De Novo Hybrid Assembled Draft Genome of Commiphora wightii (Arnott) Bhandari Reveals Key Enzymes Involved in Phytosterol Biosynthesis.

Genome sequence and identification of specific genes involved in the targeted secondary metabolite biosynthesis are two essential requirements for the improvement of any medicinal plant. Commiphora wightii (Arnott) Bhandari (family: Burseraceae), a medicinal plant native to Western India, produces a phytosterol guggulsterone, which is useful for treating atherosclerosis, arthritis, high cholesterol, acne, and obesity. For enhanced guggulsterone yield, key genes involved in its biosynthesis pathway need to be predicted, for which the genome sequence of the species is a pre-requisite. Therefore, we assembled the first-ever hybrid draft genome of C. wightii with a genome size of 1.03 Gb and 107,221 contigs using Illumina and PacBio platforms. The N50 and L50 values in this assembled genome were ~74 Kb and 3486 bp, respectively with a guanine-cytosine (GC) content of 35.6% and 98.7%. The Benchmarking Universal Single Copy Ortholog (BUSCO) value indicated good integrity of assembly. Analysis predicted the presence of 31,187 genes and 342.35 Mb repeat elements in the genome. The comparative genome analysis of C. wightii with relevant orthogroups predicted a few key genes associated with phytosterol biosynthesis and secondary metabolism pathways. The assembled draft genome and the predicted genes should help the future variety development program with improved guggulsterone contents in C. wightii.

Modulation of transcriptome and metabolome of tobacco by Arabidopsis transcription factor, AtMYB12, leads to insect resistance.

Flavonoids synthesized by the phenylpropanoid pathway participate in myriad physiological and biochemical processes in plants. Due to the diversity of secondary transformations and the complexity of the regulation of branched pathways, single gene strategies have not been very successful in enhancing the accumulation of targeted molecules. We have expressed an Arabidopsis (Arabidopsis thaliana) transcription factor, AtMYB12, in tobacco (Nicotiana tabacum), which resulted in enhanced expression of genes involved in the phenylpropanoid pathway, leading to severalfold higher accumulation of flavonols. Global gene expression and limited metabolite profiling of leaves in the transgenic lines of tobacco revealed that AtMYB12 regulated a number of pathways, leading to flux availability for the phenylpropanoid pathway in general and flavonol biosynthesis in particular. The tobacco transgenic lines developed resistance against the insect pests Spodoptera litura and Helicoverpa armigera due to enhanced accumulation of rutin. Suppression of flavonol biosynthesis by artificial microRNA reversed insect resistance of the AtMYB12-expressing tobacco plants. Our study suggests that AtMYB12 can be strategically used for developing safer insect pest-resistant transgenic plants.

The Indian Magical Herb 'Sanjeevni' (Selaginella bryopteris L.) - A Promising Anti-inflammatory Phytomedicine for the Treatment of Patients with Inflammatory Skin Diseases.

OBJECTIVES: Selaginella bryopteris L. (family: Selaginaceae), is often used in traditional Indian systems of medicine for the prevention and cure of several disorders and for the treatment of patient with spermatorrhoea, venereal disease, constipation, colitis, urinary tract infections, fever, epilepsy, leucorrhoea, beri-beri and cancer. It is also used as a strength tonic. This study aimed to evaluate the mechanisms underlying the anti-inflammatory effects of topically administered aqueous, polar and non-polar methanolic fractions (10 mg/20 µL) of Selaginella bryopteris. METHODS: An acute oral toxicity study of Selaginella bryopteris at doses from 250 to 2,000 mg/kg body weight (bw) was performed. Aqueous, polar and non-polar methanolic extracts (10 mg/20 µL) applied topically for 5 days were evaluated for their anti-inflammatory effects against 12-tetra-O-decanoyl phorbol acetate (TPA)-induced inflammation by using the redness in the ear, the ear's weight (edema), oxidative stress parameters, such as lipid-peroxide (LPO) and nitric oxide (NO), and the pro-inflammatory cytokines involved in inflammation, such as tumour necrosis factor (TNF)-α, interleukin (IL)-1ß and IL-6. Indomethacine (0.5 mg/20 µL) was used for the positive control. RESULTS: Selaginella bryopteris produced no mortalities when administered orally at doses from 250 to 2,000 mg/kg bw. Topical treatment with the non-polar methanolic fraction (10 mg/20 αL) significantly suppressed redness (2.4 ± 0.5) and edema (30.4 ± 1) and effectively reduced the LPO level (32.3 ± 3.3). The NO level was (8.07 ± 0.55), and the TNF-α, IL-1ß, and IL-6 levels were decreased to 69.6 ± 15.5, 7.7 ± 4.8 and 82.6 ± 5.9, respectively. CONCLUSION: This study demonstrated for the first time the mechanisms underlying the anti-inflammatory effect of medicinal plants like Selaginella bryopteris and quantified the pharmacological interactions between them. The present study showed this herbal product to be a promising anti-inflammatory phytomedicine for the treatment of patients with inflammatory skin diseases.

Metabolite profiling reveals abiotic stress tolerance in Tn5 mutant of Pseudomonas putida.

