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Item The Ca2+ channel CYCLIC NUCLEOTIDE GATED CHANNEL13 regulates vasculature-mediated systemic Ca2+ and jasmonate signaling on herbivory(PNAS, USA, 2026) Kumari, Misha; Prajapati, Ramgopal; Pawar, Mahendra; Sharma, Bhawana; Orlando, Marchesano Bianca Maria; Yang, Yang; Wang, Yong-Fei; Costa, Alex; Vadassery, JyothilakshmiInsect herbivory triggers rapid local and systemic signaling in plants, including membrane depolarization, cytosolic Ca2+ elevation, and activation of jasmonate-mediated defense pathways. These early events are shaped by calcium-permeable ion channels and regulatory components that generate stimulus-specific Ca2+ signatures. The glutamate receptor-like channel-centric model of long-distance Ca2+ signaling is insufficient to explain the robustness of systemic responses to diverse damage and herbivore cues, indicating the involvement of additional Ca2+ channels. Here, we identify CYCLIC NUCLEOTIDE GATED CHANNEL13 (CNGC13) as a plasma membrane-localized, vasculature-expressed Ca2+ channel that is essential for systemic jasmonate signaling and defense against lepidopteran insect pest, Spodoptera litura in Arabidopsis thaliana. CNGC13 is rapidly induced by wounding and herbivory and is required for efficient propagation of Ca2+ signals from wounded to distal leaves. Loss of CNGC13 compromises the thioglucosidase (Ricca factor)-dependent breakdown of aliphatic glucosinolates into aglucones/isothiocyanates within the vascular tissue, which are essential for wound-induced systemic Ca2+ wave propagation. Consequently, cngc13 mutants display reduced wound induced jasmonate accumulation in systemic leaves, reduced glucosinolate levels and enhanced herbivore susceptibility. CNGC13 is also required for AtPep-induced Ca2+ elevation and reactive oxygen species signaling and physically interacts with PLANT ELICITOR PEPTIDE RECEPTOR 2 (PEPR2) kinase domain. Consistently, pepr2 mutants display similar defects in glucosinolate aglucone production and systemic jasmonate accumulation. Together, our findings identify CNGC13 as a vasculature-localized Ca2+-permeable channel that integrates multiple damage-cues, including Ricca factor and AtPep-PEPR signaling, to drive and sustain systemic signaling and immune activation.Item Silicon-mediated mitigation of drought stress in crops: current understanding and future perspectives(Springer Nature Publishing AG, 2026) Jangir, Pooja; Bishnoi, Alka; Shekhawat, Pooja Kanwar; Singh, Vikram; Rathore, Rajesh Singh; Agarwal, Meenakshi; Ram, Hasthi; Soni, PraveenDrought is one of the major environmental constraints limiting crop productivity, particularly in arid and semi-arid regions. With increasing climate unpredictability and rising food demands, sustainable approaches to improve drought tolerance are needed. Silicon (Si) has emerged as a beneficial element that enhances plant resilience to drought stress. This review provides an overview of Si uptake, transport, accumulation, and fertilization strategies, followed by an evaluation of the physiological, biochemical, and molecular mechanisms underlying Si-mediated drought tolerance across diverse crop species. Si improves seed germination, growth, and yield under water-deficit conditions by enhancing plant water status, osmotic adjustment, nutrient uptake, photosynthetic efficiency, antioxidant defense, hormonal homeostasis, cellular ultrastructure, and the expression of drought-responsive genes. The review further highlights the considerable variability in Si-mediated drought tolerance arising from differences in plant species and genotype, developmental stage, Si source and formulation, application strategy, soil properties, environmental conditions, and stress scenarios. A brief perspective on silicon nanoparticles (SiNPs) is also presented. Finally, key knowledge gaps and future priorities, including elucidation of Si signaling pathways, optimization of Si application strategies, integration of multi-omics approaches, and long-term field validation, are discussed to facilitate the effective and sustainable use of Si in improving crop drought resilience.Item MicroRNA172 regulates growth under low nitrogen conditions in Arabidopsis thaliana(Springer Nature Publishing AG, 2026) Gandhi, Nidhi; Singh, Kratika; Sarkar, Ananda Kumar; Singh, Amar PalMicroRNAs (miRNAs), a class of small non-coding regulatory RNAs, play important roles in various aspects of plant growth and stress responses, including nutrient deficiency. The availability of nitrogen (N), an essential mineral for plant growth, affects the expression of several miRNAs. However, the function of miRNAs in modulating root responses under low nitrogen (LN) condition remains elusive. Here we report that LN differentially regulates miR172 expression levels, which modulate plant response. Overexpression