Browsing by Author "Samant, Sanjib Bal"

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Expression analysis of ROS-related genes during the germination of chickpea (Cicer Arietinum L.) seeds
(Springer Nature Publishing AG, 2026) Babuta, Priyanka; Samant, Sanjib Bal; Saini, Deepak; Gupta, Kapuganti Jagadis
Seed germination is a critical physiological process that transforms a quiescent seed into a metabolically active seedling and is also a crucial factor in determining maximum crop production. This transition is influenced by various intrinsic and extrinsic factors. Interestingly, reactive oxygen species (ROS) plays an important role in breaking seed dormancy by oxidation of biomolecules, weakening of the testa and degradation of endosperm. Similarly, molecular internal oxygen is also considered vital for the transition of dormancy to seed germination. However, it is essential to establish a correlation between the internal oxygen and the generation of ROS during seed germination. This chapter details protocols for imaging internal oxygen concentrations using VisiSens and fluorescent detection of ROS using H2DCFDA in chickpea seeds, complemented by qPCR analysis of key ROS-related genes (RBOH, AOX 1, UCP 1, and NADH dehydrogenase). These findings from these methods help advance our understanding of the inverse relationship between molecular oxygen and ROS dynamics during seed germination.
Measurement of reactive oxygen species and nitric oxide from tomato plants in response to abiotic and biotic stresses
(Springer Nature Publishing AG, 2024) Samant, Sanjib Bal; Manbir; Rekha; Swain, Jagannath; Singh, Pooja; Kumari, Aprajita; Gupta, Kapuganti Jagadis
Nitric oxide (NO) is a free radical molecule that has been known to influence several cellular processes such as plant growth, development, and stress responses. NO together with reactive oxygen species (ROS) play a role in signaling process. Due to extremely low half-life of these radicals in cellular environment, it is often difficult to precisely monitor them. Each method has some advantages and disadvantages; hence, it is important to measure using multiple methods. To interpret the role of each signaling molecule in numerous biological processes, sensitive and focused methods must be used. In addition to this complexity, these Reactive Oxygen Species (ROS) and NO react with each other leads to nitro-oxidative stress in plants. Using tomato as a model system here, we demonstrate stepwise protocols for measurement of NO by chemiluminescence, DAF fluorescence, nitrosative stress by western blot, and ROS measurement by NBT and DAB under stress conditions such as osmotic stress and Botrytis infection. While describing methods, we also emphasized on benefits, drawbacks, and broader applications of these methods.
Mitochondrial alternative oxidase pathway helps in nitro-oxidative stress tolerance in germinating chickpea
(Springer Nature Publishing AG, 2024) Joseph, Josepheena; Samant, Sanjib Bal; Gupta, Kapuganti Jagadis
Mitochondrial alternative oxidase (AOX) is an important protein that can help in regulating reactive oxygen species and nitric oxide in plants. The role of AOX in regulation of nitro-oxidative stress in chickpea is not known. Using germinating chickpea as a model system, we investigated the role of AOX in nitro-oxidative stress tolerance. NaCl treatment was used as an inducer of nitro-oxidative stress. Treatment of germinating seeds with 150 mM NaCl led to reduced germination and radicle growth. The AOX inhibitor SHAM caused further inhibition of germination, and the AOX inducer pyruvate improved growth of the radicle under NaCl stress. Isolated mitochondria from germinated seeds under salt stress not only increased AOX capacity but also enhanced AOX protein expression. Measurement of superoxide levels revealed that AOX inhibition by SHAM can enhance superoxide levels, whereas the AOX inducer pyruvate reduced superoxide levels. Measurement of NO by gas phase chemiluminescence revealed enhanced NO generation in response to NaCl treatment. Upon NaCl treatment there was enhanced tyrosine nitration, which is an indicator of nitrosative stress response. Taken together, our results revealed that AOX induced under salinity stress in germinating chickpea can help in mitigating nitro-oxidative stress, thereby improving germination.
