ROS management is mediated by ascorbate-glutathione-α-tocopherol triad in co-ordination with secondary metabolic pathway under cadmium stress in Withania somnifera.

Being static, plants are frequently exposed to various essential and non-essential heavy metals from the surroundings. This exposure results in considerable ROS generation leading to oxidative stress, the primary response of the plants under heavy metal stress. Withania somnifera is a reputed Indian medicinal plant in Ayurveda, having various pharmacological activities due to the presence of withanolides. The present study deals with the understanding endurance of oxidative stress caused by heavy metal exposure and its management through antioxidant partners in synchronization with secondary metabolites in W. somnifera. The quantitative assessment of enzymatic/non-enzymatic antioxidants revealed significant participation of ascorbate-glutathione-α-tocopherol triad in ROS management. Higher activities of glutathione reductase (GR), monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR) resulted in glutathione and ascorbate accumulation. In addition, superoxide dismutase (SOD), glutathione peroxidase (GPX) and peroxidase (POD) were contributed considerably in ROS homeostasis maintenance. In-situ localization and assays related to ROS generation/scavenging revealed key management of ROS status under Cd stress. Higher antioxidative and reducing power activity attributed to the tolerance capability to the plant. Increased expression of withanolide biosynthetic pathway genes such as WsHMGR, WsDXS, WsDXR and WsCAS correlated with enhanced withanolides. The present study indicated the crucial role of the ascorbate-glutathione-α-tocopherol triad in co-ordination with withanolide biosynthesis in affording the oxidative stress, possibly through a cross-talk between the antioxidant machinery and secondary metabolite biosynthesis. The knowledge may be useful in providing the guidelines for developing abiotic stress resistance in plants using conventional and molecular approaches.

Biochemical, molecular, and elemental profiling of Withania somnifera L. with response to zinc stress.

Zn stress seriously induces various toxic responses in Withania somnifera L., when accumulated above the threshold level which was confirmed by investigating the responses of protein, expression of antioxidant enzymes, and elemental profiling on accumulation of Zn. Zn was supplemented in the form of ZnSO4 (0, 25, 50, 100, and 200 µM) through MS liquid medium and allowed to grow the in vitro germinated plants for 7 and 14 days. The study revealed that when the application of Zn increased, a significant reduction of growth characteristics was noticed with alterations of proteins (both disappearance and de novo synthesis). The activity of CAT, SOD, and GPX were increased up to certain concentrations and then declined, which confirmed through in-gel activity under different treatments. RT-PCR was conducted by taking three sets of genes from CAT (RsCat, Catalase1, Cat1) and SOD (SodCp, TaSOD1.2, MnSOD) and found that gene RsCat from CAT and MnSOD from SOD have shown maximum expression of desired genes under Zn stress, which indicate plant's stress tolerance mechanisms. The proton-induced X-ray emission study confirmed an increasing order of uptake of Zn in plants by suppressing and expressing other elemental constituents which cause metal homeostasis. This study provides insights into molecular mechanisms associated with Zn causing toxicity to plants; however, cellular and subcellular studies are essential to explore molecule-molecule interaction during Zn stress in plants.

Exogenous application of IAA alleviates effects of supplemental ultraviolet-B radiation in the medicinal plant Withania somnifera Dunal.

