Synthesis, Actions, and Perspectives of Nitric Oxide in Photosynthetic Organisms

Chapter 10

Synthesis, Actions, and Perspectives of Nitric Oxide in Photosynthetic Organisms Noelia Foresi, Natalia Correa-Aragunde, Lorenzo Lamattina Institute for Biological Research, National University of Mar del Plata, National Scientific and Technical Research Council, Mar del Plata, Argentina

NITRIC OXIDE SYNTHESIS IN PHOTOSYNTHETIC ORGANISMS Structure, Diversity, and Occurrence of Nitric Oxide Synthases (NOS) in Photosynthetic Organisms: Canonical NOS Is Absent in Land Plant Genomes Nitric oxide (NO) plays multiple biological roles as signaling molecule and as cytotoxic and/or cytoprotective agent depending on its own concentration and that of the other free radical species in cells. In animals, NO is synthesized by NOS. NOS enzyme functions as homodimer and exists in mammals in three major isoforms: neuronal, endothelial, and inducible [1,2]. Mammalian NOS enzymes are expressed as a single polypeptide and share the common general organization in which the oxygenase and reductase domains (NOSoxy and NOSred, respectively) are linked via a regulatory calmodulin (CaM)–binding domain [2]. The NOSoxy domain contains a heme group at the active center and binds tetrahydrobiopterin (H4B) cofactor and the NOS substrate, l-arginine. The NOSred domain binds the cofactors NADPH, FAD, and FMN and provides electrons for l-arginine oxidation. The CaM-binding domain has a regulatory function (Fig. 10.1). On calcium-induced CaM binding, the NOSred and NOSoxy form a complex, allowing electrons to flow from NADPH to the active center for executing NOS chemistry [2]. The NOSred performs this task by transferring electrons from NADPH to FAD and FMN. In the presence of H4B, the homodimers of the enzyme are formed and oxidize l-arginine in two steps to form NO [2]. The l-arginine–dependent pathway involved in NO production was reported in several plant species. It has

Nitric Oxide. http://dx.doi.org/10.1016/B978-0-12-804273-1.00010-7 Copyright © 2017 Elsevier Inc. All rights reserved.

been inferred from measurements of NOS activity in various plant extracts [3–7], and inhibition of NO production using mammalian NOS inhibitors [3,8–10]. Although NO production from l-arginine has been detected in several plant tissues, the absence of a gene with homology to the bacterial and animal NOS plays against the evidence. Protocols and methods like spin trapping electron paramagnetic resonance (EPR) and ozone chemiluminescence provide reliable measurements of NO in biological samples and have also been used in plant samples [11–13]. The arginine–citrulline assay is a method widely used in animals whose validity, however, was questioned in Arabidopsis extracts. It has been demonstrated that, in plant extracts, an enzyme of the urea cycle uses l-arginine as a substrate and generates a product other than citrulline [14]. Therefore, the hypothesis of the presence of an enzyme that generates NO from l-arginine as a residual by-product cannot be rejected. In addition, some works have proposed a nonenzymatic NO formation through the oxidation of l-arginine by H2O2 [15–17]. In summary, in spite of all the biochemical, genetic, physiological, and pharmacological information available, considerable efforts need to be made to elucidate the identity of the enzymatic activities and/or chemical reactions responsible of the NO generation from l-arginine in plants. As stated, no gene or protein with sequence similarity to the bacteria or animal NOSs has been reported in land plants so far (Fig. 10.2). The first gene encoding one functional NOS in the plant kingdom, with resemblance to the human NOSs, has been characterized in the green alga Ostreococcus tauri (Fig. 10.2), belonging to Prasinophytes, an early diverging class within the green plant lineage [18]. More recently, Kumar et al. [19] identified NOS sequences in two

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FIGURE 10.1  Comparison of NOS architectures from mammals and the marine microalga Ostreococcus tauri. Binding domains for the cofactors H4B, FAD, FMN, NADPH, zinc (Zn), and the substrate l-arginine (l-Arg) are depicted. (Arrow with dashed lines) Not confirmed yet. CaM, Calmodulin; H4B, tetrahydrobiopterin; NOS, nitric oxide synthase.

