Brassicaceae express multiple isoforms of biotin carboxyl carrier protein in a tissue-specific manner.

Plastidial acetyl-coenzyme A carboxylase from most plants is a multi-enzyme complex comprised of four different subunits. One of these subunits, the biotin carboxyl carrier protein (BCCP), was previously proposed to be encoded by a single gene in Arabidopsis. We report and characterize here a second Arabidopsis BCCP (AtBCCP2) cDNA with 42% amino acid identity to AtBCCP1 and 75% identity to a class of oilseed rape (Brassica napus) BCCPs. Both Arabidopsis BCCP isoforms were expressed in Escherichia coli and found to be biotinylated and supported carboxylation activity when reconstituted with purified, recombinant Arabidopsis biotin carboxylase. In vitro translated AtBCCP2 was competent for import into pea (Pisum sativum) chloroplasts and processed to a 25-kD polypeptide. Extracts of Arabidopsis seeds contained biotinylated polypeptides of 35 and 25 kD, in agreement with the masses of recombinant AtBCCP1 and 2, respectively. AtBCCP1 protein was present in developing tissues from roots, leaves, flowers, siliques, and seeds, whereas AtBCCP2 protein was primarily expressed in 7 to 10 d-after-flowering seeds at levels approximately 2-fold less abundant than AtBCCP1. AtBCCP1 transcript reflected these protein expression profiles present in all developing organs and highest in 14-d leaves and siliques, whereas AtBCCP2 transcript was present in flowers and siliques. In protein blots, four different BCCP isoforms were detected in developing seeds from oilseed rape. Of these, a 35-kD BCCP was detected in immature leaves and developing seeds, whereas developing seeds also contained 22-, 25-, and 37-kD isoforms highly expressed 21 d after flowering. These data indicate that oilseed plants in the family Brassicaceae contain at least one to three seed-up-regulated BCCP isoforms, depending upon genome complexity.

A new set of Arabidopsis expressed sequence tags from developing seeds. The metabolic pathway from carbohydrates to seed oil.

Large-scale single-pass sequencing of cDNAs from different plants has provided an extensive reservoir for the cloning of genes, the evaluation of tissue-specific gene expression, markers for map-based cloning, and the annotation of genomic sequences. Although as of January 2000 GenBank contained over 220,000 entries of expressed sequence tags (ESTs) from plants, most publicly available plant ESTs are derived from vegetative tissues and relatively few ESTs are specifically derived from developing seeds. However, important morphogenetic processes are exclusively associated with seed and embryo development and the metabolism of seeds is tailored toward the accumulation of economically valuable storage compounds such as oil. Here we describe a new set of ESTs from Arabidopsis, which has been derived from 5- to 13-d-old immature seeds. Close to 28,000 cDNAs have been screened by DNA/DNA hybridization and approximately 10,500 new Arabidopsis ESTs have been generated and analyzed using different bioinformatics tools. Approximately 40% of the ESTs currently have no match in dbEST, suggesting many represent mRNAs derived from genes that are specifically expressed in seeds. Although these data can be mined with many different biological questions in mind, this study emphasizes the import of photosynthate into developing embryos, its conversion into seed oil, and the regulation of this pathway.

Stearoyl-acyl carrier protein and unusual acyl-acyl carrier protein desaturase activities are differentially influenced by ferredoxin.

Acyl-acyl carrier protein (ACP) desaturases function to position a single double bond into an acyl-ACP substrate and are best represented by the ubiquitous Delta9 18:0-ACP desaturase. Several variant acyl-ACP desaturases have also been identified from species that produce unusual monoenoic fatty acids. All known acyl-ACP desaturase enzymes use ferredoxin as the electron-donating cofactor, and in almost all previous studies the photosynthetic form of ferredoxin rather than the non-photosynthetic form has been used to assess activity. We have examined the influence of different forms of ferredoxin on acyl-ACP desaturases. Using combinations of in vitro acyl-ACP desaturase assays and [(14)C]malonyl-coenzyme A labeling studies, we have determined that heterotrophic ferredoxin isoforms support up to 20-fold higher unusual acyl-ACP desaturase activity in coriander (Coriandrum sativum), Thunbergia alata, and garden geranium (Pelargonium x hortorum) when compared with photosynthetic ferredoxin isoforms. Heterotrophic ferredoxin also increases activity of the ubiquitous Delta9 18:0-ACP desaturase 1.5- to 3.0-fold in both seed and leaf extracts. These results suggest that ferredoxin isoforms may specifically interact with acyl-ACP desaturases to achieve optimal enzyme activity and that heterotrophic isoforms of ferredoxin may be the in vivo electron donor for this reaction.

