Control of germination and lipid mobilization by COMATOSE, the Arabidopsis homologue of human ALDP

Abstract

Embryo dormancy in flowering plants is an important dispersal mechanism that promotes survival of the seed through time. The subsequent transition to germination is a critical control point regulating initiation of vegetative growth. Here we show that the Arabidopsis COMATOSE (CTS) locus is required for this transition, and acts, at least in part, by profoundly affecting the metabolism of stored lipids. CTS encodes a peroxisomal protein of the ATP binding cassette (ABC) transporter class with significant identity to the human X-linked adrenoleukodystrophy protein (ALDP). Like X-ALD patients, cts mutant embryos and seedlings exhibit pleiotropic phenotypes associated with perturbation in fatty acid metabolism. CTS expression transiently increases shortly after imbibition during germination, but not in imbibed dormant seeds, and genetic analyses show that CTS is negatively regulated by loci that promote embryo dormancy through multiple independent pathways. Our results demonstrate that CTS regulates transport of acyl CoAs into the peroxisome, and indicate that regulation of CTS function is a major control point for the switch between the opposing developmental programmes of dormancy and germination.

Fig. 1. CTS encodes an ALDP-related ABC transporter. (A) CTS gene structure. The positions of exons are indicated (boxes), and ATG and TGA codons. The cts-1 allele results from a translocation of 370 kb of chromosome V (positions 33.3–33.7 Mb) into exon 10 (data not shown). The cts-2 allele contains an insertion of T-DNA into exon 3 (codon Thr117). The positions of other known alleles in this gene are indicated (Zolman et al., 2001; Hayashi et al., 2002). (B) Positions of DNAs on chromosome IV complementing the cts-1 mutant phenotype. The CTS gene (AT4G39850) and adjacent open reading frames are shown (gene names; The Arabidopsis Genome Initiative, 2000). Clone numbers are indicated and the minimum overlapping region boxed. (C) Schematic of the CTS protein, indicating positions of two NBD and termination points of polypeptides corresponding to the cts-1 and cts-2 alleles. The most similar proteins in the SWISSPROT database to the amino terminal half of CTS (amino acids 1–690) are mouse ALD and human ALDP (Blast probability scores P = e-88). The most similar protein to the C-terminal half of CTS (691–1337), for which an experimental function has been assigned, is rat PMP70 (P = 1.1 e-69). Positions of other known alleles are indicated. (D) Western blot analysis of WT, cts-1 and cts-2. Protein extracts were from 3-day-old germinated seedlings (100 µg protein/lane). The western blot was probed for CTS, MS, ICL and KAT2 proteins.

Fig. 2. CTS is localized in the peroxisomes. Post-nuclear organellar pellet derived from leaf was separated on a sucrose density gradient (sucrose concentration, dotted line). Fractions (A) were assayed for catalase activity (continuous line), chlorophyll concentration (dashed line) and probed (B) by western blotting for CTS, KAT, ER-marker calreticulin (CAL) and ANT, a mitochondrial membrane marker. CTS co-localizes with intact peroxisomes (56% sucrose) corresponding to the first peak of catalase and thiolase.

Fig. 3. Loss of CTS in the cts-1 mutant leads to a striking failure of lipid breakdown in germinated cotyledons. Transverse electron micrograph sections were made from cotyledons germinated for 5 days in the presence of sucrose. WT and mutant seedling morphology is indistinguishable when grown under these conditions. In the WTs, lipid bodies are completely absent and mitochondria notably abundant (A, scale bar 2 µm) whilst in cts-1, lipid bodies persist and plastid morphology is abnormal (B, scale bar 2 µm). Similar peroxisomal morphology is observed in WT (C) and mutant cts-1 (D), scale bar 500 nm. Peroxisomes appear closely juxtaposed to lipid bodies in cts-1 (D). P, peroxisomes; LB, lipid bodies; M, mitochondria; C, chloroplast; V, vacuole.

Fig. 4. cts-1 and cts-2 mutants are defective in the bio-activation of 2,4DB by peroxisomal β-oxidation. Comparison of growth of WT and cts-1 and cts-2 seedlings on media with no added hormone (control) or media containing 2,4DB or 2,4D. Surrounding testa/endosperm layers were removed from seeds to facilitate germination of mutant seeds.

Fig. 5. Profiles of TAG-derived fatty acids and acyl CoA levels in WTs and cts-1 and cts-2 mutants. (A) Comparison of TAG-derived fatty acids in the WTs and cts-1 and cts-2 mutants in seeds and seedlings 0, 2 and 5 days after sowing. (B) Profile of TAG-derived fatty acids in imbibed WT and mutant seeds by chain length. (C) As (B) but profile from 5-day-old seedlings. (D) Comparison of acyl CoA levels in WTs and cts-1 and cts-2 seedlings 0, 2 and 5 days after sowing. (E) Profile of acyl CoAs in imbibed WT and mutant seeds by chain length. (F) As (E) but profile from 5-day-old seedlings. All seeds were sown on media containing 1% sucrose and held at 4°C for 4 days before transfer to germination conditions. The testa of cts mutants was disrupted to allow germination. Seedlings were at similar morphological stages of development. Bars indicate SEM.

Fig. 6. Metabolic regulation of germination in cts-1 seeds. (A) Short chain fatty acids can rescue the cts-1 mutant phenotype. Analysis of germination potential of intact after-ripened Ler or cts-1 mutant seeds either with or without sucrose (S), propionate (P) and butyrate (B). Inset; typical germination of cts-1 seeds without (control) or with (0.1 mM) propionate. Bar represents 0.5 mm. Bars indicate SEM. (B) Sucrose mobilization is restricted in the cts-1 mutant. Carbohydrate analysis of intact after-ripened Ler and cts-1 seeds was carried out under germination conditions in the absence of exogenous sucrose. Germination was 98% for Ler seeds by day 2 followed by full establishment. By comparison, cts-1 germination was 1% by day 2 and 2% at day 5 with no further growth.

Fig. 7. Genetic interactions of CTS with loci that repress germination. Germination (%) of after-ripened seed single and double mutant combinations in the absence (A) or presence (B) of exogenous sucrose is shown. Germination was measured 7 days after seed sowing. Bars indicate SEM.

Fig. 8. Analysis of CTS RNA and protein expression. (A–D) Western analysis of seeds imbibed for 7 days, using antibodies for CTS, ICL, MS and KAT. In each case, sizes of proteins are indicated. (A) After- ripened seeds (germination percentage indicated; G%). (B) Dormant seeds (G% indicated). (C) cts-1 mutant seeds (no germination). (D) WT seeds imbibed with 10 µM ABA (no germination; this concentration of ABA inhibits germination; Garciarrubio et al., 1997). (E) RT–PCR analysis of CTS gene expression in imbibed seeds.