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Abstract

Cymbopogon is an important aromatic and medicinal grass with several species of ethnopharmaceutical importance. The genus is extremely rich in secondary metabolites, monoterpenes like geraniol and citral being principal constituents, also used as biomarker for classification and identification of Cymbopogon chemotypes. In the light of this, present study involved RNA sequencing and comparison of expression profiles of four contrasting Cymbopogon species namely C. flexuosus var. Chirharit (citral rich and frost resistant), C. martinii var. PRC-1 (geraniol rich), C. pendulus var. Praman (the most stable and citral-rich genotype), and Jamrosa (a hybrid of C. nardus var. confertiflorus × C. jwarancusa (rich in geraniol and geranyl acetate)). The transcriptome profiles revealed marked differences in gene expression patterns of 28 differentially expressed genes (DEGs) of terpenoid metabolic pathways between the four Cymbopogon sp. The major DEGs were Carotenoid Cleavage Dioxygenases (CCD), Aspartate aminotransferase (ASP amino), Mevalonate E-4 hydroxy, AKR, GGPS, FDPS, and AAT. In addition, few TFs related to different regulatory pathways were also identified. The gene expression profiles of DEGs were correlated to the EO yield and their monoterpene compositions. Overall, the PRC-1 (C. martinii) shows distinguished gene expression profiles from all other genotypes. Thus, the transcriptome sequence database expanded our understanding of terpenoid metabolism and its molecular regulation in Cymbopogon species. Additionally, this data also serves as an important source of knowledge for enhancing oil yield and quality in Cymbopogon and closely related taxa. KEY MESSAGE: Unfolding the new secretes surrounding EO biosynthesis and regulation in four contrasting Cymbopogon species.

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... and geranial (48.06-49.15%) were identified in the essential oil of three citral-type (B1, B2, B3), which were higher than the rest of citral-rich plants in the world such as Litsea cubeba [10], Cymbopogon citratus [34], Ocimum gratissimum [35], Backhousia citriodra [36]. Those rare citral-type C. bodinieri varieties were identified through the scenting method from approximately 40,000 seedlings from Guizhou province, China. ...
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Essential oil isolated from lemongrass (Cymbopogon flexuosus) mutant cv. GRL-1 leaves is mainly composed of geraniol (G) and geranyl acetate (GA). The proportion of G and GA markedly fluctuates during leaf development. The proportions of GA and G in the essential oil recorded at day 10 after leaf emergence were approximately 59% and approximately 33% respectively. However, the level of GA went down from approximately 59 to approximately 3% whereas the level of G rose from approximately 33 to approximately 91% during the leaf growth period from day 10 to day 50. However, the decline in the level of GA was most pronounced in the early (day 10 to day 30) stage of leaf growth. The trend of changes in the proportion of GA and G has clearly indicated the role of an esterase that must be involved in the conversion of GA to G during leaf development. We isolated an esterase from leaves of different ages that converts GA into G and has been given the name geranyl acetate esterase (GAE). The GAE activity markedly varied during the leaf development cycle; it was closely correlated with the monoterpene (GA and G) composition throughout leaf development. GAE appeared as several isoenzymes but only three (GAE-I, GAE-II, and GAE-III) of them had significant GA cleaving activity. The GAE isoenzymes pattern was greatly influenced by the leaf developmental stages and so their GA cleaving activities. Like the GAE activity, GAE isoenzyme patterns were also found to be consistent with the monoterpene (GA and G) composition. GAE had an optimum pH at 8.5 and temperature at 30 degrees C. Besides GAE, a compound with phosphatase activity capable of hydrolyzing geranyl diphosphate (GPP) to produce geraniol has also been isolated.
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Incorporation of 13C-labelled glucose, acetate, pyruvate or erythrose allowed the determination of the origin of the carbon atoms of triterpenoids of the hopane series and/or of the ubiquinones from several bacteria (Zymomonas mobilis, Methylobacterium fujisawaense, Escherichia coli and Alicyclobacillus acidoterrestris) confirmed our earlier results obtained by incorporation of 13C-labelled acetate into the hopanoids of other bacteria and led to the identification of a novel biosynthetic route for the early steps of isoprenoid biosynthesis. The C5 framework of isoprenic units results most probably (i) from the condensation of a C2 unit derived from pyruvate decarboxylation (e.g. thiamine-activated acetaldehyde) on the C-2 carbonyl group of a triose phosphate derivative issued probably from dihydroxyacetone phosphate and not from pyruvate and (ii) from a transposition step. Although this hypothetical biosynthetic pathway resembles that of L-valine biosynthesis, this amino acid or its C5 precursors could be excluded as intermediates in the formation of isoprenic units.
