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▪ Abstract  Several reactive oxygen species (ROS) are continuously produced in plants as byproducts of aerobic metabolism. Depending on the nature of the ROS species, some are highly toxic and rapidly detoxified by various cellular enzymatic and nonenzymatic mechanisms. Whereas plants are surfeited with mechanisms to combat increased ROS levels during abiotic stress conditions, in other circumstances plants appear to purposefully generate ROS as signaling molecules to control various processes including pathogen defense, programmed cell death, and stomatal behavior. This review describes the mechanisms of ROS generation and removal in plants during development and under biotic and abiotic stress conditions. New insights into the complexity and roles that ROS play in plants have come from genetic analyses of ROS detoxifying and signaling mutants. Considering recent ROS-induced genome-wide expression analyses, the possible functions and mechanisms for ROS sensing and signaling in plants are compared with those in animals and yeast.

Formation of singlet oxygen (¹O₂) has been implicated with damaging photosystem II (PSII) that needs to undergo continuous repair to maintain photosynthetic electron transport. In addition to its damaging effect, ¹O₂ has also been shown to act as a signal that triggers stress acclimation and an enhanced stress resistance. A signaling role of ¹O₂ was first documented in the fluorescent (flu) mutant of Arabidopsis. It strictly depends on the chloroplast protein EXECUTER1 (EX1) and happens under nonphotoinhibitory light conditions. Under severe light stress, signaling is initiated independently of EX1 by ¹O₂ that is thought to be generated at the acceptor side of active PSII within the core of grana stacks. The results of the present study suggest a second source of ¹O₂ formation in grana margins close to the site of chlorophyll synthesis where EX1 is localized and the disassembly of damaged and reassembly of active PSII take place. The initiation of ¹O₂ signaling in grana margins depends on EX1 and the ATP-dependent zinc metalloprotease FtsH. As FtsH cleaves also the D1 protein during the disassembly of damaged PSII, EX1- and ¹O₂-mediated signaling seems to be not only spatially but also functionally associated with the repair of PSII.

Shortly after the release of singlet oxygen (¹O₂), drastic changes in nuclear gene expression occur in the conditional flu mutant of Arabidopsis that reveal a rapid transfer of signals from the plastid to the nucleus. In contrast to retrograde control of nuclear gene expression by plastid signals described earlier, the primary effect of ¹O₂ generation in the flu mutant is not the control of chloroplast biogenesis but the activation of a broad range of signaling pathways known to be involved in biotic and abiotic stress responses. This activity of a plastid-derived signal suggests a new function of the chloroplast, namely that of a sensor of environmental changes that activates a broad range of stress responses. Inactivation of the plastid protein EXECUTER1 attenuates the extent of ¹O₂-induced up-regulation of nuclear gene expression, but it does not fully eliminate these changes. A second related nuclear-encoded protein, dubbed EXECUTER2, has been identified that is also implicated with the signaling of ¹O₂-dependent nuclear gene expression changes. Like EXECUTER1, EXECUTER2 is confined to the plastid. Inactivation of both EXECUTER proteins in the ex1/ex2/flu triple mutant is sufficient to suppress the up-regulation of almost all ¹O₂-responsive genes. Retrograde control of ¹O₂-responsive genes requires the concerted action of both EXECUTER proteins within the plastid compartment.

Upon a dark-to-light shift, the conditional fluorescent (flu) mutant of Arabidopsis releases singlet oxygen (¹O₂) within the plastid compartment. Distinct sets of nuclear genes are activated that are different from those induced by superoxide (O[Formula: see text]) and/or hydrogen peroxide (H₂O₂), suggesting that different types of reactive oxygen species activate distinct signaling pathways. It is not known whether the pathways operate separately or interact with each other. We have addressed this problem by modulating noninvasively the level of H₂O₂ in plastids by means of a transgenic line that overexpresses the thylakoid-bound ascorbate peroxidase (tAPX). The overexpression of the H₂O₂-specific scavenger reduced strongly the activation of nuclear genes in plants treated with the herbicide paraquat that in the light leads to the enhanced generation of O[Formula: see text] and H₂O₂. In the flu mutant overexpressing tAPX, the intensity of ¹O₂-mediated cell death and growth inhibition was increased when compared with the flu parental line. Also, the expression of most of the nuclear genes that were rapidly activated after the release of ¹O₂ was significantly higher in flu plants overexpressing tAPX, whereas in wild-type plants, overexpression of tAPX did not lead to visible stress responses and had only a very minor impact on nuclear gene expression. The results suggest that H₂O₂ antagonizes the ¹O₂-mediated signaling of stress responses as seen in the flu mutant. This cross-talk between H₂O₂- and ¹O₂-dependent signaling pathways might contribute to the overall stability and robustness of wild-type plants exposed to adverse environmental stress conditions.

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.

When plant cells are under environmental stress, several chemically distinct reactive oxygen species (ROS) are generated simultaneously in various intracellular compartments and these can cause oxidative damage or act as signals. The conditional flu mutant of Arabidopsis , which generates singlet oxygen in plastids during a dark‐to‐light transition, has allowed the biological activity of singlet oxygen to be determined, and the criteria to distinguish between cytotoxicity and signalling of this particular ROS to be defined. The genetic basis of singlet‐oxygen‐mediated signalling has been revealed by the mutation of two nuclear genes encoding the plastid proteins EXECUTER (EX)1 and EX2, which are sufficient to abrogate singlet‐oxygen‐dependent stress responses. Conversely, responses due to higher cytotoxic levels of singlet oxygen are not suppressed in the ex1 / ex2 background. Whether singlet oxygen levels lower than those that trigger genetically controlled cell death activate acclimation is now under investigation.

