Differential regulation of key triterpene synthase gene under abiotic stress in Withania somnifera L. Dunal and its co-relation to sterols and withanolides.

Withania somnifera (Ashwagandha), is one of the most reputed Indian medicinal plants, having immense pharmacological activities due to the occurrence of withanolides. The withanolides are biosynthesized through triterpenoid biosynthetic pathway with the involvement of WsCAS leading to cyclization of 2, 3 oxidosqualene, which is a key metabolite to further diversify to a myriad of phytochemicals. In contrast to the available reports on the studies of WsCAS in withanolide biosynthesis, its involvement in phytosterol biosynthesis needs investigation. Present work deals with the understanding of role of WsCAS triterpenoid synthase gene in the regulation of biosynthesis of phytosterols & withanolides. Docking studies of WsCAS protein revealed Conserved amino acids, DCATE motif, and QW motif which are involved in efficient substrate binding, structure stabilization, and catalytic activity. Overexpression/silencing of WsCAS leading to increment/decline of phytosterols confers its stringent regulation in phytosterols biosynthesis. Differential regulation of WsCAS on the metabolic flux towards phytosterols and withanolide biosynthesis was observed under abiotic stress conditions. The preferential channelization of 2, 3 oxidosqualene towards withanolides and/or phytosterols occurred under heat/salt stress and cold/water stress, respectively. Stigmasterol and ß-sitosterol showed major contribution in high/low temperature and salt stress, and campesterol in water stress management. Overexpression of WsCAS in Arabidopsis thaliana led to the increment in phytosterols in general. Thus, the WsCAS plays important regulatory role in the biosynthetic pathway of phytosterols and withanolides under abiotic stress conditions.

Enhanced production of withaferin A from the hairy root culture of Withania somnifera via synergistic effect of Methyl jasmonate and ß-cyclodextrin.

Due to low amounts of withanolides produced in some plants and high demand for various applications, their biotechnological production is widely researched. The effects of two explant types (i.e., leaf and stem from the in vitro seedlings of three genotypes of Withania somnifera) and four Rhizobium strains (i.e., LBA 9402, A4, ATCC 15834, and C58C1) to improve hairy root formation efficiency was studied. Furthermore, the combined effects of ß-cyclodextrin (ß-CD) and methyl jasmonate (MeJA) on withaferin A production after 48 h exposure time was examined. Four hairy roots having the maximum percentage of induced roots and mean number of induced roots to analyze their growth kinetics and identified G3/ATCC/LEAF culture having the maximum specific growth rate (µ = 0.036 day-1) and growth index (GI = 9.18), and the shortest doubling time (Td = 18.82 day) were selected. After 48 h exposure of G3/ATCC/LEAF culture to different elicitation conditions, maximum amounts of withaferin A were produced in samples co-treated with 0.5 mM ß-CD + 100 µM MeJA (9.57 mg/g DW) and 5.0 mM ß-CD + 100 µM MeJA (17.45 mg/g DW). These outcomes represented a 6.8-fold and 12.5-fold increase, respectively, compared to the control. Similarly, combined ß-CD/MeJA elicitation increased gene expression levels of HMGR, SQS, SMT-1, and SDS/CYP710A involved in withanolides biosynthetic pathway, of which just SMT-1 had significant correlation with withaferin A production. These results demonstrated the superiority of G1-leaf explant and ATCC 15834 for hairy root induction, and revealed synergistic effect of MeJA and ß-CD on withaferin A production.

Dietary inclusion of Withania somnifera and Asparagus racemosus induces growth, activities of digestive enzymes, and transcriptional modulation of MyoD, MyoG, Myf5, and MRF4 genes in fish, Channa punctatus.

