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ABSTRACTNitric oxide (NO) is a bioactive molecule that exerts a number of diverse activities in phylogenetically distant species, as well as opposing effects in related biological systems. It was firstly described in mammals as a major messenger in the cardiovascular, immune and nervous system, in which it plays regulatory, signalling, cytoprotective and cytotoxic effects (Ignarro, Annual Review of Pharmacology and Toxicology 30, 535–560, 1990; Anbar, Experientia 51, 545–550, 1995). This versatility is mainly achieved through interactions with targets via either a redox or an additive chemistry (Stamler, Cell 78, 931–936, 1994). For this reason, metal- and thiol-containing proteins serve as major target sites for NO: these include signalling proteins, receptors, enzymes, transcription factors and DNA, among others. Furthermore, NO is a small, highly diffusible molecule. It rapidly crosses biological membranes and triggers various different processes in a short period of time. In this context, NO can co-ordinate and regulate cellular functions of microsomes and organelles such as mitochondria. The ubiquity of NO reactions, as well as the finding that the biochemical and molecular mechanisms underlying many physiological processes are well conserved between diverse species, have opened the exploration of NO chemistry in different organisms. Among these, plants were not the exception. The research in plants has been focused on three main fields: (i) the search for NO or any source of NO generation; (ii) the examination of the effects of NO upon exogenous treatments; and (iii) the search for the same molecules involved in NO-sensitive transduction pathways as in animals (e.g. cGMP, Ca2+, calmodulin). As it is evident from this review, recent progress on NO functionality in plants has been impressive. With the use of biochemistry, molecular genetics and structural biology, together with classical physiological approaches, an explosion of new discoveries will surely begin. It is certainly a good time for plant biologists. [ABSTRACT FROM AUTHOR]

Presents research demonstrating that RNA editing is also required for the correct expression of plant mitochondrial genes. Research methods.

Nitrogen (N) is the main macronutrient of plants that determines growth and productivity. Nitrate is the major source form of N in soils and its uptake and assimilatory pathway has been extensively studied. The early events that occur after the perception of nitrate is known as primary nitrate response (PNR). In this review, new findings on the redox signal that impacts PNR are discussed. We will focus on the novel role of Nitric Oxide (NO) as a signal molecule and the mechanisms that are involved to control NO homeostasis during PNR. Moreover, the role of Reactive Oxygen Species (ROS) and the possible interplay with NO in the PNR are discussed. The sources of NO during PNR will be analyzed as well as the regulation of its intracellular levels. Furthermore, we explored the relevance of the direct action of NO through the S-nitrosation of the transcription factor NLP7, one of the master regulators in the nitrate signaling cascade. This review gives rise to an interesting field with new actors to mark future research directions. This allows us to increase the knowledge of the physiological and molecular fine-tuned modulation during nitrate signaling processes in plants. The discussion of new experimental data will stimulate efforts to further refine our understanding of the redox regulation of nitrate signaling., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

The development of novel biotechnologies that promote a better use of N to optimize crop yield is a central goal for sustainable agriculture. Phytostimulation, biofertilization, and bioprotection through the use of bio-inputs are promising technologies for this purpose. In this study, the plant growth-promoting rhizobacteria Pseudomonas koreensis MME3 was genetically modified to express a nitric oxide synthase of Synechococcus SyNOS, an atypical enzyme with a globin domain that converts nitric oxide to nitrate. A cassette for constitutive expression of synos was introduced as a single insertion into the genome of P. koreensis MME3 using a miniTn7 system. The resulting recombinant strain MME3:SyNOS showed improved growth, motility, and biofilm formation. The impact of MME3:SyNOS inoculation on Brachypodium distachyon growth and N uptake and use efficiencies under different N availability situations was analyzed, in comparison to the control strain MME3:c. After 35 days of inoculation, plants treated with MME3:SyNOS had a higher root dry weight, both under semi-hydroponic and greenhouse conditions. At harvest, both MME3:SyNOS and MME3:c increased N uptake and use efficiency of plants grown under low N soil. Our results indicate that synos expression is a valid strategy to boost the phytostimulatory capacity of plant-associated bacteria and improve the adaptability of plants to N deficiency. KEY POINTS: • synos expression improves P. koreensis MME3 traits important for rhizospheric colonization • B. distachyon inoculated with MME3:SyNOS shows improved root growth • MME3 inoculation improves plant N uptake and use efficiencies in N-deficient soil., (© 2024. The Author(s).)