of miR172 (miR172OE) reduced the plant response to LN-mediated PR elongation, whereas miR172-target mimic (MIM172) lines exhibited enhanced LN-dependent PR elongation in Arabidopsis thaliana (Arabidopsis). Cellular analysis at the root tip revealed that miR172OE plants exhibited a reduced response to LN-mediated root cell elongation and meristem zone (MZ) inhibition, whereas MIM172 plants maintained LN-responsive regulation of these cellular parameters comparable to wild-type. Consistent with these findings, LN-mediated suppression of chlorophyll levels was compromised in miR172OE plants. Analysis of miR172 target genes suggests that among the known targets [SCHLAFMUTZE (SMZ), SCHNARCHZAPFEN (SNZ), APETALA2 (AP2), TARGET OF EAT1 (TOE1), TOE2, and TOE3], TOE1 and TOE2 are involved in LN-dependent elongation of PR through modulation of cellular activities at the root tip. The toe1-2;toe2-1 double mutants showed reduced response to PR elongation and compromised cell elongation under LN condition. Further, chlorate sensitivity assay data suggest that reduced response of miR172OE lines and toe1-2;toe2-1 double mutants under LN condition is associated with a reduced nitrate assimilation pathway. Together, our findings suggest the role of miR172 and its targets TOE1 and TOE2 in regulating nitrogen-responsive growth and the nitrate assimilation pathway.Item Gibberellin-dependent brassinosteroid signaling modulates plant growth by optimizing nitrogen levels during salinity stress(Oxford University Press, 2026) Gupta, Shreya; Yadav, Ritesh Kumar; Pandey, Anshika; Gandhi, Nidhi; Devi, Loitongbam Lorinda; Agarwal, Subham; Singh, Kratika; Sen, Sonali; Shamnas, V Muhammed; Sinha, Subodh Kumar; Singh, Amar PalLow nitrogen (LN) and high salt stress are major constraints for plant growth and development. Under LN stress, plants typically show root elongation to increase nitrogen foraging from the rhizosphere, whereas high salt levels cause inhibition of root growth to avoid salinity stress-induced toxicity. To date, mechanisms and strategies of root adaptations in plants under combined LN and high salt stress remain poorly defined. Here, we provide evidence that Arabidopsis plants under LN stress show increased brassinosteroid (BR) signaling activity, which in turn suppresses salt stress-responsive pathways but improves nitrogen foraging. BR signaling, through its transcriptional regulators, activates and represses nitrogen and salt-responsive genes, respectively. In contrast, salinity stress represses gibberellin (GA) signaling and leads to the accumulation of DELLA proteins. High levels of DELLAs inhibit BZR1-dependent nitrogen-responsive growth to facilitate plant adaptation under high salt stress condition. Our data suggest that GA-promoted root elongation under combined LN and high salt stress is associated with BZR1-DELLA and Salt Overly Sensitive 3 (SOS3) protein stoichiometry. Specifically, DELLAs interact with SOS3, and high levels of SOS3 in turn facilitate tolerance to salt stress by adjusting DELLA-dependent BZR1 function and nitrogen homeostasis. Taken together, our findings highlight the adaptive responses plants exert to mitigate variable nitrogen and high salt stress conditions and appear essential for balancing growth and stress response.Item An EMS mutant of rice cultivar Nagina 22 exhibits tolerance to diverse abiotic stresses(Elsevier B.V., 2026) Das, Prajna Priyadarshini; Nagalakshmi, Sanivarapu; Yugandhar, Poli; Shrivastva, Mayank; Vamshi, P.; Rao, V. Papa; Tuti, Mangal Deep; Anantha, M. S.; Barbadikar, Kalyani M.; Neeraja, C. N.; Giri, Jitender; Sundaram, R. M.; Mangrauthia, Satendra KumarRice frequently encounters diverse abiotic stresses under changing climatic conditions, including high temperature, osmotic stress, and nutrient deficiencies, which severely limit productivity. Nagina 22 (N22), an aus rice cultivar, is widely recognized as an important donor for drought and heat tolerance traits. However, its sensitivity to phosphorus deficiency poses a significant limitation, particularly under drought and high-temperature conditions, which further reduce the availability of inorganic phosphate (Pi). We hypothesized that mutagenesis of N22 would yield superior variants with enhanced agronomic performance and conserved stress-response mechanisms. One of the Ethyl methanesulfonate (EMS) mutants, NH733, outperformed N22 by maintaining higher photosynthetic efficiency, biomass, reproductive fertility, and grain yield, supported by a more efficient antioxidant machinery under high temperature and low-soil-phosphorus (P) conditions. RNA sequencing under diverse (osmotic stress, high temperature, and low-P) stresses revealed the potential roles of HSFA2D, PCF3, and GHD7 in broad-spectrum stress adaptation of NH733, thereby establishing this mutant line as a valuable genetic resource for