The present study explores growth potential of two medicinal herbs, Withania somnifera (Ashwagandha or 'A') and Asparagus racemosus (Shatavari or 'S') after their dietary inclusion in fish, Channa punctatus (13.5 ± 2 g; 11.5 ± 1 cm). Three hundred well-acclimatized fish were distributed into 10 groups- C (Control), S1 (1% S), S2 (2% S), S3 (3% S), A1 (1% A), A2 (2% A), A3 (3% A), AS1 (1% A and S), AS2 (2% A and S), and AS3 (3% A and S), each having 10 specimens. Fish were fed with these diets for 60 days. The study was performed in triplicate. Growth indices- weight gain (WG), specific growth rate percentage (SGR%), feed intake (FI), and condition factor (CF), after 30 and 60 days, were found significantly (p < 0.05) up-regulated in all the groups, except S1, when compared to the C. A significant (p < 0.05) increase in final body weight (FBW) was noticed in all the groups, except S1, after 60 days. Relative to the control group, activities of lipase and amylase in the gut tissue were elevated in all groups, at both sampling times, with the exception of lipase in S1 at 60 days, and amylase in S1 at day 30 and day 60 and S2 at day 60. The mRNA expression of myogenic regulatory factors (MRFs) was also found to be significantly (p < 0.05) up-regulated with the highest fold changes recorded in AS3 for myoD (3.93 ± 0.91); myoG (6.71 ± 0.30); myf5 (4.40 ± 0.33); MRF4 (4.94 ± 0.21) in comparison to the C.

Dynamics of reactive oxygen species and lignin biosynthesis during leaf spot disease of Withania somnifera (L.) Dunal.

Withania somnifera is an important medicinal plant, however, its cultivation and quality are compromised through infestation by leaf spot disease caused by the fungus, Alternaria alternata. To find suitable strategies against this disease, studies on post-infectional changes are important. ROS are critical as they interact with other defence signalling pathways. We analyzed ROS-generating and scavenging systems in healthy and diseased leaf samples of W. somnifera and ROS-driven downstream defence pathways. We used DAB and NBT assays for ROS detection, spectrophotometry and in-gel assays for ROS scavenging enzymes, a thioglycolic acid (TGA) based assay, histochemical staining for lignin, and qRT-PCR for transcript-level expression. Leaf spot infection in W. somnifera increased NADPH oxidase activity and ROS accumulation in infected leaves, together with enhanced antioxidant enzyme activity. Leaf spot-infected leaves had increased lignin content and higher expression of lignin biosynthesis genes. In addition, transcript levels of defence-related genes, NPR1 and PR, were also upregulated. The present work provides insights into responses to leaf spot disease through defence-related signalling in W. somnifera. It demonstrates crosstalk between ROS and lignin biosynthesis. This work identified potential targets for developing strategies to confer disease resistance against A. alternata in W. somnifera.

The growth performance, antioxidant and immune responses, and disease resistance of Litopenaeus vannamei fed on diets supplemented with Indian ginseng (Withania somnifera).

In the current study, white-leg shrimp (Litopenaeus vannamei) were fed on diets containing varying doses of Withania somnifera aqueous extract (WSAE) at a rate of 0 (control), 0.5, 1.0, and 2.0 g/kg feed for 56 days. After the feeding trial, shrimps in all groups were challenged with the exposure to Vibrio harveyi for ten days during which animals' mortality was observed. It is noted that the dietary WSAE linearly and quadratically stimulated shrimp's growth indices particularly at the treatment of 2.0 g/kg feed. Compared to the control group, the WSAE-fed L. vannamei had significantly higher villi length, villi width, and absorption area particularly in the treatment of 2.0 g/kg feed. Furthermore, L. vannamei fed on WSAE-enriched diets consumed more feed and exhibited higher total proteolytic activity, lipase, and α-amylase activities as compared with the control group. The dietary WSAE at escalating levels linearly and quadratically enhanced the antioxidant activity (serum superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), total antioxidant capacity, and reduced glutathione) and the immune response (total hemocyte counts, total protein, lysozyme, and phagocytic activity). Similarly, the mRNA expression levels of cMn-SOD, CAT, and GPx genes were linearly and quadratically upregulated in the hepatopancreas of L. vannamei fed on WSAE-enriched diets (especially in the 2.0 g/kg feed treatment), while their lowest levels were significantly observed in the control group. On the other hand, malondialdehyde levels were significantly decreased in WSAE-supplemented shrimp groups, and its highest levels were observed in animals fed on the control diet. After the bacterial exposure, the survival rates of L. vannamei fed on 1.0 and 2.0 g WSAE/kg feed (61.3% and 66.7%, respectively) were higher than those in the control animals. Taken together, the results obtained herein indicate that inclusion of WSAE in diets of L. vannamei effectively enhanced the growth, antioxidant biomarkers, immune response, and resistance to the V. harveyi infection, particularly at the treatment of 2.0 g/kg feed.

