Cytosolic ascorbate peroxidase 1 is a central component of the reactive oxygen gene network of Arabidopsis

Abstract

Reactive oxygen species (ROS), such as O2- and H2O2, play a key role in plant metabolism, cellular signaling, and defense. In leaf cells, the chloroplast is considered to be a focal point of ROS metabolism. It is a major producer of O2- and H2O2 during photosynthesis, and it contains a large array of ROS-scavenging mechanisms that have been extensively studied. By contrast, the function of the cytosolic ROS-scavenging mechanisms of leaf cells is largely unknown. In this study, we demonstrate that in the absence of the cytosolic H2O2-scavenging enzyme ascorbate peroxidase 1 (APX1), the entire chloroplastic H2O2-scavenging system of Arabidopsis thaliana collapses, H2O2 levels increase, and protein oxidation occurs. We further identify specific proteins oxidized in APX1-deficient plants and characterize the signaling events that ensue in knockout-Apx1 plants in response to a moderate level of light stress. Using a dominant-negative approach, we demonstrate that heat shock transcription factors play a central role in the early sensing of H2O2 stress in plants. Using knockout plants for the NADPH oxidase D protein (knockout-RbohD), we demonstrate that RbohD might be required for ROS signal amplification during light stress. Our study points to a key role for the cytosol in protecting the chloroplast during light stress and provides evidence for cross-compartment protection of thylakoid and stromal/mitochondrial APXs by cytosolic APX1.

Figure 1.

Deficiency in APX1 Results in H₂O₂ Accumulation and Protein Oxidation in Arabidopsis Leaves Subjected to a Moderate Level of Light Stress. (A) Accumulation of H₂O₂ in wild-type and knockout-Apx1 (KO-Apx1) plants in response to a moderate level of light stress. Compared with wild-type plants, KO-Apx1 plants are shown to accumulate higher levels of H₂O₂ (evident by darker staining of leaves at 90 and 180 min after the application of light stress). (B) Detection of proteins containing carbonyl groups (indicative of protein oxidation) in leaf extracts obtained from wild-type and KO-Apx1 plants by a protein blot assay (top). A protein gel stained with Coomassie blue is used to demonstrate equal loading of proteins (bottom). Compared with wild-type plants, KO-Apx1 plants are shown to accumulate a high level of oxidized proteins. Detection of H₂O₂ and protein oxidation was performed as described in Methods. All experiments were repeated at least three times. Representative results are shown.

Figure 2.

Deficiency in APX1 Results in a Decrease in the Steady State Level of Chloroplastic Proteins, Including Chloroplastic APXs, during a Moderate Level of Light Stress. Detection of thylakoid APX (tylAPX), stromal/mitochondrial APX (s/mAPX), APX1, Cytf (b₆f), and Rubisco (RbcS and RbcL) steady state protein levels by protein blots in leaf extracts from wild-type and knockout-Apx1 (KO-Apx1) plants subjected to a moderate level of light stress. Deficiency in APX1 is shown to result in a decrease in the steady state protein level of the chloroplastic H₂O₂-scavenging enzymes tylAPX and s/mAPX as well as in a decrease in the steady state protein level of the chloroplastic proteins Cytf (b₆f) and RbcS. Light stress experiments and protein blots were performed as described in Methods. All experiments were repeated at least three times. Representative results are shown. APX1 was detected with two different antibodies: one against purified APX1 (top panel), and one against a conserved domain from tylAPX (second panel from top).

Figure 3.

