Expanding roles of cross-talk between hydrogen sulfide and nitric oxide under abiotic stress in plants.

Abiotic stress such as salt, heavy metals, drought, temperature, and others can affect plants from seed germination to seedling growth to reproductive maturity. Abiotic stress increases reactive oxygen species and lowers antioxidant enzymes in plants resulted the plant tolerance ability against stress conditions decrease. Hydrogen sulfide (H2S) and nitric oxide (NO) are important gasotransmitters involved in seed germination, photosynthesis, growth and development, metabolism, different physiological processes and functions in plants. In plants, various enzymes are responsible for the biosynthesis of both H2S and NO via both enzymatic and non-enzymatic pathways. They also mediate post-translation modification, such as persulfidation, and nitrosylation, which are protective mechanisms against oxidative damage. They also regulate some cellular signalling pathways in response to various abiotic stress. H2S and NO also stimulate biochemical reactions in plants, including cytosolic osmoprotectant accumulation, reactive oxygen species regulation, antioxidant system activation, K+ uptake, and Na+ cell extrusion or vacuolar compartmentation. In this review, we summarize how H2S and NO interact with each other, the function of both H2S and NO, the mechanism of biosynthesis, and post-translational modification under different abiotic stress. Our main emphasis was to find the cross-talk between NO and H2S and how they regulate genes in plants under abiotic stress.

Phyto-therapeutic potential of Withania somnifera: Molecular mechanism and health implications.

Withania somnifera, the plant named Indian ginseng, Ashwagandha, or winter cherry, has been used since ancient times to cure various health ailments. Withania somnifera is rich in constituents belonging to chemical classes like alkaloids, saponins, flavonoids, phenolic acids, and withanolides. Several chemotypes were identified based on their phytochemical composition and credited for their multiple bioactivities. Besides, exhibiting neuroprotective, immunomodulatory, adaptogenic, anti-stress, bone health, plant has shown promising anti-cancer properties. Several withanolides have been reported to play a crucial role in cancer; they target cancer cells by different mechanisms such as modulating the expression of tumor suppressor genes, apoptosis, telomerase expression, and regulating cell signaling pathway. Though, many treatments are available for cancer; however, to date, no assured reliable cure for cancer is made available. Additionally, synthetic drugs may lead to development of resistance in time; therefore, focus on new and natural drugs for cancer therapeutics may prove a longtime effective alternative. This current report is a comprehensive combined analysis upto 2023 with articles focused on bio-activities of plant Withania somnifera from various sources, including national and international government sources. This review focuses on understanding of various mechanisms and pathways to inhibit uncontrolled cell growth by W. somnifera bioactives, as reported in literature. This review provides a recent updated status of the W. somnifera on pharmacological properties in general and anti-cancer in particular and may provide a guiding resource for researchers associated with natural product-based cancer research and healthcare management.

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.

Targeting Pathways and Integrated Approaches to Treat Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic symmetrical systemic disorder that not only affects joints but also other organs such as heart, lungs, kidney, and liver. Approximately there is 0.5%-1% of the total population affected by RA. RA pathogenesis still remains unclear due to which its appropriate treatment is a challenge. Further, multitudes of factors have been reported to affect its progression i.e. genetic factor, environmental factor, immune factor, and oxidative factor. Therapeutic approaches available for the treatment of RA include NSAIDs, DMARDs, enzymatic, hormonal, and gene therapies. But most of them provide the symptomatic relief without treating the core of the disease. This makes it obligatory to explore and reach the molecular targets for cure and long-term relief from RA. Herein, we attempt to provide extensive overlay of the new targets for RA treatment such as signaling pathways, proteins, and receptors affecting the progression of the disease and its severity. Precise modification in these targets such as suppressing the notch signaling pathway, SIRT 3 protein, Sphingosine-1-phosphate receptor and stimulating the neuronal signals particularly efferent vagus nerve and SIRT 1 protein may offer long term relief and potentially diminish the chronicity. To target or alter the novel molecules and signaling pathway a specific delivery system is required such as liposome, nanoparticles and micelles and many more. Present review paper discusses in detail about novel targets and delivery systems for treating RA.