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The monoterpene fraction of the lemon-scented sweet basil (Ocimum basilicum) cv Sweet Dani consists mostly of citral (a mixture of geranial and neral), with lower levels of geraniol and nerol. These compounds are stored in the peltate glands found on the leaf epidermis. Younger leaves, which have a higher density of such glands, also have a higher content of monoterpenes than older leaves. Geraniol synthase (GES) activity, generating geraniol from geranyl diphosphate, was shown to be localized exclusively or almost exclusively to glands. GES activity resides in a homodimeric protein that was purified to near homogeneity. Basil GES requires Mn2+ as a divalent metal cofactor for activity and produces only geraniol from geranyl diphosphate. Km values of 21 and 51 microM were obtained for geranyl diphosphate and Mn2+, respectively. In the presence of 18O-labeled water, GES catalyzed the formation of 18O-geraniol from geranyl diphosphate, indicating that the reaction mechanism of GES is similar to that of other monoterpene synthases and is different from the action of phosphatases. A GES cDNA was isolated based on analysis of a glandular trichome expressed sequence tag database, and the sequence of the protein encoded by this cDNA shows some similarity to sequences of other terpene synthases. The expression of the GES cDNA in Escherichia coli resulted in a protein with enzymatic activity essentially identical to that of plant-purified GES. RNA gel-blot analysis indicated that GES is expressed in glands but not in leaves of basil cv Sweet Dani, whose glands contain geraniol and citral, and not in glands or leaves of another basil variety that makes other monoterpenes but not geraniol or citral.
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The 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR; EC1.1.1.267), an NADPH-dependent reductase, plays a pivotal role in the methylerythritol 4-phosphate pathway (MEP), in the conversion of 1-deoxy-D-xylulose-5-phosphate (DXP) into MEP. The sheath and leaf of citronella (Cymbopogon winterianus) accumulates large amount of terpenes and sesquiterpenes with proven medicinal value and economic uses. Thus, sequencing of full length dxr gene and its characterization seems to be a valuable resource in metabolic engineering to alter the flux of isoprenoid active ingredients in plants. In this study, full length DXR from citronella was characterized through in silico and tissue- specific expression studies to explain its structure–function mechanism, mode of cofactor recognition and differential expression. The modelled DXR has a three-domain architecture and its active site comprised of a cofactor (NADPH) binding pocket and the substrate-binding pocket. Molecular dynamics simulation studies indicated that DXR model retained most of its secondary structure during 10 ns simulation in aqueous solution. The modelled DXR superimposes well with its closest structural homolog but subtle variations in the charge distribution over the cofactor recognition site were noticed. Molecular docking study revealed critical residues aiding tight anchoring NADPH within the active pocket of DXR. Tissue-specific differential expression analysis using semi-quantitative RT-PCR and qRT-PCR in various tissues of citronella plant revealed distinct differential expression of DXR. To our knowledge, this is the first ever report on DXR from the important medicinal plant citronella and further characterization of this gene will open up better avenues for metabolic engineering of secondary metabolite pathway genes from medicinal plants in the near future.
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Eleven elite and popular Indian cultivars of Cymbopogon aromatic grasses of essential oil trade types - citronella, palmarosa and lemongrass - were characterized by means of RAPDs to discern the extent of diversity at the DNA level between and within the oil biotypes. Primary allelic variability and the genetic bases of the cultivated germplasm were computed through parameters of gene diversity, expected heterozygosity, allele number per locus, SENA and Shannon's information indices. The allelic diversity was found to be in the order: lemongrass > palmarosa > citronella. Lemongrasses displayed higher (1.89) allelic variability per locus than palmarosa (1.63) and citronella (1.40). Also, RAPDs of diagnostic and curatorial importance were discerned as 'stand along' molecular descriptors. Principal component analysis (PCA) resolved the cultivars into four clusters: one each of citronella and palmarosa, and two of lemongrasses (one of C. flexuosus and another of C. pendulus and its hybrid with C. khasianus). Proximity of the two species-groups of lemongrasses was also revealed as they shared the same dimension in the three-dimensional PCA. The molecular distinctions are discussed in relation to oil-chemotypic variations.
