Defining the role of a caffeic acid 3-O-methyltransferase from Azadirachta indica fruits in the biosynthesis of ferulic acid through heterologous over-expression in Ocimum species and Withania somnifera.

MAIN CONCLUSION: The recombinant caffeic acid 3-O-methyltransferase gene has been cloned and characterized from Neem. The gene is involved in ferulic acid biosynthesis, a key intermediate component of lignin biosynthesis. Azadirachta indica (Neem) is a highly reputed traditional medicinal plant and is phytochemically well-known for its limonoids. Besides limonoids, phenolics are also distinctively present, which add more medicinal attributes to Neem. Caffeic acid is one of such phenolic compound and it can be converted enzymatically into another bioactive phytomolecule, ferulic acid. This conversion requires transfer of a methyl group from a donor to caffeic acid under the catalytic action of an appropriate methyltransferase. In this study, caffeic acid 3-O-methyltransferase gene from Neem (NCOMT) fruits has been isolated and heterologously expressed in E. coli. The recombinant NCOMT enzyme was purified, which exhibited efficient catalytic conversion of caffeic acid into ferulic acid, a highly potential pharmaceutical compound. The purified recombinant enzyme was physico-kinetically characterized for its catalysis. The analysis of tissue-wide expression of NCOMT gene revealed interesting pattern of transcript abundance reflecting its role in the development of fruit tissues. Further, NCOMT was heterologously overexpressed in Withania somnifera and Ocimum species, to analyze its role in ferulic acid biosynthesis in planta. Thus, the study provides insight for the endogenous role of NCOMT in ferulic acid biosynthesis en route to lignin, an important structural component. To the best of our knowledge, NCOMT pertains to be the first enzyme of the secondary metabolism that has been purified and kinetically characterized from Neem. This study may also have important prospects of applications as the observation on heterologous expression of NCOMT showed its involvement in the maintenance of the in vivo pool of ferulic acid in the plants. Thus, the study involving NCOMT opens up new dimensions of metabolic engineering approaches for the biosynthesis of potential therapeutically important phytomolecules in heterologous systems.

WRKY1-mediated regulation of tryptophan decarboxylase in tryptamine generation for withanamide production in Withania somnifera (Ashwagandha).

KEY MESSAGE: WsWRKY1-mediated transcriptional modulation of Withania somnifera tryptophan decarboxylase gene (WsTDC) helps to regulate fruit-specific tryptamine generation for production of withanamides. Withania somnifera is a highly valued medicinal plant. Recent demonstration of novel indolyl metabolites called withanamides in its fruits (berries) prompted us to investigate its tryptophan decarboxylase (TDC), as tryptophan is invariably a precursor for indole moiety. TDC catalyzes conversion of tryptophan into tryptamine, and the catalytic reaction constitutes a committed metabolic step for synthesis of an array of indolyl metabolites. The TDC gene (WsTDC) was cloned from berries of the plant and expressed in E. coli. The recombinant enzyme was purified and characterized for its catalytic attributes. Catalytic and structural aspects of the enzyme indicated its regulatory/rate-limiting significance in generation of the indolyl metabolites. Novel tissue-wise and developmentally differential abundance of WsTDC transcripts reflected its preeminent role in withanamide biogenesis in the fruits. Transgenic lines overexpressing WsTDC gene showed accumulation of tryptamine at significantly higher levels, while lines silenced for WsTDC exhibited considerably depleted levels of tryptamine. Cloning and sequence analysis of promoter of WsTDC revealed the presence of W-box in it. Follow-up studies on isolation of WsWRKY1 transcription factor and its overexpression in W. somnifera revealed that WsTDC expression was substantially induced by WsWRKY1 resulting in overproduction of tryptamine. The study invokes a key role of TDC in regulating the indolyl secondary metabolites through enabling elevated flux/supply of tryptamine at multiple levels from gene expression to catalytic attributes overall coordinated by WsWRKY1. This is the first biochemical, molecular, structural, physiological and regulatory description of a fruit-functional TDC.

Nano silver particle synthesis using Swertia paniculata herbal extract and its antimicrobial activity.

