Green synthesis and biological evaluation of glaucanic acid and dihydrocompactin acid by endophytic fungus Penicillium polonicum from Zingiber officinale.

Fungal endophytes are valued for biosynthesizing chemically diverse metabolic cascade with interesting biological activities. In the current investigation, two compounds were isolated from Penicillium polonicum, an endophyte of Zingiber officinale. The active moieties, glaucanic acid (1) and dihydrocompactin acid (2) were isolated from the ethyl acetate extract of P. polonicum and characterized by NMR and mass spectroscopy. Further, bioactive potential of the isolated compounds was evaluated by antimicrobial, antioxidant and cytotoxicity assays. Compounds 1 and 2 displayed antifungal activity against phytopathogen Colletotrichum gloeosporioides with more than 50% reduction in its growth. Both the compounds exhibited antioxidant activity against free radicals (DPPH and ABTS) and cytotoxicity activity against cancer cell lines respectively. The compounds, glaucanic acid and dihydrocompactin acid are being reported for the first time from an endophytic fungus. This is the first report on the biological activities of Dihydrocompactin acid produced by endophytic fungal strain.

Fungal endophyte bioinoculants as a green alternative towards sustainable agriculture.

Over the past half century, limited use of synthetic fertilizers, pesticides, and conservation of the environment and natural resources have become the interdependent goals of sustainable agriculture. These practices support agriculture sustainability with less environmental and climatic impacts. Therefore, there is an upsurge in the need to introduce compatible booster methods for maximizing net production. The best straightforward strategy is to explore and utilize plant-associated beneficial microorganisms and their products. Bioinoculants are bioformulations consisting of selected microbial strains on a suitable carrier used in the enhancement of crop production. Fungal endophytes used as bioinoculants confer various benefits to the host, such as protection against pathogens by eliciting immune response, mineralization of essential nutrients, and promoting plant growth. Besides, they also produce various bioactive metabolites, phytohormones, and volatile organic compounds. To design various bioformulations, transdisciplinary approaches like genomics, transcriptomics, metabolomics, proteomics, and microbiome modulation strategies like gene editing and metabolic reconstruction have been explored. These studies will refine the existing knowledge on the diversity, phylogeny and beneficial traits of the microbes. This will also help in synthesizing microbial consortia by evaluating the role of structural and functional elements of communities in a controlled manner. The present review summarizes the beneficial aspects associated with fungal endophytes for capitalizing agricultural outputs, enlists various multi-omics techniques for understanding and modulating the mechanism involved in endophytism and the generation of new bioformulations for providing novel solutions for the enhancement of crop production.

Allium cytogenetics: a critical review on the Indian taxa.

The genus Allium Linnaeus, 1753 (tribe Allieae) contains about 800 species worldwide of which almost 38 species are reported in India, including the globally important crops (onion, garlic, leek, shallot) and many wild species. A satisfactory chromosomal catalogue of Allium species is missing which has been considered in the review for the species occurring in India. The most prominent base number is x=8, with few records of x=7, 10, 11. The genome size has sufficient clues for divergence, ranging from 7.8 pg/1C to 30.0 pg/1C in diploid and 15.16 pg/1C to 41.78 pg/1C in polyploid species. Although the karyotypes are seemingly dominated by metacentrics, substantial variation in nucleolus organizing regions (NORs) is noteworthy. The chromosomal rearrangement between A.cepa Linnaeus, 1753 and its allied species has paved way to appreciate genomic evolution within Allium. The presence of a unique telomere sequence and its conservation in Allium sets this genus apart from all other Amaryllids and supports monophyletic origin. Any cytogenetic investigation regarding NOR variability, telomere sequence and genome size in the Indian species becomes the most promising field to decipher chromosome evolution against the background of species diversity and evolution, especially in the Indian subcontinent.

Cost-effective Whole Exome Sequencing discovers pathogenic variant causing Neurofibromatosis type 1 in a family from Jammu and Kashmir, India.