Cloning and homologous characterization of geranylgeranyl pyrophosphate synthase (GGPPS) from Withania somnifera revealed alterations in metabolic flux towards gibberellic acid biosynthesis.

MAIN CONCLUSION: Overexpression of a novel geranylgeranyl pyrophosphate synthase gene (WsGGPPS) in planta resulted in increased levels of gibberellic acid and decrease in withanolide content. Withania somnifera (L.) Dunal, the herb from family Solanaceae is one of the most treasured medicinal plant used in traditional medicinal systems owing to its unique stockpile of pharmaceutically active secondary metabolites. Phytochemical and pharmacological studies in this plant were well established, but the genes affecting the regulation of biosynthesis of major metabolites were not well elucidated. In this study cloning and functional characterization of a key enzyme in terpenoid biosynthetic pathway viz. geranylgeranyl pyrophosphate synthase (EC 2.5.1.29) gene from Withania somnifera was performed. The full length WsGGPPS gene contained 1,104 base pairs that encode a polypeptide of 365 amino acids. The quantitative expression analysis suggested that WsGGPPS transcripts were expressed maximally in flower tissues followed by berry tissues. The expression levels of WsGGPPS were found to be regulated by methyl jasmonate (MeJA) and salicylic acid (SA). Amino acid sequence alignment and phylogenetic studies suggested that WsGGPPS had close similarities with GGPPS of Solanum tuberosum and Solanum pennellii. The structural analysis provided basic information about three dimensional features and physicochemical parameters of WsGGPPS protein. Overexpression of WsGGPPS in planta for its functional characterization suggested that the WsGGPPS was involved in gibberellic acid biosynthesis.

Comprehensive analysis of codon usage pattern in Withania somnifera and its associated pathogens: Meloidogyne incognita and Alternaria alternata.

Meloidogyne incognita (Root-knot nematode) and Alternaria alternata (fungus) were among the dominant parasites of the medicinal plant Withania somnifera. Despite the fatal nature of their infection, a comprehensive study to explore their evolution and adaptation is lacking. The present study elucidates evolutionary and codon usage bias analysis of W. somnifera (host plant), M. incognita (root-knot nematode) and A. alternata (fungal parasite). The results of the present study revealed a weak codon usage bias prevalent in all the three organisms. Based on the nucleotide analysis, genome of W. somnifera and M. incognita was found to be A-T biased while A. alternata had GC biased genome. We found high similarity of CUB pattern between host and its nematode pathogen as compared to the fungal pathogen. Inclusively, both the evolutionary forces influenced the CUB in host and its associated pathogens. However, neutrality plot indicated the pervasiveness of natural selection on CUB of the host and its pathogens. Correspondence analysis revealed the dominant effect of mutation on CUB of W. somnifera and M. incognita while natural selection was the main force affecting CUB of A. alternata. Taken together the present study would provide some prolific insight into the role of codon usage bias in the adaptability of pathogens to the host's environment for establishing parasitic relationship.

Molecular characterization of India Ginseng Withania somnifera (L) using ISSR markers.

