Measurement of Reactive Oxygen Species and Nitric Oxide from Tomato Plants in Response to Abiotic and Biotic Stresses.

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.

Nitric oxide, energy and redox-dependent responses to hypoxia.

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.

The emerging roles of nitric oxide and its associated scavengers-phytoglobins-in plant symbiotic interactions.

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.

Metabolomic response to high light from pgrl1 and pgr5 mutants of Chlamydomonas reinhardtii.

Chlamydomonas (C.) reinhardtii metabolomic changes in cyclic electron flow-dependent mutants are still unknown. Here, we used mass spectrometric analysis to monitor the changes in metabolite levels in wild-type, cyclic electron-deficient mutants pgrl1 and pgr5 grown under high-light stress. A total of 55 metabolites were detected using GC-MS analysis. High-light stress-induced selective anaplerotic amino acids in pgr5. In addition, pgr5 showed enhancement in carbohydrate, polyamine, and polyol metabolism by 2.5-fold under high light. In response to high light, pgr5 triggers an increase in several metabolites involved in regulating osmotic pressure. Among these metabolites are glycerol pathway compounds such as glycerol-3-phosphate and glyceryl-glycoside, which increase significantly by 1.55 and 3.07 times, respectively. In addition, pgr5 also enhanced proline and putrescine levels by 2.6- and 1.36-fold under high light. On the other hand, pgrl1-induced metabolites, such as alanine and serine, are crucial for photorespiration when subjected to high-light stress. We also observed a significant increase in levels of polyols and glycerol by 1.37- and 2.97-fold in pgrl1 under high-light stress. Both correlation network studies and KEGG pathway enrichment analysis revealed that metabolites related to several biological pathways, such as amino acid, carbohydrate, TCA cycle, and fatty acid metabolism, were positively correlated in pgrl1 and pgr5 under high-light stress conditions. The relative mRNA expression levels of genes related to the TCA cycle, including PDC3, ACH1, OGD2, OGD3, IDH3, and MDH4, were significantly upregulated in pgrl1 and pgr5 under HL. In pgr5, the MDH1 level was significantly increased, while ACS1, ACS3, IDH2, and IDH3 levels were reduced considerably in pgrl1 under high-light stress. The current study demonstrates both pgr5 and prgl1 showed a differential defense response to high-light stress at the primary metabolites and mRNA expression level, which can be added to the existing knowledge to explore molecular regulatory responses of prg5 and pgrl1 to high-light stress.

Nitric oxide regulates mitochondrial biogenesis in plants.

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.

Detection of Nitric Oxide from Chickpea Using DAF Fluorescence and Chemiluminescence Methods.

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.

Alternative oxidase plays a role in minimizing ROS and RNS produced under salinity stress in Arabidopsis thaliana.

Under stress conditions, the overproduction of different reactive oxygen species (ROS) and reactive nitrogen species (RNS) causes imbalance in the redox homeostasis of the cell leading to nitro-oxidative stress in plants. Alternative oxidase (AOX) is a conserving terminal oxidase of the mitochondrial electron transport chain, which can minimize the ROS. Still, the role of AOX in the regulation of RNS during nitro-oxidative stress imposed by salinity stress is not known. Here, we investigated the role of AOX in minimizing ROS and RNS induced by 150 mM NaCl in Arabidopsis using transgenic plants overexpressing (AOX OE) and antisense lines (AOX AS) of AOX. Imposing NaCl treatment leads to a 4-fold enhanced expression of AOX accompanied by enhanced AOX capacity in WT Col-0. Further AOX-OE seedlings displayed enhanced growth compared with the AOX-AS line under stress. Examination of NO levels by DAF-FM fluorescence and chemiluminescence revealed that AOX overexpression leads to reduced levels of NO. The total NR activity was elevated under NaCl, but no significant change was observed in wild-type (WT), AOX OE, and AS lines. The total ROS, superoxide, H2 O2 levels, and lipid peroxidation were higher in the AOX-AS line than in WT and AOX-OE lines. The peroxynitrite levels were also higher in the AOX-AS line than in WT and AOX-OE lines; further, the expression of antioxidant genes was elevated in AOX-AS. Taken together, our results suggest that AOX plays an important role in the mitigation of ROS and RNS levels and enhances plant growth, thus providing tolerance against nitro-oxidative stress exerted by NaCl.

Phytoglobin-NO cycle and AOX pathway play a role in anaerobic germination and growth of deepwater rice.

