Formation Of Linalool Research Articles

Linalool synthesis related PpTPS1 and PpTPS3 are activated by transcription factor PpERF61 whose expression is associated with DNA methylation during peach fruit ripening

The monoterpene linalool is a major contributor to flavor of multiple fruit species. Although great progress has been made in identifying genes related to linalool formation, transcriptional regulation for the pathway remains largely unknown. As a super transcription factor family, roles of AP2/ERF in regulating linalool production have not been elucidated. Peach linalool is catalyzed by terpene synthases PpTPS1 and PpTPS3. Here, we observed that expression of PpERF61 correlated with these two PpTPSs during fruit ripening by transcriptome co-expression analysis. Dual-luciferase assay and EMSA results indicated that PpERF61 activated the PpTPS1 and PpTPS3 transcription by binding to the DRE/CRT motif in their promoters. Transient overexpressing PpERF61 in peach fruit significantly increased PpTPS1 and PpTPS3 expression and linalool content. Further study revealed significant correlation between PpERF61 transcripts and linalool contents across 30 peach cultivars. Besides transcriptional regulation, accumulated linalool was associated with DNA demethylation of PpERF61 during peach fruit ripening. In addition, interactions between PpERF61 and PpbHLH1 were evaluated, indicating these two transcription factors were associated with linalool production during peach fruit ripening. Overall, our results revealed a new insight into the regulation of linalool synthesis in fruit.

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.

Genome-Wide Identification and Expression Profile of TPS Gene Family in Dendrobium officinale and the Role of DoTPS10 in Linalool Biosynthesis.

Terpene synthase (TPS) is a critical enzyme responsible for the biosynthesis of terpenes, which possess diverse roles in plant growth and development. Although many terpenes have been reported in orchids, limited information is available regarding the genome-wide identification and characterization of the TPS family in the orchid, Dendrobium officinale. By integrating the D. officinale genome and transcriptional data, 34 TPS genes were found in D. officinale. These were divided into four subfamilies (TPS-a, TPS-b, TPS-c, and TPS-e/f). Distinct tempospatial expression profiles of DoTPS genes were observed in 10 organs of D. officinale. Most DoTPS genes were predominantly expressed in flowers, followed by roots and stems. Expression of the majority of DoTPS genes was enhanced following exposure to cold and osmotic stresses. Recombinant DoTPS10 protein, located in chloroplasts, uniquely converted geranyl diphosphate to linalool in vitro. The DoTPS10 gene, which resulted in linalool formation, was highly expressed during all flower developmental stages. Methyl jasmonate significantly up-regulated DoTPS10 expression and linalool accumulation. These results simultaneously provide valuable insight into understanding the roles of the TPS family and lay a basis for further studies on the regulation of terpenoid biosynthesis by DoTPS in D. officinale.

MYB21 interacts with MYC2 to control the expression of terpene synthase genes in flowers of Freesia hybrida and Arabidopsis thaliana.

Previously, linalool was found to be the most abundant component among the cocktail of volatiles released from flowers of Freesia hybrida. Linalool formation is catalysed by monoterpene synthase TPS1. However, the regulatory network developmentally modulating the expression of the TPS1 gene in Freesia hybrida remains unexplored. In this study, three regulatory genes, FhMYB21L1, FhMYB21L2, and FhMYC2, were screened from 52 candidates. Two MYB transcription factor genes were synchronously expressed with FhTPS1 and could activate its expression significantly when overexpressed, and the binding of FhMYB21L2 to the MYBCORE sites in the FhTPS1 promoter was further confirmed, indicating a direct role in activation. FhMYC2 showed an inverse expression pattern compared with FhTPS1; its expression led to a decreased binding of FhMYB21 to the FhTPS1 promoter to reduce its activation capacity when co-expressed, suggesting a role for an MYB-bHLH complex in the regulation of the FhTPS1 gene. In Arabidopsis, both MYB21 and MYC2 regulators were shown to activate the expression of sesquiterpene synthase genes, and the regulatory roles of AtMYB21 and AtMYC2 in the expression of the linalool synthase gene were also confirmed, implying conserved functions of the MYB-bHLH complex in these two evolutionarily divergent plants. Moreover, the expression ratio between MYB21 and MYC2 orthologues might be a determinant factor in floral linalool emission.