New withanolides from leaves along with alkyl glucoside from roots and stem as well as variations in withanolide contents in leaves of accessions of Withania somnifera, Ashwagandha- the Indian ginseng$.

Based on the major components in the leaves, the ashwagandha has been found to exist in several chemotypic forms in India. From the leaves of various accessions of Withania somnifera, which were maintained in our institute, three new steroids namely, 4-acetoxy-20ß-hydroxy-1-oxo-witha-2,5,24-trienolide (7), 24,25-dihydro-14α-hydroxy withanolide D (9), 5α,6ß,17α,27-tetrahydroxy-1-oxo-witha-2,24-dienolide (12) together with thirteen known withanolides were identified by spectroscopic methods. From the roots and stem of one accession and leaves of another, a new alkyl ester glucoside (4) has also been isolated. The new withanolides 7, 9 and 12 have been tentatively named as withanolide 135 A, withanolide 135B and withanolide 108, respectively.

Novel archetype in cancer therapeutics: exploring prospective of phytonanocarriers.

This paper reports various types of cancer, their incidence, and prevalence all over the globe. Along with the discovery of novel natural drugs for cancer treatment, these present a promising option which are eco-friendly, safe, and provide better acceptability in comparison to synthetic agents that carries multiple side effects. This paper provides an idea about various nanocarriers and phytochemicals, along with how their solubility and bioavailability can be enhanced in nanocarrier system. This report combines the data from various literature available on public domain including PubMed on research articles, reviews, and along with report from various national and international sites. Specialized metabolites (polyphenols, alkaloids, and steroids etc) from medicinal plants are promising alternatives to existing drugs. Studies have suggested that the treatment of cancer using plant products could be an alternative and a safe option. Studies have shown with the several cell lines as well as animal models, that phytomolecules are important in preventing/treating cancer. Phytochemicals often outperform chemical treatments by modulating a diverse array of cellular signaling pathways, promoting cell cycle arrest, apoptosis activation, and metastatic suppression, among others. However, limited water solubility, bioavailability, and cell penetration limit their potential clinical manifestations. The development of plant extract loaded nanostructures, rendering improved specificity and efficacy at lower concentrations could prove effective. Nanocarriers, such as liposomes, nanostructured lipids, polymers, and metal nanoparticles, have been tested for the delivery of plant products with enhanced effects. Recent advances have achieved improvement in the the stability, solubility, bioavailability, circulation time, and target specificity by nanostructure-mediated delivery of phytochemicals. Nanoparticles have been considered and attempted as a novel, targeted, and safe option. Newer approaches such as phyto-nanocarriers with carbohydrates, lignin, and polymers have been considered even more selective and effective modes of drug delivery in biomedical or diagnostic applications.

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.

Molecular cloning and characterization of Triterpenoid Biosynthetic Pathway Gene HMGS in Centella asiatica (Linn.).

BACKGROUND: The flux of isoprenoids and the total accumulation of triterpenoid saponins known as centellosides in C. asiatica are controlled by the key genes of the Mevalonate pathway (MVA). These genes were reported to have positive regulation of the pathway in providing isoprenoid moieties. Though, some information is available on the pathway and secondary metabolites. However, most of the pathway steps are not characterized functionally. METHODOLOGY AND RESULTS: For the study, full-length pathway gene Hydroxymethyl glutaryl-CoA-synthase (CaHMGS; GenBank accession number: MZ997833), was isolated from previously annotated transcriptome data of Centella asiatica leaves. HMGS has been successfully cloned and heterologously expressed in bacteria E. coli strain DH5α. The cloned gene has been sequenced and further characterized through in silico studies by different bioinformatics tools. Also, the gene sequences have been submitted in NCBI. In silico studies of isolated gene sequence revealed the nature, characteristics of genes. The ORF of HMGS is 1449 bp encoding 482 amino acids. Predicted molecular weight (MW) of HMGS was 48.09 kDa and theoretical pI was 5.97. Blast results and Multiple sequence alignments of the gene showing the similarity with HMGS of other plants of their respective families. The Molecular Evolutionary Genetic Analysis (MEGA) version 10.1.6 was used to construct a phylogenetic tree. Differential tissue-specific expression of different plant parts was also checked. Tissue expression patterns unveiled that the highest expression level of the CaHMGS had been seen in the roots and lowest in the node of the plant. Functional complementation experiment of the CaHMGS in Saccharomyces cerevisiae wild strain YSC1021 and haploid strain YSC1021 which lack HMGS protein confirmed that the CaHMGS gene encodes functional CaHMGS that catalyzed the biosynthesis of mevalonate in yeast. CONCLUSIONS: The gene was reported, cloned and characterized first time in Centella asiatica. Understanding this biosynthetic pathway gene will further help in the improvement of plants for enhanced secondary metabolites production.

