The Genetic Basis of Singlet Oxygen–Induced Stress Responses of Arabidopsis thaliana

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Science  12 Nov 2004:
Vol. 306, Issue 5699, pp. 1183-1185
DOI: 10.1126/science.1103178


Plants under oxidative stress suffer from damages that have been interpreted as unavoidable consequences of injuries inflicted upon plants by toxic levels of reactive oxygen species (ROS). However, this paradigm needs to be modified. Inactivation of a single gene, EXECUTER1, is sufficient to abrogate stress responses of Arabidopsis thaliana caused by the release of singlet oxygen: External conditions under which these stress responses are observed and the amounts of ROS that accumulate in plants exposed to these environmental conditions do not directly cause damages. Instead, seedling lethality and growth inhibition of mature plants result from genetic programs that are activated after the release of singlet oxygen has been perceived by the plant.

Abiotic stress conditions limit the ability of plants to use light energy for photosynthesis, often reducing their growth and productivity and causing photooxidative damages (13). The emergence of these stress symptoms has been closely associated with the enhanced production of several ROS (4, 5). Because different ROS are generated simultaneously, it is difficult to determine the biological activity and mode of action for each of these ROS separately. In order to address this problem, one would need to find conditions under which only one specific ROS is generated at a given time, within a well-defined subcellular compartment, and which also triggers a visible stress response that is easy to score.

Recently, we have isolated the conditional flu mutant of Arabidopsis thaliana that fulfills these requirements (6). The mutant generates singlet oxygen in plastids in a controlled and noninvasive manner. Immediately after the release of singlet oxygen, mature flu plants stop growing, whereas seedlings bleach and die (6). Here, we demonstrate that the two stress responses, growth inhibition and seedling lethality, do not result from physicochemical damage caused by singlet oxygen during oxidative stress but are caused by the activation of a genetically determined stress response program.

We set out to identify such a genetic program by identifying second-site mutations that abrogate either one or both of the two stress responses of the flu mutant. Three different groups of second-site mutations could be distinguished (7) (fig. S1A). One of these groups contained 15 mutants that behaved like wild type when kept under nonpermissive light-dark conditions (7) [group III (fig. S1, B to D)]. Allelism tests and mapping revealed that they were allelic, representing a single locus that was named EXECUTER1. In contrast to wild-type plants but like flu, the executer1/flu double mutant accumulated free protochlorophyllide (Pchlide) in the dark (Fig. 1, A to C, and fig. S1B). After transfer to the light, executer1/flu generated singlet oxygen in amounts similar to those of flu (Fig. 1, F to H) but grew like wild type when kept under nonpermissive light-dark cycles (Fig. 1, A to C). The second stress reaction of flu to the release of singlet oxygen is an inhibition of growth. In flu plants, the growth rate was reduced immediately after the beginning of reillumination (Fig. 1D). The executer1/flu plants, however, grew like wild-type plants (Fig. 1D). Growth inhibition of flu plants was particularly striking when plants were transferred to repeated light-dark cycles, whereas executer1/flu continued to grow like wild-type plants (Fig. 1E). All three plant lines grew equally well under continuous light (fig. S2).

Fig. 1.

A comparison of singlet oxygen production, cell death, and growth inhibition in executer1/flu, flu, and wild type (WT). Etiolated seedlings of flu (B) and executer1/flu (A) overaccumulated similar amounts of free Pchlide, as indicated by the bright red fluorescence, in contrast to etiolated WT seedlings (C). Cell death of flu seedlings (B) and growth inhibition of mature flu plants (=) grown under nonpermissive 16 hours light–8 hours dark conditions were blocked by the executer1 mutation (◼) (D and E). Results from WT control plants (▲) are also shown in (D) and (E). (F to H) Generation of singlet oxygen. WT (F), flu (G), and executer1/flu (H) were grown under continuous light until they were ready to bolt. At this stage, plants were either transferred to the dark for 8 hours or kept under light. Cut leaves were infiltrated with dansyl-2,2,5,5-tetramethyl-2,5-dehydro-1H-pyrrole and subsequently illuminated with white light (100 μmol photons m–2 s–1). Singlet oxygen trapping was measured as relative quenching of DanePy fluorescence (6). In WT controls, no difference in singlet oxygen production between plants exposed to continuous light and a dark-light shift could be found (H), whereas in leaves of flu and executer1/flu generation of singlet oxygen was enhanced in plants that had been kept in the dark before reillumination (⚫) but not in plants exposed to continuous light (◯). ex1, executer1.

