Coordinate Regulation of the Nuclear and Plastidic Genes Acetyl-Coenzyme A Carboxylase

advertisement
Plant Physiology, April 2000, Vol. 122, pp. 1057–1071, www.plantphysiol.org © 2000 American Society of Plant Physiologists
Coordinate Regulation of the Nuclear and Plastidic Genes
Coding for the Subunits of the Heteromeric
Acetyl-Coenzyme A Carboxylase1
Jinshan Ke2, Tuan-Nan Wen2, Basil J. Nikolau, and Eve Syrkin Wurtele*
Department of Botany (J.K., E.S.W.) and Department of Biochemistry, Biophysics, and Molecular
Biology (T.-N.W., B.J.N.), Iowa State University, Ames, Iowa 50011
catalyzed by the biotin-containing enzyme acetyl-CoA carboxylase (ACCase).
ACCase catalyzes a two-step reaction that requires a noncatalytic biotin-containing component and two catalytic
functions, all three of which are recognizable as conserved
structural domains. These domains are: biotin carboxylcarrier (BCC), biotin carboxylase (BCase), and carboxyltransferase (CTase). The biotin prosthetic group, which is covalently bound to the BCC subunit, is absolutely essential to
the enzymatic function of ACCase as the intermediate carrier of the carboxyl group that carboxylates acetyl-CoA
(Lane et al., 1974). The two half-reactions that result in the
carboxylation of acetyl-CoA are: (1) carboxylation of the
biotin prosthetic group, which is catalyzed by the BCase
domain, and (2) transfer of the carboxyl group from
carboxy-biotin to acetyl-CoA to form malonyl-CoA, which is
catalyzed by the CTase domain.
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 (␤-carboxyltransferase) and three nuclear-coded subunits
(biotin carboxy-carrier, biotin carboxylase, and ␣-carboxyltransferase). We report the primary structure of the Arabidopsis ␣carboxyltransferase and ␤-carboxyltransferase subunits deduced
from nucleotide sequences of the respective genes and/or cDNA.
Co-immunoprecipitation experiments confirm that the ␣-carboxyltransferase and ␤-carboxyltransferase subunits are physically
associated. The plant ␣-carboxyltransferases have gained a Cterminal 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.
Enzyme-biotin ⫹ HCO3⫺ ⫹ ATP 3 Enzyme-biotin-CO2⫺
⫹ ADP ⫹ Pi
(1)
Enzyme-biotin-CO2⫺ ⫹ acetyl-CoA 3
Enzyme-biotin ⫹ malonyl-CoA
HCO3⫺ ⫹ ATP ⫹ acetyl-CoA 3 malonyl-CoA ⫹ ADP ⫹ Pi
(2)
De novo fatty acid synthesis is a fundamental process
required for the biogenesis of membrane and storage lipids. Using acetyl-coenzyme A (CoA) as the initial primer,
this process occurs by the sequential condensation of twocarbon units, which are derived from malonyl-CoA. The
generation of malonyl-CoA from acetyl-CoA is the first
committed step in fatty acid biosynthesis. This reaction is
Two physically distinct ACCases have been identified in
plants: the heteromeric and homomeric forms (Li and Cronan, 1992d; Gornicki et al., 1993; Sasaki et al., 1993, 1995;
Alban et al., 1994; Konishi and Sasaki, 1994; Roesler et al.,
1994; Schulte et al., 1994; Shorrosh et al., 1994, 1995, 1996;
Choi et al., 1995; Yanai et al., 1995). These two enzymes
differ in the quaternary organization of their structural
domains. In the heteromeric ACCase, the three domains
occur on separate dissociable proteins, whereas in the homomeric ACCase these domains are sequentially ordered
on a single polypeptide. Heteromeric ACCases also occur
in most eubacteria (Guchhait et al., 1974; Hunaiti and Kolattukudy, 1982; Kondo et al., 1991; Li and Cronan, 1992b,
1992c), whereas homomeric ACCases occur in the cytosol
of fungi (Walid et al., 1992; Hablacher et al., 1993) and
animals (Lopez-Casillas et al., 1988; Takai et al., 1988).
In contrast to other organisms, plants contain ACCases
in two separate subcellular compartments, the plastids and
the cytosol. In most plants (excluding Graminae), the het-
1
This work was supported by the Hatch Act and State of Iowa
funds and by a U.S. Department of Agriculture-National Research
Initiative competitive grant (no. 97– 01912 to B.J.N. and E.S.W.), by
a grant from the Iowa Soybean Promotion Board (to E.S.W. and
B.J.N.), and by a research award to J.K. from the Iowa State
University Molecular, Cellular, and Developmental Biology graduate program. This is journal paper no. J–18656 of the Iowa Agriculture and Home Economics Experiment Station, Ames, project
nos. 2,997 and 2,913.
2
These authors contributed equally to the paper.
* Corresponding author; e-mail [email protected]; fax 515–294 –
1337.
1057
1058
Ke et al.
eromeric ACCase is plastidic, whereas the homomeric
ACCase is cytosolic (Kannangara and Stumpf, 1972; Li and
Cronan, 1992a; Sasaki et al., 1993; Konishi and Sasaki, 1994;
Alban et al., 1994; Choi et al., 1995; Shorrosh et al., 1995,
1996; Konishi et al., 1996). Graminae do not contain the
heteromeric ACCase, but contain homomeric isoforms in
plastids and cytosol (Egli et al., 1993; Konishi et al., 1996;
Gornicki et al., 1997). Because membranes are impermeable
to acyl-CoAs (Jacobson and Stumpf, 1972; Benjamin et al.,
1983; Banhegyi et al., 1996), these two ACCases generate
physically isolated pools of malonyl-CoA. The plastidic
malonyl-CoA pool is the precursor of de novo fatty acid
biosynthesis, which produces 16 and 18 carbon fatty acids.
The cytosolic malonyl-CoA pool is used for the elongation of
these fatty acids to 20 carbons and longer. (In aerial portions of terrestrial plants these very-long-chain fatty acids
are used for the biosynthesis of epicuticular waxes, and in
seeds of some plants they are deposited in the seed oil.) In
addition, cytosolic malonyl-CoA is utilized for the biosynthesis of flavonoids, stilbenoids, malonic acid, and malonyl derivatives (of d-amino acids, 1-aminocyclopropane
carboxylic acid, etc.) (Conn, 1981; Nikolau et al., 1984).
