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.

Spatiotemporal Regulation and Transport Engineering for Sustainable Production of Geraniol in Candida glycerinogenes.

Geraniol is an attractive natural monoterpene with significant industrial and commercial value in the fields of pharmaceuticals, condiments, cosmetics, and bioenergy. The biosynthesis of monoterpenes suffers from the availability of key intermediates and enzyme-to-substrate accessibility. Here, we addressed these challenges in Candida glycerinogenes by a plasma membrane-anchoring strategy and achieved sustainable biosynthesis of geraniol using bagasse hydrolysate as substrate. On this basis, a remarkable 2.4-fold improvement in geraniol titer was achieved by combining spatial and temporal modulation strategies. In addition, enhanced geraniol transport and modulation of membrane lipid-associated metabolism effectively promoted the exocytosis of toxic monoterpenes, significantly improved the resistance of the engineered strain to monoterpenes and improved the growth of the strains, resulting in geraniol yield up to 1207.4 mg L-1 at shake flask level. Finally, 1835.2 mg L-1 geraniol was obtained in a 5 L bioreactor using undetoxified bagasse hydrolysate. Overall, our study has provided valuable insights into the plasma membrane engineering of C. glycerinogenes for the sustainable and green production of valuable compounds.

Boosting Geranyl Diphosphate Synthesis for Linalool Production in Engineered Yarrowia lipolytica.

Linalool is a pleasant-smelling monoterpenoid widely found in the essential oils of most flowers. Due to its biologically active properties, linalool has considerable commercial potential, especially in the food and perfume industries. In this study, the oleaginous yeast Yarrowia lipolytica was successfully engineered to produce linalool de novo. The (S)-linalool synthase (LIS) gene from Actinidia argute was overexpressed to convert geranyl diphosphate (GPP) into linalool. Flux was diverted from farnesyl diphosphate (FPP) synthesis to GPP by introducing a mutated copy of the native ERG20F88W-N119W gene, and CrGPPS gene from Catharanthus roseus on its own and as part of a fusion with LIS. Disruption of native diacylglycerol kinase enzyme, DGK1, by oligo-mediated CRISPR-Cas9 inactivation further increased linalool production. The resulting strain accumulated 109.6 mg/L of linalool during cultivation in shake flasks with sucrose as a carbon source. CrGPPS expression in Yarrowia lipolytica increased linalool accumulation more efficiently than the ERG20F88W-N119W expression, suggesting that the increase in linalool production was predominantly influenced by the level of GPP precursor supply.

Identification and Characterization of a Nerol Synthase in Fungi.

Nerol, a linear monoterpenoid, is naturally found in essential oils of various plants and is widely used in the fragrance, food, and cosmetic industries. Nerol synthase, essential for nerol biosynthesis, has previously been identified only in plants that use NPP as the precursor. In this study, a novel fungal nerol synthase, named PgfB, was cloned and characterized from Penicillium griseofulvum. In vitro enzymatic assays showed that PgfB could directly convert the substrate GPP into nerol. Furthermore, the successful expression of PgfB and its homologous protein in Saccharomyces cerevisiae resulted in the heterologous production of nerol. Finally, crucial amino acid residues for PgfB's catalytic activity were identified through site-directed mutagenesis. This research broadens our understanding of fungal monoterpene synthases and presents precious gene resources for the industrial production of nerol.

Combining Protein and Organelle Engineering for Linalool Overproduction in Saccharomyces cerevisiae.

