Interorgan, intraorgan and interplant communication mediated by nitric oxide and related species.

Plant survival to a potential plethora of diverse environmental insults is underpinned by coordinated communication amongst organs to help shape effective responses to these environmental challenges at the whole plant level. This interorgan communication is supported by a complex signal network that regulates growth, development and environmental responses. Nitric oxide (NO) has emerged as a key signalling molecule in plants. However, its potential role in interorgan communication has only recently started to come into view. Direct and indirect evidence has emerged supporting that NO and related species (S-nitrosoglutathione, nitro-linolenic acid) are mobile interorgan signals transmitting responses to stresses such as hypoxia and heat. Beyond their role as mobile signals, NO and related species are involved in mediating xylem development, thus contributing to efficient root-shoot communication. Moreover, NO and related species are regulators in intraorgan systemic defence responses aiming an effective, coordinated defence against pathogens. Beyond its in planta signalling role, NO and related species may act as ex planta signals coordinating external leaf-to-leaf, root-to-leaf but also plant-to-plant communication. Here, we discuss these exciting developments and emphasise how their manipulation may provide novel strategies for crop improvement.

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.

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.

Mitochondrial alternative oxidase pathway helps in nitrooxidative stress tolerance in germinating chickpea.

Mitochondrial 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.

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.

Comparative investigation on chemical and green synthesized titanium dioxide nanoparticles against chromium (VI) stress eliciting differential physiological, biochemical, and cellular attributes in Helianthus annuus L.

Nanotechnology is a new scientific area that promotes unique concepts to comprehend the optimal mechanics of nanoparticles (NPs) in plants under heavy metal stress. The present investigation focuses on effects of synthetic and green synthesized titanium dioxide nanoparticles (TiO2 NPs and gTiO2 NPs) against Cr(VI). Green TiO2 NPs have been produced from plant leaf extract (Ricinus communis L.). Synthesis was confirmed employing an array of optical spectroscopic and electron microscopic techniques. Chromium strongly accelerated H2O2 and MDA productions by 227 % and 266 % at highest chromium concentration (60 mg/kg of soil), respectively, and also caused DNA damage, and decline in photosynthesis. Additionally, anomalies were observed in stomatal cells with gradual increment in chromium concentrations. Conversely, foliar applications of TiO2 NPs and gTiO2 NPs considerably mitigated chromium stress. Sunflower plants treated with modest amounts of green TiO2 NPs had significantly better growth index compared to chemically synthesized ones. Principal component analysis highlighted the variations among photosynthetic attributes, oxidative stress markers, and antioxidant defense systems. Notably, gTiO2 supplementation to the Cr(VI) strained plants minimized PC3 production which is a rare report so far. Conclusively, gTiO2 NPs have been identified to be promising nano-based nutrition resource for farming applications.

Co-overexpression of SWEET sucrose transporters modulates sucrose synthesis and defence responses to enhance immunity against bacterial blight in rice.

Enhancing carbohydrate export from source to sink tissues is considered to be a realistic approach for improving photosynthetic efficiency and crop yield. The rice sucrose transporters OsSUT1, OsSWEET11a and OsSWEET14 contribute to sucrose phloem loading and seed filling. Crucially, Xanthomonas oryzae pv. oryzae (Xoo) infection in rice enhances the expression of OsSWEET11a and OsSWEET14 genes, and causes leaf blight. Here we show that co-overexpression of OsSUT1, OsSWEET11a and OsSWEET14 in rice reduced sucrose synthesis and transport leading to lower growth and yield but reduced susceptibility to Xoo relative to controls. The immunity-related hypersensitive response (HR) was enhanced in the transformed lines as indicated by the increased expression of defence genes, higher salicylic acid content and presence of HR lesions on the leaves. The results suggest that the increased expression of OsSWEET11a and OsSWEET14 in rice is perceived as a pathogen (Xoo) attack that triggers HR and results in constitutive activation of plant defences that are related to the signalling pathways of pathogen starvation. These findings provide a mechanistic basis for the trade-off between plant growth and immunity because decreased susceptibility against Xoo compromised plant growth and yield.

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.

Moderate modulation by S-nitrosoglutathione of photorespiratory enzymes in pea (Pisum sativum) leaves, compared to the strong effects of high light.