In the present study, green synthesis of silver nanoparticles (AgNPs) is demonstrated using medicinal herb Swertia paniculata extract. The plant extract acted both as reducing and capping agents during synthesis process, where silver nitrate was used as silver source. Subsequent analysis revealed that particles had size range between 31 and 44 nm and were spherical in shape. Among reaction parameters, temperature and time had significantly influenced the synthesis reaction. Also, synthesized nanoparticles were found stable up to 90 days. Further, antimicrobial activity against gram negative and gram positive bacterial strains was done and results showed that synthesized AgNPs had better antimicrobial activity against Pseudomonas aeruginosa, Klebsiella pneumoniae under standard incubation conditions. Study shows that these particles can be very promising in biomedical applications in future.

Withania somnifera chemotype NMITLI 101R significantly increases the efficacy of antileishmanial drugs by generating strong IFN-γ and IL-12 mediated immune responses in Leishmania donovani infected hamsters.

BACKGROUND: Withania somnifera (L.) Dunal (Solanaceae), commonly known as Ashwagandha, is one of the most important medicinal plant in the traditional Indian medical systems. Pharmacological studies have established that root extracts of W. somnifera contain several bioactive constituents called withanolides. The plant has long been used for its several beneficial properties and recently as an immunomodulator. HYPOTHESIS/PURPOSE: A combination therapy including a potential and safe immunostimulant with lower doses of effective drug, which can reduce the parasitic burden and simultaneously can produce an enhancement of adaptive immunity, has proven to be significantly a more effective approach than immunotherapy or drug therapy alone. STUDY DESIGN: Evaluation of the immunostimulatory effect of W. somnifera chemotype NMITLI 101R when used in combination with ED50 doses of antileishmanial drugs in Leishmania donovani infected hamsters. METHODS: Infected animals were administered with chemotype 101R(30mg/kg × 15 days) either alone or in combination with ED50 doses of miltefosine (10mg/kg × 5 days), paromomycin (30mg/kg × 5 days) or amphotericin B (0.5mg/kg × 5 days). The treated animals were euthanized on days 30 and 60 post-treatment (p.t.) and checked for parasite clearance, delayed type hypersensitivity (DTH) response, cytokine and inducible nitric oxide synthase levels by real-time PCR, nitric oxide (NO) production, reactive oxygen species (ROS) generation, lymphoproliferative and antibody responses. RESULTS: The group of animals that received 101R and ED50 dose of miltefosine showed optimum inhibition of parasite multiplication (∼98%) by day 60 p.t. followed by the group that received 101R plus paromomycin (∼94%) and 101R plus amphotericin B (∼93%). The efficacy was well supported by the increased inducible NO synthase mRNA transcript, strong IFN-γand IL-12 mediated Th1 immune responses and significantly suppressed levels of Th2 cytokines (IL-4, IL-10 and TGF-ß). Additionally, same therapy also induced significant increase in the level of NO production, ROS generation, Leishmania specific IgG2 antibody along with profound DTH and strong T-cell responses as compared with all the other treated groups. CONCLUSION: Our results suggest that combination of chemotype 101R with ED50 doses of antileishmanial drugs may provide a promising alternative for the cure of visceral leishmaniasis with significant restoration of the host immune response.

RNAi and Homologous Over-Expression Based Functional Approaches Reveal Triterpenoid Synthase Gene-Cycloartenol Synthase Is Involved in Downstream Withanolide Biosynthesis in Withania somnifera.

Withania somnifera Dunal, is one of the most commonly used medicinal plant in Ayurvedic and indigenous medicine traditionally owing to its therapeutic potential, because of major chemical constituents, withanolides. Withanolide biosynthesis requires the activities of several enzymes in vivo. Cycloartenol synthase (CAS) is an important enzyme in the withanolide biosynthetic pathway, catalyzing cyclization of 2, 3 oxidosqualene into cycloartenol. In the present study, we have cloned full-length WsCAS from Withania somnifera by homology-based PCR method. For gene function investigation, we constructed three RNAi gene-silencing constructs in backbone of RNAi vector pGSA and a full-length over-expression construct. These constructs were transformed in Agrobacterium strain GV3101 for plant transformation in W. somnifera. Molecular and metabolite analysis was performed in putative Withania transformants. The PCR and Southern blot results showed the genomic integration of these RNAi and overexpression construct(s) in Withania genome. The qRT-PCR analysis showed that the expression of WsCAS gene was considerably downregulated in stable transgenic silenced Withania lines compared with the non-transformed control and HPLC analysis showed that withanolide content was greatly reduced in silenced lines. Transgenic plants over expressing CAS gene displayed enhanced level of CAS transcript and withanolide content compared to non-transformed controls. This work is the first full proof report of functional validation of any metabolic pathway gene in W. somnifera at whole plant level as per our knowledge and it will be further useful to understand the regulatory role of different genes involved in the biosynthesis of withanolides.

