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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.

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.

Nitric oxide (NO) plays a key role in plant metabolism, signaling, defense, and development. However, a fundamental question arises how the NO message is converted into a physiological response. NO-related signaling may be attributed to various NO derivatives, collectively named reactive nitrogen species (RNS). An increasing body of evidence suggests that nitroxyl (HNO) as a one-electron reduced and protonated state of NO and nitrosonium (NO+), a one-electron oxidized form of NO, may be important factors. Thus, the impact of NO in plant biology should be re-evaluated in the light of chemical properties of these compounds as they are different from that of NO. Nitroxyl, unlike NO, can interact directly with thiols, prioritize ferric rather than ferrous heme proteins and it is resistant to scavenging by superoxides. Experimental data revealed that NO+ can facilitate S-nitrosylation, while it also appears to be a key intermediate in the trans-nitrosylation reaction. In contrast to NO, it may also take part in the degradation of SNOs (S-nitrosothiols). In turn, peroxynitrite (ONOO−) is a potent oxidant and nitrating agent, generated by the reaction of nitric oxide and superoxide in one of the most rapid reactions known in biology. Overproduction of ONOO− contributes to oxidative and nitrosative stress, however, in the physiological state or under low metabolic stress, peroxynitrite triggered NO-dependent signals behave as a potent modulator of the redox regulation in various cell transduction pathways.