Linalool, a plant-derived high-value monoterpene, is widely used in the perfume, cosmetic, and pharmaceutical industries. Recently, engineering microbes to produce linalool has become an attractive alternative to plant extraction or chemical synthesis approaches. However, the low catalytic activity of linalool synthase and the shortage of precursor pools have been considered as two key factors for low yields of linalool. In this study, we rationally engineered the entrance of the substrate-binding pocket of linalool synthase (t67OMcLISM) and successfully increased the catalytic efficiency of this enzyme toward geranyl pyrophosphate. Specifically, F447E and F447A, with decreased entrance hydrophobicity and steric hindrance, increased linalool production by 2.2 and 1.9 folds, respectively. Subsequently, cytoplasm and peroxisomes were harnessed to boost linalool synthesis in Saccharomyces cerevisiae, achieving a high titer of linalool (219.1 mg/L) in shake-flask cultivation. Finally, the engineered diploid strain produced 2.6 g/L of linalool by 5 L fed-batch fermentation, which was the highest production in yeast to date. The protein engineering and biosynthetic pathway compartmentalization in the peroxisome provide references for the microbial production of other monoterpenes.

Homeotic Protein Distal-Less Regulates NlObp8 and NlCsp10 to Impact the Recognition of Linalool in the Brown Planthopper Nilaparvata lugens.

Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs), which are thought to play key roles in the olfactory recognition of insects, can be induced by the odorants they recognize, but little is known about the underlying regulatory mechanisms. Here, we found that NlOBP8 and NlCSP10 play coordinative roles in the chemoreception of brown planthoppers (BPHs) to the volatile component linalool. Also, the relative mRNA levels of NlObp8 and NlCp10 decreased upon exposure to linalool. Further, homeotic protein distal-less (Dll), which was also highly expressed in the antennae, was found to positively regulate the transcription of NlObp8 and NlCsp10 directly. Knocking down NlDll expression downregulated the expression of many additional olfactory functional genes and impaired the repellent behavior of BPHs to linalool. Our findings elucidate the direct regulatory role of Dll in BPHs' olfactory plasticity to linalool through modulating the olfactory functional gene expression and could provide guidance to sustainably control BPHs in the field.

Distinctive foliar features and volatile profiles in three Ambrosia species (Asteraceae).

MAIN CONCLUSION: Ambrosia species differ both in their trichome types and in metabolic profiles of leaf volatiles. The current study provides tools for easier taxonomic identification of ragweed species. The genus Ambrosia (Asteraceae) includes some of the most noxious allergenic invasive weeds in the world. Due to high polymorphism in this genus, identification of species is often difficult. This study focuses on microscopic investigation of foliar features and GC-MS identification of the main leaf volatile components of three Ambrosia species currently found in Israel-invasive species Ambrosia confertiflora and A. tenuifolia, and transient A. grayi. A. confertiflora and A. tenuifolia have three trichome types: non-glandular trichomes, capitate glandular trichomes and linear glandular trichomes. Their non-glandular trichomes and capitate trichomes have distinct structures and can serve as taxonomic characters. A. grayi (the least successful invader) has only very dense covering trichomes. All three Ambrosia species have secretory structures in their leaf midrib. A. confertiflora, the most problematic invasive plant in Israel, had a ten times higher volatiles content than the other two species. In A. confertiflora, the most abundant volatiles were chrysanthenone (25.5%), borneol (18%), germacrene D and (E)-caryophyllene (both around 12%). In A. tenuifolia, the most abundant volatiles were ß-myrcene (32.9%), (2E)-hexenal (13%) and 1,8-cineole (11.7%). In A. grayi, the most abundant volatiles were ß-myrcene (17.9%), germacrene D (17.8%) and limonene (14%). The three examined species have distinct trichome types and metabolic profiles. Non-glandular trichomes show structural diversification between species and are a good descriptive character. Considering the anthropocentric significance of this highly problematic genus, the current study provides tools for easier identification of ragweed species.

A novel, genetically encoded whole-cell biosensor for directed evolution of myrcene synthase in Escherichia coli.