BACKGROUND: Ashwagandha (Withania somnifera (L.) Dunal), popularly known as Indian ginseng or winter cherry is a multipurpose plant of immense therapeutic value in the ayurvedic and indigenous medicine system and distributed in wide geographic locations and exhibiting extensive phenotypic and chemical variability. METHODS AND RESULTS: The present study was carried out to assess the molecular genetic diversity among 4 CIMAP varieties and five local cultivars of ashwagandha and cluster dendrograms were created by using 20 ISSR primers. A total of 224 bands of varied length were produced, out of which 193 (86.1%) products were polymorphic and 31 (13.8%) products were monomorphic. Where each ISSR arbitrary primer had 5-16 valuable bands with an average of 11.2 bands per primer, of which 86.16% bands were polymorphic. The PIC values ranged from 0.16 to 0.36 with an average PIC value of 0.29 and RP values ranged from 2.22 to 7.99. The UPGMA cluster analysis of 20 ISSR primers grouped the nine accessions into 2 major clusters. The first and second major cluster consists of seven and two accessions respectively. CONCLUSION: Therefore, this study provides evidence that ISSR based molecular diversity assessment can be used as an efficient tool for detecting similarity and phylogenetic relationships among genotypes of Withania somnifera collected from different geographical locations. This information can be used to improve root and other characteristics of ashwagandha genotypes and there is also scope for the development of high-yielding varieties by selecting diverse parents for crossing (based on the molecular diversity) from the present accessions.

Overexpression of WssgtL3.1 gene from Withania somnifera confers salt stress tolerance in Arabidopsis.

KEY MESSAGE: Overexpression of Withania somnifera SGT gene (WssgtL3.1) in transgenic Arabidopsis improves various agronomic and physiological traits and alters conjugated sterol levels to mitigate the effect of salt stress. Sterols are essential constituents of cell membranes that are involved in several biological functions, including response to various biotic and abiotic stresses by altering membrane permeability and signaling pathways. Sterol glycosyltransferases (SGTs) are enzymes that are involved in sterol modification by converting sterols into sterol-conjugates to play essential roles in adaptive responses. However, their roles under abiotic stresses are lesser-known. Among abiotic stresses, salinity imposes serious threat to crop yield worldwide, hence the present study intends to investigate the role of WssgtL3.1-overexpressed Arabidopsis plants under salt stress indicating the crosstalk between SGT gene and salinity to develop improved crop varieties with better stress tolerance ability. The findings revealed that overexpression of WssgtL3.1 gene in A. thaliana improved the resistance against salt stress in the overexpressing lines. Transgenic lines showed significantly higher germination rate, increased plant growth with less chlorophyll damage compared to wild-type (WT) control plants. Moreover, better tolerance also correlated with enhanced osmolytes (proline and soluble sugar), better membrane integrity, decreased H2O2 production and lesser MDA accumulation and Na+/K+ ratio with more negative osmotic potential in overexpressed lines. Additionally, in sterol profiling, significant enhancement in stigmasterol was also observed in transgenic lines than WT plants. Furthermore, in expression profiling, salt responsive genes LEA 4-5, sucrose synthase, and transporter of monosaccharide (ERD) significantly upregulated in overexpressing lines as compared to WT. Thus our data strongly support the defensive role of Withania somnifera SGT gene (WssgtL3.1) against salt stress and contribute to improved salinity tolerance in plants through sterol modulation.

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.

Impact of green synthesized WcAgNPs on in-vitro plant regeneration and withanolides production by inducing key biosynthetic genes in Withania coagulans.

KEY MESSAGE: Withania coagulans (L.) Dunal bio-synthesized silver nanoparticles (WcAgNPs) worked as an abiotic elicitor or auto-catalyst that enhanced root regeneration and withanolides production in in-vitro regenerated W. coagulans. Rapid development in the production / consumption of silver nanoparticles (AgNPs) raised serious concern over its effects on the growth of natural plant community. The knowledge related to impact of AgNPs on plant growth and biocompatibility is increasing day by day, but comprehensive mechanism and gaps regarding their impacts on plant health have yet to be addressed. In the present study, we investigated the impact of Withania coagulans biosynthesized AgNPs (WcAgNPs) on in-vitro plant growth and withanolides production. Obtained results showed that the low concentrations of WcAgNPs significantly induced the plant growth by regulating oxidative stress via anti-oxidative defense system. Physiological, morphology and anatomical features also reflected healthy plant growth under low WcAgNPs exposure. While higher concentrations of WcAgNPs have a negative impact on W. coagulans plant growth due to induced lipid peroxidation, ROS accumulation, and root cell death. At lower concentrations, WcAgNPs have shown a positive effect on in-planta withanolides biosynthesis stimulating withanolide A and withaferin A up to 11.15-22.8-fold, respectively. Furthermore, the expression of withanolides biosynthetic genes were also quantified upon WcAgNPs exposure and terpenes biosynthetic genes showed over-expression. Thus, the present study concludes that the lower concentrations of WcAgNPs positively induced plant growth via improved root organogenesis and also have potential to act as an elicitor for withanolides production.

