BAHD acetyltransferase contributes to wound-induced biosynthesis of oleo-gum resin triterpenes in Boswellia.

Triterpenes (30-carbon isoprene compounds) represent a large and highly diverse class of natural products that play various physiological functions in plants. The triterpene biosynthetic enzymes, particularly those catalyzing the late-stage regio-selective modifications are not well characterized. The bark of select Boswellia trees, e.g., B. serrata exudes specialized oleo-gum resin in response to wounding, which is enriched with boswellic acids (BAs), a unique class of C3α-epimeric pentacyclic triterpenes with medicinal properties. The bark possesses a network of resin secretory structures comprised of vertical and horizontal resin canals, and amount of BAs in bark increases considerably in response to wounding. To investigate BA biosynthetic enzymes, we conducted tissue-specific transcriptome profiling and identified a wound-responsive BAHD acetyltransferase (BsAT1) of B. serrata catalyzing the late-stage C3α-O-acetylation reactions in the BA biosynthetic pathway. BsAT1 catalyzed C3α-O-acetylation of αBA, ßBA, and 11-keto-ßBA in vitro and in planta assays to produce all the major C3α-O-acetyl-BAs (3-acetyl-αBA, 3-acetyl-ßBA, and 3-acetyl-11-keto-ßBA) found in B. serrata bark and oleo-gum resin. BsAT1 showed strict specificity for BA scaffold, whereas it did not acetylate the more common C3ß-epimeric pentacyclic triterpenes. The analysis of steady-state kinetics using various BAs revealed distinct substrate affinity and catalytic efficiency. BsAT1 transcript expression coincides with increased levels of C3α-O-acetyl-BAs in bark in response to wounding, suggesting a role of BsAT1 in wound-induced biosynthesis of C3α-O-acetyl-BAs. Overall, the results provide new insights into the biosynthesis of principal chemical constituents of Boswellia oleo-gum resin.

Induced mutation breeding for qualitative and quantitative traits and varietal development in medicinal and aromatic crops at CSIR-CIMAP, Lucknow (India): past and recent accomplishment.

PURPOSE: The nature and kind of some successful mutations achieved through gamma radiation at CSIR-CIMAP, Lucknow are reviewed and described in this article. Medicinal and aromatic crops (MACs) came under the mutation-breeding program very late - perhaps during the 1980s in India. Nevertheless, successes have been glaring and unique too. The plant breeders of the Institute concerted attempts were made for the genetic restructuring of the plant-frame in Hyoscyamus niger L. (black henbane) and Cymbopogon martinii (palmarosa), enhanced alkaloid biosynthesis in henbane, weak, or lack of latex biosynthesis in Papaver somniferum L. (Opium poppy), increased productivity of seeds in Plantago ovata F. (isabgol), and new/increased formation of essential oils in Chamomila recutita [L.] Rauschert (Chamomile) and Cymbopogon winterianus (Java citronella). Several qualitative macro mutations were generated for commercial exploitation, e.g. an unbranched (Ub) and physiologically most efficient variety Aekla and high tropane alkaloid bearing variety Aela of H. niger, opium less oil-seed variety Sujata of opium poppy, a dwarf mutant and a male sterile (ms) line of palmarosa, and new compound yielding variety of chamomile and geranium. CONCLUSIONS: The ample quantitative variation was also created by reshuffling the polygenic background in both seed and vegetatively propagated MACs, and subsequently, with applying mutation breeding approach superior varieties were evolved and released after a rigorous screening in the field-evaluation or under pipeline for release. These varieties are Niharika and Mayuri of psyllium; Vallary, CIMAP Sammohak, CIM Ujjwala, and YEL (yellow) of German chamomile and Manjari and CIM Jeeva of Java citronella.

Plant growth-promoting rhizobacteria enhance wheat salt and drought stress tolerance by altering endogenous phytohormone levels and TaCTR1/TaDREB2 expression.

