Phytochemical Diversity in Relation to Cytogenetic Variability in Inula racemosa Hook.f., an Endangered Medicinal Plant of Himalayas.

Inula racemosa, a resourceful critically endangered medicinal herb in the Himalayas is traditionally utilized to cure various human disorders. The species is a wealthy source of sesquiterpene lactones has many pharmacological properties. To quantify and identify the best genetic stocks for a maximum build-up of desired metabolites (isoalantolactone and alantolactone) among existent cytotypes in the species, LC-MS/MS analysis was made. The other comprehensive experiments carried out at present included detailed meiotic examinations of different populations collected from different areas of Kashmir Himalayas. The results presented the occurrence of variable chromosome numbers as n=10 and n=20 in different populations, but the tetraploid cytotype (n=20) is new for the species. The LC-MS/MS investigation revealed significant variability in the content of sesquiterpene lactones in different plant tissues (stem, leaf, and root). An upsurge in the quantity of isoalantolactone and alantolactone was noticed with increasing ploidy levels along the increasing altitudes. Therefore, a habit to accumulate abundant quantities of secondary metabolites and increased adaptability by species/cytotypes thriving at higher altitudes is seen among tetraploid cytotypes during the present investigation. Also, the chromosomal variations seem to enhance the flexibility of polyploid species primarily at upper elevations. Thus, the present study strongly provides quantification of elite cytotypes/chemotypes with optimum concentration of secondary metabolites.

Comparative transcriptome mining for terpenoid biosynthetic pathway genes in wild and cultivated species of Plantago.

Plantagos are important economical and medicinal plants that possess several bioactive secondary metabolites, such as phenolics, iridoids, triterpenes, and alkaloids. Triterpenoids are the ubiquitous and dynamic secondary metabolites that are deployed by plants for chemical interactions and protection under biotic/abiotic stress. Plantago ovata, a cultivated species, is the source of psyllium, while Plantago major, a wild species, has significant therapeutic potential. Wild species are considered more tolerant to stressful conditions in comparison to their cultivated allies. In view of this, the present study aimed to decipher the terpenoid biosynthetic pathway operative in P. ovata and P. major using a comparative transcriptomics approach. Majority of terpenoid biosynthetic genes were observed as upregulated in P. major including rate limiting genes of MVA (HMGR) and MEP (DXR) pathways and genes (α-AS, BAS, SM, and CYP716) involved in ursolic acid biosynthesis, an important triterpenoid prevalent in Plantago species. The HPLC output further confirmed the higher concentration of ursolic acid in P. major as compared to P. ovata leaf samples, respectively. In addition to terpenoid biosynthesis, KEGG annotation revealed the involvement of differentially expressed unigenes in several metabolic pathways, aminoacyl-tRNA biosynthesis, biosynthesis of antibiotics, and biosynthesis of secondary metabolites. MYB was found as the most abundant transcription factor family in Plantago transcriptome. We have been able to generate valuable information which can help in improving terpenoid production in Plantago. Additionally, the present study has laid a strong foundation for deciphering other important metabolic pathways in Plantago.

In-vitro cytotoxicity in relation to chemotypic diversity in diploid and tetraploid populations of Gentiana kurroo Royle.

