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Nitric oxide (NO) is a critical molecule that links plant development with stress responses. Herein, new insights into the role of NO metabolism during leaf senescence in Arabidopsis are presented. A gradual decrease in NO emission accompanied dark-induced leaf senescence (DILS), and a transient wave of peroxynitrite (ONOO–) formation was detected by day 3 of DILS. The boosted ONOO– did not promote tryptophan (Trp) nitration, while the pool of 6-nitroTrp-containing proteins was depleted as senescence progressed. Immunoprecipitation combined with mass spectrometry was used to identify 63 and 4 characteristic 6-nitroTrp-containing proteins in control and individually darkened leaves, respectively. The potential in vivo targets of Trp nitration were mainly related to protein biosynthesis and carbohydrate metabolism. In contrast, nitration of tyrosine-containing proteins was intensified 2-fold on day 3 of DILS. Also, nitrative modification of RNA and DNA increased significantly on days 3 and 7 of DILS, respectively. Taken together, ONOO– can be considered a novel pro-senescence regulator that fine-tunes the redox environment for selective bio-target nitration. Thus, DILS-triggered nitrative changes at RNA and protein levels promote developmental shifts during the plant’s lifespan and temporal adjustment in plant metabolism under suboptimal environmental conditions.

Nitric oxide (NO) is an essential redox-signaling molecule operating in many physiological and pathophysiological processes. However, evidence on putative NO engagement in plant immunity by affecting defense gene expressions, including histone modifications, is poorly recognized. Exploring the effect of biphasic NO generation regulated by S-nitrosoglutathione reductase (GNSOR) activity after avr Phytophthora infestans inoculation, we showed that the phase of NO decline at 6 h post-inoculation (hpi) was correlated with the rise of defense gene expressions enriched in the TrxG-mediated H3K4me3 active mark in their promoter regions. Here, we report that arginine methyltransferase PRMT5 catalyzing histone H4R3 symmetric dimethylation (H4R3sme2) is necessary to ensure potato resistance to avr P. infestans. Both the pathogen and S-nitrosoglutathione (GSNO) altered the methylation status of H4R3sme2 by transient reduction in the repressive mark in the promoter of defense genes, R3a and HSR203J (a resistance marker), thereby elevating their transcription. In turn, the PRMT5-selective inhibitor repressed R3a expression and attenuated the hypersensitive response to the pathogen. In conclusion, we postulate that lowering the NO level (at 6 hpi) might be decisive for facilitating the pathogen-induced upregulation of stress genes via histone lysine methylation and PRMT5 controlling potato immunity to late blight.

Although peroxynitrite (ONOO−) has been well documented as a nitrating cognate of nitric oxide (NO) in plant cells, modifications of proteins, fatty acids, and nucleotides by nitration are relatively under-explored topics in plant NO research. As a result, they are seen mainly as hallmarks of redox processes or as markers of nitro-oxidative stress under unfavorable conditions, similar to those observed in human and other animal systems. Protein tyrosine nitration is the best-known nitrative modification in the plant system and can be promoted by the action of both ONOO− and related NO-derived oxidants within the cell environment. Recent progress in ‘omics’ and modeling tools have provided novel biochemical insights into the physiological and pathophysiological fate of nitrated proteins. The nitration process can be specifically involved in various cell regulatory mechanisms that control redox signaling via nitrated cGMP or nitrated fatty acids. In addition, there is evidence to suggest that nitrative modifications of nucleotides embedded in DNA and RNA can be considered as smart switches of gene expression that fine-tune adaptive cellular responses to stress. This review highlights recent advances in our understanding of the potential implications of biotargets in the regulation of intracellular traffic and plant biological processes.

Nitro-oxidative modifications of biomolecules result from the presence of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in the cellular environment. Unlike ROS/RNS-mediated modifications of proteins and fatty acids, nucleotides embedded in nucleic acids have not been a major focus of studies on nitro-oxidative metabolism in plants. However, RNA structure being more susceptible to oxidative/nitrative attack than DNA can be considered as an important functional biotarget of ROS/RNS generated under both physiological and stress conditions. The formation of 8-hydroxyguanine (8-OHG) and 8-nitroguanine (8-NG), the most common ROS- and RNS-mediated RNA modifications, respectively, could therefore affect protein synthesis to efficiently fine-tune various cellular responses. This chapter emphasizes the importance of 8-OHG and 8-NG not only as a symptom of ROS/RNS toxicity but implicates the nitro-oxidative RNA modifications in the regulation of multiple plant biological processes.

