Translocase of the Outer Mitochondrial Membrane 40 Is Required for Mitochondrial Biogenesis and Embryo Development in Arabidopsis

Abstract

In eukaryotes, mitochondrion is an essential organelle which is surrounded by a double membrane system, including the outer membrane, intermembrane space and the inner membrane. The translocase of the outer mitochondrial membrane (TOM) complex has attracted enormous interest for its role in importing the preprotein from the cytoplasm into the mitochondrion. However, little is understood about the potential biological function of the TOM complex in Arabidopsis. The aim of the present study was to investigate how AtTOM40, a gene encoding the core subunit of the TOM complex, works in Arabidopsis. As a result, we found that lack of AtTOM40 disturbed embryo development and its pattern formation after the globular embryo stage, and finally caused albino ovules and seed abortion at the ratio of a quarter in the homozygous tom40 plants. Further investigation demonstrated that AtTOM40 is wildly expressed in different tissues, especially in cotyledons primordium during Arabidopsis embryogenesis. Moreover, we confirmed that the encoded protein AtTOM40 is localized in mitochondrion, and the observation of the ultrastructure revealed that mitochondrion biogenesis was impaired in tom40-1 embryo cells. Quantitative real-time PCR was utilized to determine the expression of genes encoding outer mitochondrial membrane proteins in the homozygous tom40-1 mutant embryos, including the genes known to be involved in import, assembly and transport of mitochondrial proteins, and the results demonstrated that most of the gene expressions were abnormal. Similarly, the expression of genes relevant to embryo development and pattern formation, such as SAM (shoot apical meristem), cotyledon, vascular primordium and hypophysis, was also affected in homozygous tom40-1 mutant embryos. Taken together, we draw the conclusion that the AtTOM40 gene is essential for the normal structure of the mitochondrion, and participates in early embryo development and pattern formation through maintaining the biogenesis of mitochondria. The findings of this study may provide new insight into the biological function of the TOM40 subunit in higher plants.

Keywords: Arabidopsis; embryo development; mitochondria biogenesis; pattern formation; translocase of the outer mitochondrial membrane 40 (TOM40).

FIGURE 1

The conservation analysis of TOM40 in different species. (A) Sequence alignment of TOM40 in different species, α-helical segments and β-barrel domain were labeled according to C. glabrata. Cg, Candida glabrata; Sc, Saccharomyces cerevisiae; At, Arabidopsis thaliana; Os, Oryza sativa; Hs, Homo sapiens; Mm, Mus musculus; Zm, Zea mays; Nt, Nicotiana tabacum; Gm, Glycine max; Gh, Gossypium hirsutum; Dr, Danio rerio; Bn, Brassica napus; Sb, Sorghum bicolor. (B–D) Predicted 3-D structures of AtTOM40, ScTOM40 and OsTOM40 according to the crystal structures of their homologs in yeast. (E) The merged view of AtTOM40, ScTOM40 and OsTOM40 predicted 3-D structures.

FIGURE 2

Characterization of AtTOM40 mutation and complementation assays of tom40-1/+ and tom40-2/+ mutants in Arabidopsis. (A) Schematic diagram of the AtTOM40 gene and T-DNA positions of the two mutants. Exons and promoter are shown in black and gray boxes, and introns are shown as lines, respectively. FP and RP indicate forward and reverse primers, Bar = 200 bp. (B) Ovule phenotypes in siliques of the wild type, tom40-1/+, tom40-2/+, functionally complemented tom40-1/+ and tom40-2/+ transgenic plants. The white arrows showed the aborted ovules. Bar = 1 mm. (C) Genotypic analysis of the wild type, tom40-1/+, tom40-2/+ and the complemented mutant plants. (D) The seed abortion rates in the wild type, tom40-1/+, tom40-2/+, and the complemented mutant plants. The total numbers of seeds counted are listed on the top, and the seed abortion rates are shown on the bottom.

FIGURE 3

Status of embryo development in plants of the different genotypes. (A–D) Embryos from the globular stage to the bent cotyledon stage in wild-type ovules. (E–H) Embryos in homozygous tom40-1 ovules at parallel developmental stages with A–D. (I–L) Embryos in homozygous tom40-2 ovules at parallel developmental stages with A–D. Bar = 20 μm.

FIGURE 4

GUS Staining analysis of TOM40 in Arabidopsis. (A–E) GUS staining signals (blue staining) in different tissues of pTOM40::GUS transgenic plants. (A–D) Bar = 1mm, (E) Bar = 20 μm.

FIGURE 5

Expression analysis of TOM40 in Arabidopsis. (A–G) In situ hybridization of AtTOM40 transcripts (purple staining) accumulated in wild-type embryos and endosperm. Bar = 20 μm. (H) Quantitative real-time PCR analysis of AtTOM40 in various tissues. R, root; S, stem; L, leaf; F, flower; INF, inflorescence; 1SI–7SI, siliques at 1, 3, 5, 7 days after pollination; 7-14SE, seedlings at 7 and 14 days after germination.

FIGURE 6

(A–C) Subcellular localization of AtTOM40 protein. Fluorescent signals in mesophyll protoplasts represents AtTOM40 (green), mitochondria (red) and colocalization (merged), respectively. Bar = 10 μm. (D–G) Ultrastructure of mitochondria in wild type (D,E) and tom40-1 (F,G) embryo proper cells at 6DAP. Bar = 500 nm. (F,G) Cells in tom40-1 embryo proper at 6DAP. (D,E,G) Bar = 500 nm, (F) Bar = 1 μm. SG: starch grain; M: mitochondrion; CH: chloroplast; V: vacuole; CW: cell wall.

FIGURE 7

Relative expression levels of genes relevant to outer mitochondrial membrane (OMM) in Arabidopsis between wild type and tom40-1 albino ovules at 6DAP. (A) Expression level of AtTOM40 in the tom40-1 albino ovules was about one fifth of the expression level in the wild type. (B–J) Expression levels of genes involved in import and assembly of mitochondrion proteins. (K–L) Expression levels of genes involved in protein transport in mitochondrion. (M–O) Expression levels of genes involved in morphology and membrane dynamics. (P–Q) Expression levels of genes involved in energy and metabolism. (R–T) Expression levels of genes involved in synthetase related to the OMM. The asterisk indicates a significant difference (Student’s t-test, ^∗P < 0.05, ^∗∗P < 0.01).

FIGURE 8

Relative expression levels of genes involved in embryo cell fate decision between wild type and the tom40-1 albino ovules at 6DAP. (A) Expression level of AtTOM40 in the tom40-1 albino ovules was about one fifth of the expression level in the wild type. (B–E) Expression levels of genes involved in SAM. (F–I) Expression levels of genes involved in cotyledon. (J–M) Expression levels of genes involved in vascular primordium. (N,O) Expression levels of genes involved in protoderm. (P–T) Expression levels of genes involved in hypophysis. The asterisk indicates a significant difference (Student’s t-test, ^∗P < 0.05, ^∗∗P < 0.01).