Abiotic stresses such as salt and drought represent adverse environmental conditions that significantly damage plant growth and agricultural productivity. In this study, the mechanism of the plant growth-promoting rhizo-bacteria (PGPR)-stimulated tolerance against abiotic stresses has been explored. Results suggest that PGPR strains, Arthrobacter protophormiae (SA3) and Dietzia natronolimnaea (STR1), can facilitate salt stress tolerance in wheat crop, while Bacillus subtilis (LDR2) can provide tolerance against drought stress in wheat. These PGPR strains enhance photosynthetic efficiency under salt and drought stress conditions. Moreover, all three PGPR strains increase indole-3-acetic acid (IAA) content of wheat under salt and drought stress conditions. The SA3 and LDR2 inoculations counteracted the increase of abscisic acid (ABA) and 1-aminocyclopropane-1-carboxylate (ACC) under both salt and drought stress conditions, whereas STR1 had no significant impact on the ABA and ACC content. The impact of PGPR inoculations on these physiological parameters were further confirmed by gene expression analysis as we observed enhanced levels of the TaCTR1 gene in SA3-, STR1- and LDR2-treated wheat seedlings as compared to uninoculated drought and salt stressed plants. PGPR inoculations enhanced expression of TaDREB2 gene encoding for a transcription factor, which has been shown to be important for improving the tolerance of plants to abiotic stress conditions. Our study suggest that PGPR confer abiotic stress tolerance in wheat by enhancing IAA content, reducing ABA/ACC content, modulating expression of a regulatory component (CTR1) of ethylene signaling pathway and DREB2 transcription factor.

Scent from Jasminum grandiflorum flowers: Investigation of the change in linalool enantiomers at various developmental stages using chemical and molecular methods.

Jasminum species are among the most preferred fresh cut flowers in India since ancient times. The plant produces small and fragrant flowers, which are of great demand in the preparation of fragrant garlands and also in perfume industries. Floral volatile of Jasminum grandiflorum L. (Family: Oleaceae) was extracted using solid-phase microextraction and analyzed in enantioselective gas chromatography. Chemical classes of identified volatiles revealed the presence of terpenoids, phenylpropanoids, and fatty acid derivatives. Marker constituent of flower volatiles, linalool was selected for analytical characterization on ethyl- and acetyl-ß-cyclodextrin stationary phase. (R)-(-)-Linalool was found as major enantiomer in volatiles of floral buds whereas (S)-(+)-linalool predominated in the volatiles of matured flowers. Simultaneously, a quantitative real-time PCR was performed to find the gene expression of linalool synthase to investigate the mechanism of enantiomeric inversion. The emission pattern of (R)-(-)-linalool at different flower developmental stages was well correlated (P = 0.01) with the gene expression of the cloned linalool synthase from J. grandiflorum. We observed that the successive change in (R)- to (S)-linalool ratio from bud to mature flower was mainly due to the enantio- specific transformation and temporal decline of (R)-linalool producing gene in J. grandiflorum. This enantiomeric change also leads to the difference in flower aroma. Furthermore, this is probably the reason behind consumer's acceptance for jasmine buds rather than bloomed flowers in cut flower segments.

Transcriptomic insight into terpenoid and carbazole alkaloid biosynthesis, and functional characterization of two terpene synthases in curry tree (Murraya koenigii).

Curry tree (Murraya koenigii L.) is a rich source of aromatic terpenes and pharmacologically important carbazole alkaloids. Here, M. koenigii leaf transcriptome was generated to gain insight into terpenoid and alkaloid biosynthesis. Analysis of de novo assembled contigs yielded genes for terpene backbone biosynthesis and terpene synthases. Also, gene families possibly involved in carbazole alkaloid formation were identified that included polyketide synthases, prenyltransferases, methyltransferases and cytochrome P450s. Further, two genes encoding terpene synthases (MkTPS1 and MkTPS2) with highest in silico transcript abundance were cloned and functionally characterized to determine their involvement in leaf volatile formation. Subcellular localization using GFP fusions revealed the plastidial and cytosolic localization of MkTPS1 and MkTPS2, respectively. Enzymatic characterization demonstrated the monoterpene synthase activity of recombinant MkTPS1, which produced primarily (-)-sabinene from geranyl diphosphate (GPP). Recombinant MkTPS2 exhibited sesquiterpene synthase activity and formed (E,E)-α-farnesene as the major product from farnesyl diphosphate (FPP). Moreover, mRNA expression and leaf volatile analyses indicated that MkTPS1 accounts for (-)-sabinene emitted by M. koenigii leaves. Overall, the transcriptome data generated in this study will be a great resource and the start point for characterizing genes involved in the biosynthetic pathway of medicinally important carbazole alkaloids.