When plants are exposed to water stress, photosynthesis is downregulated due to enhanced reactive oxygen species (ROS) and nitric oxide (NO). In contrast, photorespiratory metabolism protected photosynthesis and sustained yield. Modulation of photorespiration by ROS was established, but the effect of NO on photorespiratory metabolism was unclear. We, therefore, examined the impact of externally added NO by using S-nitrosoglutathione (GSNO), a natural NO donor, in leaf discs of pea (Pisum sativum) under dark or light: moderate or high light (HL). Maximum NO accumulation with GSNO was under high light. The presence of 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), a NO scavenger, prevented the increase in NO, confirming the release of NO in leaves. The increase in S-nitrosothiols and tyrosine-nitrated proteins on exposure to GSNO confirmed the nitrosative stress in leaves. However, the changes by GSNO in the activities and transcripts of five photorespiratory enzymes: glycolate oxidase, hydroxypyruvate reductase, catalase, glycerate kinase, and phosphoglycolate phosphatase activities were marginal. The changes in photorespiratory enzymes caused by GSNO were much less than those with HL. Since GSNO caused only mild oxidative stress, we felt that the key modulator of photorespiration might be ROS, but not NO.

Unearthing the secrets of ERFVIIs: new insights into hypoxia signaling.

Group VII ethylene-responsive factor (ERFVII) transcription factors are crucial for the adaption of plants to conditions that limit oxygen availability. A recent study by Zubrycka et al. reveals new aspects of ERFVII stabilization through the PLANT CYSTEINE OXIDASE (PCO)-N degron pathway and non-autonomous regulation in response to different endogenous and exogenous cues.

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.

Lactate Dehydrogenase Superfamily in Rice and Arabidopsis: Understanding the Molecular Evolution and Structural Diversity.

Lactate/malate dehydrogenases (Ldh/Maldh) are ubiquitous enzymes involved in the central metabolic pathway of plants and animals. The role of malate dehydrogenases in the plant system is very well documented. However, the role of its homolog L-lactate dehydrogenases still remains elusive. Though its occurrence is experimentally proven in a few plant species, not much is known about its role in rice. Therefore, a comprehensive genome-wide in silico investigation was carried out to identify all Ldh genes in model plants, rice and Arabidopsis, which revealed Ldh to be a multigene family encoding multiple proteins. Publicly available data suggest its role in a wide range of abiotic stresses such as anoxia, salinity, heat, submergence, cold and heavy metal stress, as also confirmed by our qRT-PCR analysis, especially in salinity and heavy metal mediated stresses. A detailed protein modelling and docking analysis using Schrodinger Suite reveals the presence of three putatively functional L-lactate dehydrogenases in rice, namely OsLdh3, OsLdh7 and OsLdh9. The analysis also highlights the important role of Ser-219, Gly-220 and His-251 in the active site geometry of OsLdh3, OsLdh7 and OsLdh9, respectively. In fact, these three genes have also been found to be highly upregulated under salinity, hypoxia and heavy metal mediated stresses in rice.

Seedling-stage salinity tolerance in rice: Decoding the role of transcription factors.

Rice is an important staple food crop that feeds over half of the human population, particularly in developing countries. Increasing salinity is a major challenge for continuing rice production. Though rice is affected by salinity at all the developmental stages, it is most sensitive at the early seedling stage. The yield thus depends on how many seedlings can withstand saline water at the stage of transplantation, especially in coastal farms. The rapid development of "omics" approaches has assisted researchers in identifying biological molecules that are responsive to salt stress. Several salinity-responsive quantitative trait loci (QTL) contributing to salinity tolerance have been identified and validated, making it essential to narrow down the search for the key genes within QTLs. Owing to the impressive progress of molecular tools, it is now clear that the response of plants toward salinity is highly complex, involving multiple genes, with a specific role assigned to the repertoire of transcription factors (TF). Targeting the TFs for improving salinity tolerance can have an inbuilt advantage of influencing multiple downstream genes, which in turn can contribute toward tolerance to multiple stresses. This is the first comparative study for TF-driven salinity tolerance in contrasting rice cultivars at the seedling stage that shows how tolerant genotypes behave differently than sensitive ones in terms of stress tolerance. Understanding the complexity of salt-responsive TF networks at the seedling stage will be helpful to alleviate crop resilience and prevent crop damage at an early growth stage in rice.

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.

Nitric oxide regulation of plant metabolism.

Nitric 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 responses 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 that 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 also 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.

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.