Multi-functional acetyl-CoA carboxylase from Brassica napus is encoded by a multi-gene family: indication for plastidic localization of at least one isoform

Abstract

Three genes coding for different multifunctional acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) isoenzymes from Brassica napus were isolated and divided into two major classes according to structural features in their 5' regions: class I comprises two genes with an additional coding exon of approximately 300 bp at the 5' end, and class II is represented by one gene carrying an intron of 586 bp in its 5' untranslated region. Fusion of the peptide sequence encoded by the additional first exon of a class I ACCase gene to the jellyfish Aequorea victoria green fluorescent protein (GFP) and transient expression in tobacco protoplasts targeted GFP to the chloroplasts. In contrast to the deduced primary structure of the biotin carboxylase domain encoded by the class I gene, the corresponding amino acid sequence of the class II ACCase shows higher identity with that of the Arabidopsis ACCase, both lacking a transit peptide. The Arabidopsis ACCase has been proposed to be a cytosolic isoenzyme. These observations indicate that the two classes of ACCase genes encode plastidic and cytosolic isoforms of multi-functional, eukaryotic type, respectively, and that B. napus contains at least one multi-functional ACCase besides the multi-subunit, prokaryotic type located in plastids. Southern blot analysis of genomic DNA from B. napus, Brassica rapa, and Brassica oleracea, the ancestors of amphidiploid rapeseed, using a fragment of a multi-functional ACCase gene as a probe revealed that ACCase is encoded by a multi-gene family of at least five members.

Figure 1

Restriction map of genomic clones of ACCase genes from B. napus. The organization of different ACCase gene classes is shown. Clones BnACCaseg3 and 8 are overlapping and represent the complete class I.1 gene (promoter and coding sequence). Restriction fragments hybridizing with the PCR probe used for gene isolation are indicated as gray bars. Regions analyzed by sequencing are shown as white bars interrupted by black boxes describing exons of the coding region or by a gray box pointing to an intron in the 5′ untranslated region (BnACCaseg1). Exons that are confirmed by sequencing of cDNA fragments generated by reverse-transcriptase–PCR are underlined. Positions of the identified start ATG and stop TGA codons as well as the MKM biotin binding motif are indicated. The “Y” at the ends of each clone symbolizes XbaI, SacI, NotI, and SalI restriction sites.

Figure 2

Comparison between deduced N-terminal amino acid sequences of ACCase genes from B. napus [BnACCaseg8 (32); BnACCaseg1, accession no. Y10301Y10301; BnACCaseg10, accession no. Y10302Y10302] and Arabidopsis (20). The first 100 residues deduced from BnACCaseg8 and 10 are encoded by an additional 5′ exon. Amino acids that are not identical as compared with that encoded by BnACCaseg8 are indicated in reverse print.

Figure 3

Subcellular localization of green fluorescent protein (GFP S65C) with (a and b) or without (c) fusion to the peptide sequence encoded by the additional 5′ exon of the ACCase class I.1 gene in tobacco protoplasts. The additional 5′ exon was amplified by PCR and cloned into the GFP expression vector pCK GFP S65C (35). The GFP as well as the GFP fusion protein was transiently expressed in tobacco protoplasts transfected according to the protocol of Negrutiu et al. (36). Pictures depicting protoplasts 4 days upon transfection were documented either in bright field (Upper) or under blue light excitation (Lower) with filter block I3 (blue light exciter BP, 450–490 nm; beamsplitter RKP, 510 nm; emiter LP, 520 nm) from Leitz on Kodak Ektachrome 320T and Kodak Ektachrome Panther P1600X films.

Figure 4

Southern blot analysis with genomic DNA from B. napus cultivars Akela and Drakkar, B. rapa, and B. oleracea. Genomic DNA digested by EcoRI (E) was probed with the 766-bp EcoRI/XbaI fragment of clone BnACCaseg1 encompassing exon 2-4 of the class II.1 ACCase gene.

Figure 5

Dendrogram based on a comparison of deduced amino acid sequences covering BC domains or BC subunits from ACCases of different organisms using the computer program gcg pileup (47). BC domain sequences were identified showing significant similarity to the primary structure of E. coli BC. The increase in length of the horizontal lines schematically indicates the degree of decreasing similarity. Amino acid sequences of BC domains and BC subunits of ACCases are taken from following organisms: B. napus (32), Arabidopsis thaliana (20), alfalfa (28), wheat (29), maize (31), rat (48), chicken (49), yeast (50), Cyclotella cryptica (51), E. coli BC (52), and tobacco BC (21).