ß-myrcene is a high-value acyclic monoterpene. The low activity of myrcene synthase resulted to low biosynthetic titer of it. Biosensor is a promising tool applied for enzyme directed evolution. In this work, a novel genetically encoded biosensor responding to myrcene was established based on the MyrR regulator from Pseudomonas sp. Through sensing promoter characterization and engineering, the biosensor exhibiting excellent specificity and dynamic range was developed, and applied for directed evolution of myrcene synthase. After high-throughput screening of the myrcene synthase random mutation library, the best mutant R89G/N152S/D517N was obtained. Its catalytic efficiency was 1.47-fold than that of parent. Based on the mutants, the final production of myrcene reached 510.38 mg/L, which is the highest myrcene titer reported to date. This work demonstrates the great potential of whole-cell biosensor for improving enzymatic activity and the production of target metabolite.

LiMYB108 is involved in floral monoterpene biosynthesis induced by light intensity in Lilium 'Siberia'.

KEY MESSAGE: We find that the MYB family transcription factor, LiMYB108, has a novel function to regulate the floral fragrance affected by light intensity. Floral fragrance determines the commercial value of flowers and is influenced by many environmental factors, especially light intensity. However, the mechanism by which light intensity affects the release of floral fragrance is unclear. Here, we isolated an R2R3-type MYB transcription factor LiMYB108, the expression of which was induced by light intensity and located in the nucleus. Light of 200 and 600 µmol m-1 s-1 significantly increased the expression of LiMYB108, which was consistent with the improving trend of monoterpene synthesis under light. Virus-induced gene silencing (VIGS) of LiMYB108 in Lilium not only significantly inhibited the synthesis of ocimene and linalool, but also decreased the expression of LoTPS1; however, transient overexpression of LiMYB108 exerted opposite effects. Furthermore, yeast one-hybrid assays, dual-luciferase assays, and electrophoretic mobility shift assays (EMSA) demonstrated that LiMYB108 directly activated the expression of LoTPS1 by binding to the MYB binding site (MBS) (CAGTTG). Our findings demonstrate that light intensity triggered the high expression of LiMYB108, and then LiMYB108 as a transcription factor to activate the expression of LoTPS1, thus promoting the synthesis of the ocimene and linalool, which are important components of floral fragrance. These results provide new insights into the effects of light intensity on floral fragrance synthesis.

Geraniol ameliorates acute liver failure induced by lipopolysaccharide/D-galactosamine via regulating macrophage polarization and NLRP3 inflammasome activation by PPAR-γ methylation Geraniol alleviates acute liver failure.

Geraniol (Ger), a natural acyclic monoterpene alcohol, has been reported to exert protective effects through anti-inflammation in Acute liver failure (ALF). However, its specific roles and precise mechanisms underlying anti-inflammatory effects in ALF have not yet fully explored. We aimed to investigated the hepatoprotective effects and mechanisms of Ger against ALF induced by lipopolysaccharide (LPS)/D-galactosamine (GaIN). In this study, the liver tissue and serum of LPS/D-GaIN-induced mice were collected. The degree of liver tissue injury was evaluated by HE and TUNEL staining. Serum levels of liver injury markers (ALT and AST) and inflammatory factors were measured by ELISA assays. PCR and western blotting were conducted to determine the expression of inflammatory cytokines, NLRP3 inflammasome-related proteins, PPAR-γ pathway-related proteins, DNA Methyltransferases and M1/M2 polarization cytokines. Immunofluorescence staining was used to assess the localization and expression of macrophage markers (F4/80 and CD86), NLRP3 and PPAR-γ. In vitro experiments were performed in macrophages stimulated with LPS with or without IFN-γ. Purification of macrophages and cell apoptosis was analyzed using flow cytometry. We found that Ger effectively alleviated ALF in mice, specified by the attenuation of liver tissue pathological damage, inhibition of ALT, AST and inflammatory factor levels, and inactivation of NLRP3 inflammasome. Meanwhile, downregulation M1 macrophage polarization may involve in the protective effects of Ger. In vitro, Ger reduced the activation of NLRP3 inflammasome and apoptosis through regulating PPAR-γ methylation by inhibiting M1 macrophage polarization. In conclusion, Ger protects against ALF through suppressing NLRP3 inflammasome-mediated inflammation and LPS-induced macrophage M1 polarization via modulating PPAR-γ methylation.