Neuro-protective potential of a vesicular system of a standardized extract of a new chemotype of Withania somnifera Dunal (NMITLI118RT+) against cerebral stroke in rats.

Withania somnifera Dunal is an Indian medicinal plant with significant pharmacological properties, such as adaptogenic, anti-inflammatory, anti-oxidant, anti-platelet, anti-hypertensive, hypoglycemic and hypolipidemic effects. Several chemotypes of W. somnifera include NMITLI-101, NMITLI-118 and NMITLI-128. The present work elaborates the optimization and development of a liposomal delivery system for efficient delivery of NMITLI118RT+ [a standardized ethanolic extract of a new chemotype of W. somnifera Dunal (NMITLI-118) roots] against cerebral stroke in rats. Liposomal systems were prepared using thin-film hydration method and characterized on the basis of size, zeta potential, physical stability, FT-IR, DSC-TGA analysis and surface morphological studies by TEM. NMITLI118RT+ and its formulations (NMITLI118RT+LF) were evaluated for biological activity utilizing middle cerebral artery occlusion model in rats. The Z average of the developed liposomal formulation was about 142.6 ± 0.09 nm with a zeta potential of -31.20 ± 1.0 mV. Results of TEM revealed spherical particles in the range of 200 nm. The entrapment efficiency was found to be 94.603 ± 2%. The formulation was found to be physically stable over a 3-week period. Results were suggestive of the fact that both NMITLI118RT+ and its delivery system possess significant neuroprotective activity in cerebral ischemia. The liposomal system largely exhibits better performance over NMITLI118RT+ precisely in the post-treatment group. The present studies could elucidate the successful development of a delivery system for NMITLI118RT+ and demonstrate their beneficial neuro-protective potential in overcoming and reversing the consequences of I/R injury following stroke.

Comparative transcriptome analysis of different chemotypes elucidates withanolide biosynthesis pathway from medicinal plant Withania somnifera.

Withania somnifera is one of the most valuable medicinal plants synthesizing secondary metabolites known as withanolides. Despite pharmaceutical importance, limited information is available about the biosynthesis of withanolides. Chemo-profiling of leaf and root tissues of Withania suggest differences in the content and/or nature of withanolides in different chemotypes. To identify genes involved in chemotype and/or tissue-specific withanolide biosynthesis, we established transcriptomes of leaf and root tissues of distinct chemotypes. Genes encoding enzymes for intermediate steps of terpenoid backbone biosynthesis with their alternatively spliced forms and paralogous have been identified. Analysis suggests differential expression of large number genes among leaf and root tissues of different chemotypes. Study also identified differentially expressing transcripts encoding cytochrome P450s, glycosyltransferases, methyltransferases and transcription factors which might be involved in chemodiversity in Withania. Virus induced gene silencing of the sterol ∆7-reductase (WsDWF5) involved in the synthesis of 24-methylene cholesterol, withanolide backbone, suggests role of this enzyme in biosynthesis of withanolides. Information generated, in this study, provides a rich resource for functional analysis of withanolide-specific genes to elucidate chemotype- as well as tissue-specific withanolide biosynthesis. This genomic resource will also help in development of new tools for functional genomics and breeding in Withania.

Light and auxin responsive cytochrome P450s from Withania somnifera Dunal: cloning, expression and molecular modelling of two pairs of homologue genes with differential regulation.

