Heteromeric ACCase Research Articles

Expression of Acetyl Coa Carboxylase Gene from Cleome Viscosa L. and Its Seed Oil

Asian spider flower (Cleome viscosa L.) is annual herbaceous weed plant. Its seed oil has similar properties to the seed oil from Jatropha curcas L. which is the biodiesel plant. This research focused on the expression of acetyl CoA carboxylase gene in 4 stages of seeds (1-4 weeks after fruit set (WAF)) by Reverse transcriptase Polymerase Chain Reaction (RT-PCR). Acetyl CoA carboxylase (ACCase) is the essential enzyme for the first step in the biosynthesis of long-chain fatty and glycerol. The heteromeric ACCase consists of 4 subunits. accA, accB and accC gene were located in nuclear, while the accD gene was in chloroplast. The result showed that the expression of accA gene showed the most highly expressed in 3 WAF, while the expression of accC and accD showed the most highly expression in 2-3 WAF. These results indicated that co-expression of accA, accC and accD were highest in 3 WAF and down regulated in 4 WAF. In contrast, accB gene showed the high expression level in 4 weeks after fruit set. The total oil was obtained by petroleum ether soxhlet extraction. The high oil content was 3-4 WAF. These findings suggested that comparatively high expression of the Accase gene related to the persistence of oil accumulation during the late stage of seed oil formation for C. viscosa L.

Chromosome-level genome of Camellia lanceoleosa provides a valuable resource for understanding genome evolution and self-incompatibility.

The section Oleifera (Theaceae) has attracted attention for the high levels of unsaturated fatty acids found in its seeds. Here, we report the chromosome-scale genome of the sect. Oleifera using diploid wild Camellia lanceoleosa with a final size of 3.00 Gb and an N50 scaffold size of 186.43 Mb. Repetitive sequences accounted for 80.63% and were distributed unevenly across the genome. Camellia lanceoleosa underwent a whole-genome duplication event approximately 65 million years ago (65 Mya), prior to the divergence of C. lanceoleosa and Camellia sinensis (approx. 6-7 Mya). Syntenic comparisons of these two species elucidated the genomic rearrangement, appearing to be driven in part by the activity of transposable elements. The expanded and positively selected genes in C. lanceoleosa were significantly enriched in oil biosynthesis, and the expansion of homomeric acetyl-coenzyme A carboxylase (ACCase) genes and the seed-biased expression of genes encoding heteromeric ACCase, diacylglycerol acyltransferase, glyceraldehyde-3-phosphate dehydrogenase and stearoyl-ACP desaturase could be of primary importance for the high oil and oleic acid content found in C. lanceoleosa. Theanine and catechins were present in the leaves of C. lanceoleosa. However, caffeine can not be dectected in the leaves but was abundant in the seeds and roots. The functional and transcriptional divergence of genes encoding SAM-dependent N-methyltransferases may be associated with caffeine accumulation and distribution. Gene expression profiles, structural composition and chromosomal location suggest that the late-acting self-incompatibility of C. lanceoleosa is likely to have favoured a novel mechanism co-occurring with gametophytic self-incompatibility. This study provides valuable resources for quantitative and qualitative improvements and genome assembly of polyploid plants in sect. Oleifera.

The BADC and BCCP subunits of chloroplast acetyl-CoA carboxylase sense the pH changes of the light–dark cycle