Geraniol protects against cyclosporine A-induced renal injury in rats: Role of Wnt/ß-catenin and PPARγ signaling pathways.

AIMS: The nephrotoxicity of cyclosporine A (CsA) limits its use as an immunosuppressant. Wnt/ß-catenin signaling is involved in the pathogenesis of both acute and chronic kidney disease, and it is inhibited by peroxisome proliferator-activated receptor gamma (PPARγ). We aimed to evaluate if geraniol, which can modulate both PPARγ and Wnt signaling, could protect against CsA-induced nephrotoxicity. MATERIALS AND METHODS: Rats (6 groups) received the vehicle or a combination of CsA (30 mg/kg) with the vehicle, geraniol (50, 100, or 200 mg/kg), or the PPARγ agonist pioglitazone for 4 weeks. Blood pressure (BP), markers of renal injury (serum urea, serum creatinine, blood urea nitrogen, and urinary NAG), oxidative stress (glutathione peroxidase), inflammation (ICAM-1, IL-18, and NF-κB), apoptosis (caspase-3), extracellular matrix remodeling [matrix metalloproteinase-9 (MMP-9)], and fibrosis (TGF-ß1, Smad3, and Smad7) were assessed. Renal histological analysis, Wnt signaling components (Wnt-4/ß-catenin and E-cadherin), and PPARγ expression were evaluated. KEY FINDINGS: CsA group had renal injury, as well as increased BP, renal oxidative stress, inflammation, and fibrosis. The latter changes were associated with altered renal architecture, active Wnt signaling (higher Wnt-4 and ß-catenin expression and E-cadherin down-regulation), and lower PPARγ levels. Geraniol protected against kidney damage and the associated biochemical and histomorphological changes in a dose-dependent manner. The latter effects were comparable or superior to those of pioglitazone. SIGNIFICANCE: The down-regulation of Wnt/ß-catenin and the increase in PPARγ by geraniol suggest that both pathways are involved in its renoprotective potential. The study highlights geraniol as a valuable protective asset against chemically induced nephrotoxicity.

Unprecedented Biodegradable Cellulose-Derived Polyesters with Pendant Citronellol Moieties: From Monomer Synthesis to Enzymatic Degradation.

Levoglucosenone (LGO) is a cellulose-derived molecule that is present commercially on a multi-ton/year scale. Taking advantage of the α,ß-conjugated ketone of LGO, a new citronellol-containing 5-membered lactone (HBO-citro) was synthesized through a one-pot two-step pathway involving oxa-Michael addition and Baeyer-Villiger oxidation. The solvent-free treatment of HBO-citro with NaBH4 at room temperature led to the full reduction of the lactone moiety which gave a novel fully renewable triol monomer having a citronellol side chain (Triol-citro). Noticeably, by simply changing the reducing agent, temperature and reaction duration, the partial reduction of HBO-citro can be achieved to yield a mixture of 5- and 6-membered Lactol-citro molecules. Triol-citro was chosen to prepare functional renewable polyesters having citronellol pendant chains via polycondensation reactions with diacyl chlorides having different chain lengths. Good thermal stability (Td5% up to 170 °C) and low glass transition temperatures (as low as -42 °C) were registered for the polyesters obtained. The polymers were then hydrolyzed using a commercial lipase from Thermomyces lanuginosus (Lipopan® 50 BG) to assess their biodegradability. A higher degradation profile was found for the polyesters prepared using co-monomers (acyl chlorides) having longer chain lengths. This is likely due to the decreased steric hindrance around the ester bonds which allowed enhanced accessibility of the enzyme.

Yeast Surface Display for In Vitro Biosynthetic Pathway Reconstruction.