Although most studies on the ubiquitous enzyme carbonic anhydrase (CA) have indicated its significant role in plants to facilitate the diffusion of CO2 to the site of inorganic carbon fixation, it is becoming increasingly likely that carbonic anhydrase isoforms also have diverse unexplored functions in plant cells. This review lays emphasis on additional roles of CA associated with many physiological, biochemical and structural changes in plant metabolism. The presented findings have revealed essential functions of CA isoforms in plant adjustment to both abiotic and biotic agents and developmental stimuli. However, sometimes it is difficult to separate the non-photosynthetic from the photosynthetic-related role of CAs during post-stress impaired metabolism, and the preventive CA outcome might be due to the effect of these enzymes on improvement of photosynthetic capacity. Finally, taking into account the experimental evidence, the direct and indirect functional roles of CAs in mitigating negative effects of environmental conditions are presented.

In this study we proposed carbonic anhydrase (CA) as an important element of basal resistance during the potato (Solanum tuberosum L.)-Phytophthora infestans interaction. We found a different β-CA expression pattern in incompatible vs. compatible systems correlated in time with CA enzyme activity. Resistant potato leaves supplied with dorzolamide (an inhibitor of carbonate CA activity) and challenged with the pathogen showed an elevated nitric oxide (NO) synthesis, which was the most evident during the early phase of NO burst (at 3 hpi) during hypersensitive response (HR). In vitro application of dorzolamide and effective inhibitors of NO synthesis confirmed the implication of CA activity in NO metabolism during potato defense. To clarify how suppression of CA carbonate activity translates into the complexity of NO-related responses leading to potato resistance or susceptibility to an oomycete pathogen we analysed expression of NPR, PR1, and PAL.Taken together, pharmacological damping of CA activity revealed a functional link between CA and NO-dependent signaling in potato defense against P. infestans manifested by accelerated NO formation and a modified salicylic acid defense pathway. The dorzolamide-mediated effective responses for basal resistance also delayed symptoms of late blight in the susceptible potato cultivar, without overcoming HR formation in the resistant one.

In contrast to the in-depth knowledge concerning nitric oxide (NO) function, our understanding of NO synthesis in plants is still very limited. In view of the above, this paper provides a step by step presentation of the reductive pathway for endogenous NO generation involving nitrate reductase (NR) activity and nitrite implication in potato defense to Phytophthora infestans. A biphasic character of NO emission, peaking mainly at 3 and then at 24 hpi, was detected during the hypersensitive response (HR). In avr P. infestans potato leaves enhanced NR gene and protein expression was tuned with the depletion of nitrate contents and the increase in nitrite supply at 3 hpi. In the same time period a temporary down-regulation of nitrite reductase (NiR) and activity was found. The study for the link between NO signaling and HR revealed an up-regulation of used markers of effective defense, i.e. Nonexpressor of PR genes (NPR1), thioredoxins (Thx) and PR1, at early time-points (1–3 hpi) upon inoculation. In contrast to the resistant response, in the susceptible one a late overexpression (24–48 hpi) of NPR1 and PR1 mRNA levels was observed. Presented data confirmed the importance of nitrite processed by NR in NO generation in inoculated potato leaves. However, based on the pharmacological approach the potential formation of NO from nitrite bypassing the NR activity during HR response to P. infestans has also been discussed.

The effect of short-term (48 h) moderate (85 μM CdCl 2 ) and high (170 μM CdCl 2 ) cadmium (Cd) stress on endogenous peroxynitrite (ONOO − ) generation was investigated in the roots of 2-days old seedlings of soybean ( Glycine max L.). The control roots showed a relatively high level of ONOO − , whereas Cd-mediated ONOO − formation was detected only at high Cd stress. The Cd-induced ONOO − accumulation was accompanied by enhanced levels of both nitric oxide (NO) and superoxide (O 2 − ). The contrasting response appeared during moderate stress which provoked a huge NADPH-oxidase dependent O 2 − accumulation with slightly elevated reactive nitrogen species formation. The nitroproteome analysis revealed a proteins pool undergoing tyrosine nitration that were down-regulated during both moderate and high Cd stress conditions. The most pronounced changes within the pool of 3-nitrotyrosine targets in soybean roots revealed proteins involved mainly in primary metabolism that are not implicated in plant defense strategy. Moreover, sequential treatment with ONOO − and Cd was tuned with diminished Cd toxicity and up-regulation of gene coding for peroxiredoxin ( PrxII F ). Taken together, soybean seedlings mainly exposed to moderate Cd stress, showed enhanced Pxr gene expression correlated with temperate ONOO − formation what finally affected post-translational protein modification via tyrosine nitration.

