Functions of peroxynitrite in resistance of potato leaves (Solanum tuberosum L.) to Phytophthora infestans (Mont.) de Bary

Wydział BiologiiCelem badań było zweryfikowanie udziału nadtlenoazotynu (ONOO¯) oraz poznanie funkcjonalnych modyfikacji via ONOO¯ w odporności liści ziemniaka na Phytophthora infestans. Doświadczenia prowadzono na liściach ziemniaka (Solanum tuberosum L.) odmian skrajnie różniących się odpornością na P. infestans, lęgniowca będącego sprawcą zarazy ziemniaka. Przeprowadzone analizy wykazały, że zależna od reduktazy azotanowej poinfekcyjna synteza tlenku azotu wraz z akumulacją anionorodnika ponadtlenkowego prowadzi do formowania ONOO¯, przy czym kinetyka i natężenie tej reakcji były uzależnione od genotypu ziemniaka. Odmiana odporna charakteryzowała się wczesną i przejściową akumulacją ONOO¯, natomiast w odmianie podatnej podwyższony poziom tej cząsteczki obserwowano dopiero w pierwszej dobie po inokulacji. Poinfekcyjnemu generowaniu ONOO¯ towarzyszyło wzmożone nitrowanie białek oraz kwasów nukleinowych tj. RNA i mRNA. W oparciu o zastosowanie zmiataczy endogennego ONOO¯ stwierdzono, że akumulacja nitro-mRNA sprzyja programowanej śmierci komórek (PCD) podczas reakcji nadwrażliwości. Ponadto sekwencyjne traktowanie liści odmiany podatnej donorem ONOO¯ i P. infestans powodowało szybszą i bardziej efektywną indukcję genów kodujących białka PR skorelowaną z zahamowaniem rozwoju choroby. Na podstawie uzyskanych wyników stwierdza się, że wczesna i okresowa akumulacja ONOO¯ odgrywa istotną rolę w odporności na P. infestans poprzez selektywne nitrowanie biomolekuł, zaangażowanych w regulację zasięgu PCD oraz ekspresji genów PR.The aim of the study was to verify participation of peroxynitrite (ONOO¯) and the recognition of the functional modifications via ONOO¯ in potato leaves resistance to Phytophthora infestans. Experiments were conducted on the potato (Solanum tuberosum L.) leaves of cultivars, whose resistance radically differs to P. infestans, an oomycete pathogen causing late blight disease. The analyses showed that nitrite reductase-dependent nitric oxide overproduction together with the accumulation of superoxide anion leads to the formation of ONOO¯, however the kinetic and intensity of this reaction was dependent on the potato genotype. The resistant response was characterized by an early and transient formation of ONOO¯ whereas in the susceptible one, an elevated level of ONOO¯ was observed after the first 24 hours post-inoculation. The postinfectious ONOO¯ generation was accompanied by nitration phenomenon of proteins and nucleic acids, i.e. RNA and mRNA. Based on application of endogenous ONOO¯ scavengers, it was found that nitro-mRNA accumulation is conducive to programmed cell death (PCD) during the hypersensitive response. Moreover, the sequential treatment of the susceptible genotype with ONOO¯ and P. infestans provoked faster and stronger induction of genes encoding PRs correlated with inhibition of disease development. Taken together, these results indicate that the early and transient accumulation of ONOO¯ in potato leaves plays a crucial role in the resistance to P. infestans, by the selective nitration of biomolecules involved in the regulation of the PCD range and the expression of PR genes.Badania wykonano w ramach: OPUS 2011/01/B/NZ9/00243, tytuł projektu: „Poszukiwanie nowych źródeł syntezy i funkcjonowania tlenku azotu w odporności liści ziemniaka na Phytophthora infestans” Projekt finansowany przez Narodowe Centrum Nauki oraz OPUS 2014/13/B/NZ/02177, tytuł projektu: „Identyfikacja i analiza funkcjonalna zdarzeń metabolicznych zależnych od tlenku azotu u Phytophthora infestans (Mont.) de Bary” Projekt finansowany przez Narodowe Centrum Nauki

NADPH oxidase is involved in regulation of gene expression and ROS overproduction in soybean (Glycine max L.) seedlings exposed to cadmium

Cadmium-induced oxidative burst is partially mediated by NADPH oxidase. The aim of the present research was to evaluate the role of NADPH oxidase in soybeans’ response to short-term cadmium stress. The application of an NADPH oxidase inhibitor, diphenyleneiodonium chloride (DPI), affected expression of two Cd-inducible genes, encoding DOF1 and MYBZ2 transcription factors. This effect was observed after 3 h of treatment. Interestingly, Cd-dependent increases in NADPH oxidase activity occurred only after a period of time ranging from 6 and 24 h of stress. Stimulation of the enzyme correlated in time with a significant accumulation of reactive oxygen species (ROS). Further analysis revealed that pharmacological inhibition of NADPH oxidase activity during 24 h of Cd stress does not affect Cd uptake, seedling growth, or the level of lipid peroxidation. The role of NADPH oxidase in the response of soybean seedlings to short-term Cd exposure is discussed

