Plastid-localized acetyl-CoA carboxylase of bread wheat is encoded by a single gene on each of the three ancestral chromosome sets

Abstract

5'-End fragments of two genes encoding plastid-localized acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) of wheat (Triticum aestivum) were cloned and sequenced. The sequences of the two genes, Acc-1,1 and Acc-1,2, are 89% identical. Their exon sequences are 98% identical. The amino acid sequence of the biotin carboxylase domain encoded by Acc-1,1 and Acc-1,2 is 93% identical with the maize plastid ACCase but only 80-84% identical with the cytosolic ACCases from other plants and from wheat. Four overlapping fragments of cDNA covering the entire coding region were cloned by PCR and sequenced. The wheat plastid ACCase ORF contains 2,311 amino acids with a predicted molecular mass of 255 kDa. A putative transit peptide is present at the N terminus. Comparison of the genomic and cDNA sequences revealed introns at conserved sites found in the genes of other plant multifunctional ACCases, including two introns absent from the wheat cytosolic ACCase genes. Transcription start sites of the plastid ACCase genes were estimated from the longest cDNA clones obtained by 5'-RACE (rapid amplification of cDNA ends). The untranslated leader sequence encoded by the Acc-1 genes is at least 130-170 nucleotides long and is interrupted by an intron. Southern analysis indicates the presence of only one copy of the gene in each ancestral chromosome set. The gene maps near the telomere on the short arm of chromosomes 2A, 2B, and 2D. Identification of three different cDNAs, two corresponding to genes Acc-1,1 and Acc-1,2, indicates that all three genes are transcriptionally active.

Figure 1

Structure of wheat plastid ACCase cDNA and 5′-end fragments of its genes. Functional domains were identified by sequence comparison as described in ref. . BC, biotin carboxylase; BCC, biotin carboxyl carrier; CT, carboxyltransferase; cDNA pcw, coding sequence of plastid ACCase from wheat. Probe ucg1, further defined in Materials and Methods and corresponding to the genetic marker Xucg1, was used in library screening and chromosome mapping. GenBank accession numbers: pcw, AF029895; Acc-1,1, AF029897; Acc-1,2, AF029896; Wis2, X05995, X57168; pTa2, X06952; Itr1, X65875; and BARE1, Z17327.

Figure 2

Alignment of amino acid sequences of the N termini of T. aestivum plastid ACCase (Tap), Zea mays plastid ACCase (Zmp), B. napus plastid ACCase (Bnp), T. aestivum cytosolic ACCase (Tac), and the biotin carboxylase subunit of Anabaena 7120 ACCase (An). The first conserved amino acid present in all sequences (leucine, shown in boldface) identifies the beginning of the biotin carboxylase domain. The beginning of the mature ACCase (processing site) has not yet been established for any of the plastid ACCases. ∗, Amino acid identity with the T. aestivum plastid ACCase; #, an amino acid identical in all the sequences. Sequence accession numbers are listed in Table 1.

Figure 3

Southern analysis of genomic DNAs. (a) Ae. tauschii accessions TA1691, var. meyeri, and Ae. tauschii accessions TA1704, var. typica. (b) Synthetic hexaploid wheat W-7984 and hexaploid wheat variety Opata 85. cDNA probe ucg1 was used to reveal wheat plastid ACCase DNA.

Figure 4

Chromosome mapping of wheat plastid ACCase genes. (a) Southern analysis of Chinese Spring NT lines. DNA was digested with HindIII. (b) Genetic maps of chromosome 2A and 2D, and consensus physical map of wheat chromosome 2. Position of Acc-1 genes was revealed by hybridization with cDNA probe ucg1. Names of markers, distances in centimorgans (genetic maps), and distances of the deletion breakpoints of various deletion lines from the centromere as fraction of the arm length (physical map) are shown. Lines connecting the maps indicate positions of markers in common, including Xucg1(ACCp) marked with the dashed line, and the position of the centromere marked on the genetic maps with a solid oval symbol. The chromosome 2A map, obtained from the GrainGenes database (http//:wheat.pw.usda.gov/graingenes.htm/.) was published previously (36).