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American Society of Plant Biologists Arabidopsis A BOUT DE SOUFFLE, Which Is Homologous with Mammalian Carnitine Acyl Carrier, Is Required for Postembryonic Growth in the Light
a Laboratoire de Génétique Moleculaire des Plantes, Université Joseph Fourier, Unité Mixte de Recherche 5575, BP 53X, 38041 Grenoble, Cedex 09, France 2 To whom correspondence should be addressed. E-mail pierre.carol{at}ujf-grenoble.fr; fax 33-476-51-43-36
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.
From germination until the establishment of autotrophic growth (referred to here as postgerminative growth), young seedlings rely on nutrients that were stored during embryogenesis and seed development. The degradation of storage molecules provides energy and materials for the building of photosynthetic chloroplasts that, in turn, produce sugars, which are a source of carbon and energy. Oilseeds, such as Arabidopsis, store part of the energy required for postgerminative growth as lipids in the form of triacylglycerides (TAG) that are contained in oil bodies within the cotyledon cells of the embryo. When oilseeds germinate, glyoxysomes, a specialized form of peroxisome, differentiate, with TAG degradation occurring concomitantly (for review, see Olsen and Harada, 1995  ; Olsen, 1998). Plant glyoxysomes have been shown to contain the enzymes necessary for the complete degradation of the fatty acids through the process of  -oxidation (Kleiter and Gerhardt, 1998; Hayashi et al., 1999).
The pathway and location of fatty acid degradation in the plant cell differ from those in animal cells. In animal cells, the
Several CAC protein mutants have been identified in animals, including human, fruit fly, and worm. In Caenorhabditis elegans, the DIF mutation affects embryos before tissue differentiation (Ahringer, 1995
By contrast, in yeast and other fungi, fatty acid breakdown takes place exclusively in peroxisomes. In the yeast Saccharomyces cerevisiae, acetyl-CoA produced by the
It has been shown that in oleate-grown S. cerevisiae, acetylcarnitine is imported into the mitochondria by a CAC-like protein, CRC-1, which is present in the mitochondrial membrane. Mutation of the CRC-1 locus prevents growth on oleate when the citrate synthase locus also is deleted (van Roermund et al., 1999
There are many reports of the presence of carnitine in plant cells and of its role in plant metabolism, including the transport of acyl molecules across the mitochondrial membranes (for review, see Wood et al., 1992
During oilseed germination, We identified à bout de souffle (bou), an Arabidopsis mutation that results in a defective postgerminative phenotype that is light dependent. When grown in the light, mutant plants were unable to proceed to autotrophic growth. However, when seedlings were germinated in the dark, or when sugar was added to the medium, they proceeded to develop. We identified the BOU gene, which in the mutant was disrupted by a transposon. The BOU coding sequence showed homology with the CAC-like protein family that is involved in the translocation of the carnitine ester of fatty acids (acylcarnitine) or acetate (acetylcarnitine) into the mitochondria. The BOU protein is located in mitochondria. We measured storage lipid degradation in bou seedlings and BOU gene expression. Our results suggest that BOU identifies a light-regulated pathway that could be an alternative to the glyoxylate cycle during postgerminative growth.
Characterization of the Mutant Phenotype Using a modified Dissociation element [Ds(HYG)] that was introduced into Arabidopsis for insertional mutagenesis (Coupland, 1992 ; Long et al., 1993), we isolated a mutant on the basis of its pale-green adult phenotype. When mutant seeds germinated on a medium deprived of sugar, they remained arrested at the cotyledon stage, unable to produce the first pair of leaves, and did not develop any root (Figure 1A).
Arrested mutant seedlings that were transferred to a medium containing sugar resumed their development: the root elongated and leaves formed, although they were paler than those of the wild type (Figure 1B). When mutant plants grown on sugar were transferred to soil at the juvenile stage, with one pair of leaves, they grew and set seed. Mutant plants, however, were smaller than wild-type plants (Figure 1C), and their development was slower than that of the wild type, although organ formation was not altered. The progeny of mutant plants had a phenotype identical to that of the parent. From these observations, we conclude that the primary defect of the mutant is in the transition from the embryonic stage to the juvenile autotrophic stage. The small adult phenotype suggests that BOU also is required for normal adult development. Because mutant seedlings stop developing unless sugar is added, we named the mutant à bout de souffle (which means "out of breath").