Microbial cancer therapeutics: A promising approach.

The success of conventional cancer therapeutics is hindered by associated dreadful side-effects of antibiotic resistance and the dearth of antitumor drugs' selectivity and specificity. Hence, the conceptual evolution of anti-cancerous therapeutic agents that selectively target cancer cells without impacting the healthy cells or tissues, has led to a new wave of scientific interest in microbial-derived bioactive molecules. Such strategic solutions may pave the way to surmount the shortcomings of conventional therapies and raise the potential and hope for the cure of wide range of cancer in a selective manner. This review aims to provide a comprehensive summary of anti-carcinogenic properties and underlying mechanisms of bioactive molecules of microbial origin, and discuss the current challenges and effective therapeutic application of combinatorial strategies to attain minimal systemic side-effects.

Standardized root extract of Withania somnifera and Withanolide A exert moderate vasorelaxant effect in the rat aortic rings by enhancing nitric oxide generation.

ETHNO-PHARMACOLOGICAL RELEVANCE: Withania somnifera (L.) Dunal, commonly known as Ashwagandha, belongs to the family Solanaceae. In Ayurveda, Ashwagandha has been defined as one of the most important herb and is considered to be the best adaptogen. It is also an excellent rejuvenator, a general health tonic and cure for various disorders such as cerebrovascular, insomnia, asthma, ulcers, etc. Steroidal lactones (Withanolides: Withanolide A, Withaferin A, Withanolide D, Withanone, etc) isolated from this plant, possess promising medicinal properties such as anti-inflammatory, immune-stimulatory etc. Standardized root extract of the plant NMITLI-118R (NM) was prepared at CSIR-CIMAP, and was investigated for various biological activities at CSIR-CDRI. Among the notable medicinal properties, NM exhibited excellent neuroprotective activity in the middle cerebral artery occlusion (MCAO) rat model. AIM OF THE STUDY: Endothelial dysfunction is the primary event in the cerebrovascular or cardiovascular disorders, present study was thus undertaken to evaluate vasoprotective potential of NM and its biomarker compound Withanolide A (WA) using rat aortic rings and EA.hy926 endothelial cells. MATERIAL AND METHODS: Transverse aortic rings of 10 weeks old Wistar rats were used to evaluate effect of NM and WA on the vasoreactivity. While, mechanism of NM and WA mediated vasorelaxant was investigated in Ea.hy926 cell line by measuring NO generation, nitrite content, Serine 1177 phosphorylation of eNOS, reduced/oxidized biopterin levels and expression of endothelial nitric oxide synthase (eNOS) mRNA and protein. RESULTS: Fingerprinting of NM using HPLC identified presence of WA in the extract. NM as well as WA exerted moderate vasorelaxant effect in the endothelium intact rat aortic rings which was lesser than acetylcholine (ACh). NM and WA augmented ACh induced relaxation in the rat aortic rings. NM and WA dependent vasorelaxation was blocked by N-nitro-L-arginine methyl ester (L-NAME) or 1H-[1,2,4] oxadiazolo [4,3,-a]quinoxalin-1-one (ODQ), indicating role of NO/cGMP. Further Ea.hy926 cells treated with NM and WA showed accumulation of nitrite content, enhanced NO levels, eNOS expression and eNOS phosphorylation (Serine 1177). CONCLUSION: Altogether NM and WA dependent improvement in the NO availability seems to be mediated by the enhanced eNOS phosphorylation. WA, seems to be one of the active constituent of NM, and presence of other vasoactive substances cannot be ruled out. The data obtained imply that the vasorelaxant property of NM is beneficial for its neuroprotective potential.

