SHORT-ROOT Deficiency Alleviates the Cell Death Phenotype of the Arabidopsis catalase2 Mutant under Photorespiration-Promoting Conditions

Abstract

Hydrogen peroxide (H2O2) can act as a signaling molecule that influences various aspects of plant growth and development, including stress signaling and cell death. To analyze molecular mechanisms that regulate the response to increased H2O2 levels in plant cells, we focused on the photorespiration-dependent peroxisomal H2O2 production in Arabidopsis thaliana mutants lacking CATALASE2 (CAT2) activity (cat2-2). By screening for second-site mutations that attenuate the PSII maximum efficiency (Fv'/Fm') decrease and lesion formation linked to the cat2-2 phenotype, we discovered that a mutation in SHORT-ROOT (SHR) rescued the cell death phenotype of cat2-2 plants under photorespiration-promoting conditions. SHR deficiency attenuated H2O2-dependent gene expression, oxidation of the glutathione pool, and ascorbate depletion in a cat2-2 genetic background upon exposure to photorespiratory stress. Decreased glycolate oxidase and catalase activities together with accumulation of glycolate further implied that SHR deficiency impacts the cellular redox homeostasis by limiting peroxisomal H2O2 production. The photorespiratory phenotype of cat2-2 mutants did not depend on the SHR functional interactor SCARECROW and the sugar signaling component ABSCISIC ACID INSENSITIVE4, despite the requirement for exogenous sucrose for cell death attenuation in cat2-2 shr-6 double mutants. Our findings reveal a link between SHR and photorespiratory H2O2 production that has implications for the integration of developmental and stress responses.

Figure 1.

Strategy to Identify Second-Site Mutations That Alleviate the Photorespiratory Phenotype of cat2-2 Mutants. (A) PSII maximum efficiency (F_v'/F_m') decrease and cell death progression in 3-week-old plants exposed to photorespiration-promoting conditions (air-tight sealing of Petri dishes with Parafilm and transfer to continuous light). Data points represent F_v'/F_m' means from three biological replicates ±se for cat2-2 (red squares) and Col-0 (blue dots). Representative bright-field and color-coded images of F_v'/F_m' taken in parallel on days 0, 2, and 7 of the treatment are shown on the right. The F_v'/F_m' parameter was visualized with the use of a color scale ranging from black (0.0) to white (1.0) with red, orange, yellow, green, blue, and violet in between. Bar = 10 mm. (B) Schematic depiction of the forward genetic screen used to identify revertants of the photorespiratory cat2-2 phenotype. An EMS-mutagenized cat2-2 population (113,000 M2 plants) was screened under photorespiration-promoting conditions (arrows). Plants that showed lower rates of F_v'/F_m' decrease and attenuated cell death relative to the parental cat2-2 plants (red circle) were selected for further characterization.

Figure 2.

Characterization of Line 378.3. (A) Color-coded images of PSII maximum efficiency (F_v'/F_m') of 3-week-old Col-0, 378.3, and cat2-2 plants exposed to photorespiration-promoting conditions (restricted gas exchange and transfer to continuous light) for 24 h. Bar = 20 mm. (B) F_v'/F_m' decrease during the exposure to photorespiration-promoting conditions in Col-0, 378.3, and cat2-2 plants. Data points represent means of three biological replicates ±se. (C) Representative bright-field images of Col-0, cat2-2, and 378.3 before (top) and after (bottom) 7 d of exposure to photorespiration-promoting conditions. Bar = 5 mm. (D) Short-root phenotype of 378.3. Plants were grown vertically for 2 weeks on MS medium supplemented with 1% (w/v) sucrose under long-day (LD) conditions (16 h/8 h day/night) at 100 μmol m⁻² s⁻¹. Bar = 10 mm. (E) Gene model of SHR with positions of mutant alleles used in this study. The identified mutation site is marked in red. (F) Color-coded F_v'/F_m' images of 3-week-old Col-0, cat2-2, shr-6, and cat2-2 shr-6 plants exposed to photorespiration-promoting conditions for 48 h. Colors are as in Figure 1A. Bar = 10 mm. (G) Representative bright-field image of 3-week-old Col-0, cat2-2, shr-6, and cat2-2 shr-6 plants after 7 d of exposure to photorespiration-promoting conditions. Bar = 10 mm.

Figure 3.

Effect of SHR Deficiency on the Photorespiratory Phenotype of cat2-2 in Soil Conditions. Three-week-old plants grown in a high CO₂ (3000 μL L⁻¹) atmosphere at 100 μmol m⁻² s⁻¹ were transferred to ambient air and exposed to continuous high light (1000 μmol m⁻² s⁻¹). Pairs of images show representative bright-field images (left panels) of 3-week-old plants, together with color-coded F_v'/F_m' images (right panels) in the beginning (t = 0) and 24 h after the onset of the exposure to photorespiration-promoting conditions (t = 24 h). Colors are as in Figure 1A. Bar = 10 mm.