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Secretory cells were isolated from the monoterpene-producing glandular trichomes (peltate form) of peppermint as clusters of eight cells each. These isolated structures were shown to be non-specifically permeable to low-molecular-weight, water-soluble cofactors and substrates. Short incubation periods with the polar dye Lucifer yellow iodoacetamide (Mr=660) resulted in a uniform staining of the cytoplasm, with exclusion of the dye from the vacuole. The molecular-weight exclusion limit for this permeability was shown to be less than approx. 1800, based on exclusion of fluorescein-conjugated dextran (Mr ∼ 1800). Intact secretory cell clusters very efficiently incorporated [(3)H]geranyl pyrophosphate into monoterpenes. The addition of exogenous cofactors and redox substrates affected the distribution of monoterpenes synthesized from [(3)H]geranyl pyrophosphate, demonstrating that the cell clusters were permeable to these compounds and that the levels of endogenous cofactors and redox substrates were depleted in the isolated cells. When provided with the appropriate cofactors, such as NADPH, NAD(+), ATP, ADP and coenzyme A, the isolated secretory cell clusters incorporated [(14)C]sucrose into monoterpenes, indicating that these structures are capable of the de-novo biosynthesis of monoterpenes from a primary carbon source, and that they maintain a high degree of metabolic competence in spite of their permeable nature.
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At different developmental stages, citronella Java (Cymbopogon winterianus Jowitt) leaves were analyzed for essential oil content and composition. Only immature leaves (up to 75% leaf expansion) were biogenetically active to synthesize and accumulate essential oil substantially. The relative percentage of geraniol and citronellol in the oil from immature leaf increased with increase in tissue age along the leaf blade, and was accompanied by a corresponding decrease in geranyl acetate and citronellyl acetate. During leaf ontogeny, the amount of citronellal, geraniol and citronellol in the essential oil increased with leaf expansion; whereas, the amount of geranyl acetate and citronellyl acetate decreased. As the leaf matured, a significant decrease in the essential oil, citronellal and geraniol contents was observed. The essential oil level showed a highly significant linear association with the citronellal and geraniol content; whereas, the geraniol content was positively correlated with citronellol and citronellal content. Oil content was also significantly associated with leaf dry weight and chlorophyll content. The results are consistent with geraniol to citronellol and citronellol to citronellal in vivo interconversions of the monoterpene constituents.
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The essential oils of Palmarosa (Cymbopogon martinii (Roxb.) Wats. var. motia Burk) flowering herbs from three different geographical locations in India, viz. Hyderabad,Lucknow and Amarawati were analysed by high-resolution GC and GC –MS. In all three samples, geraniol (67.6 –83.6%) was the major constituent and, although the composition of the three oils was similar, quantitative differences in the concentration of some constituents were observed. The GC and GC –MS analysis of the Hyderabad, Lucknow and Amarawati oils resulted in the identification of 40, 38 and 33 constituents, representing 98.5%, 99.7% and 99.8% of the oils,respectively. Among the three oils analysed, Amarawati oil was of the highest quality due to higher geraniol (83.6%) and lower geranyl acetate (2.3%) and geranial (1.0%) content. Copyright © 2003 John Wiley & Sons, Ltd.
Article
The conversion of geraniol into citral trans in Cymbopogon flexuosus leaves is catalyzed by NADP+-dependent geraniol dehydrogenase (E.C. 1.1.1.-). The enzyme was located in cytosol, which also contained alcohol dehydrogenase activity. However, the two activities could by resolved, in an unequivocal manner, by PAGE and in situ enzyme staining. Fractionation of soluble supernatant by ammonium sulphate and 2-propanol precipitation allowed complete separation of alcohol dehydrogenase from geraniol dehydrogenase. Subsequently, chromatography on sephadex G-200 provided about 70-fold purification. The reaction catalyzed was reversible, and the enzyme had the same pH (8.0) optimum for both directions. The enzyme showed normal Michaelis-Menten kinetics. The Km values for geraniol and geranial were 0.12mM and 0.10mM, while those for NADP+ and NADPH were 19.2μM and 8.3μM, respectively. The enzyme had an apparent molecular weight of 84,000 Da. Thiol-directed reagents and N-bromosuccinamide were inhibitory, whereas imidazole and pyrazole had no effect. The redox ratio greatly influenced the enzyme activity.
Article
Nineteen Cymbopogon taxa belonging to 11 species, two varieties, one hybrid taxon and four unidentified species were analysed for their essential oil constituents and RAPD profiles to determine the extent of genetic similarity and thereby the phylogenetic relationships among them. Remarkable variation was observed in the essential oil yield ranging from 0.3% in Cymbopogon travancorensis Bor. to 1.2% in Cymbopogon martinii (Roxb.) Wats var. motia. Citral, a major essential oil constituent, was employed as the base marker for chemotypic clustering. Based on genetic analysis, elevation of Cymbopogon flexuosus var. microstachys (Hook. F.) Soenarko to species status and separate species status for C. travancorensis Bor., which has been merged under C. flexuosus (Steud.) Wats were suggested towards resolving some of the taxonomic complexes in Cymbopogon. The separate species status for the earlier proposed varieties of C. martinii (motia and sofia) is further substantiated by these analyses. The unidentified species of Cymbopogon have been observed as intermediate forms in the development of new taxa.