Elucidation of plasma protein binding, blood partitioning, permeability, CYP phenotyping and CYP inhibition studies of Withanone using validated UPLC method: An active constituent of neuroprotective herb Ashwagandha.

ETHNOPHARMACOLOGICAL RELEVANCE: Withanone (WN), an active constituent of Withania somnifera commonly called Ashwagandha has remarkable pharmacological responses along with neurological activities. However, for a better understanding of the pharmacokinetic and pharmacodynamic behavior of WN, a comprehensive in-vitro ADME (absorption, distribution, metabolism, and excretion) studies are necessary. AIM OF THE STUDY: A precise, accurate, and sensitive reverse-phase ultra-performance liquid chromatographic method of WN was developed and validated in rat plasma for the first time. The developed method was successfully applied to the in-vitro ADME investigation of WN. MATERIAL AND METHODS: The passive permeability of WN was assayed using PAMPA plates and the plasma protein binding (PPB) was performed using the equilibrium dialysis method. Pooled liver microsomes of rat (RLM) and human (HLM) were used for the microsomal stability, CYP phenotyping, and inhibition studies. CYP phenotyping was evaluated using the specific inhibitors. CYP inhibition study was performed using specific probe substrates along with WN or specific inhibitors. RESULTS: WN was found to be stable in the simulated gastric and intestinal environment and has a high passive permeability at pH 4.0 and 7.0 in PAMPA assay. The PPB of WN at 5 and 20 µg/mL concentrations were found to be high i.e. 82.01 ± 1.44 and 88.02 ± 1.15%, respectively. The in vitro half-life of WN in RLM and HLM was found to be 59.63 ± 2.50 and 68.42 ± 2.19 min, respectively. CYP phenotyping results showed that WN was extensively metabolized by CYP 3A4 and1A2 enzymes in RLM and HLM. However, the results of CYP Inhibition studies showed that none of the CYP isoenzymes were potentially inhibited by WN in RLM and HLM. CONCLUSION: The in vitro results of pH-dependent stability, plasma stability, permeability, PPB, blood partitioning, microsomal stability, CYP phenotyping, and CYP inhibition studies demonstrated that WN could be a better phytochemical for neurological disorders.

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.

Comparative transcriptome analysis to identify putative genes related to trichome development in Ocimum species.

Genus Ocimum is known to have species possessing important therapeutic essential oil. The major phytoconstituents of essential oil in Ocimum species are phenylpropanoids and terpenoids. The essential oil is accumulated in the trichomes; the specialized structures predominantly found on leaves and other tissues. The development of trichome is integrated with development of plant and leaf and also tightly coordinated with the primary and secondary metabolic pathways producing essential oil constituents. In continuation to our studies on elucidating/understanding the mechanism of biosynthesis of  essential oil pathways in Ocimum species, we have performed comparative transcriptome analysis to investigate the role of trichome-related gene expression in the regulation of biosynthetic pathways of essential oil. The essential oil biogenesis is tightly integrated with primary metabolic activities, the analysis for the expression pattern of genes related to primary metabolism and its relationship with secondary metabolism was evaluated in comparative manner. Physiological parameters in relation to primary metabolism such as photosynthetic pigment content, soluble sugar content, and invertase enzymes along with morphological parameters were analysed in O. basilicum and O. sanctum. Differential expression profiling uncovered about 8116 and 2810 differentially expressed transcripts in O. basilicum and O. sanctum, respectively. Enrichment of differentially expressed genes were analysed in relation to metabolic pathways, primary metabolism and secondary metabolism. Trichome related genes identified from the Ocimum species vis-à-vis their expression profiles suggested higher expression in O. basilicum. The findings in this study provide interesting insights into the role of trichome-related transcripts in relation to essential oil content in Ocimum species. The study is valuable as this is the first study on revealing the transcripts and their role in trichome development and essential oil biogenesis in two major species of Ocimum.

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.

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.

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.

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.

