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Author: search.filters.author.Gupta, Kapuganti JagadisSubject: search.filters.subject.Hypoxia

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    Method for the measurement of ethylene during hypoxia in rice plants
    (Springer Nature Publishing AG, 2025) Jaiswal, Rekha; Gupta, Kapuganti Jagadis; Praveen, Afsana
    Ethylene is a versatile phytohormone that is involved in the regulation of both growth and development such as senescence, and also it can act as a signaling hormone during hypoxia. Ethylene acts alone or in interaction with different phytohormones and proteins to regulate numerous cellular processes. Accumulating evidence suggest that endogenous ethylene production and emission into atmosphere are modulated by various biotic and abiotic stresses. Since it is a gaseous hormone, a precise detection, particularly under low-oxygen (hypoxic) conditions, is important for understanding its role in regulatory processes and stress signaling pathways. Currently, measurement practices such as gas chromatography, electrochemical sensing, and optical sensing are widely employed to detect ethylene. These methods are distinct from each other in terms of sensitivity, time response, selectivity, and cost. However, each method has its own advantages and limitations. Gas chromatography (GC) is one of the best techniques that is applied for the separation and measurement of ethylene due to its volatile and supersensitive nature. In this chapter, we describe a detailed GC-based procedure specifically optimized for measuring ethylene levels during hypoxic stress application in (Oryza sativa) rice plants.
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    Method for the measurement of ethylene during pathogen infection in arabidopsis
    (Springer Nature Publishing AG, 2025) Jaiswal, Rekha; Gupta, Kapuganti Jagadis; Praveen, Afsana
    Ethylene is a gaseous phytohormone that plays an important role as a signaling molecule during pathogen attack, influencing disease resistance and defense responses in plants. A precise measurement of ethylene production upon pathogen challenge is essential to elucidate its role in plant–pathogen interactions. Gas chromatography (GC) is among the most accurate and sensitive techniques for detecting and quantifying ethylene emissions due to its selectivity and effectiveness with gaseous molecules. In this chapter, we provide a detailed procedure employing GC specifically adapted for measuring ethylene levels during pathogen infection (Botrytis cinerea) in Arabidopsis leaflets. Arabidopsis leaflets infected by the necrotrophic pathogen Botrytis cinerea exhibit increased ethylene emission, facilitating the activation of defense pathways and secondary metabolites such as camalexin. The present GC method captures ethylene dynamics at early infection stages, ensuring precise quantification critical for dissecting the molecular mechanisms of plant immunity.
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    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.
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    Measurement of nitrate reductase activity in tomato (Solanum lycopersicum L.) leaves under different conditions
    (Springer Nature Publishing AG, 2020) Bulle, Mallesham; Kishorekumar, Reddy; Pathak, Pradeep K.; Wany, Aakanksha; Gupta, Kapuganti Jagadis
    Nitrogen is one of the crucial macronutrients essential for plant growth, development, and survival under stress conditions. Depending on cellular requirement, plants can absorb nitrogen mainly in multiple forms such as nitrate (NO3−) or ammonium (NH4+) or combination of both via efficient and highly regulated transport systems in roots. In addition, nitrogen-fixing symbiotic bacteria can fix atmospheric nitrogen in to NH4+ via highly regulated complex enzyme system and supply to the roots in nodules of several species of leguminous plants. If NO3− is a primary source, it is transported from roots and then it is rapidly converted to nitrite (NO2−) by nitrate reductase (NR) (EC 1.6.6.1) which is a critical and very important enzyme for this conversion. This key reaction is mediated by transfer of two electrons from NAD(P)H to NO3−. This occurs via the three redox centers comprised of two prosthetic groups (FAD and heme) and a MoCo cofactor. NR activity is greatly influenced by factors such as developmental stage and various stress conditions such as hypoxia, salinity and pathogen infection etc. In addition, light/dark dynamics plays crucial role in modulating NR activity. NR activity can be easily detected by measuring the conversion of NO3− to NO2− under optimized conditions. Here, we describe a detailed protocol for measuring relative NR enzyme activity of tomato crude extracts. This protocol offers an efficient and straightforward procedure to compare the NR activity of various plants under different conditions.
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    Localization of nitric oxide in wheat roots by DAF fluorescence
    (Springer, 2016) Wany, Aakanksha; Gupta, Kapuganti Jagadis
    Nitric oxide is a free radical signal molecule. Various methods are available for measurement of NO. Out of all methods, fluorescent probes to localize NO is very widely used method. Diaminofluorescein in diacetate form (DAF-2DA) is most widely probe for NO measurement. This method is based on application of 4,5-diaminofluorescein diacetate (DAF-2DA) which is actively diffused into cells, once taken up by cells cytoplasmic esterases cleave the acetate groups to generate 4,5-diaminofluorescein; DAF-2. The generated DAF-2 can readily react with N2O3, which is an oxidation product of NO to generate the highly fluorescent DAF-2T (triazolofluorescein). There are various advantages and disadvantages associated with this method, but to its advantage in diffusion closely to NO producing sites, it is widely used for localization studies. Here, we describe method to make sections of the roots and localization of NO in roots subjected to hypoxic stress.