The enzymes immobilized through yeast surface display (YSD) can be used in in vitro metabolic pathway reconstruction as alternatives to the enzymes isolated or purified through conventional biochemistry methods. They can be easily prepared by growing and collecting yeast cells harboring display constructs. This may provide an economical method for enriching certain enzymes for biochemistry characterization and application. Herein, we took the advantage of one-pot cascade reactions catalyzed by YSD-immobilized enzymes in the mevalonate pathway to produce geraniol in vitro. YSD-immobilized enzymes of 10 cascade reactions for geraniol production, together with optimization of catalytic components, cofactor regeneration, and byproduct removal, achieved a final yield of 7.55 mg L-1 after seven cycles. This study demonstrated that it is feasible to reconstitute a complex multi-enzymatic system for the chemical biosynthesis in vitro by exploiting YSD-immobilized cascade enzymes.

Kinetic studies and homology modeling of a dual-substrate linalool/nerolidol synthase from Plectranthus amboinicus.

Linalool and nerolidol are terpene alcohols that occur naturally in many aromatic plants and are commonly used in food and cosmetic industries as flavors and fragrances. In plants, linalool and nerolidol are biosynthesized as a result of respective linalool synthase and nerolidol synthase, or a single linalool/nerolidol synthase. In our previous work, we have isolated a linalool/nerolidol synthase (designated as PamTps1) from a local herbal plant, Plectranthus amboinicus, and successfully demonstrated the production of linalool and nerolidol in an Escherichia coli system. In this work, the biochemical properties of PamTps1 were analyzed, and its 3D homology model with the docking positions of its substrates, geranyl pyrophosphate (C10) and farnesyl pyrophosphate (C15) in the active site were constructed. PamTps1 exhibited the highest enzymatic activity at an optimal pH and temperature of 6.5 and 30 °C, respectively, and in the presence of 20 mM magnesium as a cofactor. The Michaelis-Menten constant (Km) and catalytic efficiency (kcat/Km) values of 16.72 ± 1.32 µM and 9.57 × 10-3 µM-1 s-1, respectively, showed that PamTps1 had a higher binding affinity and specificity for GPP instead of FPP as expected for a monoterpene synthase. The PamTps1 exhibits feature of a class I terpene synthase fold that made up of α-helices architecture with N-terminal domain and catalytic C-terminal domain. Nine aromatic residues (W268, Y272, Y299, F371, Y378, Y379, F447, Y517 and Y523) outlined the hydrophobic walls of the active site cavity, whilst residues from the RRx8W motif, RxR motif, H-α1 and J-K loops formed the active site lid that shielded the highly reactive carbocationic intermediates from the solvents. The dual substrates use by PamTps1 was hypothesized to be possible due to the architecture and residues lining the catalytic site that can accommodate larger substrate (FPP) as demonstrated by the protein modelling and docking analysis. This model serves as a first glimpse into the structural insights of the PamTps1 catalytic active site as a multi-substrate linalool/nerolidol synthase.

Herbivore-induced volatiles influence moth preference by increasing the ß-Ocimene emission of neighbouring tea plants.

Herbivore-induced plant volatiles prime neighbouring plants to respond more strongly to subsequent attacks. However, the key volatiles that trigger this state and their priming mechanisms remain largely unknown. The tea geometrid Ectropis obliqua is one of the most devastating leaf-feeding pests of tea plants. Here, plant-plant communication experiments demonstrated that volatiles emitted from tea plants infested by E. obliqua larvae triggered neighbouring plants to release volatiles that repel E. obliqua adult, especially mated females. Volatile analyses revealed that the quantity of eight volatiles increased dramatically when plants were exposed to volatiles emitted by infested tea plants, including (Z)-3-hexenol, linalool, α-farnesene, ß-Ocimene and (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT). The results of behavioural bioassays demonstrated that ß-Ocimene strongly repelled mated E. obliqua females. Individual volatile compound exposure experiments revealed that (Z)-3-hexenol, linalool, α-farnesene and DMNT triggered the emission of ß-Ocimene from tea plants. Chemical inhibition experiments demonstrated that the emission of ß-Ocimene induced by (Z)-3-hexenol, linalool, α-farnesene and DMNT were dependent on Ca2+ and JA signalling. These findings help us to understand how E. obliqua moths respond to volatiles emitted from tea plants and provide new insight into volatile-mediated plant-plant interactions. They have potential significance for the development of novel insect and pest control strategies in crops.