Therefore, to accommodate the multiplicity of malonylCoA-requiring processes and to regulate the supply of
malonyl-CoA for these biosynthetic processes, plants have
compartmentalized the generation of this intermediate at
the cellular and subcellular level.
The plastidic heteromeric ACCase consists of four subunits: BCC, BCase, ␣-CT, and ␤-CT. To date, genes for two
of these subunits have been characterized from Arabidopsis, the CAC1 gene, which codes for BCC (Choi et al., 1995;
Ke et al., 1997), and the CAC2 gene, which codes for BCase
(Bao et al., 1997; Sun et al., 1997). In this report, we describe
the isolation and characterization of the CAC3 gene (GenBank accession no. AF056970), which codes for ␣-CT, and
the plastidic accD gene (GenBank accession no. AF056971),
which codes for ␤-CT. To approach the regulation of
ACCase gene expression, we determined the spatial and
temporal pattern of CAC1, CAC2, CAC3, and accD mRNA
accumulation during Arabidopsis silique development.
These studies indicate that the ACCase subunit mRNAs
accumulate to highest levels in cells and tissues undergoing rapid growth and/or seed oil biogenesis. Furthermore,
these studies establish that the subunit mRNAs accumulate
at a constant molar stoichiometric ratio, which implies that
the three nuclear and one plastidic ACCase gene must
communicate in order to establish coordinate expression.
MATERIALS AND METHODS
Plant Materials
Arabidopsis Heynh (ecotype Columbia) seeds were germinated in sterile soil, and plants were grown under continuous light as described previously (Ke et al., 1997). The
first three leaves of 17-d-old seedlings, flower buds, and
open flowers were harvested and frozen in liquid N2 for
RNA isolation or processed for in situ hybridization. Developing siliques were staged relative to the day of flow-
Plant Physiol. Vol. 122, 2000
ering (DAF). After the first two flowers had opened, subsequent flowers were tagged with colored threads at the
time of flowering. Siliques were collected each day between 1 and 15 DAF, and were either frozen in liquid N2
for RNA isolation or processed for in situ hybridization.
Isolation of cDNA and Genomic Clones
The following materials were obtained from the Arabidopsis Biological Resource Center (Ohio State University,
Columbus): (a) two Arabidopsis genomic libraries, one from
the ecotype Landsberg erecta (Voytas et al., 1990) cloned in
the vector ␭FIX, and the other from the ecotype Wassilewskija cloned in the cosmid vector pOCA18 (Olszewski
et al., 1988); (b) a 3- to 6-kb size-selected cDNA library in the
vector ␭ZAP II prepared from polyadenylated RNA isolated
from 3-d-old Arabidopsis (ecotype Columbia) seedling hypocotyls (Kieber et al., 1993); and (c) the expressed sequence
tag cDNA clone GBGe16 (R. Mache, F. Quigley, F. Thomas,
and D. Yu, unpublished data).
Hybridization screening of bacteriophage and cosmid
libraries was performed according to standard methods
(Sambrook et al., 1989). Clones that hybridized to 32Plabeled probes were plaque or colony purified. DNA restriction fragments that hybridized to the probes were
isolated and subcloned into pBluescript SK for sequencing.
DNA sequencing was performed at the Iowa State University Nucleic Acids Facility with automated sequencing
equipment from Applied Biosystems (Foster City, CA).
Both strands of all DNA fragments were sequenced twice.
DNA primers used for sequencing were synthesized at the
Iowa State University Nucleic Acids Facility. Computerbased analysis of gene promoter sequences used the
PLACE database (Higo et al., 1998).
Expression of Recombinant Proteins in Escherichia coli
Recombinant CAC3- and accD-coded proteins (␣-CT and
␤-CT, respectively) were produced in E. coli using the pET30
expression vectors. This resulted in the production of chimeric recombinant polypeptides that contained a short
N-terminal “tag,” which consists of a polyhistidyl sequence
(Van Dyke et al., 1992), the S䡠Tag-peptide (Kim and Raines,
1993), and two protease cleavage sites (His䡠Tag-thrombin
cleavage site-S䡠Tag-enterokinase cleavage site).
For CAC3, the NcoI-NotI fragment encompassing nucleotides 443 to 2,853 of the CAC3 cDNA (GenBank accession
no. AF056969), which codes for the predicted mature
polypeptide (from the 67th residue) was isolated. The NcoI
overhang was filled-in with Klenow, and the DNA fragment was cloned in-frame with the N-terminal “tags” of
the pET-30c expression vector at the unique EcoRV/NotI
sites; the resulting plasmid was called pET-CAC3.
To express accD, the clone was subjected to PCR-based,
site-directed mutagenesis to create a unique NcoI site at the
translational start site. This was achieved with the primer
Coordinate Acetyl-Coenzyme A Carboxylase Gene Expression
5⬘-GGCCAGAAGCTCCATGGAAAAA-3⬘, which is complementary to the sequence around the translation start
codon, ATG, but contains an NcoI site (underlined). From
the resulting mutagenized plasmid, the 1.67-kb NcoI-SacI
accD-containing fragment was cloned in the appropriate
sites of pET30a (the SacI site is vector-derived). The resulting plasmid was called pET-accD. The pET-derived recombinant plasmids were introduced into the E. coli strain
BL21(DE3), and protein production was induced with
isopropylthio-␤-galactoside. Proteins were extracted from
the cell pellets, fractionated into soluble and insoluble fractions, and the expressed proteins were purified by affinity
chromatography with a His䡠Bind column, as described by
the manufacturer (Novagen, Madison, WI). Recombinant
expressed proteins were detected by SDS-PAGE and western analysis using S-protein alkaline phosphatase conjugate to identify the S䡠Tag.