Characterization and overexpression of sterol Δ22-desaturase, a key enzyme modulates the biosyntheses of stigmasterol and withanolides in Withania somnifera (L.) Dunal.

Withanolides constitute an extensive and vital class of metabolites displaying wide array of structural and therapeutic properties with unique side-chain modifications. These show diversified scaffolds and are promising pharmaceutical molecules with well documented anti-inflammatory and anti-cancer properties. Sterols are dynamic class of compounds and essential molecules having structural and functional significance. These contribute to the synthesis of withanolides by providing structural precursors. In this context, we have characterized sterol Δ22-desaturase from Withania somnifera and also functionally validating it by confirming its desaturase nature in conjunction with quantitative real-time expression profiling and metabolite evaluation. Further, transgenic hairy roots of W. somnifera displayed a higher accumulation of stigmasterol and withanolides. The increase in chemical constituents was concomitant with an increased gene copy number predicted via Southern blotting. Additionally, transgenic lines of tobacco over-expressing WsCYP710A11 displayed a substantial increase in its expression, corroborating well with enhanced stigmasterol content. Characterization of CYP710A11 from W. somnifera and its homologous transgenic expression has demonstrated its role in the regulation of withanolides biosynthesis. It also exhibited a differential transcriptional profile in response to exogenous elicitations. These empirical findings suggest the crucial role of CYP710A11 in stigmasterol biosynthesis. This in turn has implications for the overproduction of withanolides via pathway channelling.

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.

Production of pharmaceutical active recombinant globular adiponectin as a secretory protein in Withania Somnifera hairy root culture.

Among various in vitro plant culture systems, hairy root systems seem to be one of the most appealing methods of recombinant protein production due to their advantages in combining both whole-plant cultivation and suspension cell culture platform. This is a report on production and secretion of a recombinant pharmaceutically active protein from hairy roots cultures of Withania somnifera to improve the economic potential of this plant for the production pharmaceutical compounds. In this study, we selected and synthesized a codon-optimized globular adiponectin (gAd) gene with a calreticulin signal peptide and cloned the sequence into a plant expression binary vector containing a nptII gene as a selectable marker gene. The transgenic hairy roots were produced by Agrobacterium rhizogenes-mediated transformation protocol developed by our group. Among ten established nptII positive hairy roots lines, six colons significantly accumulated gAd protein in the biomass and extracellular medium. The presence of gAd was confirmed by western blot analysis of root extracts. The maximum level of hairy root biomass, growth rate (GR), intra- and extracellular gAd expressions were obtained after 25-26 days of culture on MS medium. The maximum level of intra- and extracellular gAd proteins were found to be 15.19 µg/gFW and 215.7 µg/L, respectively, which resulted in a significant decrease in the amount of intra- and extracellular withanolide A and withaferin A production. The addition of PVP, KNO3 and NaCl significantly increased the level of extracellular gAd by approximately 13 folds. This improvement could significantly increase the amount of intra- and extracellular withanolide A and withaferin A production, too. The recombinant gAd produced from W. somnifera is functional as proved by induction the phosphorylation of ACC in C2C12 muscle cells, as its functional amount was 5.1-fold more than gAd produced from E. coli and 45 % lower than CHO cells.

Virus-Induced Gene Silencing for Functional Genomics in Withania somnifera, an Important Indian Medicinal Plant.

