Nitric oxide synthase expression in Pseudomonas koreensis MME3 improves plant growth promotion traits.

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.

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.

Hydrogen Sulfide Increases Production of NADPH Oxidase-Dependent Hydrogen Peroxide and Phospholipase D-Derived Phosphatidic Acid in Guard Cell Signaling.

Hydrogen sulfide (H2S) 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 H2S in plants, participates in abscisic acid-induced stomatal closure. We combined pharmacological and genetic approaches to elucidate the involvement of H2S in stomatal closure and the interplay between H2S and other second messengers of the guard cell signaling network, such as hydrogen peroxide (H2O2) 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 H2S-induced stomatal closure. In vivo imaging using the cytosolic ratiometric fluorescent biosensor roGFP2-Orp1 revealed that H2S stimulates H2O2 production in Arabidopsis guard cells. Additionally, we observed an interplay between H2S and PLD activity in the regulation of reactive oxygen species production and stomatal movement. The PLDα1 and PLDδ isoforms were required for H2S-induced stomatal closure, and most of the H2S-dependent H2O2 production required the activity of PLDα1. Finally, we showed that H2S induced increases in the PLDδ-derived phosphatidic acid levels in guard cells. Our results revealed the involvement of H2S in the signaling network that controls stomatal closure, and suggest that H2S regulates NADPH oxidase and PLD activity in guard cells.

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.

The sesquiterpene botrydial from Botrytis cinerea induces phosphatidic acid production in tomato cell suspensions.

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.

Phospholipase C2 Affects MAMP-Triggered Immunity by Modulating ROS Production.

The activation of phosphoinositide-specific phospholipase C (PI-PLC) is one of the earliest responses triggered by the recognition of several microbe-associated molecular patterns (MAMPs) in plants. The Arabidopsis (Arabidopsis thaliana) PI-PLC gene family is composed of nine members. Previous studies suggested a role for PLC2 in MAMP-triggered immunity, as it is rapidly phosphorylated in vivo upon treatment with the bacterial MAMP flg22. Here, we analyzed the role of PLC2 in plant immunity using an artificial microRNA to silence PLC2 expression in Arabidopsis. We found that PLC2-silenced plants are more susceptible to the type III secretion system-deficient bacterial strain Pseudomonas syringae pv tomato (Pst) DC3000 hrcC- and to the nonadapted pea (Pisum sativum) powdery mildew Erysiphe pisi However, PLC2-silenced plants display normal susceptibility to virulent (Pst DC3000) and avirulent (Pst DC3000 AvrRPM1) P. syringae strains, conserving typical hypersensitive response features. In response to flg22, PLC2-silenced plants maintain wild-type mitogen-activated protein kinase activation and PHI1, WRKY33, and FRK1 immune marker gene expression but have reduced reactive oxygen species (ROS)-dependent responses such as callose deposition and stomatal closure. Accordingly, the generation of ROS upon flg22 treatment is compromised in the PLC2-defficient plants, suggesting an effect of PLC2 in a branch of MAMP-triggered immunity and nonhost resistance that involves early ROS-regulated processes. Consistently, PLC2 associates with the NADPH oxidase RBOHD, suggesting its potential regulation by PLC2.

Abscisic acid and nitric oxide modulate cytoskeleton organization, root hair growth and ectopic hair formation in Arabidopsis.

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.

Abscisic acid enhances cold tolerance in honeybee larvae.

The natural composition of nutrients present in food is a key factor determining the immune function and stress responses in the honeybee (Apis mellifera). We previously demonstrated that a supplement of abscisic acid (ABA), a natural component of nectar, pollen, and honey, increases honeybee colony survival overwinter. Here we further explored the role of ABA in in vitro-reared larvae exposed to low temperatures. Four-day-old larvae (L4) exposed to 25°C for 3 days showed lower survival rates and delayed development compared to individuals growing at a standard temperature (34°C). Cold-stressed larvae maintained higher levels of ABA for longer than do larvae reared at 34°C, suggesting a biological significance for ABA. Larvae fed with an ABA-supplemented diet completely prevent the low survival rate due to cold stress and accelerate adult emergence. ABA modulates the expression of genes involved in metabolic adjustments and stress responses: Hexamerin 70b, Insulin Receptor Substrate, Vitellogenin, and Heat Shock Proteins 70. AmLANCL2, the honeybee ABA receptor, is also regulated by cold stress and ABA. These results support a role for ABA increasing the tolerance of honeybee larvae to low temperatures through priming effects.