Immunological Methods
Antibodies were generated in New Zealand White female
rabbits by injecting with purified recombinant ␣-CT and
␤-CT proteins. Approximately 1 mg of purified protein was
emulsified with Freund’s complete adjuvant and injected
intradermally at multiple sites on the backs of the rabbits. A
month after the initial injection and at 2-week intervals
thereafter, the rabbits were challenged each time with muscular injections of 0.5 mg of protein emulsified in Freund’s
incomplete adjuvant. One week after each of these latter
injections, 2 to 3 mL of blood was withdrawn from the ear of
each rabbit. The blood was allowed to coagulate, and the
serum was collected after centrifugation at 4,000g for 10 min.
The serum was stored in small aliquots at ⫺20°C.
Immunological detection of proteins was carried out following SDS-PAGE (Laemmli, 1970). Proteins were electrophoretically transferred to a nitrocellulose membrane
(Kyhse-Andersen, 1984) and detected using antisera and
125
I-protein A.
For immunoinhibition studies, Arabidopsis leaf extracts
were incubated on ice for 1 h with increasing amounts of
either the anti-serum or preimmune serum. Equal aliquots
of a suspension of Protein A-agarose were added to each
reaction tube and the resulting mixtures were further incubated on ice for 1 h. Following centrifugation at 10,000g
for 1 min, a sample of each supernatant was assayed for
residual ACCase activity (Choi et al., 1995).
RNA Analysis
RNA was extracted from Arabidopsis leaves, buds, flowers, and siliques as described previously (Weaver et al.,
1995). Non-radioactive sense RNA concentration standards
of CAC1, CAC2, CAC3, and accD RNAs were obtained by in
vitro transcription from the respective pBSK full-length
cDNA clones. The RNA concentrations were determined
1059
from A280 and A260 and by comparison against standards of
known concentration following ethidium bromide staining
of gels.
Ten micrograms of RNA isolated from each Arabidopsis
tissue sample, and a range (0.01–10 pg) of CAC1, CAC2,
CAC3, and accD RNA standards were fractionated by electrophoresis in formaldehyde-containing agarose gels (Ke et
al., 1997). After transfer of the RNA to nylon membranes
(Magna Lift, MSI, Westbourough, MA), hybridizations
were conducted in a buffer containing 50% (v/v) formamide at 65°C for 12 to 16 h using 32P-labeled probes.
32
P-Labeled antisense RNA probes were transcribed from
vectors containing the following inserts: the 730 nucleotides at the 3⬘ end of the CAC1 cDNA (Choi et al., 1995; Ke
et al., 1997); the 195 nucleotides at the 5⬘ end of CAC2
cDNA (Sun et al., 1997); the CAC3 (GBGe16) cDNA; and the
1 kb at the 3⬘ end of the accD genomic clone. Hybridized
membranes were rinsed twice with 2⫻ SSC, 2% (w/v) SDS
for 10 min each time at room temperature, and then
washed twice with 0.1⫻ SSC, 0.1% (w/v) SDS for 20 min
each time at 65°C. The membranes were exposed to a
phosphor screen (Molecular Dynamics, Sunnyvale, CA) for
4 h, and the radioactivity in each band was quantified with
a phosphor imager (Storm 840, Molecular Dynamics). The
amount of each mRNA in each tissue sample was quantified by comparing the intensities of the mRNA bands with
those of the RNA concentration standards.
In Situ Hybridization
Arabidopsis siliques were harvested daily between 1 and
15 DAF. Siliques were cut into 3- to 4-mm-long pieces,
fixed, and sectioned as previously described (Wang et al.,
1995a; Ke et al., 1997). 35S-Labeled antisense and sense
RNA probes were transcribed from vectors containing the
following inserts: the 730 nucleotides at the 3⬘ end of the
CAC1 cDNA (Choi et al., 1995; Ke et al., 1997); the 195
nucleotides at the 5⬘ end of CAC2 cDNA (Sun et al., 1997);
the CAC3 (GBGe16) cDNA; and the 1 kb at the 3⬘ end of the
accD genomic clone. After hybridization and washing, the
tissue sections were coated with Kodak NTB2 emulsion
(Eastman-Kodak, Rochester, NY), exposed for 2 to 4 d, and
developed. Slides were stained with toluidine blue to detect cellular structure. Photographs were taken with a microscope (Orthopha, Leitz, Wetzlar, Germany) using
bright-field optics. In situ hybridizations were repeated
three times (using 20–30 silique sections each time). Two
sets of plant materials that had been independently processed were used, all with similar results. Control slides
containing sections of the same siliques were routinely
hybridized with the four sense RNA probes simultaneously with the antisense RNA hybridizations. Virtually
no signal was detected in the control slides. Although in
situ hybridizations were carried out using material collected every DAF through d 15, for space considerations,
data presented in figures are from alternate days only.
1060
Ke et al.
Plant Physiol. Vol. 122, 2000
Figure 1. Primary structure of ␣-CT. The amino acid sequence of the Arabidopsis ␣-CT (At.␣-CT) is compared with the
corresponding sequences from soybean (Gm.␣-CT), pea (Ps.␣-CT), Synechocystis (Sy.␣-CT), and E. coli (Ec.␣-CT). Residues
that are identical to those of the Arabidopsis sequence are shaded in black, whereas conservative substitutions are shaded
in gray. The CONSENSUS sequence identifies residues that are either conserved in the Arabidopsis and any other presented
sequence (lowercase) or are conserved in all presented sequences (uppercase).
RESULTS
The ␣-CT Subunits of Plant Heteromeric ACCase Contain a
Divergent C-Terminal Extension Relative to the Eubacterial
␣-CT Subunits
The 300-nucleotide sequence at the 5⬘ end of the Arabidopsis expressed sequence tag cDNA clone GBGe16 shows
significant sequence similarity to the E. coli accA gene that
codes for the ␣-CT subunit. Because GBGe16 is a partial
cDNA, it was used as a probe to screen an Arabidopsis
cDNA library (Kieber et al., 1993). This resulted in the
isolation of 10 hybridizing clones. The largest of these,
pCAC3, is 2,852 nucleotides in length and is a full-length
copy of the CAC3 mRNA. Beginning at nucleotide 248 of
the pCAC3 sequence is a 2,310-nucleotide open reading
frame (ORF) that codes for a polypeptide of 769 amino
acids with a calculated molecular mass of 85,305 D. This
ORF is followed by a 295-nucleotide 3⬘-untranslated region. Comparison of the deduced amino acid sequence of
the CAC3 ORF with previously published sequences indicated that the CAC3 protein shares highest sequence similarity with the ␣-CT subunit of the heteromeric ACCase of
E. coli (Li and Cronan, 1992a), pea (Shorrosh et al., 1996),
cyanobacterium (Gornicki et al., 1993), and soybean (Reverdatto et al., 1999) (Fig. 1).
