The γ-carbonic anhydrase subcomplex of mitochondrial complex I is essential for development and important for photomorphogenesis of Arabidopsis

Abstract

Complex I (NADH:ubiquinone oxidoreductase) is the entry point for electrons into the respiratory electron transport chain; therefore, it plays a central role in cellular energy metabolism. Complex I from different organisms has a similar basic structure. However, an extra structural module, referred to as the γ-carbonic anhydrase (γCA) subcomplex, is found in the mitochondrial complex I of photoautotrophic eukaryotes, such as green alga and plants, but not in that of the heterotrophic eukaryotes, such as fungi and mammals. It has been proposed that the γCA subcomplex is required for the light-dependent life style of photoautotrophic eukaryotes, but this hypothesis has not been successfully tested. We report here a genetic study of the genes γCAL1 and γCAL2 that encode two subunits of the γCA subcomplex of mitochondrial complex I. We found that mutations of γCAL1 and γCAL2 in Arabidopsis (Arabidopsis thaliana) result in defective embryogenesis and nongerminating seeds, demonstrating the functional significance of the γCA subcomplex of mitochondrial complex I in plant development. Surprisingly, we also found that reduced expression of γCAL1 and γCAL2 genes altered photomorphogenic development. The γcal1 mutant plant expressing the RNA interference construct of the γCAL2 gene showed a partial constitutive photomorphogenic phenotype in young seedlings and a reduced photoperiodic sensitivity in adult plants. The involvement of the γCA subcomplex of mitochondrial complex I in plant photomorphogenesis and the possible evolutionary significance of this plant-specific mitochondrial protein complex are discussed.

Figure 1.

γCAL1 and γCAL2 are mitochondrial proteins of the plant-specific γCA subcomplex of complex I. A, Diagram depicting the γCA subcomplex (black module indicated by arrows), the mitochondrial complex I of green algae (Polytomella spp.) and plants (Arabidopsis; Dudkina et al., 2005; Sunderhaus et al., 2006), but not in bacteria (Escherichia coli; Morgan and Sazanov, 2008), fungi (Neurospora spp.; Guénebaut et al., 1997), or mammals (bovine; Grigorieff, 1998). The structural outlines of complex I are redrawn from the published structure of the respective complex I. B to E, Confocal images showing the subcellular localization of the γCAL1-YFP and γCAL2-YFP (B–D) or γCAL1-GFP and γCAL2-GFP (E) fusion proteins in hypocotyl cells of 5-d-old seedlings (B and C) or protoplasts isolated from 3-week-old plants (E). CS16264 is a mitochondrial marker protein. Propidium iodide (PI) was used to stain the cell wall. BF, Bright field. Bars = 20 µm (B and C) and 25 µm (D). The boxed areas (B–D) are enlarged to show details. [See online article for color version of this figure.]

Figure 2.

The γCAL1 and γCAL2 subunits of the γCA subcomplex is essential for Arabidopsis development. A, The immature siliques of wild-type (WT) and rescued mutant (35S::CAL1/c1c1c2c2) parents that show uniform and green-colored ovules, and the siliques derived from C1c1c2c2 or c1c1C2c2 hemizygote parents that show segregating colorless ovules indicated by arrowheads (left). The mature siliques from the wild-type and 35S::CAL1/c1c1c2c2 parents show uniformly yellow seeds, but those from the c1c1C2c2 hemizygote parent show segregating deep-brown seeds indicated by arrowheads (right). No dark-brown seed was found in the mature siliques of the C1c1c2c2 hemizygote parents, but no double homozygous progeny were identified. Bars = 0.5 mm. B, Embryos of the segregating progeny of the c1c1C2c2 hemizygote. Ovules from a young silique of the c1c1C2c2 hemizygote parent were cleared with Hoyer’s solution and observed with a differential interference contrast microscope. The segregating white ovules arrested at various stages (c1c1c2c2) and the pale-green wild-type-looking ovule at the mature cotyledon stage from the same young silique are shown. Bars = 50 µm. C, Seeds (left) and embryos (right) of the nongerminating seeds (ngs) harvested from the C1c1c2c2 and c1c1C2c2 hemizygote parents. For the wild type, C1c1c2c2 and c1c1C2c2 seeds were placed on MS medium for 7 d. The nongerminating seeds were collected first for photographic record (left) and then dissected using a microscope. The dissected embryos corresponding to the seed (left) are shown on the right. D, Genotyping of the nongerminating seeds. Genomic DNA was isolated from the cal1 mutant, the cal2 mutant, and nongerminating seeds derived from the c1c1C2c2 parent. Genomic PCR was performed using the primers designed to amplify the wild-type γCAL1 (CAL1) and γCAL2 (CAL2) genes or the γcal1 (cal1) and γcal2 (cal2) mutant genes. [See online article for color version of this figure.]