Aims: Nitrogen (N) is a necessary nutrient for plant development and seed production, with nitrate (NO 3 - ) serving as the primary source of N in soils. Although several molecular players in plant responses to NO 3 - signaling were unraveled, it is still a complex process with gaps that require further investigation. The aim of our study is to analyze the role of nitric oxide (NO) in the primary nitrate response (PNR). Results: Using a combination of genetic and pharmacological approaches, we demonstrate that NO is required for the expression of the NO 3 - -regulated genes nitrate reductase 1 ( NIA1 ), nitrite reductase ( NIR ), and nitrate transporters (nitrate transporter 1.1 [ NRT1.1 ] and nitrate transporter 2.1 [ NRT2.1 ]) in Arabidopsis. The PNR is impaired in the Arabidopsis mutant noa1 , defective in NO production. Our results also show that PHYTOGLOBIN 1 ( PHYTOGLB1 ), involved in NO homeostasis, is rapidly induced during PNR in wild type (wt) but not in the mutants of the nitrate transceptor NTR1.1 and the transcription factor nodule inception-like protein 7 (NLP7), suggesting that the NRT1.1-NLP7 cascade modulates PHYTOGLB1 gene expression. Biotin switch experiments demonstrate that NLP7, the PNR-master regulator, is S-nitrosated in vitro . Depletion of NO during PNR intensifies the decrease in reactive oxygen species levels and the rise of catalase (CAT) and ascorbate peroxidase (APX) enzyme activity. Conclusion and Innovation: NO, a by-product of NO 3 - metabolism and a well-characterized signal molecule in plants, is an important player in the PNR.

Nitric oxide synthase (NOS) catalyzes NO formation from the substrate l-arginine (Arg). Previously, NOS with distinct biochemical properties were characterized from two photosynthetic microorganisms, the unicellular algae Ostreococcus tauri (OtNOS) and the cyanobacteria Synechococcus PCC 7335 (SyNOS). In this work we studied the effect of recombinant OtNOS and SyNOS expressed under IPTG-induced promoter in E. coli, a bacterium that lacks NOS. Results show that OtNOS and SyNOS expression promote E. coli growth in a nutrient replete medium and allow to better metabolize Arg as N source. In LB medium, OtNOS induces the expression of the NO dioxygenase hmp in E. coli, in accordance with high NO levels visualized with the probe DAF-FM DA. In contrast, SyNOS expression does not induce hmp and show a slight increase of NO production compared to OtNOS. NOS expression reduces ROS production and increases viability of E. coli cultures growing in LB. A strong nitrosative stress provoked by the addition of 1 mM of the NO donors sodium nitroprusside (SNP) and nitrosoglutathione (GSNO) inhibits bacterial growth rate. Under these conditions, the expression of OtNOS or SyNOS counteracts NO donor toxicity restoring bacterial growth. Finally, using bioinformatic tools and ligand docking analyses, we postulate that tetrahydromonapterin (MH4), an endogenous pterin found in E. coli, could act as cofactor required for NOS catalytic activity. Our findings could be useful for the development of biotechnological applications using NOS expression to improve growth in NOS-lacking bacteria., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Developing strategies to improve nitrogen (N) use efficiency (NUE) in plants is a challenge to reduce environmental problems linked to over-fertilization. The nitric oxide synthase (NOS) enzyme from the cyanobacteria Synechococcus PCC 7335 (SyNOS) has been recently identified and characterized. SyNOS catalyzes the conversion of arginine to citrulline and nitric oxide (NO), and then approximately 75 % of the produced NO is rapidly oxidized to nitrate by an unusual globin domain in the N-terminus of the enzyme. In this study, we assessed whether SyNOS expression in plants affects N metabolism, NUE and yield. Our results showed that SyNOS-expressing transgenic Arabidopsis plants have greater primary shoot length and shoot branching when grown under N-deficient conditions and higher seed production both under N-sufficient and N-deficient conditions. Moreover, transgenic plants showed significantly increased NUE in both N conditions. Although the uptake of N was not modified in the SyNOS lines, they showed an increase in the assimilation/remobilization of N under conditions of low N availability. In addition, SyNOS lines have greater N-deficiency tolerance compared to control plants. Our results support that SyNOS expression generates a positive effect on N metabolism and seed production in Arabidopsis, and it might be envisaged as a strategy to improve productivity in crops under adverse N environments., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Quantitative RT- PCR is one of the most common methods to study gene expression in response to stress. Therefore, it is crucial to have suitable reference genes (RGs) for result normalization. Although several reports describe UV-B-modulated gene expression in Solanum lycopersicum, there are no suitable RGs identified until now. The aim of this work was to evaluate the suitability of seven traditional genes: actin (ACT), tubulin (TUB), ubiquitin (UBI), glyceraldehyde- 3 phosphate dehydrogenase (GAPDH), elongation factor 1α (EF1α), phosphatase 2A catalytic subunit (PP2A) and GAGA binding transcriptional activator (GAGA); and two non-traditional genes: thioredoxin h1 (TRX h1) and UV-B RESISTANCE LOCUS 8 (UVR8), as candidate RGs for their potential use as reliable internal controls in leaves, stems and roots of tomato seedlings exposed to acute and chronic UV-B. The stability of these genes expression was evaluated using five statistical algorithms: geNorm, NormFinder, BestKeeper, Delta Ct and ANOVA. Considering the comprehensive stability ranking, we recommend ACT+TUB as the best pair of RGs for leaves, PP2A+GAPDH+TRX h1 for stems and TUB+UVR8 for roots. The reliability of the selected RGs for each tissue was verified amplifying tomato chalcone synthase 1 (CHS1) and cyclobutane pyrimidine dimer (CPD) photolyase (PHR1-LIKE). Under UV-B treatment, CHS1 was upregulated in leaves, stems and roots whereas PHR1-LIKE was only upregulated in leaves and stems. This interpretation differs when the most and least stable RGs are chosen. This is the first report regarding suitable RGs selection for accurate normalization of gene expression in tomato seedlings exposed to UV-B irradiation., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

