Irregular Monoterpenes Research Articles

Assessment of tobacco (Nicotiana tabacum) and N. benthamiana as biofactories of irregular monoterpenes for sustainable crop protection

Irregular monoterpenes are important precursors of different compounds employed in pest control such as insecticides and insect sex pheromones. Metabolically engineered plants are appealing as biofactories of such compounds, but specially as potential live biodispensers of related bioactive volatiles, which could be continuously emitted to the environment from different plant tissues. Here we assess the use of cultivated tobacco and Nicotiana benthamiana as biofactories for the irregular monoterpenes chrysanthemol and lavandulol. We evaluate the impact of high levels of constitutive metabolite production on the plant physiology and biomass, and their biosynthetic dynamics for different plant tissues and developmental stages. As an example of an active pheromone compound, we super-transformed the best lavandulol-producing tobacco line with an acetyl transferase gene to obtain a tobacco lavandulyl acetate biodispenser emitting up to 0.63 mg of lavandulyl acetate per plant every day. We estimate that with these volatile emission levels, between 200 and 500 plants per hectare would be sufficient to ensure a daily emission of pheromones comparable to commercial lures. This is an important step towards plant-based sustainable solutions for pest control, and it lays the ground for further developing biofactories for other irregular monoterpenoid pheromones, whose biosynthetic genes are yet unknown.

Engineering of a Plant Isoprenyl Diphosphate Synthase for Development of Irregular Coupling Activity.

We performed mutagenesis on a regular isoprenyl diphosphate synthase (IDS), neryl diphosphate synthase from Solanum lycopersicum (SlNPPS), that has a structurally related analogue performing non‐head‐to‐tail coupling of two dimethylallyl diphosphate (DMAPP) units, lavandulyl diphosphate synthase from Lavandula x intermedia (LiLPPS). Wild‐type SlNPPS catalyses regular coupling of isopentenyl diphosphate (IPP) and DMAPP in cis‐orientation resulting in the formation of neryl diphosphate. However, if the enzyme is fed with DMAPP only, it is able to catalyse the coupling of two DMAPP units and synthesizes two irregular monoterpene diphosphates; their structures were elucidated by the NMR analysis of their dephosphorylation products. One of the alcohols is lavandulol. The second compound is the trans‐isomer of planococcol, the first example of an irregular cyclobutane monoterpene with this stereochemical configuration. The irregular activity of SlNPPS constitutes 0.4 % of its regular activity and is revealed only if the enzyme is supplied with DMAPP in the absence of IPP. The exchange of asparagine 88 for histidine considerably enhanced the non‐head‐to‐tail coupling. While still only observed in the absence of IPP, irregular activity of the mutant reaches 13.1 % of its regular activity. The obtained results prove that regular IDS are promising starting points for protein engineering aiming at the development of irregular activities and leading to novel monoterpene structures.

Short-chain isoprenyl diphosphate synthases of lavender (Lavandula).