An important and interesting feature of rice is that it can germinate under anoxic conditions. Though several biochemical adaptive mechanisms play an important role in the anaerobic germination of rice but the role of phytoglobin-nitric oxide cycle and alternative oxidase pathway is not known, therefore in this study we investigated the role of these pathways in anaerobic germination. Under anoxic conditions, deepwater rice germinated much higher and rapidly than aerobic condition and the anaerobic germination and growth were much higher in the presence of nitrite. The addition of nitrite stimulated NR activity and NO production. Important components of phytoglobin-NO cycle such as methaemoglobin reductase activity, expression of Phytoglobin1, NIA1 were elevated under anaerobic conditions in the presence of nitrite. The operation of phytoglobin-NO cycle also enhanced anaerobic ATP generation, LDH, ADH activities and in parallel ethylene levels were also enhanced. Interestingly nitrite suppressed the ROS production and lipid peroxidation. The reduction of ROS was accompanied by enhanced expression of mitochondrial alternative oxidase protein and its capacity. Application of AOX inhibitor SHAM inhibited the anoxic growth mediated by nitrite. In addition, nitrite improved the submergence tolerance of seedlings. Our study revealed that nitrite driven phytoglobin-NO cycle and AOX are crucial players in anaerobic germination and growth of deepwater rice.

Isolation and Measurement of Respiration and Structural Studies of Purified Mitochondria from Heterotrophic Plant Tissues.

Mitochondria 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. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Isolation of mitochondria from germinating chickpea seeds, potato tubers, and cauliflower florets Basic Protocol 2: Quantification of mitochondrial protein concentration by Bradford assay Basic Protocol 3: Quantification of mitochondrial respiration using single-channel free-radical analyzer Basic Protocol 4: Staining of mitochondria and confocal imaging Basic Protocol 5: Visualization of isolated mitochondria under scanning electron microscope.

Adaptive Reprogramming During Early Seed Germination Requires Temporarily Enhanced Fermentation-A Critical Role for Alternative Oxidase Regulation That Concerns Also Microbiota Effectiveness.

Plants respond to environmental cues via adaptive cell reprogramming that can affect whole plant and ecosystem functionality. Microbiota constitutes part of the inner and outer environment of the plant. This Umwelt underlies steady dynamics, due to complex local and global biotic and abiotic changes. Hence, adaptive plant holobiont responses are crucial for continuous metabolic adjustment at the systems level. Plants require oxygen-dependent respiration for energy-dependent adaptive morphology, such as germination, root and shoot growth, and formation of adventitious, clonal, and reproductive organs, fruits, and seeds. Fermentative paths can help in acclimation and, to our view, the role of alternative oxidase (AOX) in coordinating complex metabolic and physiological adjustments is underestimated. Cellular levels of sucrose are an important sensor of environmental stress. We explored the role of exogenous sucrose and its interplay with AOX during early seed germination. We found that sucrose-dependent initiation of fermentation during the first 12 h after imbibition (HAI) was beneficial to germination. However, parallel upregulated AOX expression was essential to control negative effects by prolonged sucrose treatment. Early downregulated AOX activity until 12 HAI improved germination efficiency in the absence of sucrose but suppressed early germination in its presence. The results also suggest that seeds inoculated with arbuscular mycorrhizal fungi (AMF) can buffer sucrose stress during germination to restore normal respiration more efficiently. Following this approach, we propose a simple method to identify organic seeds and low-cost on-farm perspectives for early identifying disease tolerance, predicting plant holobiont behavior, and improving germination. Furthermore, the research strengthens the view that AOX can serve as a powerful functional marker source for seed hologenomes.

Establishing the practice of birth companion in labour ward of a tertiary care centre in India-a quality improvement initiative.

