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    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.
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    The PHYTOGLOBIN-NO cycle regulates plant mycorrhizal symbiosis
    (Elsevier B.V., 2019) Kumari, Aprajita; Pathak, Pradeep Kumar; Loake, Gary J.; Gupta, Kapuganti Jagadis
    The production of the redox-active signaling molecule, NO, has long been associated with interactions between microbes and their host plants. Emerging evidence now suggests that specific NO signatures and cognate patterns of PHYTOGLOBIN1 (PHYTOGB1) expression, a key regulator of cellular NO homeostasis, may help determine either symbiosis or pathogenicity.
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    Current approaches to measure nitric oxide in plants
    (Oxford University Press, 2019) Vishwakarma, Abhaypratap; Wany, Aakanksha; Pandey, Sonika; Bulle, Mallesham; Kumari, Aprajita; Kishorekumar, Reddy; Igamberdiev, Abir U.; Mur, Luis A.J.; Gupta, Kapuganti Jagadis
    Nitric oxide (NO), is now established as an important signalling molecule in plants where they influence growth, development and responses to stress. Despite of extensive research, the most appropriate methods to measure and localise these signalling radicals are debated and still needs investigation. Many confounding factors such as presence of other reactive intermediates, scavenging enzymes and compartmentation, influence how accurately each can be measured. Further, these signalling radicals have short half-lives ranging from seconds to minutes based on the cellular redox condition. Hence, it is necessary to use the sensitive and specific methods in order to understand the contribution of each signalling molecule to various biological processes. In this review, we will provide current state knowledge on NO measurement in plant samples, via various methods. We will also discuss advantages, limitations and wider applications of each method.
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    Moving nitrogen to the centre of plant defence against pathogens
    (Oxford University Press, 2017) Mur, Luis A.J.; Simpson, Catherine; Kumari, Aprajita; Gupta, Alok Kumar; Gupta, Kapuganti Jagadis
    Background Plants require nitrogen (N) for growth, development and defence against abiotic and biotic stresses. The extensive use of artificial N fertilizers has played an important role in the Green Revolution. N assimilation can involve a reductase series (NO–3→ NO–2 → NH+4) followed by transamination to form amino acids. Given its widespread use, the agricultural impact of N nutrition on disease development has been extensively examined. Scope: When a pathogen first comes into contact with a host, it is usually nutrient starved such that rapid assimilation of host nutrients is essential for successful pathogenesis. Equally, the host may reallocate its nutrients to defence responses or away from the site of attempted infection. Exogenous application of N fertilizer can, therefore, shift the balance in favour of the host or pathogen. In line with this, increasing N has been reported either to increase or to decrease plant resistance to pathogens, which reflects differences in the infection strategies of discrete pathogens. Beyond considering only N content, the use of NO-3 or NH+4 fertilizers affects the outcome of plant–pathogen interactions. NO-3 feeding augments hypersensitive response- (HR) mediated resistance, while ammonium nutrition can compromise defence. Metabolically, NO–3 enhances production of polyamines such as spermine and spermidine, which are established defence signals, with NH+4 nutrition leading to increased γ-aminobutyric acid (GABA) levels which may be a nutrient source for the pathogen. Within the defensive N economy, the roles of nitric oxide must also be considered. This is mostly generated from NO–2 by nitrate reductase and is elicited by both pathogen-associated microbial patterns and gene-for-gene-mediated defences. Nitric oxide (NO) production and associated defences are therefore NO-3 dependent and are compromised by NH+4. Conclusion:This review demonstrates how N content and form plays an essential role in defensive primary and secondary metabolism and NO-mediated events.
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    Chemiluminescence detection of nitric oxide from roots, leaves, and root mitochondria
    (Springer, 2016) Wany, Aakanksha; Gupta, Alok Kumar; Kumari, Aprajita; Gupta, Shika; Mishra, Sonal; Jaintu, Ritika; Pathak, Pradeep K.; Gupta, Kapuganti Jagadis
    NO is a free radical with short half-life and high reactivity; due to its physiochemical properties it is very difficult to detect the concentrations precisely. Chemiluminescence is one of the robust methods to quantify NO. Detection of NO by this method is based on reaction of nitric oxide with ozone which leads to emission of light and amount of light is proportional to NO. By this method NO can be measured in the range of pico moles to nano moles range. Using direct chemiluminescence method, NO emitted into the gas stream can be detected whereas using indirect chemiluminescence oxidized forms of NO can also be detected. We detected NO using purified nitrate reductase, mitochondria, cell suspensions, and roots; detail measurement method is described here.
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    The functional role of nitric oxide in plant mitochondrial metabolism
    (Elsevier B.V., 2016) Gupta, Alok Kumar; Kumari, Aprajita; Mishra, Sonal; Wany, Aakanksha; Gupta, Kapuganti Jagadis
    In recent years, mitochondrial nitric oxide (NO) production has attracted increasing attention. Mitochondria generate NO using nitrite as a substrate. Cytochrome c oxidase and other components of the electron transport chain also contribute to NO generation. Accumulating evidence indicates that mitochondria are scavengers of NO. Furthermore, several genes encoding mitochondrial proteins, as well as mitochondrial proteins, are regulated by NO. In this chapter, we provided an overview of the mechanisms of NO generation and scavenging in mitochondria and of the NO-dependent regulation of proteins and genes encoding mitochondrial proteins. In addition, the functional roles of NO in mitochondrial metabolism, such as inhibition of aconitase, production of ATP and induction of alternative oxidase are presented.