In the temperate climates of central Europe and North America, two distinct honeybee (Apis mellifera) populations are found in colonies: short-living summer bees emerge in spring and survive until summer, whereas long-living winter bees emerge in late August and overwinter. Besides the difference in their life spans, each of these populations fulfils a different role in the colonies and individual bees have distinct physiological and immunological adaptations depending on their roles. For instance, winter worker bees have higher vitellogenin levels and larger reserves of nutrients in the fat body than summer bees. The differences between the immune systems of both populations are well described at the constitutive level; however, our knowledge of its inducibility is still very limited. In this study, we focus on the response of 10-day-old honeybee workers to immune challenges triggered in vivo by injecting heat-killed bacteria, with particular focus on honeybees that emerge and live under hive conditions. Responses to bacterial injections differed between summer and winter bees. Winter bees exhibited a more intense response, including higher expression of antimicrobial genes and antimicrobial activity, as well as a significant decrease in vitellogenin gene expression and its concentration in the hemolymph. The intense immune response observed in winter honeybees may contribute to our understanding of the relationships between colony fitness and infection with pathogens, as well as its association with successful overwintering.
- MeSH
- imunita * MeSH
- roční období MeSH
- včely MeSH
- vitelogeniny * MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Evropa MeSH
- Severní Amerika MeSH
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
MAIN CONCLUSION: The level of resistance induced in different tomato genotypes after β-CRY treatment correlated with the upregulation of defence genes, but not sterol binding and involved ethylene and jasmonic acid signalling. Elicitins, a family of small proteins secreted by Phytophthora and Pythium spp., are the most well-known microbe-associated molecular patterns of oomycetes, a lineage of fungus-like organisms that include many economically significant crop pathogens. The responses of tomato plants to elicitin INF1 produced by Phytophthora infestans have been studied extensively. Here, we present studies on the responses of three tomato genotypes to β-cryptogein (β-CRY), a potent elicitin secreted by Phytophthora cryptogea that induces hypersensitive response (HR) cell death in tobacco plants and confers greater resistance to oomycete infection than acidic elicitins like INF1. We also studied β-CRY mutants impaired in sterol binding (Val84Phe) and interaction with the binding site on tobacco plasma membrane (Leu41Phe), because sterol binding was suggested to be important in INF1-induced resistance. Treatment with β-CRY or the Val84Phe mutant induced resistance to powdery mildew caused by the pathogen Pseudoidium neolycopersici, but not the HR cell death observed in tobacco and potato plants. The level of resistance induced in different tomato genotypes correlated with the upregulation of defence genes including defensins, β-1,3-glucanases, heveins, chitinases, osmotins, and PR1 proteins. Treatment with the Leu41Phe mutant did not induce this upregulation, suggesting similar elicitin recognition in tomato and tobacco. However, here β-CRY activated ethylene and jasmonic acid signalling, but not salicylic acid signalling, demonstrating that elicitins activate different downstream signalling processes in different plant species. This could potentially be exploited to enhance the resistance of Phytophthora-susceptible crops.
- MeSH
- cyklopentany metabolismus MeSH
- ethyleny metabolismus MeSH
- fungální proteiny metabolismus MeSH
- interakce hostitele a patogenu MeSH
- kyselina salicylová metabolismus MeSH
- listy rostlin metabolismus mikrobiologie MeSH
- nemoci rostlin mikrobiologie MeSH
- oxylipiny metabolismus MeSH
- peroxid vodíku metabolismus MeSH
- Phytophthora MeSH
- Pythium MeSH
- reaktivní formy kyslíku metabolismus MeSH
- regulátory růstu rostlin metabolismus MeSH
- signální transdukce * MeSH
- Solanum lycopersicum metabolismus mikrobiologie fyziologie MeSH
- Publikační typ
- časopisecké články 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.