Sequence similarities indicate that pCAC3 codes for the
␣-CT subunit of the heteromeric ACCase of Arabidopsis.
However, because several biotin enzymes are present in
plants (Wurtele and Nikolau, 1990; Guan et al., 1999), and
the sequences of their carboxyltransferase subunits are unknown, the enzymatic function of the CAC3-encoded protein had to be determined. Therefore, we conducted immunoprecipitation assays to confirm that CAC3 codes for the
␣-CT subunit of the heteromeric ACCase. Antiserum directed against the E. coli-expressed CAC3 protein reacts
with a single 85-kD polypeptide in Arabidopsis leaf extracts (Fig. 2A). The Mr of this polypeptide is very close to
that predicted from the CAC3 sequence. Arabidopsis leaf
extracts were incubated with increasing amounts of either
the anti-CAC3 serum or preimmune serum, and then with
protein A-agarose. Following centrifugation to remove
antigen-antibody complexes, samples of the supernatants
were assayed for residual ACCase activity. While up to 5
␮L of preimmune serum had no effect on ACCase activity,
2 ␮L of anti-CAC3 serum was sufficient to remove 50% of
the activity, and 5 ␮L of anti-CAC3 serum removed 65% of
the ACCase activity (Fig. 2B). The remaining ACCase activity was resistant to immunoprecipitation, presumably
due to the activity of the cytosolic ACCase isozyme, which
is immunologically distinct from the heteromeric ACCase.
These data demonstrate that CAC3 encodes the ␣-CT subunit of the heteromeric ACCase of Arabidopsis.
The ␣-CT protein contains an N-terminal extension of 87
amino acids, relative to the E. coli and Synechocystis accA
sequences (Fig. 1). As expected, this extension has characteristics of a plastidic stromal-targeting transit peptide: it is
Coordinate Acetyl-Coenzyme A Carboxylase Gene Expression
Figure 2. CAC3 codes for the ␣-CT subunit of the heteromeric
ACCase. A, Western-blot analysis of an Arabidopsis leaf extract
probed with antiserum directed against bacterially produced recombinant protein coded by the CAC3 cDNA. A single, 85-kD polypeptide was detected. B, Increasing amounts of preimmune (E) or antiCAC3 (F) serum were incubated with Arabidopsis leaf extracts.
Following the addition of protein A-agarose, antigen-antibody complexes were removed by centrifugation. Supernatants were assayed
for residual ACCase activity.
rich in hydroxylated residues (Ser and Thr) and in small
hydrophobic residues (Ala), but lacks acidic residues and
thus has an overall positive charge (Keegstra et al., 1989).
The PSORT algorithm (Nakai and Horton, 1999) predicts
that ␣-CT is a chloroplast stromal protein.
Comparison of the Arabidopsis, pea, soybean, Synechocystis, and E. coli ␣-CT sequences indicates that the mature
eukaryotic proteins can be divided into two domains,
which are distinguished by their degree of sequence conservation. The N-terminal domain (residues 94–426 of the
Arabidopsis ␣-CT) is highly conserved and matches the
sequence of the eubacterial ␣-CT proteins.
In contrast, the C-terminal domain (residues 426–769 of
the CAC3-coded ␣-CT) is less conserved among the plant
proteins and is absent from the eubacterial ␣-CT proteins.
The first 250 residues of this C-terminal domain (residues 426–680 in Arabidopsis) have very similar hydrophobicity profiles despite substantial sequence divergence. The
remainder of these proteins (from residue 681 of Arabidopsis) are extremely divergent with regard to both length
(Arabidopsis ⫽ 89 residues; pea ⫽ 160 residues; soybean ⫽
40 residues) and sequence. A common theme throughout
these C-terminal domains is the occurrence of nested repetitive sequences, although the specific sequences and
relative positions of these repetitions are not conserved
among the plant ␣-CTs. The largest of these repetitive
sequences occurs in the pea ␣-CT, and the duplication
partially overlaps itself (residues 453–489 are duplicated at
positions 481–517, with 83% conservation between the duplications). This repeat is flanked by a second duplication
(residues 438–458 are duplicated at positions 521–541, with
81% conservation). A shorter repeat motif that occurs in the
C-terminal domain of all three plant ␣-CTs has the sequence [hyph][E/D]-[K/R]- [hyph]- [K/R], where “hyph”
1061
stands for any hydrophobic residue. This sequence occurs
four, three, and six times in the Arabidopsis, soybean, and
pea sequences, respectively. Imperfectly repeated motifs
rich in E, L, and K residues are present seven, six, and 13
times in the Arabidopsis, soybean, and pea sequences,
respectively.
The relative lack of sequence conservation among the
plant-specific C-terminal domains, and its absence from the
eubacterial ␣-CTs, indicates that this domain may not be
required for catalysis, but, rather, may have some as-yetunknown structural function. Consistent with this hypothesis is the finding that despite the reduced sequence identity between the plant C-terminal domains, they are
predicted to share common secondary structural features,
i.e. similar hydrophobicity profiles, an overabundance of
charged residues (and thus high hydrophilicity), and
nested repetitive sequences. An additional feature consistent with these domains having a structural role is that
they are predicted by the PROTEINPREDICT algorithm
(Rost and Sander, 1993) to be rich in ␣-helices and not be
in a globular configuration. Finally, this plant-specific
C-terminal domain shares sequence similarity with myosin and other structural proteins.
␣-CT and ␤-CT Subunits Are Physically Associated
The heteromeric ACCase of E. coli readily dissociates into
three components: BCC, BCase, and CTase. The CTase
component is a heteromeric tetramer with an ␣4␤4 quaternary structure (Guchhait et al., 1974; Li and Cronan, 1992a).