We reported the functional characterization of cDNAs encoding short-chain isoprenyl diphosphate synthases that control the partitioning of precursors for lavender terpenoids. Lavender essential oil is composed of regular and irregular monoterpenes, which are derived from linear precursors geranyl diphosphate (GPP) and lavandulyl diphosphate (LPP), respectively. Although this plant strongly expresses genes responsible for the biosynthesis of both monoterpene classes, it is unclear why regular monoterpenes dominate the oil. Here, we cloned and characterized Lavandula x intermedia cDNAs encoding geranyl diphosphate synthase (LiGPPS), geranylgeranyl diphosphate synthase (LiGGPPS) and farnesyl diphosphate synthase (LiFPPS). LiGPPS was heteromeric protein, consisting of a large subunit (LiGPPS.LSU) and a small subunit for which two different cDNAs (LiGPPS.SSU1 and LiGPPS.SSU2) were detected. Neither recombinant LiGPPS subunits was active by itself. However, when co-expressed in E. coli LiGPPS.LSU and LiGPPS.SSU1 formed an active heteromeric GPPS, while LiGPPS.LSU and LiGPPS.SSU2 did not form an active protein. Recombinant LiGGPPS, LiFPPS and LPP synthase (LPPS) proteins were active individually. Further, LiGPPS.SSU1 modified the activity of LiGGPPS (to produce GPP) in bacterial cells co-expressing both proteins. Given this, and previous evidence indicating that GPPS.SSU can modify the activity of GGPPS to GPPS in vitro and in plants, we hypothesized that LiGPPS.SSU1 modifies the activity of L. x intermedia LPP synthase (LiLPPS), thus accounting for the relatively low abundance of LPP-derived irregular monoterpenes in this plant. However, LiGPPS.SSU1 did not affect the activity of LiLPPS. These results, coupled to the observation that LiLPPS transcripts are more abundant than those of GPPS subunits in L. x intermedia flowers, suggest that regulatory mechanisms other than transcriptional control of LPPS regulate precursor partitioning in lavender flowers.

Structure-Function Studies of Artemisia tridentata Farnesyl Diphosphate Synthase and Chrysanthemyl Diphosphate Synthase by Site-Directed Mutagenesis and Morphogenesis.

The amino acid sequences of farnesyl diphosphate synthase (FPPase) and chrysanthemyl diphosphate synthase (CPPase) from Artemisia tridentata ssp. Spiciformis, minus their chloroplast targeting regions, are 71% identical and 90% similar. FPPase efficiently and selectively synthesizes the "regular" sesquiterpenoid farnesyl diphosphate (FPP) by coupling isopentenyl diphosphate (IPP) to dimethylallyl diphosphate (DMAPP) and then to geranyl diphosphate (GPP). In contrast, CPPase is an inefficient promiscuous enzyme, which synthesizes the "irregular" monoterpenes chrysanthemyl diphosphate (CPP), lavandulyl diphosphate (LPP), and trace quantities of maconelliyl diphosphate (MPP) from two molecules of DMAPP, and couples IPP to DMAPP to give GPP. A.tridentata FPPase and CPPase belong to the chain elongation protein family (PF00348), a subgroup of the terpenoid synthase superfamily (CL0613) whose members have a characteristic α terpene synthase α-helical fold. The active sites of A.tridentata FPPase and CPPase are located within a six-helix bundle containing amino acids 53 to 241. The two enzymes were metamorphosed into one another by sequentially replacing the loops and helices of the six-helix bundle from enzyme with those from the other. Chain elongation was the dominant activity during the N-terminal to C-terminal metamorphosis of FPPase to CPPase, with product selectivity gradually switching from FPP to GPP, until replacement of the final α-helix, whereupon cyclopropanation and branching activity competed with chain elongation. During the corresponding metamorphosis of CPPase to FPPase, cyclopropanation and branching activities were lost upon replacement of the first helix in the six-helix bundle. Mutations of active site residues in CPPase to the corresponding amino acids in FPPase enhanced chain-elongation activity, while similar mutations in the active site of FPPase failed to significantly promote formation of significant amounts of irregular monoterpenes. Our results indicate that CPPase, a promiscuous enzyme, is more plastic toward acquiring new activities, whereas FPPase is more resistant. Mutations of residues outside of the α terpene synthase fold are important for acquisition of FPPase activity for synthesis of CPP, LPP, and MPP.

