S-nitrosation as a redox-based posttranslational modification of protein cysteine has emerged as an integral part of signaling pathways of nitric oxide across all types of organisms. Protein S-nitrosation status is controlled by two key mechanisms: by direct denitrosation performed by the thioredoxin/thioredoxin reductase system, and in an indirect way mediated by S-nitrosoglutathione reductase (GSNOR). GSNOR, which has been identified as a key component of S-nitrosothiols catabolism, catalyzes an irreversible decomposition of abundant intracellular S-nitrosothiol, S-nitrosoglutathione (GSNO) to oxidized glutathione using reduced NADH cofactor. In plants, GSNOR has been shown to play important roles in plant growth and development and plant responses to abiotic and biotic stress stimuli. In this chapter, optimized protocols of spectrophotometric measurement of GSNOR enzymatic activity and activity staining in native polyacrylamide gels in plant GSNOR are presented.

• Klíčová slova
• Nitric oxide, Plant stress, S-nitrosation, S-nitrosoglutathione reductase, S-nitrosothiols,
• MeSH
• aldehydoxidoreduktasy metabolismus   MeSH
• barvení a značení metody   MeSH
• enzymatické testy metody   MeSH
• fluorescence MeSH
• NAD chemie   MeSH
• nativní elektroforéza na polyakrylamidovém gelu MeSH
• nitrosace MeSH
• oxid dusnatý metabolismus   MeSH
• průběh práce MeSH
• rostlinné extrakty izolace a purifikace   metabolismus   MeSH
• rostliny enzymologie   MeSH
• S-nitrosoglutathion chemická syntéza   chemie   MeSH
• S-nitrosothioly metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aldehydoxidoreduktasy MeSH
• formaldehyde dehydrogenase, glutathione-independent MeSH Prohlížeč
• NAD MeSH
• oxid dusnatý MeSH
• rostlinné extrakty MeSH
• S-nitrosoglutathion MeSH
• S-nitrosothioly MeSH

S-nitrosoglutathione reductase (GSNOR) exerts crucial roles in the homeostasis of nitric oxide (NO) and reactive nitrogen species (RNS) in plant cells through indirect control of S-nitrosation, an important protein post-translational modification in signaling pathways of NO. Using cultivated and wild tomato species, we studied GSNOR function in interactions of key enzymes of reactive oxygen species (ROS) metabolism with RNS mediated by protein S-nitrosation during tomato root growth and responses to salinity and cadmium. Application of a GSNOR inhibitor N6022 increased both NO and S-nitrosothiol levels and stimulated root growth in both genotypes. Moreover, N6022 treatment, as well as S-nitrosoglutathione (GSNO) application, caused intensive S-nitrosation of important enzymes of ROS metabolism, NADPH oxidase (NADPHox) and ascorbate peroxidase (APX). Under abiotic stress, activities of APX and NADPHox were modulated by S-nitrosation. Increased production of H2O2 and subsequent oxidative stress were observed in wild Solanumhabrochaites, together with increased GSNOR activity and reduced S-nitrosothiols. An opposite effect occurred in cultivated S. lycopersicum, where reduced GSNOR activity and intensive S-nitrosation resulted in reduced ROS levels by abiotic stress. These data suggest stress-triggered disruption of ROS homeostasis, mediated by modulation of RNS and S-nitrosation of NADPHox and APX, underlies tomato root growth inhibition by salinity and cadmium stress.