The quaternary organization of the plant heteromeric
ACCase is still unresolved; however, the pea enzyme appears to dissociate into at least two components, one containing the BCC and BCase subunits and the second containing the ␣-CT and ␤-CT subunits (Sasaki et al., 1993;
Alban et al., 1994; Choi et al., 1995; Shorrosh et al., 1995,
1996). To ascertain whether the ␣-CT subunit is associated
with other subunits of the heteromeric ACCase of Arabidopsis, crude extracts and immunoprecipitates (obtained
as described for Fig. 2B) were analyzed by western blotting
using antisera against each of the heteromeric ACCase
polypeptides (Fig. 3). The ␣-CT and ␤-CT subunits, but not
the BCC or BCase subunits, were co-immunoprecipitated
by the anti-␣-CT serum. These data indicate that the ␣-CT
and ␤-CT subunits are in a more tightly associated complex
with each other than with the BCC and BCase subunits,
which is consistent with the previous characterizations of
the pea and E. coli heteromeric ACCase.
␣-CT Subunit Is a Stromal Protein Whose Localization Is
Unaffected by Illumination
The ␣-CT subunit of pea heteromeric ACCase was originally identified as a chloroplast inner envelope protein of
unknown function (Hirsch and Soll, 1995). Consistent with
this, fractionation of pea leaf extracts indicated that the
␣-CT subunit is primarily (about 90%) associated with
1062
Ke et al.
Plant Physiol. Vol. 122, 2000
mination for 10 d and then transferred to darkness for 24 h.
These extracts were fractionated by centrifugation at
100,000g for 1 h, and the supernatant and pellet (50 ␮g of
protein each) were subjected to SDS-PAGE and westernblot analysis with anti-␣-CT serum. Virtually all of the
␣-CT subunit protein was recovered in the soluble fraction,
regardless of the illumination status of the plants from
which the extract was prepared (Fig. 4). These data indicate
that the ␣-CT subunit of Arabidopsis is a soluble, stromal
protein and its solubility is unaffected by illumination.
Characterization of the CAC3 Gene
Approximately 40,000 recombinant bacteriophage from
an Arabidopsis genomic library (Voytas et al., 1990) were
screened by hybridization with the pGBGe16 cDNA. Five
of 11 hybridizing clones were plaque purified and further
analyzed. Restriction digests and Southern-blot hybridization analyses using 5⬘- and 3⬘-end-specific probes from the
CAC3 cDNA revealed that these five clones contained overlapping segments of the Arabidopsis genome, and that two
of these clones contained both ends of the CAC3 gene;
inserts from these phage were subcloned into pBluescript
SK and sequenced.
In total, more than 6-kb of a contiguous stretch of Arabidopsis genome was sequenced. Comparison of this sequence to the CAC3 cDNA sequence identified the struc-
Figure 3. Physical association between the ␣-CT and ␤-CT subunits
of the heteromeric ACCase. Preimmune or anti-CAC3 serum was
incubated with Arabidopsis leaf extracts. Antibody-antigen complexes were bound to protein A-agarose and collected by centrifugation. The resulting pellets were boiled in the presence of 2% (w/v)
SDS and, following centrifugation, aliquots of the supernatants were
subjected to western-blot analyses. These blots were sequentially
probed with anti-BCC, anti-BCase, anti-␣-CT, and anti-␤-CT sera.
The positions of the BCC, BCase, ␣-CT, and ␤-CT polypeptides are
indicated by asterisks (*).
cellular membranes (Shorrosh et al., 1996). It is not clear
how to reconcile findings that ␣-CT subunit is located in
the inner plastid membrane (Shorrosh et al., 1996), whereas
ACCase activity, BCase, and BCC are soluble (Sasaki et al.,
1993; Alban et al., 1994; Shorrosh et al., 1995; Ke et al.,
1997). Because the ␣-CT and ␤-CT subunits are in a complex (see Fig. 3), it is possible that this CTase complex is
membrane associated under some conditions in the plant,
while the BCC and BCase subunits remain soluble.
We determined directly whether the ␣-CT subunit of
Arabidopsis is a membrane-bound polypeptide. In addition, because of indications that light affects the activity of
ACCase (Sauer and Heise, 1984; Sasaki et al., 1997; Hunter
and Ohlrogge, 1998; Kozaki and Sasaki, 1999), we surmised
that light might effect a possible association of the ␣-CT
subunit with membranes. Therefore, we examined the location of the ␣-CT subunit in illuminated and in darktreated plants. Extracts were prepared from leaves of Arabidopsis plants that had either been grown under
continuous illumination for 11 d or under continuous illu-
Figure 4. ␣-CT subunit of the heteromeric ACCase is not membrane
associated. Arabidopsis plants were grown either under continuous
illumination for 11 d (L) or under continuous illumination for 10 d
and subsequently moved to a dark chamber for 1 d (D). Proteins
extracted from these two sets of plants were separated into a soluble
(S) and membrane-bound (M) fraction by centrifugation. Aliquots of
these two fractions containing equal amounts of protein (50 ␮g) were
subjected to SDS-PAGE and western analysis with anti-␣-CT serum.
Coordinate Acetyl-Coenzyme A Carboxylase Gene Expression
ture of the CAC3 gene (Fig. 5A). The CAC3 gene is
interrupted by 11 introns that range from 73 to 203 nucleotides in length. The nucleotide sequences at the intronexon-intron junctions follow characteristic patterns observed in other plant genes (Brown, 1989; Ghislain et al.,
1994). The exception to this rule is the 3⬘-end of intron 3,
which has the sequence 5⬘-TCC2G.
Interestingly, the first and last introns of the CAC3 gene
are positioned within the 5⬘- and 3⬘-untranslated regions
of the gene; this is a rather rare occurrence and its significance is still unknown. The portions of the Arabidopsis
␣-CT protein that are missing from the eubacterial ␣-CT
proteins are coded by exon 2 and exon 11 of the CAC3 gene.
