Leaf mitochondria modulate whole cell redox homeostasis, set antioxidant capacity, and determine stress resistance through altered signaling and diurnal regulation

Abstract

To explore the role of plant mitochondria in the regulation of cellular redox homeostasis and stress resistance, we exploited a Nicotiana sylvestris mitochondrial mutant. The cytoplasmic male-sterile mutant (CMSII) is impaired in complex I function and displays enhanced nonphosphorylating rotenone-insensitive [NAD(P)H dehydrogenases] and cyanide-insensitive (alternative oxidase) respiration. Loss of complex I function is not associated with increased oxidative stress, as shown by decreased leaf H(2)O(2) and the maintenance of glutathione and ascorbate content and redox state. However, the expression and activity of several antioxidant enzymes are modified in CMSII. In particular, diurnal patterns of alternative oxidase expression are lost, the relative importance of the different catalase isoforms is modified, and the transcripts, protein, and activity of cytosolic ascorbate peroxidase are enhanced markedly. Thus, loss of complex I function reveals effective antioxidant crosstalk and acclimation between the mitochondria and other organelles to maintain whole cell redox balance. This reorchestration of the cellular antioxidative system is associated with higher tolerance to ozone and Tobacco mosaic virus.

Figure 1.

N. sylvestris Wild-Type (WT) and CMSII Plants at the Rosette Stage. A 3-month-old wild-type plant is shown next to a 4-month-old CMSII mutant.

Figure 2.

In Situ Detection of Leaf H₂O₂ and O₂^.−. DAB and NBT stains were used to detect H₂O₂ and O₂^.−, respectively. The samples shown are representative of three independent experiments (six samples per experiment). In all cases, wild-type (WT) and CMSII leaf discs were harvested at the middle of the light period and stained immediately. No specific coloration was observed in controls with ascorbic acid for the DAB staining and with SOD for the NBT staining (data not shown).

Figure 3.

Quantitative Comparison of Diurnal Changes in H₂O₂ Accumulation in CMSII and Wild-Type Leaves. Leaf samples were harvested during the day/night cycle at the times indicated. The white horizontal bar indicates the light period, and the black horizontal bar indicates the dark period. Open columns indicate the wild type, and closed columns indicate CMSII. Values are means ± se from five independent experiments. The difference between wild-type and CMSII leaf H₂O₂ contents is significant in both the light (P < 0.01) and the dark (P < 0.05). FW, fresh weight.

Figure 4.

Leaf Ascorbate and Glutathione Contents and Redox States throughout the Day/Night Cycle. The white horizontal bar indicates the light period, and the black horizontal bar indicates the dark period. Open columns indicate the wild type, and closed columns indicate CMSII. Values are means ± se from two independent experiments. FW, fresh weight. (A) Ascorbate content. The increase in ascorbate content between 1 and 5 h of light is significant in CMSII. (B) Percent reduced ascorbate. (C) Glutathione content. After 5 h of light, total glutathione content was significantly lower in CMSII than in the wild type. (D) Percent reduced glutathione.

Figure 5.

Effect of the CMSII Mutation on the Abundance and Diurnal Variation of Antioxidant Transcripts. The white horizontal bar indicates the light period, and the black horizontal bar indicates the dark period. Open columns indicate the wild type, and closed columns indicate CMSII. Ten micrograms of total RNA from each sample was subjected to RNA gel blot analysis on the same blot. WT, wild type. (A) Autoradiograms were obtained using AOX, MnSOD, FeSOD, CAT1, CAT2, CAT3, chlAPX, cAPX, and chlGR probes with 18S rRNA as a standard. (B) Relative abundance of antioxidant gene transcripts in wild-type and CMSII leaves. RNA gel blot signals were scanned with the Scanalytics MasterScan software program. Results are expressed as the values of the following ratio: integrated density of the signal/integrated density of the 18S rRNA signal (arbitrary units). Values are means ± se from three independent experiments. The abundance of the following transcripts was significantly different in CMSII compared with the wild type: AOX, MnSOD, cAPX (in the light and the dark), FeSOD (in the dark), CAT2, and CAT3 (at 12 h of light).

Figure 6.