• Klíčová slova
• S-nitrosation, S-nitrosoglutathione reductase, Solanum habrochaites, Solanum lycopersicum, abiotic stress, cadmium, nitric oxide, reactive oxygen species, root growth, salinity,
• MeSH
• aldehydoxidoreduktasy metabolismus   MeSH
• askorbátperoxidasa metabolismus   MeSH
• benzamidy chemie   metabolismus   farmakologie   MeSH
• chlorid sodný farmakologie   MeSH
• fyziologický stres MeSH
• kadmium toxicita   MeSH
• kořeny rostlin účinky léků   růst a vývoj   metabolismus   MeSH
• NADPH-oxidasy metabolismus   MeSH
• nitrosace MeSH
• oxid dusnatý metabolismus   MeSH
• oxidační stres účinky léků   MeSH
• peroxid vodíku metabolismus   MeSH
• pyrroly chemie   metabolismus   farmakologie   MeSH
• reaktivní formy dusíku chemie   metabolismus   MeSH
• reaktivní formy kyslíku chemie   metabolismus   MeSH
• regulace genové exprese u rostlin účinky léků   MeSH
• rostlinné proteiny metabolismus   MeSH
• S-nitrosoglutathion farmakologie   MeSH
• S-nitrosothioly metabolismus   MeSH
• Solanum lycopersicum účinky léků   růst a vývoj   metabolismus   MeSH
• Solanum růst a vývoj   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aldehydoxidoreduktasy MeSH
• askorbátperoxidasa MeSH
• benzamidy MeSH
• chlorid sodný MeSH
• formaldehyde dehydrogenase, glutathione-independent MeSH Prohlížeč
• kadmium MeSH
• N6022 MeSH Prohlížeč
• NADPH-oxidasy MeSH
• oxid dusnatý MeSH
• peroxid vodíku MeSH
• pyrroly MeSH
• reaktivní formy dusíku MeSH
• reaktivní formy kyslíku MeSH
• rostlinné proteiny MeSH
• S-nitrosoglutathion MeSH
• S-nitrosothioly MeSH

S-nitrosation, the attachment of a nitroso group to cysteine thiols, has been recognized as an important posttranslational modification of proteins by nitric oxide and related reactive nitrogen species. Mechanisms and significance of S-nitrosation in the regulation of the structure and activity of proteins have been extensively studied in animal and plant systems. In plants, protein S-nitrosation is involved in signaling pathways of plant hormones and regulators during plant growth and development and in responses to abiotic and biotic stress stimuli. S-nitrosoglutathione reductase (GSNOR) has been identified as a key enzyme controlling the intracellular level of S-nitrosothiols. GSNOR irreversibly degrades S-nitrosoglutathione (GSNO), the major low molecular weight S-nitrosothiol involved in the formation of protein S-nitrosothiols through transnitrosylation. GSNOR level and activity in plant cells are modulated during plant development and in response to external stimuli such as pathogen infection. In this chapter, we give a detailed description of the immunochemical detection of the GSNOR protein in plant samples.

• Klíčová slova
• Nitric oxide, S-nitrosation, S-nitrosoglutathione, S-nitrosoglutathione reductase, Western blotting,
• MeSH
• aldehydoxidoreduktasy metabolismus   MeSH
• posttranslační úpravy proteinů * MeSH
• rostlinné proteiny metabolismus   MeSH
• rostliny metabolismus   MeSH
• S-nitrosoglutathion metabolismus   MeSH
• S-nitrosothioly metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• aldehydoxidoreduktasy MeSH
• formaldehyde dehydrogenase, glutathione-independent MeSH Prohlížeč
• rostlinné proteiny MeSH
• S-nitrosoglutathion MeSH
• S-nitrosothioly MeSH

Nitric oxide (NO) is considered as a signalling molecule involved in a variety of important physiological and pathological processes in plant and animal systems. The major pathway of NO reactions in vivo represents S-nitrosation of thiols to form S-nitrosothiols. S-nitrosoglutathione reductase (GSNOR) is the key enzyme in the degradation pathway of S-nitrosoglutathione (GSNO), a low-molecular weight adduct of NO and glutathione. GSNOR indirectly regulates the level of protein S-nitrosothiol in the cells. This study was focused on the dynamic regulation of the activity of plant GSNORs through reversible S-nitrosation and/or oxidative modifications of target cysteine residues. Pre-incubation with NO/NO- donors or hydrogen peroxide resulted in a decreased reductase and dehydrogenase activity of all studied plant GSNORs. Incubation with thiol reducing agent completely reversed inhibitory effects of nitrosative modifications and partially also oxidative inhibition. In biotin-labelled samples, S-nitrosation of plant GSNORs was confirmed after immunodetection and using mass spectrometry S-nitrosation of conserved Cys271 was identified in tomato GSNOR. Negative regulation of constitutive GSNOR activity in vivo by nitrosative or oxidative modifications might present an important mechanism to control GSNO levels, a critical mediator of the downstream signalling effects of NO, as well as for formaldehyde detoxification in dehydrogenase reaction mode.