1063
Exon 2 codes for the plastidic transit peptide, and would be
expected to have been acquired after the event that led to
endosymbiotic acquisition and refinement of the plastid.
Also intriguing is the indication that the entire C-terminal
domain of the plant ␣-CT subunit, which is absent from
eubacterial ␣-CT proteins, appears to have been due to a
post-endosymbiotic acquisition of a single exon (exon 11).
Southern-blot analysis of Arabidopsis genomic DNA
shows single hybridizing bands of the size predicted from
the CAC3 sequence for BamHI, EcoRI, SalI, and XhoI digests
(Fig. 5B). Furthermore, EcoRV digestion generates two hybridizing bands of different intensities, which is consistent
with the CAC3 gene sequence, which contains a unique
EcoRV site near the 3⬘-end of the gene. These data indicate
that the CAC3 gene occurs only once in the Arabidopsis
genome.
Isolation of the accD Gene Coding for the ␤-CT
Subunit of the Arabidopsis Heteromeric ACCase
To isolate the Arabidopsis plastid-encoded accD gene, a
cosmid Arabidopsis library (Olszewski et al., 1988) was
screened by hybridization with an accD-containing DNA
fragment from tobacco (the 780-bp XmnI-BamHI DNA fragment from position 59, 876–60, 655 of the tobacco chloroplast genome). This resulted in the isolation of a single
hybridizing clone, from which a 2.95-kb HindIII-KpnI accDhybridizing fragment was sequenced. This fragment contains an incomplete 98-bp ORF at its HindIII-end, identical
to the C-terminal 32 residues of rbcL (Ohyama et al., 1986;
Shinozaki et al., 1986), followed by an ORF of 1,464 bp. The
amino acid sequence of this 1,464-bp ORF shares 37%
identity with E. coli accD polypeptide, and 48%, 67%, and
91% identity with the pea, tobacco, and rape accD polypeptides, respectively (Wen, 1997). These results indicate that
this 2.95-kb fragment is from the Arabidopsis plastid genome and includes the accD gene. Antiserum made to the
expressed protein coded by accD reacts with a single 55-kD
polypeptide in a leaf extract from Arabidopsis, and this
antiserum specifically inhibits ACCase activity, directly
confirming that the gene encodes the ␤-CT subunit of the
heteromeric ACCase (Wen, 1997).
Figure 5. The CAC3 gene of Arabidopsis, which codes for the ␣-CT
subunit of the heteromeric ACCase. A, Schematic representation of a
6,280-bp Arabidopsis genomic fragment that contains the CAC3
gene. This fragment contains the potential promoter of the CAC3
gene (1,056-bp sequence 5⬘ of the CAC3 coding sequence). Positions
of the translational start (1ATG) and stop (3137TGA) codons are
indicated. Nucleotides are numbered relative to the translational start
codon. Exons are represented by shaded boxes and their positions
are: E1 (⫺450 to ⫺269; E2 (⫺65–339); E3 (441–515); E4 (649–756);
E5 (849–1,009); E6 (1,103–1,217); E7 (1,299–1,453); E8 (1,539–
1,590); E9 (1,672–1,740); E10 (1,829–1,906); E11 (1,980–3,159);
and E12 (3,244–3,494). Non-exonic sequences are represented by
solid lines. B, Arabidopsis DNA was digested with the indicated
restriction endonucleases and subjected to Southern-blot analysis.
The blot was probed with 32P-labeled CAC3 cDNA.
Coordinate Accumulation of the CAC1, CAC2,
CAC3, and accD mRNAs
As a first step in elucidating the mechanisms that regulate the heteromeric ACCase in Arabidopsis, we investigated the temporal changes in the accumulation of the
four ACCase subunit mRNAs (CAC1, CAC2, CAC3, and
accD) during silique development and compared this pattern with the accumulation of these mRNAs in young,
expanding leaves. For each mRNA, we compared the
intensity of each northern-blot hybridization band with
hybridization intensities obtained from serial dilutions of
in vitro-generated RNA standards of known concentrations. By including such standards, we were able to ab-
1064
Ke et al.
solutely quantify the accumulation of each ACCase subunit mRNA.
Hybridization of northern blots with the CAC1, CAC2,
and CAC3 antisense RNAs revealed the accumulation of
each of the corresponding mRNAs (Fig. 6A). The sizes of
these mRNAs (1.1, 2.0, and 2.8 kb, respectively) are consistent with previous characterizations (Choi et al., 1995; Bao
et al., 1997; Sun et al., 1997) and the size of the full-length
CAC3 cDNA. Hybridizations with the accD probe revealed
two mRNAs, accD-A (2.3 kb) and accD-B (1.5 kb). This was
not an unexpected finding, as multiple accD mRNAs had
previously been detected in pea and Arabidopsis leaves
(Woodbury et al., 1988; Meurer et al., 1996) and rape seed
embryos (Elborough et al., 1996). Both the accD-A
andaccD-B mRNAs are sufficiently large to encode the
Plant Physiol. Vol. 122, 2000
␤-CT subunit. The significance of multiple accD transcripts
in the regulation of ACCase expression is not clear, but
both the accD-A and accD-B mRNAs accumulate with a
similar pattern.
During silique development, there are two maxima in
the accumulation of the ACCase subunit mRNAs (Fig. 6, A
and B). The first occurs at 2 DAF, when siliques are most
rapidly expanding, and the second at 7 DAF, a time of
near-maximal oil accumulation in the embryos. Subsequently, as siliques mature (9–12 DAF), the accumulation
of these mRNAs declines to about 5% of peak levels. These
modulations in steady-state levels appear to occur coordinately for all five ACCase mRNAs.