Heavy metal pollution causes many soils to become a toxic environment not only for plants, but also microorganisms, however, little is known how heavy metal contaminated environment affects metabolism of phytopathogens and their capability of infecting host plants. In this study the oomycete Phytophthora infestans (Mont.) de Bary, the most harmful pathogen of potato, growing under moderate cadmium stress (Cd, 5 mg/L) showed nitro-oxidative imbalance associated with an enhanced antioxidant response. Cadmium notably elevated the level of nitric oxide, superoxide and peroxynitrite that stimulated nitrative modifications within the RNA and DNA pools in the phytopathogen structures. In contrast, the protein pool undergoing nitration was diminished confirming that protein tyrosine nitration is a flexible element of the oomycete adaptive strategy to heavy metal stress. Finally, to verify whether Cd is able to modify P. infestans pathogenicity, a disease index and molecular assessment of disease progress were analysed indicating that Cd stress enhanced aggressiveness of vr P. infestans towards various potato cultivars. Taken together, Cd not only affected hyphal growth rate and caused biochemical changes in P. infestans structures, but accelerated the pathogenicity as well. The nitro-oxidative homeostasis imbalance underlies the phytopathogen adaptive strategy and survival in the heavy metal contaminated environment.

The study demonstrates protein tyrosine nitration as a functional post-translational modification (PTM) in biology and pathobiology of the oomycete Phytophthora infestans (Mont.) de Bary, the most harmful pathogen of potato (Solanum tuberosum L.). Using two P. infestans isolates differing in their virulence toward potato cv. Sarpo Mira we found that the pathogen generates reactive nitrogen species (RNS) in hyphae and mature sporangia growing under in vitro and in planta conditions. However, acceleration of peroxynitrite formation and elevation of the nitrated protein pool within pathogen structures were observed mainly during the avr P. infestans MP 946-potato interaction. Importantly, the nitroproteome profiles varied for the pathogen virulence pattern and comparative analysis revealed that vr MP 977 P. infestans represented a much more diverse quality spectrum of nitrated proteins. Abundance profiles of nitrated proteins that were up- or downregulated were substantially different also between the analyzed growth phases. Briefly, in planta growth of avr and vr P. infestans was accompanied by exclusive nitration of proteins involved in energy metabolism, signal transduction and pathogenesis. Importantly, the P. infestans-potato interaction indicated cytosolic RXLRs and Crinklers effectors as potential sensors of RNS. Taken together, we explored the first plant pathogen nitroproteome. The results present new insights into RNS metabolism in P. infestans indicating protein nitration as an integral part of pathogen biology, dynamically modified during its offensive strategy. Thus, the nitroproteome should be considered as a flexible element of the oomycete developmental and adaptive mechanism to different micro-environments, including host cells.

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.

In this study peroxynitrite (ONOO−) is proposed as an important player in defence responses during the interaction of potato (Solanum tuberosum) and the oomycete pathogen Phytophthora infestans. The potato–avr P. infestans model system exhibited a transient programme of boosted ONOO− formation correlated in time with the burst of nitric oxide (NO) and superoxide during the first 6 h post-inoculation (hpi). The early ONOO− over-accumulation was not accompanied by TPx gene expression. In contrast, the compatible interaction revealed a 24 h delay of ONOO− formation; however, an enhanced level of NO and superoxide correlated with TPx up-regulation was recorded within the earlier stages of pathogen infection. Peroxynitrite over-accumulation in the susceptible potato coincided with an enhanced level of protein tyrosine nitration starting from 24 hpi. Surprisingly, the nitroproteome profile of the resistant potato did not show any visible difference after inoculation, apart from one band containing subtilisin-like protease-like proteins, which appeared 48 h after pathogen attack. An additional pharmacological approach showed that treatment of the susceptible genotype with ONOO− followed by inoculation with P. infestans contributed to slowing down of the colonization of host tissues by the pathogen via a faster and stronger up-regulation of the key defence markers, including the PR-1 gene. Taken together, the results obtained indicate that a precise control of emitted NO and superoxide in cooperation with thioredoxin-dependent redox sensors in sites of pathogen ingress could generate a sufficient threshold of ONOO−, triggering defence responses.

