Effect of nitric oxide on gene transcription – S-nitrosylation of nuclear proteins

Nitric oxide (NO) plays an important role in many different physiological processes in plants. It mainly acts by post-translationally modifying proteins. Modification of cysteine residues termed as S-nitrosylation is believed to be the most important mechanism for transduction of bioactivity of NO. The first proteins found to be nitrosylated were mainly of cytoplasmic origin or isolated from mitochondria and peroxisomes. Interestingly, it was shown that redox-sensitive transcription factors are also nitrosylated and that NO influences the redox-dependent nuclear transport of some proteins. This implies that NO plays a role in regulating transcription and/or general nuclear metabolism which is a fascinating new aspect of NO signaling in plants. In this review, we will discuss the impact of S-nitrosylation on nuclear plant proteins with a focus on transcriptional regulation, describe the function of this modification and draw also comparisons to the animal system in which S-nitrosylation of nuclear proteins is a well characterized concept

Protein Tyrosine Nitration during Development and Abiotic Stress Response in Plants

In recent years, the study of nitric oxide (NO) in plant systems has attracted the attention of many researchers. A growing number of investigations have shown the significance of NO as a signal molecule or as a molecule involved in the response against (a)biotic processes. NO can be responsible of the post-translational modifications (NO-PTM) of target proteins by mechanisms such as the nitration of tyrosine residues. The study of protein tyrosine nitration during development and under biotic and adverse environmental conditions has increased in the last decade; nevertheless, there is also an endogenous nitration which seems to have regulatory functions. Moreover, the advance in proteome techniques has enabled the identification of new nitrated proteins, showing the high variability among plant organs, development stage and species. Finally, it may be important to discern between a widespread protein nitration because of greater RNS content, and the specific nitration of key targets which could affect cell-signaling processes. In view of the above point, we present a mini-review that offers an update about the endogenous protein tyrosine nitration, during plant development and under several abiotic stress conditions.This study was supported by an ERDF grant co-financed by the Ministry of Economy and Competitiveness (project BIO2015-66390-P) and Junta de Andalucía (groups BIO286 and BIO192). Research in FJC laboratory is supported by an ERDF grant co-financed by the Ministry of Economy and Competitiveness (AGL2015-65104-P).Peer reviewedPeer Reviewe

Short-Term Low Temperature Induces Nitro-Oxidative Stress that Deregulates the NADP-Malic Enzyme Function by Tyrosine Nitration in Arabidopsis thaliana

Low temperature (LT) negatively affects plant growth and development via the alteration of the metabolism of reactive oxygen and nitrogen species (ROS and RNS). Among RNS, tyrosine nitration, the addition of an NO2 group to a tyrosine residue, can modulate reduced nicotinamide-dinucleotide phosphate (NADPH)-generating systems and, therefore, can alter the levels of NADPH, a key cofactor in cellular redox homeostasis. NADPH also acts as an indispensable electron donor within a wide range of enzymatic reactions, biosynthetic pathways, and detoxification processes, which could affect plant viability. To extend our knowledge about the regulation of this key cofactor by this nitric oxide (NO)-related post-translational modification, we analyzed the effect of tyrosine nitration on another NADPH-generating enzyme, the NADP-malic enzyme (NADP-ME), under LT stress. In Arabidopsis thaliana seedlings exposed to short-term LT (4 °C for 48 h), a 50% growth reduction accompanied by an increase in the content of superoxide, nitric oxide, and peroxynitrite, in addition to diminished cytosolic NADP-ME activity, were found. In vitro assays confirmed that peroxynitrite inhibits cytosolic NADP-ME2 activity due to tyrosine nitration. The mass spectrometric analysis of nitrated NADP-ME2 enabled us to determine that Tyr-73 was exclusively nitrated to 3-nitrotyrosine by peroxynitrite. The in silico analysis of the Arabidopsis NADP-ME2 protein sequence suggests that Tyr73 nitration could disrupt the interactions between the specific amino acids responsible for protein structure stability. In conclusion, the present data show that short-term LT stress affects the metabolism of ROS and RNS, which appears to negatively modulate the activity of cytosolic NADP-ME through the tyrosine nitration processThis research was funded by ERDF grants co-financed by the Ministry of Economy and Competitiveness (project PGC2018-096405-B-I00) and the Junta de Andalucía (group BIO286) in Spain. Research in FJ-C lab is supported by an ERDF-co-financed grant from the Ministry of Economy and Competitiveness (AGL2015-65104-P) and Junta de Andalucía (group BIO-192), Spain. Postdoctoral researcher J.B.-M. was funded by the Ministry of Economy and Competitiveness (Spain) within Juan de la Cierva-Incorporación program (IJCI-2015-23438)