Functions of peroxynitrite in resistance of potato leaves (Solanum tuberosum L.) to Phytophthora infestans (Mont.) de Bary

Wydział BiologiiCelem badań było zweryfikowanie udziału nadtlenoazotynu (ONOO¯) oraz poznanie funkcjonalnych modyfikacji via ONOO¯ w odporności liści ziemniaka na Phytophthora infestans. Doświadczenia prowadzono na liściach ziemniaka (Solanum tuberosum L.) odmian skrajnie różniących się odpornością na P. infestans, lęgniowca będącego sprawcą zarazy ziemniaka. Przeprowadzone analizy wykazały, że zależna od reduktazy azotanowej poinfekcyjna synteza tlenku azotu wraz z akumulacją anionorodnika ponadtlenkowego prowadzi do formowania ONOO¯, przy czym kinetyka i natężenie tej reakcji były uzależnione od genotypu ziemniaka. Odmiana odporna charakteryzowała się wczesną i przejściową akumulacją ONOO¯, natomiast w odmianie podatnej podwyższony poziom tej cząsteczki obserwowano dopiero w pierwszej dobie po inokulacji. Poinfekcyjnemu generowaniu ONOO¯ towarzyszyło wzmożone nitrowanie białek oraz kwasów nukleinowych tj. RNA i mRNA. W oparciu o zastosowanie zmiataczy endogennego ONOO¯ stwierdzono, że akumulacja nitro-mRNA sprzyja programowanej śmierci komórek (PCD) podczas reakcji nadwrażliwości. Ponadto sekwencyjne traktowanie liści odmiany podatnej donorem ONOO¯ i P. infestans powodowało szybszą i bardziej efektywną indukcję genów kodujących białka PR skorelowaną z zahamowaniem rozwoju choroby. Na podstawie uzyskanych wyników stwierdza się, że wczesna i okresowa akumulacja ONOO¯ odgrywa istotną rolę w odporności na P. infestans poprzez selektywne nitrowanie biomolekuł, zaangażowanych w regulację zasięgu PCD oraz ekspresji genów PR.The aim of the study was to verify participation of peroxynitrite (ONOO¯) and the recognition of the functional modifications via ONOO¯ in potato leaves resistance to Phytophthora infestans. Experiments were conducted on the potato (Solanum tuberosum L.) leaves of cultivars, whose resistance radically differs to P. infestans, an oomycete pathogen causing late blight disease. The analyses showed that nitrite reductase-dependent nitric oxide overproduction together with the accumulation of superoxide anion leads to the formation of ONOO¯, however the kinetic and intensity of this reaction was dependent on the potato genotype. The resistant response was characterized by an early and transient formation of ONOO¯ whereas in the susceptible one, an elevated level of ONOO¯ was observed after the first 24 hours post-inoculation. The postinfectious ONOO¯ generation was accompanied by nitration phenomenon of proteins and nucleic acids, i.e. RNA and mRNA. Based on application of endogenous ONOO¯ scavengers, it was found that nitro-mRNA accumulation is conducive to programmed cell death (PCD) during the hypersensitive response. Moreover, the sequential treatment of the susceptible genotype with ONOO¯ and P. infestans provoked faster and stronger induction of genes encoding PRs correlated with inhibition of disease development. Taken together, these results indicate that the early and transient accumulation of ONOO¯ in potato leaves plays a crucial role in the resistance to P. infestans, by the selective nitration of biomolecules involved in the regulation of the PCD range and the expression of PR genes.Badania wykonano w ramach: OPUS 2011/01/B/NZ9/00243, tytuł projektu: „Poszukiwanie nowych źródeł syntezy i funkcjonowania tlenku azotu w odporności liści ziemniaka na Phytophthora infestans” Projekt finansowany przez Narodowe Centrum Nauki oraz OPUS 2014/13/B/NZ/02177, tytuł projektu: „Identyfikacja i analiza funkcjonalna zdarzeń metabolicznych zależnych od tlenku azotu u Phytophthora infestans (Mont.) de Bary” Projekt finansowany przez Narodowe Centrum Nauki