FIGURE 10.2  Phylogenetic relationships and presence of complete NOS sequences among the main lineages of photosynthetic organisms. The tree topology is a composite on accepted relationships based on molecular phylogenetic evidence. The complete NOS present in different classes is indicated in red; the name of species within each class is shown below. NOS, nitric oxide synthase. Source: Adapted from Leliaert F, Smith DR, Moreau H, Herron MD, Verbruggen H, Delwiche CF, et al. Phylogeny and molecular evolution of the green algae. CRC Crit Rev Plant Sci 2012;31:1–46.

additional members of that class, Bathycoccus prasinos and Ostreococcus lucimarinus. An exciting work was followed searching NOS sequences through the analysis of 1000 plant genomes (1 KP Project) and more than 200 algae genomes. NOS with the typical NOSoxy and NOSred domains connected by a sequence with similarities to the CaM-binding domains like those characterized in O. tauri NOS (OtNOS; Fig. 10.1) was found in only 13 algae species [20]. Two of these proteins showing homology to the OtNOS were found

in Klebsormidium flaccidum (ID: Kf1000760350; 1130 aa) and in Thalassiosira oceanica (ID: EJK55330; 1245 aa). A more general structural analysis of the NOSoxy domain shows that algal NOS conserve the key features of mammalian and bacterial NOSoxy, including the proximal heme ligand Cys, the l-arginine and H4B-binding residues, and the so-called helical lariat and helical T region involved in binding the pterin cofactor and the interactions between monomers, stabilizing the homodimer [20].

Synthesis, Actions, and Perspectives of Nitric Oxide in Photosynthetic Organisms

A recently published article also reported the presence of a transcript encoding for a NOS in the diatom Pseudonitzschia multistriata but not in Pseudo-nitzschia arenysensis and Pseudo-nitzschia delicatissima [21]. A phylogenetic tree of all NOS sequences from diatoms found in NCBI and deriving from the genome sequence of the Pseudo-nitzschia showed two well-supported clades, clearly separating the sequences by their similarity with either the cyanobacteria or the green alga Ostreococcus and Homo sapiens NOS sequences. NOS sequences from diatoms confirmed their similarity with the cyanobacteria sequences together with more ancestral pennate diatoms, such as Amphiprora sp. and Cylindrotheca closterium [21]. Plants are a major group of photosynthetic eukaryotes that have played a prominent role in the global ecosystem for millions of years. An early split in their evolution gave rise to two major green lineages, one of which diversified in the world’s oceans and gave rise to a large diversity of marine and freshwater green algae (Chlorophyta), while the other gave rise to a diverse array of freshwater green algae and land plants (Streptophyta) (Fig. 10.2) [22]. Fig. 10.2 shows in red the presence of NOS complete sequences in different species of aquatic plants identified so far [18–21]. It is striking that complete canonical NOS is present in two species of streptophyte that represent the class of more evolved algae, and then it is absent in land plants. The intriguing questions are the following: Did the land plants lost the NOS gene when they emerged from the water? or Is the NOS gene still there and has evolved in such a different sequence/structure/architecture that it is difficult to identify?

Photosynthetic Organisms Do Not Synthetize the Biopterin Cofactor Required by NOS: The Role of Tetrahydrofolate Unlike higher animals, most bacteria, fungi, and plants do not synthesize H4B, but convert the first intermediate (dihydroneopterin triphosphate) into folate [23], a vitamin that serves as a coenzyme for a variety of one-carbon transfer reactions [24]. The pterin cofactor H4B plays a crucial role in NOS catalysis by acting as an electron donor.

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Genes encoding the main enzymes for H4B biosynthesis, namely 6-pyruvoyl-tetrahydropterin synthase (PTPS) and sepiapterin reductase (SR), have not been found in algae carrying NOS genes. This suggests that the algal NOS may bind a H4B analog that can support NO synthesis. Indeed, it has been demonstrated that the closely related pterin tetrahydrofolate (THF) can act as an electron donor in NOS from O. tauri [25]. This is further supported by the demonstration that NOS from bacteria, such as Deinococcus radiodurans synthesizes NO efficiently using THF [26]. As stated, plants do not synthetize H4B but produce THF. At the structural level, as highlighted earlier, the extended N-terminal portion of mammalian NOS participates in H4B binding. According to Tejero and Stuehr [27], the absence of the N-termini in the bacterial NOS allows them to bind larger pterins, such as THF. The algal NOSs contain an N-terminal extension; however, compared to mammalian NOSs, algal NOSs lack the key conserved residues in the Nterminal hook, as well as the amino acid sequence (SIM or SLV) involved in binding the dihydroxypropyl side chain of H4B in Fig. 10.3. On the other hand, a putative zinc-binding site Cys-X3-Cys (instead of Cys-X4-Cys in mammalian NOS) is present in OtNOS [28]. It is conceivable that CysX3-Cys also holds for the binding of Zn in algal NOS.