Enantioselective GC Analysis of C3-Oxygenatedp-Menthane type Indian Mentha spicata var. viridis 'Ganga' Essential Oil.

Essential oil of Mentha spicata L. var. viridis 'Ganga', an indigenously developed variety, was chemically profiled using various gas chromatographic techniques. Piperitenone oxide was characterized as the most exclusive constituent (69.7%) along with a new C3-oxygenated p-menthane alcohol,- diosphenolene (1.6%). Enantiomeric discrimination revealed (4S)-(-)-limonene, (R)-(-)-linalool and (lS,2S)-(+)-piperitenone oxide as predominant enantiomers. The oil contained mainly C3-oxygenated p-menthane monoterpenoids, which are distinctive of peppermint, instead of the characteristic C6-oxygenated class of spearmint. The present findings will aid in understanding the pathway and cause of C3-oxygenation in a spearmint taxon. The essential oil and pure piperitenone oxide showed growth inhibiting properties and thus, may be utilized in antifungal preparations for disease management of medicinal and aromatic plants.

Compositional and Enantiomeric Analysis of the Essential Oil of Taxodium distichum from India.

The leaf essential oil composition of Taxodium distichum L., collected from the foothills of Uttarakhand, India was analyzed using gas chromatography-flame ionization detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS) equipped with DB-5 (5% diphenyl-95% dimethyl polysiloxane) and ß-cyclodextrin (6-tertiarybutyldimethylsiliyl-2,3-diethyl-ß-cyclodextrin) capillary columns. Seventeen constituents, representing 90.3 to 99.4% of composition were identified in the essential oils from different seasons, viz. spring, summer, rainy, autumn and winter. The essential oil composition was mainly dominated by monoterpene hydrocarbons, represented mainly by α-pinene (81.9-94.3%). Other constituents of the oil were myrcene (0.5-4.7%), ß-pinene (2.2-2.9%), limonene (0.5-1.5%), camphene (≤ 0.03-1.5%), and α-terpineol (upto 1.6%). Chiral analysis of T. distichum essential oil on an ethyl substituted ß-cyclodextrin capillary column revealed the presence of a-pinene in racemic form, with an enantiomeric ratio of 49.3% for (1R)-(+)- and 50.7% for (1S)-(-)-α-pinene.

Compositional variability and antifungal potentials of ocimum basilicum, O. tenuiflorum, O. gratissimum and O. kilimandscharicum essential oils against Rhizoctonia solani and Choanephora cucurbitarum.

The composition of hydrodistilled essential oils of Ocimum basilicum L. (four chemovariants), O. tenuiflorum L., O. gratissimum L., and O. kilimandscharicum Guerke were analyzed and compared by using capillary gas chromatography (GC/FID) and GC-mass spectrometry (GC/MS). Phenyl propanoids (upto 87.0%) and monoterpenoids (upto 83.3%) were prevalent constituents distributed in the studied Ocimum taxa. The major constituents of the four distinct chemovariants of O. basilicum were methyl chavicol (86.3%), methyl chavicol (61.5%)/linalool (28.6%), citral (65.9%); and linalool (36.1%)/citral (28.8%). Eugenol (66.5% and 78.0%) was the major constituent of O. tenuiflorum and O. gratissimum. Eugenol (34.0%), ß-bisabolene (15.4%), (E)-α-bisabolene (10.9%), methyl chavicol (10.2%) and 1,8-cineole (8.2%) were the major constituents of O. kilimandscharicum. In order to explore the potential for industrial use, the extracted essential oils were assessed for their antifungal potential through poison food technique against two phytopathogens, Rhizoctonia solani and Choanephora cucurbitarum, which cause root and wet rot diseases in various crops. O. tenuiflorum, O. gratissimum, and O. kilimandscharicum exhibited complete growth inhibition against R. solani and C. cucurbitarum after 24 and 48 h of treatment. O. basilicum chemotypes showed variable levels of growth inhibition (63.0%-100%) against these two phytopathogens.

