Catalytic asymmetric synthesis of cannabinoids and menthol from neral.

The selective conversion of natural or synthetic neral to (1R,6S)-trans-isopiperitenol would enable and expedite sustainable routes to menthol1,2 and cannabinoids3-5. However, this reaction has been considered impossible because its product is more reactive to the required acid catalysts than its starting material, resulting in several side products6-9. We now show that an unsymmetric, strong and confined chiral acid, a highly fluorinated imino-imidodiphosphate, catalyses this process with excellent efficiency and selectivity. Expanding the method to other α,ß-unsaturated aldehydes could enable access to new cannabinoids and menthol derivatives not readily accessible previously. Mechanistic studies suggest that the confined catalyst accomplishes this reaction by binding the product in an unreactive conformation, thereby preventing its decomposition. We also show how (1R,6S)-trans-isopiperitenol can be readily converted to pharmaceutically useful cannabinoids and menthol, each in the shortest and most atom-economic routes so far.

Alternative splicing of CsbHLH133 regulates geraniol biosynthesis in tea plants.

Geraniol is one of the most abundant aromatic compounds in fresh tea leaves and contributes to the pleasant odor of tea products. Additionally, it functions as an airborne signal that interacts with other members of the ecosystem. To date, the regulation of the geraniol biosynthesis in tea plants remains to be investigated. In this study, a correlation test of the content of geraniol and its glycosides with gene expression data revealed that nudix hydrolase, CsNudix26, and its transcription factor, CsbHLH133 are involved in geraniol biosynthesis. In vitro enzyme assays and metabolic analyses of genetically modified tea plants confirmed that CsNudix26 is responsible for the formation of geraniol. Yeast one-hybrid, dual-luciferase reporter, and EMSA assays were used to verify the binding of CsbHLH133 to the CsNudix26 promoter. Overexpression of CsbHLH133 in tea leaves enhanced CsNudix26 expression and geraniol accumulation, whereas CsbHLH133 silencing reduced CsNudix26 transcript levels and geraniol content. Interestingly, CsbHLH133-AS, produced by alternative splicing, was discovered and proved to be the primary transcript expressed in response to various environmental stresses. Furthermore, geraniol release was found to be affected by various factors that alter the expression patterns of CsbHLH133 and CsbHLH133-AS. Our findings indicate that distinct transcript splicing patterns of CsbHLH133 regulate geraniol biosynthesis in tea plants in response to different regulatory factors.

Alcohol dehydrogenases regulated by a MYB44 transcription factor underlie Lauraceae citral biosynthesis.

Lineage-specific terpenoids have arisen throughout the evolution of land plants and are believed to play a role in interactions between plants and the environment. Species-specific gene clusters in plants have provided insight on the evolution of secondary metabolism. Lauraceae is an ecologically important plant family whose members are also of considerable economic value given their monoterpene contents. However, the gene cluster responsible for the biosynthesis of monoterpenes remains yet to be elucidated. Here, a Lauraceae-specific citral biosynthetic gene cluster (CGC) was identified and investigated using a multifaceted approach that combined phylogenetic, collinearity, and biochemical analyses. The CGC comprises MYB44 as a regulator and 2 alcohol dehydrogenases (ADHs) as modifying enzymes, which derived from species-specific tandem and proximal duplication events. Activity and substrate divergence of the ADHs has resulted in the fruit of mountain pepper (Litsea cubeba), a core Lauraceae species, consisting of more than 80% citral. In addition, MYB44 negatively regulates citral biosynthesis by directly binding to the promoters of the ADH-encoding genes. The aggregation of citral biosynthetic pathways suggests that they may form the basis of important characteristics that enhance adaptability. The findings of this study provide insights into the evolution of and the regulatory mechanisms involved in plant terpene biosynthesis.

Citronellol biosynthesis in pelargonium is a multistep pathway involving progesterone 5ß-reductase and/or iridoid synthase-like enzymes.

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 5ß-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.