Chemical Composition of Essentials Oils of Artemisia frigida Willd. (Asteraceae) Grown in the North and Central Asia

The compositions of essential oils found in Artemisia frigida Willd. growing within the Republic of Buryatia (Russia), Mongolia, and China are analyzed in this work. We identified 152 compounds in the essential oils of A. frigida, including acyclic, aromatic, mono-, sesqui- and diterpene compounds. The dominant compounds found in plants from the Republic of Buryatia were mostly monoterpenes, including 1,8-cineol, camphor, borneol and terpinen-4-ol. The samples from the Chita region of Russia and from Mongolia, which was drier than the Republic of Buryatia, contained a similar composition of major compounds as well as sesquiterpenes (e.g. caryophyllene, germacrene D) in addition to the above-mentioned monoterpenes. The samples from Qinghai Province, China, contained α-bisabolone oxide A, B, camphor, and 1,8-cineol as dominant compounds. There were some irregular monoterpenes (e.g., yomogi alcohol, artemisia ketone, artemisia alcohol, and lavandulol) in the Chinese samples. Eastern A. frigida populations were characterized by a high proportion of sesquiterpene compounds, while high-elevation mountain plants contain high concentrations of spathulenol and germacrene D.

Chemical Composition and Antibacterial Activity of the Bulgarian Endemic Species Achillea thracica from its Natural Habitat, and in vitro Propagated and ex vitro Established Plants

The present study reports the chemical composition of headspace volatiles (HS) and acetone extracts of the endemic Bulgarian species Achillea thracica Velen. from its natural habitat (N), in vitro propagated (IN) and ex vitro established (EX) plants. Additionally, acetone extracts were tested by a disk diffusion method for antibacterial activity. Irregular monoterpenes were the most abundant HS volatile constituents, while 0,0-dimethyl quercetin was the most abundant flavonoid in the acetone extracts. The secondary metabolites of A. thracica grown in its natural habitat (N), propagated in vitro (IN) and ex vitro established (EX) showed that the qualitative composition is mutually similar, but there are differences in the quantitative composition. Considering antibacterial activity, IN and EX samples showed moderate activity against Pseudomonas aeruginosa and Escherichia coli.

Vapour and Liquid-Phase Artemisia annua Essential Oil Activities against Several Clinical Strains of Candida.

Candida spp. are often the cause of infection in immune-compromised individuals. They are characterized by a strong resistance to antimicrobial drugs and disinfectants. The activity of Artemisia annua essential oil against Candida spp. was determined by vapour contact and microdilution assay. The oil was characterized by the presence of oxygenated monoterpenes (more than 75 % of the constituents), mainly represented by the irregular monoterpene artemisia ketone (ca. 22 %), and the widespread monoterpenes 1,8 cineole (ca. 19 %) and camphor (ca. 17 %). Other representative constituents were artemisia alcohol (5.9 %), α-pinene (5.7 %), and pinocarvone (3.0 %). Thujone, a typical toxic constituent of the Artemisia species, was not detected. The results are reported as minimum inhibitory concentration, minimum fungicidal concentration, and diameter of inhibition zone obtained by the vapour diffusion assay. We tested 10 clinical Candida strains, coming from both clinical samples and international collections. The results show that the antifungal activity of A.annua is influenced by the type of method adopted. The inhibitory action of the essential oil was, in fact, higher in the vapour than in the liquid phase. Our results show an average minimum inhibitory concentration in the liquid phase of 11.88 µL/mL, while in the vapour phase, the growth of all Candida strains tested at a concentration of 2.13 µL/cm(3) was inhibited. A strain of Candida glabrata was found to be less susceptible to the liquid medium than the vapour assay (50 µL/mL vs. 0.64 µL/cm(3), respectively). Candida albicans and Candida dubliniensis were the most susceptible to the vapour test, while Candida parapsilosis was the most resistant.

