluted fractions containing compounds with highly different concentrations. This work represents the first application and demonstration of smart active modulation (SAM) in LC×LC to simultaneously determine analytes with significant differences in concentration levels present in complex samples. The proposed method was tested with eight different strains, from which fingerprints were taken, and numerous cannabinoids and terpenes were identified in these samples. With this strategy, between 49 and 54 peaks were obtained in the LC×LC chromatograms corresponding to different strains. THCA-A was the main component in six strains, while CBDA was the main component in the other two strains. The main terpenes found were myrcene (in five strains), limonene (in two strains), and humulene (in one strain). Additionally, numerous other cannabinoids and terpenes were identified in these samples, providing valuable compositional information for growers, as well as medical and recreational users. The SAM strategy here proposed is simple and it can be extended to other complex matrices.

Compounds from Agathis dammara exert hypoglycaemic activity by enhancing glucose uptake: lignans, terpenes and others.

In this study, two new kaurane diterpenes (16, 17), together with 12 lignans (1-12), a triterpene (15), and two other compounds (13, 14) were isolated from the woods of Agathis dammara. The structure of the new compound was determined by HR ESIMS and 1D/2D NMR spectroscopy, and its absolute configuration was determined by electronic circular dichroism (ECD) exciton chirality method. Compounds 5, 11, 14 exhibit significant hypoglycaemic activity in zebrafish, and their mechanism of action is to enhance glucose uptake in zebrafish.

Combined antimicrobial action of streptomycin and terpenes against atypical mycobacteria isolated from fish.

The aim of this study was to investigate the antimycobacterial activity of 39 free terpenes and their activity in combination with streptomycin. Antimicrobial activity was first evaluated by screening 39 free terpenes at concentrations from 1.56 to 400 µg/mL. None of these exhibited positive effects against any of the nontuberculous mycobacteria (NTM) strains tested. However, six of the 39 terpenes (isoeugenol, nerol, (+)-α-terpineol, (1R)-(-)-myrtenol, (+)-terpinen-4-ol, and eugenol) were shown to enhance the activity of streptomycin against the NTM strains isolated from diseased ornamental fish.

Drought increases Norway spruce susceptibility to the Eurasian spruce bark beetle and its associated fungi.

Drought affects the complex interactions between Norway spruce, the bark beetle Ips typographus and associated microorganisms. We investigated the interplay of tree water status, defense and carbohydrate reserves with the incidence of bark beetle attack and infection of associated fungi in mature spruce trees. We installed roofs to induce a 2-yr moderate drought in a managed spruce stand to examine a maximum of 10 roof and 10 control trees for resin flow (RF), predawn twig water potentials, terpene, phenolic and carbohydrate bark concentrations, and bark beetle borings in field bioassays before and after inoculation with Endoconidiophora polonica and Grosmannia penicillata. Drought-stressed trees showed more attacks and significantly longer fungal lesions than controls, but maintained terpene resin defenses at predrought levels. Reduced RF and lower mono- and diterpene, but not phenolic concentrations were linked with increased host selection. Bark beetle attack and fungi stimulated chemical defenses, yet G. penicillata reduced phenolic and carbohydrate contents. Chemical defenses did not decrease under mild, prolonged drought in our simulated small-scale biotic infestations. However, during natural mass attacks, reductions in carbon fixation under drought, in combination with fungal consumption of carbohydrates, may deplete tree defenses and facilitate colonization by I. typographus.

Defensive glands in Stylotermitidae (Blattodea, Isoptera).

The large abundance of termites is partially achieved by their defensive abilities. Stylotermitidae represented by a single extant genus, Stylotermes, is a member of a termite group Neoisoptera that encompasses 83% of termite species and 94% of termite genera and is characterized by the presence of the frontal gland. Within Neoisoptera, Stylotermitidae represents a species-poor sister lineage of all other groups. We studied the structure of the frontal, labral and labial glands in soldiers and workers of Stylotermes faveolus, and the composition of the frontal gland secretion in S. faveolus and Stylotermes halumicus. We show that the frontal gland is a small active secretory organ in soldiers and workers. It produces a cocktail of monoterpenes in soldiers, and some of these monoterpenes and unidentified proteins in workers. The labral and labial glands are developed similarly to other termite species and contribute to defensive activities (labral in both castes, labial in soldiers) or to the production of digestive enzymes (labial in workers). Our results support the importance of the frontal gland in the evolution of Neoisoptera. Toxic, irritating and detectable monoterpenes play defensive and pheromonal functions and are likely critical novelties contributing to the ecological success of these termites.