Identification of key genes controlling monoterpene biosynthesis of Citral-type Cinnamomum bodinieri Levl. Based on transcriptome and metabolite profiling.

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.

Olfaction modulates cortical arousal independent of perceived odor intensity and pleasantness.

Throughout history, various odors have been harnessed to invigorate or relax the mind. The mechanisms underlying odors' diverse arousal effects remain poorly understood. We conducted five experiments (184 participants) to investigate this issue, using pupillometry, electroencephalography, and the attentional blink paradigm, which exemplifies the limit in attentional capacity. Results demonstrated that exposure to citral, compared to vanillin, enlarged pupil size, reduced resting-state alpha oscillations and alpha network efficiency, augmented beta-gamma oscillations, and enhanced the coordination between parietal alpha and frontal beta-gamma activities. In parallel, it attenuated the attentional blink effect. These effects were observed despite citral and vanillin being comparable in perceived odor intensity, pleasantness, and nasal pungency, and were unlikely driven by semantic biases. Our findings reveal that odors differentially alter the small-worldness of brain network architecture, and thereby brain state and arousal. Furthermore, they establish arousal as a unique dimension in olfactory space, distinct from intensity and pleasantness.

Gene isolation and enzymatic characterization of UDP-glycosyltransferases of terpene glucoside biosynthesis involved in linalyl-glycoside accumulation in Coffea arabica.

Terpenes, one of the secondary metabolites produced by plants, have diverse physiological functions. They are volatile compounds with physiological bioactivities (e.g., insect repellent, attracting enemies, and interacting with other plants). Terpenoids are also essential for flavor and aroma in plant-derived foods. In coffee, its aroma decides the value of coffee beans. Linalool, one of the volatile terpene compounds, is dominant in the coffee aroma. Coffee, with its good flavor and aroma, has high demand worldwide. Because terpenoids generally accumulate as glycosides in plant cells, glycosylation is catalyzed by UDP-glycosyltransferases (UGTs). Two linalyl-diglycosides have been identified: terpenoids reflected as necessary for coffee flavor. However, these UGTs and their action mechanisms are unknown in the Coffea genus. To obtain knowledge of terpene UGTs and elucidate the mechanism of terpene glycosylation in coffee, this study isolated terpene UGT genes and analyzed their functions. In silico screening based on the sequence of UGT85K11, which catalyzes terpene glycosylation from Camellia sinensis, was performed to obtain sequence information on five candidate UGT genes (CaUGT4, CaUGT5, CaUGT10, CaUGT15, and CaUGT20). These genes were isolated by reverse transcription-polymerase chain reaction, and the recombinant enzymes were produced with the Escherichia coli expression system. In functional analysis using radioisotopes, CaUGT4 showed critical activity against linalool, which had a higher affinity for its substrate than that of UGT85A84 from Osmanthus fragrans. Liquid chromatography-tandem mass spectrometry also revealed that CaUGT4 mainly produces linalyl glucoside. In this study, the first linalyl UGT was isolated from coffee. These findings can be used to elucidate the fundamental mechanism of the chemical defense in plants and apply aroma precursors for the plant-derived food industry in the future.

Enhancing growth and bioactive metabolites characteristics in Mentha pulegium L. via silicon nanoparticles during in vitro drought stress.