The toxic Doppelganger: on the ionic and molecular mimicry of cadmium

Cadmium is a toxic heavy metal which can cause numerous alterations in cell functioning. Exposure to cadmium leads to generation of reactive oxygen species, disorders in membrane structure and functioning, inhibition of respiration, disturbances in ion homeostasis, perturbations in cell division, and initiation of apoptosis and necrosis. This heavy metal is considered a carcinogen by the Agency for Toxic Substances and Disease Registry. At least some of the described toxic effects could result from the ability of cadmium to mimic other divalent ions and alert signal transduction networks. This review describes the role of cadmium mimicry in its uptake, reactive oxygen species generation, alterations in calmodulin, Wnt/β-catenin and estrogen signaling pathways, and modulation of neurotransmission. The last section is dedicated to the single known case of a favorable function performed by cadmium mimicry: marine diatoms, which live in zinc deficient conditions, utilize cadmium as a cofactor in carbonic anhydrase - so far the only described cadmium enzyme

The toxic Doppelganger: on the ionic and molecular mimicry of

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RNA and mRNA Nitration as a Novel Metabolic Link in Potato Immune Response to Phytophthora infestans

Peroxynitrite (ONOO-) exhibits a well-documented nitration activity in relation to proteins and lipids; however, the interaction of ONOO- with nucleic acids remains unknown in plants. The study uncovers RNA and mRNA nitration as an integral event in plant metabolism intensified during immune response. Using potato-avr/vr Phytophthora infestans systems and immunoassays we documented that potato immunity is accompanied by two waves of boosted ONOO- formation affecting guanine nucleotides embedded in RNA/mRNA and protein tyrosine residues. The early ONOO- generation was orchestrated with an elevated level of protein nitration and a huge accumulation of 8-nitroguanine (8-NO2-G) in RNA and mRNA pools confirmed as a biomarker of nucleic acid nitration. Importantly, potato cells lacking ONOO- due to scavenger treatment and attacked by the avr pathogen exhibited a low level of 8-NO2-G in the mRNA pool correlated with reduced symptoms of programmed cell death (PCD). The second burst of ONOO- coincided both with an enhanced level of tyrosine-nitrated proteins identified as subtilisine-like proteases and diminished protease activity in cells surrounding the PCD zone. Nitration of both RNA/mRNA and proteins via NO/ONOO- may constitute a new metabolic switch in redox regulation of PCD, potentially limiting its range in potato immunity to avr P. infestans

Cadmium Stress Leads to Rapid Increase in RNA Oxidative Modifications in Soybean Seedlings

Increase in the level of reactive oxygen species (ROS) is a common response to stress factors, including exposure to metals. ROS over-production is associated with oxidation of lipids, proteins, and nucleic acids. It is suggested that the products of oxidation are not solely the markers of oxidative stress but also signaling elements. For instance, it has been shown in animal models that mRNA oxidation is a selective process engaged in post-transcriptional regulation of genes expression and that it is associated with the development of symptoms of several neurodegenerative disorders. In the present study, we examined the impact of short-term cadmium (Cd) stress on the level of two RNA oxidation markers: 8-hydroxyguanosine (8-OHG) and apurinic/apyrimidinic sites (AP-sites, abasic sites). In the case of 8-OHG, a significant increase was observed after 3 h of exposure to moderate Cd concentration (10 mg/l). In turn, high level of AP-sites, accompanied by strong ROS accumulation and lipid peroxidation, was noted only after 24 h of treatment with higher Cd concentration (25 mg/l). This is the first report showing induction of RNA oxidations in plants response to stress factors. The possible signaling and gene regulatory role of oxidatively modified transcripts is discussed

Freezing Tolerance of Lolium multiflorum/Festuca arundinacea Introgression Forms is Associated with the High Activity of Antioxidant System and Adjustment of Photosynthetic Activity under Cold Acclimation

Though winter-hardiness is a complex trait, freezing tolerance was proved to be its main component. Species from temperate regions acquire tolerance to freezing in a process of cold acclimation, which is associated with the exposure of plants to low but non-freezing temperatures. However, mechanisms of cold acclimation in Lolium-Festuca grasses, important for forage production in Europe, have not been fully recognized. Thus, two L. multiflorum/F. arundinacea introgression forms with distinct freezing tolerance were used herein as models in the comprehensive research to dissect these mechanisms in that group of plants. The work was focused on: (i) analysis of cellular membranes’ integrity; (ii) analysis of plant photosynthetic capacity (chlorophyll fluorescence; gas exchange; gene expression, protein accumulation, and activity of selected enzymes of the Calvin cycle); (iii) analysis of plant antioxidant capacity (reactive oxygen species generation; gene expression, protein accumulation, and activity of selected enzymes); and (iv) analysis of Cor14b accumulation, under cold acclimation. The more freezing tolerant introgression form revealed a higher integrity of membranes, an ability to cold acclimate its photosynthetic apparatus and higher water use efficiency after three weeks of cold acclimation, as well as a higher capacity of the antioxidant system and a lower content of reactive oxygen species in low temperature