Sandy Vanderauwera

ROS signaling: the new wave?

Reactive oxygen species (ROS) play a multitude of signaling roles in different organisms from bacteria to mammalian cells. They were initially thought to be toxic byproducts of aerobic metabolism, but have now been acknowledged as central players in the complex signaling network of cells. In this review, we will attempt to address several key questions related to the use of ROS as signaling molecules in cells, including the dynamics and specificity of ROS signaling, networking of ROS with other signaling pathways, ROS signaling within and across different cells, ROS waves and the evolution of the ROS gene network.

Transcriptomic Footprints Disclose Specificity of Reactive Oxygen Species Signaling in Arabidopsis

Reactive oxygen species (ROS) are key players in the regulation of plant development, stress responses, and programmed cell death. Previous studies indicated that depending on the type of ROS (hydrogen peroxide, superoxide, or singlet oxygen) or its subcellular production site (plastidic, cytosolic, peroxisomal, or apoplastic), a different physiological, biochemical, and molecular response is provoked. We used transcriptome data generated from ROS-related microarray experiments to assess the specificity of ROS-driven transcript expression. Data sets obtained by exogenous application of oxidative stress-causing agents (methyl viologen, Alternaria alternata toxin, 3-aminotriazole, and ozone) and from a mutant (fluorescent) and transgenic plants, in which the activity of an individual antioxidant enzyme was perturbed (catalase, cytosolic ascorbate peroxidase, and copper/zinc superoxide dismutase), were compared. In total, the abundance of nearly 26,000 transcripts of Arabidopsis (Arabidopsis thaliana) was monitored in response to different ROS. Overall, 8,056, 5,312, and 3,925 transcripts showed at least a 3-, 4-, or 5-fold change in expression, respectively. In addition to marker transcripts that were specifically regulated by hydrogen peroxide, superoxide, or singlet oxygen, several transcripts were identified as general oxidative stress response markers because their steady-state levels were at least 5-fold elevated in most experiments. We also assessed the expression characteristics of all annotated transcription factors and inferred new candidate regulatory transcripts that could be responsible for orchestrating the specific transcriptomic signatures triggered by different ROS. Our analysis provides a framework that will assist future efforts to address the impact of ROS signals within environmental stress conditions and elucidate the molecular mechanisms of the oxidative stress response in plants.

Genome-Wide Analysis of Hydrogen Peroxide-Regulated Gene Expression in Arabidopsis Reveals a High Light-Induced Transcriptional Cluster Involved in Anthocyanin Biosynthesis

In plants, reactive oxygen species and, more particularly, hydrogen peroxide (H2O2) play a dual role as toxic by-products of normal cell metabolism and as regulatory molecules in stress perception and signal transduction. Peroxisomal catalases are an important sink for photorespiratory H2O2. Using ATH1 Affymetrix microarrays, expression profiles were compared between control and catalase-deficient Arabidopsis (Arabidopsis thaliana) plants. Reduced catalase levels already provoked differences in nuclear gene expression under ambient growth conditions, and these effects were amplified by high light exposure in a sun simulator for 3 and 8 h. This genome-wide expression analysis allowed us to reveal the expression characteristics of complete pathways and functional categories during H2O2 stress. In total, 349 transcripts were significantly up-regulated by high light in catalase-deficient plants and 88 were down-regulated. From this data set, H2O2 was inferred to play a key role in the transcriptional up-regulation of small heat shock proteins during high light stress. In addition, several transcription factors and candidate regulatory genes involved in H2O2 transcriptional gene networks were identified. Comparisons with other publicly available transcriptome data sets of abiotically stressed Arabidopsis revealed an important intersection with H2O2-deregulated genes, positioning elevated H2O2 levels as an important signal within abiotic stress-induced gene expression. Finally, analysis of transcriptional changes in a combination of a genetic (catalase deficiency) and an environmental (high light) perturbation identified a transcriptional cluster that was strongly and rapidly induced by high light in control plants, but impaired in catalase-deficient plants. This cluster comprises the complete known anthocyanin regulatory and biosynthetic pathway, together with genes encoding unknown proteins.