Cytochrome P450s (CYPs) catalyse a wide variety of oxygenation/hydroxylation reactions that facilitate diverse metabolic functions in plants. Specific CYP families are essential for the biosynthesis of species-specialized metabolites. Therefore, we investigated the role of different CYPs related to secondary metabolism in Withania somnifera, a medicinally important plant of the Indian subcontinent. In this study, complete complementary DNAs (cDNAs) of four different CYP genes were isolated and christened as WSCYP93Id, WSCYP93Sm, WSCYP734B and WSCYP734R. These cDNAs encoded polypeptides comprising of 498, 496, 522 and 550 amino acid residues with their deduced molecular mass of 56.7, 56.9, 59.4 and 62.2 kDa, respectively. Phylogenetic study and molecular modelling analysis of the four cloned WSCYPs revealed their categorization into two CYP families (CYP83B1 and CYP734A1) belonging to CYP71 and CYP72 clans, respectively. BLASTp searches showed similarity of 75 and 56 %, respectively, between the two CYP members of CYP83B1 and CYP734A1 with major variances exhibited in their N-terminal regions. The two pairs of homologues exhibited differential expression profiles in the leaf tissues of selected chemotypes of W. somnifera as well as in response to treatments such as methyl jasmonate, wounding, light and auxin. Light and auxin regulated two pairs of WSCYP homologues in a developing seedling in an interesting differential manner. Their lesser resemblance and homology with other CYP sequences suggested these genes to be more specialized and distinct ones. The results on chemotype-specific expression patterns of the four genes strongly suggested their key/specialized involvement of the CYPs in the biosynthesis of chemotype-specific metabolites, though their further biochemical characterization would reveal the specificity in more detail. It is revealed that WSCYP93Id and WSCYP93Sm may be broadly involved in the oxygenation reactions in the plant and, thereby, control various pathways involving such metabolic reactions in the plant. As a representative experimental validation of this notion, WSCYP93Id was heterologouly expressed in Escherichia coli and catalytic capabilities of the recombinant WSCYP93Id protein were evaluated using withanolides as substrates. Optimized assays with some major withanolides (withanone, withaferin A and withanolide A) involving spectrophotometric as well as high-pressure liquid chromatography (HPLC)-based evaluation (product detection) of the reactions showed conversion of withaferin A to a hydroxylated product. The genes belonging to other CYP group are possibly involved in some specialised synthesis such as that of brassinosteroids.

Tropine forming tropinone reductase gene from Withania somnifera (Ashwagandha): biochemical characteristics of the recombinant enzyme and novel physiological overtones of tissue-wide gene expression patterns.

Withania somnifera is one of the most reputed medicinal plants of Indian systems of medicine synthesizing diverse types of secondary metabolites such as withanolides, alkaloids, withanamides etc. Present study comprises cloning and E. coli over-expression of a tropinone reductase gene (WsTR-I) from W. somnifera, and elucidation of biochemical characteristics and physiological role of tropinone reductase enzyme in tropane alkaloid biosynthesis in aerial tissues of the plant. The recombinant enzyme was demonstrated to catalyze NADPH-dependent tropinone to tropine conversion step in tropane metabolism, through TLC, GC and GC-MS-MS analyses of the reaction product. The functionally active homodimeric ~60 kDa enzyme catalyzed the reaction in reversible manner at optimum pH 6.7. Catalytic kinetics of the enzyme favoured its forward reaction (tropine formation). Comparative 3-D models of landscape of the enzyme active site contours and tropinone binding site were also developed. Tissue-wide and ontogenic stage-wise assessment of WsTR-I transcript levels revealed constitutive expression of the gene with relatively lower abundance in berries and young leaves. The tissue profiles of WsTR-I expression matched those of tropine levels. The data suggest that, in W. somnifera, aerial tissues as well possess tropane alkaloid biosynthetic competence. In vivo feeding of U-[(14)C]-sucrose to orphan shoot (twigs) and [(14)C]-chasing revealed substantial radiolabel incorporation in tropinone and tropine, confirming the de novo synthesizing ability of the aerial tissues. This inherent independent ability heralds a conceptual novelty in the backdrop of classical view that these tissues acquire the alkaloids through transportation from roots rather than synthesis. The TR-I gene expression was found to be up-regulated on exposure to signal molecules (methyl jasmonate and salicylic acid) and on mechanical injury. The enzyme's catalytic and structural properties as well as gene expression profiles are discussed with respect to their physiological overtones.

De novo assembly, functional annotation and comparative analysis of Withania somnifera leaf and root transcriptomes to identify putative genes involved in the withanolides biosynthesis.