Supplemental (s)-UV-B radiation has adverse effects on the majority of plants. The present study was conducted to evaluate the effects of exogenous application of the growth hormone indole acetic acid (IAA) on various morphological, physiological and biochemical characteristics of Withania somnifera, an indigenous medicinal plant, subjected to s-UV-B. The s-UV-B-treated plants received ambient + 3.6 kJm-2 ·day-1 biologically effective UV-B, and IAA was applied at two doses (200 and 400 ppm) to s-UV-B-exposed plants. The plant was forced to compromise its growth, development and photosynthetic patterns to survive under s-UV-B by increasing concentrations of secondary metabolites and antioxidants (thiol, proline, ascorbic acid, α-tocopherol, ascorbate peroxidase, catalase, glutathione reductase, peroxidase, polyphenol oxidase, superoxide dismutase) to counteract oxidative stress. Increases in secondary metabolites were evidenced as increased activity of phenylpropanoid pathway enzymes: phenylalanine ammonia lyase, cinnamyl alcohol dehydrogenase, 4-coumarate CoA ligase, chalcone isomerase and dihydroflavonol reductase. Application of different IAA doses reversed the detrimental effects of s-UV-B on W. somnifera by improving growth and photosynthesis and reducing concentrations of secondary metabolites and non-enzymatic antioxidants. Antioxidant enzymes, however, had a synergistic effect on s-UV-B treatment and IAA application. The effects of s-UV-B on W. somnifera are ameliorated to varying degrees upon exogenous IAA application, and synergistic enhancement of antioxidant enzymes under s-UV-B+IAA treatment might be responsible for the partial recuperation of growth and plant protein content, as a UV-B-exposed plant is forced to allocate most of its photosynthate towards production of enzymes related to antioxidant defence.

Withania coagulans tryptophan decarboxylase gene cloning, heterologous expression, and catalytic characteristics of the recombinant enzyme.

Tryptophan decarboxylase (EC 4.1.1.28) catalyzes pyridoxal 5'-phosphate (PLP)-dependent decarboxylation of tryptophan to produce tryptamine for recruitment in a myriad of biosynthetic pathways of metabolites possessing indolyl moiety. A recent report of certain indolyl metabolites in Withania species calls for a possible predominant functional role of tryptophan decarboxylase (TDC) in the genome of Withania species to facilitate production of the indolyl progenitor molecule, tryptamine. Therefore, with this metabolic prospection, we have identified and cloned a full-length cDNA sequence of TDC from aerial tissues of Withania coagulans. The functional WcTDC gene comprises of 1506 bp open reading frame (ORF) encoding a 502 amino acid protein with calculated molecular mass and pI value of 56.38 kDa and 8.35, respectively. The gene was expressed in Escherichia coli, and the recombinant enzyme was affinity-purified to homogeneity to discern its kinetics of catalysis. The enzyme (WcTDC) exhibited much higher Km value for tryptophan than for pyridoxal 5'-phosphate and was dedicated to catalyze decarboxylation of only tryptophan or, to a limited extent, of its analogue (like 5-hydroxy tryptophan). The observed optimal catalytic functionality of the enzyme on the slightly basic side of the pH scale and at slightly higher temperatures reflected adaptability of the plant to hot and arid regions, the predominant natural habitat of the herb. This pertains to be the first report on cloning and characterization of heterologously expressed recombinant enzyme from W. coagulans and forms a starting point to further understanding of withanamide biosynthesis.

Nitrogen treatment enhances sterols and withaferin A through transcriptional activation of jasmonate pathway, WRKY transcription factors, and biosynthesis genes in Withania somnifera (L.) Dunal.

The medicinal plant Withania somnifera is researched extensively to increase the quantity of withanolides and specifically withaferin A, which finds implications in many pharmacological activities. Due to insufficient knowledge on biosynthesis and unacceptability of transgenic approach, it is preferred to follow alternative physiological methods to increase the yield of withanolides. Prior use of elicitors like salicylic acid, methyl jasmonate, fungal extracts, and even mechanical wounding have shown to increase the withanolide biosynthesis with limited success; however, the commercial viability and logistics of application are debatable. In this investigation, we tested the simple nitrogeneous fertilizers pertaining to the enhancement of withaferin A biosynthesis. Application of ammonium sulfate improved the sterol contents required for the withanolide biosynthesis and correlated to higher expression of pathway genes like FPPS, SMT1, SMT2, SMO1, SMO2, and ODM. Increased expression of a gene homologous to allene oxide cyclase, crucial in jasmonic acid biosynthetic pathway, suggested the involvement of jasmonate signaling. High levels of WRKY gene transcripts indicated transcriptional regulation of the pathway genes. Increase in transcript level could be correlated with a corresponding increase in the protein levels for WsSMT1 and WsWRKY1. The withaferin A increase was also demonstrated in the potted plants growing in the glasshouse and in the open field. These results implicated simple physiological management of nitrogen fertilizer signal to improve the yield of secondary metabolite through probable involvement of jasmonate signal and WRKY transcription factor for the first time, in W. somnifera besides improving the foliage.