BACKGROUND: Birth companion is a key component for providing respectful maternity care and has been recommended by WHO and Government of India. It is a low-cost beneficial intervention that is vital in improving quality of care during labour and delivery. LOCAL PROBLEM: Despite the available evidence on benefits of birth companion, there was no policy on allowing birth companion at our hospital in the past. METHODS AND INTERVENTIONS: We aimed to establish the practice of allowing birth companions in all eligible women in labour ward from existing 0% to 50% in 6 weeks' duration. This study was conducted in the Department of Obstetrics and Gynaecology, All India Institute of Medical Sciences, New Delhi. A quality improvement (QI) team was formed, and after obtaining the baseline data, problems were analysed using fish bone chart. A new policy of allowing birth companion was made and efforts made to sensitise and train the doctors and nurses posted in labour ward. Changed ideas were executed in multiple plan-do-study-act (PDSA) cycles. Simple interventions such as dress code for birth companions, curtains for ensuring privacy, display of posters and frequent reminders on WhatsApp groups were planned . RESULTS: The median value of women accompanied by birth companion marginally increased to 25% after the first PDSA cycle. Implementation of further changed ideas led to increase in median, which reached 66.6%. Thereafter, there was a decline, but by the end of 6 months, it was possible to attain the goal and sustain it. CONCLUSIONS: Simple steps of QI methodology can be used to address the prevalent problems in our healthcare. Implementation of any new practice comes with major challenges, but we could achieve our goal because of a motivated team working together on multiple changed ideas applied sequentially in PDSA cycles.

ROS/RNS Balancing, Aerobic Fermentation Regulation and Cell Cycle Control - a Complex Early Trait ('CoV-MAC-TED') for Combating SARS-CoV-2-Induced Cell Reprogramming.

In a perspective entitled 'From plant survival under severe stress to anti-viral human defense' we raised and justified the hypothesis that transcript level profiles of justified target genes established from in vitro somatic embryogenesis (SE) induction in plants as a reference compared to virus-induced profiles can identify differential virus signatures that link to harmful reprogramming. A standard profile of selected genes named 'ReprogVirus' was proposed for in vitro-scanning of early virus-induced reprogramming in critical primary infected cells/tissues as target trait. For data collection, the 'ReprogVirus platform' was initiated. This initiative aims to identify in a common effort across scientific boundaries critical virus footprints from diverse virus origins and variants as a basis for anti-viral strategy design. This approach is open for validation and extension. In the present study, we initiated validation by experimental transcriptome data available in public domain combined with advancing plant wet lab research. We compared plant-adapted transcriptomes according to 'RegroVirus' complemented by alternative oxidase (AOX) genes during de novo programming under SE-inducing conditions with in vitro corona virus-induced transcriptome profiles. This approach enabled identifying a major complex trait for early de novo programming during SARS-CoV-2 infection, called 'CoV-MAC-TED'. It consists of unbalanced ROS/RNS levels, which are connected to increased aerobic fermentation that links to alpha-tubulin-based cell restructuration and progression of cell cycle. We conclude that anti-viral/anti-SARS-CoV-2 strategies need to rigorously target 'CoV-MAC-TED' in primary infected nose and mouth cells through prophylactic and very early therapeutic strategies. We also discuss potential strategies in the view of the beneficial role of AOX for resilient behavior in plants. Furthermore, following the general observation that ROS/RNS equilibration/redox homeostasis is of utmost importance at the very beginning of viral infection, we highlight that 'de-stressing' disease and social handling should be seen as essential part of anti-viral/anti-SARS-CoV-2 strategies.

The role of nitrite and nitric oxide under low oxygen conditions in plants.

Plant 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 described extensively. 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. Although 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, also is 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 nonsymbiotic 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.

Using Foldscope to Monitor Superoxide Production and Cell Death During Pathogen Infection in Arabidopsis Under Different Nitrogen Regimes.

Nitrogen nutrition plays a role in plant growth development and resistance against biotic and abiotic stress. During pathogen infection various signal molecules such as reactive oxygen species, calcium, reactive nitrogen species, salicylic acid, and ethylene plays an important role. The form of nitrogen nutrition such as nitrate or ammonium plays a role in production of these molecules. Under nitrate nutrition NO is predominant. The produced NO plays a role in reacting with superoxide to generate peroxynitrite to induce cell death during hypersensitive response elicited by avirulent pathogens. Excess of ROS is also detrimental to plants and NO plays a role in regulating ROS. Hence it is important to observe superoxide production during infection. By using an avirulent Pseudomonas syringae and Arabidopsis differential N nutrition we show superoxide production in leaves using a paper microscope called Foldscope, which can be applied as a simple microscope to observe objects. The data also compared with root system infected with pathogenic Fusarium oxysporum. Taken together here we show that Foldscope is a cost-effective and powerful technique to visualize superoxide and cell death in plants during infection.

The PHYTOGLOBIN-NO Cycle Regulates Plant Mycorrhizal Symbiosis.

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.