Dual regulation of cytosolic ascorbate peroxidase (APX) by tyrosine nitration and S-nitrosylation

JBM acknowledges a PhD fellowship (F.P.U.) from the Ministry of Science and Innovation. This work was supported by an ERDF-co-financed grant from the Ministry of Science and Innovation (BIO2009-12003-C02-01, BIO2009-12003-C02-02, and BIO2012-33904) and Junta de Andalucia (group BIO286 and BIO192), Spain. LC/MS/MS analyses were carried out at the Laboratorio de Proteomica LP-CSIC/UAB, a member of the ProteoRed network. Technical and human support provided by CICT of Universidad de Jaen (UJA, MINECO, Junta de Andalucia, FEDER) is gratefully acknowledged. We acknowledge Mr Carmelo Ruiz-Torres for his excellent technical support.Post-translational modifications (PTMs) mediated by nitric oxide (NO)-derived molecules have become a new area of research, as they can modulate the function of target proteins. Proteomic data have shown that ascorbate peroxidase (APX) is one of the potential targets of PTMs mediated by NO-derived molecules. Using recombinant pea cytosolic APX, the impact of peroxynitrite (ONOO–) and S-nitrosoglutathione (GSNO), which are known to mediate protein nitration and S-nitrosylation processes, respectively, was analysed. While peroxynitrite inhibits APX activity, GSNO enhances its enzymatic activity. Mass spectrometric analysis of the nitrated APX enabled the determination that Tyr5 and Tyr235 were exclusively nitrated to 3-nitrotyrosine by peroxynitrite. Residue Cys32 was identified by the biotin switch method as S-nitrosylated. The location of these residues on the structure of pea APX reveals that Tyr235 is found at the bottom of the pocket where the haem group is enclosed, whereas Cys32 is at the ascorbate binding site. Pea plants grown under saline (150mM NaCl) stress showed an enhancement of both APX activity and S-nitrosylated APX, as well as an increase of H2O2, NO, and S-nitrosothiol (SNO) content that can justify the induction of the APX activity. The results provide new insight into the molecular mechanism of the regulation of APX which can be both inactivated by irreversible nitration and activated by reversible S-nitrosylation.Spanish GovernmentERDF from the Ministry of Science and Innovation BIO2009-12003-C02-01 BIO2009-12003-C02-02 BIO2012-33904Junta de Andalucia BIO286 BIO192CICT of Universidad de Jaen (UJA, MINECO, Junta de Andalucia, FEDER

Estudio de las especies de nitrógeno reactivo en plantas durante el proceso de estrés biótico en la interacción girasol-mildiu

En hipocótilos de plántulas de girasol sensibles y resistentes a la infección por el hongo parásito Plasmopara halstedii, responsable del mildiu, los análisis mediante quimioluminiscencia de ozono revelaron una mayor producción de óxido nítrico en la variedad sensible frente a la resistente, tanto en plantas controles como en plantas inoculadas.La inmunolocalización mediante microscopía de fluorescencia y microscopía confocal láser mostró la localización extensiva de NOS (Óxido nítrico sintasa) y Snitrosoglutation (GSNO) en células parenquimáticas. La lozalización tisular preferente del GSNO en la zona de entrada del patógeno en el hipocótilo, evidencia la posible participación del óxido nítrico en los mecanismos de defensa celulares de respuesta inmediata frente a la invasión por patógenos y antes de la inducción de la producción de óxido nítrico por las proteínas responsables de su generación.El análisis de estos resultados evidencia la presencia en células de hipocótilos de girasol de proteínas tipo NOS y sugiere la participación de la NOS, del óxido nítrico (NO·) y del GSNO en la respuesta de la planta frente al estrés biótico por el hongo Plasmopara halstedii

Differential molecular response of monodehydroascorbate reductase and glutathione reductase by nitration and S-nitrosylation