Over-expression of DXS gene enhances terpenoidal secondary metabolite accumulation in rose-scented geranium and Withania somnifera: active involvement of plastid isoprenogenic pathway in their biosynthesis.

Rose-scented geranium (Pelargonium spp.) is one of the most important aromatic plants and is well known for its diverse perfumery uses. Its economic importance is due to presence of fragrance rich essential oil in its foliage. The essential oil is a mixture of various volatile phytochemicals which are mainly terpenes (isoprenoids) in nature. In this study, on the geranium foliage genes related to isoprenoid biosynthesis (DXS, DXR and HMGR) were isolated, cloned and confirmed by sequencing. Further, the first gene of 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway, 1-deoxy-d-xylulose-5-phosphate synthase (GrDXS), was made full length by using rapid amplification of cDNA ends strategy. GrDXS contained a 2157 bp open reading frame that encoded a polypeptide of 792 amino acids having calculated molecular weight 77.5 kDa. This study is first report on heterologous expression and kinetic characterization of any gene from this economically important plant. Expression analysis of these genes was performed in different tissues as well as at different developmental stages of leaves. In response to external elicitors, such as methyl jasmonate, salicylic acid, light and wounding, all the three genes showed differential expression profiles. Further GrDXS was over expressed in the homologous (rose-scented geranium) as well as in heterologous (Withania somnifera) plant systems through genetic transformation approach. The over-expression of GrDXS led to enhanced secondary metabolites production (i.e. essential oil in rose-scented geranium and withanolides in W. somnifera). To the best of our knowledge, this is the first report showing the expression profile of the three genes related to isoprenoid biosynthesis pathways operated in rose-scented geranium as well as functional characterization study of any gene from rose-scented geranium through a genetic transformation system.

Bioremediation of petroleum contaminated soil to combat toxicity on Withania somnifera through seed priming with biosurfactant producing plant growth promoting rhizobacteria.

Soil contaminated by Petroleum oil cannot be utilized for agricultural purposes due to hydrocarbon toxicity. Oil contaminated soil induces toxicity affecting germination, growth and productivity. Several technologies have been proposed for bioremediation of oil contaminated sites, but remediation through biosurfactant producing plant growth promontory rhizobacteria (PGPR) is considered to be most promising methods. In the present study the efficacy of seed priming on growth and pigment of Withania somnifera under petroleum toxicity is explored. Seeds of W. somnifera were primed with biosurfactant producing Pseudomonas sp. AJ15 with plant growth promoting traits having potentiality to utilized petroleum as carbon source. Results indicates that plant arose from priming seeds under various petroleum concentration expressed high values for all the parameters studied namely germination, shoot length, root length, fresh and dry weight and pigments (chlorophyll and carotenoid) as compared to non primed seed. Hence, the present study signifies that petroleum degrarding biosurfactant producing PGPR could be further used for management and detoxification of petroleum contaminated soils for growing economically important crops.

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.

Interactive Effects of Growth Regulators, Carbon Sources, pH on Plant Regeneration and Assessment of Genetic Fidelity Using Single Primer Amplification Reaction (SPARS) Techniques in Withania somnifera L.