ETHNOPHARMACOLOGICAL RELEVANCE: Gentiana kurroo is a multipurpose critically endangered medicinal herb prescribed as medicine in Ayurveda in India and exhibits various pharmacological properties including anti-cancer activity. The species is rich repository of pharmacologically active secondary metabolites together with secoiridoidal glycosides. AIM OF THE STUDY: The study aimed to investigate the chemical diversity in different populations/cytotypes prevailing in G. kurroo to identify elite genetic stocks in terms of optimum accumulation/biosynthesis of desired metabolites and having higher in-vitro cytotoxicity potential in relation to chemotypic diversity. MATERIAL AND METHODS: The wild plants of the species were collected from different ranges of altitudes from the Kashmir Himalayas. For cytological evaluation, the standard meiotic analysis was performed. The standard LC-MS/MS technique was employed for phytochemical analysis based on different marker compounds viz. sweroside, swertiamarin, and gentiopicroside. Different tissues such as root-stock, aerial parts, and flowers were used for chemo-profiling. Further, the methanolic extracts of diploid and tetraploid cytotypes were assessed for cytotoxic activity by using MTT assay against four different human cancer cell lines. RESULTS: The quantification of major bioactive compounds based on tissue- and location-specific comparison, as well as in-vitro cytotoxic potential among extant cytotypes, was evaluated. The comprehensive cytomorphological studies of the populations from NW Himalayas revealed the occurrence of different chromosomal races viz. n = 13, 26. The tetraploid cytotype was hitherto unreported. The tissue-specific chemo-profiling revealed relative dominance of different phytoconstituents in root-stock. There was a noticeable increase in the quantity of the analyzed compounds in relation to increasing ploidy status along the increasing altitudes. The MTT assay of methanolic extracts of diploid and tetraploid cytotypes displayed significant cytotoxicity potential in tetraploids. The root-stock extracts of tetraploids were highly active extracts with IC50 value ranges from 5.65 to 8.53 µg/mL against HCT-116 colon cancer. CONCLUSION: The chemical evaluation of major bioactive compounds in diverse cytotypes from different plant parts along different altitudes presented an appreciable variability in sweroside, swertiamarin, and gentiopicroside contents. Additionally, the concentrations of these phytoconstituents varied for cytotoxicity potential among different screened cytotypes. This quantitative difference of active bio-constituents was in correspondence with the growth inhibition percentage of different tested cancer cell lines. Thus, the present investigation strongly alludes towards a prognostic approach for the identification of elite cytotypes/chemotypes with significant pharmacological potential.

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.

Comprehensive transcriptome analysis provides insights into metabolic and gene regulatory networks in trichomes of Nicotiana tabacum.

KEY MESSAGE: Comprehensive transcriptome analysis suggested that the primary metabolism is modulated to augment the supply of substrates towards secondary metabolism operating in the glandular trichomes of Nicotiana tabacum. The comparative gene expression and co-expression network analysis revealed that certain members of transcription factor genes belonging to the MYB, HD-ZIP, ERF, TCP, SRS, WRKY and DOF families may be involved in the regulation of metabolism and/other aspects in the glandular trichomes of N. tabacum The glandular trichomes of Nicotiana tabacum are highly productive in terms of secondary metabolites and therefore have been projected to be used as a prognostic platform for metabolic engineering of valuable natural products. For obvious reasons, detailed studies pertaining to the metabolic and gene regulatory networks operating in the glandular trichomes of N. tabacum are of pivotal significance to be undertaken. We have carried out next-generation sequencing of glandular trichomes of N. tabcaum and investigated differential gene expression among different tissues, including trichome-free leaves. We identified a total of 37,269 and 37,371 genes, expressing in trichome free leaf and glandular trichomes, respectively, at a cutoff of FPKM ≥ 1. The analysis revealed that different pathways involved with the primary metabolism are modulated in glandular trichomes of N. tabacum, providing a plausible explanation for the enhanced biosynthesis of secondary metabolism in the glandular trichomes. Further, comparative gene expression analysis revealed several genes, which display preferential expression in the glandular trichomes and thereby seem to be potential candidate genes for future studies in connection to the discovery of novel trichome specific promoters. The present study also led to the comprehensive identification of 1750 transcription factor genes expressing at a cutoff of FPKM ≥ 1 in the glandular trichomes of N. tabacum. The clustering and co-expression analysis suggested that transcription factor genes belonging to HD-ZIP, ERF, WRKY, MYB, TCP, SRS and DOF families may be the major players in the regulation of gene expression in the glandular trichomes of N. tabacum. To the best of our knowledge, the present work is the first effort towards detailed identification of genes, especially regulatory genes expressing in the glandular trichomes of N. tabacum. The data resource and the empirical findings from present work in all probability must, therefore, provide a reference and background context for future work aiming at deciphering molecular mechanism of regulation of secondary metabolism and gene expression in the glandular trichomes of N. tabacum.

Molecular characterization and overexpression analyses of secologanin synthase to understand the regulation of camptothecin biosynthesis in Nothapodytes nimmoniana (Graham.) Mabb.