Plants Possess Alternative Sources for NO Production The other pathway reported for NO synthesis is the reduction of nitrite to NO catalyzed by nitrate reductase (NR). NR is a key enzyme involved in nitrogen metabolism and NR-dependent NO production has been proved in plants, in cells and in vitro with isolated enzyme [29,30]. Accordingly, it has probably emerged as the main enzymatic source of NO in plants during the process of nitrate reduction. Nitrite also forms NO enzymatically and nonenzymatically under acidic conditions. It can be reduced to NO enzymatically [13] by chloroplasts in a reaction dependent on photosynthetic electron flow or in mitochondria under anoxia or hypoxia, via cytochrome c oxidase [31]. The enzyme xanthine oxidase/dehydrogenase (XDH) has also been suggested as a potential source for NO using nitrite and xanthine

FIGURE 10.3  Alignment of N-termini from NOS sequences. N-terminal sequence alignment of seven NOS, Cosmarium subtumidum, O. tauri, human (1, 2, 3), and mouse iNOS. Residues that make up the N-terminal hook, zinc loop, and pterin-binding elements are boxed. Conserved residues are in red and in yellow for identical residues. NOS, nitric oxide synthase. Source: Adapted from Jeandroz S, Wipf D, Stuehr DJ, Lamattina L, Melkonian M, Tian Z, et al. Occurrence, structure, and evolution of nitric oxide synthase-like proteins in the plant kingdom. Sci Signal 2016;9:re2.

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as a substrate [32]. Genetic approaches have allowed researchers to demonstrate the involvement of NR-derived NO in physiological processes of development, such as flowering in Arabidopsis [33]. The triple mutant Arabidopsis nia1nia2noa1-2, which has mutated the two NR genes (NIA/NR) and nitric oxide–associated 1 (AtNOA1), shows a very low concentration of NO [33]. NO production from nitrous acid occurs at a significant rate only at pH values below 5 [34]. Conditions that favor nonenzymatic formation of NO are rare inside the cells, but occur in the apoplast of plants [35]. In addition, NO produced by either route can generate subcellular localized concentrations sufficient to trigger a spatial/temporal specific response. Furthermore, in addition to nitrite, hydroxylamines and polyamines can also act as substrates for NO synthesis [36–38].

ACTIONS AND TARGETS OF NO IN PHOTOSYNTHETIC ORGANISMS In the past two decades, NO has gained significant importance in plant research because of its multifunctional roles in the entire plant life [39–43].

NO as a Bioactive Signaling Molecule of Stress Responses in Land Plants In higher plants, NO is a key molecule in signaling pathways involved in responses to abiotic stresses, such as UV-B radiation, cold, drought, salt, heat, iron deficiency, and heavy metals, as well as in disease resistance and apoptotic processes (for references, see Fig. 10.4) [44–75]. Most

approaches designed to decipher NO roles in plant physiology were based on the exogenous application of NO donors and/or scavengers. However, compelling genetic and biochemical results indicate that the endogenously produced and regulated NO is the biologically important messenger/ effector of physiological changes in plants. As stated, Arabidopsis mutant in NR (nia1/nia2) is impaired in NO production and NO-mediated responses, being an useful tool to functionally characterize the participation of NO and the contribution of the NR-dependent biosynthetic pathways in regulating plant development. On the contrary, endogenous NO accumulates in Arabidopsis plants harboring a loss-of-function mutation in NO overexpression 1 (NOX1), resulting in another useful tool to study the effects of high endogenous NO concentration [76]. Transgenic Arabidopsis plants harboring a bacterial NO dioxygenase (NOD) gene under the control of dexamethasone-induced promoter are also used to deplete endogenous NO and reveal its requirement in plant physiological processes [77]. Another key genetic tool is the Arabidopsis mutated in the gene coding for the enzyme nitrosoglutathione reductase (GSNOR) [78]. This mutant accumulates the endogenous NO reservoir GSNO and leads to dysregulation of total cellular S-nitrosylation (see the section “Targets and Molecular Mechanisms Underpinning NO Actions in Photosynthetic Organisms”) [79]. In this sense, many works have unambiguously confirmed the relevance of endogenous NO in regulating hormone responses like stomatal aperture [80], seed germination [81], and plant immunity. Works from different laboratories support functions of NO attenuating symptoms of abiotic and biotic stresses.