In temperate climates, low ambient temperatures in late winter and in spring can result in cold stress conditions in brood areas of weakened honey bee colonies, leading to increased levels of developmental interruptions and death of the brood. Very little is known about the physiological and molecular mechanisms that regulate honey bee brood responses to acute cold-stress. Here, we hypothesized that central regulatory pathways mediated by insulin/insulin-like peptide signalling (IIS) and adipokinetic hormone (AKH) are linked to metabolic changes in cold-stressed honey bee brood. A. mellifera brood reared at suboptimal temperatures showed diminished growth rate and arrested development progress. Notably, cold-stressed brood rapidly recovers the growth in the first 24 h after returning at control rearing temperature, sustained by the induction of compensatory mechanisms. We determined fast changes in the expression of components of IIS and AKH pathways in cold-stressed brood supporting their participation in metabolic events, growth and stress responses. We also showed that metabolic rate keeps high in brood exposed to stress suggesting a role in energy supply for growth and cell repair. Additionally, transcript levels of the uncoupling protein MUP2 were elevated in cold-stressed brood, which could indicate that this protein acts in the heat generation through mitochondrial decoupling mechanisms and/or in the ROS attenuation. Physiological, metabolic and molecular mechanisms that shape the responses to cold-stress in honey bee brood are addressed and discussed., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Nitric oxide (NO) is a messenger molecule widespread studied in plant physiology. Latter evidence supports the lack of a NO-producing system involving a NO synthase (NOS) activity in higher plants. However, a NOS gene from the unicellular marine alga Ostreococcus tauri ( OtNOS ) was characterized in recent years. Ot NOS is a genuine NOS, with similar spectroscopic fingerprints to mammalian NOSs and high NO producing capacity. We are interested in investigating whether OtNOS activity alters nitrogen metabolism and nitrogen availability, thus improving growth promotion conditions in tobacco. Tobacco plants were transformed with OtNOS under the constitutive CaMV 35S promoter. Transgenic tobacco plants expressing OtNOS accumulated higher NO levels compared to siblings transformed with the empty vector, and displayed accelerated growth in different media containing sufficient nitrogen availability. Under conditions of nitrogen scarcity, the growth promoting effect of the OtNOS expression is diluted in terms of total leaf area, protein content and seed production. It is proposed that OtNOS might possess a plant growth promoting effect through facilitating N remobilization and nitrate assimilation with potential to improve crop plants performance., (Copyright © 2020 Nejamkin, Foresi, Mayta, Lodeyro, Del Castello, Correa-Aragunde, Carrillo and Lamattina.)

