Arabidopsis A BOUT DE SOUFFLE, which is homologous with mammalian carnitine acyl carrier, is required for postembryonic growth in the light

Abstract

The degradation of storage compounds just after germination is essential to plant development, providing energy and molecules necessary for the building of a photosynthetic apparatus and allowing autotrophic growth. We identified à bout de souffle (bou), a new Arabidopsis mutation. Mutant plants stopped developing after germination and degraded storage lipids, but they did not proceed to autotrophic growth. Neither leaves nor roots developed in the mutant. However, externally added sugar or germination in the dark could bypass this developmental block and allowed mutant plants to develop. The mutated gene was cloned using the transposon Dissociation as a molecular tag. The gene coding sequence showed similarity to those of the mitochondrial carnitine acyl carriers (CACs) or CAC-like proteins. In animals and yeast, these transmembrane proteins are involved in the transport of lipid-derived molecules across mitochondrial membranes for energy and carbon supply. The data presented here suggest that BOU identifies a novel mitochondrial pathway that is necessary to seedling development in the light. The BOU pathway would be an alternative to the well-known glyoxylate pathway.

Figure 1.

Phenotype of the bou Mutant. (A) The bou phenotype is apparent after germination on mineral medium. 0+, seeds that had imbibed (magnification ×30); 1, 1 day of growth (magnification ×40); 2 to 6, 2 to 6 days of growth (magnification ×15). Top row, wild-type (WT) seedlings; bottom row, bou seedlings. (B) Top row, wild-type plants (left) and mutant plants (right) grown for 20 days on sugar-containing medium. Bottom row, stable bou mutant (left) and two bou mutants showing an unstable phenotype with wild-type sectors (right). The arrows indicate the presence of a revertant sector seen as dark green on a pale green background. Bar = 1 cm. (C) Wild-type (left) and bou (right) plants grown for 2 weeks on Murashige and Skoog (1962) medium and then on soil for 1 more week. Bar = 1 cm. (D) Dark indicates seedlings germinated in the dark for 4 days and then transferred to long-day conditions for 15 days. Light indicates mutants kept in long-day conditions for 17 days (magnification ×7.5).

Figure 2.

Root Sensitivity to 2,4-DB. (A) Root elongation of plants (n = 10) grown on ATG medium containing a range of concentrations of 2,4-DB. (B) Root elongation of plants (n = 10) grown on ATG medium containing a range of concentrations of the active compound 2,4-D. Plants were grown for 1 week on a vertically orientated plate before root length measurement. Root length is expressed as a percentage of the length of wild-type and bou roots grown in the absence of 2,4-DB and 2,4-D, respectively. bou, mutant plants; WT, wild-type plants. Bars represent the standard deviation.

Figure 3.

Lipid Degradation and Polar Lipid Accumulation during Early Arabidopsis Seedling Development. Storage lipids (TAG) and polar lipids were measured each day during seed germination. The arrows indicate the apparent completion of germination (protrusion of the radicle from the seed coat). The germination of bou was delayed by 1 day compared with that of the wild type (WT). Seeds were germinated in vitro on ATG medium (without sugar). 0, dry seeds; 0⁺, seeds allowed to imbibe overnight at 4°C before germination; 1 to 5, days of culture at 24°C in the light. Bars indicate the standard deviation from an average of three experiments (n = 10). Polar lipids were measured from a pool of 20 seedlings.

Figure 4.

Molecular Analysis of the bou Mutation. (A) DNA gel blot analysis of the bou mutation. DNA extracted from wild-type (WT) and heterozygous (het) hygromycin-resistant lines was digested with EcoRI, transferred to a nitrocellulose membrane, and probed with the BOU cDNA. Two extra genomic DNA fragments were revealed (arrows) in the heterozygote samples. M, molecular mass markers. (B) RNA gel blot showing a BOU transcript in wild-type seedlings (arrow) that was absent from the mutant sample. RNA extracted from 2-week-old seedlings was probed with the BOU cDNA. Hybridization to the 25S rRNA was used as a loading control. (C) Sequence analysis of the transposon insertion. The nucleotides and the deduced amino acids that differed from the wild-type sequence are shown in boldface. (a) Relevant sequence of the BOU gene. (b) Sequence of the insertion allele. (c) to (h) Sequences of six revertant alleles. (i) Sequence of a stable mutant with an insertion resulting in frameshifting and a truncated reading frame.