Accumulation of palmitate in Arabidopsis mediated by the acyl-acyl carrier protein thioesterase FATB1.

The acyl-acyl carrier protein thioesterase B1 from Arabidopsis (AtFATB1) was previously shown to exhibit in vitro hydrolytic activity for long chain acyl-acyl carrier proteins (P. Dörmann, T.A. Voelker, J.B. Ohlrogge [1995] Arch Biochem Biophys 316: 612-618). In this study, we address the question of which role in fatty acid biosynthesis this enzyme plays within the plant. Over-expression of the AtFATB1 cDNA under a seed-specific promoter resulted in accumulation of high amounts of palmitate (16:0) in seeds. RNA and protein-blot analysis in Arabidopsis and rapeseed (Brassica napus) showed that the endogenous AtFATB1 expression was highest in flowers and lower in leaves. All floral tissues of wild-type plants contained elevated amounts of 16:0, and in the polar lipid fraction of flowers close to 50 mol % of the fatty acids were 16:0. Therefore, flowers contain polar lipids with an unusually high amount of saturated fatty acids as compared to all other plant tissues. Antisense expression of the AtFATB1 cDNA under the cauliflower mosaic virus 35S promoter resulted in a reduction of seed and flower 16:0 content, but no changes in leaf fatty acids. We conclude that the AtFATB1 thioesterase contributes to 16:0 production particularly in flowers, but that additional factors are involved in leaves.

Biotin carboxyl carrier protein isoforms in Brassicaceae oilseeds.

De novo fatty acid biosynthesis occurs predominantly in plastids. The committed step for this pathway is the production of malonyl-CoA catalysed by acetyl-CoA carboxylase (ACCase). In most plants, plastidial ACCase is a multisubunit complex minimally comprised of four polypeptides, which catalyse two reactions. In the simple oilseed plant, Arabidopsis thaliana, two cDNAs encoding biotin carboxyl carrier protein (BCCP) isoforms have been identified. The remaining three subunits of ACCase appear to be single gene members in A. thaliana [Mekhedov, Martinez de Ilarduya and Ohlrogge (2000) Plant Physiol. 122, 389-401]. Transcript and protein analyses indicate that BCCP isoform 1 is constitutively expressed while isoform 2 is predominantly expressed in developing seeds. The apparent masses of constitutive and seed-enriched BCCP isoforms agree with the apparent masses of recombinantly expressed isoforms 1 and 2, respectively. In a related oilseed, Brassica napus, multiple putative BCCP polypeptides were also observed in developing seeds. The presence of a divergent class of BCCP genes in A. thaliana and B. napus, coincident with appropriately sized biotin-containing proteins expressed specifically in developing seeds, suggests that these BCCPs play an evolutionarily conserved role in oil deposition.

Isoforms of acyl carrier protein involved in seed-specific fatty acid synthesis.

Seeds of coriandrum sativum (coriander) and Thunbergia alata (black-eyed Susan vine) produce unusual monoenoic fatty acids which constitute over 80% of the total fatty acids of the seed oil. The initial step in the formation of these fatty acids is the desaturation of palmitoyl-ACP (acyl carrier protein) at the delta(4) or delta(6) positions to produce delta(4)-hexadecenoic acid (16:1(delta(4)) or delta(6)-hexadecenoic acid (16:1(delta(6)), respectively. The involvement of specific forms of ACP in the production of these novel monoenoic fatty acids was studied. ACPs were partially purified from endosperm of coriander and T. alata and used to generate 3H- and 14C-labelled palmitoyl-ACP substrates. In competition assays with labelled palmitoyl-ACP prepared from spinach (Spinacia oleracea), delta(4)-acyl-ACP desaturase activity was two- to threefold higher with coriander ACP than with spinach ACP. Similarly, the T. alata delta(6) desaturase favoured T. alata ACP over spinach ACP. A cDNA clone, Cs-ACP-1, encoding ACP was isolated from a coriander endosperm cDNA library. Cs-ACP-1 mRNA was predominantly expressed in endosperm rather than leaves. The Cs-ACP-1 mature protein was expressed in E. coli and comigrated on SDS-PAGE with the most abundant ACP expressed in endosperm tissues. In in vitro delta(4)-palmitoyl-ACP desaturase assays, the Cs-ACP-1 expressed from E. coli was four- and 10-fold more active than spinach ACP or E. coli ACP, respectively, in the synthesis of delta(4)-hexadecenoic acid from palmitoyl-ACP. In contrast, delta(9)-stearoyl-ACP desaturase activity from coriander endosperm did not discriminate strongly between different ACP species. These results indicate that individual ACP isoforms are specifically involved in the biosynthesis of unusual seed fatty acids and further suggest that expression of multiple ACP isoforms may participate in determining the products of fatty acid biosynthesis.