Acetyl-CoA carboxylase (ACCase) catalyzes the first committed step in the de novo synthesis of fatty acids. The multisubunit ACCase in the chloroplast is activated by a shift to pH 8 upon light adaptation and is inhibited by a shift to pH 7 upon dark adaptation. Here, titrations with the purified ACCase biotin attachment domain-containing (BADC) and biotin carboxyl carrier protein (BCCP) subunits from Arabidopsis indicated that they can competently and independently bind biotin carboxylase (BC) but differ in responses to pH changes representing those in the plastid stroma during light or dark conditions. At pH 7 in phosphate buffer, BADC1 and BADC2 gain an advantage over BCCP1 and BCCP2 in affinity for BC. At pH 8 in KCl solution, however, BCCP1 and BCCP2 had more than 10-fold higher affinity for BC than did BADC1. The pH-modulated shifts in BC preferences for BCCP and BADC partners suggest they contribute to light-dependent regulation of heteromeric ACCase. Using NMR spectroscopy, we found evidence for increased intrinsic disorder of the BADC and BCCP subunits at pH 7. We propose that this intrinsic disorder potentially promotes fast association with BC through a "fly-casting mechanism." We hypothesize that the pH effects on the BADC and BCCP subunits attenuate ACCase activity by night and enhance it by day. Consistent with this hypothesis, Arabidopsis badc1 badc3 mutant lines grown in a light-dark cycle synthesized more fatty acids in their seeds. In summary, our findings provide evidence that the BADC and BCCP subunits function as pH sensors required for light-dependent switching of heteromeric ACCase activity.

Two Independent Plastid accD Transfers to the Nuclear Genome of Gnetum and Other Insights on Acetyl-CoA Carboxylase Evolution in Gymnosperms.

Acetyl-CoA carboxylase (ACCase) is the key regulator of fatty acid biosynthesis. In most plants, ACCase exists in two locations (cytosol and plastids) and in two forms (homomeric and heteromeric). Heteromeric ACCase comprises four subunits, three of them (ACCA–C) are nuclear encoded (nr) and the fourth (ACCD) is usually plastid encoded. Homomeric ACCase is encoded by a single nr-gene (ACC). We investigated the ACCase gene evolution in gymnosperms by examining the transcriptomes of newly sequenced Gnetum ula, combined with 75 transcriptomes and 110 plastomes of other gymnosperms. AccD-coding sequences are elongated through the insertion of repetitive DNA in four out of five cupressophyte families (except Sciadopityaceae) and were functionally transferred to the nucleus of gnetophytes and Sciadopitys. We discovered that, among the three genera of gnetophytes, only Gnetum has two copies of nr-accD. Furthermore, using protoplast transient expression assays, we experimentally verified that the nr-accD precursor proteins in Gnetum and Sciadopitys can be delivered to the plastids. Of the two nr-accD copies of Gnetum, one dually targets plastids and mitochondria, whereas the other potentially targets plastoglobuli. The distinct transit peptides, gene architectures, and flanking sequences between the two Gnetum accDs suggest that they have independent origins. Our findings are the first account of two distinctly targeted nr-accDs of any green plants and the most comprehensive analyses of ACCase evolution in gymnosperms to date.

Seed-Specific Expression of AtLEC1 Increased Oil Content and Altered Fatty Acid Composition in Seeds of Peanut (Arachis hypogaea L.).

Peanut (Arachis hypogaea L.) is one of the major oil crops and is the fifth largest source of plant oils in the world. Numerous genes participate in regulating the biosynthesis and accumulation of the storage lipids in seeds or other reservoir organs, among which several transcription factors, such as LEAFY COTYLEDON1 (AtLEC1), LEC2, and WRINKLED1 (WRI1), involved in embryo development also control the lipid reservoir in seeds. In this study, the AtLEC1 gene was transferred into the peanut genome and expressed in a seed-specific manner driven by the NapinA full-length promoter or its truncated 230-bp promoter. Four homozygous transgenic lines, two lines with the longer promoter and the other two with the truncated one, were selected for further analysis. The AtLEC1 mRNA level and the corresponding protein accumulation in different transgenic overexpression lines were altered, and the transgenic plants grew and developed normally without any detrimental effects on major agronomic traits. In the developing seeds of transgenic peanuts, the mRNA levels of a series of genes were upregulated. These genes are associated with fatty acid (FA) biosynthesis and lipid accumulation. The former set of genes included the homomeric ACCase A (AhACC II), the BC subunit of heteromeric ACCase (AhBC4), ketoacyl-ACP synthetase (AhKAS II), and stearoyl-ACP desaturase (AhSAD), while the latter ones were the diacylglycerol acyltransferases and oleosins (AhDGAT1, AhDGAT2, AhOle1, AhOle2, and AhOle3). The oil content and seed weight increased by 4.42–15.89% and 11.1–22.2%, respectively, and the levels of major FA components including stearic acid, oleic acid, and linoleic acid changed significantly in all different lines.