Camptothecin is a high-value anti-cancerous compound produced in many taxonomically unrelated species. Its biosynthesis involves a complex network of pathways and a diverse array of intermediates. Here, we report the functional characterization and regulation of secologanin synthase (NnCYP72A1), a cytochrome P450 involved in camptothecin biosynthesis from Nothapodytes nimmoniana. It comprises an open reading frame of 1566 bp in length. Heterologous expression in Saccharomyces cerevisiae and in vitro enzymatic assays using loganin as substrate confirmed the formation of secologanin. In planta transient overexpression analysis of NnCYP72A1 resulted in 4.21- and 2.73-fold increase in transcript levels of NnCYP72A1 on days 3 and 6 respectively. Phytochemical analysis of transformed tissues revealed ~ 1.13-1.43- and 2.02-2.86-fold increase in secologanin and CPT accumulation, respectively. Furthermore, promoter analysis of NnCYP72A1 resulted in the identification of several potential cis-regulatory elements corresponding to different stress-related components. Methyl jasmonate, salicylic acid, and wounding treatments resulted in considerable modulation of mRNA transcripts of NnCYP72A1 gene. Chemical analysis of elicitor-treated samples showed a significant increase in CPT content which was concordant with the mRNA transcript levels. Overall, the functional characterization and overexpression of NnCYP72A1 may plausibly enhance the pathway intermediates and serve as prognostic tool for enhancing CPT accumulation.

Metabolic and transcriptional analyses in response to potent inhibitors establish MEP pathway as major route for camptothecin biosynthesis in Nothapodytes nimmoniana (Graham) Mabb.

BACKGROUND: Nothapodytes nimmoniana, a plant of pivotal medicinal significance is a source of potent anticancer monoterpene indole alkaloid (MIA) camptothecin (CPT). This compound owes its potency due to topoisomerase-I inhibitory activity. However, biosynthetic and regulatory aspects of CPT biosynthesis so far remain elusive. Production of CPT is also constrained due to unavailability of suitable in vitro experimental system. Contextually, there are two routes for the biosynthesis of MIAs: the mevalonate (MVA) pathway operating in cytosol and the methylerythritol phosphate (MEP) pathway in the plastids. Determination of relative precursor flux through either of these pathways may provide a new vista for manipulating the enhanced CPT production. RESULTS: In present study, specific enzyme inhibitors of MVA (lovastatin) and MEP pathways (fosmidomycin) were used to perturb the metabolic flux in N. nimmoniana. Interaction of both these pathways was investigated at transcriptional level by using qRT-PCR and at metabolite level by evaluating secologanin, tryptamine and CPT contents. In fosmidomycin treated plants, highly significant reduction was observed in both secologanin and CPT accumulation in the range 40-57% and 64-71.5% respectively, while 4.61-7.69% increase was observed in tryptamine content as compared to control. Lovastatin treatment showed reduction in CPT (7-11%) and secologanin (7.5%) accumulation while tryptamine registered slight increase (3.84%) in comparison to control. These inhibitor mediated changes were reflected at transcriptional level via altering expression levels of deoxy-xylulose-5-phosphate reductoisomerase (DXR) and hydroxymethylglutaryl-CoA reductase (HMG). Further, mRNA expression of four more genes downstream to DXR and HMG of MEP and MVA pathways respectively were also investigated. Expression analysis also included secologanin synthase (SLS) and strictosidine synthase (STR) of seco-iridoid pathway. Present investigation also entailed development of an efficient in vitro multiplication system as a precursor to pathway flux studies. Further, a robust Agrobacterium-mediated transformed hairy root protocol was also developed for its amenability for up-scaling as a future prospect. CONCLUSIONS: Metabolic and transcriptional changes reveal differential efficacy of cytosolic and plastidial inhibitors in context to pathway flux perturbations on seco-iridoid end-product camptothecin. MEP pathway plausibly is the major precursor contributor towards CPT production. These empirical findings allude towards developing suitable biotechnological interventions for enhanced CPT production.

Gene Silencing and Over-Expression Studies in Concurrence With Promoter Specific Elicitations Reveal the Central Role of WsCYP85A69 in Biosynthesis of Triterpenoids in Withania somnifera (L.) Dunal.