Neurofibromatosis type 1 (NF1) is a multisystemic hereditary disorder associated with an increased risk of benign and malignant tumor formation predominantly on the skin, bone, and peripheral nervous system. It has been reported that out of all the NF1 cases, more than 95% cases develop the disease due to heterozygous loss-of-function variants in Neurofibromin (NF1) gene. However, identification of NF1 causative variants by presently recommended method of gene-targeted Sanger sequencing is challenging and cost-intensive due to the large size of the NF1gene with 60 exons spanning about 350 kb. Further, conducting the genetic studies is difficult in low resource regions and among families with the limited financial capabilities, restricting them from availing diagnostic as well as proper disease management measures. Here, we studied a three-generation family from Jammu and Kashmir state in India, with multiple affected family members showing clinical indications of NF1. We combinedly used two applications, Whole Exome Sequencing (WES) and Sanger sequencing, for this study and discovered a nonsense variant NM_000267.3:c.2041C>T (NP_000258.1:p.Arg681Ter*) in exon 18 of NF1 gene in a cost effective manner. In silico analyses further substantiated the pathogenicity of this novel variant. The study also emphasized on the role of Next Generation Sequencing (NGS) as a cost-effective method for the discovery of pathogenic variants in disorders with known phenotypes found in large sized candidate genes. The current study is the first study based on the genetic characterization of NF1 from Jammu and Kashmir-India, highlighting the importance of the described methodology adopted for the identification and understanding of the disease in low resource region. The early diagnosis of genetic disorders would open the door to appropriate genetic counseling, reducing the disease burden in the affected families and the general population at large.

A Brief Review of Plant Cell Transfection, Gene Transcript Expression, and Genotypic Integration for Enhancing Compound Production.

Plants possess a plethora of important secondary metabolites, which are unique sources of natural pigments, pharmaceutical compounds, food additives, natural pesticides, and other industrial components. The commercial significance of such metabolites/compounds has directed the research toward their production and exploration of methods for enhancement of production. Biotechnological tools are critical in selecting, integrating, multiplying, improving, and analyzing medicinal plants for secondary metabolite production. Out of many techniques that are being explored to enhance secondary metabolite production, "plant cell transfection" is the latest tool to achieve maximum output from the plant source. It is based upon the introduction of foreign DNA into the plant cell relying on physical treatment such as electroporation, cell squeezing, sonoporation, optical transfection nanoparticles, magnetofection, and chemical treatment or biological treatment that depends upon carrier. One of the promising tools that have been exploited is CRISPR-Cas9. Overall, the abovementioned tools focus on the stable transfection of desired gene transcripts. Since the integration and continuous expression of transfected gene of particular trait represents stable transfection of host cell genome, resulting from transfer of required trait to daughter cells ultimately leading to enhanced production of secondary metabolites of interest. This chapter will review a set of biotechnological tools that are candidates for achieving the enhanced bioactive compound production indicated here to be used for drug discovery.

The complexities of migraine: A debate among migraine researchers: A review.

Migraine is indeed a neurovascular disorder for which several genes have been identified in this era of Genome-Wide Association Studies (GWAS) and neuroimaging studies have already revealed structural changes and different mechanisms that cause migraine, but the exact cause of this debilitating and disabling neurovascular disorder remained unclear. Low neuronal hyperexcitability ("the migrainous brain") is set and hindered by genetic and environmental factors, respectively. Migraine is also found to be associated with different diseases (co-morbidity). There is still a subject of contention: is migraine a disease of evolution or disease of pathology? This research review seeks to provide a brief overview on the genetics of disorders, structural abnormalities in the brain, CSD-like symptoms, and faulty Trigeminovascular System activation for migraine pain phenotype. This review briefly covered here to provide some ideas that may also be utilized in migraine research and to serve as motivation for future research.

Algorithm for sample availability prediction in a hospital-based epidemiological study spreadsheet-based sample availability calculator.

Looking at the population's behavior by taking samples is quite uncertain due to its big and dynamic structure and unimaginable variability. All quantitative sampling approaches aim to draw a representative sample from the population so that the results of the studying samples can then be generalized back to the population. The probability of detecting a true effect of a study largely depends on the sample size and if taking small samples will give lowers statistical power, higher risk of missing a meaningful underlying difference. The probability of rejecting the null hypothesis i.e., finding significant difference using the sample largely depends upon the statistical power. There are a lot of online tools used for calculating the sample size, but none tell us about the availability of samples from single site in a fixed span. This study aims to provide an efficient calculation method for the availability of samples during a specific period of a research study which is an important question to be answered during the research study design. So, we have designed a spreadsheet-based sample availability calculator tool implemented in MS-Excel 2007.