In our experimental approach we investigated how post-infection nitric oxide-dependent signaling activated in potato leaves was related to defense against avirulent (avr) and virulent (vr) races of Phytophthora infestans. Results revealed that only in an incompatible response, early NO and superoxide (O2 •-) generation led to peroxynitrite (ONOO−) formation and together with hydrogen peroxide (H2O2) production synchronized with SOD activity induced effective defense against avr pathogen. Early oxidative and nitrosative bursts triggered an imbalance in redox homeostasis in inoculated tissue. To counteract that effect, a total antioxidative capacity, ascorbate and sulfhydryl (-SH) group compounds increased both synergistically and markedly, confirming the precise mechanism of redox re-adjustment in avr oomycete -potato interaction. Moreover, the NO-coded message was stored and converted into an enhanced total SNO pool and particular S-nitrosylation of targeted proteins. Overall, we identified 104 proteins typed for S-nitrosylation in mock- or P. infestans-inoculated potato leaves. The S-nitrosoproteome structure comprised a wide repertoire of proteins, i.e. defense- and redox-related. Finally, only in the incompatible interaction, NO-based signal was re-written on the rapid PR-1 gene and PR-2 protein activation and was tuned with a limitation of late blight disease symptoms.

This study demonstrates the sequence of enhanced generation of signal molecules such as phytohormones, i.e. jasmonic acid (JA), ethylene (ET), salicylic acid (SA), and a relatively stable free radical, nitric oxide (NO), in response of Pisum sativum L. cv. Cysterski seedling leaves to the infestation of pea aphid Acyrthosiphon pisum (Harris) at a varied population size. In time from 0 to 96 h after A. pisum infestation these signal molecules accumulated transiently. Moreover, the convergence of these signaling pathways occurred. JA and its methyl derivative MeJA reached the first maximum of generation at 24th hour of infestation. An increase in ET and NO generation was observed at 48th hour of infestation. The increase in SA, JA/MeJA and ET concentrations in aphid-infested leaves occurred from the 72nd to 96th hour. In parallel, an increase was demonstrated for the activities of enzymes engaged in the biosynthesis of SA, such as phenylalanine ammonia-lyase (PAL) and benzoic acid 2-hydroxylase (BA2H). Additionally, a considerable post-infestation accumulation of transcripts for PAL was observed. An increase in the activity of lipoxygenase (LOX), an important enzyme in the biosynthesis of JA was noted. This complex signaling network may contribute to the coordinated regulation of gene expression leading to specific defence responses.

The phenomenon of cross-resistance allows plants to acquire resistance to a broad range of stresses after previous exposure to one specific factor. Although this stress–response relationship has been known for decades, the sequence of events that underpin cross-resistance remains unknown. Our experiments revealed that susceptible potato (Solanum tuberosum L. cv. Bintje) undergoing aluminum (Al) stress at the root level showed enhanced defense responses correlated with reduced disease symptoms after leaf inoculation with Phytophthora infestans. The protection capacity of Al to subsequent stress was associated with the local accumulation of H2O2 in roots and systemic activation of salicylic acid (SA) and nitric oxide (NO) dependent pathways. The most crucial Al-mediated changes involved coding of NO message in an enhanced S-nitrosothiol formation in leaves tuned with an abundant SNOs accumulation in the main vein of leaves. Al-induced distal NO generation was correlated with the overexpression of PR-2 and PR-3 at both mRNA and protein activity levels. In turn, after contact with a pathogen we observed early up-regulation of SA-mediated defense genes, e.g. PR1, PR-2, PR-3 and PAL, and subsequent disease limitation. Taken together Al exposure induced distal changes in the biochemical stress imprint, facilitating more effective responses to a subsequent pathogen attack. Electronic supplementary material The online version of this article (doi:10.1007/s00425-013-2008-8) contains supplementary material, which is available to authorized users.

Summary An explosion of research in plant nitric oxide (NO) biology during the last two decades has revealed that NO is a key signal involved in plant development, abiotic stress responses and plant immunity. During the course of evolutionary changes, microorganisms parasitizing plants have developed highly effective offensive strategies, in which NO also seems to be implicated. NO production has been demonstrated in several plant pathogens, including fungi, but the origin of NO seems to be as puzzling as in plants. So far, published studies have been spread over multiple species of pathogenic microorganisms in various developmental stages; however, the data clearly indicate that pathogen-derived NO is an important regulatory molecule involved not only in developmental processes, but also in pathogen virulence and its survival in the host. This review also focuses on the search for potential mechanisms by which pathogens convert NO messages into a physiological response or detoxify both endo- and exogenous NO. Finally, taking into account the data available from model bacteria and yeast, a basic draft for the mode of NO action in phytopathogenic microorganisms is proposed.

