Search

Nitric oxide (NO) is a redox active gaseous signal uniformly present in eukaryotes, but its formation, signalling and effects are specific within the plant kingdom in several aspects. NO synthesis in algae proceeds by mechanisms similar to that in mammals, but there are different pathways in higher plants. Beyond synthesis, the regulatory processes to maintain steady-state NO levels are also integral for the projection of NO function. As a key redox molecule, NO exhibits a number of pivotal molecular interactions, for example, with reactive oxygen species, hydrogen sulphide and calcium, with these molecular interplays largely underpinning NO bioactivity. In this context, NO has emerged as a key regulator in plant growth, development and environmental interactions. In this special issue, a collection of reviews discuss the current state-of-the-art and possible future directions related to the biology and chemistry of plant NO function.

Nitric oxide (NO) is involved in a vast number of physiologically important processes in plants, such as organ development, stress resistance, and immunity. Transduction of NO bioactivity is generally achieved by post-translational modification of proteins, with S-nitrosation of cysteine residues as the predominant form. While traditionally the subcellular location of the factors involved was of lesser importance, recent studies identified the connection between NO and transcriptional activity and thereby raised the question about the route of NO into the nuclear sphere. Identification of NO-affected transcription factors and chromatin-modifying histone deacetylases implicated the important role of NO signaling in the plant nucleus as a regulator of epigenetic mechanisms and gene transcription. Here, we discuss the relationship between NO and its directly regulated protein targets in the nuclear environment, focusing on S-nitrosated chromatin modulators and transcription factors.

Nitric oxide (NO) is a multifunctional gaseous signal that modulates the growth, development and stress tolerance of higher plants. NO donors have been used to boost plant endogenous NO levels and to activate NO-related responses, but this strategy is often hindered by the relative instability of donors. Alternatively, nanoscience offers a new, promising way to enhance NO delivery to plants, as NO-releasing nanomaterials (e.g. S-nitrosothiol-containing chitosan nanoparticles) have many beneficial physicochemical and biochemical properties compared to non-encapsulated NO donors. Nano NO donors are effective in increasing tissue NO levels and enhancing NO effects both in animal and human systems. The authors believe, and would like to emphasize, that new trends and technologies are essential for advancing plant NO research and nanotechnology may represent a breakthrough in traditional agriculture and environmental science. Herein, we aim to draw the attention of the scientific community to the potential of NO-releasing nanomaterials in both basic and applied plant research as alternatives to conventional NO donors, providing a brief overview of the current knowledge and identifying future research directions. We also express our opinion about the challenges for the application of nano NO donors, such as the environmental footprint and stakeholder's acceptance of these materials., NO research in the Kolbert lab is financed by NRDI Office (grant no. K135303). NO research in the Seabra lab is financed by Fapesp (2018/08194-2, 2020/03646-2) and CNPq (404815/2018-9, 313117/2019-5). NO research in the Oliveira’s lab is financed by Fundacao Araucaria de Apoio ao Desenvolvimento Científico do Parana (18.017.997-6), Fapesp (2019/15095-3) and CNPq (311034/2020-9). NMS and RVR acknowledge the financial support by Fapesp (2012/19167-0, 2015/21546-7, 2017/11279- 7, 2018/08194-2, 2020/03646-2), CNPq (302460/2018–7) and National Program of Post-Doctorate (PNPD, Capes). Research by FJC and JMP is supported by a European Regional Development Fund cofinanced from the Ministry of Economy and Competitiveness (PID 2019-103924 GB-I00) and Junta de Andalucía, Spain.

In the past, reactive nitrogen species (RNS) were supposed to be stress-induced by-products of disturbed metabolism that cause oxidative damage to biomolecules. However, emerging evidence demonstrates a substantial role of RNS as endogenous signals in eukaryotes. In plants, S-nitrosoglutathione (GSNO) is the dominant RNS and serves as the •NO donor for S-nitrosation of diverse effector proteins. Remarkably, the endogenous GSNO level is tightly controlled by S-nitrosoglutathione reductase (GSNOR) that irreversibly inactivates the glutathione-bound NO to ammonium. Exogenous feeding of diverse RNS, including GSNO, affected chromatin accessibility and transcription of stress-related genes, but the triggering function of RNS on these regulatory processes remained elusive. Here, we show that GSNO reductase-deficient plants (gsnor1-3) accumulate S-adenosylmethionine (SAM), the principal methyl donor for methylation of DNA and histones. This SAM accumulation triggered a substantial increase in the methylation index (MI = [SAM]/[S-adenosylhomocysteine]), indicating the transmethylation activity and histone methylation status in higher eukaryotes. Indeed, a mass spectrometry-based global histone profiling approach demonstrated a significant global increase in H3K9me2, which was independently verified by immunological detection using a selective antibody. Since H3K9me2-modified regions tightly correlate with methylated DNA regions, we also determined the DNA methylation status of gsnor1-3 plants by whole-genome bisulfite sequencing. DNA methylation in the CG, CHG, and CHH contexts in gsnor1-3 was significantly enhanced compared to the wild type. We propose that GSNOR1 activity affects chromatin accessibility by controlling the transmethylation activity (MI) required for maintaining DNA methylation and the level of the repressive chromatin mark H3K9me2.