Chemical analysis of volatile oils from West Himalayan Pindrow Fir Abies pindrow.

The essential oil composition of needle and stem oils of Abies pindrow (Royle ex D.Don) Royle, commonly known as Pindrow or West Himalayan Fir, were analyzed by gas chromatography with flame ionization detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS). Fifty-six constituents, accounting for 96.0% of needle and 83.5% of stem oil composition, were identified. The oils were characterized by a high content of monoterpenoids (68.9%-79.9%), mainly comprised by limonene (21.0%-34.4%), camphene (0.5%-19.9%), alpha-pinene (13.8%-16.8%), myrcene (6.7%-8.3%) and beta-pinene (6.5%-8.6%). Monoterpene hydrocarbons were predominant in both oils, but the quantitative and qualitative composition of the volatile constituents was specific for each part of the tree; and considerable variations in their terpenoid production pattern were also noticed. Results were compared with earlier reported fir species from different geographic regions.

4-coumarate: CoA ligase partitions metabolites for eugenol biosynthesis.

Biosynthesis of eugenol shares its initial steps with that of lignin, involving conversion of hydroxycinnamic acids to their corresponding coenzyme A (CoA) esters by 4-coumarate:CoA ligases (4CLs). In this investigation, a 4CL (OS4CL) was identified from glandular trichome-rich tissue of Ocimum sanctum with high sequence similarity to an isoform (OB4CL_ctg4) from Ocimum basilicum. The levels of OS4CL and OB4CL_ctg4-like transcripts were highest in O. sanctum trichome, followed by leaf, stem and root. The eugenol content in leaf essential oil was positively correlated with the expression of OS4CL in the leaf at different developmental stages. Recombinant OS4CL showed the highest activity with p-coumaric acid, followed by ferulic, caffeic and trans-cinnamic acids. Transient RNA interference (RNAi) suppression of OS4CL in O. sanctum leaves caused a reduction in leaf eugenol content and trichome transcript level, with a considerable increase in endogenous p-coumaric, ferulic, trans-cinnamic and caffeic acids. A significant reduction in the expression levels was observed for OB4CL_ctg4-related transcripts in suppressed trichome compared with transcripts similar to the other four isoforms (OB4CL_ctg1, 2, 3 and 5). Sinapic acid and lignin content were also unaffected in RNAi suppressed leaf samples. Transient expression of OS4CL-green fluorescent protein fusion protein in Arabidopsis protoplasts was associated with the cytosol. These results indicate metabolite channeling of intermediates towards eugenol by a specific 4CL and is the first report demonstrating the involvement of 4CL in creation of virtual compartments through substrate utilization and committing metabolites for eugenol biosynthesis at an early stage of the pathway.

Molecular docking and ADME studies of natural compounds of Agarwood oil for topical anti-inflammatory activity.

Aquilaria agallocha Roxb. family, Thymelaeaceae, is an evergreen plant of South-East Asia, commonly described as aloe wood or agarwood. Traditionally, the bark, root and heartwood are used for their medicinal properties as a folk medicine for hundreds of years. Chemical analyses revealed that the bulk of the oil is constituted by agarospirol (12.5%), jinkoh-eremol (11.8%) and hinesol (8.9%) as major contributor. In the present work, a QSAR model for antiinflammatory activity of 10-epi-γ-Eudesmol, jinkoh-eremol, agarospirol and other compounds has been developed by multiple linear regression method. The r(2) and rCV(2) of a model were 0.89 and 0.81 respectively. In silico molecular docking study suggests that compound 10-epi-γ-Eudesmol, jinkoh-eremol and agarospirol are preferentially more active than other identified compounds with strong binding affinity to major anti-inflammatory and immunomodulatory receptors. The oil displayed a significant and dose dependent reduction of 12-O-tetradecanoylphorobol-13 acetate (TPA)- induced ear edema and MDA activity when compared with vehicle treated mice. Pro-inflammatory cytokines (IL-1ß, IL-6 and TNF-α) were also reduced significantly in a dose dependent manner in all the TPA treated groups as compared to control. The present study indicates that agarwood oil significantly reduced the skin thickness, ear weight, oxidative stress and pro-inflammatory cytokines production in TPA-induced mouse ear inflammation model and contributed towards validation of its traditional use to treat inflammation related ailments.