An improved and methodical in vitro shoot morphogenic approach through axillary bud multiplication was established in a drug yielding plant, Withania somnifera L. Effects of plant growth regulators [6-benzyladenine (BA), kinetin (Kin), 2-isopentenyladenine (2iP), and thidiazuron (TDZ)] either singly or in combination with α-napthalene acetic acid (NAA), indole-3-butyric acid (IBA), and indole-3-acetic acid (IAA) in Murashige and Skoog (MS) medium were tested. The highest regeneration frequency (90 %) with optimum number of shoots (32 ± 0.00)/explant were obtained on MS medium fortified with 2.5 µM 6-benzyladenine (BA) and 0.5 µM NAA and 30 g/l sucrose at pH 5.8. Among the tried TDZ concentrations, 0.5 µM resulted in maximum number of shoots (20.4 ± 0.40)/explant after 4 weeks of exposure. The proliferating shoot cultures established by repeated subculturing of the mother explants on the hormone-free medium produced the highest shoot number (29.4 ± 0.40) with shoot length (6.80 ± 0.12 cm)/explant at fourth subculture passage, which a decline in shoot proliferation was recorded. Different concentrations of NAA were tested for ex vitro rooting of microshoots. The maximum percentage of rooting 100 % with maximum roots (18.3 ± 0.1) was achieved in soilrite when basal portion of the microshoots were treated with 200 µM (NAA) for 15 min per shoot. The plantlets went through hardening phase in a growth chamber, prior to ex vitro transfer. The PCR-based single primer amplification reaction (SPAR) methods which include random amplified polymorphic DNA (RAPD) and direct amplification of minisatellite DNA (DAMD) markers has been used for assessment of genetic stability of micropropagated plantlets. No variation was observed in DNA fingerprinting patterns among the micropropagated and the donor plants illustrating their genetic uniformity.

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.

Molecular characterization of two A-type P450s, WsCYP98A and WsCYP76A from Withania somnifera (L.) Dunal: expression analysis and withanolide accumulation in response to exogenous elicitations.

BACKGROUND: Pharmacological investigations position withanolides as important bioactive molecules demanding their enhanced production. Therefore, one of the pivotal aims has been to gain knowledge about complete biosynthesis of withanolides in terms of enzymatic and regulatory genes of the pathway. However, the pathway remains elusive at the molecular level. P450s monooxygenases play a crucial role in secondary metabolism and predominantly help in functionalizing molecule core structures including withanolides. RESULTS: In an endeavor towards identification and characterization of different P450s, we here describe molecular cloning, characterization and expression analysis of two A-type P450s, WsCYP98A and WsCYP76A from Withania somnifera. Full length cDNAs of WsCYP98A and WsCYP76A have open reading frames of 1536 and 1545 bp encoding 511 (58.0 kDa) and 515 (58.7 kDa) amino acid residues, respectively. Entire coding sequences of WsCYP98A and WsCYP76A cDNAs were expressed in Escherichia coli BL21 (DE3) using pGEX4T-2 expression vector. Quantitative real-time PCR analysis indicated that both genes express widely in leaves, stalks, roots, flowers and berries with higher expression levels of WsCYP98A in stalks while WsCYP76A transcript levels were more obvious in roots. Further, transcript profiling after methyl jasmonate, salicylic acid, and gibberellic acid elicitations displayed differential transcriptional regulation of WsCYP98A and WsCYP76A. Copious transcript levels of both P450s correlated positively with the higher production of withanolides. CONCLUSIONS: Two A-types P450 WsCYP98A and WsCYP76A were isolated, sequenced and heterologously expressed in E. coli. Both P450s are spatially regulated at transcript level showing differential tissue specificity. Exogenous elicitors acted as both positive and negative regulators of mRNA transcripts. Methyl jasmonate and salicylic acid resulted in copious expression of WsCYP98A and WsCYP76A. Enhanced mRNA levels also corroborated well with the increased accumulation of withanolides in response to elicitations. The empirical findings suggest that elicitors possibly incite defence or stress responses of the plant by triggering higher accumulation of withanolides.

Enhanced biosynthesis of withanolides by elicitation and precursor feeding in cell suspension culture of Withania somnifera (L.) Dunal in shake-flask culture and bioreactor.