The high annual losses of managed honey bees ( Apis mellifera ) has attracted intensive attention, and scientists have dedicated much effort trying to identify the stresses affecting bees. There are, however, no simple answers; rather, research suggests multifactorial effects. Several works have been reported highlighting the relationship between bees' immunosuppression and the effects of malnutrition, parasites, pathogens, agrochemical and beekeeping pesticides exposure, forage dearth and cold stress. Here we analyze a possible connection between immunity-related signaling pathways that could be involved in the response to the stress resulted from Varroa -virus association and cold stress during winter. The analysis was made understanding the honey bee as a superorganism, where individuals are integrated and interacting within the colony, going from social to individual immune responses. We propose the term "Precision Nutrition" as a way to think and study bees' nutrition in the search for key molecules which would be able to strengthen colonies' responses to any or all of those stresses combined.

In temperate climates, beekeeping operations suffer colony losses and colony depopulation of Apis mellifera during overwintering, which are associated with biotic and abiotic stressors that impact bees' health. In this work, we evaluate the impacts of abscisic acid (ABA) dietary supplementation on honey bee colonies kept in Langstroth hives. The effects of ABA were evaluated in combination with two different beekeeping nutritional strategies to confront overwintering: "honey management" and "syrup management". Specifically, we evaluated strength parameters of honey bee colonies (adult bee and brood population) and the population dynamics of Nosema (prevalence and intensity) associated with both nutritional systems and ABA supplementation during the whole study (late autumn-winter-early spring). The entire experiment was designed and performed with a local group of beekeepers, "Azahares del sudeste", who showed interest in answering problems associated with the management of honey bee colonies during the winter. The results indicated that the ABA supplementation had positive effects on the population dynamics of the A. mellifera colonies during overwintering and on the nosemosis at colony level (prevalence) in both nutritional strategies evaluated.

Nitric oxide (NO) is an essential signal molecule to maintain cellular homeostasis in uni and pluricellular organisms. Conceptually, NO intervenes as much in sustaining basal metabolic processes, as in firing cellular responses to changes in internal and external conditions, and also in guiding the return to basal conditions. Behind these unusual capabilities of NO is the chemistry of this molecule, an unstable, reactive, free radical and short half-life gas. It is a lipophilic molecule that crosses all the barriers that biological membranes can impose. The extraordinary impact that the elucidation of physiological processes regulated by NO has had on plants, is comparable to the consequences of the discovery in 1986 that NO is present in animal tissues, and the following deep studies that demonstrated its biological activity regulating blood pressure. In this review, we have summarized and discuss the main discoveries that have emerged at Mar del Plata University over the past 20 years, and that have contributed to understand part of the biology of NO in plants. Besides, these findings are put in context with the progress made by other research groups, and in perspective, emphasizing that the history of NO in plants has just begun., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Abscisic acid (ABA) and nitric oxide (NO) are two plant growth regulators that participate in many signaling cascades in different organs all along the plant life. Here, we were interested in deciphering the effects of ABA and NO on the cytoskeleton organization in a model of polarized cell growth like root hairs. Arabidopsis roots were exposed to different concentrations of ABA, and the length of primary root, epidermal cells and root hairs were measured. The NO concentration was detected with the NO-specific fluorescent probe DAF-FM DA. To quantify the effects of ABA and NO on cytoskeleton, Arabidopsis seedlings expressing GFP-MAP4 were used to analyze microtubules (MTs) orientation. Changes in cytoplasmic streaming were quantified through fluorescence recovery after photobleaching (FRAP) experiments using confocal laser scanning microscopy (CLSM) and the probe fluorescein diacetate (FDA). Results indicate that ABA decreases root hair length and induces the differentiation of atrichoblasts into trichoblasts, increasing root hair density. ABA also triggers an increase of NO level in root hairs. Both, ABA and NO affect MT organization in root hairs. While root hairs show MT orientation close to the longitudinal axis in control roots, ABA and NO treatments induce the oblique orientation of MTs. In parallel, cytoplasmic flow, executed by actin cytoskeleton, is enhanced by NO, in an ABA-independent manner. For all experimental conditions assayed, basal levels of NO are required to keep MT organization and cytoplasmic streaming. Our findings support ABA and NO as key modulators of growth and ectopic formation of root hairs through actions on cytoskeleton functions., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The F-box proteins (FBPs) TIR1/AFBs are the substrate recognition subunits of SKP1-cullin-F-box (SCF) ubiquitin ligase complexes and together with Aux/IAAs form the auxin co-receptor. Although tremendous knowledge on auxin perception and signaling has been gained in the last years, SCF TIR1/AFBs complex assembly and stabilization are emerging as new layers of regulation. Here, we investigated how nitric oxide (NO), through S-nitrosylation of ASK1 is involved in SCF TIR1/AFBs assembly. We demonstrate that ASK1 is S-nitrosylated and S-glutathionylated in cysteine (Cys) 37 and Cys118 residues in vitro. Both, in vitro and in vivo protein-protein interaction assays show that NO enhances ASK1 binding to CUL1 and TIR1/AFB2, required for SCF TIR1/AFB2 assembly. In addition, we demonstrate that Cys37 and Cys118 are essential residues for proper activation of auxin signaling pathway in planta. Phylogenetic analysis revealed that Cys37 residue is only conserved in SKP proteins in Angiosperms, suggesting that S-nitrosylation on Cys37 could represent an evolutionary adaption for SKP1 function in flowering plants. Collectively, these findings indicate that multiple events of redox modifications might be part of a fine-tuning regulation of SCF TIR1/AFBs for proper auxin signal transduction., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