• Klíčová slova
• Nitric oxide, Post-translational modifications, Redox regulation, S-nitrosation, S-nitrosoglutathione reductase,
• MeSH
• aldehydoxidoreduktasy antagonisté a inhibitory   chemie   metabolismus   MeSH
• cystein chemie   metabolismus   MeSH
• donory oxidu dusnatého farmakologie   MeSH
• nitrosace MeSH
• oxid dusnatý metabolismus   MeSH
• oxidace-redukce MeSH
• peroxid vodíku farmakologie   MeSH
• posttranslační úpravy proteinů MeSH
• rekombinantní proteiny chemie   genetika   metabolismus   MeSH
• rostlinné proteiny antagonisté a inhibitory   chemie   metabolismus   MeSH
• S-nitrosoglutathion metabolismus   MeSH
• S-nitrosothioly metabolismus   MeSH
• signální transdukce MeSH
• Solanum lycopersicum genetika   růst a vývoj   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aldehydoxidoreduktasy MeSH
• cystein MeSH
• donory oxidu dusnatého MeSH
• formaldehyde dehydrogenase, glutathione-independent MeSH Prohlížeč
• oxid dusnatý MeSH
• peroxid vodíku MeSH
• rekombinantní proteiny MeSH
• rostlinné proteiny MeSH
• S-nitrosoglutathion MeSH
• S-nitrosothioly MeSH

We studied the effects of the H(2)S donor Na(2)S on the mean arterial blood pressure (MAP) and heart and breathing rates of anesthetized Wistar rats in the presence and absence of captopril. Bolus administration of Na(2)S (1-4 micromol/kg) into the right jugular vein transiently decreased heart and increased breathing rates; at 8-30 micromol/kg, Na(2)S had a biphasic effect, transiently decreasing and increasing MAP, while transiently decreasing heart rate and increasing and decreasing breathing rate. These results may indicate independent mechanisms by which H(2)S influences MAP and heart and breathing rates. The effect of Na(2)S in decreasing MAP was less pronounced in the presence of captopril (2 micromol/l), which may indicate that the renin-angiotensin system is partially involved in the Na(2)S effect. Captopril decreased H(2)S-induced NO release from S-nitrosoglutathione, which may be related to some biological activities of H(2)S. These results contribute to the understanding of the effects of H(2)S on the cardiovascular system.

• MeSH
• antagonismus léků MeSH
• dechová frekvence fyziologie   účinky záření   MeSH
• kaptopril aplikace a dávkování   MeSH
• krevní tlak účinky léků   fyziologie   MeSH
• krysa rodu Rattus MeSH
• lékové interakce MeSH
• oxid dusnatý metabolismus   MeSH
• potkani Wistar MeSH
• S-nitrosoglutathion metabolismus   MeSH
• sulfan metabolismus   MeSH
• sulfidy aplikace a dávkování   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kaptopril MeSH
• oxid dusnatý MeSH
• S-nitrosoglutathion MeSH
• sodium sulfide MeSH Prohlížeč
• sulfan MeSH
• sulfidy MeSH

Various nitric oxide modulators (NO donors--SNP, GSNO, DEA NONOate and scavengers--PTIO, cPTIO) were tested to highlight the role of NO under Cd excess in various ontogenetic stages of chamomile (Matricaria chamomilla). Surprisingly, compared to Cd alone, SNP and PTIO elevated Cd uptake (confirmed also by PhenGreen staining) but depleted glutathione (partially ascorbic acid) and phytochelatins PC2 and PC3 in both older plants (cultured hydroponically) and seedlings (cultured in deionised water). Despite these anomalous impacts, fluorescence staining of NO and ROS confirmed predictable assumptions and revealed reciprocal changes (decrease in NO but increase in ROS after PTIO addition and the opposite after SNP application). Subsequent tests using alternative modulators and seedlings confirmed changes to NO and ROS after application of GSNO and DEA NONOate as mentioned above for SNP while cPTIO altered only NO level (depletion). On the contrary to SNP and PTIO, GSNO, DEA NONOate and cPTIO did not elevate Cd content and phytochelatins (PC2, PC3) were rather elevated. These data provide evidence that various NO modulators are useful in terms of NO and ROS manipulation but interactions with intact plants affect metal uptake and must therefore be used with caution. In this view, cPTIO and DEA NONOate revealed the less pronounced side impacts and are recommended as suitable NO scavenger/donor in plant physiological studies under Cd excess.