A new irregular monoterpene acetate along with eight known compounds with antifungal potential from the aerial parts of Artemisia incisa Pamp (Asteraceae)

A new compound named as santolinylol-3-acetate (4-(2-hydroxypropan-2-yl)-2-methylhexa-1,5-dien-3-yl acetate) (3), along with seven known compounds; linoleic acid (1), benzoic acid (2), santolinylol (4), ethyl-(E)-p-hydroxy cinnamate (5), scopoletin (6), esculetin (7) isofraxidin (8) and eupatorin (9), were isolated from the aerial parts (ethanolic extract) of endangered species: Artemisia incisa Pamp (Asteraceae). The compounds’ structures were determined through modern spectroscopic techniques, and comparison of data (physicochemical constants) with the literature. The relative stereochemistry of santolinylol-3-acetate (3) was determined by comparing its data of NOESY, and specific rotation with its diol analogue; santolinylol (4), isolated from the same plant; A. incisa. The results of the antifungal activity showed that coumarins are as whole less active compounds. Compounds 3 (25 and 300 μg/mL), and 4 (12.5 and 300 μg/mL), showed good activities against Candida albicans, and Aspergillus flavus, respectively, which justifies A. incisa as a traditional medicine for curing the said fungal infections.

Unexpected Irregular Monoterpene “Yomogi Alcohol” in the Volatiles of the <i>Lathyrus</i> L. species (Leguminosae) of Cyprus

Lathyrus species including L. ochrus and L. sativus are known for their food, feed and horticultural uses. Despite their widespread uses and cultivation, there is limited information on their chemistry. Previously, only the essential oil composition of L. rotundifolius, L. vernus and volatiles of L. odoratus have been reported. In the present research, volatiles of seven Lathyrus L. species, namely, L. aphaca, L. ochrus, L. cicera, L. sativus, L. gorgonei, L. saxatilis and L. blepharicarpos var. cyprius were analyzed by SPME GC-MS for the first time. Plant materials were collected from five different locations in Cyprus (February-March 2012). The main components of L. aphaca volatiles from four locations were yomogi alcohol 26.1-16.5%, camphor 21.6-10.1%, tetradecane 14.3-0%; L. cicera from five locations were yomogi alcohol 20.3-3.0%, camphor 18.7-2.0%; L. gorgonei from two locations were yomogi alcohol 24.5-13.1%, camphor 17.1-9.0% and L. sativus was yomogi alcohol 11.4%, camphor 9.0%. Yomogi alcohol was not present as the major compound in L. ochrus (2-methyl butanoic acid 7.2%), L. saxatilis (hexanal 7.7%) and L. blepharicarpos var. cyprius ((Z)-3-hexenal 8.6%) volatiles. The volatiles of the Lathyrus species were also compared with each other quantitative and qualitatively using AHC analysis to find out differences among the species. The irregular monoterpene yomogi alcohol is reported from the Lathyrus and the Leguminosae family for the first time. The existence of yomogi alcohol in Lathyrus volatiles points out that the irregular monoterpenes are not restricted solely to Asteraceae family.

Essential oil composition of four Artemisia species from Ethiopia

The essential oil composition of four Artemisia species, namely A. schimperi Sch. Bip. ex Engl. A. abyssinica Sch. Bip. ex A. Rich., A. afra Jacq. ex Willd., and A. absinthium L. (previously called A. rehan) from Ethiopia has been studied. The essential oil obtained from A. absinthium (seedling from Europe) grown in two places in Ethiopia (Addis Ababa and Butajira) was also analyzed for comparison. Morphological study on the leaves of A. absinthium L. from Ethiopia (previously called A. rehan) and A. absinthium (from Europe) was also conducted. The essential oils were obtained by hydrodistillation using a Clevenger apparatus and analyzed by capillary GC and GC/MS. Forty three compounds representing 76 to 94% of the oils were identified. The composition of the essential oils of A. schimperi, A. afra and A. abyssinica are mainly dominated by irregular monoterpenes: yogomi alcohol (13.5-37.6%), artemisyl acetate (12.7-35.5%), and artemisia ketone (2.3-13.2%). The composition of the oil of A. absinthium (previously A. rehan) however, differs from the other three species in having camphor (21.2-28.3%) and davanone (21.3-26.5%) as major components. The composition of A. absinthum (Europe) was found to have β-thujone (42.3-66.4%) and chamazulene (11.3-24.2%) as major components. The study indicated that the composition of the essential oil of A. absinthium (previously A. rehan) is not only different from the other three species but also from A. absinthium from Europe and does not belong to any of the chemotypes described for the species in the literature. The morphological study on the leaves also showed that it differs from that of A. absinthium from Europe. KEY WORDS: Artemisia schimperi, A. afra, A. abyssinica, A. absinthium, A. rehan, Asteraceae, Essential oil, Lyratyl isovalerate, Camphor, Davanone Bull. Chem. Soc. Ethiop. 2015, 29(1), 123-128DOI: http://dx.doi.org/10.4314/bcse.v29i1.11

