The tiny giant of the sea, Ostreococcus's unique adaptations.

Ostreococcus spp. are unicellular organisms with one of the simplest cellular organizations. The sequencing of the genomes of different Ostreococcus species has reinforced this status since Ostreococcus tauri has one most compact nuclear genomes among eukaryotic organisms. Despite this, it has retained a number of genes, setting it apart from other organisms with similar small genomes. Ostreococcus spp. feature a substantial number of selenocysteine-containing proteins, which, due to their higher catalytic activity compared to their selenium-lacking counterparts, may require a reduced quantity of proteins. Notably, O. tauri encodes several ammonium transporter genes, that may provide it with a competitive edge for acquiring nitrogen (N). This characteristic makes it an intriguing model for studying the efficient use of N in eukaryotes. Under conditions of low N availability, O. tauri utilizes N from abundant proteins or amino acids, such as L-arginine, similar to higher plants. However, the presence of a nitric oxide synthase (L-arg substrate) sheds light on a new metabolic pathway for L-arg in algae. The metabolic adaptations of O. tauri to day and night cycles offer valuable insights into carbon and iron metabolic configuration. O. tauri has evolved novel strategies to optimize iron uptake, lacking the classic components of the iron absorption mechanism. Overall, the cellular and genetic characteristics of Ostreococcus contribute to its evolutionary success, making it an excellent model for studying the physiological and genetic aspects of how green algae have adapted to the marine environment. Furthermore, given its potential for lipid accumulation and its marine habitat, it may represent a promising avenue for third-generation biofuels.

Redox regulation in primary nitrate response: Nitric oxide in the spotlight.

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.

Nitric Oxide Is Required for Primary Nitrate Response in Arabidopsis: Evidence for S-Nitrosation of NLP7.

Aims: Nitrogen (N) is a necessary nutrient for plant development and seed production, with nitrate (NO3-) serving as the primary source of N in soils. Although several molecular players in plant responses to NO3- 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 NO3--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 NO3- metabolism and a well-characterized signal molecule in plants, is an important player in the PNR.

Nitric oxide synthases from photosynthetic organisms improve growth and confer nitrosative stress tolerance in E. coli. Insights on the pterin cofactor.

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.

Cyanobacterial NOS expression improves nitrogen use efficiency, nitrogen-deficiency tolerance and yield in Arabidopsis.

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.

Nitrogen Depletion Blocks Growth Stimulation Driven by the Expression of Nitric Oxide Synthase in Tobacco.

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. OtNOS 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.

The era of nitric oxide in plant biology: Twenty years tying up loose ends.

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.

A singular nitric oxide synthase with a globin domain found in Synechococcus PCC 7335 mobilizes N from arginine to nitrate.

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.

The NOS-like protein from the microalgae Ostreococcus tauri is a genuine and ultrafast NO-producing enzyme.

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.

Analysis of the Expression and Activity of Nitric Oxide Synthase from Marine Photosynthetic Microorganisms.

Nitric oxide (NO) functions as a signaling molecule in many biological processes in species belonging to all kingdoms of life. In animal cells, NO is synthesized primarily by NO synthase (NOS), an enzyme that catalyze the NADPH-dependent oxidation of L-arginine to NO and L-citrulline. Three NOS isoforms have been identified, the constitutive neuronal NOS (nNOS) and endothelial NOS (eNOS) and one inducible (iNOS). Plant NO synthesis is complex and is a matter of ongoing investigation and debate. Despite evidence of an Arg-dependent pathway for NO synthesis in plants, no plant NOS homologs to animal forms have been identified to date. In plants, there is also evidence for a nitrate-dependent mechanism of NO synthesis, catalyzed by cytosolic nitrate reductase. The existence of a NOS enzyme in the plant kingdom, from the tiny single-celled green alga Ostreococcus tauri was reported in 2010. O. tauri shares a common ancestor with higher plants and is considered to be part of an early diverging class within the green plant lineage.In this chapter we describe detailed protocols to study the expression and characterization of the enzymatic activity of NOS from O. tauri. The most used methods for the characterization of a canonical NOS are the analysis of spectral properties of the oxyferrous complex in the heme domain, the oxyhemoglobin (oxyHb) and citrulline assays and the NADPH oxidation for in vitro analysis of its activity or the use of fluorescent probes and Griess assay for in vivo NO determination. We further discuss the advantages and drawbacks of each method. Finally, we remark factors associated to the measurement of NOS activity in photosynthetic organisms that can generate misunderstandings in the interpretation of results.

Expression of the tetrahydrofolate-dependent nitric oxide synthase from the green alga Ostreococcus tauri increases tolerance to abiotic stresses and influences stomatal development in Arabidopsis.

Nitric oxide (NO) is a signaling molecule with diverse biological functions in plants. NO plays a crucial role in growth and development, from germination to senescence, and is also involved in plant responses to biotic and abiotic stresses. In animals, NO is synthesized by well-described nitric oxide synthase (NOS) enzymes. NOS activity has also been detected in higher plants, but no gene encoding an NOS protein, or the enzymes required for synthesis of tetrahydrobiopterin, an essential cofactor of mammalian NOS activity, have been identified so far. Recently, an NOS gene from the unicellular marine alga Ostreococcus tauri (OtNOS) has been discovered and characterized. Arabidopsis thaliana plants were transformed with OtNOS under the control of the inducible short promoter fragment (SPF) of the sunflower (Helianthus annuus) Hahb-4 gene, which responds to abiotic stresses and abscisic acid. Transgenic plants expressing OtNOS accumulated higher NO concentrations compared with siblings transformed with the empty vector, and displayed enhanced salt, drought and oxidative stress tolerance. Moreover, transgenic OtNOS lines exhibited increased stomatal development compared with plants transformed with the empty vector. Both in vitro and in vivo experiments indicate that OtNOS, unlike mammalian NOS, efficiently uses tetrahydrofolate as a cofactor in Arabidopsis plants. The modulation of NO production to alleviate abiotic stress disturbances in higher plants highlights the potential of genetic manipulation to influence NO metabolism as a tool to improve plant fitness under adverse growth conditions.