Alternative Oxidase (AOX) Senses Stress Levels to Coordinate Auxin-Induced Reprogramming From Seed Germination to Somatic Embryogenesis-A Role Relevant for Seed Vigor Prediction and Plant Robustness.

Somatic embryogenesis (SE) is the most striking and prominent example of plant plasticity upon severe stress. Inducing immature carrot seeds perform SE as substitute to germination by auxin treatment can be seen as switch between stress levels associated to morphophysiological plasticity. This experimental system is highly powerful to explore stress response factors that mediate the metabolic switch between cell and tissue identities. Developmental plasticity per se is an emerging trait for in vitro systems and crop improvement. It is supposed to underlie multi-stress tolerance. High plasticity can protect plants throughout life cycles against variable abiotic and biotic conditions. We provide proof of concepts for the existing hypothesis that alternative oxidase (AOX) can be relevant for developmental plasticity and be associated to yield stability. Our perspective on AOX as relevant coordinator of cell reprogramming is supported by real-time polymerase chain reaction (PCR) analyses and gross metabolism data from calorespirometry complemented by SHAM-inhibitor studies on primed, elevated partial pressure of oxygen (EPPO)-stressed, and endophyte-treated seeds. In silico studies on public experimental data from diverse species strengthen generality of our insights. Finally, we highlight ready-to-use concepts for plant selection and optimizing in vivo and in vitro propagation that do not require further details on molecular physiology and metabolism. This is demonstrated by applying our research & technology concepts to pea genotypes with differential yield performance in multilocation fields and chickpea types known for differential robustness in the field. By using these concepts and tools appropriately, also other marker candidates than AOX and complex genomics data can be efficiently validated for prebreeding and seed vigor prediction.

Nitric oxide accelerates germination via the regulation of respiration in chickpea.

Seed germination is crucial for the plant life cycle. We investigated the role of nitric oxide (NO) in two chickpea varieties that differ in germination capacity: Kabuli, which has a low rate of germination and germinates slowly, and Desi, which shows improved germination properties. Desi produced more NO than Kabuli and had lower respiratory rates. As a result of the high respiration rates, Kabuli had higher levels of reactive oxygen species (ROS). Treatment with the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) reduced respiration in Kabuli and decreased ROS levels, resulting in accelerated germination rates. These findings suggest that NO plays a key role in the germination of Kabuli. SNAP increased the levels of transcripts encoding enzymes involved in carbohydrate metabolism and the cell cycle. Moreover, the levels of amino acids and organic acids were increased in Kabuli as a result of SNAP treatment. 1H-nuclear magnetic resonance analysis revealed that Kabuli has a higher capacity for glucose oxidation than Desi. An observed SNAP-induced increase in 13C incorporation into soluble alanine may result from enhanced oxidation of exogenous [13C]glucose via glycolysis and the pentose phosphate pathway. A homozygous hybrid that originated from a recombinant inbred line population of a cross between Desi and Kabuli germinated faster and had increased NO levels and a reduced accumulation of ROS compared with Kabuli. Taken together, these findings demonstrate the importance of NO in chickpea germination via the control of respiration and ROS accumulation.

Nitrite and nitric oxide are important in the adjustment of primary metabolism during the hypersensitive response in tobacco.

Nitrate and ammonia deferentially modulate primary metabolism during the hypersensitive response in tobacco. In this study, tobacco RNAi lines with low nitrite reductase (NiRr) levels were used to investigate the roles of nitrite and nitric oxide (NO) in this process. The lines accumulate NO2-, with increased NO generation, but allow sufficient reduction to NH4+ to maintain plant viability. For wild-type (WT) and NiRr plants grown with NO3-, inoculation with the non-host biotrophic pathogen Pseudomonas syringae pv. phaseolicola induced an accumulation of nitrite and NO, together with a hypersensitive response (HR) that resulted in decreased bacterial growth, increased electrolyte leakage, and enhanced pathogen resistance gene expression. These responses were greater with increases in NO or NO2- levels in NiRr plants than in the WT under NO3- nutrition. In contrast, WT and NiRr plants grown with NH4+ exhibited compromised resistance. A metabolomic analysis detected 141 metabolites whose abundance was differentially changed as a result of exposure to the pathogen and in response to accumulation of NO or NO2-. Of these, 13 were involved in primary metabolism and most were linked to amino acid and energy metabolism. HR-associated changes in metabolism that are often linked with primary nitrate assimilation may therefore be influenced by nitrite and NO production.