Chemical Composition and Aroma Evaluation of Essential Oils from Evolvulus alsinoides L.

The aim of this study was to investigate the chemical composition and the odor-active compounds of the essential oils from Evolvulus alsinoides, which is a well-known edible and medicinal plant. The volatile compounds in the oils were identified by hydrodistillation (HD) and solvent-assisted flavor evaporation (SAFE) in combination with GC, GC/MS, GC/O (=olfactometry), aroma extract dilution analysis (AEDA), and relative flavor activities (RFA values). The most abundant compound in the HD oil was cis-α-necrodol (12.62%), an irregular monoterpene with a cyclopentane skeleton, which is very unusual in the plant kingdom. In the SAFE oil, the main components included 2-butoxyethanol (9.01%), benzyl alcohol (8.01%), and γ-butyrolactone (7.37%). Through sensory analysis, 21 aroma-active compounds were identified by GC/O. The most intense aroma-active compounds in the HD oil were hexan-1-ol and γ-nonalactone, both of which showed high RFA values. α-Methyl-γ-butyrolactone and dimethyl sulfone contributed more strongly to the aroma of the SAFE oil. These results imply that the essential oils of E. alsinoides deserve further investigation in the food industry.

Essential oil of Tanacetum dumosum as a new source of fragranol

Tanacetum (tansy) belongs to the tribe Anthemideae, which is one of the largest tribes of Asteraceae family, comprising about 111 genera and ca. 1800 species worldwide [1]. In the flora of Iran, this genus is represented by 31 annual and perennial species, of which 15 are endemic [2]. The essential oils of several Iranian tansies, including T. bachtiaricum Mozaff. [3], T. fruticulosum Ledeb. [4], T. canescens and T. turcomanicum [5], and T. polycephalum Schultz-Bip. [6], were analyzed by different authors and found to have monoterpenoids as major constituents. Among them, pinenes, 1,8-cineol, camphor, chrysanthenol derivatives, and thujones are detected more frequently. In addition to the above regular monoterpenoids, irregular monoterpenes such as artemisia ketone, santolina triene, and yomogi alcohol are found in some tansy species, including T. kotschyi [7] and T. santolinoides [8]. To the best of our knowledge, this is the first report on characterization of the chemical composition of the essential oil of T. dumosum. The aerial parts of Tanacetum dumosum Boiss. were collected from Dena mountain at an altitude of 3200 m in July 2011. The plant material was identified by one of us (MA), and a voucher specimen (No. PC-87-113) was deposited at the herbarium of our institute (MNCRS), Shiraz, Iran. The essential oil of the leaves and flowers of the plant (70 g) was extracted by hydrodistillation to yield 0.2 mL of pale-yellow oil, dried over anhydrous sodium sulfate, and analyzed by gas chromatography-mass spectrometry (GC-MS) and GC-flame ionization detector (GC-FID). Twenty seven monoand sesquiterpenoids were identified in the oil using GC retention indices (RRI) and mass spectral (MS) data, obtained from GC and GC-MS experiments, and comparison of these data to those reported for the standard volatiles in the literature [9, 10] (Table 1). Among them, the monoterpene hydrocarbons santolina triene (2.2%) and -pinene (2.2%) and the oxygenated monoterpenes fragranol (26.7%), 1,8-cineol (5.7%), fragranyl acetate (23.6%), and trans-carvyl acetate (10.1%) were detected as the major constituents. Helifolenol A, -sesquiphellandrene, caryophyllene oxide, and germacrene