Peroxisomal Hydrogen Peroxide Is Coupled to Biotic Defense Responses by ISOCHORISMATE SYNTHASE1 in a Daylength-Related Manner

While it is well established that reactive oxygen species can induce cell death, intracellularly generated oxidative stress does not induce lesions in the Arabidopsis (Arabidopsis thaliana) photorespiratory mutant cat2 when plants are grown in short days (SD). One interpretation of this observation is that a function necessary to couple peroxisomal hydrogen peroxide (H2O2)-triggered oxidative stress to cell death is only operative in long days (LD). Like lesion formation, pathogenesis-related genes and camalexin were only induced in cat2 in LD, despite less severe intracellular redox perturbation compared with SD. Lesion formation triggered by peroxisomal H2O2 was modified by introducing secondary mutations into the cat2 background and was completely absent in cat2 sid2 double mutants, in which ISOCHORISMATE SYNTHASE1 (ICS1) activity is defective. In addition to H2O2-induced salicylic acid (SA) accumulation, the sid2 mutation in ICS1 abolished a range of LD-dependent pathogen responses in cat2, while supplementation of cat2 with SA in SD activated these responses. Nontargeted transcript and metabolite profiling identified clusters of genes and small molecules associated with the daylength-dependent ICS1-mediated relay of H2O2 signaling. The effect of oxidative stress in cat2 on resistance to biotic challenge was dependent on both growth daylength and ICS1. We conclude that (1) lesions induced by intracellular oxidative stress originating in the peroxisomes can be genetically reverted; (2) the isochorismate pathway of SA synthesis couples intracellular oxidative stress to cell death and associated disease resistance responses; and (3) camalexin accumulation was strictly dependent on the simultaneous presence of both H2O2 and SA signals.

Silencing of poly(ADP-ribose) polymerase in plants alters abiotic stress signal transduction

Transgenic plants with reduced poly(ADP-ribose) polymerase (PARP) levels have broad-spectrum stress-resistant phenotypes. Both Arabidopsis thaliana and oilseed rape (Brassica napus) lines overexpressing RNA interference-PARP constructs were more resistant to various abiotic stress treatments in laboratory and greenhouse experiments without negative effects on growth, development, and seed production. This outperforming stress tolerance was initially attributed solely to a maintained energy homeostasis due to reduced NAD+ consumption. We show that in PARP2-deficient Arabidopsis plants, the observed abiotic stress resistance can also be explained by alterations in abscisic acid levels that facilitate the induction of a wide set of defense-related genes.

Extranuclear protection of chromosomal DNA from oxidative stress

Eukaryotic organisms evolved under aerobic conditions subjecting nuclear DNA to damage provoked by reactive oxygen species (ROS). Although ROS are thought to be a major cause of DNA damage, little is known about the molecular mechanisms protecting nuclear DNA from oxidative stress. Here we show that protection of nuclear DNA in plants requires a coordinated function of ROS-scavenging pathways residing in the cytosol and peroxisomes, demonstrating that nuclear ROS scavengers such as peroxiredoxin and glutathione are insufficient to safeguard DNA integrity. Both catalase (CAT2) and cytosolic ascorbate peroxidase (APX1) play a key role in protecting the plant genome against photorespiratory-dependent H2O2-induced DNA damage. In apx1/cat2 double-mutant plants, a DNA damage response is activated, suppressing growth via a WEE1 kinase-dependent cell-cycle checkpoint. This response is correlated with enhanced tolerance to oxidative stress, DNA stress-causing agents, and inhibited programmed cell death.

ERF115 controls root quiescent center cell division and stem cell replenishment.

The Root of the Problem The quiescent center (QC) within the root meristem plays a key role as a stem cell organizer to sustain the root stem cell niche. The QC cells execute a dual role: prevention of the differentiation of neighboring stem cells, and maintenance of the root structure by undergoing only occasional cell division. The mechanisms that account for the low QC proliferation are unclear, although the anaphase-promoting complex/cyclosome (APC/C) E3 ubiquitin ligase is known to suppress QC cell division. Through a systematic functional analysis of APC/C-copurifying proteins, Heyman et al. (p. 860) characterized a transcription factor ERF115 as a rate-limiting factor for QC cell division. ERF115 needs to be destroyed to retain QC cells in a resting state. ERF115 operates in a brassinosteroid-dependent manner and controls QC cell division through transcriptional activation of phytosulfokine signaling. Restrained growth of a key root tip region involves an interplay between hormonal activation and transcription factor levels. The quiescent center (QC) plays an essential role during root development by creating a microenvironment that preserves the stem cell fate of its surrounding cells. Despite being surrounded by highly mitotic active cells, QC cells self-renew at a low proliferation rate. Here, we identified the ERF115 transcription factor as a rate-limiting factor of QC cell division, acting as a transcriptional activator of the phytosulfokine PSK5 peptide hormone. ERF115 marks QC cell division but is restrained through proteolysis by the APC/CCCS52A2 ubiquitin ligase, whereas QC proliferation is driven by brassinosteroid-dependent ERF115 expression. Together, these two antagonistic mechanisms delimit ERF115 activity, which is called upon when surrounding stem cells are damaged, revealing a cell cycle regulatory mechanism accounting for stem cell niche longevity.