Regulation of spinach chloroplast acetyl-CoA carboxylase.

We have investigated several factors which influence acetyl-CoA carboxylase (ACCase) activity in lysed spinach chloroplasts. (1) When assayed after rapid lysis of light-incubated chloroplasts, ACCase activity was 2-fold higher than activity from dark-incubated chloroplasts. Within 5 min after lysis, activity from dark-incubated chloroplasts increased, suggesting a transient inactivation or inhibition of ACCase in the dark. (2) When lysed chloroplast suspensions were incubated with 30 to 100 microM acetyl-CoA before starting assays, activity was 4-fold higher than if suspensions were not preincubated with acetyl-CoA. CoA, malonyl-CoA, propionyl-CoA, and butyryl-CoA also activated ACCase. Full acetyl-CoA activation required MgATP and was essentially complete after 8 min. ACCase activity decreased upon removal of acetyl-CoA by gel filtration and was partially restored by readdition of acetyl-CoA. Thus, ACCase activation by acetyl-CoA was reversible. (3) Dithiothreitol and thioredoxin stimulated ACCase activity, but only in preparations where ACCase activity was low. (4) ACCase was assayed in concentrations of ATP, ADP, NADPH, NADP+, Mg2+, and CO2/HCO-3, which are estimated to occur in the stroma of chloroplasts under illumination or darkness. ACCase activity from lysed chloroplast suspensions was 10-fold higher when illuminated conditions were used. However, this activity was still 5-fold to 10-fold lower than the rates required to sustain known in vivo rates of fatty acid synthesis and in vitro rates achieved under optimum assay conditions with saturating substrates.

Structural homology of spinach acyl carrier protein and Escherichia coli acyl carrier protein based on NMR data.

Acyl carrier proteins (ACPs) from spinach and from Escherichia coli have been used to demonstrate the utility of proton NMR for comparison of homologous structures. The structure of E. coli ACP had been previously determined and modeled as a rapid equilibrium among multiple conformational forms (Kim and Prestegard, Biochemistry 28:8792-8797, 1989). Spinach ACP showed two slowly exchanging forms and could be manipulated into one form for structural study. Here we compare this single form to postulated multiple forms of E. coli ACP using the limited amount of NOE data available for the spinach protein. A number of long-range NOE contacts were present between homologous residues in both spinach and E. coli ACP, suggesting tertiary structural homology. To allow a more definitive structural comparison, a method was developed to use spinach ACP NOE constraints to search for regions of structural divergence from two postulated forms of E. coli ACP. The homologous regions of the two protein sequences were aligned, additional distance constraints were extracted from the E. coli structure, and these were mapped onto the spinach sequence. These distance constraints were combined with experimental NOE constraints and a distance geometry simulated annealing protocol was used to test for compatibility of the constraints. All of the experimental spinach NOE constraints could be successfully combined with the E. coli data, confirming the general hypothesis of structural homology. A better fit was obtained with one form, suggesting a preferential stabilization of that form in the spinach case.

Isolation and characterization of an Arabidopsis biotin carboxylase gene and its promoter.

In the plastids of most plants, acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) is a multisubunit complex consisting of biotin carboxylase (BC), biotin-carboxyl carrier protien (BCCP), and carboxytransferase (alpha-CT, beta-CT) subunits. To better understand the regulation of this enzyme, we have isolated and sequenced a BC genomic clone from Arabidopsis and partially characterized its promoter. Fifteen introns were identified. The deduced amino acid sequence of the mature BC protein is highly conserved between Arabidopsis and tobacco (92.6% identity). BC expression was evaluated using northern blots and BC/GUS fusion constructs in transgenic Arabidopsis. GUS activity in the BC/GUS transgenics as well as transcript level of the native gene were both found to be higher in silique and flower than in root and leaf. Analysis of tobacco suspension cells transformed with truncated BC promoter/GUS gene fusions indicated the region from -140 to +147 contained necessary promoter elements which supported basal gene expression. A positive regulatory region was found to be located between -2100 and -140, whereas a negative element was possibly located in the first intron. In addition, several conserved regulatory elements were identified in the BC promoter. Surprisingly, although BC is a low-abundance protein, the expression of BC/GUS fusion constructs was similar to 35S/GUS constructs.