The development and production of secondary metabolites from priceless medicinal plants are restricted by drought stress. Mentha pulegium L. belongs to the Lamiaceae family and is a significant plant grown in the Mediterranean region for its medicinal and aesthetic properties. This study investigated the effects of three polyethylene glycol (PEG) (0, 5, and 10%) as a drought stress inducer and four silicon nanoparticle (SiNP) (0, 25, 50, and 100 ppm) concentrations as an elicitor to overcome the adverse effect of drought stress, on the growth parameters and bioactive chemical composition of M. pulegium L. plants grown in vitro. The experiment was performed as a factorial experiment using a completely randomized design (CRD) consisting of 12 treatments with two factors (3 PEG × 4 SiNPs concentrations), 6 replicates were used for each treatment for a total of 72 experimental units.The percentage of shoot formation was inversely proportional to the PEG concentration; for the highest PEG concentration, the lowest percentage of shoot formation (70.26%) was achieved at 10% PEG. SiNPs at 50 ppm enhanced shoot formation, the number of shoots, shoot height, fresh and dry weight, rosmarinic acid, total phenols, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity. The methanol extract from M. pulegium revealed the presence of significant secondary metabolites using gas chromatography‒mass spectrometry (GC-MS). The principal constituents of the extract were limonene (2.51, 2.99%), linalool (3.84, 4.64%), geraniol (6.49, 8.77%), menthol (59.73, 65.43%), pulegone (3.76, 2.76%) and hexadecanoic acid methyl ester or methyl palmitate (3.2, 4.71%) for the 0 ppm SiNPs, PEG 0% and 50 ppm SiNPs, and PEG 10%, respectively. Most of the chemical components identified by GC‒MS in the methanol extract were greater in the 50 ppm SiNP and 10% PEG treatment groups than in the control group. SiNP improves drought tolerance by regulating biosynthesis and accumulating some osmolytes and lessens the negative effects of polyethylene glycol-induced drought stress.Based on the results, the best treatment for most of the parameters was 50 ppm SiNPs combined with 10% PEG, the morphological and chemical characteristics were inversely proportional to the PEG concentration, as the highest PEG concentration (10%) had the lowest results. Most parameters decreased at the highest SiNP concentration (100 ppm), except for the DPPH scavenging percentage, as there was no significant difference between the 50 and 100 ppm SiNPs.

Females smell differently: characteristics and significance of the most common olfactory sensilla of female silkmoths.

In the silkmoth Bombyx mori, the role of male sensilla trichodea in pheromone detection is well established. Here we study the corresponding female sensilla, which contain two olfactory sensory neurons (OSNs) and come in two lengths, each representing a single physiological type. Only OSNs in medium trichoids respond to the scent of mulberry, the silkworm's exclusive host plant, and are more sensitive in mated females, suggesting a role in oviposition. In long trichoids, one OSN is tuned to (+)-linalool and the other to benzaldehyde and isovaleric acid, both odours emitted by silkworm faeces. While the significance of (+)-linalool detection remains unclear, isovaleric acid repels mated females and may therefore play a role in avoiding crowded oviposition sites. When we examined the underlying molecular components of neurons in female trichoids, we found non-canonical co-expression of Ir8a, the co-receptor for acid responses, and ORco, the co-receptor of odorant receptors, in long trichoids, and the unexpected expression of a specific odorant receptor in both trichoid sensillum types. In addition to elucidating the function of female trichoids, our results suggest that some accepted organizational principles of the insect olfactory system may not apply to the predominant sensilla on the antenna of female B. mori.

LiNAC100 contributes to linalool biosynthesis by directly regulating LiLiS in Lilium 'Siberia'.

MAIN CONCLUSION: The 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.

Identification and functional analysis of floral terpene synthase genes in Curcuma alismatifolia.

