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

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