Overexpression of Heteromeric GhACCase Subunits Enhanced Oil Accumulation in Upland Cotton

Acetyl-CoA carboxylase (ACCase) catalyzes acetyl-CoA to produce malonyl-CoA, which is the essential first step in the biosynthesis of de novo fatty acids. The heteromeric ACCase of higher plants, such as upland cotton, is composed of four subunits: biotin carboxyl carrier protein (BCCP), biotin carboxylase (BC), α-subunit of carboxyltransferase (CTα), and β-subunit of carboxyltransferase (CTβ). In this study, four subunits encoding heteromeric ACCase were amplified and the structures were analyzed further. The result showed that 7, 16, 10, and 1 exon(s) were identified from GhBCCP1, GhBC1, GhCTα2, and GhCTβ, respectively. The expression pattern analysis showed that the transcripts of these four subunits were ubiquitous in all the tested tissues and may have a positive correlation between the four subunits and oil accumulation. The four subunits coordinate to enhance oil content. The expression patterns and levels varied under ABA, MeJA treatments, and cold stress. Furthermore, under the control of a seed-specific AGP promoter, overexpression of the four subunits separately in Upland cotton demonstrated that GhBCCP1 transgenic plants showed a significant increase of 21.92% in oil content in cotton seeds, while about a 17% increase was noted for GhBC1 and GhCTβ, respectively. So overexpression of each subunit of heteromeric GhACCase may effectively increase seed oil content in Upland cotton.

Analysis of Arabidopsis Accessions Hypersensitive to a Loss of Chloroplast Translation.

Natural accessions of Arabidopsis (Arabidopsis thaliana) differ in their ability to tolerate a loss of chloroplast translation. These differences can be attributed in part to variation in a duplicated nuclear gene (ACC2) that targets homomeric acetyl-coenzyme A carboxylase (ACCase) to plastids. This functional redundancy allows limited fatty acid biosynthesis to occur in the absence of heteromeric ACCase, which is encoded in part by the plastid genome. In the presence of functional ACC2, tolerant alleles of several nuclear genes, not yet identified, enhance the growth of seedlings and embryos disrupted in chloroplast translation. ACC2 knockout mutants, by contrast, are hypersensitive. Here we describe an expanded search for hypersensitive accessions of Arabidopsis, evaluate whether all of these accessions are defective in ACC2, and characterize genotype-to-phenotype relationships for homomeric ACCase variants identified among 855 accessions with sequenced genomes. Null alleles with ACC2 nonsense mutations, frameshift mutations, small deletions, genomic rearrangements, and defects in RNA splicing are included among the most sensitive accessions examined. By contrast, most missense mutations affecting highly conserved residues failed to eliminate ACC2 function. Several accessions were identified where sensitivity could not be attributed to a defect in either ACC2 or Tic20-IV, the chloroplast membrane channel required for ACC2 uptake. Overall, these results underscore the central role of ACC2 in mediating Arabidopsis response to a loss of chloroplast translation, highlight future applications of this system to analyzing chloroplast protein import, and provide valuable insights into the mutational landscape of an important metabolic enzyme that is highly conserved throughout eukaryotes.

Phylogenetic Analysis of Nucleus-Encoded Acetyl-CoA Carboxylases Targeted at the Cytosol and Plastid of Algae.