MAIN CONCLUSION: CaTPS2 and CaTPS3 were significantly expressed in flowers of Curcuma alismatifolia 'Shadow' and demonstrated bifunctional enzyme activity, CaTPS2 generated linalool and nerolidol as products, and CaTPS3 catalyzed ß-myrcene and ß-farnesene formation. This study presents the discovery and functional characterization of floral terpene synthase (TPS) genes in Curcuma alismatifolia 'Shadow', a cultivar renowned for its unique fragrance. Addressing the gap in understanding the genetic basis of floral scent in this species, we identified eight TPS genes through comprehensive transcriptome sequencing. Among these, CaTPS2 and CaTPS3 were significantly expressed in floral tissues and demonstrated bifunctional enzyme activity corresponding to the major volatile compounds detected in 'Shadow'. Functional analyses, including in vitro assays complemented with rigorous controls and alternative identification methods, elucidated the roles of these TPS genes in terpenoid biosynthesis. In vitro studies were conducted via heterologous expression in E. coli, followed by purification of the recombinant protein using affinity chromatography, enzyme assays were performed with GPP/FPP as the substrate, and volatile products were inserted into the GC-MS for analysis. Partially purified recombinant protein of CaTPS2 catalyzed GPP and FPP to produce linalool and nerolidol, respectively, while partially purified recombinant protein of CaTPS3 generated ß-myrcene and ß-farnesene with GPP and FPP as substrates, respectively. Real-time quantitative PCR further validated the expression patterns of these genes, correlating with terpenoid accumulation in different plant tissues. Our findings illuminate the molecular mechanisms underpinning floral fragrance in C. alismatifolia and provide a foundation for future genetic enhancements of floral scent in ornamental plants. This study, therefore, contributes to the broader understanding of terpenoid biosynthesis in plant fragrances, paving the way for biotechnological applications in horticulture plant breeding.

Metabolic engineering of Pichia pastoris for overproduction of cis-trans nepetalactol.

Monoterpene indole alkaloids (MIAs) are a group of plant-derived natural products with high-value medicinal properties. However, their availability for clinical application is limited due to challenges in plant extraction. Microbial production has emerged as a promising strategy to meet the clinical demands for MIAs. The biosynthetic pathway of cis-trans nepetalactol, which serves as the universal iridoid scaffold for all MIAs, has been successfully identified and reconstituted. However, bottlenecks and challenges remain to construct a high-yielding platform strain for cis-trans nepetalactol production, which is vital for subsequent MIAs biosynthesis. In the present study, we focused on engineering of Pichia pastoris cell factories to enhance the production of geraniol, 8-hydroxygeraniol, and cis-trans nepetalactol. By targeting the biosynthetic pathway from acetyl-CoA to geraniol in both peroxisomes and cytoplasm, we achieved comparable geraniol titers in both compartments. Through protein engineering, we found that either G8H or CPR truncation increased the production of 8-hydroxygeraniol, with a 47.8-fold and 14.0-fold increase in the peroxisomal and cytosolic pathway strain, respectively. Furthermore, through a combination of dynamical control of ERG20, precursor and cofactor supply engineering, diploid engineering, and dual subcellular compartmentalization engineering, we achieved the highest ever reported production of cis-trans nepetalactol, with a titer of 4429.4 mg/L using fed-batch fermentation in a 5-L bioreactor. We anticipate our systematic metabolic engineering strategies to facilitate the development of P. pastoris cell factories for sustainable production of MIAs and other plant natural products.

Efficient production of (S)-limonene and geraniol in Saccharomyces cerevisiae through the utilization of an Erg20 mutant with enhanced GPP accumulation capability.

Monoterpenes and monoterpenoids such as (S)-limonene and geraniol are valuable chemicals with a wide range of applications, including cosmetics, pharmaceuticals, and biofuels. Saccharomyces cerevisiae has proven to be an effective host to produce various terpenes and terpenoids. (S)-limonene and geraniol are produced from geranyl pyrophosphate (GPP) through the enzymatic actions of limonene synthase (LS) and geraniol synthase (GES), respectively. However, a major hurdle in their production arises from the dual functionality of the Erg20, a farnesyl pyrophosphate (FPP) synthase, responsible for generating GPP. Erg20 not only synthesizes GPP by condensing isopentenyl pyrophosphate (IPP) with dimethylallyl pyrophosphate but also catalyzes further condensation of IPP with GPP to produce FPP. In this study, we have tackled this issue by harnessing previously developed Erg20 mutants, Erg20K197G (Erg20G) and Erg20F96W, N127W (Erg20WW), which enhance GPP accumulation. Through a combination of these mutants, we generated a novel Erg20WWG mutant with over four times higher GPP accumulating capability than Erg20WW, as observed through geraniol production levels. The Erg20WWG mutant was fused to the LS from Mentha spicata or the GES from Catharanthus roseus for efficient conversion of GPP to (S)-limonene and geraniol, respectively. Further improvements were achieved by localizing the entire mevalonate pathway and the Erg20WWG-fused enzymes in peroxisomes, while simultaneously downregulating the essential ERG20 gene using the glucose-sensing HXT1 promoter. In the case of (S)-limonene production, additional Erg20WWG-LS was expressed in the cytosol. As a result, the final strains produced 1063 mg/L of (S)-limonene and 1234 mg/L of geraniol by fed-batch biphasic fermentations with ethanol feeding. The newly identified Erg20WWG mutant opens doors for the efficient production of various other GPP-derived chemicals including monoterpene derivatives and cannabinoids.