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.

Endophytic Fungi-Mediated Biocatalysis and Biotransformations Paving the Way Toward Green Chemistry.

Catalysis is a process carried out in the presence of a heterogenous catalyst for accelerating the rate of a chemical reaction. It plays a pivotal role in transition from take, make, and dispose technology to sustainable technology via chemo- and biocatalytic processes. However, chemocatalyzed reactions are usually associated with copious amounts of perilous/hazardous environmental footprints. Therefore, whole-cell biotransformations or enzyme cocktails serve as cleaner biocatalytic alternatives in replacing the classical chemical procedures. These benchmark bioconversion reactions serve as important key technology in achieving the goals of green chemistry by eliminating waste generation at source. For this, nature has always been a driving force in fuelling natural product discovery and related applications. The fungal endophytic community, in particular, has undergone co-evolution with their host plant and has emerged as a powerful tool of genetic diversity. They can serve as a treasure trove of biocatalysts, catalyzing organic transformations of a wide range of substances into enantiopure compounds with biotechnological relevance. Additionally, the biocatalytic potential of endophytic fungi as whole-intact organisms/isolated enzyme systems has been greatly expanded beyond the existing boundaries with the advancement in high-throughput screening, molecular biology techniques, metabolic engineering, and protein engineering. Therefore, the present review illustrates the promising applications of endophytic fungi as biocatalysts for the synthesis of new structural analogs and pharmaceutical intermediates and refinement of existing proteins for novel chemistries.

Engineering Host Microbiome for Crop Improvement and Sustainable Agriculture.

Dynamic consortium of microbial communities (bacteria, fungi, protists, viruses, and nematodes) colonizing multiple tissue types and coevolving conclusively with the host plant is designated as a plant microbiome. The interplay between plant and its microbial mutualists supports several agronomic functions, establishing its crucial role in plant beneficial activities. Deeper functional and mechanistic understanding of plant-microbial ecosystems will render many "ecosystem services" by emulating symbiotic interactions between plants, soil, and microbes for enhanced productivity and sustainability. Therefore, microbiome engineering represents an emerging biotechnological tool to directly add, remove, or modify properties of microbial communities for higher specificity and efficacy. The main goal of microbiome engineering is enhancement of plant functions such as biotic/abiotic stresses, plant fitness and productivities, etc. Various ecological-, biochemical-, and molecular-based approaches have come up as a new paradigm for disentangling many microbiome-based agromanagement hurdles. Furthermore, multidisciplinary approaches provide a predictive framework in achieving a reliable and sustainably engineered plant-microbiome for stress physiology, nutrient recycling, and high-yielding disease-resistant genotypes.

Transcriptome-wide identification of squalene epoxidase genes from Glycyrrhiza glabra L.: expression analysis and heterologous expression of GgSQE1 suggest important role in terpenoid biosynthesis.

Squalene epoxidase (SQE) is a crucial regulatory enzyme for the biosynthesis of several important classes of compounds including sterols and triterpenoids. The present paper identified and characterised five SQE genes (GgSQE1 to GgSQE5) from Glycyrrhiza glabra through transcriptome data mining and homology-based cloning, for the first time. The phylogenetic analysis implied their functional divergence. The ORF corresponding to one of the five SQEs, namely, GgSQE1, was cloned and studied for its function in a heterologous system, following transient and stable expressions. The transient expression followed by GgSQE1 encoding protein purification suggested approximately 58.0-kDa protein following the predicted molecular mass of the deduced protein. The gene expression profiling based on qRT-PCR indicated its highest expression (6.4-folds) in the 10-month-old roots. Furthermore, ABA (12.4-folds) and GA3 (2.47) treatments upregulated the expression of GgSQE1 in the shoots after 10 and 12 hours, respectively, which was also reflected in glycyrrhizin accumulation. The inductive effects of ABA and GA3 over GgSQE1 expression were also confirmed through functional analysis of GgSQE1 promoters using GUS fusion construct. Stable constitutive expression of GgSQE1 in Nicotiana tabacum modulated the sterol contents. The study could pave the way for understanding the metabolic flux regulation concerning biosynthesis of related sterols and triterpenoids.