The ascorbate–glutathione cycle is a metabolic pathway that detoxifies hydrogen peroxide and involves enzymatic and non-enzymatic antioxidants. Proteomic studies have shown that some enzymes in this cycle such as ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), and glutathione reductase (GR) are potential targets for post-translational modifications (PMTs) mediated by nitric oxide-derived molecules. Using purified recombinant pea peroxisomal MDAR and cytosolic and chloroplastic GR enzymes produced in Escherichia coli, the effects of peroxynitrite (ONOO–) and S-nitrosoglutathione (GSNO) which are known to mediate protein nitration and S-nitrosylation processes, respectively, were analysed. Although ONOO– and GSNO inhibit peroxisomal MDAR activity, chloroplastic and cytosolic GR were not affected by these molecules. Mass spectrometric analysis of the nitrated MDAR revealed that Tyr213, Try292, and Tyr345 were exclusively nitrated to 3-nitrotyrosine by ONOO–. The location of these residues in the structure of pea peroxisomal MDAR reveals that Tyr345 is found at 3.3 Å of His313 which is involved in the NADPbinding site. Site-directed mutagenesis confirmed Tyr345 as the primary site of nitration responsible for the inhibition of MDAR activity by ONOO–. These results provide new insights into the molecular regulation of MDAR which is deactivated by nitration and S-nitrosylation. However, GR was not affected by ONOO– or GSNO, suggesting the existence of a mechanism to conserve redox status by maintaining the level of reduced GSH. Under a nitro-oxidative stress induced by salinity (150 mM NaCl), MDAR expression (mRNA, protein, and enzyme activity levels) was increased, probably to compensate the inhibitory effects of S-nitrosylation and nitration on the enzyme. The present data show the modulation of the antioxidative response of key enzymes in the ascorbate–glutathione cycle by nitric oxide (NO)- PTMs, thus indicating the close involvement of NO and reactive oxygen species metabolism in antioxidant defence against nitro-oxidative stress situations in plants.Spanish GovernmentERDF - Ministry of Economy and Competitiveness BIO2012-33904Junta de Andalucía BIO286 BIO19

Oxidative Stress in Plants

Environmental stresses negatively affect plant growth, development and crop productivity [...

Computational Prediction of Candidate Proteins for S-Nitrosylation in <i>Arabidopsis thaliana</i>

<div><p>Nitric oxide (NO) is an important signaling molecule that regulates many physiological processes in plants. One of the most important regulatory mechanisms of NO is S-nitrosylation—the covalent attachment of NO to cysteine residues. Although the involvement of cysteine S-nitrosylation in the regulation of protein functions is well established, its substrate specificity remains unknown. Identification of candidates for S-nitrosylation and their target cysteine residues is fundamental for studying the molecular mechanisms and regulatory roles of S-nitrosylation in plants. Several experimental methods that are based on the biotin switch have been developed to identify target proteins for S-nitrosylation. However, these methods have their limits. Thus, computational methods are attracting considerable attention for the identification of modification sites in proteins. Using GPS-SNO version 1.0, a recently developed S-nitrosylation site-prediction program, a set of 16,610 candidate proteins for S-nitrosylation containing 31,900 S-nitrosylation sites was isolated from the entire <i>Arabidopsis</i> proteome using the medium threshold. In the compartments “chloroplast,” “CUL4-RING ubiquitin ligase complex,” and “membrane” more than 70% of the proteins were identified as candidates for S-nitrosylation. The high number of identified candidates in the proteome reflects the importance of redox signaling in these compartments. An analysis of the functional distribution of the predicted candidates showed that proteins involved in signaling processes exhibited the highest prediction rate. In a set of 46 proteins, where 53 putative S-nitrosylation sites were already experimentally determined, the GPS-SNO program predicted 60 S-nitrosylation sites, but only 11 overlap with the results of the experimental approach. In general, a computer-assisted method for the prediction of targets for S-nitrosylation is a very good tool; however, further development, such as including the three dimensional structure of proteins in such analyses, would improve the identification of S-nitrosylation sites.</p></div

Prediction of <i>Arabidopsis</i> candidate proteins for S-nitrosylation using the GPS-SNO 1.0 software.

<p><i>Arabidopsis</i> amino acid sequences were extracted from TAIR 10 database (<a href="http://www.arabidopsis.org" target="_blank">www.arabidopsis.org</a>) and analysed by GPS-SNO 1.0 software using medium threshold condition. The 10% of predicted sites with the highest prediction confidence were determined by ranking the prediction results according to the raw score divided by the threshold (Cutoff) for a particular cluster.</p><p>Prediction of <i>Arabidopsis</i> candidate proteins for S-nitrosylation using the GPS-SNO 1.0 software.</p

Subcellular compartment classification of <i>Arabidopsis</i> proteins.

<p>Total number of proteins, number of predicted candidates for <i>S</i>-nitrosylation, and the number of candidates with the highest 10% prediction confidence were assigned to their subcellular localization according to gene ontology cellular component classification. The prediction confidence was calculated by dividing the raw score value by the cutoff value of a particular cluster.</p><p>Subcellular compartment classification of <i>Arabidopsis</i> proteins.</p