Nitric oxide is a ubiquitous signal for maintaining redox balance in plant cells: regulation of ascorbate peroxidase as a case study.

Oxidative and nitrosative stresses and their respective antioxidant responses are common metabolic adjustments operating in all biological systems. These stresses result from an increase in reactive oxygen species (ROS) and reactive nitrogen species (RNS) and an imbalance in the antioxidant response. Plants respond to ROS and RNS accumulation by increasing the level of the antioxidant molecules glutathione and ascorbate and by activating specific antioxidant enzymes. Nitric oxide (NO) is a free radical considered to be toxic or protective depending on its concentration, combination with ROS compounds, and subcellular localization. In this review we focus on the mechanisms of NO action in combination with ROS on the regulation of the antioxidant system in plants. In particular, we describe the redox post-translational modifications of cytosolic ascorbate peroxidase and its influence on enzyme activity. The regulation of ascorbate peroxidase activity by NO as a redox sensor of acute oxidative stress or as part of a hormone-induced signalling pathway leading to lateral root development is presented and discussed.

Auxin induces redox regulation of ascorbate peroxidase 1 activity by S-nitrosylation/denitrosylation balance resulting in changes of root growth pattern in Arabidopsis.

S-Nitrosylation of Cys residues is one of the molecular mechanisms driven by nitric oxide (NO) for regulating biological functions of key proteins. While the studies on S-nitrosylation of Cys residues have served for identifying SNO proteomes, the physiological relevance of protein S-nitrosylation/denitrosylation remains poorly understood. In this study, it is shown that auxin influences the balance of S-nitrosylated/denitrosylated proteins in roots of Arabidopsis seedlings. 2D-PAGE allowed the identification of ascorbate peroxidase 1 (APX1) as target of auxin-induced denitrosylation in roots. Auxin causes APX1 denitrosylation and partial inhibition of APX1 activity in Arabidopsis roots. In agreement, the S-nitrosylated form of recombinant APX1 expressed in Escherichia coli is more active than the denitrosylated form. Consistently, Arabidopsis apx1 mutants have increased H2O2 accumulation in roots, shorter roots, and less sensitivity to auxin than the wild type. It is postulated that an auxin-regulated counterbalance of APX1 S-nitrosylation/denitrosylation contributes to a fine-tuned control of root development and determination of root architecture.

Characterization of a nitric oxide synthase from the plant kingdom: NO generation from the green alga Ostreococcus tauri is light irradiance and growth phase dependent.

The search for a nitric oxide synthase (NOS) sequence in the plant kingdom yielded two sequences from the recently published genomes of two green algae species of the Ostreococcus genus, O. tauri and O. lucimarinus. In this study, we characterized the sequence, protein structure, phylogeny, biochemistry, and expression of NOS from O. tauri. The amino acid sequence of O. tauri NOS was found to be 45% similar to that of human NOS. Folding assignment methods showed that O. tauri NOS can fold as the human endothelial NOS isoform. Phylogenetic analysis revealed that O. tauri NOS clusters together with putative NOS sequences of a Synechoccocus sp strain and Physarum polycephalum. This cluster appears as an outgroup of NOS representatives from metazoa. Purified recombinant O. tauri NOS has a K(m) for the substrate l-Arg of 12 ± 5 µM. Escherichia coli cells expressing recombinant O. tauri NOS have increased levels of NO and cell viability. O. tauri cultures in the exponential growth phase produce 3-fold more NOS-dependent NO than do those in the stationary phase. In O. tauri, NO production increases in high intensity light irradiation and upon addition of l-Arg, suggesting a link between NOS activity and microalgal physiology.

Phosphatidic acid formation is required for extracellular ATP-mediated nitric oxide production in suspension-cultured tomato cells.

*In animals and plants, extracellular ATP exerts its effects by regulating the second messengers Ca(2+), nitric oxide (NO) and reactive oxygen species (ROS). In animals, phospholipid-derived molecules, such as diacylglycerol, phosphatidic acid (PA) and inositol phosphates, have been associated with the extracellular ATP signaling pathway. The involvement of phospholipids in extracellular ATP signaling in plants, as it is established in animals, is unknown. *In vivo phospholipid signaling upon extracellular ATP treatment was studied in (32)P(i)-labeled suspension-cultured tomato (Solanum lycopersicum) cells. *Here, we report that, in suspension-cultured tomato cells, extracellular ATP induces the formation of the signaling lipid phosphatidic acid. Exogenous ATP at doses of 0.1 and 1 mM induce the formation of phosphatidic acid within minutes. Studies on the enzymatic sources of phosphatidic acid revealed the participation of both phospholipase D and C in concerted action with diacylglycerol kinase. *Our results suggest that extracellular ATP-mediated nitric oxide production is downstream of phospholipase C/diacylglycerol kinase activation.