Influence of Technological Parameters on the Isomerization of Geraniol Using Sepiolite

In the current study, the isomerization of geraniol over a natural sepiolite as a catalyst was investigated and optimized. Prior to application in the isomerization process, the physical and chemical properties of sepiolite were characterized using a battery of instrumental techniques, including XRD, nitrogen adsorption at 77 K, SEM, EDXRF, UV–Vis and FT-IR. Results indicated that geraniol isomerization is very complicated due to the large number of reactions taking place. The catalytic studies showed that the main reaction products were β-pinene, ocimenes, linalool, nerol, citrals, thunbergol and isocembrol; all chemical products with commercial applications. The quantity of each of these products depended on the temperature, catalyst content and reaction time employed in the isomerization process. During the current study, these parameters were varied in a step-wise approach over the ranges 80–150 °C (temperature), 5–15 wt% catalyst content and reaction time of 15–1440 min. As linalool is one of the most commercially important reaction products, the geraniol isomerization method was studied to identify conditions producing the highest selectivity for this compound. The most beneficial conditions for geraniol conversion and linalool formation were established as a temperature of 120 °C, catalyst content of 10 wt% and a reaction time of 3 h.Graphic

Differential Accumulation of Aroma Compounds in Normal Green and Albino-Induced Yellow Tea (Camellia sinensis) Leaves.

Tea (Camellia sinensis) cultivars with green leaves are the most widely used for making tea. Recently, tea mutants with white or yellow young shoots have attracted increasing interest as raw materials for making “high-quality” tea products. Albino teas are generallycharacterized as having metabolites of relatively high amino acid content and lower catechin content. However, little is known about aroma compounds in albino tea leaves. Herein, we compared original normal leaves (green) and light-sensitive albino leaves (yellow) of cv. Yinghong No. 9. GC-MS was employed to analyze endogenous tea aroma compounds and related precursors. Quantitative real time PCR was used to measure expression levels of genes involved in biosyntheses of tea aromas.The total contents of most endogenous free tea aromas, including aroma fatty acid derivatives, aroma terpenes, and aroma phenylpropanoids/benzenoids, and their glycosidically bound aroma compounds, were lower in yellow leaves than in green leaves. The content of the key precursor geranyl diphosphate (GDP) and expression levels of key synthetic genes involved in the formation of linalool, a major aroma compound in cv. Yinghong No. 9, were investigated. Linalool content was lower in albino-induced yellow leaves, which was due to the lower GDP content compared with normal green leaves.

Identification and characterization of terpene synthase genes accounting for volatile terpene emissions in flowers of Freesia x hybrida.

The development of flower scents was a crucial event in biological evolution, providing olfactory signals by which plants can attract pollinators. In this study, bioinformatics, metabolomics, and biochemical and molecular methodologies were integrated to investigate the candidate genes involved in the biosynthesis of volatile components in two cultivars of Freesia x hybrida, Red River® and Ambiance, which release different categories of compounds. We found that terpene synthase (TPS) genes were the pivotal genes determining spatiotemporal release of volatile compounds in both cultivars. Eight FhTPS genes were isolated and six were found to be functional: FhTPS1 was a single-product enzyme catalyzing the formation of linalool, whereas the other four FhTPS proteins were multi-product enzymes, among which FhTPS4, FhTPS6, and FhTPS7 could recognize geranyl diphosphate and farnesyl diphosphate simultaneously. The FhTPS enzymatic products closely matched the volatile terpenes emitted from flowers, and significant correlations were found between release of volatile terpenes and FhTPS gene expression. Graphical models based on these results are proposed that summarize the biosynthesis of Freesia floral volatile terpenes. The characterization of FhTPS genes paves the way to decipher their roles in the speciation and fitness of Freesia, and this knowledge could also be used to introduce or enhance scent in other plants.

Characterization of two monoterpene synthases involved in floral scent formation in Hedychium coronarium.

Hedychium coronarium, a perennial herb belonging to the family Zingiberaceae, is cultivated as a garden plant or cut flower as well as for medicine and aromatic oil. Its flowers emit a fresh and inviting scent, which is mainly because of monoterpenes present in the profile of the floral volatiles. However, fragrance produced as a result of monoterpenes has not been well studied. In the present study, two novel terpene synthase (TPS) genes (HcTPS7 and HcTPS8) were isolated to study the biosynthesis of monoterpenes in H. coronarium. In vitro characterization showed that the recombinant HcTPS7 was capable of generating sabinene as its main product, in addition to nine sub-products from geranyl diphosphate (GPP). Recombinant HcTPS8 almost specifically catalyzed the formation of linalool from GPP, while it converted farnesyl diphosphate (FPP) to α-bergamotene, cis-α-bisabolene, β-farnesene and other ten sesquiterpenes. Subcellular localization experiments revealed that HcTPS7 and HcTPS8 were located in plastids. Real-time PCR analyses showed that HcTPS7 and HcTPS8 genes were highly expressed in petals and sepals, but were almost undetectable in vegetative organs. The changes of their expression levels in petals were positively correlated with the emission patterns of sabinene and linalool, respectively, during flower development. The results indicated that HcTPS7 and HcTPS8 were involved in the biosynthesis of sabinene and linalool in H. coronarium flowers. Results on these two TPSs first characterized from H. coronarium provide new insights into molecular mechanisms of terpene biosynthesis in this species and also lay the basis for biotechnological modification of floral scent profile in Hedychium.