Identification and Evaluation of Aromatic Volatile Compounds in 26 Cultivars and 8 Hybrids of Freesia hybrida.

Freesia hybrida is a group of cultivars in the genus Freesia with a strong floral scent composed of diverse volatile organic compounds (VOCs). In this study, the VOCs of 34 F. hybrida were extracted and analyzed by headspace solid phase microextraction and gas chromatography mass spectrometry (HS-SPME-GC-MS). A total of 164 VOCs whose relative contents were higher than 0.05% were detected. The numbers of VOCs in all germplasms differed between 11 to 38, and the relative contents ranged from 32.39% to 94.28%, in which most germplasms were higher than 80%. Terpenoids, especially monoterpenes, were the crucial type of VOCs in most germplasms, of which linalool and D-limonene were the most frequently occurring. Principal component analysis (PCA) clearly separated samples based on whether linalool was the main component, and hierarchical clustering analysis (HCA) clustered samples into 4 groups according to the preponderant compounds linalool and (E)-ß-ocimene. Comparison of parental species and hybrids showed heterosis in three hybrids, and the inherited and novel substances suggested that monoterpene played an important role in F. hybrida floral scent. This study established a foundation for the evaluation of Freesia genetic resources, breeding for the floral aroma and promoting commercial application.

Engineering of Yeast Old Yellow Enzyme OYE3 Enables Its Capability Discriminating of (E)-Citral and (Z)-Citral.

The importance of yeast old yellow enzymes is increasingly recognized for direct asymmetric reduction of (E/Z)-citral to (R)-citronellal. As one of the most performing old yellow enzymes, the enzyme OYE3 from Saccharomyces cerevisiae S288C exhibited complementary enantioselectivity for the reduction of (E)-citral and (Z)-citral, resulting in lower e.e. value of (R)-citronellal in the reduction of (E/Z)-citral. To develop a novel approach for the direct synthesis of enantio-pure (R)-citronellal from the reduction of (E/Z)-citral, the enzyme OYE3 was firstly modified by semi-rational design to improve its (R)-enantioselectivity. The OYE3 variants W116A and S296F showed strict (R)-enantioselectivity in the reduction of (E)-citral, and significantly reversed the (S)-enantioselectivity in the reduction of (Z)-citral. Next, the double substitution of OYE3 led to the unique variant S296F/W116G, which exhibited strict (R)-enantioselectivity in the reduction of (E)-citral and (E/Z)-citral, but was not active on (Z)-citral. Relying on its capability discriminating (E)-citral and (Z)-citral, a new cascade reaction catalyzed by the OYE3 variant S296F/W116G and glucose dehydrogenase was developed, providing the enantio-pure (R)-citronellal and the retained (Z)-citral after complete reduction of (E)-citral.

Studies on the Catalytic Properties of Crude Freeze-Dried Preparations of Yarrowia lipolytica Extracellular Lipases for Geranyl Ester Derivative Synthesis.