To investigate the effect of high atmospheric NO concentrations on crop plants and the role of phytoglobins under these conditions, we performed a long-term study on barley ‘Golden Promise’ wild type (WT), class 1 phytoglobin knockdown (HvPgb1.1−) and class 1 phytoglobin overexpression (HvPgb1.1+) lines. Plants were cultivated with nitrogen-free nutrient solution during the entire growth period and were fumigated with different NO concentration (ambient, 800, 1500, and 3000 ppb). Analysis of fresh weight, stem number, chlorophyll content, and effective quantum yield of PSII showed that NO fumigation promoted plant growth and tillering significantly in the HvPgb1.1+ line. After 80 d of NO fumigation, dry matter weight, spikes number, kernel number, and plant kernel weight were significantly increased in HvPgb1.1+ plants with increasing NO concentration. In contrast, yield decreased in WT and HvPgb1.1− plants the higher the NO level. Application of atmospheric 15NO and 15NO2 demonstrated NO specificity of phytoglobins. 15N from 15NO could be detected in RNA, DNA, and proteins of barley leaves and the 15N levels were significantly higher in HvPgb1.1+ plants in comparison with HvPgb1.1− and WT plants. Our results demonstrate that overexpression of phytoglobins allows plants to more efficiently use atmospheric NO as N source., Overexpression of phytoglobins enables a better use of atmospheric NO as a nitrogen source in barley. Such a NO-fixation capability might serve as a new economically important breeding trait to increase N use efficiency.

Nitric oxide (NO) is a signaling molecule with multiple regulatory functions in plant physiology and stress response. In addition to direct effects on transcriptional machinery, NO executes its signaling function via epigenetic mechanisms. We report that light intensity-dependent changes in NO correspond to changes in global histone acetylation (H3, H3K9, and H3K9/K14) in Arabidopsis (Arabidopsis thaliana) wild-type leaves, and that this relationship depends on S-nitrosoglutathione reductase and histone deacetylase 6 (HDA6). The activity of HDA6 was sensitive to NO, demonstrating that NO participates in regulation of histone acetylation. Chromatin immunoprecipitation sequencing and RNA-seq analyses revealed that NO participates in the metabolic switch from growth and development to stress response. This coordinating function of NO might be particularly important in plant ability to adapt to a changing environment, and is therefore a promising foundation for mitigating the negative effects of climate change on plant productivity.

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 70% of the produced NO is rapidly oxidized to nitrate by an unusual globin domain in its 5'-terminus. In this study, we assessed whether SyNOS expression in plants affects N metabolism improving NUE and yield. Our results showed that transgenic Arabidopsis plants had higher primary shoot length and shoot branching when grown in N-deficient conditions and higher seed production in N-sufficient and -deficient conditions. Moreover, transgenic plants showed significantly increased NUE in both N conditions. No differences were observed in N uptake for SyNOS lines. However, SyNOS lines presented an increase in N assimilation/remobilization under low N conditions. In addition, SyNOS lines had greater N-deficiency tolerance compared to wt 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.

The perception and transduction of nitric oxide (NO) signal is achieved by NO-dependent posttranslational modifications (PTMs) among which S-nitrosation and tyrosine nitration has biological significance. In plants, 100-1000 S-nitrosated and tyrosine nitrated proteins have been identified so far by mass spectrometry. The determination of NO-modified protein targets/amino acid residues is often methodologically challenging. In the past decade, the growing demand for the knowledge of S-nitrosated or tyrosine nitrated sites has motivated the introduction of bioinformatics tools. For predicting S-nitrosation seven computational tools have been developed (GPS-SNO, SNOSite, iSNO-PseACC, iSNO-AAPAir, PSNO, PreSNO, RecSNO). Four predictors have been developed for indicating tyrosine nitration sites (GPS-YNO2, iNitro-Tyr, PredNTS, iNitroY-Deep), and one tool (DeepNitro) predicts both NO-dependent PTMs. The advantage of these computational tools is the fast provision of large amount of information. In this review, the available software tools have been tested on plant proteins in which S-nitrosated or tyrosine nitrated sites have been experimentally identified. The predictors showed distinct performance and there were differences from the experimental results partly due to the fact that the three-dimensional protein structure is not taken into account by the computational tools. Nevertheless, the predictors excellently establish experiments, and it is suggested to apply all available tools on target proteins and compare their results. In the future, computational prediction must be developed further to improve the precision with which S-nitrosation/tyrosine nitration-sites are identified.