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.

Publisher Correction: A genetic analysis identifies a haplotype at adiponectin locus: Association with obesity and type 2 diabetes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Tissue-specific transcriptional regulation and metabolite accumulation in tomato (Solanum lycopersicum L.).

Tomato is an excellent model for studying fruit development, ripening, and other secondary metabolic pathways such as carotenoid biosynthetic pathway, flavonoid pathway, and many more. Tomato fruit development and ripening occurs under tight genetic control and involves the expression of thousands of genes affecting fruit quality and accumulation of pigments and metabolites. Here, we have described the development of a microarray platform that has allowed establishment of a framework for quantification of the expression of large number of genes and transcription factors possibly regulating various secondary metabolic pathways in tomato. To unravel the molecular mechanisms of fruit development and ripening, a tomato 60-mer oligonucleotide 44 K microarray along with the custom array for many genes and transcription factors was designed and validated in the fruit and leaf tissues. Comparative profiling of gene expression studies has allowed us to identify a large number of differentially expressed genes and transcription factors. Gene ontology revealed the involvement of these genes in various biological, cellular, and molecular processes like isoprenoid, terpenoid, pigment, ethylene biosynthesis, phytohormone signaling, and fruit ripening. Further, correlation, as well as differential expression studies, has revealed that several transcription factors like RIN, AGAMOUS, TAGL1, MYB, MADS-box etc. could be the possible regulators of various secondary metabolic pathways. The present study has identified various metabolites, their biosynthetic pathways and genes which may possibly be controlled by different transcription factors. The present findings have laid a base for understanding the transcriptional and metabolic shifts which occur in parallel during programmed fruit ripening and developmental processes in tomato.

A genetic analysis identifies a haplotype at adiponectin locus: Association with obesity and type 2 diabetes.

Adiponectin is a prime determinant of the status of insulin resistance. Association studies between adiponectin (ADIPOQ) gene single nucleotide polymorphisms (SNPs) and metabolic diseases have been reported earlier. However, results are ambiguous due to apparent contradictions. Hence, we investigated (1) the association between ADIPOQ SNPs: -11377C/G, +10211T/G, +45T/G and +276G/T for the risk towards type 2 diabetes (T2D) and, (2) genotype-phenotype association of these SNPs with various biochemical parameters in two cohorts. Genomic DNA of diabetic patients and controls from Gujarat and, Jammu and Kashmir (J&K) were genotyped using PCR-RFLP, TaqMan assay and MassArray. Transcript levels of ADIPOQ were assessed in visceral adipose tissue samples, and plasma adiponectin levels were estimated by qPCR and ELISA respectively. Results suggest: (i) reduced HMW adiponectin/total adiponectin ratio in Gujarat patients and its association with +10211T/G and +276G/T, and reduced ADIPOQ transcript levels in T2D, (ii) association of the above SNPs with increased FBG, BMI, TG, TC in Gujarat patients and (iii) increased GGTG haplotype in obese patients of Gujarat population and, (iv) association of -11377C/G with T2D in J&K population. Reduced HMW adiponectin, in the backdrop of obesity and ADIPOQ genetic variants might alter metabolic profile posing risk towards T2D.

A Comprehensive Transcriptome-Wide Identification and Screening of WRKY Gene Family Engaged in Abiotic Stress in Glycyrrhiza glabra.