The pea chloroplast membrane-associated protein, IEP96, is a subunit of acetyl-CoA carboxylase.

Two forms of acetyl-CoA carboxylase (ACCase) have been characterized in pea (Pisum sativum L.) leaves; a heteromeric chloroplast enzyme and a homomeric, presumably cytosolic enzyme. The biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and beta-carboxyltransferase (CT) subunits of the plastidial-ACCase have recently been characterized and cloned. To further characterize the carboxyltransferase, an improved assay for CT was developed and used to follow its partial purification. CT activity co-purifies with ACCase activity during gel permeation chromatography. However, upon anion-exchange chromatography or native PAGE, CT separates from the BC and BCCP subunits of plastidial-ACCase and ACCase activity is lost. In addition, it is demonstrated that a previously sequenced pea chloroplast cDNA of unknown function (IEP96) with a predicted molecular weight of 91 kDa encodes the alpha-CT subunit of the MS-ACCase. Antibodies raised against the first 404 amino acids of IEP96 protein detected a polypeptide with molecular weight of 91 kDa that co-eluted during gel permeation chromatography with plastidial CT and ACCase activities. These antibodies also immunoprecipitated the activities of both ACCase and CT with the concomitant precipitation of the beta-CT subunit. Furthermore, antibodies against beta-CT immunoprecipitated the IEP96 protein. Two-dimensional PAGE and DEAE purification of ACCase protein demonstrated that the beta-CT forms a tight association with the IEP96 protein. Pea leaf was fractionated into soluble and membrane fractions and the alpha-CT subunit was primarily associated with the membrane fraction. Together, these data demonstrate that IEP96 is the alpha-CT subunit of pea chloroplast ACCase.

Characterization of an acyl-CoA-binding protein from Arabidopsis thaliana.

A cDNA clone was obtained from Arabidopsis thaliana that encodes a protein containing 92 amino acid residues with high sequence identity (57%) to bovine acyl-CoA-binding protein (ACBP). The coding sequence of this clone was expressed in Escherichia coli and the gene product (10.4 kDa) was purified. The recombinant A. thaliana ACBP (rAthACBP) was shown to bind acyl-CoA esters and protect acyl-CoAs from degradation by microsomal acyl-hydrolases. Antibodies that were raised to rAthACBP recognized the native Arabidopsis ACBP and also cross-reacted with a number of other plant ACBPs, including rapeseed (Brassica napus) ACBP. The pattern of expression and level of the gene product were examined in various tissues of Arabidopsis and Brassica using Western blotting. A. thaliana tissues contained between 3 and 143 micrograms AthACBP g(-1) FW depending on the tissue (0.4 to 14 nmol g(-1) FW). Developing B. napus seeds underwent a 12-fold increase in ACBP levels during seed maturation (20 to 250 micrograms ACBP g(-1) FW); the highest concentration occurring near the peak of triacylglycerol accumulation (26 nmol g(-1) FW.

Evidence That Isolated Chloroplasts Contain an Integrated Lipid-Synthesizing Assembly That Channels Acetate into Long-Chain Fatty Acids.

High rates of light-dependent fatty acid synthesis from acetate were measured in isolated chloroplasts that were permeabilized to varying extents by resuspension in hypotonic reaction medium. The reactions in hypotonic medium unsupplemented with cofactors were linear with time and were directly proportional to chlorophyll concentration, suggesting that the enzymes and cofactors of fatty acid synthesis remained tightly integrated and thylakoid associated within disrupted chloroplasts. Permeabilized chloroplasts expanded to at least twice the volume of intact chloroplasts, lost about 50% of their stromal proteins in the medium, and metabolized exogenous nucleotides. However, neither acetyl-coenzyme A (CoA) nor malonyl-CoA inhibited fatty acid synthesis from acetate; nor were [1-14C]acetyl-CoA and [14C]malonyl-CoA significantly incorporated into fatty acids. Fatty acid synthesis from acetate was independent of added cofactors but was totally light dependent. Changes in the products of fatty acid synthesis were consistent with the loss of endogenous glycerol-3-phosphate from permeabilized chloroplasts. However, in appropriately supplemented medium, the products of acetate incorporation by spinach (Spinacia oleracea) chloroplasts were similar when reactions were carried out in either isotonic or hypotonic medium. Taken together, the results of this study suggest that the enzymes of fatty acid synthesis with chloroplasts are organized into a multienzyme assembly that channels acetate into long-chain fatty acids, glycerides, and CoA esters.