Article
The aerial parts (leaves) of lemongrass ( Cymbopogon flexuosus Nees ex Steud) wats (cultivar OD-19) on steam-distillation yield an essential oil rich in acyclic monoterpenes, citral (83%). To specifically locate the sites of citral accumulation in lemongrass we employed Schiff’s reagent, which upon reaction with aldehydes (citral) gives a purple-red colouration. Using this technique, single oil-accumulating cells were detected in the adaxial side of leaf mesophyll commonly adjacent to non-photosynthetic tissue and between vascular bundles. In addition to cultivar OD-19, a citral lacking cultivar GRL-1 (geraniol rich) leaf sections, also stained with schiff’s reagent and compared with that of lemongrass cultivar OD-19 leaf sections. In lemongrass mutant GRL-1, these specialized cells, however, could not be stained due to lack of citral. Hence, it is confirm that the observed schiff’s staining is associated with accumulated citral.
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This issue contains:- "The discovery of a mevalonate-independent pathway for isoprenoid biosynthesis in bacteria, algae and higher plants", M. Rohmer, p. 565-574; "Lantibiotics: biosynthesis, mode of action and applications", C. van Kraaij*, W. M. de Vos, R. J. Siezen, O, P. Kuipers, p. 575-588; "Bioactive natural products with carbon-phosphorus bonds and their biosynthesis", H. Seto*, T. Kuzuyama, p. 589-596; "Nitrile-containing natural products", F. F. Fleming, p. 597-606; "Steroids: reactions and partial synthesis", J. R. Hanson, p. 607-618; and "Diterpenoid and steroidal alkaloids", Atta-ur-Rahman*, M. I. Choudhary, p. 618-636..
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The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data.
Article
Geraniol may accumulate up to 86-98% of the leaf essential oils in geraniol chemotypes of the evergreen camphor tree Cinnamomum tenuipilum. A similarity-based cloning strategy yielded a cDNA clone that appeared to encode a terpene synthase and which could be phylogenetically grouped within the angiosperm monoterpene synthase/subfamily. After its expression in Escherichia coli and enzyme assay with prenyl diphosphates as substrates, the enzyme encoded by the putative C. tenuipilum monoterpene synthase gene was shown to specifically convert geranyl diphosphate to geraniol as a single product by GC-MS analysis. Biochemical characterization of the partially purified recombinant protein revealed a strong dependency for Mg2+ and Mn2+, and an apparent Michaelis constant of 55.8 microM for geranyl diphosphate. Thus, a new member of the monoterpene synthase family was identified and designated as CtGES. The genome contains a single copy of CtGES gene. Expression of CtGES was exclusively observed in the geraniol chemotype of C. tenuipilum. Furthermore, in situ hybridization analysis demonstrated that CtGES mRNA was localized in the oil cells of the leaves.
Article
Geraniol synthases were isolated from five pure strains of Perilla citriodora and Perilla frutescens which vary in essential oil type, the main compounds of which were citral, elsholtziaketone, perillaketone, and perillene, respectively. This result supports the putative biosynthetic pathways of these three furylalkenes which are all produced by way of citral. Nucleotide sequences of geraniol synthases from three oil types of P. citriodora were identical, and almost the same as the sequence from P. frutescens, a species with twice the chromosome number of P. citriodora. This identity in sequence between P. citriodora and P. frutescens, together with other previous results, indicates that P. frutescens was formed as an amphidiploid of P. citriodora and an unknown wild species.
Essential Oil Plants and Their Cultivation
  • A Husain
A. Husain. Essential Oil Plants and Their Cultivation. Central Inst. Medicinal Aromatic Plants, 1994, pp. 58-66p..
Changes in the essential oil content and composition during palmarosa (Cymbopogon martini Roxb. Wats var. motia) in- florescence development
  • Dubey
Chirharit-a high yielding lemongrass variety with frost resistance, stay-green habit and new chromosomal ploidy status
  • Patra
Chirharit-a high yielding lemongrass variety with frost resistance, stay-green habit and new chromosomal ploidy status
  • N K Patra
  • S Kumar
  • S P Khanuja
  • A K Shasany
  • M P Darokar
  • A Kalra
  • P Ram
  • H B Singh
  • H P Singh
  • V R Singh
  • B Kumar
N.K. Patra, S. Kumar, S.P. Khanuja, A.K. Shasany, M.P. Darokar, A. Kalra, P. Ram, H.B. Singh, H.P. Singh, V.R. Singh, B. Kumar, Chirharit-a high yielding lemongrass variety with frost resistance, stay-green habit and new chromosomal ploidy status, J. Med. Arom. Pl. Sci. 23 (2001) 137-140.