Sensitivity to 2,4-Dichlorophenoxybutyric Acid
Wild-type plants germinated on 2,4-DB showed arrested development, because the 2,4-D produced caused abnormal cell growth and division. In ped mutants, glyoxysomal
Lipid Degradation Analysis of the lipid content of wild-type and bou seeds showed similar composition of total fatty acids from TAG (data not shown). TAG represented 90% of the lipid content in dry seeds. Seeds that were harvested from soil-grown bou plants had approximately half of the wild-type lipid content. When seeds were harvested from a mutant bou plant grown on sugar-supplemented medium, the lipid content was similar to that of the wild type (Table 1). Whatever their seed lipid content, all bou seedlings showed the arrested phenotype.
In the wild type, storage lipid degradation was low during the first day of growth, when germination occurred, but increased dramatically thereafter, at the postgermination stage. Less than 2% of the TAG remained after 5 days, indicating the mobilization of storage lipids from the storage oil bodies. In mutant seedlings, storage lipids were degraded more slowly (Figure 3). In wild-type plants, polar lipids, which are present in membranes, were being synthesized when TAG were being degraded after germination was completed. In mutant seedlings, very few polar lipids were synthesized (Figure 3).
Because TAG degradation and -oxidation occur in mutant seedlings, it is likely that the primary defect in bou is not in the degradation of lipid storage molecules. It is more likely that bou seedlings are defective in the use of the products of fatty acid degradation.
Genetic Characterization of the bou Mutation It is clear from these experiments that the Ds(HYG) transposon is linked tightly to the mutation. Furthermore, the bou phenotype results from a recessive monogenic trait that is associated with the Ds(HYG) transposon and is most likely caused by the transposon. Heterozygous plants, which carry an active transposase gene, were screened for the loss of the mutant phenotype. An average of 2% of the mutant plants showed a sectored phenotype in which wild-type leaf tissue developed on a mutant background (Figure 1B). Six of the mutants showed a reversion phenotype that encompassed the floral meristem, giving rise to a revertant floral branch. The progeny of the revertant branches segregated for wild-type plants. This finding shows that the phenotypic reversion is transmissible genetically and is not merely a phenotypic variation. This behavior is typical of a transposon-caused mutation.
Cloning of BOU
The sequence of the mutated bou gene showed an 8-bp footprint at the Ds(HYG) insertion site, as expected from a Ds family transposable element (Figure 4C, sequence b). We determined the BOU sequences of 10 revertant plants of wild-type phenotype that were obtained either from revertant branches (as described above) or from the progeny of mutants in which revertants occur at low frequency. In all cases, Ds was not found. The sequences of four independent revertants showed a wild-type DNA sequence at the point of insertion. The sequences of six other independent revertants showed a footprint of 3 to 6 bp at the insertion site, restoring the open reading frame (Figure 4C, sequences c to h).
The footprint did not seem to be a remnant of the 8-bp duplication created upon the insertion of the transposon; rather, it appeared to be a rearrangement of the DNA upon transposon excision. A similar effect has been observed in Arabidopsis and tomato (Keddie et al., 1996 In addition, we obtained a new mutant allele in which the Ds upon excision left a footprint causing the disruption of the reading frame (Figure 4C, sequence i). The unstable mutant phenotype we observed, and the restoration of the gene sequence in revertant alleles, confirmed that the bou mutation is caused by the Ds(HYG) element.
BOU Gene Expression
In adult plants, BOU was expressed more highly in leaves and flowers and developing fruits (siliques) than in roots and maturing siliques (Figure 5C). The expression profile of BOU mRNA with higher levels in green (photosynthetic) tissues, which are known for light-regulated gene expression, suggested that BOU mRNA level could be modulated by light. A strong BOU mRNA signal was detected in light-grown seedlings, whereas dark-grown seedlings showed much less BOU mRNA (Figure 5D). When dark-grown seedlings were placed in light growth conditions, a rapid increase in BOU mRNA was detected (Figure 5E).
Light Modulates the bou Phenotype
In long-day conditions (Figure 6A), seedlings showed the arrested phenotype. A few seedlings (8%; Figure 6B) became established when germinated for 24 h in the dark. Lengthening of the dark period increased the percentage of established seedlings (Figure 6C). Up to 80% of the seedlings developed and became established when seeds were germinated for 48 h in the dark (Figure 6D). Fewer seedlings (66%) became established if a 24-h light period was applied during the first day of germination (Figure 6E). A short-day light regime during and after germination allowed the establishment of approximately half of the seedlings (Figure 6F). In conclusion, the presence or absence of light during and just after germination modulated the bou phenotype. Therefore, it seems that BOU function, which is vital for the establishment of seedlings in the light, is not necessary for seedling establishment in the dark.