Figure 4.

Influence of Exogenous Sucrose on the Tolerance of SHR-Deficient Plants to the Photorespiration-Promoting Conditions. Plants were germinated and grown for 18 d on a nylon mesh placed on a 1× MS sucrose-supplemented medium (1% w/v) and subsequently transferred either to a medium with a similar sucrose concentration or to a sucrose-free medium. Three days after the transfer, plants were exposed to photorespiration-promoting conditions. (A) and (B) Representative bright-field images of plants exposed to photorespiration-promoting conditions for 7 d on medium containing 1% (w/v) sucrose (A) and on medium with no sucrose (B). Bar = 20 mm. (C) and (D) Photorespiration-triggered F_v'/F_m' changes over time in Col-0 (closed circles), cat2-2 (closed squares), shr-6 (open circles), and cat2-2 shr-6 (open squares) plants. Data points represent means of three biological replicates ±se.

Figure 5.

Cellular Redox Alterations in the Absence of Functional SHR. (A) Glutathione and ascorbate content in 3-week-old Col-0, cat-2, shr-6, and cat2-2 shr-6 plants before (Control) and after (Photorespiratory stress) 24 h of exposure to photorespiration-promoting conditions. White bars indicate GSH or AA content, and black bars indicate GSSG or dehydroascorbate (DHA) content averaged from four biological replicates ±se. Asterisk indicates significant difference of all mutant genotypes relative to Col-0 at P < 0.05, and + indicates significant difference between cat2-2 and cat2-2 shr-6 at P < 0.05 for total glutathione (GSH + GSSG) and ascorbate (AA + DHA). (B) Comparison of expression patterns of a subset of ROS-responsive genes that are found in the transcriptome signatures of Col-0, cat2-2, shr-6, and cat2-2 shr-6 plants upon exposure to photorespiration-promoting conditions. The following experiments were used in the analysis: exposure of seedlings to ozone for 6 h (6 h O₃, E-MEXP-342); treatment of seedlings with 50 µM antimycin A for 3 h (3 h AA, GSE41136); reillumination of the conditional flu mutant for 2 h after a dark acclimation (2 h flu, GSE10812); treatment of seedlings with 10 mM H₂O₂ for 24 h (24 h H₂O₂, unpublished data), and treatment of seedlings with oligomycin for 4 h (4 h OM, GSE38965). ROS marker genes published previously (Gadjev et al., 2006) are shown alongside the heat map.

Figure 6.

Effect of SHR Deficiency on Photosynthetic CO₂ Assimilation and Photorespiratory Metabolism. (A) Impact of SHR deficiency on the activities of the peroxisomal photorespiratory enzymes glycolate oxidase and catalase. Enzyme activities were extracted from 2-week-old rosettes of Col-0 and shr-6 plants and are expressed as a percentage of Col-0 values. Bars represent means of three biological replicates ±se. Asterisks (*P < 0.05, **P < 0.01, and ***P < 0.001) indicate significance according to Student’s t test. (B) CO₂ assimilation of 3-week-old in vitro-grown Col-0, cat2-2, shr-6, and cat2-2 shr-6 plants. Values represent means of eight biological replicates ±se. Asterisks (*P < 0.05, **P < 0.01, and ***P < 0.001) show significant differences relative to Col-0 identified by a one-way ANOVA followed by Dunnett’s multiple comparisons post hoc test. (C) Levels of glycolate, glycine, and serine under control and photorespiration-promoting conditions (24 h) in 3-week-old in vitro-grown plants. Glycine and serine levels were quantified based on the abundance of 3-trimethylsilyl (TMS) and 2-TMS derivatives, respectively. Values represent means of five biological replicates ±se. Data were analyzed with a two-way ANOVA with treatment (photorespiration-promoting conditions versus control conditions) and genotype as main factors, followed by a Tukey’s multiple comparison post hoc test. Asterisks (*P < 0.05, **P < 0.01, and ***P < 0.001) show significant differences to Col-0 within the respective conditions.

Figure 7.

Influence of SCR on the Photorespiratory Phenotype of cat2-2. (A) and (B) The photorespiratory phenotypes of cat2-2 and cat2-2 scr-3. Representative bright-field images illustrating cell death lesion formation in cat2-2 and cat2-2 scr-3 plants 1 week following the onset of exposure to photorespiration-promoting conditions (A), together with changes of F_v'/F_m' over time (B). Data points represent means of three biological replicates ±se. Bar = 10 mm. (C) and (D) Performance of independent scr alleles (scr-3 and scr-1) during exposure to photorespiration-promoting conditions. Representative bright-field images illustrating cell death lesion formation in cat2-2 and cat2-2 scr-3 plants 1 week after the onset of treatment (C), together with changes of F_v'/F_m' over time (D). Data points represent means of three biological replicates ±se. Bar = 10 mm.