Withania somnifera (Ashwagandha) synthesizes a wide spectrum of triterpenoids that are produced via an intricate isoprenoid pathway whose biosynthetic and regulatory mechanism remains elusive. Their pharmacological examination position them as potent bioactive molecules, hence demanding their copious production. Previous investigations have revealed that P450 monooxygenases are pivotal enzymes involved in the biosynthetic machinery of various metabolites and assist in decorating their core skeletal structures. The present study entails the isolation and functional characterization of castasterone synthase (CYP85A69) from W. somnifera. The full length WsCYP85A69, having an open reading frame of 1413 bp, encodes 470 amino acid residues. Further, in vitro conversion of 6-deoxocastasterone into castasterone validated its oxidative functionality. Product formation was confirmed using LC-PDA-MS with a m/z value of 506 [M+ACN]+. In planta transient over-expression of WsCYP85A69 significantly enhanced castasterone, stigmasterol and withanolides (WS-I, WS-II, WS-III). Artificial micro-RNA mediated silencing of WsCYP85A69 resulted in the reduced accumulation of castasterone, stigmasterol and withanolides (WS-I, WS-II, WS-III). Altogether, these non-complementary approaches plausibly suggest a key role of WsCYP85A69 in the biosynthesis of castasterone and the accumulation of withanolides and stigmasterol. Furthermore, a promoter analysis of WsCYP85A69 resulted in the identification of several potential cis-regulatory elements. Elicitations, given on the basis of identified cis-regulatory elements, demonstrated methyl jasmonate as an effective inducer of WsCYP85A69. Overall, these empirical findings suggest that functional characterization of WsCYP85A69 may conceivably be helpful to unravel the mechanism of brassinosteroids biosynthesis and could also pave the way for targeted metabolic engineering.

Jasmonate responsive transcription factor WsMYC2 regulates the biosynthesis of triterpenoid withanolides and phytosterol via key pathway genes in Withania somnifera (L.) Dunal.

KEY MESSAGE: Functional characterization of WsMYC2 via artificial microRNA mediated silencing and transient over-expression displayed significant regulatory role vis-à-vis withanolides and stigmasterol biosyntheses in Withania somnifera. Further, metabolic intensification corroborated well with higher expression levels of putative pathway genes. Additionally, copious expression of WsMYC2 in response to exogenous elicitors resulted in enhanced withanolides production. Withania somnifera, a high value multipurpose medicinal plant, is a rich reservoir of structurally diverse and biologically active triterpenoids known as withanolides. W. somnifera has been extensively pursued vis-à-vis pharmacological and chemical studies. Nonetheless, there exists fragmentary knowledge regarding the metabolic pathway and the regulatory aspects of withanolides biosynthesis. Against this backdrop, a jasmonate-responsive MYC2 transcription factor was identified and functionally characterized from W. somnifera. In planta transient over-expression of WsMYC2 showed significant enhancement of mRNA transcript levels which corroborated well with the enhanced content of withanolides and stigmasterol. Further, a comparative analysis of expression levels of some of the genes of triterpenoid pathway viz. WsCAS, WsCYP85A, WsCYP90B and WsCYP710A in corroboration with the over-expression and silencing of WsMYC2 suggested its positive influence on their regulation. These corroboratory approaches suggest that WsMYC2 has cascading effect on over-expression of multiple pathway genes leading to the increased triterpenoid biosynthesis in infiltered plants. Further, the functional validation of WsMYC2 was carried out by artificial micro-RNA mediated silencing. It resulted in significant reduction of withanolides and stigmasterol levels, indicative of crucial role of WsMYC2 in the regulation of their biosyntheses. Taken together, these non-complementary approaches provided unambiguous understanding of the regulatory role of WsMYC2 in context to withanolides and stigmasterol biosyntheses. Furthermore, the upstream promoter of WsMYC2 presented several cis-regulatory elements primarily related to phytohormone responsiveness. WsMYC2 displayed inducible nature in response to MeJA. It had substantial influence on the higher expression of WsMYC2 which was in consonance with enhanced accumulation of withanolides.

Rheum australe, an endangered high-value medicinal herb of North Western Himalayas: a review of its botany, ethnomedical uses, phytochemistry and pharmacology.

Rheum australe (Himalayan Rhubarb) is a multipurpose, endemic and endangered medicinal herb of North Western Himalayas. It finds extensive use as a medicinal herb since antiquity in different traditional systems of medicine to cure a wide range of ailments related to the circulatory, digestive, endocrine, respiratory and skeletal systems as well as to treat various infectious diseases. The remedying properties of this plant species are ascribed to a set of diverse bioactive secondary metabolite constituents, particularly anthraquinones (emodin, chrysophanol, physcion, aloe-emodin and rhein) and stilbenoids (piceatannol, resveratrol), besides dietary flavonoids known for their putative health benefits. Recent studies demonstrate the pharmacological efficacy of some of these metabolites and/or their derivatives as lead molecules for the treatment of various human diseases. Present review comprehensively covers the literature available on R. australe from 1980 to early 2018. The review provides up-to-date information available on its botany for easy identification of the plant, and origin and historical perspective detailing its trade and commerce. Distribution, therapeutic potential in relation to traditional uses and pharmacology, phytochemistry and general biosynthesis of major chemical constituents are also discussed. Additionally, efficient and reproducible in vitro propagation studies holding vital significance in preserving the natural germplasm of the plant and for its industrial exploitation have also been highlighted. The review presents a detailed perspective for future studies to conserve and sustainably make use of this endangered plant species at a commercial scale.