breeding climate-resilient rice varieties. Functional validation of the genes and regulators identified in this study will further facilitate their exploitation in genome editing or genomic assisted breeding–driven crop improvement programs.Item Establishment of a CRISPR-Cas9 library for indica rice and identification of OsOPR5 (LOC_Os06g11210) as a regulator of root architecture(John Wiley & Sons, 2026) Chowdhury, Sankhadeep; Nayak, Suprava Priyadarsini; Pattanayak, Rajashree; Sardar, Shaswati; Majhi, Ashis; Yadav, Banita; Dasari, Abhilash; Mandlik, Rushil; Sonah, Humira; Deshmukh, Rupesh; Gupta, Ishaan; Bauer, Petra; Ram, HasthiFunctional characterization of a large number of rice genes remains a major challenge despite the availability of genome sequences and large-scale transcriptomic datasets. CRISPR-Cas9 library is a powerful approach for high-throughput targeted mutagenesis; however, its application in indica rice cultivars remains limited due to low transformation and regeneration efficiencies. In this study, we developed a CRISPR-Cas9 library targeting 12,000 rice genes and evaluated its utility for functional genomics in the indica cultivar MTU-1010. Sanger sequencing and NGS analysis of the plasmid library revealed high sgRNA coverage and more than 80% accuracy. Transformation of the developed library into the indica cultivar MTU-1010 resulted in a high target editing efficiency, with 90% of analyzed transgenic plants carrying mutations at the intended target site. Functional analysis of one homozygous mutant identified a previously uncharacterized role for OsOPR5 (LOC_Os06g11210), a member of the 12-oxophytodienoate reductase family in root architecture. The opr5 mutants exhibited significant reductions in lateral root number, seminal and crown root number, and root length, demonstrating that OsOPR5 positively regulates root system architecture in rice. Notably, endogenous jasmonic acid (JA) and JA-isoleucine levels were not significantly altered in the mutant, suggesting potential functional specialization or redundancy among rice OPR family members for JA accumulation. The root system architecture is a key determinant of water and nutrient acquisition; our results suggest that OsOPR5 may play an important role in adaptation under adverse environmental conditions. Collectively, this study establishes an efficient genome-editing platform for indica rice and identifies OsOPR5 as a novel regulator of root development.Item Genome-wide association study delineating genomic regions contributing to heat stress tolerance at vegetative stage in mung bean [Vigna radiata (L.) R. Wilczek](Springer Nature Publishing AG, 2026) Jha, Uday Chand; Shafi, Sadiah; Mohanty, Jitendra Kumar; Nayyar, Harsh; Yadava, Yashwant Kumar; Ciampitti, Ignacio Antonio; Parida, Swarup Kumar; Siddique, Kadambot Hamza Mohamed; Prasad, Pagadala Venkata VaraIntensifying climate change and increasing heat stress pose a growing challenge to global food crop production, including mung bean [Vigna radiata (L.) R. Wilczek]. Mung bean is a vital protein-rich pulse crop with essential vitamins and micronutrients. However, rising heat stress increasingly constrains its production worldwide. Despite extensive studies on physiological and biochemical responses, genomic resources for heat tolerance in mung bean remain limited. This study employed a genome-wide association study (GWAS) using 5,758 high-quality SNPs to identify genomic regions and candidate genes associated with heat stress tolerance at the vegetative stage. A globally diverse panel of 396 genotypes was evaluated under optimal (34/25 °C) and heat stress (42/30 °C) conditions, assessing six major morpho-physiological traits including leaf chlorophyll index measured by SPAD, photosystem II maximum quantum efficiency (Fv/Fm), membrane damage, plant height, days to flowering and plant biomass. Marker-trait association (MTA) analysis identified nine significant MTAs under optimal conditions and 12 under heat stress. Under heat stress, two MTAs for days to flowering were detected on chromosome (Chr) 3 (S3_8626095) and Chr5 (S5_26149549). These SNPs explained 2.3% to 4.3% of the phenotypic variance explained (PVE). Eleven putative candidate genes were identified, including LOC106757788 encoding phytochrome A-like proteins, LOC106770897 encoding leucine-rich repeat extension-like proteins (maintaining structural integrity under heat stress), and LOC106761867 encoding calcium ATPases (maintaining low cytoplasmic calcium and preventing cytotoxicity). These findings identify key genomic regions and pathways associated with heat stress tolerance, offering valuable resources for marker-assisted breeding of heat-resilient mung bean cultivars. Further validation of candidate genes will enable designing of improved climate resilient varieties. This article supports United Nations’ Sustainable Development Goal: SDG-2 (Zero Hunger) by advancing molecular and cytogenetic research for the genetic improvement of mung bean.Item In vitro evaluation of effectiveness of antifungal agents against Macrophomina phaseolina(Springer Nature Publishing AG, 2026) Acharya, Krishna Ramakrishnan; Chilakala, Aswin Reddy; Senthil-Kumar, MuthappaAntifungal agents provide both preventive and curative effects by protecting plant surfaces and eliminating existing infections within plant tissues. Evaluating their impact against phytopathogenic fungi is critical for sustainable agriculture. Macrophomina phaseolina is a highly destructive soil-borne pathogen with a broad host range, causing charcoal rot and related diseases that result in severe yield losses in crops such as soybean, sorghum, maize, and legumes. Several methods are employed to assess the in vitro antifungal efficacy of various substances, including broth microdilution, disk diffusion, gradient screening, and agar-based screening. However, these conventional techniques are generally slower, more labor-intensive, and limited by reproducibility issues. The adoption of microplate reader-based antifungal efficacy testing has enhanced efficiency and scalability, enabling high-throughput evaluation of multiple antifungal agents across a range of concentrations while providing a streamlined platform for preliminary screening. This chapter presents a convenient and cost-effective protocol for assessing the activity of antifungal agents against fungi that produce microsclerotia, with emphasis on standardization and quality control measures. The antifungal efficacy testing and cell death assay are employed to determine inhibitory concentrations via spectrophotometry and to quantify hyphal death through fluorescence microscopy, respectively. In addition, detailed procedures for inoculum preparation, antifungal agent dilution, incubation conditions, and statistical analysis are described.Item Quantification of mitochondrial pyrroline-5-carboxylate in Arabidopsis under Pseudomonas Syringae infection(Springer Nature Publishing AG, 2026) Qamar, Aarzoo; Senthil-Kumar, MuthappaPyrroline-5 carboxylic acid (P5C) is an intermediary metabolite formed during proline synthesis and catabolism. Proline catabolism in two steps occurs in mitochondria where proline dehydrogenase (ProDH) catabolizes proline into P5C, and finally, pyrroline-5 carboxylate dehydrogenase catabolizes P5C into glutamate. Estimating mitochondrial P5C is essential to know how the metabolism of P5C in mitochondria via proline catabolism genes is responsible for causing disease and defense reaction in the case of Pseudomonas syringae–Arabidopsis interaction. There is no protocol available in the literature to estimate mitochondrial P5C due to which its role is masked and not properly deciphered. Here, we describe the protocol for estimating mitochondrial P5C from Arabidopsis leaves infected with bacterial pathogen P. syringae. Mitochondria was isolated from Arabidopsis leaf samples by percoll gradient centrifugation method. Mitochondrial amino acids and their derivatives were extracted in 80% methanol and without derivatization directly used for LC-MS analysis with standard labeled amino acids acting as internal control. P5C was detected as the daughter ion of the parent ion proline and quantified from the internally labeled proline standard.Item Isolation of AtSWEET membrane proteins from Arabidopsis leaves for host-pathogen interaction studies(Springer Nature Publishing AG, 2026) Anjali, Anjali; Khan, Wajahat Ali; Arockiasamy, Arulandu; Senthil-Kumar, MuthappaOptimizing the conditions for the overexpression and purification of membrane proteins for functional and structural studies is a complex and tedious process, which demands considerable time and effort. Membranes are made up of phospholipid bilayer embedded with proteins, cholesterol, and carbohydrates. While cholesterol regulates the fluidity, proteins and carbohydrates provide functionality to the membranes. Membrane proteins are responsible for transport, signaling, adhesion, and catalysis and have membrane spanning domains that tether them to the lipid bilayer. Due to the hydrophobic nature of these membrane spanning domains, isolation, and biochemical analysis becomes challenging. For an extended period, researchers have refined techniques to achieve the highest purity in plasma membrane vesicle preparations derived from crude plant extracts. While, conventional detergent-based isolation methods can solubilize both globular and membrane proteins, an enrichment step is necessary to remove cytosolic or aggregated contaminants. The current protocol outlines a method for membrane purification utilizing ultracentrifugation and detergents, which facilitates the isolation of membrane proteins in their native conformational state.