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. 2013 Oct;25(10):4195-208.
doi: 10.1105/tpc.113.118018. Epub 2013 Oct 22.

The Importance of Cardiolipin Synthase for Mitochondrial Ultrastructure, Respiratory Function, Plant Development, and Stress Responses in Arabidopsis

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The Importance of Cardiolipin Synthase for Mitochondrial Ultrastructure, Respiratory Function, Plant Development, and Stress Responses in Arabidopsis

Bernard Pineau et al. Plant Cell. .
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Abstract

Cardiolipin (CL) is the signature phospholipid of the mitochondrial inner membrane. In animals and yeast (Saccharomyces cerevisiae), CL depletion affects the stability of respiratory supercomplexes and is thus crucial to the energy metabolism of obligate aerobes. In eukaryotes, the last step of CL synthesis is catalyzed by CARDIOLIPIN SYNTHASE (CLS), encoded by a single-copy gene. Here, we characterize a cls mutant in Arabidopsis thaliana, which is devoid of CL. In contrast to yeast cls, where development is little affected, Arabidopsis cls seedlings are slow developing under short-day conditions in vitro and die if they are transferred to long-day (LD) conditions. However, when transferred to soil under LD conditions under low light, cls plants can reach the flowering stage, but they are not fertile. The cls mitochondria display abnormal ultrastructure and reduced content of respiratory complex I/complex III supercomplexes. The marked accumulation of tricarboxylic acid cycle derivatives and amino acids demonstrates mitochondrial dysfunction. Mitochondrial and chloroplastic antioxidant transcripts are overexpressed in cls leaves, and cls protoplasts are more sensitive to programmed cell death effectors, UV light, and heat shock. Our results show that CLS is crucial for correct mitochondrial function and development in Arabidopsis under both optimal and stress conditions.