Nitric oxide (NO) is a signaling molecule with multiple regulatory functions in plant physiology and stress response. Besides direct effects on the transcriptional machinery, NO can fulfill its signaling function via epigenetic mechanisms.We report that light intensity-dependent changes in NO correlate with changes in global histone acetylation (H3, H3K9 and H3K9/K14) in Arabidopsis thaliana wild-type leaves and that this correlation depends on S-nitrosoglutathione reductase and histone deacetylase 6. The activity of histone deacetylase 6 was sensitive to NO, which demonstrates that NO participates in regulation of histone acetylation. ChIP-seq and RNA-seq analyses revealed that NO is involved in the metabolic switch from growth and development to stress response. This coordinating function of NO might be of special importance in adaptation to a changing environment and could therefore be a promising starting point to mitigating the negative effects of climate change on plant productivity.

Nitric oxide (NO) is perfectly suited for the role of a redox signalling molecule. A key route for NO bioactivity occurs via protein S-nitrosation, and involves the addition of a NO moiety to a protein cysteine (Cys) thiol (–SH) to form an S-nitrosothiol (SNO). This process is thought to underpin a myriad of cellular processes in plants that are linked to development, environmental responses and immune function. Here we collate emerging evidence showing that NO bioactivity regulates a growing number of diverse post-translational modifications including SUMOylation, phosphorylation, persulfidation and acetylation. We provide examples of how NO orchestrates these processes to mediate plant adaptation to a variety of cellular cues., Research in the GJL laboratory has been supported by BBSRC grant BB/DO11809/1

Nitric oxide (NO) emerged as a key signal molecule in plants. During the last two decades impressive progress has been made in plant NO research. This small, redox-active molecule is now known to play an important role in plant immunity, stress responses, environmental interactions, plant growth and development. To more accurately and robustly establish the full spectrum of NO bioactivity in plants, it will be essential to apply methodological best practice. In addition, there are some instances of conflicting nomenclature within the field, which would benefit from standardisation. In this context, we attempt to provide some helpful guidance for best practice associated with NO research and also suggestions for the cognate terminology. This article is protected by copyright. All rights reserved.

Nitric oxide (center dot NO) acts as signaling molecule in plants being involved in diverse physiological processes such as germination, root growth, stomata closing and response to biotic and abiotic stress. S-Nitrosoglutathione (GSNO) is the storage and transport form of. NO and has a very important function in. NO signaling since it can transfer its. NO moiety to other proteins (trans-nitrosylation). The level of GSNO and thus the level of S-nitrosylated proteins are regulated by GSNO-reductase (GSNOR). In this way, this enzyme regulates the S-nitrosothiol levels and plays a balancing role in fine-tuning. NO signaling. Interestingly, oxidative post-translationally modification of GSNOR inhibited the activity of this enzyme suggesting a direct crosstalk between ROS-and RNS-signaling. In this review article the regulatory effects of ROS on GSNOR are highlighted and their physiological function in context of crosstalk between ROS and. NO and species in plants are discussed.

Nitric oxide (NO) is a key signaling molecule in all kingdoms. In plants, NO is involved in the regulation of various processes of growth and development as well as biotic and abiotic stress response. It mainly acts by modifying protein cysteine or tyrosine residues or by interacting with protein bound transition metals. Thereby, the modification of cysteine residues known as protein S-nitrosation is the predominant mechanism for transduction of NO bioactivity. Histone acetylation on N-terminal lysine residues is a very important epigenetic regulatory mechanism. The transfer of acetyl groups from acetyl-coenzyme A on histone lysine residues is catalyzed by histone acetyltransferases. This modification neutralizes the positive charge of the lysine residue and results in a loose structure of the chromatin accessible for the transcriptional machinery. Histone deacetylases, in contrast, remove the acetyl group of histone tails resulting in condensed chromatin with reduced gene expression activity. In plants, the histone acetylation level is regulated by S-nitrosation. NO inhibits HDA complexes resulting in enhanced histone acetylation and promoting a supportive chromatin state for expression of genes. Moreover, methylation of histone tails and DNA are important epigenetic modifications, too. Interestingly, methyltransferases and demethylases are described as targets for redox molecules in several biological systems suggesting that these types of chromatin modifications are also regulated by NO. In this review article, we will focus on redox-regulation of histone acetylation/methylation and DNA methylation in plants, discuss the consequences on the structural level and give an overview where NO can act to modulate chromatin structure.