BOU Shows Similarity to CAC
The comparison of BOU with the human acylcarnitine carrier and the yeast acetylcarnitine carrier proteins, whose functions are known, revealed similar features (Figure 7C). The CAC-like proteins have six transmembrane domains (TM). Comparing the predicted TM relative spacing of CAC and BOU, we found that five of six TM are colinear, but TM4 is predicted to be at a different position in BOU. The predicted relocation of TM4 corresponds to an amino acid insertion sequence unique to BOU that is not found in other CAC-related proteins.
Location of BOU
bou and Peroxisome Function The primary phenotype associated with bou was the inability to proceed from embryonic to juvenile autotrophic growth. This phenotype is similar to that of ped mutant plants, which are defective in glyoxysomal thiolase activity and are unable to produce acetate through the -oxidation of fatty acids. In Arabidopsis, up to 5 µg of TAG was stored in the seed. Quantification of TAG throughout seedling development showed that TAG remained for 24 h, when germination occurred, before being degraded completely during the subsequent 2 days. We conclude that lipid degradation is not used for germination per se. It is likely that the energy and molecules generated by lipid degradation are used at the postgerminative stage, when the seedling embryonic structure develops photosynthetic tissues, leading to an autotrophic plant.
bou mutant seedlings displayed an arrested growth phenotype whether the seeds had a normal complement of storage lipids (the progeny of mutant plants grown on sugar) or half of the wild-type lipid content (the progeny of a soil-grown mutant plant). The mutant phenotype also was observed in the progeny of heterozygous plants. We found that bou mutants could degrade storage lipids. This was confirmed by the biological assay for Mutant plants that remained arrested for a few days were able to resume development when moved to sugar-containing medium. Sugar was needed until the first leaves appeared. Thereafter, mutant plants could sustain autotrophic growth. From these observations, we conclude that the defect in bou mutants affects neither germination nor storage lipid degradation. Most likely, the bou mutation prevents postgerminative plant development or the use of storage compound degradation products. The germination of mutant seedlings in the dark alleviated the developmental block. This light-dependent phenotype suggests that BOU performs a function that is necessary only in the light, and another different pathway is sufficient for development in the dark.
BOU Expression During seedling development, when storage lipids sustain cell growth and differentiation, embryonic cotyledons undergo a metabolic transition from storage degradation to photosynthetic tissue. BOU expression was light induced in these developing seedlings. Later, after 4 days, when cotyledons became senescent, there was a decrease of BOU expression. BOU was detected again in newly formed photosynthetic leaves. Although BOU mRNA was detected first after 36 h and reached a maximum after 2 days of growth, BOU protein was detected after 2 days and reached a maximum at 3 days of growth. It is possible that a translational mechanism regulates BOU production. The accumulation of BOU in the wild type coincided with the stage at which the bou phenotype was first detected in the mutant (i.e., after germination). BOU gene expression also was correlated with the light-dependent phenotype of the mutant. The bou phenotype appeared in the light growth conditions that corresponded to the highest BOU accumulation in the wild type. In the dark, when BOU was expressed at a low level, the function was not necessary for seedling growth, because the mutants were able to develop.
