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.

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.

Biosystematic studies of genus Withania Pauquy in Egypt.

Withania (Solanaceae, Solanoideae) is a widespread genus. Comparative macro-, micro-morphological, anatomical, and molecular features of this genus in Egypt were examined using light and scanning electron microscopy to reassess the conflicted taxonomic relationships between the two studied species. The most significant morphological differences that have been found were: the shape of the lamina, apex, anther, and stigma, and the ratio of calyx tube/lobe; anatomical examination of taxonomic interest are as follows: number of vascular bundles, presence of ears and distribution of accessory vascular bundles in petiole and shape of spongy cells, and number of lower parenchyma in the midrib region of the leaf; trichomes of both species showed no significant differences; pollen, and seed characters are of taxonomic significance in differentiation and characterization between them. Protein profiling revealed that W. somnifera has only conserved proteins, while W. obtusifolia possessed both conserved and additional proteins in their SDS-PAGE banding patterns. Eleven starts codon-targeted (ScoT) primers were applied and produced 96 amplicons with an average of 70.83% polymorphism/primer. W. obtusifolia generated more polymorphic bands and maintained monomorphic ones. SDS-PAGE disclosed that both Withania species were 50% related. While Scot-Dendrogram revealed that both Withania species were poorly related. So, protein and molecular analyses showed considerable genetic variations between these two species.

Withania somnifera chemotypes NMITLI 101R, NMITLI 118R, NMITLI 128R and withaferin A protect Mastomys coucha from Brugia malayi infection.

Withania somnifera is an ayurvedic Indian medicinal plant whose immunomodulatory activities have been widely used as a home remedy for several ailments. We recently observed immunostimulatory properties in the root extracts of chemotypes NMITLI-101, NMITLI-118, NMITLI-128 and pure withanolide, withaferin A. In the present study, we evaluated the potential immunoprophylactic efficacies of these extracts against an infective pathogen. Our results show that administration of aqueous ethanol extracts (10 mg/kg) and withaferin A (0·3 mg/kg), 7 days before and after challenge with human filarial parasite Brugia malayi, offers differential protection in Mastomys coucha with chemotype 101R offering best protection (53·57%) as compared to other chemotypes. Our findings also demonstrate that establishment of B. malayi larvae was adversely affected by pretreatment with withaferin A as evidenced by 63·6% reduction in adult worm establishment. Moreover, a large percentage of the established female worms (66·2%) also showed defective embryogenesis. While the filaria-specific immunological response induced by withaferin A and NMITLI-101 showed a mixed Th1/Th2 phenotype, 118R stimulated production of IFN-γ and 128R increased levels of IL-4. Taken together, our findings reveal potential immunoprophylactic properties of W. somnifera, and further studies are needed to ascertain the benefits of this plant against other pathogens as well.

Functional characterization and differential expression studies of squalene synthase from Withania somnifera.

Squalene synthase (SQS: EC 2.5.1.21) is a potential branch point regulatory enzyme and represents the first committed step to diverge the carbon flux from the main isoprenoid pathway towards sterol biosynthesis. In the present study, cloning and characterization of Withania somnifera squalene synthase (WsSQS) cDNA was investigated subsequently followed by its heterologous expression and preliminary enzyme activity. Two different types of WsSQS cDNA clones (WsSQS1and WsSQS2) were identified that contained an open reading frames of 1,236 and 1,242 bp encoding polypeptides of 412 and 414 amino acids respectively. Both WsSQS isoforms share 99 % similarity and identity with each other. WsSQS deduced amino acids sequences, when compared with SQS of other plant species, showed maximum similarity and identity with Capsicum annuum followed by Solanum tuberosum and Nicotiana tabacum. To obtain soluble recombinant enzymes, 24 hydrophobic amino acids were deleted from the carboxy terminus and expressed as 6X His-Tag fusion protein in Escherichia coli. Approximately 43 kDa recombinant protein was purified using Ni-NTA affinity chromatography and checked on SDS-PAGE. Preliminary activity of the purified enzymes was determined and the products were analyzed by gas chromatograph-mass spectrometer (GC-MS). Quantitative real-time PCR (qRT-PCR) analysis showed that WsSQS expresses more in young leaves than mature leaves, stem and root.