Functional and Mechanistic Characterization of the 4,5-diepi-Isoishwarane Synthase from the Liverwort Radula lindenbergiana.

The microbial type sesquiterpene synthase RlMTPSL4 from the liverwort Radula lindenbergiana was investigated for its products, showing the formation of several sesquiterpene hydrocarbons. The main product was structurally characterized as the new compound 4,5-diepi-isoishwarane, while the side products included the known hydrocarbons germacrene A, α-selinene, eremophilene and 4,5-diepi-aristolochene. The cyclization mechanism towards 4,5-diepi-isoishwarane catalyzed by RlMTPSL4 was investigated through isotopic labeling experiments, revealing the stereochemical course for the deprotonation step to the neutral intermediate germacrene A, a reprotonation for its further cyclization, and a 1,2-hydride shift along the cascade. The absolute configuration of 4,5-diepi-isoishwarane was determined using a stereoselective deuteration approach, revealing an absolute configuration typically observed for a microbial type sesquiterpene.

Populus MYC2 orchestrates root transcriptional reprogramming of defence pathway to impair Laccaria bicolor ectomycorrhizal development.

The jasmonic acid (JA) signalling pathway plays an important role in the establishment of the ectomycorrhizal symbiosis. The Laccaria bicolor effector MiSSP7 stabilizes JA corepressor JAZ6, thereby inhibiting the activity of Populus MYC2 transcription factors. Although the role of MYC2 in orchestrating plant defences against pathogens is well established, its exact contribution to ECM symbiosis remains unclear. This information is crucial for understanding the balance between plant immunity and symbiotic relationships. Transgenic poplars overexpressing or silencing for the two paralogues of MYC2 transcription factor (MYC2s) were produced, and their ability to establish ectomycorrhiza was assessed. Transcriptomics and DNA affinity purification sequencing were performed. MYC2s overexpression led to a decrease in fungal colonization, whereas its silencing increased it. The enrichment of terpene synthase genes in the MYC2-regulated gene set suggests a complex interplay between the host monoterpenes and fungal growth. Several root monoterpenes have been identified as inhibitors of fungal growth and ECM symbiosis. Our results highlight the significance of poplar MYC2s and terpenes in mutualistic symbiosis by controlling root fungal colonization. We identified poplar genes which direct or indirect control by MYC2 is required for ECM establishment. These findings deepen our understanding of the molecular mechanisms underlying ECM symbiosis.

Two Sesterterpene Synthases from Lentzea atacamensis Demonstrate the Role of Conformational Variability in Terpene Biosynthesis.

Mining of two multiproduct sesterterpene synthases from Lentzea atacamensis resulted in the identification of the synthases for lentzeadiene (LaLDS) and atacamatriene (LaATS). The main product of LaLDS (lentzeadiene) is a new compound, while one of the side products (lentzeatetraene) is the enantiomer of brassitetraene B and the other side product (sestermobaraene F) is known from a surprisingly distantly related sesterterpene synthase. LaATS produces six new compounds, one of which is the enantiomer of the known sesterterpene Bm1. Notably, for both enzymes the products cannot all be explained from one and the same starting conformation of geranylfarnesyl diphosphate, demonstrating the requirement of conformational flexibility of the substrate in the enzymes' active sites. For lentzeadiene an intriguing thermal [1,5]-sigmatropic rearrangement was discovered, reminiscent of the biosynthesis of vitamin D3. All enzyme reactions and the [1,5]-sigmatropic rearrangement were investigated through isotopic labeling experiments and DFT calculations. The results also emphasize the importance of conformational changes during terpene cyclizations.