Cezary Waszczak

Sulfenome mining in Arabidopsis thaliana

Significance When oxygen gets incompletely reduced, reactive oxygen species (ROS) are generated. These ROS molecules can harm the building blocks of the cell but are also important signaling molecules. Until now, the ROS language of the cell has not been understood and a clear view is needed on how the cell differentiates metabolic ROS noise from ROS that allows signaling, regulation, and protection. To address this question, we focused on Arabidopsis thaliana and identified the proteins that react with hydrogen peroxide on the thiol of the amino acid cysteine, which after reaction forms a sulfenic acid. The characterization of the plant sulfenome improves the understanding of important ROS signaling pathways. 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.

Oxidative post-translational modifications of cysteine residues in plant signal transduction

In plants, fluctuation of the redox balance by altered levels of reactive oxygen species (ROS) can affect many aspects of cellular physiology. ROS homeostasis is governed by a diversified set of antioxidant systems. Perturbation of this homeostasis leads to transient or permanent changes in the redox status and is exploited by plants in different stress signalling mechanisms. Understanding how plants sense ROS and transduce these stimuli into downstream biological responses is still a major challenge. ROS can provoke reversible and irreversible modifications to proteins that act in diverse signalling pathways. These oxidative post-translational modifications (Ox-PTMs) lead to oxidative damage and/or trigger structural alterations in these target proteins. Characterization of the effect of individual Ox-PTMs on individual proteins is the key to a better understanding of how cells interpret the oxidative signals that arise from developmental cues and stress conditions. This review focuses on ROS-mediated Ox-PTMs on cysteine (Cys) residues. The Cys side chain, with its high nucleophilic capacity, appears to be the principle target of ROS. Ox-PTMs on Cys residues participate in various signalling cascades initiated by plant stress hormones. We review the mechanistic aspects and functional consequences of Cys Ox-PTMs on specific target proteins in view of stress signalling events.

Reactive Oxygen Species in the Regulation of Stomatal Movements

Reactive oxygen species play an important role in guard cell signaling leading to stomatal closure and regulate signal amplification and specificity. Guard cells form stomatal pores that optimize photosynthetic carbon dioxide uptake with minimal water loss. Stomatal movements are controlled by complex signaling networks that respond to environmental and endogenous signals. Regulation of stomatal aperture requires coordinated activity of reactive oxygen species (ROS)-generating enzymes, signaling proteins, and downstream executors such as ion pumps, transporters, and plasma membrane channels that control guard cell turgor pressure. Accumulation of ROS in the apoplast and chloroplasts is among the earliest hallmarks of stomatal closure. Subsequent increase in cytoplasmic Ca2+ concentration governs the activity of multiple kinases that regulate the activity of ROS-producing enzymes and ion channels. In parallel, ROS directly regulate the activity of multiple proteins via oxidative posttranslational modifications to fine-tune guard cell signaling. In this review, we summarize recent advances in the role of ROS in stomatal closure and discuss the importance of ROS in regulation of signal amplification and specificity in guard cells.

Cysteines under ROS attack in plants: a proteomics view

Plants generate reactive oxygen species (ROS) as part of their metabolism and in response to various external stress factors, potentially causing significant damage to biomolecules and cell structures. During the course of evolution, plants have adapted to ROS toxicity, and use ROS as signalling messengers that activate defence responses. Cysteine (Cys) residues in proteins are one of the most sensitive targets for ROS-mediated post-translational modifications, and they have become key residues for ROS signalling studies. The reactivity of Cys residues towards ROS, and their ability to react to different oxidation states, allow them to appear at the crossroads of highly dynamic oxidative events. As such, a redox-active cysteine can be present as S-glutathionylated (-SSG), disulfide bonded (S-S), sulfenylated (-SOH), sulfinylated (-SO2H), and sulfonylated (-SO3H). The sulfenic acid (-SOH) form has been considered as part of ROS-sensing pathways, as it leads to further modifications which affect protein structure and function. Redox proteomic studies are required to understand how and why cysteines undergo oxidative post-translational modifications and to identify the ROS-sensor proteins. Here, we update current knowledge of cysteine reactivity with ROS. Further, we give an overview of proteomic techniques that have been applied to identify different redox-modified cysteines in plants. There is a particular focus on the identification of sulfenylated proteins, which have the potential to be involved in plant signal transduction.