Characterization of volatile components of Zingiber roseum essential oil using capillary GC on modified cyclodextrins.

The essential oil from different parts of Zingiber roseum plants was extracted by hydrodistillation, and analyzed using enantio-GC, capillary-GC and GC-MS. Two chiral selectors, 6-tert-butyldimethylsilyl-2,3-diethyl-beta-cyclodextrin (TBDE-beta-CD), and 2,3,6-methyl-beta-cyclodextrin (PM-beta-CD) doped into 14% cyanopropylphenyl/86% dimethylpolysiloxane, and 35% diphenyl/65% dimethylpolysiloxane, respectively were compared in order to clarify the stereochemistry and enantioselectivity of terpenoids using chiral gas chromatography. The enantiomeric excess for (1R)-(+)-alpha-pinene, (1R)-(+)-beta-pinene, and (R)-(+)-limonene were characteristic for the rhizome. In TBDE-beta-cyclodextrin coated chiral columns, a significant increase in separation factor (alpha) for beta-pinene, limonene, linalool and alpha-terpineol enantiomers was observed when compared with methyl substituted beta-cyclodextrin. The increase in chain length of the alkyl substituents may be the possible cause for enantiomer separation in beta-cyclodextrin cavity. In addition, enantioreversal of alpha-pinene enantiomers in 6-tert-butyldimethylsilyl-2,3-diethyl-beta-cyclodextrin was noticed as a unique feature. The enantiomeric compositions of Z. roseum fruit and flower essential oils were similar, but, in contrast, the rhizome oil contained an entirely different composition. Therefore, these results aid in the authentication of the natural origin of Z. roseum essential oils.

Characterization of cytochrome P450 monooxygenases isolated from trichome enriched fraction of Artemisia annua L. leaf.

CYPs have major role in the biosynthesis and modification of secondary metabolites. Predicting the possible involvement of CYPs in secondary metabolism, 20 partial sequences were amplified from the cDNA of trichome enriched tissue of Artemisia annua. Seven CYPs were converted to full length and assigned to different families based on sequence homology. These were co-expressed with CPR in Saccharomyces cerevisiae and microsome fractions were assayed for conversion of sesquiterpenes, phenols and fatty acid substrates. CIM_CYP02(c73) and CIM_CYP05(c81) converted trans-cinnamic acid to p-coumaric acid; and capric acid, lauric acid to their hydroxylated products, respectively. Higher expression of CIM_CYP71AV1, CIM_CYP03(c72a), CIM_CYP06(c72b), CIM_CYP02(c73) and CIM_CYP04(c83) was observed in the mature leaf, whereas expression of CIM_CYP05(c81) was more in the seedling. CIM_CYP71AV1, CIM_CYP02(c73) and CIM_CYP04(c83) expressed more in the flower bud compared to the leaf, with minor expression in stem. All CYPs' expression increased progressively with time after wounding except for CIM_CYP07(c92). These results relate involvement of CIM_CYP02(c73) to phenyl-propanoid metabolism in the leaf and CIM_CYP05(c81) to fatty acid metabolism in the seedling. Expression of CIM_CYP71AV1 and CIM_CYP02(c73) significantly increased when sprayed with trans-cinnamic acid indicating a relationship between phenylpropanoid and artemisinic acid pathways.

Variation in the volatile constituents of different plant parts of Ligusticopsis wallichiana from western Himalaya, India.