FIGURE 10.4  Summary of the biotic and abiotic stresses that land plants and aquatic photosynthetic microorganisms are confronting during their life cycle, in which NO is acting in acclimation responses. Numbers in square brackets are references supporting the statements.

Synthesis, Actions, and Perspectives of Nitric Oxide in Photosynthetic Organisms

This was confirmed through the use of transgenic plants expressing the NOS gene that results in increased endogenous NO levels and improved tolerance of transgenic plants to confront stresses [25,52,64,82]. As mentioned earlier, canonical NOS gene from the unicellular marine algae O. tauri (OtNOS) has been characterized [18]. 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 [83], which responds to abiotic stresses and the plant hormone abscisic acid (ABA). Transgenic plants expressing OtNOS displayed enhanced salt, drought, and oxidative stress tolerance and accelerated the germination rate [25]. Other approaches to express NOS in plants included transformation of Arabidopsis [64], tobacco [52], and rice [82] with a cDNA encoding the rat brain neuronal NOS (nNOS) under the control of the constitutive promoter cauliflower mosaic virus (35S CaMV). Several Arabidopsis transgenic lines exhibiting NOS activity and increased NO levels were shown to be more tolerant than their wild-type (WT) counterpart to drought and salt stress, and to infection with a virulent pathogen [64]. Transcriptome analysis in those transgenic lines allowed the identification of genes that were differentially expressed as a consequence of drought stress and nNOS gene expression [84]. Analyses of metabolic pathways and gene ontology (GO) terms enrichment revealed that genes involved in photosynthesis, redox balance, stress, phytohormone signaling, and secondary metabolism were greatly influenced by the expression of the nNOS transgene and the high level of NO. Two ABA receptors AtPYL4 and AtPYL5 were found upregulated by both the expression of the nNOS transgene and ABA treatment. These observations added new information for the understanding of the NO role in drought stress responses in Arabidopsis [84]. Tobacco transgenic lines expressing mammalian NOS were also more resistant to different lifestyle pathogens, and it was mediated by NO-dependent buildup of salicylic acid (SA) levels [52].

Implications of NO in the Stress Responses of the Aquatic Photosynthetic Microorganisms In the marine environment, organisms are constantly exposed to NO, whose levels are 104 times higher than those present in the atmosphere [85]. In the sea, NO can be derived from nitrification/denitrification processes of the nitrogen cycle, plant emission fluxes, as well as sunlight photolysis of nitrate and strong nitrate deposits coming from croplands due to increasing fertilization activities [86]. Increasing evidence suggests that NO is also involved in the stress responses of marine photosynthetic organisms (MPOs), as occurs in plants (Fig. 10.4). High amounts of NO were produced at high temperatures in the zooxanthellae

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S. microadriaticum, commonly found in symbiotic association with reef-building corals [73]. Increasing NOS activity leading to an increased production of NO was associated with high water temperature during the coral bleaching process. It was hypothesized that NO acts as a cytotoxic molecule either by inactivating metabolic pathways or by causing damage to macromolecules and irreversible inhibition of mitochondrial respiration [72]. In dinoflagellate, a positive correlation between increase in NO levels, caspase 3-like activity, and high temperature was found, suggesting that NO triggers cell death probably through apoptosis [71]. On the other hand, aforementioned findings in higher plants led to the question whether NO formation may also be advantageous for algae exposed to stresses causing high and toxic ROS production. Hydrogen peroxide (H2O2) and NO can be produced in the nitrate-grown photosynthesizing cells at the same time [69]. A concentration-dependent toxicity of H2O2 was observed on growth yield, chlorophyll content, and chlorophyll fluorescence of the green micro alga Scenedesmus obliquus. Interestingly, the addition of the NO donor sodium nitroprusside (SNP) prevented chlorophyll losses and the inhibition of growth, as well as the maximum quantum yield of photosystem II (PSII) and the light-adapted quantum yield of PSII, which were significantly reduced. The protective actions of SNP were arrested in cultures where SNP was supplemented in combination with 2-(4-carboxyphenyl)4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), a specific scavenger of NO [69]. In the same sense, it was demonstrated that high light intensities induce OtNOS expression and NO generation in the unicellular alga O. tauri, suggesting that increased levels of NO are required under those conditions [18]. Other studies carried out with species of the marine phytoplankton S. costatum and P. subcordiforms showed that NO promoted their growth under different abiotic stress agents, such as selenium, lead, pesticides, and UV lights. It has been speculated that this protective effect is related to the antioxidant capability of NO [75]. Both NO and Fe have been shown to play significant roles in plant metabolism probably through the formation of dinitrosyl iron complexes (DNICs) involved in the storage and delivery of NO and Fe [87]. In our lab, we have found that iron deficiency induces the NO generation in O. tauri (Fig. 10.5), like occurs in land plants [88]. This is consistent with the finding that NO was able to positively influence Fe availability during growth processes in the phytoplankton [75]. Thus, it appears that the NO ability to promote growth in phytoplankton under Fe-limited conditions is in accordance with the earlier assays demonstrating that NO mitigates the adverse effects of Fe deficiency in land plants, as reported by Graziano and Lamattina [56] (Fig. 10.4). Altogether, this evidence shows that NO is a ubiquitous molecule in photosynthetic organisms, and can induce or inhibit the growth depending on the physiological situations, the level of ROS, and the relative concentration of NO in cells.