Structural analysis, plastid localization, and expression of the biotin carboxylase subunit of acetyl-coenzyme A carboxylase from tobacco.

Acetyl-coenzyme A carboxylase (ACCase, EC 6.4.1.2) catalyzes the synthesis of malonyl-coenzyme A, which is utilized in the plastid for de novo fatty acid synthesis and outside the plastid for a variety of reactions, including the synthesis of very long chain fatty acids and flavonoids. Recent evidence for both multifunctional and multisubunit ACCase isozymes in dicot plants has been obtained. We describe here the isolation of a tobacco (Nicotiana tabacum L. cv bright yellow 2 [NT1]) cDNA clone (E3) that encodes a 58.4-kD protein that shares 80% sequence similarity and 65% identity with the Anabaena biotin carboxylase subunit of ACCase. Similar to other biotin carboxylase subunits of acetyl-CoA carboxylase, the E3-encoded protein contains a putative ATP-binding motif but lacks a biotin-binding site (methionine-lysine-methionine or methionine-lysine-leucine). The deduced protein sequence contains a putative transit peptide whose function was confirmed by its ability to direct in vitro chloroplast uptake. The subcellular localization of this biotin carboxylase has also been confirmed to be plastidial by western blot analysis of pea (Pisum sativum), alfalfa (Medicago sativa L.), and castor (Ricinus communis L.) plastid preparations. Northern blot analysis indicates that the plastid biotin carboxylase transcripts are expressed at severalfold higher levels in castor seeds than in leaves.

Analysis of in vivo levels of acyl-thioesters with gas chromatography/mass spectrometry of the butylamide derivative.

The analysis of the in vivo level and composition of acyl-thioester pools, such as acyl-ACP, has been accomplished by selective formation of acyl-butylamides and subsequent analysis by gas chromatography/mass spectrometry. The acyl-butylamide derivative was synthesized by direct aminolysis with n-butylamine. The reaction was specific for thioester-linked acyl groups and 90% conversion was achieved with acyl-ACP and acyl-CoA in aqueous solution. Electron ionization mass spectra exhibited two intense diagnostic ions m/z 115 and 128 common to butylamides of saturated and monounsaturated fatty acids. Mixtures of butylamides with fatty acid moieties ranging between C4 and C20 were analyzed by selective ion monitoring gas chromatography/mass spectrometry set to 115 and 128 amu. The limit for the quantitative analysis of the long-chain 18:0- and 18:1-butylamides was 1.5 pmol and the detection limit was less than 0.5 pmol. The utility of this method was demonstrated by analysis of two model systems: standard acyl-CoA mixtures and in vivo levels of spinach leaf acyl-ACP. A purification protocol based on DE 52 anion exchange chromatography was necessary in order to separate spinach acyl-ACP and acyl-CoA from tissue extracts. The acyl composition obtained from total spinach leaf acyl-ACP by selective ion monitoring gas chromatography/mass spectrometry of the butylamide derivatives matched with the direct analysis of the same sample by urea-polyacrylamide gel electrophoresis and subsequent scanning densitometry of the anti-spinach ACP immunoblot.

Cloning and expression in Escherichia coli of a novel thioesterase from Arabidopsis thaliana specific for long-chain acyl-acyl carrier proteins.

An Arabidopsis thaliana partial cDNA was previously identified with a sequence similar to the lauroyl-acyl carrier protein (ACP) thioesterase from Umbellularia california (Grellet et al., 1993, Plant Physiol. Biochem. 31, 599-602). Using this DNA fragment, we isolated a 1.8-kb cDNA coding for a 412-amino-acid preprotein. The deduced amino acid sequence is 51% identical to the lauroyl-ACP thioesterase but only 39% identical to safflower oleoyl-ACP thioesterase. The cDNA was expressed in Escherichia coli and the gene product showed thioesterase activity for long-chain acyl-ACPs (14:0, 16:0, 18:0, 18:1 delta 9cis). When expressed in beta-oxidation mutants of E. coli, lipid analysis revealed that cells transformed with the thioesterase produced high amounts of free fatty acids that mostly consisted of 16:0 and some 14:0, 16:1 delta 9cis, and 18:1 delta 11cis. Antibodies were raised to the recombinant protein and used to determine tissue-specific and developmental expression in A. thaliana and Brassica napus. A 40-kDa protein was detected by immunoblots in A. thaliana siliques, leaves, and roots. A maximal expression of the B. napus protein between 18 and 31 days after flowering was found, which correlates with the rapid accumulation of triacylglycerols in the seeds. Based upon these results, we suggest that this long-chain acyl-ACP thioesterase may be a ubiquitous enzyme in plants which is involved in the synthesis of long-chain fatty acids.