In our experimental approach we examined how potato leaves exposed to a chemical agent might induce nitric oxide (NO) dependent biochemical modifications for future mobilization of an effective resistance to Phytophthora infestans. After potato leaf treatment with one of the following SAR inducers, i.e. β-aminobutyric acid (BABA), 2,6-dichloroisonicotinic acid (INA) or Laminarin, we observed enhanced NO generation concomitant with biochemical changes related to a slight superoxide anion (O2(-)) and hydrogen peroxide (H2O2) accumulation dependent on minimal NADPH oxidase and peroxidase activities, respectively. These rather normoergic changes, linked to the NO message, were mediated by the temporary down-regulation of S-nitrosoglutathione reductase (GSNOR). In turn, after challenge inoculation signal amplification promoted potato resistance manifested in the up-regulation of GSNOR activity tuned with the depletion of the SNO pool, which was observed by our team earlier (Floryszak-Wieczorek et al., 2012). Moreover, hyperergic defense responses related to an early and rapid O2(-)and H2O2 overproduction together with a temporary increase in NADPH oxidase and peroxidase activities were noted. BABA treatment was the most effective against P. infestans resulting in the enhanced activity of β-1,3-glucanase and callose deposition. Our results indicate that NO-mediated biochemical modifications might play an important role in creating more potent defense responses of potato to a subsequent P. infestans attack.

The aim of this study was to investigate the effect of exogenous polyamines (PAs) on the membrane status and proline level in roots of water stressed cucumber (Cucumis sativus cv. Dar) seedlings. It was found that water shortage resulted in an increase of membrane injury, lipoxygenase (LOX) activity, lipid peroxidation and proline concentration in cucumber roots during progressive dehydration. PA pretreatment resulted in a distinct reduction of the injury index, and this effect was reflected by a lower stress-evoked LOX activity increase and lipid peroxide levels at the end of the stress period. In contrast, PA-supplied stressed roots displayed a higher proline accumulation. The presented results suggest that exogenous PAs are able to alleviate water deficit-induced membrane permeability and diminish LOX activity. Observed changes were accompanied by an accumulation of proline, suggesting that the accumulation of this osmolyte might be another possible mode of action for PAs to attain higher membrane stability, and in this way mitigate water deficit effects in roots of cucumber seedlings.

The aim of this study was to investigate the effect of exogenous nitric oxide (NO), i.e., S-nitrosoglutathione (GSNO) and sodium nitroprusside (SNP), on the metabolic status of Pisum sativum L. cv. Cysterski leaves infested by Acyrthosiphon pisum Harris, population demographic parameters and A. pisum feeding activity. A reduction in the level of semiquinone radicals in pea seedling leaves pretreated with exogenous NO occurred 24 h after A. pisum infestation, which was earlier than in non-pretreated leaves. A decrease in the level of O2•− was observed in leaves pretreated with GSNO and infested by aphids at 48 and 72 h post-infestation (hpi). Directly after the pretreatment with GSNO, an increase in the level of metal ions was recorded. NO considerably induced the relative mRNA levels for phenylalanine ammonia-lyase in 24-h leaves pretreated with NO donors, both non-infested and infested. NO stimulated the accumulation of pisatin in leaves until 24 h. The Electrical Penetration Graph revealed a reduction in the feeding activity of the pea aphid on leaves pretreated with NO. The present study showed that foliar application of NO donors induced sequentially defense reactions of pea against A. pisum and had a deterrent effect on aphid feeding and limited the population growth rate.

Peroxynitrite (ONOO(-)) is a potent oxidant and nitrating species, generated by the reaction of nitric oxide and superoxide in one of the most rapid reactions known in biology. It is widely accepted that an enhanced ONOO(-) formation contributes to oxidative and nitrosative stress in various biological systems. However, an increasing number of studies have reported that ONOO(-) cannot only be considered as a mediator of cellular dysfunction, but also behaves as a potent modulator of the redox regulation in various cell signal transduction pathways. Although the formation of ONOO(-) has been demonstrated in vivo in plant cells, the relevance of this molecule during plant physiological responses is still far from being clarified. Admittedly, the detection of protein tyrosine nitration phenomena provides some justification to the speculations that ONOO() is generated during various plant stress responses associated with pathophysiological mechanisms. On the other hand, it was found that ONOO(-) itself is not as toxic for plant cells as it is for animal ones. Based on the concepts of the role played by ONOO(-) in biological systems, this review is focused mainly on the search for potential functions of ONOO(-) in plants. Moreover, it is also an attempt to stimulate a discussion on the significance of protein nitration as a paradigm in signal modulation, since the newest reports identified proteins associated with signal transduction cascades within the plant nitroproteome.

