gurke and pasticcino3 mutants affected in embryo development are impaired in acetyl-CoA carboxylase

Abstract

Normal embryo development is required for correct seedling formation. The Arabidopsis gurke and pasticcino3 mutants were isolated from different developmental screens and the corresponding embryos exhibit severe defects in their apical region, affecting bilateral symmetry. We have recently identified lethal acc1 mutants affected in acetyl-CoA carboxylase 1 (ACCase 1) that display a similar embryo phenotype. A series of crosses showed that gk and pas3 are allelic to acc1 mutants, and direct sequencing of the ACC1 gene revealed point mutations in these new alleles. The isolation of leaky acc1 alleles demonstrated that ACCase 1 is essential for correct plant development and that mutations in ACCase affect cellular division in plants, as is the case in yeast. Interestingly, significant metabolic complementation of the mutant phenotype was obtained by exogenous supply of malonate, suggesting that the lack of cytosolic malonyl-CoA is likely to be the initial factor leading to abnormal development in the acc1 mutants.

Figure 1

Identification of gksC, pas3-1 and pas3-2 mutations in ACC1 gene. (A) Structure of ACC1 gene and position of the point mutations identified in gk-SC, pas3-1 and pas3-2 mutants; acc1-1 and acc1-2 mutations. Closed boxes represent exons, whereas open boxes stand for untranslated regions (UTRs). Nucleotide positions are relative to the translational start site. (B) Characterization of abnormal ACC1 transcripts in gk-SC embryos. Primer sets were used to amplify EF (EF1.up and EF1.low; control) and ACC1 (ACC1.up and ACC1.low) on cDNA from excised Ler and gk-SC embryos aged 15 DAA and on Ler DNA. ACC1.up was designed in the first exon of ACC1 gene, and ACC1.low in the second exon. (C) Amino-acid sequence alignments of a conserved motif in the CT domain of ACCases. Bold residues indicate positions where amino acids are highly conserved. The asterisk indicates the conserved amino-acid residue mutated in pas3-1. (D) Amino-acid sequence alignments of a conserved motif in the CT domain of ACCases. Bold residues indicate positions where amino acids are highly conserved. The asterisk indicates the conserved amino-acid residue mutated in pas3-2. AtACC1, A. thaliana ACCase 1 (At1g36160); ZmACC, Zea mays ACCase (U19183); TaACC, Triticum aestivum ACCase (U10187); GgACC, Gallus gallus ACCase (J03541); HsACC, Homo sapiens ACCase 1 (U19822); ScFAS3, S. cerevisiae FAS3 (M92156).

Figure 2

Multifunctional ACCase detection in excised pas3 and gksC embryos. Fresh seeds were removed from heterozygous siliques and mutant embryos excised from severely wrinkled seeds were sampled. Total proteins of excised embryos aged 15 DAA were submitted to SDS–polyacrylamide gel electrophoresis on a 4–15% polyacrylamide gel. A nitrocellulose blot was probed with peroxidase-conjugated streptavidin, which binds biotin cofactor. Biotinylated polypeptides such as methylcrotonoyl-CoA carboxylase (MCCase) or ACCase were thus revealed.

Figure 3

Fatty acid composition of mature mutant seeds. Total lipid extraction and fatty acid transmethylation were carried out on batches of 20 seeds. Total fatty acid composition (in mol%) of seeds was determined by GC analyses. Results are the mean of three independent measurements; s.d. was always less than 1% of the mean. Fatty acid composition of WT Ws (acc1-1 background), Ler (gk background) and Col0 (pas3 background) seeds was analysed; the results presented for the Ws seeds are representative of the three ecotypes.

Figure 4

Complementation of acc1 mutations by exogenous supply of malonate. (A) Response of immature acc1-1 mutant embryos. Arrested embryos were removed from immature siliques of heterozygous plants at 15 DAA, plated on basal medium, treated every 2 days with malonate and observed after several weeks of culture. Pictures on the bottom row are close-ups of embryos or plantlets presented in pictures on the top row. The development of WT Ws embryos was unaffected by malonate supply (not shown). (B) Response of pas3-1 embryos during germination. Col0 and pas3-1 seeds were germinated on basal and enriched (1 mM malonate) medium and observed after several weeks of culture. Scale bars, 5 mm.