Geraniol attenuates oxygen-glucose deprivation/reoxygenation-induced ROS-dependent apoptosis and permeability of human brain microvascular endothelial cells by activating the Nrf-2/HO-1 pathway.

Blood-brain barrier breakdown and ROS overproduction are important events during the progression of ischemic stroke aggravating brain damage. Geraniol, a natural monoterpenoid, possesses anti-apoptotic, cytoprotective, anti-oxidant, and anti-inflammatory activities. Our study aimed to investigate the effect and underlying mechanisms of geraniol in oxygen-glucose deprivation/reoxygenation (OGD/R)-induced human brain microvascular endothelial cells (HBMECs). Apoptosis, caspase-3 activity, and cytotoxicity of HBMECs were evaluated using TUNEL, caspase-3 activity, and CCK-8 assays, respectively. The permeability of HBMECs was examined using FITC-dextran assay. Reactive oxygen species (ROS) production was measured using the fluorescent probe DCFH-DA. The protein levels of zonula occludens-1 (ZO-1), occludin, claudin-5, ß-catenin, nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) were determined by western blotting. Geraniol showed no cytotoxicity in HBMECs. Geraniol and ROS scavenger N-acetylcysteine (NAC) both attenuated OGD/R-induced apoptosis and increase of caspase-3 activity and the permeability to FITC-dextran in HBMECs. Geraniol relieved OGD/R-induced ROS accumulation and decrease of expression of ZO-1, occludin, claudin-5, and ß-catenin in HBMECs. Furthermore, we found that geraniol activated Nrf2/HO-1 pathway to inhibit ROS in HBMECs. In conclusion, geraniol attenuated OGD/R-induced ROS-dependent apoptosis and permeability in HBMECs through activating the Nrf2/HO-1 pathway.

Evaluation of antifungal and apoptotic effects of linalool, citral, and carvacrol separately and in combination with nystatin against clinical isolates of Pichia kudriavzevii.

Pichia kudriavzevii (formerly Candida krusei) poses a significant threat to immunocompromised patients due to its inherent resistance to various antifungal drugs. This study explored the anticandidal potential of citral, linalool, and carvacrol in combination with nystatin against P. kudriavzevii strains.Using the microdilution method following CLSI guidelines, Minimum Inhibitory Concentrations (MICs) and fungicidal concentrations (MFCs) were determined. Citral exhibited MIC values ranging from 50 to 100 µg/ml, averaging 70.24 ± 16.99 µg/ml, while carvacrol had MIC values of 50 to 100 µg/ml, averaging 86.90 ± 16.99 µg/ml. Linalool demonstrated weaker antifungal activity, with MIC values between 100 and 200 µg/ml, averaging 150 ± 38.73 µg/ml. The study assessed the synergistic effectsof these phenols with nystatin through fractional inhibitory concentration indices (FICIS). In addition, flow cytometry was employed to assess apoptosis induction in P. kudriavzevii cells.Carvacrol displayed a remarkable synergistic effect in combination with nystatin against all 21 isolates tested. Conversely, linalool showed synergy in 17 isolates, while citral exhibited synergy in only 2 isolates. These findings highlight distinct patterns of synergy between the different compounds and nystatin against P. kudriavzevii. Also, Carvacrol emerged as the most potent inducer of apoptosis across all P. kudriavzevii strains, followed by citral and linalool. This suggests that carvacrol not only possesses a stronger antifungal effect but also has a more pronounced ability to trigger programmed cell death in P. kudriavzevii. In conclusion, the study supports the potential of carvacrol, citral and linalool, as anticandidal agents, suggesting their supplementation with nystatin for treating P. kudriavzevii infections.