The understanding of algal phylogeny is being impeded by an unknown number of events of horizontal gene transfer (HGT), and primary and secondary/tertiary endosymbiosis. Through these events, previously heterotrophic eukaryotes developed photosynthesis and acquired new biochemical pathways. Acetyl-CoA carboxylase (ACCase) is a key enzyme in the fatty acid synthesis and elongation pathways in algae, where ACCase exists in two locations (cytosol and plastid) and in two forms (homomeric and heteromeric). All algae contain nucleus-encoded homomeric ACCase in the cytosol, independent of the origin of the plastid. Nucleus-encoded homomeric ACCase is also found in plastids of algae that arose from a secondary/tertiary endosymbiotic event. In contrast, plastids of algae that arose from a primary endosymbiotic event contain heteromeric ACCase, which consists of three nucleus-encoded and one plastid-encoded subunits. These properties of ACCase provide the potential to inform on the phylogenetic relationships of hosts and their plastids, allowing different hypothesis of endosymbiotic events to be tested. Alveolata (Dinoflagellata and Apicomplexa) and Chromista (Stramenopiles, Haptophyta and Cryptophyta) have traditionally been grouped together as Chromalveolata, forming the red lineage. However, recent genetic evidence groups the Stramenopiles, Alveolata and green plastid containing Rhizaria as SAR, excluding Haptophyta and Cryptophyta. Sequences coding for plastid and cytosol targeted homomeric ACCases were isolated from Isochrysis aff. galbana (TISO), Chromera velia and Nannochloropsis oculata, representing three taxonomic groups for which sequences were lacking. Phylogenetic analyses show that cytosolic ACCase strongly supports the SAR grouping. Conversely, plastidial ACCase groups the SAR with the Haptophyta, Cryptophyta and Prasinophyceae (Chlorophyta). These two ACCase based, phylogenetic relationships suggest that the plastidial homomeric ACCase was acquired by the Haptophyta, Cryptophyta and SAR, before the photosynthetic Rhizaria acquired their green plastid. Additionally, plastidial ACCase was derived by HGT from an ancestor or relative of the Prasinophyceae and not by duplication of cytosolic ACCase.

Insights into Molecular Assembly of ACCase Heteromeric Complex in Chlorella variabilis—A Homology Modelling, Docking and Molecular Dynamic Simulation Study

Acetyl-CoA carboxylase (ACCase), a biotin-dependent enzyme that catalyses the first committed step of fatty acid biosynthesis, is considered as a potential target for improving lipid accumulation in oleaginous feedstocks, including microalgae. ACCase is composed of three distinct conserved domains, and understanding the structural details of each catalytic domain assumes great significance to gain insights into the molecular basis of the complex formation and mechanism of biotin transport. In the absence of a crystal structure for any single heteromeric ACCase till date, here we report the first heteromeric association model of ACCase from an oleaginous green microalga, Chlorella variabilis, using a combination of homology modelling, docking and molecular dynamic simulations. The binding site of the docked biotin carboxylase (BC) and carboxyltransferase (CT) were predicted to be contiguous but distinct in biotin carboxyl carrier protein (BCCP) molecule. Simulation studies revealed considerable flexibility for the BC and CT domains in the BCCP-bound forms, thus indicating the adaptive behaviour of BCCP. Further, principal component analysis revealed that in the presence of BCCP, the BC and CT domains exhibited an open-state conformation via the outward clockwise rotation of the binding helices. These conformational changes might be responsible for binding of BCCP domain and its translocation to the respective active sites. Various rearrangements of inter-domain hydrogen bonds (H-bonds) contributed to conformational changes in the structures. H-bond interactions between the interacting residue pairs involving Glu201BCCP/Arg255BC and Asp224BCCP/Gln228CT were found to be essential for the intermolecular assembly. The present findings are consistent with previous biochemical studies.