De novo transcriptome analyses reveals putative pathway genes involved in biosynthesis and regulation of camptothecin in Nothapodytes nimmoniana (Graham) Mabb.

KEY MESSAGE: Comprehensive transcriptome analysis of leaf and root tissues of Nothapodytes nimmoniana unravels several putative pathway genes, transcription factors and CYPs related to camptothecin (CPT) biosynthesis. Additionally, post-transcriptional suppression by artificial microRNA (aMIR) of NnCYP76B6 (geraniol 10-hydroxylase) suggests its role in CPT biosynthesis. Tissue-specific LC-MS/MS analysis revealed the presence of secologanin as the central intermediate of MIA pathway in N. nimmoniana. Nothapodytes nimmoniana is a rich source of potent anticancer drug camptothecin (CPT) whose biosynthetic pathway is unresolved due to the lack of genomic and transcriptomic information. Present investigation entails deep transcriptome analysis of N. nimmoniana which led to identification of putative pathway genes and regulatory components involved in CPT biosynthesis. Using Illumina HiSeq 2500 sequencing platform a total of 31,172,889 (6.23 Gb) and 31,218,626 (6.24 Gb) raw reads were generated from leaf and root wood, respectively. These were assembled de novo into 138,183 unique contigs. Additionally, 16 cytochrome P450 transcripts related to secondary metabolism were also identified. Further, transcriptome data pool presented 1683 putative transcription factors of which transcripts corresponding to WRKY TFs were the most abundant (14.14%). A total of 2741 transcripts were differentially expressed out of which 478 contigs showed downregulation in root wood and 2263 contigs were up-regulated. Further, comparative analyses of 17 genes involved in CPT biosynthetic pathway were validated by qRT-PCR. On basis of intermediates, two distinct seco-iridoid pathways are involved in the biosynthesis of monoterpene indole alkaloids either through multiple isomers of strictosidinic acid or strictosidine. Tissue-specific LC-MS/MS analysis revealed the presence of secologanin as the central intermediate of MIA pathway in N. nimmoniana. Geraniol-10 hydroxylase (NnCYP76B6) an important enzyme in CPT biosynthesis which specifically shunts geraniol into the secologanin pathway was also cloned from the trancriptome resource. In planta transient expression of NnCYP76B6 showed a significant enhancement in mRNA transcript levels coincident with enhanced CPT accumulation. Further, artificial microRNA (aMIR) mediated downregulation of NnCYP76B6 resulted in reduction of mRNA transcript levels as well as CPT content in comparison to control. These empirical results suggest a plausible regulatory role for NnCYP76B6 in CPT biosynthesis and also establish a valuable repository for deciphering various structural, rate limiting and regulatory genes of CPT biosynthetic pathway.

Molecular and functional characterization of two isoforms of chalcone synthase and their expression analysis in relation to flavonoid constituents in Grewia asiatica L.

Chalcone synthase constitutes a functionally diverse gene family producing wide range of flavonoids by catalyzing the initial step of the phenylpropanoid pathway. There is a pivotal role of flavonoids in pollen function as they are imperative for pollen maturation and pollen tube growth during sexual reproduction in flowering plants. Here we focused on medicinally important fruit-bearing shrub Grewia asiatica. It is a rich repository of flavonoids. The fruits are highly acclaimed for various putative health benefits. Despite its importance, full commercial exploitation is hampered due to two drawbacks which include short shelf life of its fruits and larger seed volume. To circumvent these constraints, seed abortion is one of the viable options. Molecular interventions tested in a number of economic crops have been to impair male reproductive function by disrupting the chalcone synthase (CHS) gene activity. Against this backdrop the aim of the present study included cloning and characterization of two full-length cDNA clones of GaCHS isoforms from the CHS multigene family. These included GaCHS1 (NCBI acc. KX129910) and GaCHS2 (NCBI acc. KX129911) with an ORF of 1176 and 1170 bp, respectively. GaCHSs were heterologously expressed and purified in E. coli to validate their functionality. Functionality of CHS isoforms was also characterized via enzyme kinetic studies using five different substrates. We observed differential substrate specificities in terms of their Km and Vmax values. Accumulation of flavonoid constituents naringenin and quercetin were also quantified and their relative concentrations corroborated well with the expression levels of GaCHSs. Further, our results demonstrate that GaCHS isoforms show differential expression patterns at different reproductive phenological stages. Transcript levels of GaCHS2 were more than its isoform GaCHS1 at the anthesis stage of flower development pointing towards its probable role in male reproductive maturity.