• MeSH
• antioxidancia chemie   MeSH
• cyklické N-oxidy chemie   MeSH
• donory oxidu dusnatého chemie   MeSH
• fluorescenční mikroskopie MeSH
• glutathion chemie   MeSH
• heřmánek, heřmánkovec, rmen, rmenec účinky léků   MeSH
• imidazoly chemie   MeSH
• kadmium chemie   MeSH
• konfokální mikroskopie MeSH
• kyselina askorbová chemie   MeSH
• nitroprusid chemie   MeSH
• oxid dusnatý chemie   MeSH
• reaktivní formy kyslíku chemie   MeSH
• S-nitrosoglutathion chemie   MeSH
• semena rostlinná účinky léků   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide MeSH Prohlížeč
• antioxidancia MeSH
• cyklické N-oxidy MeSH
• donory oxidu dusnatého MeSH
• glutathion MeSH
• imidazoly MeSH
• kadmium MeSH
• kyselina askorbová MeSH
• nitroprusid MeSH
• oxid dusnatý MeSH
• reaktivní formy kyslíku MeSH
• S-nitrosoglutathion MeSH

Carbonyl reductase 1 (CBR1 or SDR21C1) is a ubiquitously-expressed, cytosolic, monomeric, and NADPH-dependent enzyme. CBR1 participates in apoptosis, carcinogenesis and drug resistance, and has a protective role in oxidative stress, cancer and neurodegeneration. S-Nitrosoglutathione (GSNO) represents the newest addition to its diverse substrate spectrum, which includes a wide range of xenobiotics and endogenous substances. GSNO has also been shown to covalently modify and inhibit CBR1. The aim of the present study was to quantify and characterize the resulting modifications. Of five candidate cysteines for modification by 2 mM GSNO (positions 26, 122, 150, 226, 227), the last four were analyzed using MALDI-TOF/TOF mass spectrometry and then quantified using the Selected Reaction Monitoring Approach on hyphenated HPLC with a triple quadrupole mass spectrometer. The analysis confirmed GSNO concentration-dependent S-glutathionylation of cysteines at positions 122, 150, 226, 227 which was 2-700 times higher compared to wild-type CBR1 (WT-CBR1). Moreover, a disulfide bond between neighboring Cys-226 and Cys-227 was detected. We suggest a role of these two cysteines as a redox-sensitive cysteine pair. The catalytic properties of wild-type and enzyme modified with 2 mM GSNO were also investigated by steady state kinetic experiments with various substrates. GSNO treatment of CBR1 resulted in a 2-5-fold decrease in kcat with menadione, 4-benzoylpyridine, 2,3-hexanedione, daunorubicin and 1,4-naphthoquinone. In contrast, the same treatment increased kcat for substrates containing a 1,2-diketo group in a ring structure (1,2-naphthoquinone, 9,10-phenanthrenequinone, isatin). Except for 9,10-phenanthrenequinone, all changes in kcat were at least in part compensated for by a similar change in Km, overall yielding no drastic changes in catalytic efficiency. The findings indicate that GSNO-induced covalent modification of cysteine residues affects the kinetic mechanism of CBR1 both in terms of substrate binding and turnover rate, probably by covalent modification of Cys-226 and/or Cys-227.

