31595469 OR Measurement of S-Nitrosoglutathione Reductase Activity in Plants
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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.
- 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
S-nitrosoglutathione reductase (GSNOR) is considered a key enzyme in the regulation of intracellular levels of S-nitrosoglutathione and protein S-nitrosylation. As a part of nitric oxide catabolism, GSNOR catalyzes the irreversible decomposition of GSNO to oxidized glutathione. GSNOR is involved in the regulation of plant growth and development, mediated by NO-dependent signaling mechanisms, and is known to play important roles in plant responses to various abiotic and biotic stress conditions. Here we present optimized protocols to determine GSNOR enzyme activities in plant samples by spectrophotometric measurements and by activity staining after the native polyacrylamide gel electrophoresis.
Cellular homeostasis of S-nitrosoglutathione (GSNO), a major cache of nitric oxide bioactivity in plants, is controlled by the NADH-dependent S-nitrosoglutathione reductase (GSNOR) belonging to the family of class III alcohol dehydrogenases (EC 1.1.1.1). GSNOR is a key regulator of S-nitrosothiol metabolism and is involved in plant responses to abiotic and biotic stresses. This study was focused on GSNOR from two important crop plants, cauliflower (Brassica oleracea var. botrytis, BoGSNOR) and lettuce (Lactuca sativa, LsGSNOR). Both purified recombinant GSNORs were characterized in vitro and found to exists as dimers, exhibit high thermal stability and substrate preference towards GSNO, although both enzymes have dehydrogenase activity with a broad range of long-chain alcohols and ω-hydroxy fatty acids in presence of NAD+. Data on enzyme affinities to their cofactors NADH and NAD+ obtained by isothermal titration calorimetry suggest the high affinity to NADH might underline the GSNOR capacity to function in the intracellular environment. GSNOR activity and gene expression peak during early developmental stages of lettuce and cauliflower at 20 and 30 days after germination, respectively. GSNOR activity was also measured in four other Lactuca spp. genotypes with different degree of resistance to biotrophic pathogen Bremia lactucae. Higher GSNOR activities were found in non-infected plants of susceptible genotypes L. sativa UCDM2 and L. serriola as compared to resistant genotypes. GSNOR and GSNO were localized by confocal laser scanning microscopy in vascular bundles and in epidermal and parenchymal cells of leaf cross-sections. The presented results bring new insight in the role of GSNOR in the regulation of S-nitrosothiol levels in plant growth and development.