Day length is a key regulator of transcriptomic responses to both CO2 and H2O2 in Arabidopsis

Growth day length, CO(2) levels and H(2)O(2) all impact plant function, but interactions between them remain unclear. Using a whole-genome transcriptomics approach, we identified gene expression patterns responding to these three factors in Arabidopsis Col-0 and the conditional catalase-deficient mutant, cat2. Plants grown for 5 weeks at high CO(2) in short days (hCO(2)) were transferred to air in short days (SD air) or long days (LD air), and microarray data produced were subjected to three independent studies. The first two analysed genotype-independent responses. They identified 1549 genes differentially expressed after transfer from hCO(2) to SD air. Almost half of these, including genes modulated by sugars or associated with redox, stress or abscisic acid (ABA) functions, as well as light signalling and clock genes, were no longer significant after transfer to air in LD. In a third study, day length-dependent H(2)O(2)-responsive genes were identified by comparing the two genotypes. Two clearly independent responses were observed in cat2 transferred to air in SD and LD. Most H(2)O(2) -responsive genes were up-regulated more strongly in SD air. Overall, the analysis shows that both CO(2) and H(2)O(2) interact with day length and photoreceptor pathways, indicating close networking between carbon status, light and redox state in environmental responses.

The Arabidopsis SIAMESE-RELATED Cyclin-Dependent Kinase Inhibitors SMR5 and SMR7 Regulate the DNA Damage Checkpoint in Response to Reactive Oxygen Species

Reactive oxygen species (ROS) cause DNA damage. In this work, two SIAMESE/SIAMESE-RELATED (SIM/SMR) genes that encode cyclin-dependent kinase inhibitors are described as being part of a signaling pathway that arrests cell proliferation in response to ROS, revealing a novel cell cycle checkpoint-signaling cascade. Whereas our knowledge about the diverse pathways aiding DNA repair upon genome damage is steadily increasing, little is known about the molecular players that adjust the plant cell cycle in response to DNA stress. By a meta-analysis of DNA stress microarray data sets, three family members of the SIAMESE/SIAMESE-RELATED (SIM/SMR) class of cyclin-dependent kinase inhibitors were discovered that react strongly to genotoxicity. Transcriptional reporter constructs corroborated specific and strong activation of the three SIM/SMR genes in the meristems upon DNA stress, whereas overexpression analysis confirmed their cell cycle inhibitory potential. In agreement with being checkpoint regulators, SMR5 and SMR7 knockout plants displayed an impaired checkpoint in leaf cells upon treatment with the replication inhibitory drug hydroxyurea (HU). Surprisingly, HU-induced SMR5/SMR7 expression depends on ATAXIA TELANGIECTASIA MUTATED (ATM) and SUPPRESSOR OF GAMMA RESPONSE1, rather than on the anticipated replication stress-activated ATM AND RAD3-RELATED kinase. This apparent discrepancy was explained by demonstrating that, in addition to its effect on replication, HU triggers the formation of reactive oxygen species (ROS). ROS-dependent transcriptional activation of the SMR genes was confirmed by different ROS-inducing conditions, including high-light treatment. We conclude that the identified SMR genes are part of a signaling cascade that induces a cell cycle checkpoint in response to ROS-induced DNA damage.

A subcellular localization compendium of hydrogen peroxide‐induced proteins

The signal transduction mechanisms of the oxidative stress response in plants remain largely unexplored. Previously, increased levels of cellular hydrogen peroxide (H(2)O(2)) had been shown to drastically affect the plant transcriptome. Genome-wide transcriptome analyses allowed us to build a comprehensive inventory of H(2)O(2)-induced genes in plants. Here, the primary objective was to determine the subcellular localization of these genes and to assess potential trafficking during oxidative stress. After high-throughput cloning in Gateway-derived vectors, the subcellular localization of 49 proteins fused to the green fluorescent protein (GFP) was identified in a transient assay in tobacco (Nicotiana benthamiana) by means of agro-infiltration and confirmed for a selection of genes in transgenic Arabidopsis thaliana plants. Whereas eight of the GFP-tagged proteins are exclusively localized in the nucleus, 23 reside both in the nucleus and cytosol, in which several classes of known transcription factors and proteins of unknown function can be recognized. In this study, the mapping of the subcellular localization of H(2)O(2) -induced proteins paves the way for future research to unravel the H(2)O(2) responses in plants. Furthermore, the effect of increased H(2)O(2) levels on the subcellular localization of a subset of proteins was assessed.