Genes encoding the biotin carboxylase subunit of acetyl-CoA carboxylase from Brassica napus and parental species: cloning, expression patterns, and evolution

Comparative genomics is a useful tool to investigate gene and genome evolution. Biotin carboxylase (BC), an important subunit of heteromeric acetyl-CoA carboxylase (ACCase) that is a rate-limiting enzyme in fatty acid biosynthesis in dicots, catalyzes ATP, biotin carboxyl carrier protein, and CO2 to form carboxybiotin carboxyl carrier protein. In this study, we cloned four genes encoding BC from Brassica napus L. (namely BnaC.BC.a, BnaC.BC.b, BnaA.BC.a, and BnaA.BC.b), and two were cloned from each of the two parental species Brassica rapa L. (BraA.BC.a and BraA.BC.b) and Brassica oleracea L. (BolC.BC.a and BolC.BC.b). Sequence analyses revealed that in B. napus the genes BnaC.BC.a and BnaC.BC.b were from the C genome of B. oleracea, whereas BnaA.BC.a and BnaA.BC.b were from the A genome of B. rapa. Comparative and cluster analysis indicated that these genes were divided into two major groups, BnaC.BC.a, BnaA.BC.a, BraA.BC.a, and BolC.BC.a in group-1 and BnaC.BC.b, BnaA.BC.b, BraA.BC.b, and BolC.BC.b in group-2. The divergence of group-1 and group-2 genes occurred in their common ancestor 13-17 million years ago (MYA), soon after the divergence of Arabidopsis and Brassica (15-20 MYA). This time of divergence is identical to the previously reported triplicated time of paralogous subgenomes of diploid Brassica species and the divergence date of group-1 and group-2 genes of α-carboxyltransferase, another subunit of heteromeric ACCase, in Brassica. Reverse transcription PCR revealed that the expression level of group-1 and group-2 genes varied in different organs, and the expression patterns of the two groups of genes were similar in different organs, except in flower. However, two paralogs of group-2 BC genes from B. napus could express differently in mature plants tested by generating BnaA.BC.b and BnaC.BC.b promoter-β-glucuronidase (GUS) fusions. The amino acid sequences of proteins encoded by these genes were highly conserved, except the sequence encoding predicted plastid transit peptides. The plastid transit peptides on the BC precursors of Brassica (71-72 amino acid residues) were predicted based on AtBC protein, compared, and confirmed by fusion with green fluorescent protein. Our results will be helpful in elucidating the evolution and the regulation of ACCase in the genus Brassica.

Molecular cloning and expression of heteromeric ACCase subunit genes from Jatropha curcas

Acetyl-CoA carboxylase (ACCase) catalyzes the biotin-dependent carboxylation of acetyl-CoA to produce malonyl-CoA, which is the essential first step in the biosynthesis of long-chain fatty acids. ACCase exists as a multi-subunit enzyme in most prokaryotes and the chloroplasts of most plants and algae, while it is present as a multi-domain enzyme in the endoplasmic reticulum of most eukaryotes. The heteromeric ACCase of higher plants consists of four subunits: an α-subunit of carboxyltransferase (α-CT, encoded by accA gene), a biotin carboxyl carrier protein (BCCP, encoded by accB gene), a biotin carboxylase (BC, encoded by accC gene) and a β-subunit of carboxyltransferase (β-CT, encoded by accD gene). In this study, we cloned and characterized the genes accA, accB1, accC and accD that encode the subunits of heteromeric ACCase in Jatropha ( Jatropha curcas), a potential biofuel plant. The full-length cDNAs of the four subunit genes were isolated from a Jatropha cDNA library and by using 5′ RACE, whereas the genomic clones were obtained from a Jatropha BAC library. They encode a 771 amino acid (aa) α-CT, a 286-aa BCCP1, a 537-aa BC and a 494-aa β-CT, respectively. The single-copy accA, accB1 and accC genes are nuclear genes, while the accD gene is located in chloroplast genome. Jatropha α-CT, BCCP1, BC and β-CT show high identity to their homologues in other higher plants at amino acid level and contain all conserved domains for ACCase activity. The accA, accB1, accC and accD genes are temporally and spatially expressed in the leaves and endosperm of Jatropha plants, which are regulated by plant development and environmental factors.