• MeSH
• alkoholoxidoreduktasy metabolismus   MeSH
• cystein metabolismus   MeSH
• daunomycin metabolismus   farmakologie   MeSH
• hexanony metabolismus   farmakologie   MeSH
• kinetika MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• naftochinony metabolismus   farmakologie   MeSH
• oxidace-redukce účinky léků   MeSH
• pyridiny metabolismus   farmakologie   MeSH
• S-nitrosoglutathion metabolismus   farmakologie   MeSH
• sekvence aminokyselin MeSH
• vitamin K 3 metabolismus   farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 1,4-naphthoquinone MeSH Prohlížeč
• 2,3-hexanedione MeSH Prohlížeč
• 4-benzoylpyridine MeSH Prohlížeč
• alkoholoxidoreduktasy MeSH
• CBR1 protein, human MeSH Prohlížeč
• cystein MeSH
• daunomycin MeSH
• hexanony MeSH
• naftochinony MeSH
• pyridiny MeSH
• S-nitrosoglutathion MeSH
• vitamin K 3 MeSH

BACKGROUND AND AIMS: Nitric oxide (NO) is involved in the signalling and regulation of plant growth and development and responses to biotic and abiotic stresses. The photoperiod-sensitive mutant 7B-1 in tomato (Solanum lycopersicum) showing abscisic acid (ABA) overproduction and blue light (BL)-specific tolerance to osmotic stress represents a valuable model to study the interaction between light, hormones and stress signalling. The role of NO as a regulator of seed germination and ABA-dependent responses to osmotic stress was explored in wild-type and 7B-1 tomato under white light (WL) and BL. METHODS: Germination data were obtained from the incubation of seeds on germinating media of different composition. Histochemical analysis of NO production in germinating seeds was performed by fluorescence microscopy using a cell-permeable NO probe, and endogenous ABA was analysed by mass spectrometry. KEY RESULTS: The NO donor S-nitrosoglutathione stimulated seed germination, whereas the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) had an inhibitory effect. Under WL in both genotypes, PTIO strongly suppressed germination stimulated by fluridone, an ABA inhibitor. The stimulatory effect of the NO donor was also observed under osmotic stress for 7B-1 seeds under WL and BL. Seed germination inhibited by osmotic stress was restored by fluridone under WL, but less so under BL, in both genotypes. This effect of fluridone was further modulated by the NO donor and NO scavenger, but only to a minor extent. Fluorescence microscopy using the cell-permeable NO probe DAF-FM DA (4-amino-5-methylamino-2',7'-difluorofluorescein diacetate) revealed a higher level of NO in stressed 7B-1 compared with wild-type seeds. CONCLUSIONS: As well as defective BL signalling, the differential NO-dependent responses of the 7B-1 mutant are probably associated with its high endogenous ABA concentration and related impact on hormonal cross-talk in germinating seeds. These data confirm that light-controlled seed germination and stress responses include NO-dependent signalling.

• MeSH
• biologické modely MeSH
• cyklické N-oxidy farmakologie   MeSH
• donory oxidu dusnatého farmakologie   MeSH
• fluoresceiny analýza   MeSH
• fyziologický stres * účinky léků   účinky záření   MeSH
• imidazoly farmakologie   MeSH
• kinetika MeSH
• klíčení * účinky léků   účinky záření   MeSH
• kyselina abscisová metabolismus   MeSH
• mutace MeSH
• osmóza účinky léků   účinky záření   MeSH
• oxid dusnatý farmakologie   MeSH
• pyridony farmakologie   MeSH
• regulace genové exprese u rostlin účinky léků   účinky záření   MeSH
• regulátory růstu rostlin metabolismus   MeSH
• S-nitrosoglutathion farmakologie   MeSH
• scavengery volných radikálů farmakologie   MeSH
• semena rostlinná účinky léků   genetika   fyziologie   účinky záření   MeSH
• signální transdukce účinky léků   účinky záření   MeSH
• Solanum lycopersicum účinky léků   genetika   fyziologie   účinky záření   MeSH
• světlo * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide MeSH Prohlížeč
• 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate MeSH Prohlížeč
• cyklické N-oxidy MeSH
• donory oxidu dusnatého MeSH
• fluoresceiny MeSH
• fluridone MeSH Prohlížeč
• imidazoly MeSH
• kyselina abscisová MeSH
• oxid dusnatý MeSH
• pyridony MeSH
• regulátory růstu rostlin MeSH
• S-nitrosoglutathion MeSH
• scavengery volných radikálů MeSH