Belowground insect herbivory induces systemic volatile emissions that strengthen neighbouring plant resistance aboveground.

Plants transmit ecologically relevant messages to neighbouring plants through chemical cues. For instance, insect herbivory triggers the production of herbivore-induced plant volatiles (HIPVs), which can enhance neighbouring plant defences. HIPVs are emitted from directly damaged plant tissues and from systemic, nondamaged tissues. Although volatile-mediated interplant interactions have been observed both above- and belowground, it remains unknown whether belowground herbivory induces systemic HIPVs aboveground that influence neighbouring plants. To explore how belowground herbivory affects interplant interactions aboveground, we characterised systemic HIPVs from squash induced by belowground striped cucumber beetle (Acalymma vittatum) larval herbivory. We exposed squash 'receiver plants' to systemic HIPVs or volatiles from nondamaged plants. We then measured herbivore resistance by challenging 'receiver plants' with aboveground-feeding herbivores: adult beetles (A. vittatum) or squash bugs (Anasa tristis). We discovered belowground-damaged plants emitted more (E)-ß-ocimene, a key volatile from the systemic HIPV blend, than nondamaged controls, and that exposure to systemic HIPVs enhanced neighbouring plant resistance to aboveground squash bugs, but not adult beetles. Further investigations into the mechanism of interplant interaction revealed ß-ocimene alone can elicit plant resistance against squash bugs. Overall, our findings reveal a novel form of volatile-mediated interactions between plants spanning across aboveground-belowground plant systems.

Communication between undamaged plants can elicit changes in volatile emissions from neighbouring plants, thereby altering their susceptibility to aphids.

Plant volatiles play an important role in intra- and interspecific plant communication, inducing direct and indirect defenses against insect pests. However, it remains unknown whether volatile interactions between undamaged cultivars alter host plant volatile emissions and their perception by insect pests. Here, we tested the effects of exposure of a spring barley, Hordeum vulgare L., cultivar, Salome, to volatiles from other cultivars: Fairytale and Anakin. We found that exposing Salome to Fairytale induced a significantly higher emission of trans-ß-ocimene and two unidentified compounds compared when exposed to Anakin. Aphids were repelled at a higher concentration of trans-ß-ocimene. Salome exposure to Fairytale had significant repulsive effects on aphid olfactory preference, yet not when Salome was exposed to Anakin. We demonstrate that volatile interactions between specific undamaged plants can induce changes in volatile emission by receiver plants enhancing certain compounds, which can disrupt aphid olfactory preferences. Our results highlight the significant roles of volatiles in plant-plant interactions, affecting plant-insect interactions in suppressing insect pests. This has important implications for crop protection and sustainable agriculture.

Plant-derived monoterpene S-linalool and ß-ocimene generated by CsLIS and CsOCS-SCZ are key chemical cues for attracting parasitoid wasps for suppressing Ectropis obliqua infestation in Camellia sinensis L.