Transcription factor repertoire in Ashwagandha (Withania somnifera) through analytics of transcriptomic resources: Insights into regulation of development and withanolide metabolism.

Transcription factors (TFs) are important regulators of cellular and metabolic functions including secondary metabolism. Deep and intensive RNA-seq analysis of Withania somnifera using transcriptomic databases provided 3532 annotated transcripts of transcription factors in leaf and root tissues, belonging to 90 different families with major abundance for WD-repeat (174 and 165 transcripts) and WRKY (93 and 80 transcripts) in root and leaf tissues respectively, followed by that of MYB, BHLH and AP2-ERF. Their detailed comparative analysis with Arabidopsis thaliana, Capsicum annum, Nicotiana tabacum and Solanum lycopersicum counterparts together gave interesting patterns. However, no homologs for WsWDR representatives, LWD1 and WUSCHEL, were observed in other Solanaceae species. The data extracted from the sequence read archives (SRA) in public domain databases were subjected to re-annotation, re-mining, re-analysis and validation for dominant occurrence of WRKY and WD-repeat (WDR) gene families. Expression of recombinant LWD1 and WUSCHEL proteins in homologous system led to enhancements in withanolide content indicating their regulatory role in planta in the biosynthesis. Contrasting expression profiles of WsLWD1 and WsWUSCHEL provided tissue-specific insights for their participation in the regulation of developmental processes. The in-depth analysis provided first full-spectrum and comparative characteristics of TF-transcripts across plant species, in the perspective of integrated tissue-specific regulation of metabolic processes including specialized metabolism.

Reciprocal loss of CArG-boxes and auxin response elements drives expression divergence of MPF2-Like MADS-box genes controlling calyx inflation.

Expression divergence is thought to be a hallmark of functional diversification between homologs post duplication. Modification in regulatory elements has been invoked to explain expression divergence after duplication for several MADS-box genes, however, verification of reciprocal loss of cis-regulatory elements is lacking in plants. Here, we report that the evolution of MPF2-like genes has entailed degenerative mutations in a core promoter CArG-box and an auxin response factor (ARF) binding element in the large 1(st) intron in the coding region. Previously, MPF2-like genes were duplicated into MPF2-like-A and -B through genome duplication in Withania and Tubocapsicum (Withaninae). The calyx of Withania grows exorbitantly after pollination unlike Tubocapsicum, where it degenerates. Besides inflated calyx syndrome formation, MPF2-like transcription factors are implicated in functions both during the vegetative and reproductive development as well as in phase transition. MPF2-like-A of Withania (WSA206) is strongly expressed in sepals, while MPF2-like-B (WSB206) is not. Interestingly, their combined expression patterns seem to replicate the pattern of their closely related hypothetical progenitors from Vassobia and Physalis. Using phylogenetic shadowing, site-directed mutagenesis and motif swapping, we could show that the loss of a conserved CArG-box in MPF2-like-B of Withania is responsible for impeding its expression in sepals. Conversely, loss of an ARE in MPF2-like-A relaxed the constraint on expression in sepals. Thus, the ARE is an active suppressor of MPF2-like gene expression in sepals, which in contrast is activated via the CArG-box. The observed expression divergence in MPF2-like genes due to reciprocal loss of cis-regulatory elements has added to genetic and phenotypic variations in the Withaninae and enhanced the potential of natural selection for the adaptive evolution of ICS. Moreover, these results provide insight into the interplay of floral developmental and hormonal pathways during ICS development and add to the understanding of the importance of polyploidy in plants.

Chemistry and pharmacology of Withania coagulans: an Ayurvedic remedy.

OBJECTIVES: The use of Withania coagulans, a member of the solanaceae family, has been highlighted in Ayurveda. We have reviewed the chemical constituents and pharmacological properties of W. coagulans, as well as its morphology. This has included therapeutic effects of the whole plant and its extracts, fractions and isolated withanolides. The hepatoprotective, anti-inflammatory, antihyperglycaemic, hypolipidaemic, free radical scavenging, antimicrobial, cardiovascular, central nervous system depressant, immunomodulating, antitumour and cytotoxic activities of W. coagulans have been described. KEY FINDINGS: Research carried out using different biological testing in-vitro and in-vivo techniques supported the claims. SUMMARY: This review has covered the morphology, chemistry and pharmacology of the plant. It has described 37 compounds containing 46 references.