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FIGURE 10.5  Iron deficiency induces NO generation in the unicellular green alga Ostreococcus tauri. (A) Fe3+ detection by calcein probe and (B) NO detection by DAF-FM DA fluorescence probe, after treatment with the iron chelator desferroxamine (DFO). (C) O. tauri cells treated or not with 300 µM DFO in the presence of DAF-FM DA. NO fluorescence was visualized using fluorescence microscopy. Bar = 50 µm. DAF, Diaminofluorescein; RFU, relative fluorescence units.

NO Is a Key Player in Auxin-Mediated Processes Leading to Root Growth and Development Roots are plant organs that help in acquiring water and nutrients from soil and in plant anchorage. Root development is under the control of a regulated cell proliferation

within a morphogenesis program, where auxin is the master plant hormone that governs these processes [89–92]. As stated, NO has emerged as a secondary chemical messenger playing key roles in a broad spectrum of plant developmental processes. Among them, NO participates in the signal transduction pathways that determine root architecture. First findings reporting NO requirement in the auxin

Synthesis, Actions, and Perspectives of Nitric Oxide in Photosynthetic Organisms

signal transduction pathway were obtained in Lamattina’s lab in 2002. It was demonstrated that NO mediates the auxin response leading to the adventitious root formation in cucumber (Cucumis sativus) [93]. Two NO donors, SNP and S-nitroso-N-acetylpenicillamine (SNAP), applied to hypocotyl cuttings (primary root removed) of cucumber were able to mimic the effect of the auxin indole acetic acid (IAA) in inducing de novo root organogenesis [93]. A transient increase of endogenous NO concentration was shown to be rapidly produced in cucumber explants on auxin addition [93]. Besides, many findings confirmed that NO and auxin are important components in the process of lateral root formation in tomato, rice, pea, and Arabidopsis [94–97]. The participation and requirement of NO in lateral root development is widely accepted, and supported by experimental evidence from many plants where increased NO levels in roots correlate with higher lateral root density [25,92,98,99]. The most common and sensitive method used to detect and visualize NO outside and inside living cells, close to its production sites, is based on fluorescence compounds. Diaminofluorescein (DAF) is the most commonly used probe, but despite that, more than a thousand publications using DAF in the past decade and in different experimental models of botany, medicine, and zoology, scope and reliability of this method is strongly discussed. These compounds are available as cell-permeable diacetates (DAF2DA and DAF FM-DA) that are hydrolyzed and trapped inside the cell. The basic reaction implies the nitration of the indicator by an oxidation product of NO (NO2) to give the highly fluorescing and more stable DAF-2 triazole (DAF-2T). Considering that the oxidation products of NO are dependent on variations in peroxidase activities and ROS, and that DAF can also react with ascorbic acid and dehydroascorbic acid to give the fluorescence product DAF-2T, it is conceivable that the strict dependence of the detected fluorescence and that emitted by DAF-2T might not correspond to the actual concentration of NO [100]. Consequently, detection/measurement of NO by more than one method is strongly recommended, as was done in the earlier work, where NO production in explants of cucumber treated with the auxin IAA was demonstrated by DAF and EPR [93]. NO is also involved in root hair growth and development. The root epidermis is composed of two cell types: trichoblasts (or hair cells) and atrichoblasts (nonhair cells). In Arabidopsis, as well as in lettuce, the NO scavenger cPTIO blocked the auxin-induced root hair elongation indicating that NO is involved in the hormone-signaling cascade leading to root hair growth [101]. NO concentrates, especially in trichoblasts, with respect to atrichoblasts and they are detected inside the vacuole in immature growing root hairs and in the cytoplasm in mature root hairs [102]. Genetic and pharmacological evidence showed that, during