The enzyme nitric oxide synthase (NOS) oxidizes L-arginine to NO and citrulline. In this work, we characterise the NOS from the cyanobacteria Synechococcus PCC 7335 (SyNOS). SyNOS possesses a canonical mammalian NOS architecture consisting of oxygenase and reductase domains. In addition, SyNOS possesses an unusual globin domain at the N-terminus. Recombinant SyNOS expressed in bacteria is active, and its activity is suppressed by the NOS inhibitor L-NAME. SyNOS allows E. coli to grow in minimum media containing L-arginine as the sole N source, and has a higher growth rate during N deficiency. SyNOS is expressed in Synechococcus PCC 7335 where NO generation is dependent on L-arginine concentration. The growth of Synechococcus is dramatically inhibited by L-NAME, suggesting that SyNOS is essential for this cyanobacterium. Addition of arginine in Synechococcus increases the phycoerythrin content, an N reservoir. The role of the novel globin domain in SyNOS is discussed as an evolutionary advantage, conferring new functional capabilities for N metabolism.

Main Conclusion: The phytotoxin botrydial triggers PA production in tomato cell suspensions via PLD and PLC/DGK activation. PLC/DGK-derived PA is partially required for botrydial-induced ROS generation. Phosphatidic acid (PA) is a phospholipid second messenger involved in the induction of plant defense responses. It is generated via two distinct enzymatic pathways, either via phospholipase D (PLD) or by the sequential action of phospholipase C and diacylglycerol kinase (PLC/DGK). Botrydial is a phytotoxic sesquiterpene generated by the necrotrophic fungus Botrytis cinerea that induces diverse plant defense responses, such as the production of reactive oxygen species (ROS). Here, we analyzed PA and ROS production and their interplay upon botrydial treatments, employing tomato (Solanum lycopersicum) cell suspensions as a model system. Botrydial induces PA production within minutes via PLD and PLC/DGK. Either inhibition of PLC or DGK diminishes ROS generation triggered by botrydial. This indicates that PLC/DGK is upstream of ROS production. In tomato, PLC is encoded by a multigene family constituted by SlPLC1-SlPLC6 and the pseudogene SlPLC7. We have shown that SlPLC2-silenced plants have reduced susceptibility to B. cinerea. In this work, we studied the role of SlPLC2 on botrydial-induced PA production by silencing the expression of SlPLC2 via a specific artificial microRNA. Upon botrydial treatments, SlPLC2-silenced-cell suspensions produce PA levels similar to wild-type cells. It can be concluded that PA is a novel component of the plant responses triggered by botrydial.