This coordination is even more evident from analysis of
the data, as shown in Figure 6C. For each sample point
Figure 6. Coordinate temporal changes in accumulation of mRNAs coding the heteromeric ACCase subunits. A, RNA blots
were hybridized with CAC1-, CAC2-, CAC3-, and accD-specific 32P-labeled antisense RNA probes. RNA was isolated from
young expanding leaves (L), flower buds (B), flowers (F), and developing siliques at the indicated DAF. B, The concentration
of the CAC1, CAC2, CAC3, and accD mRNAs was determined by comparing the intensity of hybridization of each mRNA
to the hybridization intensity obtained with RNA concentration standards, as described in “Materials and Methods.” The
average values from three independent determinations are shown. The SD for each determination ranged between 10% and
15% of the indicated values. CAC1 (red), CAC2 (green), CAC3 (yellow), accD-A (blue), or accD-B (purple) mRNAs. C, For
each of the tissues analyzed in A and B, the concentrations of the CAC1 (red), CAC3 (yellow), accD-A (blue), or accD-B
(purple) mRNAs are plotted against the concentration of the CAC2 mRNA. Linear regression analyses indicate that there is
a linear relationship between the concentrations of each of these mRNAs with correlation coefficients of greater than 0.85.
Coordinate Acetyl-Coenzyme A Carboxylase Gene Expression
shown in Figure 6B, the concentrations of the CAC1, CAC3,
accD-A, and accD-B mRNAs are plotted against the concentration of the CAC2 mRNA (Fig. 6C). These plots reveal the
linear relationship between the concentration of the CAC1,
CAC3, accD-A, or accD-B mRNAs, and the concentration of
the CAC2 mRNA, indicating that the ACCase subunit
mRNAs accumulate at a constant molar ratio. The molar
ratio between these mRNAs was calculated to be CAC1:
CAC2:CAC3:accD-A:accD-B ⫽ 0.14:1.0:0.17:0.04:0.02. This
ratio is also maintained in organs other than developing siliques, namely, expanding leaves, flower buds, and
flowers.
In addition, we used the quantitative data in Figure 6 to
calculate the absolute abundance of each of the ACCase
mRNAs. These range from 0.5 to 170 fmol/mg total RNA.
Presuming that an average RNA is 2,000 bases, 10 fmol/mg
of total RNA is equivalent to 0.00066 mol % of total RNA.
(If 1% of the total RNA is mRNA, this figure translates to
0.066 mol % of cellular mRNA. At its highest levels, the
CAC2 mRNA accumulates at up to 1 mol % of mRNA.)
Silique Development Induces Coordinate Changes in the
Spatial and Temporal Patterns of CAC1, CAC2,
CAC3, and accD mRNA Accumulation
To obtain more detailed insights into the regulation of
ACCase expression, we investigated the spatial distribution of the CAC1, CAC2, CAC3, and accD mRNAs in developing siliques by in situ hybridization (Fig. 7). These analyses indicated that silique development induced both
quantitative and qualitative changes in the accumulation of
the ACCase subunit mRNAs. Most dramatically, development coordinately affected changes in the spatial distribution of all of these ACCase mRNAs.
In very young siliques (1 DAF; Fig. 7, A–D), which are
rapidly expanding, all four of the ACCase mRNAs are
evenly distributed among the silique tissues, including the
cells of the silique walls, central septum, and developing
ovules (integuments and endosperm). In 3 DAF siliques
(Fig. 7, E–H), which are almost fully expanded, the accumulation of ACCase mRNAs is reduced in the silique
walls, integuments of the ovules, and the central septum
compared with 1 DAF siliques. In these 3 DAF siliques, the
embryo within the ovule has grown to the globular stage of
development and is of sufficient size that it is readily
apparent; the accumulation of the ACCase mRNAs is concentrated within these embryos. By 5 DAF, the embryos
have developed to the heart stage and are beginning to
accumulate oil; the ACCase mRNAs are highly concentrated within these embryos (Fig. 7, I–L). In the other
silique tissues (silique walls, integuments of the ovules,
and the central septum), the accumulation of the ACCase
mRNAs is barely above background. From 7 DAF and
later, ACCase mRNAs are not detectable in the nonembryo tissues of the siliques, and the entire hybridization
signal for each mRNA is concentrated solely within the
embryo (Fig. 7, M–P). Maximal accumulation of the
ACCase mRNAs within the developing embryo occurs at
the torpedo stage of development (i.e. in siliques at 7 DAF).
At this stage, the rate of oil accumulation in the embryos is
1065
maximal. Subsequently, as the siliques mature, the embryos within develop to the walking stick stage by 9 DAF
(Fig. 7, Q–T) and to maturity by 12 DAF (Fig. 7, U–Y), just
prior to the initiation of desiccation. During these later
stages of development, the accumulation of all of the
ACCase mRNAs declines in the embryo.
DISCUSSION
In plants, de novo fatty acid biosynthesis from acetylCoA occurs solely in plastids. Although many studies have
attempted to ascertain whether ACCase has a major role in
controlling plant fatty acid biosynthesis, it is not yet clear
how this process is regulated. This is in part due to the fact
that plants contain two ACCases that are differentially
compartmentalized at the subcellular level. Furthermore,
at least in non-Graminae species, these two ACCase
isozymes are structurally distinct and the plastidic isozyme
is heteromeric, unstable, and dissociates during isolation
(e.g. Sasaki et al., 1993; Alban et al., 1995; Shorrosh et al.,
1996). These complicating characteristics, which have become apparent only in the last 6 years, have precluded the
direct demonstration that ACCase regulates fatty acid biosynthesis in plants.
To address the questions of whether and how ACCase
may regulate de novo fatty acid biosynthesis, we have
focused on the characterization of the heteromeric ACCase
of Arabidopsis. We report the isolation of the CAC3 and
accD genes and the CAC3 cDNA. In combination with
previous characterizations of the CAC1 (Choi et al., 1995;
Ke et al., 1997) and CAC2 (Bao et al., 1997; Sun et al., 1997)
genes, this work completes the isolation of all four genes
that code for the heteromeric ACCase of Arabidopsis. This
body of work shows that in Arabidopsis, each of the four
ACCase subunits is probably encoded by single-copy
genes, three of which are nuclear and one of which is
plastidic. This simplified genetic organization compared
with other plant species (Shorrosh et al., 1995; Elborough et
al., 1996; Reverdatto et al., 1999) should facilitate the molecular dissection of the mechanism by which ACCase is
regulated.