Just imagine a Plant Science professor in front of a classroom full of interested and attentive students. Imagine what their answers to this intriguing question would be: “What are, according to you, the functions ensured by the plant cell nucleus?” It would be very surprising if some of them would answer cell signaling in response to biotic and abiotic stresses or developmental processes. Most of them would probably answer according to a classical point of view: DNA replication or gene expression. Hence it is still admitted in recent publications (see for instance Fedorenko et al., 2010) that molecules smaller than 40 kDa can diffuse freely across the nuclear envelope pores. However, Pauly et al. (2000) showed by studying nuclear Ca2+ signaling that elevations in the extranuclear Ca2+ concentration do not induce an automatic increase of nuclear [Ca2+] as it could be expected. Hence Ca2+ does not freely diffuse across the nuclear envelop pores, indicating that its transport is finely regulated. Then it becomes evident that we should now consider the nucleus as a key component of cell signaling processes leading to the regulation of specific sets of genes. The aim of this topic was to point out how nuclear post-translational modifications (PTMs) play fundamental roles in the signaling pathways initiated in response to environmental changes. Two major points were assessed. First, different papers clearly demonstrate that the nucleus is fully equipped to perform the main PTMs: (de)phosphorylation (Bigeard and Hirt; Krysan and Colcombet), (de)acetylation (Luo et al.; Fül et al.; Ramirez-Prado et al.), oxidoreduction (Martins et al.) or SUMOylation/ubiquitination (Mazur et al.; Serrano et al.). Hence the nucleus can easily integrate a complex network of second messengers including changes in Ca2+ concentration, reactive oxygen species or nitric oxide. Martins et al. nicely exemplified how changes in the nuclear redox status regulate fundamental processes such as cell cycle, protein transport or transcription via S-nitrosylation or S-glutationylation. Activation of nuclear PTMs can also be achieved by the translocation of enzymes which are both substrates and effectors of these PTMs. This is the case for mitogen-activated protein kinases (MAPKs) that in some specific contexts, translocate from the cytosol to the nucleus upon their activation by their corresponding MAPK kinases (Bigeard and Hirt). Relocation of proteins in response to or through PTMs can also be considered at the intranuclear level. Hence in response to SUMO conjugation several Arabidopsis transcription factors were shown to be re-localized in certain nuclear foci (Mazur et al.). PTMs can also affect the behavior of nuclear proteins and in fine their activity. Serrano et al. highlight how the ubiquitin-proteasome system contributes to the nuclear proteome plasticity. Focusing on E3-Ub-ligases, they illustrate how these enzymes attenuate the signaling pathway once the stress has ceased and how they control the homeostasis of nuclear proteins (transcription factors, immune receptors). The second major point of this topic concerns the target proteins of these PTMs. Of course histones are a piece of choice. The review by Ramirez-Prado et al. illustrates how removing or adding marks (phosphorylation, acetylation, methylation, or ubiquitination) on specific histone lysine residues associated to defense genes (WRKYs, pathogenesis-related proteins, etc.) mostly under the control of salicylic acid or jasmonic acid/ethylene signaling pathways, will control the outcome of the plant-microorganism interaction. A second interesting aspect in this review is the illustration of how pathogens manipulate the chromatin regulatory network of the host to achieve their infection process through for example the production of toxins inhibiting histone deacetylases (HDACs), leading to plant susceptibility. PTMs on histones are also of major importance in the response to abiotic stresses. Luo et al reviewed how HDACs, by deacetylating specific lysine residues (mainly H3K9, H3K14, and H4K15) of specific genes regulate responses to salt, drought, cold or heat. However, chromatin remodeling and in fine regulation of gene expression is not only linked to histone modifications. Fül et al. as a perspective in this topic remind us that subunits of key chromatin remodelers (such as MED12, MED13, and MED19A) or transcription factors like Yin Yang 1 in the response to abscisic acid are also targeted and regulated by lysine deacetylases. Hence lysine (de)acetylation regulates gene expression by acting on histones, but also on the whole transcriptional machinery. In conclusion this topic clearly illustrates the diversity of nuclear PTMs, the crosstalks between some of them, and finally their major roles in the regulation of gene expression. A first fascinating challenge is now to decipher at the molecular level the ways cells are using to translocate signals from cytoplasm to the nucleus that regulate nuclear PTMs. A second one is to go further in the characterization of their target proteins: as it is now nicely exemplified by the interplay of histone marks in epigenetics, this is the sine qua non condition to fully understand the power of nuclear PTMs.