Here, we review information on how plants face redox imbalance caused by climate change, and focus on the role of nitric oxide (NO) in this response. Life on Earth is possible thanks to greenhouse effect. Without it, temperature on Earth's surface would be around -19°C, instead of the current average of 14°C. Greenhouse effect is produced by greenhouse gasses (GHG) like water vapor, carbon dioxide (CO 2 ), methane (CH 4 ), nitrous oxides (N x O) and ozone (O 3 ). GHG have natural and anthropogenic origin. However, increasing GHG provokes extreme climate changes such as floods, droughts and heat, which induce reactive oxygen species (ROS) and oxidative stress in plants. The main sources of ROS in stress conditions are: augmented photorespiration, NADPH oxidase (NOX) activity, β-oxidation of fatty acids and disorders in the electron transport chains of mitochondria and chloroplasts. Plants have developed an antioxidant machinery that includes the activity of ROS detoxifying enzymes [e.g., superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), glutathione peroxidase (GPX), and peroxiredoxin (PRX)], as well as antioxidant molecules such as ascorbic acid (ASC) and glutathione (GSH) that are present in almost all subcellular compartments. CO 2 and NO help to maintain the redox equilibrium. Higher CO 2 concentrations increase the photosynthesis through the CO 2 -unsaturated Rubisco activity. But Rubisco photorespiration and NOX activities could also augment ROS production. NO regulate the ROS concentration preserving balance among ROS, GSH, GSNO, and ASC. When ROS are in huge concentration, NO induces transcription and activity of SOD, APX, and CAT. However, when ROS are necessary (e.g., for pathogen resistance), NO may inhibit APX, CAT, and NOX activity by the S-nitrosylation of cysteine residues, favoring cell death. NO also regulates GSH concentration in several ways. NO may react with GSH to form GSNO, the NO cell reservoir and main source of S-nitrosylation. GSNO could be decomposed by the GSNO reductase (GSNOR) to GSSG which, in turn, is reduced to GSH by glutathione reductase (GR). GSNOR may be also inhibited by S-nitrosylation and GR activated by NO. In conclusion, NO plays a central role in the tolerance of plants to climate change.

Hydrogen sulfide (H 2 S) is an important gaseous signaling molecule in plants that participates in stress responses and development. l-Cys desulfhydrase 1, one of the enzymatic sources of H 2 S in plants, participates in abscisic acid-induced stomatal closure. We combined pharmacological and genetic approaches to elucidate the involvement of H 2 S in stomatal closure and the interplay between H 2 S and other second messengers of the guard cell signaling network, such as hydrogen peroxide (H 2 O 2 ) and phospholipase D (PLD)-derived phosphatidic acid in Arabidopsis ( Arabidopsis thaliana ). Both NADPH oxidase isoforms, respiratory burst oxidase homolog (RBOH)D and RBOHF, were required for H 2 S-induced stomatal closure. In vivo imaging using the cytosolic ratiometric fluorescent biosensor roGFP2-Orp1 revealed that H 2 S stimulates H 2 O 2 production in Arabidopsis guard cells. Additionally, we observed an interplay between H 2 S and PLD activity in the regulation of reactive oxygen species production and stomatal movement. The PLDα1 and PLDδ isoforms were required for H 2 S-induced stomatal closure, and most of the H 2 S-dependent H 2 O 2 production required the activity of PLDα1. Finally, we showed that H 2 S induced increases in the PLDδ-derived phosphatidic acid levels in guard cells. Our results revealed the involvement of H 2 S in the signaling network that controls stomatal closure, and suggest that H 2 S regulates NADPH oxidase and PLD activity in guard cells., (© 2018 American Society of Plant Biologists. All Rights Reserved.)

The exponential increase of genomes' sequencing has revealed the presence of NO-Synthases (NOS) throughout the tree of life, uncovering an extraordinary diversity of genetic structure and biological functions. Although NO has been shown to be a crucial mediator in plant physiology, NOS sequences seem present solely in green algae genomes, with a first identification in the picoplankton species Ostreococcus tauri. There is no rationale so far to account for the presence of NOS in this early-diverging branch of the green lineage and its absence in land plants. To address the biological function of algae NOS, we cloned, expressed and characterized the NOS oxygenase domain from Ostreococcus tauri (OtNOSoxy). We launched a phylogenetic and structural analysis of algae NOS, and achieved a 3D model of OtNOSoxy by homology modeling. We used a combination of various spectroscopies to characterize the structural and electronic fingerprints of some OtNOSoxy reaction intermediates. The analysis of OtNOSoxy catalytic activity and kinetic efficiency was achieved by stoichiometric stopped-flow. Our results highlight the conserved and particular features of OtNOSoxy structure that might explain its ultrafast NO-producing capacity. This integrative Structure-Catalysis-Function approach could be extended to the whole NOS superfamily and used for predicting potential biological activity for any new NOS., (Copyright © 2017 Elsevier B.V. All rights reserved.)