Withania somnifera is one of the most valuable medicinal plants used in Ayurvedic and other indigenous medicine systems due to bioactive molecules known as withanolides. As genomic information regarding this plant is very limited, little information is available about biosynthesis of withanolides. To facilitate the basic understanding about the withanolide biosynthesis pathways, we performed transcriptome sequencing for Withania leaf (101L) and root (101R) which specifically synthesize withaferin A and withanolide A, respectively. Pyrosequencing yielded 8,34,068 and 7,21,755 reads which got assembled into 89,548 and 1,14,814 unique sequences from 101L and 101R, respectively. A total of 47,885 (101L) and 54,123 (101R) could be annotated using TAIR10, NR, tomato and potato databases. Gene Ontology and KEGG analyses provided a detailed view of all the enzymes involved in withanolide backbone synthesis. Our analysis identified members of cytochrome P450, glycosyltransferase and methyltransferase gene families with unique presence or differential expression in leaf and root and might be involved in synthesis of tissue-specific withanolides. We also detected simple sequence repeats (SSRs) in transcriptome data for use in future genetic studies. Comprehensive sequence resource developed for Withania, in this study, will help to elucidate biosynthetic pathway for tissue-specific synthesis of secondary plant products in non-model plant organisms as well as will be helpful in developing strategies for enhanced biosynthesis of withanolides through biotechnological approaches.

Purification and physicokinetic characterization of a gluconolactone inhibition-insensitive ß-glucosidase from Andrographis paniculata nees. Leaf.

A gluconolactone inhibition-insensitive ß-glucosidase from Andrographis paniculata (Acanthaceae) leaves has been isolated, homogeneity purified, and characterized for its physicokinetic properties. The purified enzyme appeared to be a monomeric structure with native molecular weight about 60 kD. The enzyme exhibited optimum pH 5.5 and pI 4.0, meso-thermostability and high temperature optimum (55°C) for catalytic activity, with activation energy of 6.8 kcal Mol(-1). The substrate saturation kinetics studies of the enzyme revealed a Michaelis-Menten constant (Km) of 0.25 mM for pNPG and catalytic efficiency (Kcat/Km) of 52,400 M (-1) s(-1), respectively. Substrate specificity of the enzyme was restricted to ß-linked gluco-, manno- and fuco-conjugates. The gluconolactone inhibition insensitivity was evident from its very low inhibition at millimolar inhibitor concentrations. Interestingly, the enzyme showed geraniol transglucosylating activity with pNPG as glucosyl donor but not with cellobiose. The catalytic activity of the enzyme has been reported to be novel with respect to its activity and preferences from a medicinal plant resource.

Activated charcoal-mediated RNA extraction method for Azadirachta indica and plants highly rich in polyphenolics, polysaccharides and other complex secondary compounds.

BACKGROUND: High quality RNA is a primary requisite for numerous molecular biological applications but is difficult to isolate from several plants rich in polysaccharides, polyphenolics and other secondary metabolites. These compounds either bind with nucleic acids or often co-precipitate at the final step and many times cannot be removed by conventional methods and kits. Addition of vinyl-pyrollidone polymers in extraction buffer efficiently removes polyphenolics to some extent, but, it failed in case of Azadirachta indica and several other medicinal and aromatic plants. FINDINGS: Here we report the use of adsorption property of activated charcoal (0.03%-0.1%) in RNA isolation procedures to remove complex secondary metabolites and polyphenolics to yield good quality RNA from Azadirachta indica. We tested and validated our modified RNA isolation method across 21 different plants including Andrographis paniculata, Aloe vera, Rosa damascena, Pelargonium graveolens, Phyllanthus amarus etc. from 13 other different families, many of which are considered as tough system for isolating RNA. The A260/280 ratio of the extracted RNA ranged between 1.8-2.0 and distinct 28S and 18S ribosomal RNA bands were observed in denaturing agarose gel electrophoresis. Analysis using Agilent 2100 Bioanalyzer revealed intact total RNA yield with very good RNA Integrity Number. CONCLUSIONS: The RNA isolated by our modified method was found to be of high quality and amenable for sensitive downstream molecular applications like subtractive library construction and RT-PCR. This modified RNA isolation procedure would aid and accelerate the biotechnological studies in complex medicinal and aromatic plants which are extremely rich in secondary metabolic compounds.