Virus-induced gene silencing (VIGS) has emerged as a fast and efficient reverse and forward genetics tool to study gene function in model plants as well as in agriculturally important plants. In addition, VIGS approach has been successfully used to provide insights into the role of several genes and regulators involved in plant secondary metabolism. Ashwagandha (Withania somnifera) is an important Indian medicinal plant that accumulates pharmacologically important triterpenoid steroidal lactones, which are collectively termed as withanolides. W. somnifera being a highly recalcitrant plant for genetic transformation, Tobacco rattle virus (TRV)-mediated VIGS was established by our group to facilitate understanding of withanolides' pathway. Here, we describe a detailed procedure to carry out VIGS for gene function studies in W. somnifera.

In silico mining and functional analysis of AP2/ERF gene in Withania somnifera.

Withania somnifera owing to its strong and remarkable stress tolerance property is a reliable candidate for the determination of genes involved in mechanism of adaption/tolerance of various stress conditions. 187 AP2/ERF gene related transcripts (GRTs) were identified during comprehensive search in W. somnifera transcriptome repertoire. Major hits in homology search were observed from the model plant Arabidopsis and members of Solanaceae family. Cloning, expression analysis of the gene and genetic transient transformation with the gene (WsAP2) were performed to predict its functional role in planta. Enhanced expression of some of the pathway genes for terpenoid biosynthesis was observed in transformed tissues in comparison to the control tissues. It is speculated that WsAP2 gene crucially regulates the expression of GGPPS gene in addition to the regulation of other important genes of terpenoid pathway via induction of expression of other genes such as HMGR, CAS, DXS and DXR. To the best of our knowledge, this is the first report representing detailed study of AP2/ERF gene family in W. somnifera. It is also suggested from the study that gene might have role in eliciting responses to combat stress and attribute the strong stress tolerant property associated with the plant.

Authentication of the market samples of Ashwagandha by DNA barcoding reveals that powders are significantly more adulterated than roots.

ETHNOPHARMACOLOGICAL RELEVANCE: Ashwagandha, also known as Indian Ginseng, is a highly traded medicinal plant, which is used in Ayurveda, Siddha and Unani systems of medicine to improve cognitive function, decrease inflammation, and to counter the ill-effects of aging. Withanolide A and Withaferin A from Ashwagandha were shown to improve immunity and have anti-cancer property, respectively. AIM OF THE STUDY: Here, we aimed to create reference DNA barcodes for W. somnifera and to authenticate root and powder samples of Ashwagandha collected from markets. MATERIALS AND METHODS: Three plant specimen of W. somnifera were collected, and reference DNA barcodes were generated using rbcL, matK, trnH-psbA, and ITS2 DNA barcode markers. Market samples in the form of root (n = 33) and powder (n = 70) were collected and authenticated using ITS2 and trnH-psbA DNA barcodes. RESULTS: Genomic DNA was successfully isolated from all plant specimens and market samples. DNA barcoding showed that 77% of samples were authentic. About 22% of non-authentic samples were powder samples and only 1% were root samples. Among the non-authentic samples, 18% were completely substituted with single species (Mucuna pruriens (L.) DC., Trigonella foenum-graceum L., or Senna auriculata (L.) Roxb.) and 82% were mixed samples containing more than one species. About 63% of the mixed samples contained Ashwagandha as the major ingredient. Furthermore, we identified that six taxonomically divergent plant species from four families were present as adulterants in the mixed samples. CONCLUSION: DNA barcoding revealed that botanical adulteration in the market samples of Ashwagandha is significant. Powder samples are more prone to adulteration than root samples. The adulterated samples contained plant material that is not related to Ashwagandha, which warrants strict quantity control and market surveillance to derive the true medicinal benefits of this medicinal plant.

Elucidation of genotype-environment interactions and genetic stability parameters for yield, quality and agromorphological traits in ashwagandha (Withania somnifera (L.) Dunal).