Characterization of Knockout Plants Deficient in Stromal/Mitochondrial APX. (A) Photograph of wild-type (WT1 and WT2 for Columbia and Wassilewskija [Ws], respectively), knockout-Apx1 (KO-Apx1, Ws), and knockout stromal/mitochondrial APX (KO-s/mApx, Columbia) plants grown under controlled conditions. No visible phenotype is shown to be associated with the lack of s/mAPX under these conditions. (B) Protein blot analysis showing the lack of the s/mAPX proteins in KO-s/mApx plants. (C) Measurements of photosynthetic activity (CO₂ gas exchange) in WT1, WT2, KO-Apx1, and KO-s/mApx plants. In contrast with KO-Apx1 (APX1) plants, the photosynthetic activity of KO-s/mApx (s/mAPX) plants is shown not to be suppressed. Conditions for photosynthetic measurements were as follows: CO₂, 400ppm; light intensity, 1000 μmol m⁻² s⁻¹; temperature, 21°C. (D) Accumulation of H₂O₂ in wild-type and knockout-s/mApx (KO-s/mApx) plants in response to a moderate level of light stress. Compared with wild-type or KO-Apx1 plants (Figure 1A), KO-s/mApx are shown not to accumulate high levels of H₂O₂ in response to a moderate level of light stress. (E) Inhibition of root growth in 5-d-old seedlings grown on agar plates in the presence of different concentrations of the superoxide-generating compound paraquat. In contrast with seedlings of KO-Apx1 plants that show high sensitivity to paraquat (top panel), the root growth of KO-s/mApx seedlings is shown to be less sensitive to the paraquat treatment. (F) Detection of thylakoid APX (tylAPX), stromal/mitochondrial APX (s/mAPX), APX1, Cytf (b₆f), and Rubisco (RbcL) steady state protein levels by protein blots in leaf extracts from wild-type and KO-s/mApx plants subjected to a moderate level of light stress. Compared with wild-type or KO-Apx1 plants (Figure 2), the steady state protein levels of tylAPX and Cytf (b₆f) are shown not to be suppressed in response to a moderate level of light stress. Detection of H₂O₂ in leaves, protein blots, and measurements of photosynthetic activity were performed as described in Methods. No differences were found between the sensitivity of Ws and Columbia cultivars to the treatments shown in Figures 1 to 3 (data not shown). All experiments were repeated at least three times. Representative results are shown.

Figure 4.

Microarray Analysis of Knockout-Apx1 Plants Subjected to a Moderate Level of Light Stress. (A) RNA gel blots showing an increase in the steady state level of transcripts encoding APX1 (Apx1), the zinc-finger protein Zat12 (Zat12), HSF21, NADPH oxidase D (RbohD), catalase (Cat1), iron superoxide dismutase (FeSOD), and heat shock protein 70 (Hsp70) in plants subjected to a moderate level of light stress. RNA gel blots showing the transcript level of transcripts encoding the large subunit of Rubisco (RbcL) as well as a photograph of total RNA are shown to demonstrate equal loading of RNA. Experiments were repeated at least six times. Representative results are shown. (B) Changes in steady state transcript level of transcripts encoding all members of the APX gene family (APX1-7, tylAPX, and s/mAPX) in wild-type (control) and knockout-Apx1 (knockout) plants in response to a moderate level of light stress. (C) Changes in steady state transcript level of transcripts encoding four HSFs (HSF21, HSF5, HSF4, and a putative HSF [pHSF]) all with an increase in expression of more than twofold during the light stress treatment. (D) Changes in steady state level of transcripts encoding all members of the NADPH oxidase gene family (RbohA to J) in control and knockout-Apx1 (knockout) plants in response to a moderate level of light stress. Time-course microarray analsysis ([B] to [D]) was repeated twice with similar results, and representative results are shown. RNA gel blots and microarray analysis were performed as described in Methods. Visualization of transcript levels in (B), (C), and (D) was performed with ArrayAssist. Statistical analysis of microarray results was performed as described in Methods. Transcripts that were significantly elevated in KO-Apx1 compared with wild-type plants are shown in Table 2 and in the supplemental data online.

Figure 5.

Functional Analysis of HSF21 and RbohD in Transgenic and Knockout Arabidopsis Plants. (A) A scheme describing the structure of the HSF21 dominant-negative construct expressed in transgenic plants. (B) RNA gel blots showing the steady state transcript level of Apx1, Zat12, HSF21, and HSP70 in wild-type plants and transgenic plants expressing the dominant-negative construct for HSF21 (DN-HSF21) plants in response to a moderate level of light stress. (C) RNA gel blots showing the steady state transcript level of Apx1 and Cat1 in wild-type plants and knockout plants lacking RbohD in response to a moderate level of light stress. (D) A model showing the putative signal transduction pathway that controls the expression of Apx1 during a moderate level of light stress in Arabidopsis. Construction of transgenic plants, light stress experiments, and RNA gel blots were performed as described in Methods.