As a first step toward the functional characterization of EXECUTER1, we used a map-based cloning strategy to isolate the EXECUTER1 gene. EXECUTER1 was genetically mapped on chromosome IV on a genomic fragment of about 90 kb (Fig. 2A). A contig consisting of 11 cosmid clones that encompassed this chromosomal region was generated (Fig. 2A), and the ability to complement the executer1 mutation was tested (7). Seedlings of the double mutant transformed with the genomic DNA of the cosmid clone 44 that contained a wild-type copy of EXECUTER1 died like flu seedlings when grown under nonpermissive dark-light conditions, whereas seedlings of plants transformed with genomic DNA of other cosmid clones grew like seedlings of the original executer1/flu parental line (Fig. 2B).

Fig. 2.

Identification of the EXECUTER1 gene. (A) Genetic and physical map of the DNA region on chromosome IV of A. thaliana that contains the EXECUTER1 gene (arrow). The region between markers F17M5 and T16L1 is encompassed by 11 cosmid clones that were used to complement the executer1-7/flu double mutant. Cosmid clones 44 and 76, marked by stars, contained genomic DNA fragments that largely overlapped. The DNA fragment of cosmid 44 restored the cell death of seedlings (B) and the growth inhibition of mature plants of the parental flu line (C) when grown under nonpermissive light-dark cycles. Similar results were obtained with cosmid 76 (15).

The second test was done with mature T2 plants transformed with DNA of cosmid clone 44. The original executer1/flu mutant plants were not visibly affected by a shift to light-dark cycles and continued to grow like wild-type plants, but executer1/flu plants complemented with the cosmid 44 DNA were indistinguishable from the flu mutant in that they ceased growth and their leaves developed necrotic lesions (Fig. 2C). Both assays demonstrate that the rapid bleaching of flu seedlings and the inhibition of growth after the release of singlet oxygen are not because of the toxicity of this ROS and do not reflect photooxidative damage and injury, but instead result from the activation of genetically controlled responses that require the activity of the EXECUTER1 gene.

The complementing genomic fragment of cosmid clone 44 has a size of about 14,000 base pairs (bp) and contains three open reading frames (ORFs). One of them, At4g33630, was identified as EXECUTER1 (7) (fig. S3A). The ORF of the EXECUTER1 cDNA predicts a protein of 684 amino acids with a molecular mass of 76,534.9 daltons. It is unrelated to known proteins, but its N-terminal part resembles import signal sequences of nuclear-encoded plastid proteins (fig. S3B). This prediction was confirmed experimentally (fig. S4, A to C). In all higher plants for which expressed sequence tag sequence data were available, including major crop plants, EXECUTER1 homologs could be found (fig. S3C). Thus, EXECUTER1 represents a highly conserved plastid protein that seems to enable higher plants to perceive singlet oxygen as a stress signal that activates a genetically determined stress response program.

The physiological role of the EXECUTER1 gene in wild-type plants was assessed by first isolating executer1 mutant plants that no longer carried copies of the mutated flu gene (7). Wild-type and executer1 plants were then exposed to higher light intensities in the presence of 3-(3, 4-dichlorphenyl)-1,1-dimethylurea (DCMU), which is known to stimulate the release of singlet oxygen (8). The effect of the executer1 mutation on singlet oxygen–mediated cell death was assessed by floating cut leaves on solutions with increasing concentrations of DCMU, ranging from 25 nM to 25 μM, first in the dark for 30 min and then under light (950 μmol photons m–2 s–1) for 24 hours. Inhibition of photosystem II (PSII) by DCMU was similar in wild types and executer1 mutants, as indicated by the concurrent decline of the maximum efficiency of PSII at increasing DCMU concentrations in both leaf samples (Fig. 3A).

Fig. 3.

