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

Nitro-fatty acids in plant signaling: Nitro-linolenic acid induces the molecular chaperone network in Arabidopsis

Nitro-fatty acids (NO-FAs) are the product of the reaction between reactive nitrogen species derived of nitric oxide (NO) and unsaturated fatty acids. In animal systems, NO-FAs are considered novel signaling mediators of cell function based on a proven antiinflammatory response. Nevertheless, the interaction of NO with fatty acids in plant systems has scarcely been studied. Here, we examine the endogenous occurrence of nitro-linolenic acid (NO-Ln) in Arabidopsis and the modulation of NO-Ln levels throughout this plant’s development by mass spectrometry. The observed levels of this NO-FA at picomolar concentrations suggested its role as a signaling effector of cell function. In fact, a transcriptomic analysis by RNA-seq technology established a clear signaling role for this molecule, demonstrating that NO-Ln was involved in plant defense response against different abiotic-stress conditions, mainly by inducing heat shock proteins and supporting a conserved mechanism of action in both animal and plant defense processes. Bioinformatics analysis revealed that NO-Ln was also involved in the response to oxidative stress conditions, mainly depicted by HO, reactive oxygen species, and oxygen-containing compound responses, with a high induction of ascorbate peroxidase expression. Closely related to these results, NO-Ln levels significantly rose under several abiotic-stress conditions such as wounding or exposure to salinity, cadmium, and low temperature, thus validating the outcomes found by RNA-seq technology. Jointly, to our knowledge, these are the first results showing the endogenous presence of NO-Ln in Arabidopsis (Arabidopsis thaliana) and supporting the strong signaling role of these molecules in the defense mechanism against different abiotic-stress situations.C.M.-P. thanks the University of Jaén for funding the Ph.D. fellowship. LC-MS/MS analyses were carried out at the Technical Services Department of the University of Granada, Spain. ACSCs were kindly provided by Dr. Juan Bautista Arellano from the Institute of Natural Resources and Agrobiology (IRNASA-CSIC, Salamanca, Spain).Peer Reviewe

Functions of nitric oxide-mediated post-translational modifications under abiotic stress

Environmental conditions greatly impact plant growth and development. In the current context of both global climate change and land degradation, abiotic stresses usually lead to growth restriction limiting crop production. Plants have evolved to sense and respond to maximize adaptation and survival; therefore, understanding the mechanisms involved in the different converging signaling networks becomes critical for improving plant tolerance. In the last few years, several studies have shown the plant responses against drought and salinity, high and low temperatures, mechanical wounding, heavy metals, hypoxia, UV radiation, or ozone stresses. These threats lead the plant to coordinate a crosstalk among different pathways, highlighting the role of phytohormones and reactive oxygen and nitrogen species (RONS). In particular, plants sense these reactive species through post-translational modification (PTM) of macromolecules such as nucleic acids, proteins, and fatty acids, hence triggering antioxidant responses with molecular implications in the plant welfare. Here, this review compiles the state of the art about how plant systems sense and transduce this crosstalk through PTMs of biological molecules, highlighting the S-nitrosylation of protein targets. These molecular mechanisms finally impact at a physiological level facing the abiotic stressful traits that could lead to establishing molecular patterns underlying stress responses and adaptation strategies

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

Transcriptomic profiling of linolenic acid-responsive genes in ROS signaling from RNA-seq data in Arabidopsis

Linolenic acid (Ln) released from chloroplast membrane galactolipids is a precursor of the phytohormone jasmonic acid (JA). The involvement of this hormone in different plant biological processes, such as responses to biotic stress conditions, has been extensively studied. However, the role of Ln in the regulation of gene expression during abiotic stress situations mediated by cellular redox changes and/or by oxidative stress processes remains poorly understood. An RNA-seq approach has increased our knowledge of the interplay among Ln, oxidative stress and ROS signaling that mediates abiotic stress conditions. Transcriptome analysis with the aid of RNA-seq in the absence of oxidative stress revealed that the incubation of Arabidopsis thaliana cell suspension cultures (ACSC) with Ln resulted in the modulation of 7525 genes, of which 3034 genes had a 2-fold-change, being 533 up- and 2501 down-regulated genes, respectively. Thus, RNA-seq data analysis showed that an important set of these genes were associated with the jasmonic acid biosynthetic pathway including lypoxygenases (LOXs) and Allene oxide cyclases (AOCs). In addition, several transcription factor families involved in the response to biotic stress conditions (pathogen attacks or herbivore feeding), such as WRKY, JAZ, MYC, and LRR were also modified in response to Ln. However, this study also shows that Ln has the capacity to modulate the expression of genes involved in the response to abiotic stress conditions, particularly those mediated by ROS signaling. In this regard, we were able to identify new targets such as galactinol synthase 1 (GOLS1), methionine sulfoxide reductase (MSR) and alkenal reductase in ACSC. It is therefore possible to suggest that, in the absence of any oxidative stress, Ln is capable of modulating new sets of genes involved in the signaling mechanism mediated by additional abiotic stresses (salinity, UV and high light intensity) and especially in stresses mediated by ROS.CP would like to thank the University of Jaén for funding the Ph.D. fellowship. This study was supported by an ERDF grant co-financed by the Ministry of Economy and Competitiveness (projects BIO2012-33904 and RECUPERA2020) and the Junta de Andalucía (groups BIO286 and BIO192) in Spain.Peer reviewedPeer Reviewe

Nitro-fatty acids in plant signaling: New key mediators of nitric oxide metabolism

Recent studies in animal systems have shown that NO can interact with fatty acids to generate nitro-fatty acids (NO-FAs). They are the product of the reaction between reactive nitrogen species and unsaturated fatty acids, and are considered novel mediators of cell signaling based mainly on a proven anti-inflammatory response. Although these signaling mediators have been described widely in animal systems, NO-FAs have scarcely been studied in plants. Preliminary data have revealed the endogenous presence of free and protein-adducted NO-FAs in extra-virgin olive oil (EVOO), which appear to be contributing to the cardiovascular benefits associated with the Mediterranean diet. Importantly, new findings have displayed the endogenous occurrence of nitro-linolenic acid (NO-Ln) in the model plant Arabidopsis thaliana and the modulation of NO-Ln levels throughout this plant's development. Furthermore, a transcriptomic analysis by RNA-seq technology established a clear signaling role for this molecule, demonstrating that NO-Ln was involved in plant-defense response against different abiotic-stress conditions, mainly by inducing the chaperone network and supporting a conserved mechanism of action in both animal and plant defense processes. Thus, NO-Ln levels significantly rose under several abiotic-stress conditions, highlighting the strong signaling role of these molecules in the plant-protection mechanism. Finally, the potential of NO-Ln as a NO donor has recently been described both in vitro and in vivo. Jointly, this ability gives NO-Ln the potential to act as a signaling molecule by the direct release of NO, due to its capacity to induce different changes mediated by NO or NO-related molecules such as nitration and S-nitrosylation, or by the electrophilic capacity of these molecules through a nitroalkylation mechanism. Here, we describe the current state of the art regarding the advances performed in the field of NO-FAs in plants and their implication in plant physiology.CM-P would like to thank the University of Jaén for funding the Ph.D. fellowship. This study was supported by the ERDF grants co-financed by the Ministry of Economy and Competitiveness (projects BIO2015-66390-P and AGL2015-65104-P) and the Junta de Andalucía (groups BIO286 and BIO192) in Spain.Peer Reviewe