Genoproteomics-assisted improvement of Andrographis paniculata: toward a promising molecular and conventional breeding platform for autogamous plants affecting the pharmaceutical industry.

Andrographis paniculata (Burm. f.) Wall. ex Nees. (AP) is a hermaphroditic, self-compatible, and habitual inbreeding plant. Its main bioactive component is andrographolide, which is capable of inducing autophagic cell death in some human cancer cells and helps fight HIV/AIDS. Increasing the andrographolide content by investigating the genetic mechanisms controlling its biosynthesis in order to improve and develop high-yielding cultivars are the main breeding targets for AP. However, there might exist some limitations or barriers for crossability within AP accessions. Recently, this problem was addressed in AP by using a combination of crossbreeding and biotechnology-aided genetic methods. This review emphasizes that development of a breeding platform in a hard-to-breed plant, such as AP, requires the involvement of a broad range of methods from classical genetics to molecular breeding. To this end, a phenological stage (for example, flowering and stigma development) can be simplified to a quantitative morphological trait (for example, bud or stigma length) to be used as an index to express the highest level of receptivity in order to manage outcrossing. The outcomes of the basic crossability research can be then employed in diallel mating and crossbreeding. This review explains how genomic data could produce useful information regarding genetic distance and its influence on the crossability of AP accessions. Our review indicates that co-dominant DNA markers, such as microsatellites, are also capable of resolving the evolutionary pathway and cryptic features of plant populations and such information can be used to select the best breeding strategy. This review also highlights the importance of proteomic analysis as a breeding tool. In this regard, protein diversification, as well as the impact of normal and stress-responsive proteins on morphometric and physiological behaviors, could be used in breeding programs. These findings have immense potential for improving plant production and, therefore, can be regarded as prospective breeding platforms for medicinal plants that have an autogamous mode of reproduction. Finally, this review suggests that novel site-directed genome editing approaches such as TALENs (Transcription Activator-Like Effector Nucleases) and CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein-9 nuclease) systems together with other new plant breeding technologies (NPBT) should simultaneously be taken into consideration for improvement of pharmaceutical plants.

Functional Promiscuity of Two Divergent Paralogs of Type III Plant Polyketide Synthases.

Plants effectively defend themselves against biotic and abiotic stresses by synthesizing diverse secondary metabolites, including health-protective flavonoids. These display incredible chemical diversity and ubiquitous occurrence and confer impeccable biological and agricultural applications. Chalcone synthase (CHS), a type III plant polyketide synthase, is critical for flavonoid biosynthesis. It catalyzes acyl-coenzyme A thioesters to synthesize naringenin chalcone through a polyketidic intermediate. The functional divergence among the evolutionarily generated members of a gene family is pivotal in driving the chemical diversity. Against this backdrop, this study was aimed to functionally characterize members of the CHS gene family from Rheum emodi, an endangered and endemic high-altitude medicinal herb of northwestern Himalayas. Two full-length cDNAs (1,179 bp each), ReCHS1 and ReCHS2, encoding unique paralogs were isolated and characterized. Heterologous expression and purification in Escherichia coli, bottom-up proteomic characterization, high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry analysis, and enzyme kinetic studies using five different substrates confirmed their catalytic potential. Phylogenetic analysis revealed the existence of higher synonymous mutations in the intronless divergents of ReCHS. ReCHS2 displayed significant enzymatic efficiency (Vmax/Km) with different substrates. There were significant spatial and altitudinal variations in messenger RNA transcript levels of ReCHSs correlating positively with metabolite accumulation. Furthermore, the elicitations in the form of methyl jasmonate, salicylic acid, ultraviolet B light, and wounding, chosen on the basis of identified cis-regulatory promoter elements, presented considerable differences in the transcript profiles of ReCHSs. Taken together, our results demonstrate differential propensities of CHS paralogs in terms of the accumulation of flavonoids and their relative substrate selectivities.

