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A micropropagation system through leaf explant culture has been developed for Withania coagulans. Shoot bud proliferation occurred through both adventitious and de novo routes depending on the hormonal regime of the culture medium. Green compact nodular organogenic callus developed on Murashige and Skoog (MS) medium supplemented with 2.3 lM kinetin (Kn) and lower levels of 6-benzyladenine (BA) (13.3 lM) while multiple adventitious shoot bud differentiation occurred on medium fortified with 2.3 lM kinetin (Kn) and higher levels of BA (22.2 lM). Shoot buds were transferred to proliferation medium containing 2.2 lM BA, 2.3 lM Kn, and 3.9 lM phloroglucinol (PG) for further growth and development of shoot system. Elongated shoots were rooted using a two-step procedure involving pulse treatment of 7 days in a medium containing 71.6 lM choline chloride (CC) and 3.9 lM PG and then transferred to rooting medium containing MS, 1.2 lM IBA, 3.6 lM PAA, and 14.3 lM CC for 3 weeks. Well-rooted plants were transferred to a greenhouse for hardening and further growth. Random amplification of polymorphic DNA (RAPD) showed monomorphic bands in all the plants thereby confirming clonality of the regenerants. Thin layer chromatography (TLC) showed the presence of withanolides in the regenerated plants. Quantification through reverse-phase HPLC revealed increased concentration of withanolides in the regenerated plants compared to the field-grown mother plant. Accumulation of withaferin A and withanolide A increased up to twofold and that of withanone up to tenfold. Direct regeneration via leaf explants will be useful for Agrobacterium-mediated genetic transformation, and will facilitate pathway manipulation using metabolic engineering for bioactive withanolides.

Summary Pathogen‐/microbe‐associated molecular patterns (PAMPs/MAMPs) initiate complex defense responses by reorganizing the biomolecular dynamics of the host cellular machinery. The extracellular matrix (ECM) acts as a physical scaffold that prevents recognition and entry of phytopathogens, while guard cells perceive and integrate signals metabolically. Although chitosan is a known MAMP implicated in plant defense, the precise mechanism of chitosan‐triggered immunity (CTI) remains unknown. Here, we show how chitosan imparts immunity against fungal disease. Morpho‐histological examination revealed stomatal closure accompanied by reductions in stomatal conductance and transpiration rate as early responses in chitosan‐treated seedlings upon vascular fusariosis. Electron microscopy and Raman spectroscopy showed ECM fortification leading to oligosaccharide signaling, as documented by increased galactose, pectin and associated secondary metabolites. Multiomics approach using quantitative ECM proteomics and metabolomics identified 325 chitosan‐triggered immune‐responsive proteins (CTIRPs), notably novel ECM structural proteins, LYM2 and receptor‐like kinases, and 65 chitosan‐triggered immune‐responsive metabolites (CTIRMs), including sugars, sugar alcohols, fatty alcohols, organic and amino acids. Identified proteins and metabolites are linked to reactive oxygen species (ROS) production, stomatal movement, root nodule development and root architecture coupled with oligosaccharide signaling that leads to Fusarium resistance. The cumulative data demonstrate that ROS, NO and eATP govern CTI, in addition to induction of PR proteins, CAZymes and PAL activities, besides accumulation of phenolic compounds downstream of CTI. The immune‐related correlation network identified functional hubs in the CTI pathway. Altogether, these shifts led to the discovery of chitosan‐responsive networks that cause significant ECM and guard cell remodeling, and translate ECM cues into cell fate decisions during fusariosis.

Extracellular matrix (ECM) is the unique organelle that perceives stress signals and reprograms molecular events of host cell during patho-stress. However, our understanding of how ECM dictates plant immunity is largely unknown. Vascular wilt caused by the soil borne filamentous fungus Fusarium oxysporum is a major impediment for global crop productivity. To elucidate the role of ECM proteins and molecular mechanism associated with cell wall mediated immunity, the temporal changes of ECM proteome was studied in vascular wilt resistant chickpea cultivar upon F. oxysporum infection. The 2DE protein profiling coupled with mass spectrometric analysis identified 166 immune responsive proteins (IRPs) involved in variety of functions. Our data suggest that wall remodeling; protein translocation, stabilization, and chitin triggered immunity; and extracellular ATP signaling are major players in early, middle, and later phases of ECM signaling during fungal attack. Furthermore, we interrogated the proteome data using network analysis that identified modules enriched in known and novel immunity-related prognostic proteins centered around nascent aminopolypeptide complex (NAC), amine oxidase, thioredoxin, and chaperonin. This study for the first time provides an insight into the complex network operating in the ECM and impinges on the surveillance mechanism of innate immunity during patho-stress in crop plant.