Interspecies comparative features of trichomes in Ocimum reveal insights for biosynthesis of specialized essential oil metabolites.

Ocimum species commonly referred to as "Tulsi" are well-known for their distinct medicinal and aromatic properties. The characteristic aroma of Ocimum species and cultivars is attributed to their specific combination of volatile phytochemicals mainly belonging to terpenoid and/or phenylpropanoid classes in their essential oils. The essential oil constituents are synthesized and sequestered in specialized epidermal secretory structures called as glandular trichomes. In this comparative study, inter- and intra-species diversity in structural attributes and profiles of expression of selected genes related to terpenoid and phenylpropanoid biosynthetic pathways have been investigated. This is performed to seek relationship of variations in the yield and phytochemical composition of the essential oils. Microscopic analysis of trichomes of O. basilicum, O. gratissimum, O. kilimandscharicum, and O. tenuiflorum (green and purple cultivars) revealed substantial variations in density, size, and relative proportions of peltate and capitate trichomes among them. The essential oil yield has been observed to be controlled by the population, dominance, and size of peltate and capitate glandular trichomes. The essential oil sequestration in leaf is controlled by the dominance of peltate glandular trichome size over its number and is also affected by the capitate glandular trichome size/number with variations in leaf area albeit at lower proportions. Comprehension and comparison of results of GC-MS analysis of essential oils showed that most of the Ocimum (O. basilicum, O. tenuiflorum, and O. gratissimum) species produce phenylpropanoids (eugenol, methyl chavicol) as major volatiles except O. kilimandscharicum, which is discrete in being monoterpenoid-rich species. Among the phenylpropanoid-enriched Ocimum (O. basilicum, O. gratissimum, O. tenuiflorum purple, O. tenuiflorum green) as well, terpenoids were important constituents in imparting characteristic aroma. Further, comparative abundance of transcripts of key genes of phenylpropanoid (PAL, C4H, 4CL, CAD, COMT, and ES) and terpenoid (DXS and HMGR) biosynthetic pathways was evaluated vis-à-vis volatile oil constituents. Transcript abundance demonstrated that richness of their essential oils with specific constituent(s) of a chemical group/subgroup was manifested by the predominant upregulation of phenylpropanoid/terpenoid pathway genes. The study provides trichomes as well as biosynthetic pathway-based knowledge for genetic improvement in Ocimum species for essential oil yield and quality.

HMG-CoA reductase from Camphor Tulsi (Ocimum kilimandscharicum) regulated MVA dependent biosynthesis of diverse terpenoids in homologous and heterologous plant systems.

Ocimum kilimandscharicum is unique in possessing terpenoids whereas other Ocimum species are renowned for phenylpropanoids as major constituents of essential oil. The key enzyme of MVA/terpenoid metabolic pathway viz 3-hydroxy-3-methylglutaryl Co-A reductase (OkHMGR) of 1.7-Kb ORF encoding ~60-kDa protein was cloned from O. kilimandscharicum and its kinetic characteristics revealed the availability of HMG-CoA as a control point of MVA-pathway. Transcript profiling of the OkHMGR elucidated tissue-specific functions of the gene in flower and leaf tissues in accumulation of terpenoidal essential oil. OkHMGR was differentially regulated in response to exposure to methyl-jasmonate, salicylic-acid, and stress conditions such-as salt and temperature stress, demonstrating its key role in managing signaling and stress-responses. To elucidate its functional role, OkHMGR was transiently over-expressed in homologous and heterologous plants such as O. sanctum, O. basilicum, O. gratissimum, Withania somnifera and Artemisia annua. The over-expression and inhibition dual strategy revealed that the additional OkHMGR in-planta could afford endogenous flow of isoprenoid units towards synthesis of terpenoids. The present study provides in-depth insight of OkHMGR in regulation of biosynthesis of non-plastidal isoprenoids. This is first report on any gene of MVA/isoprenoid pathway from under-explored Camphor Tulsi belonging to genus Ocimum. Studies also suggested that OkHMGR could be a potential tool for attempting metabolic engineering for enhancing medicinally important terpenoidal metabolites in plants.

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.