Arabidopsis phosphatidylinositol-phospholipase C2 (PLC2) is required for female gametogenesis and embryo development.

MAIN CONCLUSION: AtPLC2 is an essential gene in Arabidopsis, since it is required for female gametogenesis and embryo development. AtPLC2 might play a role in cell division during embryo-sac development and early embryogenesis. Phosphoinositide-specific phospholipase C (PI-PLC) plays an important role in signal transduction during plant development and in the response to various biotic- and abiotic stresses. The Arabidopsis PI-PLC gene family is composed of nine members, named PLC1 to PLC9. Here, we report that PLC2 is involved in female gametophyte development and early embryogenesis. Using two Arabidopsis allelic T-DNA insertion lines with different phenotypic penetrations, we observed both female gametophytic defects and aberrant embryos. For the plc2-1 mutant (Ws background), no homozygous plants could be recovered in the offspring from self-pollinated plants. Nonetheless, plc2-1 hemizygous mutants are affected in female gametogenesis, showing embryo sacs arrested at early developmental stages. Allelic hemizygous plc2-2 mutant plants (Col-0 background) present reduced seed set and embryos arrested at the pre-globular stage with abnormal patterns of cell division. A low proportion (0.8%) of plc2-2 homozygous mutants was found to escape lethality and showed morphological defects and disrupted megagametogenesis. PLC2-promoter activity was observed during early megagametogenesis, and after fertilization in the embryo proper. Immunolocalization studies in early stage embryos revealed that PLC2 is restricted to the plasma membrane. Altogether, these results establish a role for PLC2 in both reproductive- and embryo development, presumably by controlling mitosis and/or the formation of cell-division planes.

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.

S-nitrosylation influences the structure and DNA binding activity of AtMYB30 transcription factor from Arabidopsis thaliana.

MYB proteins are a family of transcription factors that play an important role in plant development and regulatory defense processes. Arabidopsis thaliana MYB30 (AtMYB30), a member of this protein family, is involved in cell death processes during the hypersensitive response (HR) of plants. HR is characterized by a vast production of reactive oxygen species (ROS) and nitric oxide (NO). NO may thus influence the binding of AtMYB30 to DNA. In this work we evaluated the effect of NO on AtMYB30 DNA binding activity, and also in the protein structural properties. A fully active minimal DNA-binding domain (DBD) of AtMYB30 (residues 11-116) containing two cysteine residues (C49 and C53) was overexpressed and purified. Site-directed mutagenesis was used to obtain AtMYB30 DBD mutants C49A and C53A. The DNA binding activity of AtMYB30 DBD, and Cys single mutants is clearly inhibited upon incubation with a NO donor, and S-nitrosylation was confirmed by the biotin switch assay. Finally, in order to understand the mechanism of NO effect on AtMYB30 DNA binding activity we performed circular dichroism analysis, to correlate the observed protein function inhibition and a potential structural impairment on AtMYB30 DBD. Indeed, NO modification of C49 and C53 residues promotes a subtle modification on the secondary structure of this transcription factor. We thus demonstrated, using various techniques, the in vitro effect of NO on AtMYB30 DBD, and thus the potential consequences of NO activity on plant metabolism influenced by this transcription factor.

Ultraviolet-B-induced stomatal closure in Arabidopsis is regulated by the UV RESISTANCE LOCUS8 photoreceptor in a nitric oxide-dependent mechanism.