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Fruits of angiosperms evolved intricate regulatory machinery for sensorial attributes and storage quality after harvesting. Organic acid composition of storage organs forms the molecular and biochemical basis of organoleptic and nutritional qualities with metabolic specialization. Of these, oxalic acid (OA), determines the post-harvest quality in fruits. Tomato (Solanum lycopersicum) fruit has distinctive feature to undergo a shift from heterotrophic metabolism to carbon assimilation partitioning during storage. We have earlier shown that decarboxylative degradation of OA by FvOXDC leads to acid homeostasis besides increased fungal tolerance in E8.2-OXDC tomato. Here, we elucidate the metabolic consequences of oxalate down-regulation and molecular mechanisms that determine organoleptic features, signaling and hormonal regulation in E8.2-OXDC fruit during post-harvest storage. A comparative proteomics approach has been applied between wild-type and E8.2-OXDC tomato in temporal manner. The MS/MS analyses led to the identification of 32 and 39 differentially abundant proteins associated with primary and secondary metabolism, assimilation, biogenesis, and development in wild-type and E8.2-OXDC tomatoes, respectively. Next, we interrogated the proteome data using correlation network analysis that identified significant functional hubs pointing toward storage related coinciding processes through a common mechanism of function and modulation. Furthermore, physiochemical analyses exhibited reduced oxalic acid content with concomitant increase in citric acid, lycopene and marginal decrease in malic acid in E8.2-OXDC fruit. Nevertheless, E8.2-OXDC fruit maintained an optimal pH and a steady state acid pool. These might contribute to reorganization of pectin constituent, reduced membrane leakage and improved fruit firmness in E8.2-OXDC fruit with that of wild-type tomato during storage. Collectively, our study provides insights into kinetically controlled protein network, identified regulatory module for pathway formulation and provide basis toward understanding the context of storage quality maintenance as a consequence of oxalate downregulation in the sink organ.

Fruit is an assimilator of metabolites, nutrients, and signaling molecules, thus considered as potential target for pathogen attack. In response to patho-stress, such as fungal invasion, plants reorganize their proteome, and reconfigure their physiology in the infected organ. This remodeling is coordinated by a poorly understood signal transduction network, hormonal cascades, and metabolite reallocation. The aim of the study was to explore organ-based proteomic alterations in the susceptibility of heterotrophic fruit to necrotrophic fungal attack. We conducted time-series protein profiling of Sclerotinia rolfsii invaded tomato (Solanum lycopersicum) fruit. The differential display of proteome revealed 216 patho-stress responsive proteins (PSRPs) that change their abundance by more than 2.5-fold. Mass spectrometric analyses led to the identification of 56 PSRPs presumably involved in disease progression; regulating diverse functions viz. metabolism, signaling, redox homeostasis, transport, stress-response, protein folding, modification and degradation, development. Metabolome study indicated differential regulation of organic acid, amino acids, and carbohydrates paralleling with the proteomics analysis. Further, we interrogated the proteome data using network analysis that identified two significant functional protein hubs centered around malate dehydrogenase, T-complex protein 1 subunit gamma, and ATP synthase beta. This study reports, for the first-time, kinetically controlled patho-stress responsive protein network during post-harvest storage in a sink tissue, particularly fruit and constitute the basis toward understanding the onset and context of disease signaling and metabolic pathway alterations. The network representation may facilitate the prioritization of candidate proteins for quality improvement in storage organ.

The extracellular matrix (ECM) has a molecular machinery composed of diverse proteins and proteoforms that combine properties of tensile strength with extensibility exhibiting growth-regulatory functions and self- and non-self-recognition. The identification of ECM proteoforms is the prerequisite towards a comprehensive understanding of biological functions accomplished by the outermost layer of the cell. Regulatory mechanisms of protein functions rely on post-translational modifications, phosphorylation in particular, affecting enzymatic activity, interaction, localization and stability. To investigate the ECM proteoforms, we have isolated the cell wall proteome and phosphoproteome of a tuberous crop, potato (Solanum tuberosum). LC-MS/MS analysis led to the identification of 38 proteins and 35 phosphoproteins of known and unknown functions. The findings may provide a better understanding of biochemical machinery and the integrated protein and phosphoprotein network of ECM for future functional studies of different developmental pathways and guidance cues in mechanosensing and integrity signaling.