Serine hydroxymethyltransferase6 is involved in growth and resistance against pathogens via ethylene and lignin production in Arabidopsis.

Photorespiratory serine hydroxymethyltransferases (SHMTs) are important enzymes of cellular one-carbon metabolism. In this study, we investigated the potential role of SHMT6 in Arabidopsis thaliana. We found that SHMT6 is localized in the nucleus and expressed in different tissues during development. Interestingly SHMT6 is inducible in response to avirulent, virulent Pseudomonas syringae and to Fusarium oxysporum infection. Overexpression of SHMT6 leads to larger flowers, siliques, seeds, roots, and consequently an enhanced overall biomass. This enhanced growth was accompanied by increased stomatal conductance and photosynthetic capacity as well as ATP, protein, and chlorophyll levels. By contrast, a shmt6 knockout mutant displayed reduced growth. When challenged with Pseudomonas syringae pv tomato (Pst) DC3000 expressing AvrRpm1, SHMT6 overexpression lines displayed a clear hypersensitive response which was characterized by enhanced electrolyte leakage and reduced bacterial growth. In response to virulent Pst DC3000, the shmt6 mutant developed severe disease symptoms and becomes very susceptible, whereas SHMT6 overexpression lines showed enhanced resistance with increased expression of defense pathway associated genes. In response to Fusarium oxysporum, overexpression lines showed a reduction in symptoms. Moreover, SHMT6 overexpression lead to enhanced production of ethylene and lignin, which are important components of the defense response. Collectively, our data revealed that SHMT6 plays an important role in development and defense against pathogens.

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.

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

Hypoxia occurs when 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 (H2O2) 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 the hyponastic response. NO and H2O2 participate in local and systemic signaling pathways that facilitate acclimation to changing energetic requirements, controlling glycolytic fermentation, the γ-aminobutyric acid (GABA) shunt, and amino acid synthesis. NO enhances antioxidant capacity and contributes to the recycling of redox equivalents in energy metabolism through the phytoglobin (Pgb)-NO cycle. Here, we summarize current knowledge of the central role of NO and redox regulation in adaptive responses that prevent hypoxia-induced death in challenging conditions such as flooding.

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.

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.

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.

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.

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.

Alternative oxidase is an important player in the regulation of nitric oxide levels under normoxic and hypoxic conditions in plants.

Plant mitochondria possess two different pathways for electron transport from ubiquinol: the cytochrome pathway and the alternative oxidase (AOX) pathway. The AOX pathway plays an important role in stress tolerance and is induced by various metabolites and signals. Previously, several lines of evidence indicated that the AOX pathway prevents overproduction of superoxide and other reactive oxygen species. More recent evidence suggests that AOX also plays a role in regulation of nitric oxide (NO) production and signalling. The AOX pathway is induced under low phosphate, hypoxia, pathogen infections, and elicitor treatments. The induction of AOX under aerobic conditions in response to various stresses can reduce electron transfer through complexes III and IV and thus prevents the leakage of electrons to nitrite and the subsequent accumulation of NO. Excess NO under various stresses can inhibit complex IV; thus, the AOX pathway minimizes nitrite-dependent NO synthesis that would arise from enhanced electron leakage in the cytochrome pathway. By preventing NO generation, AOX can reduce peroxynitrite formation and tyrosine nitration. In contrast to its function under normoxia, AOX has a specific role under hypoxia, where AOX can facilitate nitrite-dependent NO production. This reaction drives the phytoglobin-NO cycle to increase energy efficiency under hypoxia.

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.

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.

Current approaches to measure nitric oxide in plants.

Nitric oxide (NO) is now established as an important signalling molecule in plants where it influences growth, development, and responses to stress. Despite extensive research, the most appropriate methods to measure and localize these signalling radicals are debated and still need investigation. Many confounding factors such as the 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 sensitive and specific methods in order to understand the contribution of each signalling molecule to various biological processes. In this review, we summarize the current knowledge on NO measurement in plant samples, via various methods. We also discuss advantages, limitations, and wider applications of each method.

Nitrate nutrition influences multiple factors in order to increase energy efficiency under hypoxia in Arabidopsis.