UV RESISTANCE LOCUS8 (UVR8) signaling involves CONSTITUTIVELY PHOTOMORPHOGENIC1, the ELONGATED HYPOCOTYL5 (HY5) transcription factor, and the closely related HY5 HOMOLOG. Some UV-B responses mediated by UVR8 are also regulated by nitric oxide (NO), a bioactive molecule that orchestrates a wide range of processes in plants. In this study, we investigated the participation of the UVR8 pathway and its interaction with NO in UV-B-induced stomatal movements in Arabidopsis (Arabidopsis thaliana). Stomata in abaxial epidermal strips of Arabidopsis ecotype Landsberg erecta closed in response to increasing UV-B fluence rates, with maximal closure after 3-h exposure to 5.46 µmol m⁻² s⁻¹ UV-B. Both hydrogen peroxide (H2O2) and NO increased in response to UV-B, and stomatal closure was maintained by NO up to 24 h after the beginning of exposure. Stomata of plants expressing bacterial NO dioxygenase, which prevents NO accumulation, did not close in response to UV-B, although H2O2 still increased. When the uvr8-1 null mutant was exposed to UV-B, stomata remained open, irrespective of the fluence rate. Neither NO nor H2O2 increased in stomata of the uvr8-1 mutant. However, the NO donor S-nitrosoglutathione induced closure of uvr8-1 stomata to the same extent as in the wild type. Experiments with mutants in UVR8 signaling components implicated CONSTITUTIVELY PHOTOMORPHOGENIC1, HY5, and HY5 HOMOLOG in UV-B-induced stomatal closure. This research provides evidence that the UVR8 pathway regulates stomatal closure by a mechanism involving both H2O2 and NO generation in response to UV-B exposure.

Hydrogen sulfide generated by L-cysteine desulfhydrase acts upstream of nitric oxide to modulate abscisic acid-dependent stomatal closure.

Abscisic acid (ABA) is a well-studied regulator of stomatal movement. Hydrogen sulfide (H2S), a small signaling gas molecule involved in key physiological processes in mammals, has been recently reported as a new component of the ABA signaling network in stomatal guard cells. In Arabidopsis (Arabidopsis thaliana), H2S is enzymatically produced in the cytosol through the activity of l-cysteine desulfhydrase (DES1). In this work, we used DES1 knockout Arabidopsis mutant plants (des1) to study the participation of DES1 in the cross talk between H2S and nitric oxide (NO) in the ABA-dependent signaling network in guard cells. The results show that ABA did not close the stomata in isolated epidermal strips of des1 mutants, an effect that was restored by the application of exogenous H2S. Quantitative reverse transcription polymerase chain reaction analysis demonstrated that ABA induces DES1 expression in guard cell-enriched RNA extracts from wild-type Arabidopsis plants. Furthermore, stomata from isolated epidermal strips of Arabidopsis ABA receptor mutant pyrabactin-resistant1 (pyr1)/pyrabactin-like1 (pyl1)/pyl2/pyl4 close in response to exogenous H2S, suggesting that this gasotransmitter is acting downstream, although acting independently of the ABA receptor cannot be ruled out with this data. However, the Arabidopsis clade-A PROTEIN PHOSPHATASE2C mutant abscisic acid-insensitive1 (abi1-1) does not close the stomata when epidermal strips were treated with H2S, suggesting that H2S required a functional ABI1. Further studies to unravel the cross talk between H2S and NO indicate that (1) H2S promotes NO production, (2) DES1 is required for ABA-dependent NO production, and (3) NO is downstream of H2S in ABA-induced stomatal closure. Altogether, data indicate that DES1 is a unique component of ABA signaling in guard cells.

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 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.

Nitric oxide function in plant biology: a redox cue in deconvolution.

Nitric oxide (NO), a gaseous, redox-active small molecule, is gradually becoming established as a central regulator of growth, development, immunity and environmental interactions in plants. A major route for the transfer of NO bioactivity is S-nitrosylation, the covalent attachment of an NO moiety to a protein cysteine thiol to form an S-nitrosothiol (SNO). This chemical transformation is rapidly emerging as a prototypic, redox-based post-translational modification integral to the life of plants. Here we review the myriad roles of NO and SNOs in plant biology and, where known, the molecular mechanisms underpining their activity.

Nitric oxide is required for determining root architecture and lignin composition in sunflower. Supporting evidence from microarray analyses.