Lack of GLYCOLATE OXIDASE1, but Not GLYCOLATE OXIDASE2, Attenuates the Photorespiratory Phenotype of CATALASE2-Deficient Arabidopsis

Arabidopsis GOX1 and GOX2 have distinct roles under photorespiration-promoting conditions. The genes coding for the core metabolic enzymes of the photorespiratory pathway that allows plants with C3-type photosynthesis to survive in an oxygen-rich atmosphere, have been largely discovered in genetic screens aimed to isolate mutants that are unviable under ambient air. As an exception, glycolate oxidase (GOX) mutants with a photorespiratory phenotype have not been described yet in C3 species. Using Arabidopsis (Arabidopsis thaliana) mutants lacking the peroxisomal CATALASE2 (cat2-2) that display stunted growth and cell death lesions under ambient air, we isolated a second-site loss-of-function mutation in GLYCOLATE OXIDASE1 (GOX1) that attenuated the photorespiratory phenotype of cat2-2. Interestingly, knocking out the nearly identical GOX2 in the cat2-2 background did not affect the photorespiratory phenotype, indicating that GOX1 and GOX2 play distinct metabolic roles. We further investigated their individual functions in single gox1-1 and gox2-1 mutants and revealed that their phenotypes can be modulated by environmental conditions that increase the metabolic flux through the photorespiratory pathway. High light negatively affected the photosynthetic performance and growth of both gox1-1 and gox2-1 mutants, but the negative consequences of severe photorespiration were more pronounced in the absence of GOX1, which was accompanied with lesser ability to process glycolate. Taken together, our results point toward divergent functions of the two photorespiratory GOX isoforms in Arabidopsis and contribute to a better understanding of the photorespiratory pathway.

Bound by Fate: The Role of Reactive Oxygen Species in Receptor-Like Kinase Signaling

Receptor-like kinases and reactive oxygen species are intricately entangled and have central roles in controlling many processes in plants, but their interaction is still insufficiently understood. In plants, receptor-like kinases (RLKs) and extracellular reactive oxygen species (ROS) contribute to the communication between the environment and the interior of the cell. Apoplastic ROS production is a frequent result of RLK signaling in a multitude of cellular processes; thus, by their nature, these two signaling components are inherently linked. However, it is as yet unclear how ROS signaling downstream of receptor activation is executed. In this review, we provide a broad view of the intricate connections between RLKs and ROS signaling and describe the regulatory events that control and coordinate extracellular ROS production. We propose that concurrent initiation of ROS-dependent and -independent signaling linked to RLKs might be a critical element in establishing cellular responses. Furthermore, we discuss the possible ROS sensing mechanisms in the context of the biochemical environment in the apoplast. We suggest that RLK-dependent modulation of apoplastic and intracellular conditions facilitates ROS perception and signaling. Based on data from plant and animal models, we argue that specific RLKs could be components of the ROS sensing machinery or ROS sensors. The importance of the crosstalk between RLK and ROS signaling is discussed in the context of stomatal immunity. Finally, we highlight challenges in the understanding of these signaling processes and provide perspectives for future research.

A Cryptic Cytoplasmic Male Sterility Unveils a Possible Gynodioecious Past for Arabidopsis thaliana