Suppression of cell death in wild-type plants by the inactivation of EXECUTER1. (A) The effects of different concentrations of DCMU on the maximum efficiency of PSII in WT and executer1-48 plants grown for 3 weeks under 16 hours light–8 hours dark conditions. (B) Selective suppression of cell death by executer1 in DCMU-treated leaves kept at 950 μmol photons m–2 s–1. The progression of cell death was determined by measuring the electrolyte leakage of cut leaves at different lengths of illumination. The ion leakage of leaves boiled for 25 min was taken as 100%. At concentrations of up to 0.25 μM DCMU, cell death was suppressed in executer1, whereas in WT plants it steadily increased with increasing DCMU concentrations. In leaves of the flu mutant kept in the dark for 8 hours before the experiment, the extent of ion leakage after 24 hours of reillumination at 100 μmol photons m–2 s–1 reached about 50% (9).

Cell death and membrane damage in cut leaves were estimated by measuring ion leakage. When leaves of wild type and executer1 were tested in the absence of DCMU, ion leakage reached about 10% of the maximum after boiling the leaves (Fig. 3B). In the presence of 25 nM and 250 nM DCMU, ion leakage in leaves of wild type increased strongly to 40 and 55%, respectively, whereas in leaves of the executer1 mutant ion leakage remained at a low level, similar to that in the water control (Fig. 3B). Only when the concentration of DCMU was further increased to 25 μM were leaves of both wild type and executer1 almost completely damaged, with ion leakage being close to 100% after 24 hours of illumination (Fig. 3B). However, even under these harsher conditions, the increase of cell damage over time in DCMU-treated leaves of wild type occurred much faster than the damage in leaves of executer1 (9).

In most of the second-site mutants of flu that have been identified during our suppressor screen, only one of the two major singlet oxygen–mediated stress reactions was abolished, i.e., either growth inhibition or seedling lethality (fig. S1). In the executer1/flu mutant line, however, both visible stress responses were abrogated. One could argue that a mutated form of the EXECUTER1 protein may act as a scavenger of singlet oxygen and in this way may eliminate detrimental toxic effects of this ROS. However, in such a case, singlet oxygen should no longer be detectable in the executer1/flu mutant line. Furthermore, suppression of stress responses in flu by the executer1 mutation was not confined to allelic lines that synthesized modified EXECUTER1 proteins with single amino acid exchanges that could turn the mutated protein into a scavenger of singlet oxygen but occurred also in executer1 allelic lines that were no longer able to synthesize this protein. Lastly, a mutation that confers an increased scavenging capacity to the executer1/flu mutant should be transmitted as a dominant trait, whereas all executer1 alleles represent recessive mutations.

Inactivation of the EXECUTER1 protein did not only suppress the induction of death in flu seedlings grown under light-dark cycles but also in wild-type plants that were treated with DCMU. DCMU is known to stimulate the release of singlet oxygen in chloroplasts. It inhibits PSII and mimics photoinhibition by binding to the secondary quinone electron acceptor of PSII, QB, and inhibiting forward electron transport. Charge recombination in PSII favors the formation of a chlorophyll (Chl) triplet state that reacts with ground-state triplet oxygen to form 1O2 (10).

Carotenoids of light-harvesting complexes effectively quench triplet Chl and singlet oxygen (11), but the β-carotenes bound to PSII reaction center fail to do so because they are localized too far away from the P680 Chl (12). Thus, continuous 1O2 production seems to be an inherent property of PSII even under low-light conditions. The quenching of this singlet oxygen has been linked to the turnover of the D1 protein that is oxidized by singlet oxygen and apparently serves as a scavenger of this ROS (13, 14). Excess amounts of singlet oxygen that cannot be quenched by the D1 protein and that interact with other targets within the vicinity of PSII may be the trigger that initiates singlet oxygen–mediated stress responses in wild-type plants (14). So far, these stress responses have been attributed to the toxicity of this ROS (1, 2). However, as shown in our present work, the intensity and quality of these responses to light stress may range from necrotic reactions resulting from severe photooxidative damage to the activation of a genetically controlled cell death. At lower DCMU concentrations, ion leakage and membrane damage seem to result from the activation of the EXECUTER1-dependent cell-death program. At higher DCMU concentrations, this genetically controlled cell-death reaction is gradually masked by an EXECUTER1-independent cell-death reaction that seems primarily caused by the toxicity of elevated levels of singlet oxygen. In the past, it was not possible to distinguish between these two cell death reactions, because the genetically determined part remained unnoticed. With the identification of the executer1 mutation, it is now possible to define conditions under which the genetically controlled cell death prevails.

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