Figure 5.

RNA Gel Blot Analysis of BOU Expression. (A) Detection of BOU mRNA during seed germination from 12 to 48 h. (B) Detection of BOU mRNA after germination in seedlings grown in the light for 2 to 5 days. (C) Detection of BOU mRNA in Arabidopsis organs. R, roots; L, leaves; C, 3-day-old cotyledons; FB, flower buds; F, flowers; S, siliques; S1, immature developing siliques (up to 7 days after fertilization); S2, developing siliques (1 to 2 weeks after fertilization); S3, maturing siliques (2 to 3 weeks after fertilization). (D) Detection of BOU mRNA in light-grown seedlings. 3L, 3 days in the light; 3D, 3 days in the dark; 2D1L, 2 days in the dark followed by 1 day in the light; 2L1D, 2 days in the light followed by 1 day in the dark. (E) Effect of light treatment (0, 3, or 6 h) on BOU RNA accumulation in 3-day-old dark-grown seedlings. The CAB gene was used as a control for light-induced gene expression. In all experiments, the loading control was the 25S rRNA hybridization signal.

Figure 6.

Scheme of the Light-Dependent bou Phenotype. Before growth, bou seeds that had imbibed were germinated for 2 days in the light or in the dark for various time periods. The time scale represents the germination of bou seedlings, starting at 0 h. Germinated seedlings were grown for 15 days, and the phenotype was scored as arrested if the seedlings had not developed and as established if the seedlings had developed roots and leaves. At left ([A] to [F]), the light periods (L) are shown with open boxes, and the dark periods (D) are shown with black boxes. At right, for each light regime, the percentage of seedlings that were arrested (black) is compared with the percentage of established seedlings (gray). Results shown are averages of two independent experiments with 100 seeds each. A control sample of wild-type seeds always showed normal development (100% established) in all of the described conditions.

Figure 7.

The BOU Gene and Its Coding Sequence. (A) Structure of the BOU gene. Exons are represented as boxes, and introns are represented as a line. The relative position of the Ds transposon is indicated. Bar = 100 bp. (B) Nucleotide and amino acid sequences of BOU cDNA. The arrowheads indicate the positions of the introns in the gene. The triangle indicates the insertion point of the Ds transposon. The 8-bp sequence duplicated upon Ds insertion is underlined. (C) Comparison of the BOU protein with CAC-like proteins. CAC-Hs, CAC from human; CRC1-Sc, acetylcarnitine carrier CRC-1 from S. cerevisiae. Identical amino acids are shaded in black, and similar amino acids are shaded in gray. Putative transmembrane domains (TM1 to TM6) are underlined. The sequences were aligned using the Genetics Computer Group computer package version 8.1 (Madison, WI). The transmembrane domains were predicted using TMpred (Hofmann and Stoffel, 1993) (http://www.ch.embnet.org/).

Figure 8.

Immunodetection of BOU, a Mitochondrial Protein. (A) to (D) Immunodetection of BOU in total plant protein extracts. M, molecular mass markers. (A) Immunodetection of BOU in wild-type (WT) and mutant (bou) protein extracts. (B) Coomassie blue staining of the samples. (C) Immunodetection of BOU during seed germination, from 1 to 4 days of seedling development. (D) Immunodetection of BOU in light-grown (L) and dark-grown (D) 3-day-old seedlings. (E) and (F) Immunodetection of BOU in Arabidopsis organelles. (E) Comparison of total (T) proteins and mitochondrial (M) and peroxisome (P) fractions from leaf tissue of 3-week-old Arabidopsis. NAD9 was used as a mitochondria-specific control, and PMP22 was used as a peroxisome-specific control. (F) Immunodetection of BOU in purified mitochondrial membrane (MM) and total proteins (T) from Arabidopsis cells. (G) Comparison of total proteins (T) and chloroplast fraction (C) from leaf tissue of 3-week-old Arabidopsis. The detection of RPL4 chloroplast protein was used as a control.