The present study investigated the biosynthesis of major and minor withanolides of Withania somnifera in cell suspension culture using shake-flask culture and bioreactor by exploiting elicitation and precursor feeding strategies. Elicitors like cadmium chloride, aluminium chloride and chitosan, precursors such as cholesterol, mevalonic acid and squalene were examined. Maximum total withanolides detected [withanolide A (7606.75 mg), withanolide B (4826.05 mg), withaferin A (3732.81 mg), withanone (6538.65 mg), 12 deoxy withanstramonolide (3176.63 mg), withanoside IV (2623.21 mg) and withanoside V (2861.18 mg)] were achieved in the combined treatment of chitosan (100 mg/l) and squalene (6 mM) along with 1 mg/l picloram, 0.5 mg/l KN, 200 mg/l L-glutamine and 5% sucrose in culture at 4 h and 48 h exposure times respectively on 28th day of culture in bioreactor. We obtained higher concentrations of total withanolides in shake-flask culture (2.13-fold) as well as bioreactor (1.66-fold) when compared to control treatments. This optimized protocol can be utilized for commercial level production of withanolides from suspension culture using industrial bioreactors in a short culture period.

[Plant regeneration of Withania somnifera].

OBJECTIVE: To study tissue culture and plant regeneration of Withania somnifera. METHOD: Leaves of W. somnifera were used for explants, effects of different plant growth substances on callus and shoot induction were studied, different medium and plant growth substances for rooting induction was optimized. RESULT AND CONCLUSION: The best plant growth substances combination for callus induction was MS + 1.0 mg x L(-1) 2,4-D + 0.1 mg x L(-1) KT. The optimal medium for germination was MS + 1.0 mg x L(-1) 6-BA + 0.1 mg x L(-1) NAA. The best medium and plant growth substances combination for rooting induction was 1/2MS + 0.5 mg x L(-1) NAA, transplant survival rate of plantlet reached 92% in humus soil-pearlite (1:1).

Coagulanolide, a withanolide from Withania coagulans fruits and antihyperglycemic activity.

One new withanolide, (17S,20S,22R)-14alpha,15alpha,17beta,20beta-tetrahydroxy-1-oxowitha-2,5,24-trienolide) named coagulanolide (4) along with four known withanolides 1-3 and 5 have been isolated from Withania coagulans fruits and their structures were elucidated by spectroscopic techniques. The compounds 1-5 showed significant inhibition on postprandial rise in hyperglycemia post-sucrose load in normoglycemic rats as well as streptozotocin-induced diabetic rats. The compound 5 also showed significant fall on fasting blood glucose profile and improved the glucose tolerance of db/db mice. Further compound 5 showed antidyslipidemic activity in db/db mice. The median effective dose of the compound 5 was determined to be around 25 mg/kg in streptozotocin-induced diabetic rats, which is comparable to the standard antidiabetic drug metformin. Our results provide further support to explain the traditional use of W. coagulans as antihyperglycemic cum antidyslipidemic agent by the traditional medical practitioners.

Purification and characterization of a novel glucosyltransferase specific to 27beta-hydroxy steroidal lactones from Withania somnifera and its role in stress responses.

Sterol glycosyltransferases catalyze the synthesis of diverse glycosterols in plants. Withania somnifera is a medically important plant, known for a variety of pharmacologically important withanolides and their glycosides. In this study, a novel 27beta-hydroxy glucosyltransferase was purified to near homogeneity from cytosolic fraction of W. somnifera leaves and studied for its biochemical and kinetic properties. The purified enzyme showed activity with UDP-glucose but not with UDP-galactose as sugar donor. It exhibited broad sterol specificity by glucosylating a variety of sterols/withanolides with beta-OH group at C-17, C-21 and C-27 positions. It transferred glucose to the alkanol at C-25 position of the lactone ring, provided an alpha-OH was present at C-17 in the sterol skeleton. A comparable enzyme has not been reported earlier from plants. The enzyme is distinct from the previously purified W. somnifera 3beta-hydroxy specific sterol glucosyltransferase and does not glucosylate the sterols at C-3 position; though it also follows an ordered sequential bisubstrate reaction mechanism, in which UDP-glucose and sterol are the first and second binding substrates. The enzyme activity with withanolides suggests its role in secondary metabolism in W. somnifera. Results on peptide mass fingerprinting showed its resemblance with glycuronosyltransferase like protein. The enzyme activity 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.