Each living cell of a plant produces photons in certain conditions. Under normal physiological conditions, cell photon emission is stationary and minimal. Disturbance in the oxidative homeostasis by biotic stress is manifested by increased ‘biophoton’ production. Such biophoton responses of plants may be used as an integral indicator of the degree of oxidative homeostasis misbalance. Our results demonstrate that biophoton generation has been much higher in a resistant potato variety than in a susceptible one till 10 h after Phytophthora infestans inoculation. In contrast, ultra-weak luminescence from detached susceptible potato and moderately resistant pelargonium leaves increased from 1–4 to 4–5 days after inoculation with Phytophthora infestans or Botrytis cinerea, respectively. Pre-treatment of susceptible potato leaves with a defence inducer, arachidonic acid, resulted in a transient burst of light in response to P. infestans lasting for 30–45 h post inoculation (hpi). This study presents the potential adaptation of functional imaging of ultra-weak luminescence to monitor time-dependent free radical processes during disease development and its application to draw conclusions on plant resistance to pathogens of different lifestyle. Moreover, it has been shown that imaging of temporal biophoton generation from potato leaves treated with arachidonic acid might be a helpful marker in mapping oxidative changes leading to systemic acquired resistance (SAR).

In the present work, we tested known nitric oxide (NO) modulators generating the NO+ (sodium nitroprusside, SNP) and NO˙ forms (S-nitroso-N-acetyl-D-penicillamine, SNAP and nitrosoglutathione, GSNO). This allowed us to compare downstream NO-related physiological effects on proteins found in leaves of pelargonium (Pelargonium peltatum L.). Protein modification via NO donors generally affects plant metabolism in a distinct manner, manifested by a lower thiobarbituric acid reactive substance (TBARS) content and lipoxygenase (LOX) activity in response to SNAP and GSNO. This is in contrast to the response observed for SNP treatment. Most changes in enzyme activity (GR, glutathione reductase; GST, glutathione-S-transferase; GPX, glutathione peroxidase) are most spectacular and repeatable during the first 8 h of incubation, which is explained by the half-life of the applied donors. In particular, a close dependence was found between the time-course of NO emission from the applied donors and the temporary inhibition of antioxidant enzymes, such as catalase (CAT) and ascorbate peroxidase (APX). The observed changes were accompanied by time-dependent alterations in protein accumulation as analysed by two-dimensional gel electrophoresis (2-DE) in pelargonium leaves treated with NO donors (SNP, SNAP and GSNO). Using proteomics, different proteins were found to be down- and up-regulated. However, no new protein spots characteristic of all three donors were found. These results indicate that the form of NO emitted from the donor structure plays a key role in switching on appropriate metabolic modifications. It has been noted that several NO-affected metabolomic changes induced by the used donors were not comparable, which confirms the need to maintain caution when interpreting results obtained using the pharmacological approach with different NO modulator compounds.

The effect of water deficit on nitric oxide (NO) generation was investigated in cucumber ( Cucumis sativus cv. Dar) seedling roots using bio-imaging with an NO-selective fluorophor, diaminofluorescein-2-diacetate (DAF-2DA). Roots subjected to mild (5 and 10 h) water deficit showed slightly enhanced NO synthesis in cells of root tips and in the surrounding elongation zone. However, severe (17 h) stress resulted in an intensive NO production localized mainly in and above the elongation zone. Water stress-induced NO generation was blocked by a specific NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) as well as nitrate reductase (NR) and partially by nitric oxide synthase (NOS-like) inhibitors. A pharmacological approach was used in order to verify the capacity of NO to induce adaptive responses of cucumber roots to water deficit. A positive correlation was found between NO donor (SNP 100 μM and GSNO 100 μM) pretreatment and plant hydration status, measured as relative water content (RWC) during progressive dehydration. At an early stage (5 h) of stress duration NO caused a periodical increase in lipoxygenase (LOX) activity, correlated with time-dependent enhancement of lipid peroxidation. Beginning from 10 h up to severe stress (17 h) exogenous NO was able to diminish LOX activity and alleviate water deficit-induced membrane permeability and lipid peroxidation, measured as TBARS content and visualised by histochemical staining in situ . Observed changes via NO were accompanied by a significant reduction of proline level, suggesting that the accumulation of this osmolyte might not be essential in water stress tolerance. Obtained results clearly indicate that NO augmentation is likely to help the plant at the initial stage of tissue dehydration to trigger efficient mechanisms, mitigating severe water deficit effects in roots of cucumber seedlings.

