Pathogenesis-related protein 1 (PR-1) is an antimicrobial protein involved in systemic acquired resistance (SAR) in plants, but its regulatory role and interactions with other pathways remain unclear. In this study, we functionally characterize WsPR-1 gene of Withania somnifera in Nicotiana tabacum to elucidate its role in plant defense, growth, and development. Interestingly, transgenic tobacco plants with increased levels of cytokinin (CK) and decreased gibberellins (GAs) exhibited stunted shoot growth, an underdeveloped root system, modified leaf morphology, reduced seed pod production, and delayed leaf senescence. Transcriptional analysis revealed that WsPR-1 overexpression downregulated the GA 20-oxidase (GA20ox) gene involved in GA biosynthesis while upregulating GA 2-oxidase (GA2ox), a GA catabolic enzyme. Moreover, transcript levels of FRUITFULL (FUL) and LEAFY (NFL2) flowering genes exhibited a decrease in WsPR-1 plants, which could explain the delayed flowering and reduced seed pod development in transgenic plants. Confocal microscopy confirmed increased lignin deposition in stem cross-sections of WsPR-1 transgenic plants, supported by gene expression analysis and lignin content quantification. Additionally, our findings also suggest the involvement of Knotted1-like homeobox (KNOX) gene in enhancing cytokinin levels. This study highlights PR-1's regulatory role in plant growth and development, with potential to boost crop yields and enhance resilience.

Neuroinflammation is a major risk factor associated with the pathogenesis of neurodegenerative diseases. Conventional non-steroidal anti-inflammatory drugs are prescribed but their long term use is associated with adverse effects. Thus, herbal based medicines are attracting major attraction worldwide as potential therapeutic candidates. Tylophora indica (Burm. f) Merrill is a valuable medicinal plant well known in Ayurvedic practices for its immunomodulatory, anti-oxidant, anti-asthmatic and antirheumatic activities. The present study aimed to elucidate the anti-neuroinflammatory potential of water and hydroalcoholic leaf extracts of micropropagated plants of T. indica using BV-2 microglia activated with lipopolysaccharide as an in vitro model system and development of an efficient reproducible protocol for its in vitro cloning. Non cytotoxic doses of the water and hydroalcoholic extracts (0.2μg/ml and 20μg/ml, respectively) were selected using MTT assay. α-Tubulin, Iba-1 and inflammatory cascade proteins like NFκB, AP1 expression was studied using immunostaining to ascertain the anti-neuroinflammatory potential of these extracts. Further, anti-migratory activity was also analyzed by Wound Scratch Assay. Both extracts effectively attenuated lipopolysaccharide induced microglial activation, migration and the production of nitrite via regulation of the expression of NFκB and AP1 as the possible underlying target molecules. An efficient and reproducible protocol for in vitro cloning of T. indica through multiple shoot proliferation from nodal segments was established on both solid and liquid Murashige and Skoog's (MS) media supplemented with 15μM and 10μM of Benzyl Amino Purine respectively. Regenerated shoots were rooted on both solid and liquid MS media supplemented with Indole-3-butyric acid (5-15μM) and the rooted plantlets were successfully acclimatized and transferred to open field conditions showing 90% survivability. The present study suggests that T. indica may prove to be a potential anti-neuroinflammatory agent and may be further explored as a potential therapeutic candidate for the management of neurodegenerative diseases. Further, the current study will expedite the conservation of T. indica ensuring ample supply of this threatened medicinal plant to fulfill its increasing demand in herbal industry.

Leaf spot disease caused by Alternaria alternata severely affects the growth and yield of a high repute medicinal plant, Withania somnifera(L.) Dunal. Thus, to substantiate the growing demand of this plant, it is critical to develop suitable strategies to combat the effects of leaf spot disease. Reactive oxygen species (ROS) modulation is one of the key mechanisms for achieving biotic stress tolerance as it evokes multiple stress adaptive pathways in plants. Hence, in the present investigation, ROS generating and scavenging systems have been evaluated together in leaf spot disease resistant and susceptible accessions of W. somnifera. The levels of various types of ROS have been detected using different staining methods and biochemical assays have been performed to estimate the enzymatic activities of ROS generating and scavenging enzymes. The results of enzymatic activities have been consolidated at the transcript level using real-time PCR. Results show that leaf spot resistant accession has higher basal levels of hydrogen peroxide due to the increased enzymatic activities of nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase and superoxide dismutase (SOD) enzymes. Hydrogen peroxide is well known as a signaling molecule that regulates a plethora of biotic stress-responsive pathways. The threshold levels of this molecule are maintained by the action of a series of antioxidant enzymes which also showed increased levels in the present investigation. Different oxidative stress markers including proline and malondialdehyde (MDA) have also been analyzed. Our study demonstrates that ROS dynamics in leaf spot resistant accession of W. somnifera due to the coordinate action of ROS generating and scavenging system could be one of the reasons for resistance against A. alternata. Hence, modulation of ROS machinery could a feasible approach for achieving leaf spot disease resistance in W. somnifera.

Screening of 20,000 clones of a fosmid gene bank, constructed from DNA extracted from North West Himalaya (NWH) glacier soil sample, using functional approach identified 10 esterase/lipase-producing clones. Of these, a clone designated pFG43 with an insert size of 45 kb which produced the highest concentration of enzyme (467.43 U/mg) was sequenced. Clone pFG43 contained 61 open reading frames (ORF) and of these an ORF of 1155 bp designated ME-003, was found to be closely related to a hydrolase from Acidobacteria sps (77% sequence identity and E value = 1e-164) and subsequently identified as a putative cocaine esterase. ORF ME-003 was amplified and sub-cloned using a TA vector system into E. coli (DH5α). The purified recombinant enzyme with a molecular weight of 43 kDa had optimal activity at 40 °C, pH 6 and the highest activity with shorter chain fatty acids than with higher chain length fatty acids. There is insignificant effect of inhibitors on the enzyme activity of ME-003, except PMSF which completely inhibited its activity. ME-003 activity was also inhibited in the presence of copper oxide but remained stable in presence of other metal ions. The enzyme activity was also inhibited in the presence of organic solvents; however, in the presence of 10% isopropanol, 12% of enzymatic activity was retained. Among various detergents, SDS completely inhibited enzymatic activity. The recombinant enzyme also shows enantio-specific activity against the racemic drug intermediates/precursors and exhibited 90% ee against racemic 1-phenyl ethanol and fluoxetine.