The present study was undertaken to delineate genotype-environment interactions and stability status of 16 genotypes of ashwagandha (Withania somnifera (L.) Dunal) in context to the 12 characters, namely plant height, number of primary branches, number of secondary branches, days to flowering, days to maturity, number of berries, number of seeds/berry, root length, root diameter, root branches, dry root yield and total alkaloid content (%). Experiment was carried out in a randomized complete block design with three replicationsover three different locations (S. K. Nagar, Jagudan and Bhiloda) in north Gujarat for three years (2016-17, 2017-18 and 2018-19). Pooled analysis of variance revealed that the mean squares due to genotypes and genotype 9 environment interaction along with linear and nonlinear components were highly significant (P<0.01) for most of the traits under study. Stability parameters for component traits through Eberhart and Russell model showed that genotypes that can be used directly in breeding programme are SKA-4 for early flowering, SKA-21 for early maturity and SKA-1, SKA-4, SKA-6 and SKA-17 for shorter plant height. Further, SKA-21 could be used for improving number of primary branches per plant, SKA-11 and SKA-17 for number of secondary branches per plant, SKA-19 for number of berries per plant, SKA-6, SKA-21, SKA-27 and AWS-1 for root branches and SKA-17 for root length as these genotypes were found to be moststable across the environments for mentioned traits. The result revealed that some reliable predictions about genotype 9 environment interaction and its unpredictable components were involved significantly in determining the stability of genotypes. Hence, the present investigation can be exploited for the identification of more productive genotypes in specific environments, leading to significant increase in root productivity of ashwagandha.

Berry transcriptome: insights into a novel resource to understand development dependent secondary metabolism in Withania somnifera (Ashwagandha).

Withania somnifera (Ashwagandha) is considered as Rasayana in Indian systems of medicine. This study reports a novel transcriptome of W. somnifera berries, with high depth, quality and coverage. Assembled and annotated transcripts for nearly all genes related with the withanolide biosynthetic pathway were obtained. Tissue-wide gene expression analysis reflected almost similar definitions for the terpenoid pathway in leaf, root and berry tissues with relatively higher abundance of transcripts linked to steroid, phenylpropanoid metabolism as well as flavonoid metabolism in berries. The metabolome map generated from the data embodied transcripts from 143 metabolic pathways connected together and mediated collectively by about 1792 unique enzyme functions specific to berry, leaf and root tissues, respectively. Transcripts specific to cytochrome p450 (CYP450), methyltransferases and glycosyltransferases were distinctively located in a tissue specific manner and exhibited a complex network. Significant distribution of transcription factor genes such as MYB, early light inducible protein (ELI), minichromosome maintenance1, agamous, deficiens and serum response factor (MADS) and WRKY etc. was observed, as the major transcriptional regulators of secondary metabolism. Validation of the assembly was ascertained by cloning WsELI, which has a light dependent regulatory role in development. Quantitative expression of WsELI was observed to be considerably modulated upon exposure to abiotic stress and elicitors. Co-relation of over-expression of WsELI, may provide new aspects for the functional role of ELI proteins in plants linked to secondary metabolism. The study offers the first comprehensive and comparative evaluation of W. somnifera transcriptome data between the three tissues and across other members of Solanaceae (Nicotiana, Solanum and Capsicum) with respect to major pathways and their metabolome regulation.

Characterization of Withania somnifera chloroplast genome and its comparison with other selected species of Solanaceae.

Withania somnifera (L) Dunal, a wonder herb of family Solanaceae, has multiple medicinal properties. Here, we reported the chloroplast genome sequence of Withania somnifera (154,386 bp) which comprises of a large single copy region (85,688 bp), and a small single copy region (18,464 bp), separated by a pair of large inverted repeats (25,117 bp). The chloroplast genome has 132 genes including 86 protein-coding, 37 tRNAs and 8 rRNAs. Comparison of chloroplast genomes of Withania somnifera with four other Solanaceae species revealed similarities in genomic features, including structure, nucleotide content, codon usage, RNA editing sites, simple sequence repeats (SSRs), oligonucleotide repeats, and tandem repeats. We identified 147 simple sequence repeats in protein-coding, and 229 in non-protein-coding regions. We observed numerous post-transcriptional substitutions of Serine to Leucine, specifically at the second nucleotide position of the codon. Maximum likelihood and maximum parsimony tree reconstructed displayed Withania somnifera a sister taxon of Physalis peruviana.