Reactive oxygen species and organellar signaling.

The evolution of photosynthesis and its associated metabolic pathways has been crucial to the successful establishment of plants, but has also challenged plant cells in the form of production of reactive oxygen species (ROS). Intriguingly, multiple forms of ROS are generated in virtually every plant cell compartment through diverse pathways. As a result, a sophisticated network of ROS detoxification and signaling that is simultaneously tailored to individual organelles and safeguards the entire cell is necessary. Here we take an organelle-centric view on the principal sources and sinks of ROS across the plant cell and provide insights into the ROS-induced organelle to nucleus retrograde signaling pathways needed for operational readjustments during environmental stresses.

Protein Promiscuity in H2O2 Signaling.

SIGNIFICANCE: Decrypting the cellular response to oxidative stress relies on a comprehensive understanding of the redox signaling pathways stimulated under oxidizing conditions. Redox signaling events can be divided into upstream sensing of oxidants, midstream redox signaling of protein function, and downstream transcriptional redox regulation. Recent Advances: A more and more accepted theory of hydrogen peroxide (H2O2) signaling is that of a thiol peroxidase redox relay, whereby protein thiols with low reactivity toward H2O2 are instead oxidized through an oxidative relay with thiol peroxidases. CRITICAL ISSUES: These ultrareactive thiol peroxidases are the upstream redox sensors, which form the first cellular port of call for H2O2. Not all redox-regulated interactions between thiol peroxidases and cellular proteins involve a transfer of oxidative equivalents, and the nature of redox signaling is further complicated through promiscuous functions of redox-regulated "moonlighting" proteins, of which the precise cellular role under oxidative stress can frequently be obscured by "polygamous" interactions. An ultimate goal of redox signaling is to initiate a rapid response, and in contrast to prokaryotic oxidant-responsive transcription factors, mammalian systems have developed redox signaling pathways, which intersect both with kinase-dependent activation of transcription factors, as well as direct oxidative regulation of transcription factors through peroxiredoxin (Prx) redox relays. FUTURE DIRECTIONS: We highlight that both transcriptional regulation and cell fate can be modulated either through oxidative regulation of kinase pathways, or through distinct redox-dependent associations involving either Prxs or redox-responsive moonlighting proteins with functional promiscuity. These protein associations form systems of crossregulatory networks with multiple nodes of potential oxidative regulation for H2O2-mediated signaling.

In vivo detection of protein cysteine sulfenylation in plastids.

Protein cysteine thiols are post-translationally modified under oxidative stress conditions. Illuminated chloroplasts are one of the important sources of hydrogen peroxide (H2 O2 ) and are highly sensitive to environmental stimuli, yet a comprehensive view of the oxidation-sensitive chloroplast proteome is still missing. By targeting the sulfenic acid YAP1C-trapping technology to the plastids of light-grown Arabidopsis cells, we identified 132 putatively sulfenylated plastid proteins upon H2 O2 pulse treatment. Almost half of the sulfenylated proteins are enzymes of the amino acid metabolism. Using metabolomics, we observed a reversible decrease in the levels of the amino acids Ala, Asn, Cys, Gln, Glu, His, Ile, Leu, Lys, Phe, Ser, Thr and Val after H2 O2 treatment, which is in line with an anticipated decrease in the levels of the glycolysis and tricarboxylic acid metabolites. Through the identification of an organelle-tailored proteome, we demonstrated that the subcellular targeting of the YAP1C probe enables us to study in vivo cysteine sulfenylation at the organellar level. All in all, the identification of these oxidation events in plastids revealed that several enzymes of the amino acid metabolism rapidly undergo cysteine oxidation upon oxidative stress.

European contribution to the study of ROS: A summary of the findings and prospects for the future from the COST action BM1203 (EU-ROS).

The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associated with oxidative stress established the theory of oxidative stress as a trigger of diseases that can be corrected by antioxidant therapy. However, while experimental studies support this thesis, clinical studies still generate controversial results, due to complex pathophysiology of oxidative stress in humans. For future improvement of antioxidant therapy and better understanding of redox-associated disease progression detailed knowledge on the sources and targets of RONS formation and discrimination of their detrimental or beneficial roles is required. In order to advance this important area of biology and medicine, highly synergistic approaches combining a variety of diverse and contrasting disciplines are needed.

Redox Strategies for Crop Improvement.

SIGNIFICANCE: Recently, the agro-biotech industry has been driven by overcoming the limitations imposed by fluctuating environmental stress conditions on crop productivity. A common theme among (a)biotic stresses is the perturbation of the redox homeostasis. RECENT ADVANCES: As a strategy to engineer stress-tolerant crops, many approaches have been centered on restricting the negative impact of reactive oxygen species (ROS) accumulation. CRITICAL ISSUES: In this study, we discuss the scientific background of the existing redox-based strategies to improve crop performance and quality. In this respect, a special focus goes to summarizing the current patent landscape because this aspect is very often ignored, despite constituting the forefront of applied research. FUTURE DIRECTIONS: The current increased understanding of ROS acting as signaling molecules has opened new avenues to exploit redox biology for crop improvement required for sustainable food security.

DYn-2 Based Identification of Arabidopsis Sulfenomes.

Identifying the sulfenylation state of stressed cells is emerging as a strategic approach for the detection of key reactive oxygen species signaling proteins. Here, we optimized an in vivo trapping method for cysteine sulfenic acids in hydrogen peroxide (H2O2) stressed plant cells using a dimedone based DYn-2 probe. We demonstrated that DYn-2 specifically detects sulfenylation events in an H2O2 dose- and time-dependent way. With mass spectrometry, we identified 226 sulfenylated proteins after H2O2 treatment of Arabidopsis cells, residing in the cytoplasm (123); plastid (68); mitochondria (14); nucleus (10); endoplasmic reticulum, Golgi and plasma membrane (7) and peroxisomes (4). Of these, 123 sulfenylated proteins have never been reported before to undergo cysteine oxidative post-translational modifications in plants. All in all, with this DYn-2 approach, we have identified new sulfenylated proteins, and gave a first glance on the locations of the sulfenomes of Arabidopsis thaliana.

Sulfenome mining in Arabidopsis thaliana.

Reactive oxygen species (ROS) have been shown to be potent signaling molecules. Today, oxidation of cysteine residues is a well-recognized posttranslational protein modification, but the signaling processes steered by such oxidations are poorly understood. To gain insight into the cysteine thiol-dependent ROS signaling in Arabidopsis thaliana, we identified the hydrogen peroxide (H2O2)-dependent sulfenome: that is, proteins with at least one cysteine thiol oxidized to a sulfenic acid. By means of a genetic construct consisting of a fusion between the C-terminal domain of the yeast (Saccharomyces cerevisiae) AP-1-like (YAP1) transcription factor and a tandem affinity purification tag, we detected ∼ 100 sulfenylated proteins in Arabidopsis cell suspensions exposed to H2O2 stress. The in vivo YAP1-based trapping of sulfenylated proteins was validated by a targeted in vitro analysis of dehydroascorbate reductase2 (DHAR2). In DHAR2, the active site nucleophilic cysteine is regulated through a sulfenic acid-dependent switch, leading to S-glutathionylation, a protein modification that protects the protein against oxidative damage.