Photosynthetic organisms must achieve a delicate balance between the light energy absorbed by chlorophyll and their capacity to channel that energy into productive photochemical reactions. Release of excess absorbed energy in the cell can cause lethal photooxidative damage. We identified a basic helix-loop-helix (bHLH) transcription factor, designated PHYTOCHROME-INTERACTING FACTOR 1 (PIF1), that negatively regulates chlorophyll biosynthesis. pif1 mutant seedlings accumulate excess free protochlorophyllide when grown in the dark, with consequent lethal bleaching upon exposure to light. PIF1 interacts specifically with the photoactivated conformer of phytochromes A and B, suggesting a signaling pathway by which chlorophyll biosynthetic rates are tightly controlled during the critical initial emergence of seedlings from subterranean darkness into sunlight.

Environmental stress often leads to an increased production of reactive oxygen species that are involved in plastid-to-nucleus retrograde signaling. Soon after the release of singlet oxygen (¹O ₂) in chloroplasts of the flu mutant of Arabidopsis , reprogramming of nuclear gene expression reveals a rapid transfer of signals from the plastid to the nucleus. We have identified extraplastidic signaling constituents involved in ¹O ₂-initiated plastid-to-nucleus signaling and nuclear gene activation after mutagenizing a flu line expressing the luciferase reporter gene under the control of the promoter of a ¹O ₂-responsive AAA-ATPase gene (At3g28580) and isolating second-site mutations that lead to a constitutive up-regulation of the reporter gene or abrogate its ¹O ₂-dependent up-regulation. One of these mutants, caa39 , turned out to be a weak mutant allele of the Topoisomerase VI (Topo VI) A-subunit gene with a single amino acid substitution. Transcript profile analysis of flu and flu caa39 mutants revealed that Topo VI is necessary for the full activation of AAA-ATPase and a set of ¹O ₂-responsive transcripts in response to ¹O ₂. Topo VI binds to the promoter of the AAA-ATPase and other ¹O ₂-responsive genes, and hence could directly regulate their expression. Under photoinhibitory stress conditions, which enhance the production of ¹O ₂ and H ₂O ₂, Topo VI regulates ¹O ₂-responsive and H ₂O ₂-responsive genes in a distinct manner. These results suggest that Topo VI acts as an integrator of multiple signals generated by reactive oxygen species formed in plants under adverse environmental conditions.

The conditional fluorescent (flu) mutant of Arabidopsis accumulates the photosensitizer protochlorophyllide in the dark. After a dark-to-light shift, the generation of singlet oxygen, a nonradical reactive oxygen species, starts within the first minute of illumination and was shown to be confined to plastids. Immediately after the shift, plants stopped growing and developed necrotic lesions. These early stress responses of the flu mutant do not seem to result merely from physicochemical damage. Peroxidation of chloroplast membrane lipids in these plants started rapidly and led to the transient and selective accumulation of a stereospecific and regiospecific and isomer of hydroxyoctadecatrieonic acid, free (13S)-HOTE, that could be attributed almost exclusively to the enzymatic oxidation of linolenic acid. Within the first 15 min of reillumination, distinct sets of genes were activated that were different from those induced by superoxide/hydrogen peroxide. Collectively, these results demonstrate that singlet oxygen does not act primarily as a toxin but rather as a signal that activates several stress-response pathways. Its biological activity in Arabidopsis exhibits a high degree of specificity that seems to be derived from the chemical identity of this reactive oxygen species and/or the intracellular location at which it is generated.

During seedling development chloroplast formation marks the transition from heterotrophic to autotrophic growth. The development and activity of chloroplasts may differ in cotyledons that initially serve as a storage organ and true leaves whose primary function is photosynthesis. A genetic screen was used for the identification of genes that affect selectively chloroplast function in cotyledons of Arabidopsis thaliana. Several mutants exhibiting pale cotyledons and green true leaves were isolated and dubbed snowy cotyledon (sco). One of the mutants, sco1, was characterized in more detail. The mutated gene was identified using map-based cloning. The mutant contains a point mutation in a gene encoding the chloroplast elongation factor G, leading to an amino acid exchange within the predicted 70S ribosome-binding domain. The mutation results in a delay in the onset of germination. At this early developmental stage embryos still contain undifferentiated proplastids, whose proper function seems necessary for seed germination. In light-grown sco1 seedlings the greening of cotyledons is severely impaired, whereas the following true leaves develop normally as in wild-type plants. Despite this apparent similarity of chloroplast development in true leaves of mutant and wild-type plants various aspects of mature plant development are also affected by the sco1 mutation such as the onset of flowering, the growth rate, and seed production. The onset of senescence in the mutant and the wild-type plants occurs, however, at the same time, suggesting that in the mutant this particular developmental step does not seem to suffer from reduced protein translation efficiency in chloroplasts.