Browsing by Author "Yadav, Nidhi"

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Detection of nitric oxide from chickpea using DAF fluorescence and chemiluminescence methods
(John Wiley & Sons, 2022) Kumari, Aprajita; Bhatoee, Manbir; Singh, Pooja; Kaladhar, Vemula Chandra; Yadav, Nidhi; Paul, Debarati; Loake, Gary J.; Gupta, Kapuganti Jagadis
The free radical nitric oxide (NO) has emerged as an important signal molecule in plants, due to its involvement in various plant growth, development, and stress responses. For elucidating the role of NO, it is very important to precisely determine, localize, and quantify NO levels. Due to a relatively short half-life and its rapid, complex reactivity with other radicals, together with its capacity to diffuse from the source of production, the quantification of NO in whole plants, tissues, organelles, and extracts is notoriously difficult. Hence, it is essential to employ sensitive procedures for precise detection of NO. Currently available methods can fulfill many requirements to precisely determine NO, but each method has several advantages and pitfalls. In this article, we describe a detailed procedure for the measurement of NO by diaminofluorescein (DAF) in cell-permeable forms (DAF-FM-DA). In this method, the tissues are immersed in DAF-FM DA, leading to their diffusion from the plasma membrane to the inside of the cell, where intracellular esterases cleave the ester bonds, leading to DAF-FM release. The resulting DAF-FM reacts with intracellularly generated NO and forms highly fluorescent triazolofluorescein (DAF-FMT), which can be localized and monitored by fluorescence or confocal microscopy, and can also be detected via fluorimetry and flow cytometry. DAF dyes are very popular as they are non-invasive, relatively easy to handle, and commercially available. Another precise and very sensitive method is chemiluminescence detection of NO, where NO reacts with ozone (O3), leading to emission of a quantum of light from which NO can be calculated. Using chickpea seedlings, we describe in detail the measurement of NO using DAF-FM-DA and chemiluminescence methods. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Measurement of nitric oxide from chickpea seedlings using DAF-FM DA fluorescence with fluorescence and confocal microscopy Basic Protocol 2: Chemiluminescence detection of nitric oxide from chickpea seedlings
The emerging roles of nitric oxide and its associated scavengers-phytoglobins-in plant symbiotic interactions
(Oxford University Press, 2024) Pathak, Pradeep Kumar; Yadav, Nidhi; Kaladhar, Vemula Chandra; Jaiswal, Rekha; Kumari, Aprajita; Igamberdiev, Abir U.; Loake, Gary J.; Gupta, Kapuganti Jagadis
A key feature in the establishment of symbiosis between plants and microbes is the maintenance of the balance between the production of the small redox-related molecule, nitric oxide (NO), and its cognate scavenging pathways. During the establishment of symbiosis, a transition from a normoxic to a microoxic environment often takes place, triggering the production of NO from nitrite via a reductive production pathway. Plant hemoglobins [phytoglobins (Phytogbs)] are a central tenant of NO scavenging, with NO homeostasis maintained via the Phytogb–NO cycle. While the first plant hemoglobin (leghemoglobin), associated with the symbiotic relationship between leguminous plants and bacterial Rhizobium species, was discovered in 1939, most other plant hemoglobins, identified only in the 1990s, were considered as non-symbiotic. From recent studies, it is becoming evident that the role of Phytogbs1 in the establishment and maintenance of plant–bacterial and plant–fungal symbiosis is also essential in roots. Consequently, the division of plant hemoglobins into symbiotic and non-symbiotic groups becomes less justified. While the main function of Phytogbs1 is related to the regulation of NO levels, participation of these proteins in the establishment of symbiotic relationships between plants and microorganisms represents another important dimension among the other processes in which these key redox-regulatory proteins play a central role.
New insights into nitric oxide biosynthesis underpin lateral root development
(Elsevier B.V., 2024) Gupta, Kapuganti Jagadis; Yadav, Nidhi; Kumari, Aprajita; Loake, Gary J.
In recent years, nitric oxide (NO) has emerged as a key redox signaling molecule in plants functioning in the regulation of key developmental programs and the orchestration of responses to a plethora of environmental cues (Kolbert et al., 2019). The predominant route for the transfer of NO bioactivity is through S-nitrosylation, the addition of an NO moiety to a protein cysteine thiol to form an S-nitrosothiol (Yun et al., 2011). Specificity for this process is established by the structural constraints imposed by tertiary protein structure in gating access to given cysteine redox switches and associated proteins that can either facilitate the addition or removal of the NO moiety at these residues (Umbreen et al., 2018).
Nitric oxide regulates mitochondrial biogenesis in plants
(John Wiley & Sons, 2023) Kumari, Aprajita; Kaladhar, Vemula Chandra; Yadav, Nidhi; Singh, Pooja; Reddy, Kishorekumar; Gupta, Kapuganti Jagadis
The site of nitric oxide (NO) production in mitochondrial cytochrome c oxidase and the role of NO in mitochondrial biogenesis are not known in plants. By imposing osmotic stress and recovery on Arabidopsis seedlings we investigated the site of NO production and its role in mitochondrial biogenesis. Osmotic stress reduced growth and mitochondrial number while increasing NO production. During the recovery phase the mitochondrial number increased and this increase was higher in wild type and the high NO-producing Pgb1 silencing line in comparison to the NO-deficient nitrate reductase double mutant (nia1/nia2). Application of nitrite stimulated NO production and mitochondrial number in the nia1/nia2 mutant. Osmotic stress induced COX6b−3 and COA6-L genes encoding subunits of COX. The mutants cox6b-3 and coa6-l were impaired both in NO production and mitochondrial number during stress to recovery suggesting the involvement of these subunits in nitrite-dependent NO production. Transcripts encoding the mitochondrial protein import machinery showed reduced expression in cox6b-3 and coa6-l mutants. Finally, COX6b-3 and COA6-L interacted with the VQ27 motif-containing protein in the presence of NO. The vq27 mutant was impaired in mitochondrial biogenesis. Our results suggest the involvement of COX derived NO in mitochondrial biogenesis.
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.