D are the major sesquiterpenes detected in this oil, but they are still in smaller quantities compared to the monoterpenoids fraction (Table 1). Fragranol, the E isomer of 2-isopropenyl-1-methylcyclobutane ethanol, was reported previously in the oils of Achillea santolina [9], A. nobilis [11], and A. falcata [12], but to the best of our knowledge for the first time in tansy species. Previously, fragranol was reported by one of us (ARJ) in the oil of Artemisia haussknechtii [13], a member of Anthemideae, which grows nearby T. dumosum. Grandisol, the Z-isomer of fragranol, is reported to be an aggregation and sex-attractant pheromone for several weevil insects [14, 15], and recently together with camphor and 1,8-cineol as the major constituent of an anti-insect essential oil of Artemisia vestita [16]. The antibacterial and insecticidal activity of the essential oils of the above plant species with similar chemical composition reported here for T. dumosum suggests that this plant is a potential medicinal plant to be explored in the future [9, 11, 12]. The good smell of the plant may be due to the presence of fragranol and its acetate ester and suggests that this plant can be used in the perfume industry. GC-MS and FID Analyses. GC-MS analysis was carried out on an Agilent 7890A GC coupled to an HP-6890 mass spectrometer operating in EI mode at 70 eV. The GC was equipped with a J&W DB-5 ms Scientific column (30 m 0.25 mm i.d., 0.25 m film thickness). The oven temperature was programmed from 60 C to 220 C at 5 C/min and kept for 10 min at 220 C. The carrier gas was helium (He) with a flow rate of 1 mL/min, and the injector temperature was set at 260 C in the split mode for the pure essential oil (1:100). The injection volume was 0.1 L. The GC-FID analysis was performed using the above instrument coupled to a FID with the same analytical conditions, but the column was replaced by a DB1 column with the same dimension as that used in GC-MS with the temperature of the FID set at 260 C.

The Biosynthetic Origin of Irregular Monoterpenes in Lavandula

Lavender essential oils are constituted predominantly of regular monoterpenes, for example linalool, 1,8-cineole, and camphor. However, they also contain irregular monoterpenes including lavandulol and lavandulyl acetate. Although the majority of genes responsible for the production of regular monoterpenes in lavenders are now known, enzymes (including lavandulyl diphosphate synthase (LPPS)) catalyzing the biosynthesis of irregular monoterpenes in these plants have not been described. Here, we report the isolation and functional characterization of a novel cis-prenyl diphosphate synthase cDNA, termed Lavandula x intermedia lavandulyl diphosphate synthase (LiLPPS), through a homology-based cloning strategy. The LiLPPS ORF, encoding for a 305-amino acid long protein, was expressed in Escherichia coli, and the recombinant protein was purified by nickel-nitrilotriacetic acid affinity chromatography. The approximately 34.5-kDa bacterially produced protein specifically catalyzed the head-to-middle condensation of two dimethylallyl diphosphate units to LPP in vitro with apparent Km and kcat values of 208 ± 12 μm and 0.1 s(-1), respectively. LiLPPS is a homodimeric enzyme with a sigmoidal saturation curve and Hill coefficient of 2.7, suggesting a positive co-operative interaction among its catalytic sites. LiLPPS could be used to modulate the production of lavandulol and its derivatives in plants through metabolic engineering.