Figure 3.

Genotyping showing the complementation of the γcal1γcal2 (c1c2) double mutant. A, The c1c1C2c2 plants were transformed with the 35S::CAL1 transgene. The genomic DNA was isolated from arbitrarily selected T2 Basta-resistant plants and PCR amplified using the primers specific to the CAL2 gene or the cal2 mutant locus. B, The individuals identified as the cal2 mutant in A were further genotyped for the cal1 mutant locus and the 35S::CAL1 transgene. Primers specific for the CAL2, cal2, CAL1, and cal1 loci as well as the 35S::CAL1 transgene are indicated on the right. WT, Wild type.

Figure 4.

The delayed germination phenotype of the c1c2i knockdown seeds. Seeds of the indicated genotypes (wild type [WT], cal1, and c1c2i) were placed on MS medium, and the photographs were taken at the times indicated. Seeds or seedlings of independent c1c2i mutant transgenic lines show similar phenotypes of delayed germination or retarded growth, respectively. Bars = 1 mm. [See online article for color version of this figure.]

Figure 5.

The growth retardation phenotypes of transgenic lines expressing the CAL2-RNAi construct in the cal1 mutant background (c1c2i). A, Quantitative PCR showing decreased mRNA expression of the CAL1 (left) and CAL2 (right) genes in independent transgenic lines expressing the CAL2-RNAi construct in the cal1 mutant background (c1c2i). B, Phenotypes of the c1c2i mutant transgenic seedlings grown for 7 d in a long-day (16 h of light/8 h of dark) photoperiod (left) or a short-day (8 h of light/16 h of dark) photoperiod (right). C, Phenotypes of the c1c2i mutant transgenic seedlings grown for 11 d in a long-day photoperiod. WT, Wild type. [See online article for color version of this figure.]

Figure 6.

The wavelength-independent hypersensitive light responses of the c1c2i knockdown seedlings. A, Representative seedling phenotypes of the indicated genotypes. Independent lines of the c1c2i seedlings and the controls were grown in the dark or in continuous blue light (25 µmol m⁻² s⁻¹), far-red light (2.5 µmol m⁻² s⁻¹), or red light (15 µmol m⁻² s⁻¹) for 5 d. Seedlings of four independent c1c2i lines (lines 1, 5, 9, and 20) are shown. B, Hypocotyl lengths of seedlings of the indicated genotypes. Seedlings were grown for 5 d under the different fluence rates indicated, and sd values are shown (n ≥ 20). C, Quantitative PCR results showing the increased expression of the light-induced CHS gene in c1c2i seedlings. Wild-type (WT) and c1c2i seedlings were grown in the dark (D7–D9) or under continuous blue light (B7–B9) for 7, 8, or 9 d before sample harvest for RNA isolation. The relative expression of CHS is normalized by UBQ5. [See online article for color version of this figure.]

Figure 7.

The photoperiod-dependent delay of floral initiation of the c1c2i knockdown plants. A, Plants of the indicated genotypes were grown in a long-day (LD) photoperiod (16 h of light/8 h of dark; left). The flowering time was measured as the days to flowering and the numbers of rosette leaves at the time of flowering (right). B, Plants of the indicated genotypes grown in a short-day (SD) photoperiod (8 h of light/16 h of dark; left). The flowering times were measured as days to flowering and the number of rosette leaves, and sd values are shown (n ≥ 20). C, Quantitative PCR results showing the mRNA expression of the indicated flowering-time genes in wild-type (WT) and c1c2i plants. Plants were grown in continuous white light, and tissues were collected at the indicated days after germination for RNA isolation and quantitative PCR analysis. The relative expression of the indicated genes is normalized to the level of the UBQ5 control. [See online article for color version of this figure.]