Cloning and expression of a plastid-encoded subunit, beta-carboxyltransferase gene ( accD) and a nuclear-encoded subunit, biotin carboxylase of acetyl-CoA carboxylase from oil palm ( Elaeis guineensis Jacq.)

Palm oil is the second largest traded oil or fat in the world market and palm is the most important crop grown mainly for its oil. Identified varieties of Elaeis guineensis Jacq., with a high oil content and produced through a selective breeding program, are desirable for improving the yield of oil, subsequently enhancing the economic feasibility of using oil palm in various applications, including bio-diesel. We have cloned the gene of biotin carboxylase ( accD) from E. guineensis Jacq. This gene encodes a plastid-coded subunit of heteromeric acetyl-CoA carboxylase (ACCase). The cDNA of accD gene (accession number DQ004687) has an open reading frame of 1479 bp that encodes a putative protein of 492 amino acid residues ( AAY86362) with a predicted molecular mass of 55.47 kDa. The heteromeric form of ACCase is important as it catalyzes the first committed step of fatty acid synthesis. There is evidence that collectively suggests that the expression of accD in plastids is crucial to the levels of heteromeric ACCase and in turn, to the amount of seed oil in plant. Here we support the hypothesis that the expression level of accD is correlated with the oil palm production by using a semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time polymerase chain reaction (real-time PCR) analysis. Moreover, we observed the similar expression profile in nuclear-encoded subunit, biotin carboxylase ( accC). This finding represents the genetic background of the expressed genes that correlate to high yield in plant and that ACCase can be used as a marker in the breeding program.

Plant acetyl-CoA carboxylase: structure, biosynthesis, regulation, and gene manipulation for plant breeding.

Acetyl-CoA carboxylase (ACCase) catalyzes the first committed step of fatty acid synthesis, the carboxylation of acetyl-CoA to malonyl-CoA. Two physically distinct types of enzymes are found in nature. Heteromeric ACCase composed of four subunits is usually found in prokaryotes, and homomeric ACCase composed of a single large polypeptide is found in eukaryotes. Most plants have both forms, the heteromeric form in plastids, in which de novo fatty acids are synthesized, and the homomeric form in cytosol. This review focuses on the structure and regulation of plant heteromeric ACCase and its manipulation for plant breeding.

Coordinate regulation of the nuclear and plastidic genes coding for the subunits of the heteromeric acetyl-coenzyme A carboxylase.

Plastidic acetyl-coenzyme A (CoA) carboxylase (ACCase) catalyzes the first committed reaction of de novo fatty acid biosynthesis. This heteromeric enzyme is composed of one plastid-coded subunit (beta-carboxyltransferase) and three nuclear-coded subunits (biotin carboxy-carrier, biotin carboxylase, and alpha-carboxyltransferase). We report the primary structure of the Arabidopsis alpha-carboxyltransferase and beta-carboxyltransferase subunits deduced from nucleotide sequences of the respective genes and/or cDNA. Co-immunoprecipitation experiments confirm that the alpha-carboxyltransferase and beta-carboxyltransferase subunits are physically associated. The plant alpha-carboxyltransferases have gained a C-terminal domain relative to eubacteria, possibly via the evolutionary acquisition of a single exon. This C-terminal domain is divergent among plants and may have a structural function rather than being essential for catalysis. The four ACCase subunit mRNAs accumulate to the highest levels in tissues and cells that are actively synthesizing fatty acids, which are used either for membrane biogenesis in rapidly growing tissues or for oil accumulation in developing embryos. Development coordinately affects changes in the accumulation of the ACCase subunit mRNAs so that these four mRNAs maintain a constant molar stoichiometric ratio. These data indicate that the long-term, developmentally regulated expression of the heteromeric ACCase is in part controlled by a mechanism(s) that coordinately affects the steady-state concentrations of each subunit mRNA.