In this year there is the 40th anniversary of the first publication of plant nitric oxide (NO) emission by Lowell Klepper. In the decades since then numerous milestone discoveries have revealed that NO is a multifunctional molecule in plant cells regulating both plant development and stress responses. Apropos of the anniversary, these authors aim to review and discuss the developments of past concepts in plant NO research related to NO metabolism, NO signaling, NO's action in plant growth and in stress responses and NO's interactions with other reactive compounds. Despite the long-lasting research efforts and the accumulating experimental evidences numerous questions are still needed to be answered, thus future challenges and research directions have also been drawn up., Financial background of this work was provided by the National Research, Development and Innovation Fund (Grant no. NKFI-6, K120383 and NKFI-8, KH129511). ZSK was supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences (Grant no. BO/00751/16/8) and by UNKP-18-4 New National Excellence Program of the Ministry of Human Capacities.

Nitric oxide (NO) is an endogenous signaling molecule in plants. Sodium nitroprusside (SNP), an established NO donor used in plant science research, simultaneously releases NO, cyanide (CN−) and iron (Fe) in solution. Since cyanide and iron mask NO effect of SNP, its use in NO research is debatable. Deciphering the action of SNP through NO, CN− or Fe has been undertaken in the present work. Cotyledons from salt stressed sunflower seedlings grown in the presence of NO donors were subjected to spectrofluorometric analysis of NO, CN− and Fe contents, and proteome and biochemical analyses. Diethylenetriamine NONOate (DETA) proved to be a better NO source since SNP enhanced ROS accumulation in the tissue. Abundance of 127 proteins is modulated by salt stress. SNP and exhausted SNP (exSNP) alter the abundance of 117 and 129 proteins, respectively. These proteins belong to primary metabolism, stress-response, transport, translation, proteolysis, chaperone, regulatory, and storage. Salt-responsive proteins, such as, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK) and isocitrate dehydrogenase are negatively modulated. DETA and SNP lower the activities of GAPDH and S-adenosylmethionine synthase (SAMS). Abundance of heat shock 70 kDa protein and actin are sensitive to both NaCl and NO. SNP affects plant growth by modulating proteome though iron, cyanide and NO. Its use only as an NO donor is thus debatable. exSNP control also releases substantial amount of cyanide and iron, thus questioning its use as control in NO research.

Nitric oxide (NO) is an important signalling molecule that is involved in many different physiological processes in plants. Here, we report about a NO-fixing mechanism in Arabidopsis, which allows the fixation of atmospheric NO into nitrogen metabolism. We fumigated Arabidopsis plants cultivated in soil or as hydroponic cultures during the whole growing period with up to 3 ppmv of NO gas. Transcriptomic, proteomic and metabolomic analyses were used to identify non-symbiotic haemoglobin proteins as key components of the NO-fixing process. Overexpressing non-symbiotic haemoglobin 1 or 2 genes resulted in fourfold higher nitrate levels in these plants compared with NO-treated wild-type. Correspondingly, rosettes size and weight, vegetative shoot thickness and seed yield were 25, 40, 30, and 50% higher, respectively, than in wild-type plants. Fumigation with 250 ppbv 15NO confirmed the importance of non-symbiotic haemoglobin 1 and 2 for the NO-fixation pathway, and we calculated a daily uptake for non-symbiotic haemoglobin 2 overexpressing plants of 250 mg N/kg dry weight. This mechanism is probably important under conditions with limited N supply via the soil. Moreover, the plant-based NO uptake lowers the concentration of insanitary atmospheric NOx, and in this context, NO-fixation can be beneficial to air quality.

Nitric oxide (NO) is an important signaling molecule involved in various plant physiological processes. The main effect of NO arises from its reaction with proteins. S-Nitrosation is the most studied NO-mediated protein posttranslational modification in plants. Via S-nitrosation, NO derivatives react with thiol groups (SHs) of protein cysteine residues and produce nitrosothiol groups (SNOs). From the time of discovering the biological function of NO in plants, an interesting case of study has been the detection of the endogenous S-nitrosated proteins in different plants, tissues, organelles, and various conditions. Maps of S-nitrosated proteins provide hints for deeper studies on the function of this modification in specific proteins, biochemical pathways, and physiological processes. Many functions of NO have been found to be related to plant defense; on the other hand the involvement of nuclear proteins in regulation of plant defense reactions is well studied. Here, an approach is described in which the Arabidopsis cell cultures first are treated with P. syringae, afterward their bioactive nuclear proteins are extracted, then the nuclear proteins are subjected to biotin switch assay in which S-nitrosated proteins are specifically converted to S-biotinylated proteins. The biotin switch technique (BST) which was introduced by Jaffrey et al. (Nat Cell Biol 3:193-197, 2001) solves the instability issue of SNOs. Additionally, it provides detection and purification of biotinylated proteins by anti-biotin antibody and affinity chromatography, respectively.