Adaptive evolution of the chrysanthemyl diphosphate synthase gene involved in irregular monoterpene metabolism

BackgroundChrysanthemyl diphosphate synthase (CDS) is a key enzyme in biosynthetic pathways producing pyrethrins and irregular monoterpenes. These compounds are confined to plants of the tribe Anthemideae of the Asteraceae, and play an important role in defending the plants against herbivorous insects. It has been proposed that the CDS genes arose from duplication of the farnesyl diphosphate synthase (FDS) gene and have different function from FDSs. However, the duplication time toward the origin of CDS and the evolutionary force behind the functional divergence of the CDS gene are still unknown.ResultsTwo duplication events were detected in the evolutionary history of the FDS gene family in the Asteraceae, and the second duplication led to the origin of CDS. CDS occurred after the divergence of the tribe Mutisieae from other tribes of Asteraceae but before the birth of the Anthemideae tribe. After its origin, CDS accumulated four mutations in sites homologous to the substrate-binding and catalysis sites of FDS. Of these, two sites were involved in the binding of the nucleophilic substrate isopentenyl diphosphate in FDS. Maximum likelihood analyses showed that some sites in CDS were under positive selection and were scattered throughout primary sequences, whereas in the three-dimensional structure model they clustered in the large central cavity.ConclusionPositive selection associated with gene duplication played a major role in the evolution of CDS.

Lavandula luisieri essential oil as a source of antifungal drugs

This work reports the antifungal activity of Lavandula luisieri essential oils against yeast, dermatophyte and Aspergillus strains responsible for human infections and food contamination. The oil’s cytotoxicity and its effect on the yeast-mycelium transition in Candida albicans, an important virulence factor, were also evaluated.Analyses by GC and GC/MS showed a peculiar composition of irregular monoterpenes. Significant differences between the samples occurred in the amounts of 1,8-cineole, fenchone and trans-α-necrodyl acetate. The oil with higher amounts of irregular monoterpenes was the most effective. The influence of the oils on the dimorphic transition in C. albicans was also studied through the germ tube inhibition assay. Filamentation was completely inhibited at concentrations sixteen times lower than the minimal inhibitory concentration.The results support the use of L. luiseiri essential oils in the development of new phytopharmaceuticals and food preservatives and emphasise its antifungal properties at concentrations not cytotoxic or with very low detrimental effects on mammalian cells.

Cloning and functional analysis of CDS_CCI2: a Tanacetum cinerariaefolium chrysanthemyl diphosphate synthase gene

Although irregular monoterpenes are important and common in the Asteraceae family, little is known about their biosynthesis at the genetic level via the MEP pathway. Chrysanthemyl diphosphate (CPP) is an intermediate in the biosynthesis of pyrethrins which are irregular monoterpenes with excellent insecticidal activity in Tanacetum cinerariaefolium (T. cinerariaefolium). In this study, a chrysanthemyl diphosphate synthase (CDS) gene named CDS_CCI2 (GenBank accession no. HQ235057) was isolated from T. cinerariaefolium. It was homologous to T. cinerariaefolium CPP gene family, and proved to be located in the plastid by the in situ subcellular localization. CDS_CCI2 was found to be expressed in roots, stems, leaves, buds and flowers. Moreover, the expression of CDS_CCI2 can be up-regulated by abscisic acid (ABA), methyl jasmonate and ethrel treatment. Phenotypic and molecular analysis showed that overexpression of CDS_CCI2 in micro-tom tomato resulted in dwarf phenotypes characterized with infertile flowers and seedless fruits. Furthermore, overexpression of CDS_CCI2 altered the production of endogenous secondary metabolites. Our data indicate that CPP affects the synthesis of gibberellic acid (GA) and ABA.

Chemical Compositions of the Essential Oils of the Aerial Parts of Chamaemelum mixtum (L.) Alloni

The chemical compositions of the aerial parts essential oils of Chamaemelum mixtum (L.) Alloni from Corsica and Sardinia were investigated employing gas chromatography and gas chromatography-mass spectrometry (GC-MS). The structure of (Z)-heptadeca-9,16-dien-7-one, a natural compound not previously described, was elucidated by GC-MS (electron impact and chemical ionization) and one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy. The variation in C. mixtum essential oil was studied, and statistical analysis showed the clustering of oil samples into three groups according to the amount of oxygenated compounds; these groups correlated to the harvest area. The strong biological activity of the oxygenated fraction (minimum inhibitory concentration of <0.1 mg/mL) of the Corsican oil against Candida albicans , Citrobacter frendii , Enterococcus faecalis , Escherichia coli , Klebsiella pneumoniae , Listeria monocytogenes , and Staphyllococcus aureus can be attributed to the presence of irregular monoterpene alcohols and (Z)-heptadeca-9,16-dien-7-one.