Tomato terpene synthases TPS5 and TPS39 account for a monoterpene linalool production in tomato fruits

Recombinant tomato terpene synthases, TPS5/37/39, catalyze the formation of linalool or nerolidol in vitro. However, little is known about their actual biological activities in tomato plants, especially in their fruits. Here, when all three TPSs were induced in tomato fruits by a chemical elicitor, geraniol, a significant linalool peak was detected in fruit tissues but not in control fruits. Considering the compartments of these TPS proteins and available substrates, the linalool peak induced by geraniol might be attributed to TPS5 and TPS37, both of them putatively localized in the plastids where high levels of monoterpene substrate geranyl diphosphate exist. In addition, application of geraniol also triggered jasmonic acid (JA)-related defense genes suggesting that the inducible TPSs might be correlated with JA-signaled defense responses.

Enantiospecific (S)-(+)-Linalool Formation from β-Myrcene by Linalool Dehydratase-Isomerase

The linalool dehydratase-isomerase from Castellaniella defragrans strain 65Phen catalyzes in the thermodynamically unfavourable direction the hydration of betamyrcene to linalool and further the isomerization to geraniol, the initial steps in anaerobic beta-myrcene biodegradation. We have now investigated the stereochemistry of this reaction. (S)-(+)-Linalool is formed with an enantiomeric excess of at least 95.4%. (R)-(-)-Linalool was not detected. This indicates an introduction of the hydroxy group on the si-face of beta-myrcene.

Enantiospecific (S)-(+)-Linalool Formation from beta-Myrcene by Linalool Dehydratase-Isomerase

Enantiospecific (S)-(+)-Linalool Formation from beta-Myrcene by Linalool Dehydratase-Isomerase

Linalool Dehydratase-Isomerase, a Bifunctional Enzyme in the Anaerobic Degradation of Monoterpenes

Castellaniella (ex Alcaligenes) defragrans strain 65Phen mineralizes monoterpenes in the absence of oxygen. Soluble cell extracts anaerobically catalyzed the isomerization of geraniol to linalool and the dehydration of linalool to myrcene. The linalool dehydratase was present in cells grown on monoterpenes, but not if grown on acetate. We purified the novel enzyme ∼1800-fold to complete homogeneity. The native enzyme had a molecular mass of 160 kDa. Denaturing gel electrophoresis revealed one single protein band with a molecular mass of 40 kDa, which indicated a homotetramer as native conformation. The aerobically purified enzyme was anaerobically activated in the presence of 2 mm DTT. The linalool dehydratase catalyzed in vitro two reactions in both directions depending on the thermodynamic driving forces: a water secession from the tertiary alcohol linalool to the corresponding acyclic monoterpene myrcene and an isomerization of the primary allylalcohol geraniol in its stereoisomer linalool. The specific activities (V(max)) were 140 nanokatals mg(-1) for the linalool dehydratase and 410 nanokatals mg(-1) for the geraniol isomerase, with apparent K(m) values of 750 μm and 500 μm, respectively. The corresponding open reading frame was identified and revealed a precursor protein with a signal peptide for a periplasmatic location. The amino acid sequence did not affiliate with any described enzymes. We suggest naming the enzyme linalool dehydratase-isomerase according to its bifunctionality and placing it as a member of a new protein family within the hydrolyases (EC 4.2.1.X).

Two nearly identical terpene synthases catalyze the formation of nerolidol and linalool in snapdragon flowers

Terpenoids emitted from snapdragon flowers include three monoterpenes derived from geranyl diphosphate (GPP), myrcene, (E)-beta-ocimene and linalool, and a sesquiterpene, nerolidol, derived from farnesyl diphosphate (FPP). Using a functional genomics approach, we have isolated and biochemically characterized two nearly identical nerolidol/linalool synthases, AmNES/LIS-1 and AmNES/LIS-2, two enzymes responsible for the terpenoid profile of snapdragon scent remaining to be characterized. The AmNES/LIS-2 protein has an additional 30 amino acids in the N-terminus, and shares 95% amino acid sequence identity with AmNES/LIS-1, with only 23 amino acid substitutions distributed across the homologous regions of the proteins. Although these two terpene synthases have very similar catalytic properties, and synthesize linalool and nerolidol as specific products from GPP and FPP, respectively, they are compartmentally segregated. GFP localization studies and analysis of enzyme activities in purified leucoplasts, together with our previous feeding experiments, revealed that AmNES/LIS-1 is localized in cytosol, and is responsible for nerolidol biosynthesis, whereas AmNES/LIS-2 is located in plastids, and accounts for linalool formation. Our results show that subcellular localization of bifunctional enzymes, in addition to the availability of substrate, controls the type of product formed. By directing nearly identical bifunctional enzymes to more than one cellular compartment, plants extend the range of available substrates for enzyme utilization, thus increasing the diversity of the metabolites produced.