The study aimed to evaluate the impact of selected factors of the freeze-drying process on the hydrolytic and synthetic activity of the extracellular lipases of Y. lipolytica KKP 379 and to attempt the use of the crude enzyme preparation as a biocatalyst in the synthesis of geranyl 4-hydroxyphenylpropanoate. Antioxidant and antibacterial properties of the geranyl ester derivative were also investigated in order to evaluate their usefulness as a novel food additive. The studies confirmed that freeze-drying was an effective method of dehydrating yeast supernatant and allowed for obtaining lyophilizates with low water activity from 0.055 to 0.160. The type and concentration of the additive (2-6% whey protein hydrolyzate, 0.5% and 1% ammonium sulphate) had a significant effect on the hydrolytic activity of enzyme preparations, while the selected variants of drying temperature during the freeze-drying process were not significant (10 °C and 50 °C). Low yield of geranyl 4-hydroxyphenylopropionate was shown when the lyophilized supernatant was used (5.3%), but the yield of ester synthesis increased when the freeze-dried Y. lipolytica yeast biomass was applied (47.9%). The study confirmed the antioxidant properties of the synthesized ester by the DPPH• and CUPRAC methods, as well as higher antibacterial activity against tested bacteria than its precursor with 0.125 mM MIC (minimal inhibitory concentration) against L. monocytogenes.

Herbivore-induced and constitutive volatiles are controlled by different oxylipin-dependent mechanisms in rice.

Despite the importance of volatile organic compounds (VOCs) for plants, control mechanisms for their basal and stress-induced biosynthesis and release remain unclear. We sampled and characterized headspace and internal leaf volatile pools in rice (Oryza sativa), after a simulated herbivory treatment, which triggers an endogenous jasmonate burst. Certain volatiles, such as linalool, were strongly upregulated by simulated herbivory stress. In contrast, other volatiles, such as ß-caryophyllene, were constitutively emitted and fluctuated according to time of day. Transcripts of the linalool synthase gene transiently increased 1-3 h after exposure of rice to simulated herbivory, whereas transcripts of caryophyllene synthase peaked independently at dawn. Unexpectedly, although emission and accumulation patterns of rice inducible and constitutive VOCs were substantially different, both groups of volatiles were compromised in jasmonate-deficient hebiba mutants, which lack the allene oxide cyclase (AOC) gene. This suggests that rice employs at least two distinct oxylipin-dependent mechanisms downstream of AOC to control production of constitutive and herbivore-induced volatiles. Levels of the JA precursor, 12-oxo-phytodienoic acid (OPDA), were correlated with constitutive volatile levels suggesting that OPDA or its derivatives could be involved in control of volatile emission in rice.

The methyl jasmonate-responsive transcription factor DobHLH4 promotes DoTPS10, which is involved in linalool biosynthesis in Dendrobium officinale during floral development.

Linalool is an aromatic monoterpene produced in the Chinese medicinal plant Dendrobium officinale, but little information is available on the regulation of linalool biosynthesis. Here, a novel basic helix-loop-helix (bHLH) transcription factor, DobHLH4 from D. officinale, was identified and functionally characterized. The expression profile of DobHLH4 was positively correlated with that of DoTPS10 (R2 = 0.985, p < 0.01), which encodes linalool synthase that is responsible for linalool production, during floral development. DobHLH4 was highly expressed in petals, and was significantly induced by methyl jasmonate. Analysis of subcellular localization showed that DobHLH4 was located in the nucleus. Yeast one-hybrid and dual-luciferase assays indicated that DobHLH4 bound directly to the DoTPS10 promoter harboring the G-box element, and up-regulated DoTPS10 expression. A yeast two-hybrid screen confirmed that DobHLH4 physically interacted with DoJAZ1, suggesting that DobHLH4 might function in the jasmonic acid-mediated accumulation of linalool. Furthermore, transient overexpression of DobHLH4 in D. officinale petals significantly increased linalool production by triggering linalool biosynthetic pathway genes, especially DoTPS10. We suggest a hypothetical model that depicts how jasmonic acid signaling may regulate DoTPS10 by interacting with DobHLH4 and DoJAZ1. In doing so, the formation of linalool is controlled. Our results indicate that DobHLH4 is a positive regulator of linalool biosynthesis and may be a promising target for in vitro-based metabolic engineering to produce linalool.