delta 6 Hexadecenoic acid is synthesized by the activity of a soluble delta 6 palmitoyl-acyl carrier protein desaturase in Thunbergia alata endosperm.

delta 6 Hexadecenoic acid (16:1 delta 6) composes more than 80% of the seed oil of Thunbergia alata. Studies were conducted to determine the biosynthetic origin of the double bond of this unusual fatty acid. Assays of fractions of developing T. alata seed endosperm with [1-14C]palmitoyl (16:0)-acyl carrier protein (ACP) revealed the presence of a soluble delta 6 desaturase activity. This activity was greatest when 16:0-ACP was provided as a substrate, whereas no desaturation of the coenzyme A ester of this fatty acid was detected. In addition, delta 6 16:0-ACP desaturase activity in T. alata endosperm extracts was dependent on the presence of ferredoxin and molecular oxygen and was stimulated by catalase. To further characterize this enzyme, a cDNA encoding a diverged acyl-ACP desaturase was isolated from a T. alata endosperm cDNA library using polymerase chain reaction with degenerate oligonucleotides corresponding to conserved amino acid sequences in delta 9 stearoyl (18:0)- and delta 4 16:0-ACP desaturases. The primary structure of the mature peptide encoded by this cDNA shares 66% identity with the mature castor delta 9 18:0-ACP desaturase and 57% identity with the mature coriander delta 4 16:0-ACP desaturase. Extracts of Escherichia coli that express the T. alata cDNA catalyzed the delta 6 desaturation of 16:0-ACP. These results demonstrate that 16:1 delta 6 in T. alata endosperm is formed by the activity of a soluble delta 6 16:0-ACP desaturase that is structurally related to the delta 9 18:0- and delta 4 16:0-ACP desaturases. Implications of this work to an understanding of active site structures of acyl-ACP desaturases are discussed.

Structure and expression of an Arabidopsis acetyl-coenzyme A carboxylase gene.

Acetyl-coenzyme A carboxylase (ACCase) catalyzes the formation of malonyl-coenzyme A, which is used in the plastid for fatty acid synthesis and in the cytosol for several pathways including fatty acid elongation and flavonoid synthesis. Two overlapping Arabidopsis genomic clones were isolated and sequenced to determine the entire ACCase-coding region. Thirty introns with an average size of 94 bp were identified by comparison with an alfalfa ACCase cDNA sequence. The 10-kb Arabidopsis ACCase gene encodes a 251-kD polypeptide, which has 80% amino acid sequence identity with alfalfa ACCase and about 40% identity with ACCase of rat, chicken, yeast, and the diatom Cyclotella. No chloroplast transit peptide sequence was observed, suggesting that this Arabidopsis gene encodes a cytosolic ACCase isozyme. ACCase gene transcripts were detected by RNase protection assays in Arabidopsis root, leaf, silique, and seed. Genomic DNA blot analysis revealed the presence of a second related Arabidopsis ACCase gene.

Molecular cloning, characterization, and elicitation of acetyl-CoA carboxylase from alfalfa.

Acetyl-CoA carboxylase [ACCase; acetyl-CoA:carbon-dioxide ligase (ADP-forming), EC 6.4.1.2] catalyzes the ATP-dependent carboxylation of acetyl CoA to produce malonyl CoA. In plants, malonyl CoA is needed for plastid localized fatty acid biosynthesis and for a variety of pathways in the cytoplasm including flavonoid biosynthesis. We have determined the full nucleotide sequence of an ACCase from alfalfa, which appears to represent a cytoplasmic isozyme. Partial cDNAs were isolated from a cDNA library of suspension culture cells that had been elicited for isoflavonoid phytoalexin synthesis. The full-length sequence was obtained by primer extension and amplification of the cDNA with synthetic primers. The sequence codes for a protein of 2257 amino acids with a calculated M(r) of 252,039. The biotin carboxylase, biotin carboxyl carrier protein, and carboxyltransferase domains, respectively, show approximately 72%, 50%, and 65% sequence similarity to those of animal, diatom, and yeast ACCase sequences. ACCase enzyme activity and transcripts are induced severalfold upon addition of yeast or fungal elicitors to alfalfa cell cultures.