Effect of heterogeneous and homogeneous pathways on selectivity of pinane-2-ol to linalool isomerization

The regularities of pinane-2-ol isomerization over block catalyst under reagent pressure of 2–45 Torr and within a temperature range of 450–620 °C were studied. A scheme of key product—linalool, as well as side products—hydrocarbons and 5-membered cyclic alcohols formation was suggested. Light hydrocarbons C 1–C 6 are formed by gas phase free radical pathway of pinane-2-ol destruction, hydrocarbons C 10 by linalool dehydration over catalyst surface acid centers, 5-membered cyclic alcohols by linalool intermolecular cyclization. The reaction rate constants of cis/ trans-pinane-2-ol conversion to linalool and its following cyclization to 5-membered alcohols were evaluated. The linalool formation mechanism including the intermolecular interaction of OH group proton resulting in pinane ring opening was suggested.

Changes of volatile compounds during heating of bacuri pulp.

The formation of volatile compounds from precursors or through chemical rearrangement during heat treatment of bacuri pulp at fruit natural pH were studied using simultaneous distillation/extraction (SDE) technique. An increase of the quantities of oxygenated and hydrocarbon terpenes and, to a lesser degree, aldehydes, was observed after SDE at pH 3, relative to the other extraction methods used, SDE at neutral pH and solid phase extraction (SPE). More particularly, linalool, linalool furanoxides, alpha-terpineol, hotrienol, nerol oxide, nerol, and geraniol were isolated in more important quantities after the first treatment than after the others. These results can be partially explained by the hydrolysis of glycosidically bound compounds previously identified in bacuri. Other pathways such as polyol rearrangements were also involved. The formation of linalool and alpha-terpineol was probably the result of the rearrangement of 2,6-dimethyloct-1-ene-3,7-diol. Moreover, it was assumed that oxidation reactions occurred during SDE at pH 3; more particularly, linalool pyranoxides partially resulted from nonenzymatic oxidation of linalool. When SDE was performed at pH 3, an increase of furfural and 4-methoxy-2,5-dimethyl-3(2H)-furanone was noticed. The modifications of the concentration of aliphatic aldehydes, known as lipid oxidation compounds, and of fatty acid esters were in good agreement with the observed decrease of palmitic and linoleic acid concentrations during this treatment. Moreover, important amounts of 2-acetyl-1-pyrroline were found in the SDE extract recovered at pH 7.

Kinetics of 2-Pinanol Isomerization to Linalool on the Monolith Carbon-Containing Catalyst

Isomerization of 2-pinanol into linalool is studied on a monolith carbon-containing catalyst at 733–893 and a total pressure of 2–40 torr. The rate constants of cis(trans)-2-pinanol transformation into linalool and linalool cyclization are determined. Experimental data are described by the consecutive kinetic scheme. The mechanism of linalool formation is proposed according to which pinane ring opening occurs in an intramolecular reaction. A highly selective process of linalool synthesis by the thermal isomerization of 2-pinanol is shown to be possible.

Linalyl Acetate Is Metabolized by Pseudomonas incognita with the Acetoxy Group Intact

Metabolism of linalyl acetate by Pseudomonas incognita isolated by enrichment culture on the acyclic monoterpene alcohol linalool was studied. Biodegradation of linalyl acetate by this strain resulted in the formation of linalool, linalool-8-carboxylic acid, oleuropeic acid, and Delta-4-acetoxy-4-methyl hexenoic acid. Cells adapted to linalyl acetate metabolized linalyl acetate-8-aldehyde to linalool-8-carboxylic acid, linalyl acetate-8-carboxylic acid, Delta-4-acetoxy-4-methyl hexenoic acid, and geraniol-8-carboxylic acid. Resting cell suspensions previously grown with linalyl acetate oxidized linalyl acetate-8-aldehyde to linalyl acetate-8-carboxylic acid, Delta-4-acetoxy-4-methyl hexenoic acid, and pyruvic acid. The crude cell-free extract (10,000 g of supernatant), obtained from the sonicate of linalyl acetate-grown cells, was shown to contain enzyme systems responsible for the formation of linalyl acetate-8-carboxylic acid and linalool-8-carboxylic acid from linalyl acetate. The same supernatant contained NAD-linked alcohol and aldehyde dehydrogenases involved in the formation of linalyl acetate-8-aldehyde and linalyl acetate-8-carboxylic acid, respectively. On the basis of various metabolites isolated from the culture medium, resting cell experiments, growth and manometric studies carried out with the isolated metabolites as well as related synthetic analogs, and the preliminary enzymatic studies performed with the cell-free extract, a probable pathway for the microbial degradation of linalyl acetate with the acetoxy group intact is suggested.