BACKGROUND AND AIMS: Nitrogen (N) levels vary between ecosystems, while the form of available N has a substantial impact on growth, development and perception of stress. Plants have the capacity to assimilate N in the form of either nitrate (NO3-) or ammonium (NH4+). Recent studies revealed that NO3- nutrition increases nitric oxide (NO) levels under hypoxia. When oxygen availability changes, plants need to generate energy to protect themselves against hypoxia-induced damage. As the effects of NO3- or NH4+ nutrition on energy production remain unresolved, this study was conducted to investigate the role of N source on group VII transcription factors, fermentative genes, energy metabolism and respiration under normoxic and hypoxic conditions. METHODS: We used Arabidopsis plants grown on Hoagland medium with either NO3- or NH4+ as a source of N and exposed to 0.8 % oxygen environment. In both roots and seedlings, we investigated the phytoglobin-nitric oxide cycle and the pathways of fermentation and respiration; furthermore, NO levels were tested using a combination of techniques including diaminofluorescein fluorescence, the gas phase Griess reagent assay, respiration by using an oxygen sensor and gene expression analysis by real-time quantitative reverse transcription-PCR methods. KEY RESULTS: Under NO3- nutrition, hypoxic stress leads to increases in nitrate reductase activity, NO production, class 1 phytoglobin transcript abundance and metphytoglobin reductase activity. In contrast, none of these processes responded to hypoxia under NH4+ nutrition. Under NO3- nutrition, a decreased total respiratory rate and increased alternative oxidase capacity and expression were observed during hypoxia. Data correlated with decreased reactive oxygen species and lipid peroxidation levels. Moreover, increased fermentation and NAD+ recycling as well as increased ATP production concomitant with the increased expression of transcription factor genes HRE1, HRE2, RAP2.2 and RAP2.12 were observed during hypoxia under NO3- nutrition. CONCLUSIONS: The results of this study collectively indicate that nitrate nutrition influences multiple factors in order to increase energy efficiency under hypoxia.

Interaction of nitric oxide with the components of the plant mitochondrial electron transport chain.

Mitochondria 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 receiving 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 a 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 a supercomplex with complex III under hypoxia. Complex II is a target for NO which, by inhibiting Fe-S centres, regulates reactive oxygen species (ROS) generation. 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 the 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.

Nitric oxide is essential for the development of aerenchyma in wheat roots under hypoxic stress.

In response to flooding/waterlogging, plants develop various anatomical changes including the formation of lysigenous aerenchyma for the delivery of oxygen to roots. Under hypoxia, plants produce high levels of nitric oxide (NO) but the role of this molecule in plant-adaptive response to hypoxia is not known. Here, we investigated whether ethylene-induced aerenchyma requires hypoxia-induced NO. Under hypoxic conditions, wheat roots produced NO apparently via nitrate reductase and scavenging of NO led to a marked reduction in aerenchyma formation. Interestingly, we found that hypoxically induced NO is important for induction of the ethylene biosynthetic genes encoding ACC synthase and ACC oxidase. Hypoxia-induced NO accelerated production of reactive oxygen species, lipid peroxidation, and protein tyrosine nitration. Other events related to cell death such as increased conductivity, increased cellulase activity, DNA fragmentation, and cytoplasmic streaming occurred under hypoxia, and opposing effects were observed by scavenging NO. The NO scavenger cPTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt) and ethylene biosynthetic inhibitor CoCl2 both led to reduced induction of genes involved in signal transduction such as phospholipase C, G protein alpha subunit, calcium-dependent protein kinase family genes CDPK, CDPK2, CDPK 4, Ca-CAMK, inositol 1,4,5-trisphosphate 5-phosphatase 1, and protein kinase suggesting that hypoxically induced NO is essential for the development of aerenchyma.

Moving nitrogen to the centre of plant defence against pathogens.

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 ( NO3- → NO2- → NH4+ ) 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 NO3- or NH4+ fertilizers affects the outcome of plant-pathogen interactions. NO3- feeding augments hypersensitive response- (HR) mediated resistance, while ammonium nutrition can compromise defence. Metabolically, NO3- enhances production of polyamines such as spermine and spermidine, which are established defence signals, with NH4+ 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 NO2- 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 NO3- dependent and are compromised by NH4+ . CONCLUSION: This review demonstrates how N content and form plays an essential role in defensive primary and secondary metabolism and NO-mediated events.

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.

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.

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.