Cloning and functional characterization of 3-hydroxy-3-methylglutaryl coenzyme A reductase gene from Withania somnifera: an important medicinal plant.

Withania somnifera (L.) Dunal is one of the most valuable medicinal plants synthesizing a large number of pharmacologically active secondary metabolites known as withanolides, the C28-steroidal lactones derived from triterpenoids. Though the plant has been well characterized in terms of phytochemical profiles as well as pharmaceutical activities, not much is known about the biosynthetic pathway and genes responsible for biosynthesis of these compounds. In this study, we have characterized the gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR; EC 1.1.1.34) catalyzing the key regulatory step of the isoprenoid biosynthesis. The 1,728-bp full-length cDNA of Withania HMGR (WsHMGR) encodes a polypeptide of 575 amino acids. The amino acid sequence homology and phylogenetic analysis suggest that WsHMGR has typical structural features of other known plant HMGRs. The relative expression analysis suggests that WsHMGR expression varies in different tissues as well as chemotypes and is significantly elevated in response to exposure to salicylic acid, methyl jasmonate, and mechanical injury. The functional color assay in Escherichia coli showed that WsHMGR could accelerate the biosynthesis of carotenoids, establishing that WsHMGR encoded a functional protein and may play a catalytic role by its positive influence in isoprenoid biosynthesis.

Cloning and characterization of 2-C-methyl-D-erythritol-4-phosphate pathway genes for isoprenoid biosynthesis from Indian ginseng, Withania somnifera.

Withania somnifera (L.) is one of the most valuable medicinal plants used in Ayurvedic and other indigenous medicines. Pharmaceutical activities of this herb are associated with presence of secondary metabolites known as withanolides, a class of phytosteroids synthesized via mevalonate (MVA) and 2-C-methyl-D-erythritol-4-phosphate pathways. Though the plant has been well characterized in terms of phytochemical profiles as well as pharmaceutical activities, not much is known about the genes responsible for biosynthesis of these compounds. In this study, we have characterized two genes encoding 1-deoxy-D-xylulose-5-phosphate synthase (DXS; EC 2.2.1.7) and 1-deoxy-D-xylulose-5-phosphate reductase (DXR; EC 1.1.1.267) enzymes involved in the biosynthesis of isoprenoids. The full-length cDNAs of W. somnifera DXS (WsDXS) and DXR (WsDXR) of 2,154 and 1,428 bps encode polypeptides of 717 and 475 amino acids residues, respectively. The expression analysis suggests that WsDXS and WsDXR are differentially expressed in different tissues (with maximal expression in flower and young leaf), chemotypes of Withania, and in response to salicylic acid, methyl jasmonate, as well as in mechanical injury. Analysis of genomic organization of WsDXS shows close similarity with tomato DXS in terms of exon-intron arrangements. This is the first report on characterization of isoprenoid biosynthesis pathway genes from Withania.

Comprehensive metabolic fingerprinting of Withania somnifera leaf and root extracts.

Profiling of metabolites is a rapidly expanding area of research for resolving metabolic pathways. Metabolic fingerprinting in medicinally important plants is critical to establishing the quality of herbal medicines. In the present study, metabolic profiling of crude extracts of leaf and root of Withania somnifera (Ashwagandha), an important medicinal plant of Indian system of medicine (ISM) was carried out using NMR and chromatographic (HPLC and GC-MS) techniques. A total of 62 major and minor primary and secondary metabolites from leaves and 48 from roots were unambiguously identified. Twenty-nine of these were common to the two tissues. These included fatty acids, organic acids, amino acids, sugars and sterol based compounds. Eleven bioactive sterol-lactone molecules were also identified. Twenty-seven of the identified metabolites were quantified. Highly significant qualitative and quantitative differences were noticed between the leaf and root tissues, particularly with respect to the secondary metabolites.

Withanolide A is inherently de novo biosynthesized in roots of the medicinal plant Ashwagandha (Withania somnifera).