Nitric oxide (NO) is an important signaling molecule involved in various plant physiological processes. The main effect of NO arises from its reaction with proteins. S-Nitrosation is the most studied NO-mediated protein posttranslational modification in plants. Via S-nitrosation, NO derivatives react with thiol groups (SHs) of protein cysteine residues and produce nitrosothiol groups (SNOs). From the time of discovering the biological function of NO in plants, an interesting case of study has been the detection of the endogenous S-nitrosated proteins in different plants, tissues, organelles, and various conditions. Maps of S-nitrosated proteins provide hints for deeper studies on the function of this modification in specific proteins, biochemical pathways, and physiological processes. Many functions of NO have been found to be related to plant defense; on the other hand the involvement of nuclear proteins in regulation of plant defense reactions is well studied. Here, an approach is described in which the Arabidopsis cell cultures first are treated with P. syringae, afterward their bioactive nuclear proteins are extracted, then the nuclear proteins are subjected to biotin switch assay in which S-nitrosated proteins are specifically converted to S-biotinylated proteins. The biotin switch technique (BST) which was introduced by Jaffrey et al. (Nat Cell Biol 3:193-197, 2001) solves the instability issue of SNOs. Additionally, it provides detection and purification of biotinylated proteins by anti-biotin antibody and affinity chromatography, respectively.

This article comments on: Calvo-Begueria L, Rubio MC, Martínez JI, Pérez-Rontomé C, Delgado MJ, Bedmar EJ, Becana M. 2018. Redefining nitric oxide production in legume nodules through complementary insights from electron paramagnetic resonance spectroscopy and specific fluorescent probes. Journal of Experimental Botany 69, 3703-3714.

Nitric oxide (NO) is a free radical gas regulating a wide range of biological processes in plants. Proteins are the main reaction target of NO inside the cells. The relevance of S-nitrosation as one of the NO-mediated protein posttranslational modifications has been studied in detail. S-nitrosylation causes alterations of the activity/function, sub-cellular localization or interaction partners of proteins. Up to present, a large number of S-nitrosation candidates have been detected in plants. Recombinant proteins are widely used to show or confirm the protein posttranslational modifications. Here, using recombinant proteins subjected to biotin switch assay, the S-nitrosation of some nuclear candidates of Arabidopsis is verified. Proteins usually contain several cysteine residues which each might involve in structure of protein active sites. So, an important question is: which cysteine residue is the target of S-nitrosation and does it belong to an active site? Here, using the approach of substitution of cysteines by serines on recombinant proteins, the NO-sensitive cysteine residue of an Arabidopsis nuclear protein is identified. The next step could be to investigate the effect of S-nitrosation on protein activity/function and further to test the role of target cysteines and S-nitrosation of them in protein activity/function.

Ragweed pollen is the main cause of allergenic diseases in Northern America, and the weed has become a spreading neophyte in Europe. Climate change and air pollution are speculated to affect the allergenic potential of pollen. The objective of this study was to investigate the effects of NO2 , a major air pollutant, under controlled conditions, on the allergenicity of ragweed pollen. Ragweed was exposed to different levels of NO2 throughout the entire growing season, and its pollen further analysed. Spectroscopic analysis showed increased outer cell wall polymers and decreased amounts of pectin. Proteome studies using two-dimensional difference gel electrophoresis and liquid chromatography-tandem mass spectrometry indicated increased amounts of several Amb a 1 isoforms and of another allergen with great homology to enolase Hev b 9 from rubber tree. Analysis of protein S-nitrosylation identified nitrosylated proteins in pollen from both conditions, including Amb a 1 isoforms. However, elevated NO2 significantly enhanced the overall nitrosylation. Finally, we demonstrated increased overall pollen allergenicity by immunoblotting using ragweed antisera, showing a significantly higher allergenicity for Amb a 1. The data highlight a direct influence of elevated NO2 on the increased allergenicity of ragweed pollen and a direct correlation with an increased risk for human health.