The essential oil composition of the leaves, stem, flowers and roots of Ligusticopsis wallichiana (DC.) Pimenov & Kljuykov were analyzed by GC-FID and GC-MS methods. Forty-five constituents, forming 93.2%-97.8% of the oil compositions, were dominated by acetylenic (31.5%-92.8%) compounds and sesquiterpenoids (0.3%-44.4%). The leaf essential oil was mainly composed 3,5-nonadiyne (35.8%), beta-selinene (20.9%), alpha-funebrene (10.1%) and (Z)-falcarinol (6.1%). The stem oil was dominated by acetylenic compounds (73.8%) represented by 3,5-nonadiyne (67.8%) and (Z)-falcarinol (5.7%). On the contrary, the major components of the flower essential oil were sesquiterpenoids (37.5%), such as germacrene D (16.6%), alpha-funebrene (7.4%), and acetylenic compounds (31.5%), such as (Z)-falcarinol (21.0%) and 3,5-nonadiyne (10.0%). Monoterpenoids constituted 23.9% of the flower oil with limonene (19.9%) as the single major constituent. The essential oil of the roots was dominated by 3,5-nonadiyne (90.5%). The results showed considerable qualitative and quantitative variations in the essential oil compositions of the different plant parts of L. wallichiana. (Z)-Falcarinol (1.9%-21.0%) and alpha-funebrene (0.1%-10.1%) were reported for the first time from the essential oils of L. wallichiana.

Chemical composition of leaf and root essential oils of Boenninghausenia albiflora Reichb. from northern India.

The leaf and root essential oil composition of Boenninghausenia albiflora Reichb and Meissner (family: Rutaceae), collected from Uttarakhand, India, was analysed by capillary gas chromatography and gas chromatography-mass spectrometry. The major constituents identified in the leaf essential oil were ß-myrcene, (Z)-ß-guaiene, (Z)-ß-ocimene and ß-caryophyllene, whereas bicyclogermacrene, α-terpinyl acetate, geijerene and ß-copaene-4α-ol were identified as the major constituents of the root essential oil. This is the first time that the chemical compositions of leaf and root essential oils of B. albiflora have been investigated in detail. The results show significant qualitative and quantitative variations in leaf and root oil composition.

Chemical composition and antimicrobial activity of the inflorescence essential oil of Capillipedium parviflorum (R. Br.) Stapf. from India.

Essential oil isolated from the inflorescences of Capillipedium parviflorum (R. Br.) Stapf., collected from Kumaon region of Western Himalaya, India, was investigated by GC and GC-MS. A total of 45 constituents representing 99.0% of the essential oil were identified. The major components of this oil were 4-undecanone (33.2%), 4-undecanol (29.7%), 4-nonanol (13.9%), α-muurolol (5.3%), 4-tridecanone (3.6%), methyl-2-oxo-nonanoate (3.1%), trans-2-hexenyl butanoate (1.1%) and 1-tetradecanol (1.0%). The antimicrobial activity of the essential oil was determined against eight Gram-positive and Gram-negative bacterial strains, as well as two fungal strains. The bioassay showed that the essential oil possessed good antibacterial activity.

2-undecanone rich leaf essential oil from Zanthoxylum armatum.

The leaf essential oils of Zanthoxylum armatum DC (Rutaceae) from Kumaon, India, extracted by hydrodistillation, were analyzed by capillary gas chromatography (GC-FID) and gas chromatography-mass spectrometry (GC-MS). The major classes of compounds found in the leaf oils were acyclic and menthane monoterpenoids as well as simple alcohols, aldehydes and ketones. The high proportion of non-terpenic acyclic ketones, notably 2-undecanone and 2-tridecanone, and the low abundance of undec-10-en-1-al and p-phellandren-8-ol make the composition entirely new. Other constituents present in significant amounts were oxygenated monoterpenes, which include 1,8-cineole, linalool, terpinen-4-ol, and alpha-terpineol, and sesquiterpene hydrocarbons represented mainly by trans-caryophyllene, a-humulene and germacrene D. On the contrary, the oil distilled from the leaves on the second day of distillation was characterized by a high content of 2-tridecanone (27.1%) and trans-caryophyllene (7.4%), as compared with 3.5% and 4.6%, respectively, for the fresh leaves; a slight decrease in pH of the distillate was also significant. Moreover, the presence of a high 2-undecanone content followed by 2-tridecanone is being reported for the first time for Z. armatum from this region. In terms of molecular diversity, the simple acyclic ketones dominate the essential oils as compared with linalool that was reported in several previous studies on Z. armatum. Therefore, the two acyclic ketones may be utilized to establish the origin and authenticity of the material.