A Decade of Molecular Understanding of Withanolide Biosynthesis and In vitro Studies in Withania somnifera (L.) Dunal: Prospects and Perspectives for Pathway Engineering.

Withania somnifera, a multipurpose medicinal plant is a rich reservoir of pharmaceutically active triterpenoids that are steroidal lactones known as withanolides. Though the plant has been well-characterized in terms of phytochemical profiles as well as pharmaceutical activities, limited attempts have been made to decipher the biosynthetic route and identification of key regulatory genes involved in withanolide biosynthesis. This scenario limits biotechnological interventions for enhanced production of bioactive compounds. Nevertheless, recent emergent trends vis-à-vis, the exploration of genomic, transcriptomic, proteomic, metabolomics, and in vitro studies have opened new vistas regarding pathway engineering of withanolide production. During recent years, various strategic pathway genes have been characterized with significant amount of regulatory studies which allude toward development of molecular circuitries for production of key intermediates or end products in heterologous hosts. Another pivotal aspect covering redirection of metabolic flux for channelizing the precursor pool toward enhanced withanolide production has also been attained by deciphering decisive branch point(s) as robust targets for pathway modulation. With these perspectives, the current review provides a detailed overview of various studies undertaken by the authors and collated literature related to molecular and in vitro approaches employed in W. somnifera for understanding various molecular network interactions in entirety.

Secalonic Acid-D Represses HIF1α/VEGF-Mediated Angiogenesis by Regulating the Akt/mTOR/p70S6K Signaling Cascade.

Tumor angiogenesis is a validated target for therapeutic intervention, but agents that are more disease selective are needed. Here, we report the isolation of secalonic acid-D (SAD), a mycotoxin from a novel source that exhibits potent antiangiogenic antitumor activity. SAD inhibited multiple HIF1α/VEGF-arbitrated angiogenesis dynamics as scored in human umbilical vascular endothelial cells and human MCF-7 breast tumor xenografts. Similarly, SAD suppressed VEGF-induced microvessel sprouting from rat aortic ring and blood vessel formation in the Matrigel plug assay in C57/BL6J mice. Under normoxic or hypoxic conditions, SAD inhibited cell survival through the Akt/mTOR/p70S6K pathway, with attendant effects on key proangiogenesis factors, including HIF1α, VEGFR, and MMP-2/MMP-9. These effects were reversed by cotreatment with the Akt inhibitors perifosine and GSK69069 or by the addition of neutralizing VEGF antibodies. The apoptotic properties of SAD were determined to be both extrinsic and intrinsic in nature, whereas the cell-cycle inhibitory effects were mediated by altering the level of key G1-S transition-phase proteins. In experimental mouse models of breast cancer, SAD dosing produced no apparent toxicities (either orally or intraperitoneal) at levels that yielded antitumor effects. Taken together, our findings offered a preclinical validation and mechanistic definition of the antiangiogenic activity of a novel mycotoxin, with potential application as a cancer-selective therapeutic agent.

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.

A phenylalanine ammonia-lyase ortholog (PkPAL1) from Picrorhiza kurrooa Royle ex. Benth: molecular cloning, promoter analysis and response to biotic and abiotic elicitors.

Picrorhiza kurrooa Royle ex Benth. is a highly reputed medicinal herb utilised in the preparation of a number of herbal drug formulations, principally due to the presence of novel monoterpene iridoid glycosides kenned as picrosides. Phenylalanine ammonia-lyase catalyses an important rate-limiting step in phenylpropanoid pathway and supplies precursors like cinnamic acid, vanillic acid, ferulic acid, etc., to a variety of secondary metabolites including picrosides. The imperilled status of P. kurrooa coupled with lack of information regarding biogenesis of picrosides necessitates deciphering the biosynthetic pathway for picrosides. In the present study, a PAL gene, designated PkPAL1 was isolated from P. kurrooa. The cDNA is 2312 bp in length, consisting of an ORF of 2142 bp encoding for a 713 amino acid protein having a predicted molecular weight of 77.66 kDa and an isoelectric point of pH 6.82. qRT-PCR analysis of various tissues of P. kurrooa showed that PkPAL1 transcript levels were highest in the leaves, consistent with picroside accumulation pattern. Using Genome walking, a 718 bp promoter region was also isolated resulting in identification of distinct cis-regulatory elements including TGA-element, TGACG-motif, CGTCA-motif, etc. qRT-PCR indicated up-regulation of PkPAL1 by methyl jasmonate, salicylic acid, 2,4-dicholorophenoxy acetic acid and UV-B elicitations that corroborated positively with the identified cis-elements within the promoter region. Moreover, altitude was found to have a positive effect on the PkPAL1 transcript levels, driving the expression of PkPAL1 abundantly. Based on docking analysis, we identified eight residues as potentially essential for substrate binding in PkPAL1.

