Ethylene Response Factor 6 Is a Regulator of Reactive Oxygen Species Signaling in Arabidopsis

et al. (2013) Ethylene Response Factor 6 Is a Regulator of Reactive Oxygen Species Signaling in Arabidopsis. PLoS ONE 8(8): e70289. doi:10.1371/journal.pone.0070289 Ethylene Response Factor 6 Is a Regulator of Reactive Oxygen Species Signaling in Arabidopsis Nasser Sewelam 0 Kemal Kazan 0 Skye R. Thomas-Hall 0 Brendan N. Kidd 0 John M. Manners 0 Peer M. Schenk 0 Shin-Han Shiu, Michigan State University, United States of America 0 1 Plant-Microbe Interactions Laboratory, School of Agriculture and Food Sciences, The University of Queensland , Brisbane, Queensland , Australia , 2 Commonwealth Scientific and Industrial Research Organization Plant Industry, Queensland Bioscience Precinct , Brisbane, Queensland , Australia Reactive oxygen species (ROS) are produced in plant cells in response to diverse biotic and abiotic stresses as well as during normal growth and development. Although a large number of transcription factor (TF) genes are up- or down-regulated by ROS, currently very little is known about the functions of these TFs during oxidative stress. In this work, we examined the role of ERF6 (ETHYLENE RESPONSE FACTOR6), an AP2/ERF domain-containing TF, during oxidative stress responses in Arabidopsis. Mutant analyses showed that NADPH oxidase (RbohD) and calcium signaling are required for ROS-responsive expression of ERF6. erf6 insertion mutant plants showed reduced growth and increased H2O2 and anthocyanin levels. Expression analyses of selected ROS-responsive genes during oxidative stress identified several differentially expressed genes in the erf6 mutant. In particular, a number of ROS responsive genes, such as ZAT12, HSFs, WRKYs, MAPKs, RBOHs, DHAR1, APX4, and CAT1 were more strongly induced by H2O2 in erf6 plants than in wild-type. In contrast, MDAR3, CAT3, VTC2 and EX1 showed reduced expression levels in the erf6 mutant. Taken together, our results indicate that ERF6 plays an important role as a positive antioxidant regulator during plant growth and in response to biotic and abiotic stresses. - Funding: This work was supported by the University of Queensland, the Australian Research Council (DP1094749) and the Egyptian Government (Ministry of Higher Education). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Reactive oxygen species (ROS) are produced constantly during normal plant growth and development (e.g. during photosynthesis) and they also fulfill essential roles as highly specific signaling molecules under stress conditions. However, due to their highly toxic nature, ROS are also constantly scavenged by complex and redundant antioxidant defenses. Under various biotic and abiotic stress conditions such as high-light, drought, heat or pathogen attack, excessive amounts of ROS are produced and the balance between ROS production and degradation is disturbed, with potentially damaging consequences to cellular machinery [4,14]. Given the importance of ROS as both damaging and signaling molecules, a better understanding of plant processes involved in ROS generation, signaling and scavenging is of significant importance in both basic plant biology and crop improvement. In plants, ROS are produced through multiple pathways which include photosynthetic and respiratory electron transport chains, photorespiration, amine oxidases, cell wall-bound peroxidases, and membrane-bound NADPH oxidases (reviewed by Mittler et al., [43]). Membrane-bound NADPH oxidases also known as respiratory burst oxidase homologs (Rboh) are a group of enzymes that catalyze the production of superoxide radicals in both animals and plants (reviewed by Suzuki et al., [66]). Recent studies also show intimate links between ROS and plant hormones [43]. In stomatal guard cells, for instance, the plant hormone ABA activates ROS production through the NADPH oxidase RbohD and this leads to stomatal closure [21,25]. Another study has shown that DELLA proteins with roles in GA-signaling regulate plant growth and stress tolerance through modulation of ROS levels [2]. Furthermore, other plant hormones such as auxin and plant defense hormones salicylic (SA) and jasmonic acid (JA) modulate the plants ROS status [43]. These studies suggest that plants expediently integrate signals from multiple endogenous and exogenous cues that lead to the modulation of cellular ROS levels. Emerging evidence also indicates that both the level and subcellular location of ROS can induce specific cellular processes. For instance, ROS required for maintaining normal growth and development is produced at low levels and specifically where it is needed such as in root tip cells [28,60]. In contrast, higher amounts of ROS produced under stress conditions can negatively affect plant growth. During challenge by an incompatible pathogen, ROS is specifically generated in the extra-cellular spaces of cells undergoing programmed cell death [68]. This hypersensitive-type (HR) response is genetically controlled by the plant and is often considered to be a useful evolutionary trait against the threat by biotrophic pathogens [62]. However, necrotrophic pathogens as part of their infection strategy, deliberately induce the production of ROS and cell death which facilitates subsequent tissue colonization [9,67]. Similarly, under severe abiotic stress conditions, excessive amounts of ROS are generated as a result of cellular damage. Therefore, plants have also evolved mechanisms to protect themselves from the danger posed by ROS through various antioxidant defenses. Indeed, ROS coordinately activate the expression of genes encoding enzymes for ROS scavenging or synthesis of antioxidant enzymes or molecules required to counteract the potentially damaging effects of ROS. At least ten major cellular mechanisms involved in ROS removal are known (reviewed by Mittler [41]). These include several enzymatic mechanisms that involve the action of antioxidant enzymes such as superoxide dismutase (SOD), which converts O.22 to H2O2, and catalases and peroxidases, which remove H2O2. The harmful effects of ROS can also be neutralized by non-enzymatic means through antioxidant molecules such as ascorbic acid, glutathione, carotenoids, and a-tocopherol. Furthermore, different ROS (such as superoxide radicals, H2O2 or singlet oxygen 1O2) produced in different subcellular compartments (e.g. plastids, mitochondria and peroxisomes) induce specific adaptive responses. For example, cytosolic H2O2 induces the expression of heat shock proteins during light stress [57]. In contrast, peroxisomal photorespirationdependent H2O2 has a negative effect on the high-light stress induction of transcripts within the biosynthetic pathway for antioxidant anthocyanins [70]. Specific ROS sensors are not known; however, after perception, ROS signals are transmitted to downstream components by the action of secondary (...truncated)