Nitric oxide (NO) is a signal molecule involved in several physiological processes in plants, including root development. Despite the importance of NO as a root growth regulator, the knowledge about the genes and metabolic pathways modulated by NO in this process is still limited. A constraint to unravel these pathways has been the use of exogenous applications of NO donors that may produce toxic effects. We have analyzed the role of NO in root architecture through the depletion of endogenous NO using the scavenger cPTIO. Sunflower seedlings growing in liquid medium supplemented with cPTIO showed unaltered primary root length while the number of lateral roots was deeply reduced; indicating that endogenous NO participates in determining root branching in sunflower. The transcriptional changes induced by NO depletion have been analyzed using a large-scale approach. A microarray analysis showed 330 genes regulated in the roots (p≤0.001) upon endogenous NO depletion. A general cPTIO-induced up-regulation of genes involved in the lignin biosynthetic pathway was observed. Even if no detectable changes in total lignin content could be detected, cell walls analyses revealed that the ratio G/S lignin increased in roots treated with cPTIO. This means that endogenous NO may control lignin composition in planta. Our results suggest that a fine tuning regulation of NO levels could be used by plants to regulate root architecture and lignin composition. The functional implications of these findings are discussed.

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 influences auxin signaling through S-nitrosylation of the Arabidopsis TRANSPORT INHIBITOR RESPONSE 1 auxin receptor.

Previous studies have demonstrated that auxin (indole-3-acetic acid) and nitric oxide (NO) are plant growth regulators that coordinate several plant physiological responses determining root architecture. Nonetheless, the way in which these factors interact to affect these growth and developmental processes is not well understood. The Arabidopsis 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. A broad spectrum of NO-mediated protein modifications are known in eukaryotic cells. Here, we provide evidence that NO donors increase auxin-dependent gene expression while NO depletion blocks Aux/IAA protein degradation. NO also enhances TIR1-Aux/IAA interaction as evidenced by pull-down and two-hybrid assays. In addition, we provide evidence for NO-mediated modulation of auxin signaling through S-nitrosylation of the TIR1 auxin receptor. S-nitrosylation of cysteine is a redox-based post-translational modification that contributes to the complexity of the cellular proteome. We show that TIR1 C140 is a critical residue for TIR1-Aux/IAA interaction and TIR1 function. These results suggest that TIR1 S-nitrosylation enhances TIR1-Aux/IAA interaction, facilitating Aux/IAA degradation and subsequently promoting activation of gene expression. Our findings underline the importance of NO in phytohormone signaling pathways.

Nitric oxide regulation of leaf phosphoenolpyruvate carboxylase-kinase activity: implication in sorghum responses to salinity.

Nitric oxide (NO) is a signaling molecule that mediates many plant responses to biotic and abiotic stresses, including salt stress. Interestingly, salinity increases NO production selectively in mesophyll cells of sorghum leaves, where photosynthetic C4 phosphoenolpyruvate carboxylase (C4 PEPCase) is located. PEPCase is regulated by a phosphoenolpyruvate carboxylase-kinase (PEPCase-k), which levels are greatly enhanced by salinity in sorghum. This work investigated whether NO is involved in this effect. NO donors (SNP, SNAP), the inhibitor of NO synthesis NNA, and the NO scavenger cPTIO were used for long- and short-term treatments. Long-term treatments had multifaceted consequences on both PPCK gene expression and PEPCase-k activity, and they also decreased photosynthetic gas-exchange parameters and plant growth. Nonetheless, it could be observed that SNP increased PEPCase-k activity, resembling salinity effect. Short-term treatments with NO donors, which did not change photosynthetic gas-exchange parameters and PPCK gene expression, increased PEPCase-k activity both in illuminated leaves and in leaves kept at dark. At least in part, these effects were independent on protein synthesis. PEPCase-k activity was not decreased by short-term treatment with cycloheximide in NaCl-treated plants; on the contrary, it was decreased by cPTIO. In summary, NO donors mimicked salt effect on PEPCase-k activity, and scavenging of NO abolished it. Collectively, these results indicate that NO is involved in the complex control of PEPCase-k activity, and it may mediate some of the plant responses to salinity.