Measurement of S-Nitrosoglutathione Reductase Activity in Plants.

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.

Tomato Root Growth Inhibition by Salinity and Cadmium Is Mediated By S-Nitrosative Modifications of ROS Metabolic Enzymes Controlled by S-Nitrosoglutathione Reductase.

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.

Diverse responses of wild and cultivated tomato to BABA, oligandrin and Oidium neolycopersici infection.

Background and Aims: Current strategies for increased crop protection of susceptible tomato plants against pathogen infections include treatment with synthetic chemicals, application of natural pathogen-derived compounds or transfer of resistance genes from wild tomato species within breeding programmes. In this study, a series of 45 genes potentially involved in defence mechanisms was retrieved from the genome sequence of inbred reference tomato cultivar Solanum lycopersicum 'Heinz 1706'. The aim of the study was to analyse expression of these selected genes in wild and cultivated tomato plants contrasting in resistance to the biotrophic pathogen Oidium neolycopersici , the causative agent of powdery mildew. Plants were treated either solely with potential resistance inducers or by inducers together with the pathogen. Methods: The resistance against O. neolycopersici infection as well as RT-PCR-based analysis of gene expression in response to the oomycete elicitor oligandrin and chemical agent ß-aminobutyric acid (BABA) were investigated in the highly susceptible domesticated inbred genotype Solanum lycopersicum 'Amateur' and resistant wild genotype Solanum habrochaites . Key Results: Differences in basal expression levels of defensins, germins, ß-1,3-glucanases, heveins, chitinases, osmotins and PR1 proteins in non-infected and non-elicited plants were observed between the highly resistant and susceptible genotypes. Moreover, these defence genes showed an extensive up-regulation following O. neolycopersici infection in both genotypes. Application of BABA and elicitin induced expression of multiple defence-related transcripts and, through different mechanisms, enhanced resistance against powdery mildew in the susceptible tomato genotype. Conclusions: The results indicate that non-specific resistance in the resistant genotype S. habrochaites resulted from high basal levels of transcripts with proven roles in defence processes. In the susceptible genotype S. lycopersicum 'Amateur', oligandrin- and BABA-induced resistance involved different signalling pathways, with BABA-treated leaves displaying direct activation of the ethylene-dependent signalling pathway, in contrast to previously reported jasmonic acid-mediated signalling for elicitins.

Nitric oxide and reactive oxygen species regulate the accumulation of heat shock proteins in tomato leaves in response to heat shock and pathogen infection.

Heat shock proteins (HSP) are produced in response to various stress stimuli to prevent cell damage. We evaluated the involvement of nitric oxide (NO) and reactive oxygen species (ROS) in the accumulation of Hsp70 proteins in tomato leaves induced by abiotic and biotic stress stimuli. A model system of leaf discs was used with two tomato genotypes, Solanum lycopersicum cv. Amateur and Solanum chmielewskii, differing in their resistance to fungal pathogen Oidium neolycopersici. Leaf discs were exposed to stress factors as heat shock and pathogen infection alone or in a combination, and treated with substances modulating endogenous NO and ROS levels. Two proteins of Hsp70 family were detected in stressed tomato leaf discs: a heat-inducible 72 kDa protein and a constitutive 75 kDa protein. The pathogenesis and mechanical stress influenced Hsp75 accumulation, whereas heat stress induced mainly Hsp72 production. Treatment with NO donor and NO scavenger significantly modulated the level of Hsp70 in variable manner related to the genotype resistance. Hsp70 accumulation correlated with endogenous NO level in S. lycopersicum and ROS levels in S. chmielewskii. We conclude NO and ROS are involved in the regulation of Hsp70 production and accumulation under abiotic and biotic stresses in dependence on plant ability to trigger its defence mechanisms.

The role of nitric oxide in the germination of plant seeds and pollen.

Two complex physiological processes, with opposite positions in the plant's life-cycle, seed and pollen germination, are vital to the accomplishment of successful plant growth and reproduction. This review summarizes the current state of knowledge of the intersection of NO signalling with the signalling pathways of ABA, GA, and ethylene; plant hormones that control the release of plant seeds from dormancy and germination. The cross-talk of NO and ROS is involved in the light- and hormone-specific regulation of seeds' developmental processes during the initiation of plant ontogenesis. Similarly to seed germination, the mechanisms of plant pollen hydration, germination, tube growth, as well as pollen-stigma recognition are tightly linked to the proper adjustment of NO and ROS levels. The interaction of NO with ROS and secondary messengers such as Ca(2+), cAMP and cGMP discovered in pollen represent a common mechanism of NO signalling. The involvement of NO in both breakpoints of plant physiology, as well as in the germination of spores within fungi and oomycetes, points toward NO as a component of an evolutionary conserved signalling pathway.

Local and systemic production of nitric oxide in tomato responses to powdery mildew infection.

Various genetic and physiological aspects of resistance of Lycopersicon spp. to Oidium neolycopersici have been reported, but limited information is available on the molecular background of the plant-pathogen interaction. This article reports the changes in nitric oxide (NO) production in three Lycopersicon spp. genotypes which show different levels of resistance to tomato powdery mildew. NO production was determined in plant leaf extracts of L. esculentum cv. Amateur (susceptible), L. chmielewskii (moderately resistant) and L. hirsutum f. glabratum (highly resistant) by the oxyhaemoglobin method during 216 h post-inoculation. A specific, two-phase increase in NO production was observed in the extracts of infected leaves of moderately and highly resistant genotypes. Moreover, transmission of a systemic response throughout the plant was observed as an increase in NO production within tissues of uninoculated leaves. The results suggest that arginine-dependent enzyme activity was probably the main source of NO in tomato tissues, which was inhibited by competitive reversible and irreversible inhibitors of animal NO synthase, but not by a plant nitrate reductase inhibitor. In resistant tomato genotypes, increased NO production was localized in infected tissues by confocal laser scanning microscopy using the fluorescent probe 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate. NO production observed in the extracts from pathogen conidia, together with elevated NO production localized in developing pathogen hyphae, demonstrates a complex role of NO in plant-pathogen interactions. Our results are discussed with regard to a possible role of increased NO production in pathogens during pathogenesis, as well as local and systemic plant defence mechanisms.