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root hair growth, NO is required for endocytosis, vesicle formation, and trafficking [102]. Similar results regarding NO modulation of vesicle trafficking and dynamic of the actin cytoskeleton were reported studying maize root growth [103]. Altogether, accumulating evidence along the past decade indicates that NO is able not only to mimic the effect of auxins on several processes associated with root growth and development but also to influence auxin itself [104].

Targets and Molecular Mechanisms Underpinning NO Actions in Photosynthetic Organisms Besides the involvement of NO in keeping the homeostasis of basic cellular functions, NO represents a second messenger in many signaling pathways. In animals, NO mediates its effects through either cGMP-dependent or cGMPindependent pathways. The first pathway implies guanylate cyclase (GC) activation and increased production of cGMP, thus affecting downstream targets of this second messenger. The second one involves the reaction of reactive nitrogen species (RNS) with the amino acid residues tyrosine and cysteine in proteins, resulting in nitration or S-nitrosylation of proteins, respectively, which affect protein activities and, consequently, cellular functions [105,106]. Understanding the molecular mechanisms by which NO exerts its biological functions in plants has been the subject of considerable effort in the past decade. In plants, NO is an extensive signal molecule acting through cellular components that participate downstream in many cascades, involving ion channels, protein kinases, receptors of phytohormones, and transcription factors and coactivators that target genes [107]. More and more compelling findings support NO operating through posttranslational modification (PTM) of proteins in plants notably via S-nitrosylation and tyrosine nitration. Proteome-wide scale analyses led to the identification of numerous protein candidates for S-nitrosylation in plants [108–111]. Subsequent biochemical analysis and in silico structural studies revealed the precise targeted Cys residues in proteins through which S-nitrosylation impacts their functions. As stated, auxin (indole-3-acetic acid) and NO are plant growth regulators [112] that coordinate several plant physiological responses determining, for instance, root architecture [93,94]. The A. thaliana F-box proteins transport inhibitor response 1/auxin signaling F-box (TIR1/ AFB) are auxin receptors that mediate degradation of auxin/indole-3-acetic acid (Aux/IAA) repressors to induce auxin-regulated responses. Terrile et al. [113] provided evidence that NO increases auxin-dependent reporter gene expression while NO depletion blocks Aux/IAA repressor degradation. NO also enhances the interaction between the auxin receptor TIR1 with the hormone as evidenced by

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pull-down and two-hybrid assays. The authors provided evidence for the NO-mediated modulation of the auxin signaling through the S-nitrosylation of the Cys140 and, to a less extent Cys480, of the TIR1 auxin receptor. These results suggest that TIR1 S-nitrosylation enhances TIR1– Aux/IAA interaction, facilitating the Aux/IAA repressor degradation and subsequently promoting activation of auxin-regulated gene expression [113]. Correa-Aragunde et al. [114] showed 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 a target of auxin-induced denitrosylation in roots. Auxin causes APX1 denitrosylation and the partial inhibition of its activity in Arabidopsis roots with the consequent increase of H2O2. In agreement, the S-nitrosylated form of the recombinant APX1 expressed in Escherichia coli is more active than the denitrosylated form. It is postulated that an auxin-regulated counterbalance between the two forms of APX1 (S-nitrosylated/denitrosylated) contributes to a finetuned control of H2O2 concentration, which directly affects the root growth and determination of its architecture [114]. Moreover, auxin and NO modulate positively the activity of thioredoxin reductases (TrxR), thus affecting the balance of oxidized/reduced thioredoxin (Trx), which is a key input for the APX denitrosylation [115]. Under stress conditions, NO regulates the expression and activity of antioxidant enzymes, which can cause alterations of the cellular redox status [116]. For instance, chronic NO production during salt stress induces the antioxidant system thereby increasing salt tolerance in various plants. In contrast, rapid NO accumulation in response to strong stress stimuli was occasionally linked to inhibition of antioxidant enzymes and a subsequent rise in H2O2 levels [117]. In biotic stresses, during the incompatible A. thaliana–Pseudomonas syringae interaction, ROS burst and cell death progression were shown to be terminated by S-nitrosylation–triggered NO-mediated inhibition of NADPH oxidases [118], further highlighting the multiple roles of NO during redox signaling [54]. In chemical reactions between ROS and RNS, new molecules arise with characteristics different from their precursors [117]. Nonsymbiotic hemoglobins (nsHbs) and other oxidase-like enzymes act in NO degradation. nsHbs are also capable of catalyzing protein nitration through a nitrite- and H2O2-dependent process [117]. The chemical affinity between NO and Fe not only creates metal-nitrosyl complexes but also affects the activity of ferrous enzymes, as well as the requirement of Fe for the formation of the potent oxidant hydroxyl (OH) [119]. Also, the biological activity of proteins can be affected through the NO reactions with heme and Fe-sulfur groups. Many findings support the regulatory role of NO in cell redox balance by acting as either oxidant or antioxidant, depending on cell redox status [120].