Ashwagandha (Withania somnifera Dunal., Solanaceae) is one of the most reputed medicinal plants of Ayurveda, the traditional medical system. Several of its traditionally proclaimed medicinal properties have been corroborated by recent molecular pharmacological investigations and have been shown to be associated with its specific secondary metabolites known as withanolides, the novel group of ergostane skeletal phytosteroids named after the plant. Withanolides are structurally distinct from tropane/nortropane alkaloids (usually found in Solanaceae plants) and are produced only by a few genera within Solanaceae. W. somnifera contains many structurally diverse withanolides in its leaves as well as roots. To date, there has been little biosynthetic or metabolism-related research on withanolides. It is thought that withanolides are synthesized in leaves and transported to roots like the tropane alkaloids, a group of bioactive secondary metabolites in Solanaceae members known to be synthesized in roots and transported to leaves for storage. To examine this, we have studied incorporation of (14)C from [2-(14)C]-acetate and [U-(14)C]-glucose into withanolide A in the in vitro cultured normal roots as well as native/orphan roots of W. somnifera. Analysis of products by thin layer chromatography revealed that these primary metabolites were incorporated into withanolide A, demonstrating that root-contained withanolide A is de novo synthesized within roots from primary isoprenogenic precursors. Therefore, withanolides are synthesized in different parts of the plant (through operation of the complete metabolic pathway) rather than imported.

Withanolide A biogeneration in in vitro shoot cultures of ashwagandha (Withania somnifera DUNAL), a main medicinal plant in Ayurveda.

Multiple shoot cultures of two experimental lines of Withania somnifera plants (RS-Selection-1 and RS-Selection-2) were established using nodal segments as explants. The hormonal combinations of benzyl adenine and kinetin not only influenced their morphogenetic response but also differentially modulated the level of biogeneration of withanolide A in the in vitro shoots of the two lines. Interestingly, withanolide-A, that was hardly detectable in the aerial parts of field-grown Withania somnifera (explant source), accumulated considerably in the in vitro shoot cultures of the plant. The productivity of withanolide A in the cultures varied considerably (ca. 10-fold, 0.014 to 0.14 mg per gram fresh weight) with the change in the hormone composition of the culture media as well as genotype used as source of the explant. The shoot culture of RS-Selection-1 raised at 1.00 ppm of BAP and 0.50 ppm of kinetin displayed the highest concentration of withanolide A in the green shoots of 0.238 g per 100 g dry weight tissue. This was a more analytical concentration keeping in view the isolation yields so far reported from the dried roots of the field-grown plant (ca. 0.015 g per 100 g dry weight), even if isolation losses are considered during purification. The enhanced de novo biogenesis of withanolide A in shoot cultures was corroborated with radiolabel incorporation studies using [2-(14)C] acetate as a precursor. Production of withaferin A was also found in the in vitro shoot cultures. As this compound is a predominant withanolide of native shoots as well and has been already reported to be accumulated in in vitro shoot cultures, its biogeneration observed in these shoot cultures is not discussed in detail.

Molecular cloning and characterization of one member of 3beta-hydroxy sterol glucosyltransferase gene family in Withania somnifera.

Sterol glycosides are constituents of plant cell membranes. Glucosylations of the sterols are catalyzed by sterol glucosyltransferases (SGTs), which are members of family 1 glycosyltransferases. We have identified the family of SGT genes expressed in the leaves of a medicinal plant Withania somnifera. One member (SGTL1) of this gene family was cloned. The full-length cDNA sequence of SGTL1 represents 2532 bp, comprising untranslated regions (UTRs) of 337 and 89 bp at the 5' and 3' ends, respectively. The amino acid sequence deduced from the 2103 bp open reading frame (ORF) showed homology (67-45%) to the reported plant SGTs. The presence of two putative transmembrane domains suggested the association of SGTL1 with membrane. The SGTL1 was expressed in Escherichia coli and recombinant enzyme from the supernatant was partially purified and biochemically characterized. The relative activity and kinetic properties of SGTL1 for different sterols were compared with a recombinant SGT (GenBank Accession No. Z83833) of Arabidopsis thaliana (AtSGT). Both the recombinant enzymes showed activity with 3-beta-OH sterols. The distribution of SGTL1 transcript in W. somnifera, as determined by quantitative PCR, showed higher expression in roots and mature leaves. Expression of the SGTL1 transcript in the leaves of W. somnifera was enhanced following the application of salicylic acid. In contrast, it decreased rapidly on exposure of the plants to heat shock, suggesting functional role of the enzyme in biotic and abiotic stresses.