Natalia Correa-Aragunde

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

This study characterizes a nitric oxide synthase (NOS) from Ostreococcus tauri, a marine green alga. Like animal NOS sequences, O. tauri NOS contains an oxygenase and reductase domain. The recombinant protein is a functional enzyme. NO production in O. tauri cell cultures is induced in the presence of the NOS substrate l-Arg and 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 Km 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.

Arginase-Negative Mutants of Arabidopsis Exhibit Increased Nitric Oxide Signaling in Root Development

Mutation of either arginase structural gene (ARGAH1 or ARGAH2 encoding arginine [Arg] amidohydrolase-1 and -2, respectively) resulted in increased formation of lateral and adventitious roots in Arabidopsis (Arabidopsis thaliana) seedlings and increased nitric oxide (NO) accumulation and efflux, detected by the fluorogenic traps 3-amino,4-aminomethyl-2′,7′-difluorofluorescein diacetate and diamino-rhodamine-4M, respectively. Upon seedling exposure to the synthetic auxin naphthaleneacetic acid, NO accumulation was differentially enhanced in argah1-1 and argah2-1 compared with the wild type. In all genotypes, much 3-amino,4-aminomethyl-2′,7′-difluorofluorescein diacetate fluorescence originated from mitochondria. The arginases are both localized to the mitochondrial matrix and closely related. However, their expression levels and patterns differ: ARGAH1 encoded the minor activity, and ARGAH1-driven β-glucuronidase (GUS) was expressed throughout the seedling; the ARGAH2∷GUS expression pattern was more localized. Naphthaleneacetic acid increased seedling lateral root numbers (total lateral roots per primary root) in the mutants to twice the number in the wild type, consistent with increased internal NO leading to enhanced auxin signaling in roots. In agreement, argah1-1 and argah2-1 showed increased expression of the auxin-responsive reporter DR5∷GUS in root tips, emerging lateral roots, and hypocotyls. We propose that Arg, or an Arg derivative, is a potential NO source and that reduced arginase activity in the mutants results in greater conversion of Arg to NO, thereby potentiating auxin action in roots. This model is supported by supplemental Arg induction of adventitious roots and increased NO accumulation in argah1-1 and argah2-1 versus the wild type.

Nitric oxide: an active nitrogen molecule that modulates cellulose synthesis in tomato roots

Nitric oxide (NO) is a bioactive molecule involved in several growth and developmental processes in plants. These processes are mostly characterized by changes in primary and secondary metabolism. Here, the effect of NO on cellulose synthesis in tomato (Solanum lycopersicum) roots was studied. The phenotype of roots, cellulose content, the incorporation of 14C-glucose into cellulosic fraction and the expression of tomato cellulose synthase (CESA) transcripts in roots treated with the NO donor sodium nitroprusside (SNP) were analysed. Nitric oxide affected cellulose content in roots in a dose dependent manner. Low concentrations of SNP (pmoles of NO) increased cellulose content in roots while higher concentrations of SNP (nmoles of NO) had the opposite effect. This result correlated with assays of 14C-glucose incorporation into cellulose in roots. The effect of NO on 14C-glucose incorporation into cellulose was transient and reversible. Microscopic analysis of roots suggested that NO affected primary cell wall cellulose synthesis. Three tomato cellulose synthase (SICESA) transcripts were identified. Reverse transcriptase polymerase chain reaction experiments were carried out and indicated that SICESA1 and SICESA3 levels were affected by high NO concentrations. Together, these results support the hypothesis that variations in NO levels influence cellulose synthesis and content in roots.

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.

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.

Nitric Oxide and Plant Growth Promoting Rhizobacteria: Common Features Influencing Root Growth and Development

Abstract Nitric oxide (NO) is a gas produced by prokaryotes and eukaryotes as part of their N metabolism that profoundly influences the physiology of the cells. In plants, the biological implications of NO as a signal molecule modulating physiological responses have been elucidated in the last decade. The NO action as an intermediary in auxin‐regulated signaling cascades influencing root growth and developmental processes is probably one of the most important functions in plant biology. Here we describe the signaling pathways and the cellular messengers involved in the NO induction of adventitious root formation, lateral root development, and root hair formation. We also review the first evidence supporting the NO role in the induction of adventitious and lateral root development by plant growth promoting rhizobacteria (PGPR). Finally, it is presented and discussed as an overview of the putative and potential biosynthetic pathways of NO and their close dependence on the different N sources in PGPR.

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.

Structure diversity of nitric oxide synthases (NOS): the emergence of new forms in photosynthetic organisms

Humans have enormously increased thelevel of nitrogen (N) circulating in thetroposphere and the earth surface duringthe last century, correlating with the pop-ulation increase. As an undesirable con-sequence, high levels of reactive N arepolluting the environment where humansinhabit. Nitric oxide (NO) is one of thereactive N species with both positive andnegative impact on life. NO synthases(NOSs) are enzymes that oxidize arginineto citrulline and generate the denitrify-ingintermediateNOwhichcanbesubse-quently reduced to N

Nitric Oxide Functions as Intermediate in Auxin, Abscisic Acid, and Lipid Signaling Pathways

Nitric oxide (NO) is a chemical messenger that actively operates in the plant kingdom. In recent years, NO has been shown to be involved in many and diverse growth, developmental, and physiological processes in plants. It has been shown that NO takes part in different hormone signaling pathways and also acts in concert with well-characterized second messengers. In this chapter, we discuss findings that contribute to the understanding of the role/s of NO during root organogenesis and stomatal movement, focusing on the interrelations between NO and the phytohormones auxin and abscisic acid. We emphasize the requirement of calcium as an essential intermediate present in the NO-mediated responses. Finally, novel data concerning the cross-talk between NO and phosphatidic acid signaling pathways in response to (a)biotic stresses are presented and discussed.

Nitric oxide is required for the auxin-induced activation of NADPH-dependent thioredoxin reductase and protein denitrosylation during root growth responses in arabidopsis

BACKGROUND AND AIMS Auxin is the main phytohormone controlling root development in plants. This study uses pharmacological and genetic approaches to examine the role of auxin and nitric oxide (NO) in the activation of NADPH-dependent thioredoxin reductase (NTR), and the effect that this activity has on root growth responses in Arabidopsis thaliana. METHODS Arabidopsis seedlings were treated with auxin with or without the NTR inhibitors auranofin (ANF) and 1-chloro-2, 4-dinitrobenzene (DNCB). NTR activity, lateral root (LR) formation and S-nitrosothiol content were measured in roots. Protein S-nitrosylation was analysed by the biotin switch method in wild-type arabidopsis and in the double mutant ntra ntrb. KEY RESULTS The auxin-mediated induction of NTR activity is inhibited by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO), suggesting that NO is downstream of auxin in this regulatory pathway. The NTR inhibitors ANF and DNCB prevent auxin-mediated activation of NTR and LR formation. Moreover, ANF and DNCB also inhibit auxin-induced DR5 : : GUS and BA3 : : GUS gene expression, suggesting that the auxin signalling pathway is compromised without full NTR activity. Treatment of roots with ANF and DNCB increases total nitrosothiols (SNO) content and protein S-nitrosylation, suggesting a role of the NTR-thioredoxin (Trx)-redox system in protein denitrosylation. In agreement with these results, the level of S-nitrosylated proteins is increased in the arabidopsis double mutant ntra ntrb as compared with the wild-type. CONCLUSIONS The results support for the idea that NTR is involved in protein denitrosylation during auxin-mediated root development. The fact that a high NO concentration induces NTR activity suggests that a feedback mechanism to control massive and unregulated protein S-nitrosylation could be operating in plant cells.