Figures

Figure 1.
Figure 1.
Characterization of T-DNA Insertions in the Arabidopsis CLS Gene. (A) Positions of the T-DNA insertions for the three cls mutants we identified. (B) PCR products of genomic DNA extracted from Col-0 as well as SALK_49835 (cls1), SALK_4984 (cls2), and SALK_2263 (cls3) homozygous mutant seedlings using primers surrounding the T-DNA insertions (cls.5 and cls.3). (C) PCR products of genomic DNA extracted from SALK_49835 (cls1), SALK_4984 (cls2), and SALK_2263 (cls3) mutant seedlings using primers surrounding the T-DNA insertions (cls.5 and cls.3) and T-DNA primer LBb1. (D) Estimation of transcript levels of CLS was done by RT-PCR using cDNA obtained from Col-0 and SALK_49835 (cls1), SALK_4984 (cls2), and SALK_2263 (cls3) mutant seedlings and ACTIN2 (ACT2) as the reference gene. For each experiment, roughly 50 seedlings were used for DNA or RNA extraction.
Figure 2.
Figure 2.
Phenotypes of cls Mutant Plants. (A) to (C) Seedlings of SALK_49835 (cls1; [A]) SALK_4984 (cls2; [B]) and SALK_2263 (cls3; [C]) lines grown in vitro under continuous light and under a very low light intensity of 30 μmol m−2 s−1. (D) Seedlings of the SALK_49835 line grown for 4 weeks under SD (80 μmol m−2 s−1) conditions. (E) and (F) cls1 plantlets grown for 3 weeks under SD (E) or transferred to LD (80 μmol m−2 s−1) conditions for 1 additional week (F). (G) Adult Col-0 and cls1 plants transferred to soil in LD conditions and grown under low illumination (∼50 μmol m−2 s−1). Insets show siliques (arrows) of Col-0 and the cls1 mutant.
Figure 3.
Figure 3.
Lipid Quantification of cls1 Mutant Plants. Seedlings were grown in vitro in continuous light at very low intensity (30 μmol m−2 s−1) for 3 weeks and transferred to soil for 2 additional weeks. (A) Quantification of CL and total polar lipids in Col-0 and cls1 mutants. FW, Fresh weight. (B) Variation of lipid content (%) between cls1 and Col-0 seedlings. PA, Phosphatidic acid; PC, phosphatidylcholine; PI, phosphatidylinositol; PS, phosphatidylserine. All measurements were done in duplicate on three independent samples. Error bars represent se.
Figure 4.
Figure 4.
Confocal Microscopy of Col-0 and cls1 Cells and Flow Cytometry of NAO Intensity in Protoplasts. (A) to (F) Leaves. In Col-0 mesophyll (A) and epidermal (B) cells, the Nernst potential-sensitive dyes MitoTracker Red (red) and DiOC6(5) (green) revealed punctate (arrowhead) or fusiform mitochondria ∼1 µm in length; chloroplasts are coded blue. Occasional clusters of mitochondria were also observed (arrows). In cls1 mesophyll (D) and epidermal (E) cells, both very small punctate mitochondria (arrowhead) and large reticulate mitochondria (arrows) were labeled with MitoTracker Red, and immobile globular structures were observed with DiOC6(5). The putatively potential-insensitive mitochondrial dye NAO (green) revealed a similar range of mitochondrial forms in Col-0 mesophyll ([C]; chloroplasts coded red); in cls1, punctate mitochondria were observed in epidermal cells ([F]; arrowhead), and large globular mitochondria ([F]; arrows) were present in stomata. The lipidic osteole was also labeled by NAO. Bars = 5 µm. (G) to (N) Protoplasts isolated from young in vitro–grown seedlings. In Col-0 protoplasts, MitoTracker Red revealed typical small punctate mitochondria with high contrast ([G] and [H]). Both small punctate mitochondria and large organelles (arrows) were labeled in cls1 protoplasts ([I] and [J]). Chloroplasts are coded in blue in (G) and (I), whereas (H) and (J) are single-color micrographs. NAO (green) labeled tiny punctate mitochondria and larger organelles in Col-0 protoplasts ([K] and [L]); in cls1 protoplasts ([M] and [N]), both punctate mitochondria and a whole range of structures, such as cytoplasmic membranes and the chloroplast periphery (arrow in [N]), were labeled by NAO. Bars = 5 µm. (O) Flow cytometry of Col-0 and cls1 protoplasts to quantify NAO uptake. Histograms indicate the relative intensity of NAO charge in protoplasts after 5 min of staining with 100 nM NAO and counterstaining with PI. The capacity to label with NAO was measured after storing the protoplasts at room temperature 2 h (left histogram) and 4 h (right histogram). The geometric mean of NAO fluorescence was determined for Col-0 (shaded histogram) and cls1 protoplasts (unshaded histogram) using analytical cytometry to exclude PI-permeant protoplasts. For each histogram, the geometric mean was obtained and then reexpressed in arbitrary units (au), assigning 100 to the mean of the wild type at 2 h. Unstained protoplasts were <3 arbitrary units.
Figure 5.
Figure 5.
Electron Micrographs of Col-0 and cls1 Leaf Mitochondria and Plastids. (A) and (B) Col-0 mitochondria (m) and plastid (pl). (C) cls1 mitochondrion with similar size and internal structure to Col-0 mitochondria. (D) Giant cls1 mitochondrion. (E) to (G) Irregular cls1 mitochondria with poorly developed cristae. (H) Giant cls1 mitochondrion and plastid. The same magnification was used in all figures except in (H), with one-third less magnification. Bars = 500 nm.
Figure 6.
Figure 6.
Consequences of the Absence of CLS Expression for the Composition of the Mitochondrial Respiratory Chain. CI and CI/CIII were separated by blue native electrophoresis of protein extracts, followed by either NADH/NBT staining or immunodetection using antisera directed against the CI NAD9 subunit and the COXII subunit of complex IV. Compared with Col-0 plants, the accumulation of CI and CI/CIII is impaired in the cls1 mutant. (A) Proteins from dodecyl maltoside (DDM)–solubilized membranes. (B) Proteins from membranes solubilized by digitonin, which preserves CI/CIII association (Pineau et al., 2008).
Figure 7.
Figure 7.
Changes in Antioxidant Gene Expression Levels in the cls1 Mutant. The accumulation of transcripts related to oxidative stress in mitochondria (AOX1a and NDB2), plastids (BAP1 and FER1), cytosols (APX1 and APX2), or catalases (CAT1, CAT2, and CAT3) was quantified by real-time PCR in Col-0 and cls1 seedlings. Results shown are mean relative mRNA levels ± se from at least three independent samples. Asterisks indicate significant differences between cls1 and Col-0 values.
Figure 8.
Figure 8.
Leaf Metabolic Content Is Altered in the cls1 Mutant. TCA-derived metabolites, amino acids, and total sugars are from GC-TOF-MS analyses. Results shown are mean metabolite contents according to fresh weight (arbitrary units) ± se from three independent samples. Asterisks indicate significant differences between cls1 and Col-0 values. TCA-fumarate indicates all TCA-derived organic acids except fumarate, by far the most abundant organic acid in Arabidopsis.
Figure 9.
Figure 9.
Effects of UV-C Light and Heat Shock on the Extent of Cell Death in cls1 and Col-0 Protoplasts. (A) Protoplasts subjected to UV-C irradiation (10 kJ m−2). (B) Protoplasts subjected to heat shock (HS; 55°C, 10 min). In both cases, the percentage of dead protoplasts was calculated 0, 2, 4, and 6 h after treatment using Evans Blue (0.04%). Data represent means ± se of four independent experiments, with a minimum of 100 protoplasts being counted per sample.

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