Publications of NIPGR Scientists
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Item 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 JagadisMitochondrial 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.Item Nitric oxide regulates mitochondrial biogenesis in plants(John Wiley & Sons, 2023) Kumari, Aprajita; Kaladhar, Vemula Chandra; Yadav, Nidhi; Singh, Pooja; Reddy, Kishorekumar; Gupta, Kapuganti JagadisThe 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.Item Nitric oxide regulation of plant metabolism(Elsevier B.V., 2022) Gupta, Kapuganti Jagadis; Kaladhar, Vemula Chandra; Fitzpatrick, Teresa B; Fernie, Alisdair R; Møller, Ian Max; Loake, Gary JNitric oxide (NO) has emerged as an important signal molecule in plants, having myriad roles in plant development. In addition, NO also orchestrates both biotic and abiotic stress responses, during which intensive cellular metabolic reprogramming occurs. Integral to these response is the location of NO biosynthetic and scavenging pathways in diverse cellular compartments, enabling plants to effectively organize signal transduction pathways. NO regulates plant metabolism and in turn, metabolic pathways reciprocally regulate NO accumulation and function. Thus, these diverse cellular processes are inextricably linked. This review addresses the numerous redox pathways, located in the various subcellular compartments, which produce NO, in addition to the mechanisms underpinning NO scavenging. We focus on how this molecular dance is integrated into the metabolic state of the cell. Within this context, a reciprocal relationship between NO accumulation and metabolite production is often apparent. We additionally showcase cellular pathways including those associated with nitrate reduction that provide evidence for this integration of NO function and metabolism. Finally, we discuss the potential importance of the biochemical reactions governing NO levels in determining plant responses to a changing environment.Item Isolation and measurement of respiration and structural studies of purified mitochondria from heterotrophic plant tissues(John Wiley & Sons, 2021) Pandey, Sonika; Kumari, Aprajita; Singh, Pooja; Gupta, Kapuganti JagadisMitochondria are the power houses of eukaryotic cells. These organelles contain various oxidoreductase complexes. Electron transfer from different reducing equivalents channeled via these complexes drives proton translocation across the inner mitochondrial membrane, leading to ATP generation. Plant mitochondria contain alternative NAD(P)H dehydrogenases, alternative oxidase, and uncoupling protein, and TCA cycle enzymes are located in their matrix. Apart from ATP production, mitochondria are also involved in synthesis of vitamins and cofactors and participate in fatty acid, nucleotide, photorespiratory, and antioxidant metabolism. Recent emerging evidence suggests that mitochondria play a role in redox signaling and generation of reactive oxygen and nitrogen species. For mitochondrial studies, it is essential to isolate physiologically active mitochondria with good structural integrity. In this article, we explain a detailed procedure for isolation of mitochondria from various heterotrophic tissues, such as germinating chickpea seeds, potato tubers, and cauliflower florets. This procedure requires discontinuous Percoll gradient centrifugation and can give a good yield of mitochondria, in the range of 4 to 8 mg per 50 g tissue with active respiratory capacity. After MitoTracker staining, isolated mitochondria can be visualized by using a confocal microscope. The structure of mitochondria can be monitored by scanning electron microscopy.Item The power of the phytoglobin-NO cycle in the regulation of nodulation and symbiotic nitrogen fixation(John Wiley & Sons, Inc., 2020) Singh, Pooja; Kumari, Aprajita; Foyer, Christine H.; Gupta, Kapuganti JagadisRecent years have seen a large increase in our understanding of the multifaceted roles of nitric oxide (NO) in plant biology, particularly in the regulation of symbiotic interactions with soil microorganisms. NO serves key functions in the initiation and maintenance of legume–rhizobium symbiosis. Extensive molecular crosstalk between the host legume and the soil rhizobium results in the initiation of symbiosis and the establishment of a highly specialized organ called the nodule, which provides the conditions required for bacterial nitrogen fixation. Within the nodules, the rhizobium bacteria are housed in bacteroids, in which the oxygen is maintained at very low levels allowing the bacterial nitrogenase to function. This enzyme is oxygen sensitive (half maximal inhibitory concentration (IC50) oxygen for nitrogenase is 2.9 µM; Goldberg et al., 1987). Hence, a key feature of the nodule is the maintenance of a strong oxygen diffusion barrier, in which leghaemoglobin is used to transfer oxygen to the plant mitochondria to sustain respiration.Item Recommendations on terminology and experimental best practice associated with plant nitric oxide research(John Wiley & Sons, 2020) Gupta, Kapuganti Jagadis; Hancock, John T.; Petrivalsky, Marek; Kolbert, Zsuzsanna; Lindermayr, Christian; Durner, Jorg; Barroso, Juan B.; Palma, José M.; Brouquisse, Renaud; Wendehenne, David; Corpas, Francisco J.; Loake, Gary J.Nitric oxide (NO) emerged as a key signal molecule in plants. During the last two decades impressive progress has been made in plant NO research. This small, redox‐active molecule is now known to play an important role in plant immunity, stress responses, environmental interactions, plant growth and development. To more accurately