Pseudomonas is an efficient plant growth-promoting rhizobacteria (PGPR); however, intolerance to drought and high temperature limit its application in agriculture as a bioinoculant. Transposon 5 (Tn5) mutagenesis was used to generate a stress tolerant mutant from a PGPR Pseudomonas putida NBRI1108 isolated from chickpea rhizosphere. A mutant NBRI1108T, selected after screening of nearly 10,000 transconjugants, exhibited significant tolerance towards high temperature and drought. Southern hybridization analysis of EcoRI and XhoI restricted genomic DNA of NBRI1108T confirmed that it had a single Tn5 insertion. The metabolic changes in the polar and non-polar extracts of NBRI1108 and NBRI1108T were examined using 1H, 31P nuclear magnetic resonance (NMR) spectroscopy and gas chromatography-mass spectrometry (GC-MS). Thirty six chemically diverse metabolites consisting of amino acids, fatty acids and phospholipids were identified and quantified. Insertion of Tn5 influenced amino acid and phospholipid metabolism and resulted in significantly higher concentration of aspartic acid, glutamic acid, glycinebetaine, glycerophosphatidylcholine (GPC) and putrescine in NBRI1108T as compared to that in NBRI1108. The concentration of glutamic acid, glycinebetaine and GPC increased by 34%, 95% and 100%, respectively in the NBRI1108T as compared to that in NBRI1108. High concentration of glycerophosphatidylethanolamine (GPE) and undetected GPC in NBRI1108 indicates that biosynthesis of GPE may have taken place via the methylation pathway of phospholipid biosynthesis. However, high GPC and low GPE concentration in NBRI1108T suggest that methylation pathway and phosphatidylcholine synthase (PCS) pathway of phospholipid biosynthesis are being followed in the NBRI1108T. Application of multivariate principal component analysis (PCA) on the quantified metabolites revealed clear variations in NBRI1108 and NBRI1108T in polar and non-polar metabolites. Identification of abiotic stress tolerant metabolites from the NBRI1108T suggest that Tn5 mutagenesis enhanced tolerance towards high temperature and drought. Tolerance to drought was further confirmed in greenhouse experiments with maize as host plant, where NBRI1108T showed relatively high biomass under drought conditions.

Characterization of Withania somnifera leaf transcriptome and expression analysis of pathogenesis-related genes during salicylic acid signaling.

Withania somnifera (L.) Dunal is a valued medicinal plant with pharmaceutical applications. The present study was undertaken to analyze the salicylic acid induced leaf transcriptome of W. somnifera. A total of 45.6 million reads were generated and the de novo assembly yielded 73,523 transcript contig with average transcript contig length of 1620 bp. A total of 71,062 transcripts were annotated and 53,424 of them were assigned GO terms. Mapping of transcript contigs to biological pathways revealed presence of 182 pathways. Seventeen genes representing 12 pathogenesis-related (PR) families were mined from the transcriptome data and their pattern of expression post 17 and 36 hours of salicylic acid treatment was documented. The analysis revealed significant up-regulation of all families of PR genes by 36 hours post treatment except WsPR10. The relative fold expression of transcripts ranged from 1 fold to 6,532 fold. The two families of peroxidases including the lignin-forming anionic peroxidase (WsL-PRX) and suberization-associated anionic peroxidase (WsS-PRX) recorded maximum expression of 377 fold and 6532 fold respectively, while the expression of WsPR10 was down-regulated by 14 fold. Additionally, the most stable reference gene for normalization of qRT-PCR data was also identified. The effect of SA on the accumulation of major secondary metabolites of W. somnifera including withanoside V, withaferin A and withanolide A was also analyzed and an increase in content of all the three metabolites were detected. This is the first report on expression patterns of PR genes during salicylic acid signaling in W. somnifera.

Enhanced methanol production in plants provides broad spectrum insect resistance.

Plants naturally emit methanol as volatile organic compound. Methanol is toxic to insect pests; but the quantity produced by most of the plants is not enough to protect them against invading insect pests. In the present study, we demonstrated that the over-expression of pectin methylesterase, derived from Arabidopsis thaliana and Aspergillus niger, in transgenic tobacco plants enhances methanol production and resistance to polyphagous insect pests. Methanol content in the leaves of transgenic plants was measured using proton nuclear spectroscopy (1H NMR) and spectra showed up to 16 fold higher methanol as compared to control wild type (WT) plants. A maximum of 100 and 85% mortality in chewing insects Helicoverpa armigera and Spodoptera litura larvae was observed, respectively when fed on transgenic plants leaves. The surviving larvae showed less feeding, severe growth retardation and could not develop into pupae. In-planta bioassay on transgenic lines showed up to 99 and 75% reduction in the population multiplication of plant sap sucking pests Myzus persicae (aphid) and Bemisia tabaci (whitefly), respectively. Most of the phenotypic characters of transgenic plants were similar to WT plants. Confocal microscopy showed no deformities in cellular integrity, structure and density of stomata and trichomes of transgenic plants compared to WT. Pollen germination and tube formation was also not affected in transgenic plants. Cell wall enzyme transcript levels were comparable with WT. This study demonstrated for the first time that methanol emission can be utilized for imparting broad range insect resistance in plants.

Comprehensive metabolic fingerprinting of Withania somnifera leaf and root extracts.

Profiling of metabolites is a rapidly expanding area of research for resolving metabolic pathways. Metabolic fingerprinting in medicinally important plants is critical to establishing the quality of herbal medicines. In the present study, metabolic profiling of crude extracts of leaf and root of Withania somnifera (Ashwagandha), an important medicinal plant of Indian system of medicine (ISM) was carried out using NMR and chromatographic (HPLC and GC-MS) techniques. A total of 62 major and minor primary and secondary metabolites from leaves and 48 from roots were unambiguously identified. Twenty-nine of these were common to the two tissues. These included fatty acids, organic acids, amino acids, sugars and sterol based compounds. Eleven bioactive sterol-lactone molecules were also identified. Twenty-seven of the identified metabolites were quantified. Highly significant qualitative and quantitative differences were noticed between the leaf and root tissues, particularly with respect to the secondary metabolites.