THE POTENTIAL OF NOS TO IMPROVE THE FITNESS OF CROP PLANTS As stated earlier, Foresi et al. [25] showed that the precise regulation of NO generation in plants expressing the OtNOS gene under the control of a stress-inducible promoter provides enhanced tolerance to various abiotic stresses. Several plant traits were taken into consideration, and the evidence obtained for all of them reinforces the notion that a controlled and temporally precise bonus of endogenous NO production may assist plants to confront a stress situation after perceiving it. As a consequence, NO appears as a molecule with potential to improve crop agricultural performance, and OtNOS manipulation may therefore represent a useful tool in plant genetic engineering programs, even though precautions must be taken with regard to the disadvantages arising from the potential increase in atmospheric NO. Nevertheless, Gregg et al. [48] demonstrated that trees grow better in New York City (NYC) than in rural sites, due to the higher concentration of ozone (O3) in rural sites, with detrimental effects on plant growth parameters. Authors found that the NOx produced by regular urban activities depletes O3 in the NYC, resulting in a lower urban concentration of O3 and a higher plant biomass generation compared to that in rural sites. Anyway, soil microorganisms are greater contributors than plants to global NO emissions. Indeed, plants behave mostly as scavengers of NO, due to the activity of nsHg that have been proposed to deplete plant endogenous NO concentrations and even the level of NO in the surrounding environment [121]. In summary, the possibility that plants themselves generate NO when required under stress situations represents an enormous advantage compared to strategies involving exogenous application of NO donors.

CONCLUDING REMARKS AND PERSPECTIVES Since its identification as an endothelium-derived relaxing factor (EDRF) in the 1980s, NO has become the source of intensive and exciting research in animals. In contrast, the role of NO in plants has received attention just 10 years later. Since then, a new era of research in plants has started. There, an exciting rediscovery of the pillars of the plant physiology processes occurred, now revisited and considering the participation of NO. These findings allowed the emergency of a body of evidence for sustaining that many signal transduction pathways involving NO are shared between plants and animals. The identification of different NOS architectures in aquatic photosynthetic organisms forwards the hypothesis of a rapid evolution of NOS-coding gene sequences in the ocean microorganisms. An interesting case is diatoms where many species displayed two transcripts, one coding for a

Synthesis, Actions, and Perspectives of Nitric Oxide in Photosynthetic Organisms

canonical NOS sequence with similarity to the animal NOS sequences and the other one that has similarity with the cyanobacterial NOS sequences, either lacking or not having an extra globin domain. In addition, when different datasets for the same strain of the aquatic microorganism that is grown in different conditions are available, the NOS transcript was not retrieved in all environmental growth conditions, indicating an adaptive regulation of NOS expression [21]. The presence of alternative splicing of human iNOS mRNA was described in alveolar macrophages and several human tissues. Four sites of alternative splicing were identified by sequence analysis from the deduced amino acid sequences of the iNOS. Because iNOS is active as a dimer, the novel forms of alternatively spliced iNOS may be involved in regulation of NO synthesis [122]. This represents a yet unexplored field in the expression of NOS in photosynthetic organisms. The activity of the NOS from O. tauri (OtNOS) is very high like that found in mammalian NOS [18]. It would be interesting to go further into the understanding of the hidden secrets held by that enzyme and use this knowledge for genetic engineering programs concerning the mammalian NOS.

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