- 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
Reactive oxygen species (ROS) have been recognized as important signaling compoundsof major importance in a number of developmental and physiological processes in plants. Theexistence of cellular compartments enables efficient redox compartmentalization and ensuresproper functioning of ROS-dependent signaling pathways. Similar to other organisms, theproduction of individual ROS in plant cells is highly localized and regulated bycompartment-specific enzyme pathways on transcriptional and post-translational level. ROSmetabolism and signaling in specific compartments are greatly affected by their chemicalinteractions with other reactive radical species, ROS scavengers and antioxidant enzymes. Adysregulation of the redox status, as a consequence of induced ROS generation or decreasedcapacity of their removal, occurs in plants exposed to diverse stress conditions. During stresscondition, strong induction of ROS-generating systems or attenuated ROS scavenging can lead tooxidative or nitrosative stress conditions, associated with potential damaging modifications of cellbiomolecules. Here, we present an overview of compartment-specific pathways of ROS productionand degradation and mechanisms of ROS homeostasis control within plant cell compartments.
- Publikační typ
- časopisecké články MeSH
- přehledy 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.
MAIN CONCLUSION: Resistant Lactuca spp. genotypes can efficiently modulate levels of S-nitrosothiols as reactive nitrogen species derived from nitric oxide in their defence mechanism against invading biotrophic pathogens including lettuce downy mildew. S-Nitrosylation belongs to principal signalling pathways of nitric oxide in plant development and stress responses. Protein S-nitrosylation is regulated by S-nitrosoglutathione reductase (GSNOR) as a key catabolic enzyme of S-nitrosoglutathione (GSNO), the major intracellular S-nitrosothiol. GSNOR expression, level and activity were studied in leaves of selected genotypes of lettuce (Lactuca sativa) and wild Lactuca spp. during interactions with biotrophic mildews, Bremia lactucae (lettuce downy mildew), Golovinomyces cichoracearum (lettuce powdery mildew) and non-pathogen Pseudoidium neolycopersici (tomato powdery mildew) during 168 h post inoculation (hpi). GSNOR expression was increased in all genotypes both in the early phase at 6 hpi and later phase at 72 hpi, with a high increase observed in L. sativa UCDM2 responses to all three pathogens. GSNOR protein also showed two-phase increase, with highest changes in L. virosa-B. lactucae and L. sativa cv. UCDM2-G. cichoracearum pathosystems, whereas P. neolycopersici induced GSNOR protein at 72 hpi in all genotypes. Similarly, a general pattern of modulated GSNOR activities in response to biotrophic mildews involves a two-phase increase at 6 and 72 hpi. Lettuce downy mildew infection caused GSNOR activity slightly increased only in resistant L. saligna and L. virosa genotypes; however, all genotypes showed increased GSNOR activity both at 6 and 72 hpi by lettuce powdery mildew. We observed GSNOR-mediated decrease of S-nitrosothiols as a general feature of Lactuca spp. response to mildew infection, which was also confirmed by immunohistochemical detection of GSNOR and GSNO in infected plant tissues. Our results demonstrate that GSNOR is differentially modulated in interactions of susceptible and resistant Lactuca spp. genotypes with fungal mildews and uncover the role of S-nitrosylation in molecular mechanisms of plant responses to biotrophic pathogens.
- MeSH
- aldehydoxidoreduktasy metabolismus MeSH
- konfokální mikroskopie MeSH
- nemoci rostlin mikrobiologie MeSH
- odolnost vůči nemocem fyziologie MeSH
- oomycety patogenita MeSH
- polymerázová řetězová reakce MeSH
- regulace genové exprese u rostlin MeSH
- S-nitrosothioly metabolismus MeSH
- salát (hlávkový) enzymologie fyziologie MeSH
- western blotting MeSH
- Publikační typ
- časopisecké články MeSH
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.
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.
- 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
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 β-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, β-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.
- MeSH
- aminobutyráty farmakologie MeSH
- Ascomycota fyziologie MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- nemoci rostlin imunologie mikrobiologie MeSH
- odolnost vůči nemocem MeSH
- regulace genové exprese u rostlin * MeSH
- rostlinné proteiny genetika metabolismus MeSH
- sekvenční analýza DNA MeSH
- seskviterpeny farmakologie MeSH
- Solanum lycopersicum genetika imunologie mikrobiologie MeSH
- Solanum genetika imunologie mikrobiologie MeSH
- upregulace MeSH
- Publikační typ
- časopisecké články MeSH