Summary Superoxide dismutases (SODs) are differentially inhibited by peroxynitrite-mediated tyrosine nitration. Tyr63 is the main target responsible for inactivation of MnSOD1. This mechanism seems to be evolutionarily conserved in multicellular organisms., Despite the importance of superoxide dismutases (SODs) in the plant antioxidant defence system little is known about their regulation by post-translational modifications. Here, we investigated the in vitro effects of nitric oxide derivatives on the seven SOD isoforms of Arabidopsis thaliana. S-nitrosoglutathione, which causes S-nitrosylation of cysteine residues, did not influence SOD activities. By contrast, peroxynitrite inhibited the mitochondrial manganese SOD1 (MSD1), peroxisomal copper/zinc SOD3 (CSD3), and chloroplastic iron SOD3 (FSD3), but no other SODs. MSD1 was inhibited by up to 90% but CSD3 and FSD3 only by a maximum of 30%. Down-regulation of these SOD isoforms correlated with tyrosine (Tyr) nitration and both could be prevented by the peroxynitrite scavenger urate. Site-directed mutagenesis revealed that—amongst the 10 Tyr residues present in MSD1—Tyr63 was the main target responsible for nitration and inactivation of the enzyme. Tyr63 is located nearby the active centre at a distance of only 5.26 Å indicating that nitration could affect accessibility of the substrate binding pocket. The corresponding Tyr34 of human manganese SOD is also nitrated, suggesting that this might be an evolutionarily conserved mechanism for regulation of manganese SODs.

Nitric oxide (NO) and various reactive nitrogen species (RNS) produced in cells in normal growth conditions and their enhanced production under stress conditions, are responsible for a variety of biochemical aberrations. Present findings demonstrate that sunflower seedling roots exhibit high sensitivity to salt stress in terms of nitrite accumulation. A significant reduction in S-nitrosoglutathione reductase (GSNOR) activity is evident in response to salt stress. Restoration of GSNOR activity with dithioerythritol (DTT) shows that the enzyme is reversibly inhibited under conditions of 120 mM NaCl. Salt stress mediated S-nitrosylation of cytosolic proteins was analyzed in roots and cotyledons using biotin switch assay. LC-MS/MS analysis revealed opposite patterns of S-nitrosylation in seedling cotyledons and roots. Salt stress enhances S-nitrosylation of proteins in cotyledons whereas roots exhibit denitrosylation of proteins. Highest number of proteins having undergone S-nitrosylation belonged to the category of carbohydrate metabolism followed by other metabolic proteins. Of the total 61 proteins observed to be regulated by S-nitrosylation, 17 are unique to cotyledons, 4 are unique to roots whereas 40 are common to both. Eighteen S-nitrosylated proteins are being reported for the first time in plant systems, including pectinesterase, phospholipase D alpha and calmodulin. Further physiological analysis of glyceraldehyde-3-phosphate dehydrogenase and monodehydro-ascorbate reductase showed that salt stress leads to a reversible inhibition of both these enzymes in cotyledons. However, seedling roots exhibit enhanced enzyme activity under salinity stress. These observations implicate the role of S-nitrosylation and denitrosylation in NO signaling thereby regulating various enzyme activities under salinity stress in sunflower seedlings.

In the present study, we performed a large-scale protein analysis based on 2-DE DIGE to examine the effects of ozone on the leaves of juvenile European beech ( Fagus sylvatica L.), one of the most important deciduous tree species in Central Europe. To this end, beech trees were grown under field conditions and subjected to ambient and twice ambient ozone concentrations during the vegetation periods of four consecutive years. The twice ambient ozone concentration altered the abundance of 237 protein spots, which showed relative ratios higher than 30% compared to the ambient control trees. A total of 74 protein spots were subjected to mass spectrometry identification (LC–MS/MS), followed by homology-driven searches. The differentially expressed proteins participate in key biological processes including the Calvin cycle and photosynthesis, carbon metabolism, defense- and stress-related responses, detoxification mechanisms, protein folding and degradation, and mechanisms involved in senescence. The ozone-induced responses provide evidence of a changing carbon metabolism and counteraction against increased levels of reactive oxygen species. Biological significance This study provides useful information on how European beech, an economically and ecologically important tree species, reacts on the molecular level to increased ozone concentrations expected in the near future. The main emphasis in the present study was placed on identifying differentially abundant proteins after long-term ozone exposure under climatically realistic settings, rather than short-term responses or reactions under laboratory conditions. Additionally, using nursery-grown beech trees, we took into account the natural genotypic variation of this species. As such, the results presented here provide information on molecular responses to ozone in an experimental plant system at very close to natural conditions. Furthermore, this proteomic approach was supported by previous studies on the present experiment. Ultimately, the combination of this proteomic approach with several approaches including transcriptomics, analysis of non-structural carbohydrates, and morphological effects contributes to a more global picture of how beech trees react under increased ozone concentrations.