The subject of this study was the participation of nitric oxide (NO) in plant responses to wounding, promoted by nicking of pelargonium (Pelargonium peltatum L.) leaves. Bio-imaging with the fluorochrome 4,5-diaminofluorescein diacetate (DAF-2DA) and electrochemical in situ measurement of NO showed early (within minutes) and transient (2 h) NO generation after wounding restricted to the site of injury. In order to clarify the functional role of NO in relation to modulation of the redox balance during wounding, a pharmacological approach was used. A positive correlation was found between NO generation and regulation of the redox state. NO caused a slight restriction of post-wounded O(2) (-) production, in contrast to the periodic and marked increase in H(2)O(2) level. The observed changes were accompanied by time-dependent inhibition of catalase (CAT) and ascorbate peroxidase (APX) activity. The effect was specific to NO, since the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5 tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) reversed the inhibition of CAT and APX, as well as temporarily enhancing H(2)O(2) synthesis. Finally, cooperation of NO/H(2)O(2) restricted the depletion of the low-molecular weight antioxidant pool (i.e. ascorbic acid and thiols) was positively correlated with sealing and reconstruction changes in injured pelargonium leaves (i.e. lignin formation and callose deposition). The above results clearly suggest that NO may promote restoration of wounded tissue through stabilisation of the cell redox state and stimulation of the wound scarring processes.

The effect of polyamines (PAs) on nitric oxide (NO) generation was investigated in cucumber (Cucumis sativus cv. Dar) primary leaves using bio-imaging with an NO-selective fluorophor, DAF-2DA. Seedlings pretreated with PAs and subjected to water deficit showed early (after 5 h) and transient NO production. The amplitude of the response depended on the form of the applied polyamine. Spermine (1.0 mM) and spermidine (1.0 mM) induced higher NO-dependent fluorescence compared with putrescine (1.0 mM) and the control. The NO production was blocked by tungstate, an inhibitor of nitrate reductase, and partially by an inhibitor of nitric oxide synthase (NOS-like) enzymes. NO donor administration preceding drought had no effect on endogenous PA levels but was positively correlated with an alleviation of water deficit-induced membrane permeability and lipid peroxidation. Application of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), a membrane-permeable NO scavenger, markedly reversed the NO donor effects. Similarly, pretreating seedlings with PAs resulted in lower ion leakage from the membrane and modified lipid peroxidation. The results indicate that NO may act downstream of PAs in cucumber seedlings under water stress.

In the course of evolutionary changes pathogens have developed many invasion strategies, to which the host organisms responded with a broad range of defense reactions involving endogenous signaling molecules, such as nitric oxide (NO). There is evidence that pathogenic microorganisms, including two most important groups of eukaryotic plant pathogens, also acquired the ability to synthesize NO via non-unequivocally defined oxidative and/or reductive routes. Although the both kingdoms Chromista and Fungi are remarkably diverse, the experimental data clearly indicate that pathogen-derived NO is an important regulatory molecule controlling not only developmental processes, but also pathogen virulence and its survival in the host. An active control of mitigation or aggravation of nitrosative stress within host cells seems to be a key determinant for the successful invasion of plant pathogens representing different lifestyles and an effective mode of dispersion in various environmental niches.

The functional role of nitric oxide (NO) produced endogenously in plants affected by cadmium (Cd) has been thoroughly studied over the past decade. However, the fundamental question of the fate of the NO message during the heavy metal (HM) stress is still open. So far, the dual role of NO in Cd toxicity has been demonstrated in various model plants. On the one hand, NO may buffer cell homoeostasis and constitutes a key element in the expression of gene sets responsible for stress tolerance. Conversely, NO contributes to Cd toxicity by promoting cellular redox misbalance, Cd uptake and metal-induced reduction of root growth. Trying to resolve how NO switches plant responses to cope with HM stress, the current review is focused mainly on potential costs and benefits arising from NO accumulation during Cd exposure.

Our research provides new insights into how the low and steady-state levels of nitric oxide (NO) and reactive oxygen species (ROS) in potato leaves are altered after the challenge with the hemibiotroph Phytophthora infestans or the necrotroph Botrytis cinerea, with the subsequent rapid and invader-dependent modification of defense responses with opposite effects. Mainly in the avirulent (avr) P. infestans–potato system, NO well balanced with the superoxide level was tuned with a battery of SA-dependent defense genes, leading to the establishment of the hypersensitive response (HR) successfully arresting the pathogen. Relatively high levels of S-nitrosoglutathione and S-nitrosothiols concentrated in the main vein of potato leaves indicated the mobile function of these compounds as a reservoir of NO bioactivity. In contrast, low-level production of NO and ROS during virulent (vr) P. infestans-potato interactions might be crucial in the delayed up-regulation of PR-1 and PR-3 genes and compromised resistance to the hemibiotrophic pathogen. In turn, B. cinerea triggered huge NO overproduction and governed inhibition of superoxide production by blunting NADPH oxidase. Nevertheless, a relatively high level of H2O2 was found owing to the germin-like activity in cooperation with NO-mediated HR-like cell death in potato genotypes favorable to the necrotrophic pathogen. Moreover, B. cinerea not only provoked cell death, but also modulated the host redox milieu by boosting protein nitration, which attenuated SA production but not SA-dependent defense gene expression. Finally, based on obtained data the organismal cost of having machinery for HR in plant resistance to biotrophs is also discussed, while emphasizing new efforts to identify other components of the NO/ROS cell death pathway and improve plant protection against pathogens of different lifestyles.