The study reports 147 full-length WRKY genes based on the transcriptome analysis of Glycyrrhiza genus (G. glabra and G. uralensis). Additional motifs in G. glabra included DivIVA (GgWRKY20) and SerS Superfamily (GgWRKY21) at the C-terminal, and Coat family motifs (GgWRKY55) at the N-terminal of the proteins, while Exo70 exo cyst complex subunit of 338 amino acid (GuWRKY9) was present at the N-terminal of G. uralensis only. Plant Zn cluster super-family domain (17 WRKYs) and bZIP domain (2 WRKYs) were common between the two species. Based on the number of WRKY domains, sequence alignment and phylogenesis, the study identified GuWRKY27 comprising of 3 WRKY domains in G. uralensis and a new subgroup-IIf (10 members), having novel zinc finger pattern (C-X4-C-X22-HXH) in G. glabra. Multiple WRKY binding domains (1-11) were identified in the promoter regions of the GgWRKY genes indicating strong interacting network between the WRKY proteins. Tissue-specific expression of 25 GgWRKYs, under normal and treated conditions, revealed 11 of the 18 induction factor triggered response corroborating to response observed in AtWRKYs. The study identified auxin-responsive GgWRKY 55 & GgWRKY38; GA3 responsive GgWRKYs15&59 in roots and GgWRKYs8, 20, 38, 57 &58 in the shoots of the treated plant. GgWRKYs induced under various stresses included GgWRKY33 (cold), GgWRKY4 (senescence), GgWRKYs2, 28 & 33 (salinity) and GgWRKY40 (wounding). Overall, 23 GgWRKYs responded to abiotic stress, and 17 WRKYs were induced by hormonal signals. Of them 13 WRKYs responded to both suggesting inter-connection between hormone signalling and stress response. The present study will help in understanding the transcriptional reprogramming, protein-protein interaction and cross-regulation during stress and other physiological processes in the plant.

Carbohydrate Modifications of Neoandrographolide for Improved Reactive Oxygen Species-Mediated Apoptosis through Mitochondrial Pathway in Colon Cancer.

Modifications at the carbohydrate moiety of neoandrographolide, isolated from the medicinal plant Andrographis paniculata, result in more potent and less toxic derivatives, namely, 4',6'-benzylidene neoandrographolide (2b) and 4'6'-p-methoxybenzylidene neoandrographolide (2c). These showed improved cytotoxicity against SW-620, PC-3, and A549 cancer cell lines. Nuclear morphology studies were conducted on compound 2b by 4',6-diamidino-2-phenylidole staining and detection of intracellular reactive oxygen species (ROS) accumulation. It showed an increase in the generation of cellular and mitochondrial ROS level. The probable relation of B-cell lymphoma-2 (Bcl-2, an apoptosis inhibitor) to B-cell lymphoma-2-associated X protein (Bax, an apoptosis promoter) ratio with caspase-3 (apoptosis coordination enzyme) in the colon cancer cell line SW-620 was investigated, and it was discovered that upon 2b treatment, the expression of caspase-3 Bax increased remarkably. However, in 2b-treated cells, the expression of Bcl-2 was downregulated as compared to untreated cells.

Identification and in silico characterization of cis-acting elements of genes involved in carotenoid biosynthesis in tomato.

Carotenoids, the widespread and structurally diverse class of pigments, accumulate in the fruits of tomato plants in a tissue specific manner. The carotenoid biosynthetic pathway genes have been cloned and characterized in tomato and other plants, however, its regulation is still obscure. We collected and analyzed forty different accessions of tomato for the present study. HPLC analysis revealed differential accumulation of major carotenoids (lycopene and ß-carotene) in the ripe fruit tissue. In order to understand the underlying regulatory mechanisms in carotenoid biosynthesis and accumulation, we sequenced the cis-acting elements i.e. promoter, 5' and 3' untranslated regions of the carotenoid pathway genes, in all accessions, followed by their in silico validation. Major differences observed in the CAAT Box, Opaque-2 Box and L-box in the promoters of carotenoid isomerase and lycopene-beta cyclase genes, respectively, along with the variations in musashi binding element of 5' untranslated regions of the carotenoid isomerase gene, suggest their differential role in regulating the carotenogenesis process in tomato. The binding sites for various transcription factors namely RIN, AGAMOUS, CRY, RAP2.2 and PIF1 on the promoters of important carotenoid pathway genes were predicted in silico. We propose that expression of carotenoid genes and also the formation of protein product in ripe tomato fruits, is regulated efficiently by the binding of these transcription factors at selected sites in the promoter region. Finally, the differential expression of the above-mentioned genes in different developmental tissues supports the possible involvement of promoters and untranslated regions in carotenoid biosynthesis and accumulation process. The present study has generated significant information concerning regulatory players involved in the carotenoid biosynthesis in tomato.

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.