Antimicrobial peptides are a promising complement to common antibiotics, development of resistance to which is a growing problem. Here we present a de novo-designed peptide, SP1-1 (RKKRLKLLKRLL-NH2), with antimicrobial activity against multiresistant Staphylococcus aureus (minimal inhibitory concentration: 6.25 μM). Elucidation of the mode of action of this peptide revealed a strong interaction with RsbW kinase (Kd: 6.01±2.73 nM), a serine kinase negatively regulating the activity of the transcription factor σB (SigB). SP1-1 binding and functional modulation of RsbW were shown in vitro by a combination of biochemical, molecular, and biophysical methods, which were further genetically evidenced in vivo by analysis of S. aureus ΔsigB deletion mutants. Intracellular localization of the peptide was demonstrated using nanometer-scaled secondary ion mass spectrometry. Moreover, microarray analysis revealed that transcription of numerous genes, involved in cell wall and amino acid metabolism, transport mechanisms, virulence, and pigmentation, is affected. Interestingly, several WalR binding motif containing genes are induced by SP1-1. In sum, the designed peptide SP1-1 seems to have multiple modes of action, including inhibition of a kinase, and therefore might contribute to the development of new antibacterial compounds, giving bacterial kinase inhibition a closer inspection.-Dangel, A., Ackermann, N., Abdel-Hadi, O., Maier, R., Önder, K., Francois, P., Müller, C. W., Pané-Farré, J., Engelmann, S., Schrenzel, J., Heesemann, J., Lindermayr, C. A de novo-designed antimicrobial peptide with activity against multiresistant Staphylococcus aureus acting on RsbW kinase.

Nitrogen oxide (NOx) is naturally present in the atmosphere as part of the Earth’s nitrogen cycle and has a variety of natural sources. NOx gases are formed whenever combustion occurs in the presence of nitrogen—as in an air-breathing engine; they also are produced naturally by lightning. However, also human activities such as agriculture, fossil fuel combustion, wastewater management, and industrial processes are increasing the amount of NOx in the atmosphere resulting in significant air pollution. But nitrogen is also an essential nutrient required for plant growth and development. Nitrogen can be taken up by plants through ammonium and nitrate or as nitrogen gas through plant-associated microorganisms in root nodules. Interestingly, a plant growth-promoting effect is also described for NO and NO2. In this chapter we want to highlight the positive effect of NO and NO2 on plant growth and development and on postharvest effects on fruits and flowers. Moreover, the NO-scavenging function of phytoglobins is discussed in this context.

Researchers have been examining the biological function(s) of isoprene in isoprene-emitting (IE) species for two decades. There is overwhelming evidence that leaf-internal isoprene increases the thermotolerance of plants and protects them against oxidative stress, thus mitigating a wide range of abiotic stresses. However, the mechanisms of abiotic stress mitigation by isoprene are still under debate. Here, we assessed the impact of isoprene on the emission of nitric oxide (NO) and the S-nitroso-proteome of IE and non-isoprene-emitting (NE) gray poplar (Populus × canescens) after acute ozone fumigation. The short-term oxidative stress induced a rapid and strong emission of NO in NE compared with IE genotypes. Whereas IE and NE plants exhibited under nonstressful conditions only slight differences in their S-nitrosylation pattern, the in vivo S-nitroso-proteome of the NE genotype was more susceptible to ozone-induced changes compared with the IE plants. The results suggest that the nitrosative pressure (NO burst) is higher in NE plants, underlining the proposed molecular dialogue between isoprene and the free radical NO. Proteins belonging to the photosynthetic light and dark reactions, the tricarboxylic acid cycle, protein metabolism, and redox regulation exhibited increased S-nitrosylation in NE samples compared with IE plants upon oxidative stress. Because the posttranslational modification of proteins via S-nitrosylation often impacts enzymatic activities, our data suggest that isoprene indirectly regulates the production of reactive oxygen species (ROS) via the control of the S-nitrosylation level of ROS-metabolizing enzymes, thus modulating the extent and velocity at which the ROS and NO signaling molecules are generated within a plant cell.

Nitric oxide (NO) is a reactive free radical with pleiotropic function that is not only involved in regulation of plant growth and development, but also in the response to biotic and abiotic stressors. It mainly acts by posttranslationally modifying proteins. The most important mode of action of NO is protein S-nitrosylation, the covalent attachment of an NO group to the thiol side of protein cysteine residues. Other major types of NO-dependent modifications are metal nitrosylation and tyrosine nitration. NO can regulate gene expression at different levels. On one side, it can initiate signaling cascades or modify proteins involved in signal transduction pathways. On the other side, redox-sensitive transcription factors can be also target for S-nitrosylation, and NO can also affect redox-dependent nuclear transport of some proteins. This suggests that NO plays a pivotal role in regulating transcription and/or general nuclear metabolism in plants.