The transcript of the PR1 gene accumulation as an informative marker of systemic acquired resistance (SAR) was analyzed in β-aminobutyric acid (BABA) primed potato in the short-lasting (3 days) and long-lasting (28 days) time periods after induction and in the vegetative descendants of primed plants derived from tubers and from in vitro seedlings. BABA pretreatment resulted either in minimal or no PR1 gene expression, but sequential treatment with BABA followed by virulent Phytophthora infestans provided data on the imprint of post-stress information and its duration until fertilization, in the form of an enhanced PR1 transcript accumulation and a transient increase of basal resistance to the late blight disease. The primed state for defense of the susceptible potato cultivar was transmitted to its vegetative progeny as a potentiated PR1 mRNA accumulation following challenge inoculation. However, variation was observed between vegetative accessions of the BABA-primed potato genotype in responsiveness to disease. In contrast to plants derived from tubers, potato propagated through in vitro seedlings largely lost inducible resistance traits, although itretained primed PR1 gene expression.

Huge advances achieved recently in elucidating the role of NO in plants have been made possible by the application of NO donors. However, the application of NO to plants in various forms and doses should be subjected to detailed verification criteria. Not all metabolic responses induced by NO donors are reliable and reproducible in other experimental designs. The aim of the presented studies was to investigate the half-life of the most frequently applied donors (SNP, SNAP and GSNO), the rate of NO release under the influence of light and reducing agents. At a comparable donor concentration (500 microM) and under light conditions the highest rate of NO generation was found for SNAP, followed by GSNO and SNP. The measured half-life of the donor in the solution was 3 h for SNAP, 7 h for GSNO and 12 h for SNP. A temporary lack of light inhibited NO release from SNP, both in the solution and SNP-treated leaf tissue, which was measured by the electrochemical method. Also a NO, selective fluorescence indicator DAF-2DA in leaves supplied with different donors showed green fluorescence spots in the epidermal cells mainly in the light. SNP as a NO donor was the most photosensitive. The activity of PAL, which plays an important role in plant defence, was also activated by SNP in the light, not in the dark. S-nitrosothiols (SNAP and GSNO) also underwent photodegradation, although to a lesser degree than SNP. Additionally, NO generation capacity from S-nitrosothiols was shown in the presence of reducing agents, i.e. ascorbic acid and GSH, and the absence of light. The authors of this paper would like to polemicize with the commonly cited statement that "donors are compounds that spontaneously break down to release NO" and wish to point out the fact that the process of donor decomposition depends on the numerous external factors. It may be additionally stimulated or inhibited by live plant tissue, thus it is necessary to take into consideration these aspects and monitor the amount of NO released by the donor.

Benzothiadiazole (BTH) was found to be highly effective in increasing resistance of two poinsettia cultivars — ‘Coco White’ and ‘Malibu Red’, moderately susceptible to the fungus Botrytis cinerea. BTH applied at a concentration of 0.3 mM on the discs cut out from the leaves of these poinsettia cultivars reduced disease symptoms by more than 60 % in comparison to the control discs treated with water and exposed to infection.

In this study the proportion of lignin in wood tissue was analysed in 36 Scots pines differing in their form of dead bark. Material for analyses came from pure pine stands aged 89-91 years located in northern Poland. Chemical analyses were performed in the mature wood zone comprising the last 10 diameter increments located at a height of 1.3 m (DBH). Results indicate differences in lignin content and growth rate in pines whose stems were characterized by different forms of dead bark. The study showed significant differences in lignin contents between trees with shell and scaly dead bark and trees with ropy bark form. Pines with ropy bark were characterized by the lowest lignin content in relation to trees having the other 2 forms of dead bark. Moreover, a relationship was observed between diameter growth rate and lignin content in wood tissue.

Local treatment of potato plants with arachidonic acid caused a repeated increase of ribonuclease and proteinase activities in leaves that were directly sprayed with this inducer. Immunization caused no significant systemic changes in the enzyme activity of other potato leaves.