Putative Transport Function of BOU
The transport function of BOU remains to be assessed. Plant cells do contain carnitine (for review, see Wood et al., 1992
Hypothesizing an Alternative to the Glyoxylate Cycle in Plants
Surprisingly, it has been shown that Arabidopsis icl mutants, which are defective for the ICL gene coding for the enzyme ICL from the glyoxylate cycle, are able to germinate and develop into autotrophic photosynthetic plants in the light (Eastmond et al., 2000
This is similar to the metabolism of yeast, in which fatty acid
The possible occurrence of an acetylcarnitine pathway in Arabidopsis has been speculated (Eastmond and Graham, 2001
In addition to its role during seedling development, BOU also is required during the entire plant life cycle. Adult bou plants are shorter and paler green than wild-type plants, and they accumulate fewer storage fatty acids. It can be speculated that the BOU transport function is not involved directly in lipid biosynthesis, because it has been shown that the pool of acetate is not a primary source of carbon for lipid biosynthesis in leaves (Bao et al., 2000
Plant Growth Conditions The Arabidopsis thaliana Landsberg erecta Ds(Hyg)-containing line has been described previously (Coupland, 1992 ; Long et al., 1993), and the T-DNA insertion carrying the Ds(Hyg) transposon was mapped on chromosome V (Long et al., 1997). Detection of plants carrying the transposase construct was performed by monitoring the -glucuronidase activity associated with 35S::-glucuronidase (Long et al., 1993). Antibiotic selection of Ds(Hyg), namely hygromycin resistance and streptomycin resistance (associated with Ds excision from the T-DNA), was described by Long et al. (1993). The Arabidopsis Landsberg erecta line carrying the Activator transposase gene fused to the 35S promoter of Cauliflower mosaic virus (35S::TPase) has been described previously (Swinburne et al., 1992).
Plants were grown on standard Murashige and Skoog (1962)
Lipid Analysis The total fatty acids of seeds were analyzed by gas chromatography. Thereafter, triacylglycerides (TAG) were computed from the measured C20:1 value in a total lipid extract. Because C20:1 is a typical storage fatty acid found in TAG, it can be used as a marker to determine the overall content of TAG in Arabidopsis seedlings, because the overall fatty acid composition of TAG fatty acids does not vary over 4 days after germination (data not shown). Heneicosanoic acid (C21:0) was used as an internal standard. Gas chromatography was performed using a capillary column (BPX70; Scientific Glass Engineering, Victoria, Australia).
Cloning of BOU
DNA and RNA Analysis To isolate DNA from revertants, PCR was performed using the primers H (5'-TCAAATCTAAAAACAGAGAGG-3') and C (5'-GAA-ATGGCGGATGCGTGG-3') using 10 ng of plant DNA as a template, followed by 4 min of denaturation at 94°C and 30 cycles of amplification (1 min at 94°C, 1 min at 55°C, and 1 min at 72°C). The PCR product was purified and sequenced using the PCR sequencing method with primers M (5'-GCTTGTCCTACCGAGTTG-3') and N (5'-TTC-ACGGGCGAATGTAGG-3').
Immunoblot Analysis After transfer, the filters were blocked in TBS (20 mM Tris-HCl, pH 7.6, and 137 mM NaCl) with 5% nonfat powdered milk, and the primary antibody was incubated overnight at 4°C in TBS. The filters were rinsed three times in TBS before incubating with the secondary antibody anti-rabbit goat IgG-peroxidase (Bio-Rad, Hercules, CA) for 1 h at room temperature. The signal was developed using the enhanced chemiluminescence kit (Amersham Pharmacia Biotech).
Isolation of Organelles Peroxisomes were collected from the top of the 57 and 52% layers and mitochondria were collected from the top of the 42% layer. The organelles were fractionated once more using the same gradient and centrifugation conditions, diluted in extraction buffer, and pelleted for 2 h at 72,000g. All of the steps were performed at 4°C. A catalase activity assay (the decrease of H2O2 by absorbance at 240 nm in 0.1 M phosphate buffer, pH 7, 10% Triton X-100, and 0.01% H2O2) confirmed the identification of the peroxisome fraction.
Mitochondria were identified by immunoreaction with anti-NAD9 serum (Lamattina et al., 1993 Upon request, all novel materials described in this article will be made available in a timely manner for noncommercial research purposes. No restrictions or conditions will be placed on the use of any materials described in this article that would limit their use for noncommercial research purposes.
Accession Numbers
The authors thank Jean-Pierre Alcaraz for the DNA sequence data, Gérard Clabault for his help with computers, Mireille Jaubert for her help with the photographs, and Daniel Salvi for his help with lipid analysis. Alison Baker, Florence Courtois, Roland Douce, and Ian Graham are thanked for their helpful discussion on the metabolism of glyoxysomes. We thank Jacques Bourguignon for his kind gift of pure Arabidopsis mitochondrial membranes and Rachel Carol for correcting the manuscript.
Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.002485.
1 Current address: Max-Planck-Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, D-50829 Köln, Germany. Received February 20, 2002; accepted May 29, 2002.
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Abstract
INTRODUCTION
1.2 kb in the wild-type sample that was undetectable in RNA extracted from mutant plants (
and 
