Catalase and NO CATALASE ACTIVITY1 promote autophagy-dependent cell death in Arabidopsis

Abstract

Programmed cell death often depends on generation of reactive oxygen species, which can be detoxified by antioxidative enzymes, including catalases. We previously isolated catalase-deficient mutants (cat2) in a screen for resistance to hydroxyurea-induced cell death. Here, we identify an Arabidopsis thaliana hydroxyurea-resistant autophagy mutant, atg2, which also shows reduced sensitivity to cell death triggered by the bacterial effector avrRpm1. To test if catalase deficiency likewise affected both hydroxyurea and avrRpm1 sensitivity, we selected mutants with extremely low catalase activities and showed that they carried mutations in a gene that we named NO CATALASE ACTIVITY1 (NCA1). nca1 mutants showed severely reduced activities of all three catalase isoforms in Arabidopsis, and loss of NCA1 function led to strong suppression of RPM1-triggered cell death. Basal and starvation-induced autophagy appeared normal in the nca1 and cat2 mutants. By contrast, autophagic degradation induced by avrRpm1 challenge was compromised, indicating that catalase acted upstream of immunity-triggered autophagy. The direct interaction of catalase with reactive oxygen species could allow catalase to act as a molecular link between reactive oxygen species and the promotion of autophagy-dependent cell death.

Figure 1.

Hydroxyurea Resistance of Autophagy and Immunity Mutants. Seedlings germinated in the presence of 3 mM hydroxyurea. Unfolded green cotyledons indicate resistance to cell death induced by hydroxyurea. Ws, Wassilewskija. [See online article for color version of this figure.]

Figure 2.

Characterization of the nca1 Mutant Phenotype. (A) to (C) Wild-type (wt), nca1, and cat2 seedlings grown on one-half-strength MS supplemented with 3 mM hydroxyurea (A), one-half-strength MS at ambient CO₂ level (B), and one-half-strength MS at high CO₂ level (C). (D) to (G) Material from 12-d-old seedlings. Student's t test (two means) or one-way analysis of variance (ANOVA) and Dunnett's multiple comparison test was used for P value calculations. (D) Comparison of catalase activity levels. All mutants differed significantly from the wild type (P < 0.0001). Both nca1 mutants differed significantly from cat2-2 (*P < 0.05 and **P < 0.01). (E) Comparison of catalase transcript levels. White, Col-0 wild type; light gray, cat2-2; dark gray, nca1-1. nd, not detected. Only CAT1 expression in the nca1 background was significantly different from the wild type (*P < 0.05). Error bars indicate se. (F) Comparison of catalase protein levels as determined by immunoblotting. Three biological replicates are shown for each genotype. (G) Zymogram showing activities of the different catalase isoforms. Three biological replicates were used, and a representative gel is shown. (H) NCA1 gene structure. Exons are shown as boxes. Untranslated regions are shown in gray. Mutations are indicated by triangles. See also Supplemental Figures 1 and 2 and Supplemental Tables 2 and 3 online. [See online article for color version of this figure.]

Figure 3.

NCA1 and CAT2 Protein Localization and Mutant Phenotypes. (A) Localization of the fusion proteins indicated in the top left corner in the genetic backgrounds shown in the bottom left corner. The NCA1-eYFP and CAT2-eYFP fusion proteins were driven by their native promoters, and PTS1-GFP was expressed from the cauliflower mosaic virus 35S promoter. Bars = 10 µm. (B) Four-week-old plants grown in short days on soil. (C) Response to photorespiration-promoting growth conditions. The three leftmost panels show maximum quantum efficiency of photosystem II (Fv/Fm) on a heat map scale where red represents the lowest values. The experiment was performed in six replicates, and a representative plate is shown. (D) Expression of oxidative stress marker genes. Fold changes with respect to the untreated Col-0 sample are shown. Gray bars indicate expression prior to photorespiratory stress treatment. Blue bars show expression after 24 h of treatment. Three replicates were used, and error bars indicate se. Two-way ANOVA and Dunnett's multiple comparison test were used for calculating P values indicating the probability that an expression value was identical to the Col-0 value at the given time point (*P < 0.05 and **P < 0.01). (E) Log₂ fold changes in protein abundances as determined by iTRAQ proteomics are plotted. Fold change values are based on three biological and four technical replicates. All quantified proteins were included in the plot. wt, wild type.

Figure 4.

Suppression of Pst DC3000 avrRpm1–Mediated Cell Death in Catalase- and Autophagy-Deficient Mutants. Ion leakage measurements of the wild type (Col-0), rpm1-3, cat2-2, nca1-1, and atg2-1 after inoculation with a (OD₆₀₀ = 0.2) culture of avirulent Pst DC3000 expressing avrRpm1. (A) Four-week-old short-day-grown plants. (B) Four-week-old short-day-grown plants shifted to long days 3 d prior to infection. Mean and se were calculated from four leaf discs per treatment, with four replicates per experiment.

Figure 5.

Pst DC3000 avrRpm1 Resistance Characteristics of Catalase and Autophagy-Deficient Mutants. Growth of Pst DC3000 avrRpm1 in leaves of 5-week-old plants at 0 and 2 d after infiltration with 1 × 10⁵ CFU mL⁻¹. Log-transformed values are means ± sd (n = 3). Two-way ANOVA and Dunnett's multiple comparison test were used for calculating P values indicating the probability that a CFU value was identical to the Col-0 value at the given time point (****P < 0.0001).

Figure 6.

Catalase and Autophagy Interactions. (A) Prolonged darkness assay. Left panel, prior to dark treatment; middle panel, after 10 d in darkness; right panel, after 10 d in darkness and 10 d of recovery in light. wt, the wild type. (B) Detached leaves incubated on moist filter paper for 3 d. (C) Catalase activities of immune effector and autophagy mutants. Error bars show se, and three replicates were used. P values indicating the probabilities that the activity levels were identical to the wild-type level were calculated using a one-way ANOVA followed by Dunnett's test for multiple comparisons. (*P < 0.05 and ****P < 0.0001). (D) NBR1 immunoblot on extracts from plants prior to and 7 h after inoculation (hpi) with Pst DC3000 avrRpm1. Arrowhead indicates the position of the NBR1 band. The bottom panel shows the Ponceau-stained membrane. (E) Ubiquitin immunoblot on extracts from plants prior to and 7 h after inoculation with Pst DC3000 avrRpm1. The top panel shows ubiquitinylated proteins. The top and bottom arrowheads indicate sizes of 55 and 60 kD, respectively. The bottom panel shows the Ponceau-stained membrane. [See online article for color version of this figure.]

Figure 7.

Tentative Model for Catalase Integration of PCD Signals. In our screen for hydroxyurea resistance, the death of wild-type plants at germination coincides with ROS generation as a byproduct of β-oxidation of fatty acids during seed storage oil mobilization. These ROS activate catalase, which interacts directly with hydroxyurea to produce an autophagy-dependent PCD response. AvrRpm1 challenge causes a HR comprising an oxidative burst. The ROS produced activate catalase, which reacts with unknown compounds to generate an autophagy-dependent PCD response. Resistance to Pst DC3000 avrRpm1 is induced independently of the PCD response mediated by catalase and autophagy proteins. Cell death inducers (red) and genetic components (blue) investigated in this study are highlighted.