Gynodioecy, the coexistence of hermaphrodites and females (i.e. male-sterile plants) in natural plant populations, most often results from polymorphism at genetic loci involved in a particular interaction between the nuclear and cytoplasmic genetic compartments (cytonuclear epistasis): cytoplasmic male sterility (CMS). Although CMS clearly contributes to the coevolution of involved nuclear loci and cytoplasmic genomes in gynodioecious species, the occurrence of CMS genetic factors in the absence of sexual polymorphism (cryptic CMS) is not easily detected and rarely taken in consideration. We found cryptic CMS in the model plant Arabidopsis thaliana after crossing distantly related accessions, Sha and Mr-0. Male sterility resulted from an interaction between the Sha cytoplasm and two Mr-0 genomic regions located on chromosome 1 and chromosome 3. Additional accessions with either nuclear sterility maintainers or sterilizing cytoplasms were identified from crosses with either Sha or Mr-0. By comparing two very closely related cytoplasms with different male-sterility inducing abilities, we identified a novel mitochondrial ORF, named orf117Sha, that is most likely the sterilizing factor of the Sha cytoplasm. The presence of orf117Sha was investigated in worldwide natural accessions. It was found mainly associated with a single chlorotype in accessions belonging to a clade predominantly originating from Central Asia. More than one-third of accessions from this clade carried orf117Sha, indicating that the sterilizing-inducing cytoplasm had spread in this lineage. We also report the coexistence of the sterilizing cytoplasm with a non-sterilizing cytoplasm at a small, local scale in a natural population; in addition a correlation between cytotype and nuclear haplotype was detected in this population. Our results suggest that this CMS system induced sexual polymorphism in A. thaliana populations, at the time when the species was mainly outcrossing.

Mapping of the ms8 male sterility gene in sweet pepper (Capsicum annuum L.) on the chromosome P4 using PCR-based markers useful for breeding programmes

The nuclear male sterility gene ms8 is expected to facilitate the production of sweet pepper (Capsicum annuum L.) hybrids as it provides means for hybridization without the labor-intensive hand emasculation of female inbred lines. The development of molecular markers linked to ms8 locus will help the breeding practice for the selection of hybrid parental lines. In this study, F2 population resulting from a cross between the sweet pepper male sterile line 320 and the male fertile variety Elf was used to identify DNA markers linked to the ms8 locus. With the use of RAPD–BSA technique, seven markers linked to the ms8 locus were found. Four of them were converted into SCAR markers. In addition, two COSII/CAPS markers linked to the ms8 locus were identified. Comparative mapping with reference pepper maps indicated that the ms8 locus is located on the lower arm of the pepper chromosome P4. Identified markers are useful for molecular breeding, however, at present markers tightly linked to ms8 locus are still lacking. Identification of molecular markers linked to the ms8 locus and determination of its chromosomal localization are useful for fine mapping and also provide the perspective for ms8 gene cloning.

ROS signalling in a destabilised world: A molecular understanding of climate change

Climate change results in increased intensity and frequency of extreme abiotic and biotic stress events. In plants, reactive oxygen species (ROS) accumulate in proportion to the level of stress and are major signalling and regulatory metabolites coordinating growth, defence, acclimation and cell death. Our knowledge of ROS homeostasis, sensing, and signalling is therefore key to understanding the impacts of climate change at the molecular level. Current research is uncovering new insights into temporal-spatial, cell-to-cell and systemic ROS signalling pathways, particularly how these affect plant growth, defence, and more recently acclimation mechanisms behind stress priming and long term stress memory. Understanding the stabilising and destabilising factors of ROS homeostasis and signalling in plants exposed to extreme and fluctuating stress will concomitantly reveal how to address future climate change challenges in global food security and biodiversity management.

Reactive Oxygen Species in Plant Signaling

As fixed organisms, plants are especially affected by changes in their environment and have consequently evolved extensive mechanisms for acclimation and adaptation. Initially considered by-products from aerobic metabolism, reactive oxygen species (ROS) have emerged as major regulatory molecules in plants and their roles in early signaling events initiated by cellular metabolic perturbation and environmental stimuli are now established. Here, we review recent advances in ROS signaling. Compartment-specific and cross-compartmental signaling pathways initiated by the presence of ROS are discussed. Special attention is dedicated to established and hypothetical ROS-sensing events. The roles of ROS in long-distance signaling, immune responses, and plant development are evaluated. Finally, we outline the most challenging contemporary questions in the field of plant ROS biology and the need to further elucidate mechanisms allowing sensing, signaling specificity, and coordination of multiple signals.