Effect of the CMSII Mutation on the Abundance of AOX, CAT, and cAPX Proteins throughout the Day/Night Cycle. The white horizontal bar indicates the light period, and the black horizontal bar indicates the dark period. Protein gel blot analysis was performed on total proteins (10 μg) extracted from wild-type (W) and CMSII (C) leaf samples. Results are representative of three independent experiments. The bands correspond to the following approximate molecular masses: 35 kD (AOX), 30 kD (cAPX), and 50 kD (CAT). The large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RBCL; 55 kD) was used as a control. (A) AOX and RBCL proteins visualized by the chemiluminescence method. (B) cAPX, CAT, and RBCL proteins visualized by the peroxidase reaction.

Figure 7.

CAT, GR, and APX Activities in CMSII and Wild-Type Plants throughout the Day/Night Cycle. The white horizontal bar indicates the light period, and the black horizontal bar indicates the dark period. Open columns indicate the wild type, and closed columns indicate CMSII. Values are means ± se from three to six independent experiments. (A) Total CAT activity (μmol H₂O₂·min⁻¹·mg⁻¹ protein). (B) Total GR activity (μmol NADPH oxidized·min⁻¹·mg⁻¹ protein). (C) Total APX activity (nmol ascorbate oxidized·min⁻¹·mg⁻¹ protein). (D) Ascorbate depletion-insensitive APX (nonchloroplastic APX) activity (nmol ascorbate oxidized·min⁻¹·mg⁻¹ protein).

Figure 8.

The CMSII Mutant Is More Resistant Than the Wild Type to Ozone and TMV. (A) Differences in symptoms in wild-type (WT) and CMSII (CMS) plants subjected to ozone (1 treatment = 1000 parts per billion, 4 h per day). (B) Relative abundance and size of necrotic lesions developed on N. sylvestris wild-type and CMSII leaves after two or three ozone treatments. Only the wild-type leaves show extensive damage, although arrows indicate small necrotic points on the CMSII leaf after the third treatment. (C) Induction of AOX and cAPX transcripts by ozone exposure. Ozone treatments were as in (A) and (B). 1T indicates one ozone treatment, and 3T indicates three ozone treatments. Samples were harvested 1 day after the third ozone exposure from the middle part of three successive leaves: the leaf immediately younger than the most damaged leaf (a); the most damaged leaf (b); and the leaf immediately older than the most damaged leaf (c). Ten micrograms of total RNA from each sample was subjected to RNA gel blot analysis on the same blot. AOX and cAPX were as in Figure 5. 18S rRNA was used as a standard. (D) Hypersensitive response induced by the U1 strain of TMV in N. sylvestris × N. tabacum hybrid plants carrying the N gene of resistance with either the wild-type [WT(N)] or the mutant [CMS(N)] cytoplasm. All of the developed leaves of plants at the pre-floral-bud stage were inoculated with virus (1 μg/mL). The middle-rank leaf (5; see [F]) is shown, photographed 7 days after inoculation. (E) Immunodetection of the TMV coat protein (CP) in inoculated WT(N) and CMS(N) plants. At each time point, protein was extracted from 10 lesions on inoculated leaf number 3. RBCL, large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. (F) Average TMV-induced lesion diameter in consecutive leaves from bottom (bottom leaf) to top (top leaf). At all ranks, lesion size was reduced significantly in CMS(N) compared with WT(N). Results are representative of two independent experiments, with at least 50 lesions measured per experiment.

Figure 9.

Whole Cell Redox Signaling by Leaf Mitochondria: Hypothesis. The scheme proposes that alterations in redox status in the mitochondria (Mit), possibly manifested as increased AOS, are detected by as yet unidentified sensors that may involve thiol-disulfide exchange (SH/SS). These mitochondrion-specific signals are relayed to the nucleus by transducers (dotted line), leading to the effects observed in CMSII. These effects are as follows: increased abundance of transcripts of antioxidative enzymes that process locally generated AOS species (MnSOD) or decrease their rate of production (AOX) in mitochondria [1]; and whole cell induction of transcripts that encode antioxidative enzymes located in compartments (CAT1, CAT3, cAPX, and FeSOD) external to the mitochondria [2]. Together, these acclimatory responses trigger decreased whole cell reactive oxygen species (ROS) accumulation and enhance stress resistance [3].