and robustly establish the full spectrum of NO bioactivity in plants, it will be essential to apply methodological best practice. In addition, there are some instances of conflicting nomenclature within the field, which would benefit from standardisation. In this context, we attempt to provide some helpful guidance for best practice associated with NO research and also suggestions for the cognate terminology.Item The role of nitrite and nitric oxide under low oxygen conditions in plants(Oxford University Press, 2020) Gupta, Kapuganti Jagadis; Mur, Luis A.J.; Wany, Aakanksha; Kumari, Aprajita; Fernie, Alisdair R.; Ratcliffe, R. GeorgePlant tissues, particularly roots, can be subjected to periods of hypoxia due to environmental circumstances. Plants have developed various adaptations in response to hypoxic stress and these have been extensively described. Less well-appreciated is the body of evidence demonstrating that scavenging of nitric oxide (NO) and the reduction of nitrate/nitrite regulate important mechanisms that contribute to tolerance to hypoxia. Whilst ethylene controls hyponasty and aerenchyma formation, NO production apparently regulates hypoxic ethylene biosynthesis. In the hypoxic mitochondrion, cytochrome c oxidase, which is a major source of NO, is also inhibited by NO, thereby reducing the respiratory rate and enhancing local oxygen concentrations. Nitrite can maintain ATP generation under hypoxia by coupling its reduction to the translocation of protons from the inner side of mitochondria and generating an electrochemical gradient. This reaction can be further coupled to a reaction whereby non-symbiotic haemoglobin oxidizes NO to nitrate. In addition to these functions, nitrite has been reported to influence mitochondrial structure and supercomplex formation, as well as playing a role in oxygen sensing via the N-end rule pathway. These studies establish that nitrite and NO perform multiple functions during plant hypoxia and suggest that further research into the underlying mechanisms is warranted.Item 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 JagadisNitric 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.Item Interaction of nitric oxide with the components of plant mitochondrial electron transport chain(Oxford University Press, 2018) Gupta, Kapuganti Jagadis; Kumari, Aprajita; Florez-Sarasa, Igor; Fernie, Alisdair R; Igamberdiev, Abir UMitochondria are not only major sites for energy production but also participate in several alternative functions, among these generation of nitric oxide (NO) and its different impacts on this organelle is increasing attention. The inner mitochondrial membrane contains the chain of protein complexes, and electron transfer via oxidation of various organic acids and reducing equivalents leads to generation of proton gradient that results in energy production. Recent evidence suggests that these complexes are sources and targets for NO. Complex I and rotenone-insensitive NAD(P)H dehydrogenases regulate hypoxic NO production, while complex I also participates in the formation of supercomplex with complex III under hypoxia. Complex II is a target for NO which by inhibiting Fe-S centres regulates ROS generation, and complex III is one of the major sites for NO production and the produced NO participates in the phytoglobin-NO cycle that leads to the maintenance of redox level and limited energy production under hypoxia. Expression of the alternative oxidase (AOX) is induced by NO under various stress conditions, and evidence exists that AOX can regulate mitochondrial NO production. Complex IV is another major site for NO production which can also be linked to ATP generation via the phytoglobin-NO cycle. Inhibition of complex IV by NO can prevent oxygen depletion at the frontier of anoxia. The NO production and action on various complexes play a major role in NO signalling and energy metabolism.Item Pathways of nitric oxide metabolism and operation of phytoglobins in legume nodules: missing links and future directions(John Wiley & Sons, 2018) Berger, Antoine; Brouquisse, Renaud; Pathak, Pradeep Kumar; Hichri, Imène; Singh, Inderjit; Bhatia, Sabhyata; Boscari, Alexandre; Igamberdiev, Abir U.; Gupta, Kapuganti JagadisThe interaction between legumes and rhizobia leads to the establishment of a beneficial symbiotic relationship. Recent advances in legume - rhizobium symbiosis revealed that various reactive oxygen and nitrogen species including nitric oxide (NO) play important roles during this process. Nodule development occurs with a transition from a normoxic environment during the establishment of symbiosis to a microoxic environment in functional nodules. Such oxygen dynamics are required for activation and repression of various NO production and scavenging pathways. Both the plant and bacterial partners participate in the synthesis and degradation of NO. However, the pathways of NO production and degradation as well as their cross-talk and involvement in the metabolism are still a matter of debate. The plant-originated reductive pathways are known to contribute to the NO production in nodules under hypoxic conditions. Non-symbiotic hemoglobin (phytoglobin) (Pgb) possesses high NO oxygenation capacity, buffers and scavenges NO. Its operation, through a respiratory cycle called Pgb-NO cycle, leads to the maintenance of redox and energy balance in nodules. The role of Pgb/NO cycle under fluctuating NO production from soil needs further investigation for complete understanding of NO regulatory mechanism governing nodule development to attain optimal food security under changing environment.