Modulation of nitric oxide mediated by Phytoglobin1 plays a role in salinity tolerance via reduced nitro-oxidative stress in Arabidopsis
(Elsevier B.V., 2026) Swain, Jagannath; Babuta, Priyanka; Pandey, Sonika; Samant, Sanjib Bal; Yadav, Reena; Manbir; Hebelstrup, Kim H.; Igamberdiev, Abir U.; Singla-Pareek, Sneh Lata; Pareek, Ashwani; Gupta, Kapuganti Jagadis
Salinity is one of the major abiotic stresses that induces nitro-oxidative stress, which severely diminishes plant growth, development, and survival by altering various metabolic pathways. Phytoglobin (Pgb) is a nitric oxide (NO) scavenger that plays an important role in various stresses. However, the role of differential levels of phytoglobin1 in regulation of salinity stress induced nitro-oxidative stress in plants is not known. Here we characterized the role of Pgb-mediated NO in salinity tolerance by regulation of nitro-oxidative stress using Pgb1 overexpressing (Pgb1-OE) and silencing lines (pgb1-AS) of Arabidopsis. We found that imposing salinity leads to enhanced expression of Pgb1. NO measurement by both chemiluminescence and DAF-FM-DA suggested that salinity stress induces NO production. Pgb1-OE lines showed reduced levels of NO which is accompanied by reduced ROS, superoxide and H2O2 levels. On the contrary, pgb1-AS lines showed increased NO and ROS under salt stress. Further, gene expression analysis revealed an elevated expression of antioxidant genes in Pgb1-OE line in comparison to WT and pgb1-AS lines under salinity stress. Pgb1-OE lines showed enhanced survival which is correlated with reduced peroxynitrite and tyrosine nitration and opposing effect was observed in pgb1-AS lines along with increased cell death. Taken together, our study revealed that modulation of Pgb1 enhances tolerance to salinity-induced nitro-oxidative stress.
Nitric oxide, energy and redox-dependent responses to hypoxia
(Oxford University Press, 2024) Samant, Sanjib Bal; Yadav, Nidhi; Swain, Jagannath; Joseph, Josepheena; Kumari, Aprajita; Praveen, Afsana; Sahoo, Ranjan Kumar; Manjunatha, Girigowda; Seth, Chandra Shekar; Singla-Pareek, Sneh Lata; Foyer, Christine H; Pareek, Ashwani; Gupta, Kapuganti Jagadis
Hypoxia occurs when the oxygen levels fall below the levels required for mitochondria to support respiration. Regulated hypoxia is associated with quiescence, particularly in storage organs (seeds) and stem cell niches. In contrast, environmentally-induced hypoxia poses significant challenges for metabolically-active cells that are adapted to aerobic respiration. The perception of oxygen availability through cysteine oxidases, which function as oxygen-sensing enzymes in plants that control the N-degron pathway, and the regulation of hypoxia-responsive genes and processes is essential to survival. Functioning together with reactive oxygen species (ROS), particularly hydrogen peroxide and reactive nitrogen species (RNS), such as nitric oxide (•NO), nitrogen dioxide (•NO2), S‐nitrosothiols (SNOs), and peroxynitrite (ONOO−), hypoxia signaling pathways trigger anatomical adaptations such as formation of aerenchyma, mobilization of sugar reserves for anaerobic germination, formation of aerial adventitious roots and hyponastic response. NO and hydrogen peroxide (H2O2) participate in local and systemic signaling pathways that facilitate acclimation to changing energetic requirements, controlling glycolytic fermentation, the GABA shunt and amino acid synthesis. NO enhances antioxidant capacity and contributes to the recycling of redox equivalents energy metabolism through the phytoglobin (Pgb)-NO cycle. Here, we summarize current knowledge, highlighting the central role of NO and redox regulation in adaptive responses that prevent hypoxia-induced death in challenging conditions such as flooding.
Overexpression of phytoglobin1 in rice leads to enhanced nitrogen use efficiency via modulation of nitric oxide
(John Wiley & Sons, 2025) Samant, Sanjib Bal; Swain, Jagannath; Yadav, Nidhi; Yadav, Reena; Singh, Pooja; Rai, Preeti; Sheri, Vijay; Sreeman, Sheshshayee; Subramanyam, Rajagopal; Pareek, Ashwani; Gupta, Kapuganti Jagadis
Nitric oxide (NO) is one of the byproducts of nitrogen metabolism. Excess amount of NO is scavenged by phytoglobins. The role of phytoglobin mediated NO homoeostasis in modulation of nitrate transporters was investigated using NO scavenger cPTIO, phytoglobin overexpressing rice and Arabidopsis. Growing plants under low nitrate leads to generation of reduced levels of NO accompanied by elevated expression of high affinity transporters (HATs) such as NRT2.1, NRT2.3 and NRT2.4. Scavenging of NO by cPTIO under optimal nitrate caused enhanced HATs expression. Phytoglobin overexpressing Arabidopsis showed improved growth and enhanced expression of HATs under low nitrogen in comparison to WT. Pretreatment of optimal nitrate grown plants with NO scavenger cPTIO enhanced HATs expression and shifting of these primed plants from optimal to low nitrate leads to further elevation of HATs expression accompanied by enhanced nitrogen uptake and its accumulation with positive effect on growth. Phytoglobin overexpression in rice leads to enhanced HATs expression, improved growth, nitrogen accumulation under low nitrate. Pgb OE lines showed enhanced accumulation of amino acids. Taken together our results suggest an important role of phytoglobins in nitrogen uptake and assimilation.