Insect-induced plant volatile organic compounds (VOCs) may function as either direct defence molecules to deter insects or indirect defence signals to attract the natural enemies of the invading insects. Tea (Camellia sinensis L.), an important leaf-based beverage crop, is mainly infested by Ectropis obliqua which causes the most serious damage. Here, we report a mechanistic investigation of tea plant-derived VOCs in an indirect defence mechanism against E. obliqua. Parasitoid wasp Parapanteles hyposidrae, a natural enemy of E. obliqua, showed strong electrophysiological response and selection behaviour towards S-linalool and ß-ocimene, two monoterpenes with elevated emission from E. obliqua-damaged tea plants. Larvae frass of E. obliqua, which also released S-linalool and ß-ocimene, was found to attract both mated female or male Pa. hyposidrae according to gas chromatography-electroantennogram detection and Y-tube olfactometer assays. In a field setting, both S-linalool and ß-ocimene were effective in recruiting both female and male Pa. hyposidrae wasps. To understand the molecular mechanism of monoterpenes-mediated indirect defence in tea plants, two novel monoterpene synthase genes, CsLIS and CsOCS-SCZ, involved in the biosynthesis of S-linalool or ß-ocimene, respectively, were identified and biochemically characterised. When the expression of these two genes in tea plants was inhibited by antisense oligodeoxynucleotide, both volatile emission and attraction of wasps were reduced. Furthermore, gene expression analysis suggested that the expression of CsLIS and CsOCS-SCZ is regulated by the jasmonic acid signalling pathway in the tea plant.

Specific decorations of 17-hydroxygeranyllinalool diterpene glycosides solve the autotoxicity problem of chemical defense in Nicotiana attenuata.

The native diploid tobacco Nicotiana attenuata produces abundant, potent anti-herbivore defense metabolites known as 17-hydroxygeranyllinalool diterpene glycosides (HGL-DTGs) whose glycosylation and malonylation biosynthetic steps are regulated by jasmonate signaling. To characterize the biosynthetic pathway of HGL-DTGs, we conducted a genome-wide analysis of uridine diphosphate glycosyltransferases (UGTs) and identified 107 family-1 UGT members. The transcript levels of three UGTs were highly correlated with the transcript levels two key HGL-DTG biosynthetic genes: geranylgeranyl diphosphate synthase (NaGGPPS) and geranyllinalool synthase (NaGLS). NaGLS's role in HGL-DTG biosynthesis was confirmed by virus-induced gene silencing. Silencing the Uridine diphosphate (UDP)-rhamnosyltransferase gene UGT91T1 demonstrated its role in the rhamnosylation of HGL-DTGs. In vitro enzyme assays revealed that UGT74P3 and UGT74P4 use UDP-glucose for the glucosylation of 17-hydroxygeranyllinalool (17-HGL) to lyciumoside I. Plants with stable silencing of UGT74P3 and UGT74P5 were severely developmentally deformed, pointing to a phytotoxic effect of the aglycone. The application of synthetic 17-HGL and silencing of the UGTs in HGL-DTG-free plants confirmed this phytotoxic effect. Feeding assays with tobacco hornworm (Manduca sexta) larvae revealed the defensive functions of the glucosylation and rhamnosylation steps in HGL-DTG biosynthesis. Glucosylation of 17-HGL is therefore a critical step that contributes to the resulting metabolites' defensive function and solves the autotoxicity problem of this potent chemical defense.

Citral and triclosan synergistically silence quorum sensing and potentiate antivirulence response in Pseudomonas aeruginosa.

Among the ESKAPE pathogens, Pseudomonas aeruginosa is an extensively notorious superbug that causes difficult-to-treat infections. Since quorum sensing (QS) directly promotes pseudomonal virulence, targeting QS circuits is a promising approach for disarming phenotypic virulence. Hence, this study scrutinizes the anti-QS, antivirulence, and anti-biofilm potential of citral (CiT; phytochemical) and triclosan (TcN; disinfectant), alone and in combination, against P. aeruginosa PAO1/PA14. The findings confirmed synergism between CiT and TcN and revealed their quorum quenching (QQ) potential. At sub-inhibitory levels, CiT-TcN combination significantly impeded pyocyanin, total bacterial protease, hemolysin, and pyochelin production alongside inhibiting biofilm formation in P. aeruginosa. Moreover, the QQ and antivirulence potential of CiT and TcN was positively correlated by molecular docking studies that predicted strong associations of the drugs with QS receptors of P. aeruginosa. Collectively, the study identifies CiT-TcN as an effective drug combination that harbors QQ, antivirulence, and anti-biofilm prospects against P. aeruginosa.