Cloning and functional characterization of three branch point oxidosqualene cyclases from Withania somnifera (L.) dunal.

Oxidosqualene cyclases (OSCs) positioned at a key metabolic subdividing junction execute indispensable enzymatic cyclization of 2,3-oxidosqualene for varied triterpenoid biosynthesis. Such branch points present favorable gene targets for redirecting metabolic flux toward specific secondary metabolites. However, detailed information regarding the candidate OSCs covering different branches and their regulation is necessary for the desired genetic manipulation. The aim of the present study, therefore, was to characterize members of OSC superfamily from Withania somnifera (Ws), a medicinal plant of immense repute known to synthesize a large array of biologically active steroidal lactone triterpenoids called withanolides. Three full-length OSC cDNAs, ß-amyrin synthase (WsOSC/BS), lupeol synthase (WsOSC/LS), and cycloartenol synthase (WsOSC/CS), having open reading frames of 2289, 2268, and 2277 bp, were isolated. Heterologous expression in Schizosaccharomyces pombe, LC-MS analyses, and kinetic studies confirmed their monofunctionality. The three WsOSCs were found to be spatially regulated at transcriptional level with WsOSC/CS being maximally expressed in leaf tissue. Promoter analysis of three WsOSCs genes resulted in identification of distinct cis-regulatory elements. Further, transcript profiling under methyl jasmonate, gibberellic acid, and yeast extract elicitations displayed differential transcriptional regulation of each of the OSCs. Changes were also observed in mRNA levels under elicitations and further substantiated with protein expression levels by Western blotting. Negative regulation by yeast extract resulted in significant increase in withanolide content. Empirical evidence suggests that repression of competitive branch OSCs like WsOSC/BS and WsOSC/LS possibly leads to diversion of substrate pool toward WsOSC/CS for increased withanolide production.

An inducible NADPH-cytochrome P450 reductase from Picrorhiza kurrooa - an imperative redox partner of cytochrome P450 enzymes.

Picrorhiza kurrooa synthesizes a large array of pharmacologically important monoterpenoid iridoid glycosides called picrosides. Although chemical profile and pharmacological activities of P. kurrooa have been extensively studied, limited attempts have been made to decipher the biosynthetic route and to identify the key regulatory genes involved in picroside biosynthesis. In the present study, NADPH-cytochrome P450 reductase, a key enzyme involved in electron transfer to cytochrome P450s was identified from P. kurrooa. The full length cDNA (2679 bp) contained an open reading frame of 2133 bp, corresponding to 710 amino acids. PkCPR was heterologously expressed in Escherichia coli and the kinetic parameters of the recombinant enzyme were determined. Specific activity, V max and K m of PkCPR were found to be 5.8 ± 0.05 µmol min(-1) mg(-1), 8.1 ± 0.12 µmol min(-1) mg(-1) and 7.8 µM, respectively. PkCPR was found to be spatially regulated at transcript level, being maximally expressed in leaf tissues. Altitude was found to have a positive effect on the picroside concentration and the picroside content positively correlated with the PkCPR transcript levels in samples collected at varied altitudes. Further, transcript profiling under methyl jasmonate, salicylic acid, 2,4-dicholorophenoxy acetic acid and UV-B elicitations displayed differential transcriptional regulation of PkCPR that fully corroborated with the identified cis-elements within the PkCPR promoter. Expression of PkCPR was inducible by UV-B and phytohormone elicitation, indicating that the PkCPR is possibly related to defence reactions, including biosynthesis of secondary metabolites. Present study is so far the only report of identification and functional characterization of CPR ortholog from P. kurrooa.