Variation in the volatile constituents of Artemisia annua var. CIM-Arogya during plant ontogeny.

The essential oils yield and composition of the aerial parts of A. annua var. CIM-Arogya grown in Uttarakhand, India were analyzed and compared by capillary GC and GC-MS at different stages of development. The analysis led to the identification of 81 constituents forming 91.0%-97.1% of the essential oils compositions. The essential oil content of the aerial parts was found to vary from 0.3% to 0.7% at different stages of growth. A. annua crop harvested at full flowering and seed setting stage gave higher yield of essential oil (0.6%, 0.7%) than that harvested at pre flowering (0.5%), late vegetative (0.4%, 0.5%), mid vegetative (0.4%, 0.4%) and early vegetative stages (0.3%, 0.3%). The essential oils at different stages of growth showed monoterpenoids (38.5%-72.0%) and sesquiterpenoids (22.2%-48.2%) as major grouped constituents. The major constituents identified were camphor (22.8%-42.6%), 1,8-cineole (3.7%-8.4%), linalool (<0.1%-11.9%), beta-caryophyllene (2.0%-9.2%), (E)-beta-farnesene (1.3%-8.5%), germacrene D (0.5%-7.3%) and 1-epi-cubenol (0.7%-5.2%) in essential oil samples collected at different crop stages.

Metabolic pathway reconstruction of eugenol to vanillin bioconversion in Aspergillus niger.

Identification of missing genes or proteins participating in the metabolic pathways as enzymes are of great interest. One such class of pathway is involved in the eugenol to vanillin bioconversion. Our goal is to develop an integral approach for identifying the topology of a reference or known pathway in other organism. We successfully identify the missing enzymes and then reconstruct the vanillin biosynthetic pathway in Aspergillus niger. The procedure combines enzyme sequence similarity searched through BLAST homology search and orthologs detection through COG & KEGG databases. Conservation of protein domains and motifs was searched through CDD, PFAM & PROSITE databases. Predictions regarding how proteins act in pathway were validated experimentally and also compared with reported data. The bioconversion of vanillin was screened on UV-TLC plates and later confirmed through GC and GC-MS techniques. We applied a procedure for identifying missing enzymes on the basis of conserved functional motifs and later reconstruct the metabolic pathway in target organism. Using the vanillin biosynthetic pathway of Pseudomonas fluorescens as a case study, we indicate how this approach can be used to reconstruct the reference pathway in A. niger and later results were experimentally validated through chromatography and spectroscopy techniques.

Chemical diversity in the genus Alpinia (Zingiberaceae): comparative composition of four Alpinia species grown in Northern India.

The essential-oil compositions of leaves, flowers, and rhizomes of Alpinia galanga (L.) Willd., Alpinia calcarata Rosc., Alpinia speciosa K. Schum., and Alpinia allughas Rosc. were examined and compared by capillary GC and GC/MS. Monoterpenoids were the major oil constituents identified. 1,8-Cineole, alpha-terpineol, (E)-methyl cinnamate, camphor, terpinen-4-ol, and alpha- and beta-pinenes were the major constituents commonly distributed in leaf and flower essential oils. The presence of endo-fenchyl acetate, exo-fenchyl acetate, and endo-fenchol was the unique feature of rhizome essential oils of A. galanga, A. calcarata, and A. speciosa. On contrary, the rhizome oil of A. allughas was dominated by beta-pinene. Significant qualitative and quantitative variations were observed in essential-oil compositions of different parts of Alpinia species growing in subtemperate and subtropical regions of Northern India. Cluster analysis was performed to find similarities and differences in essential-oil compositions based on representative molecular skeletons. Monoterpenoids, viz., 1,8-cineole, terpinen-4-ol, camphor, pinenes, (E)-methyl cinnamate, and fenchyl derivatives, were used as chemotaxonomic markers.