Transformation of (±)-lavandulol and (±)-tetrahydrolavandulol by a fungal strain Rhizopus oryzae

Biotransformation of an irregular monoterpene alcohol, (±)-lavandulol [(±)-5-methyl-2-(1-methylethenyl)-4-hexen-1-ol] (I) and its tetrahydro derivative, (±)-tetrahydrolavandulol [(±)-2-isopropyl-5-methylhexan-1-ol] (II) were studied using a soil isolated fungal strain Rhizopus oryzae. Five metabolites, 2-((3,3-dimethyloxiran-2-yl)methyl)-3-methylbut-3-en-1-ol (Ia), 2-methyl-5-(prop-1-en-2-yl)hex-2-ene-1,6-diol (Ib), 2-methyl-5-(prop-1-en-2-yl)hexane-1,6-diol (Ic), 2-(3-methylbut-2-enyl)-3-methylenebutane-1,4-diol (Id), 5-methyl-2-(2-methyloxiran-2-yl)hex-4-en-1-ol (Ie) have been isolated from the fermentation medium and characterized with lavandulol as a substrate. When tetrahydrolavandulol used as a substrate, two metabolites 2-isopropyl-5-methylhexane-1,5-diol (IIa) and 2-isopentyl-3-methylbutane-1,3-diol (IIb) have been isolated from the fermentation medium. Biotransformation studies with R. oryzae clearly indicate that the organism initiates the transformation either by hydroxylation at allylic methyl groups or epoxidation of double bond. GC and GCMS analyses indicated that both (R)- and (S)-enantiomers of I and II have been transformed into corresponding hydroxylated or epoxy derivatives, when racemic I and II were used as substrates.

Differential induction, purification and characterization of cold active lipase from Yarrowia lipolytica NCIM 3639

The production, purification and characterization of cold active lipases by Yarrowia lipolytica NCIM 3639 is described. The study presents a new finding of production of cell bound and extracellular lipase activities depending upon the substrate used for growth. The strain produced cell bound and extracellular lipase activity when grown on olive oil and Tween 80, respectively. The organism grew profusely at 20°C and at initial pH of 5.5, producing maximum extracellular lipase. The purified lipase has a molecular mass of 400kDa having 20 subunits forming a multimeric native protein. Further the enzyme displayed an optimum pH of 5.0 and optimum temperature of 25°C. Peptide mass finger printing reveled that some peptides showed homologues sequence (42%) to Yarrowia lipolytica LIP8p. The studies on hydrolysis of racemic lavandulyl acetate revealed that extracellular and cell bound lipases show preference over the opposite antipodes of irregular monoterpene, lavandulyl acetate.

Megastigmane glucosides and an unusual monoterpene from the leaves of Cananga odorata var. odorata, and absolute structures of megastigmane glucosides isolated from C. odorata var. odorata and Breynia officinalis

From a 1-BuOH-soluble fraction of a MeOH extract of Cananga odorata var. odorata, collected at the Botanical Garden of Chiang Mai University, a new megastigmane glucoside, named canangaionoside, and an irregular monoterpene were isolated. A known compound, breyniaionoside A, which has been obtained from the leaves of Breynia officinalis, was also isolated, and its absolute structure was substantiated for the first time in this study. On this occasion, the absolute stereochemistries of structurally related megastigmane glucosides, breyniaionosides B and C, isolated from B. officinalis were examined.