Investigations to date indicate that ACCase regulation is
complex (Post-Beittenmiller et al., 1991, 1992; Shintani and
Ohlrogge, 1995; Ke et al., 1997; Roesler et al., 1997;
Roughan, 1997; Sun et al., 1997; Caffrey et al., 1998) and
may encompass many different levels of control. Potential
post-translational regulatory mechanisms that affect
ACCase activity on a short time frame include the biotinylation of the BCC subunit (as is the case for the biotincontaining subunit of 3-methylcrotonyl-CoA carboxylase;
Wang et al., 1995b); the targeting, import, and assembly of
the three nuclear encoded subunits (BCC, BCase, and ␣-CT)
with the plastid encoded subunit (␤-CT); phosphorylation
(Savage and Ohlrogge, 1999); and the biochemical modulation of enzymatic activity (Eastwell and Stumpf, 1983;
Sasaki et al., 1997; Hunter and Ohlrogge, 1998; Kozaki and
Sasaki, 1999).
1066
Ke et al.
Plant Physiol. Vol. 122, 2000
Figure 7. (Continues on facing page.)
In contrast, developmentally induced modulations of
ACCase occur in a longer time frame and are likely to be
the consequence of the regulation of pre-translational processes. During seed maturation, fatty acids are rapidly
biosynthesized and deposited as seed oils. To gain insight
into how ACCase expression is regulated during this development, we examined the temporal and spatial patterns
of ACCase mRNA accumulation in developing siliques.
These data indicate that the CAC1, CAC2, CAC3, and
accD mRNAs accumulate to maximal levels in cells just
prior to or at the stage when they would be expected to be
actively synthesizing fatty acids. Furthermore, the accumulation of these mRNAs declines when fatty acid biosynthesis would be expected to be decreasing. Specifically, siliques undergo rapid growth and expansion early in their
development, between 1 and 2 DAF (Bowman, 1994). Dur-
Coordinate Acetyl-Coenzyme A Carboxylase Gene Expression
Figure 7. (Continued from facing page.)
Cellular distribution of mRNAs coding the heteromeric ACCase subunits in developing siliques of Arabidopsis. Silique
sections containing ovules and enclosed embryos were hybridized with 35S-labeled antisense RNA probes as described in
“Materials and Methods.” Hybridizations depict the accumulation of: CAC1 mRNA (A, E, I, M, Q, and U); CAC2 mRNA (B,
F, J, N, R, and V); CAC3 mRNA (C, G, K, O, S, and W); and accD mRNA (D, H, L, P, T, and X). Siliques are sampled at 1
DAF (A–D); 3 DAF (E–H); 5 DAF (I–L); 7 DAF (M–P); 9 DAF (Q–T); and 12 DAF (U–X). Sections are stained with toluidine
blue O. Hybridization signal is observed as black spots. w, Silique wall; ii, inner integument of ovule; oi, outer integument
of ovule; o, ovule; ge, globular embryo; he, heart embryo; te, torpedo embryo;ce, curled embryo; me, mature embryo; s,
central septum. Bar ⫽ 65 ␮m.
1067
1068
Ke et al.
ing this time, the cells of the silique walls, central septum,
and integuments would be expected to be synthesizing
fatty acids, which would be used for the deposition of
membranes to support the growth of the silique. Thus, the
finding that the maximal accumulation of the ACCase
mRNAs in the non-embryonic cells of the silique occurs at
1 DAF is consistent with the expected demand of malonylCoA generation needed for membrane lipid biogenesis by
these cells.
By 3 DAF, when the growth of the silique and associated
membrane deposition decrease, the accumulation of the
ACCase mRNAs declines in the non-embryonic cells of the
siliques. In contrast, between 3 and 9 DAF, the embryos
within the developing seeds undergo transition from
globular embryos of several hundred cells to mature embryos containing thousands of cells. Juxtaposed on this
embryonic growth is the accumulation of oil, which occurs at maximal rates at between 5 and 8 DAF (Mansfield
and Briarty, 1991, 1992; Bowman, 1994). By maturity,
about 30% of the fresh weight of the seed is oil (H.R. Qian,
E. Wurtele, and B. Nikolau, unpublished data). Consistent
with the expected demand for malonyl-CoA generation in the embryos, the accumulation of the
ACCase mRNAs in the embryos is substantial between 3
and 5 DAF and peaks by 7 DAF. Subsequently, as the rate
of oil deposition declines, the accumulation of these mRNAs decreases. Thus, the highest accumulation of the
ACCase mRNAs occurs in cells undergoing rapid cell
division and growth and/or accumulating large quantities of oil.
The spatial and temporal pattern of accumulation of the
ACCase mRNAs contrasts with patterns of mRNAs coding
for enzymes not related to fatty acid biosynthesis. These
include mRNAs coding for seed storage proteins (Raynal et
al., 1999), and the metabolic enzymes methylcrotonyl-CoA
carboxylase (J. Ke, B. Nikolau, and E. Wurtele, unpublished
data), acetyl-CoA synthetase, homomeric acetyl-CoA carboxylase, ATP-citrate lyase, and biotin synthase (Ke, 1997).
However, the accumulation pattern of ACCase mRNAs is
nearly identical to that of plastidic pyruvate dehydrogenase, which is consistent with a role of this enzyme in
generating acetyl-CoA for fatty acid biosynthesis in seeds
(Ke, 1997; Wurtele et al., 1997).
These analyses indicate that during silique development,
the temporal and spatial patterns of CAC1, CAC2, CAC3,
and accD mRNA accumulation are coordinately altered.
This finding contrasts with earlier work finding that the
accumulation of BCase, BCC, and ␣-CT subunit proteins
does not maintain a strict stoichiometric ratio when the
expression of the BCase is up-regulated or down-regulated
in leaves of transgenic tobacco (Shintani et al., 1997). The
difference in the conclusions of these two studies may be
due to differences in experimental systems and/or could
reflect more complex layered regulatory mechanisms.
The quantitative analyses presented herein indicate that
CAC2 mRNA accumulates to levels about 7-fold higher
than the CAC1 and CAC3 mRNAs, and 15-fold higher than
the accD mRNAs (accD-A plus accD-B). If the stoichiometric
ratio of these mRNAs reflects even in part the levels of the
respective subunit proteins, the BCase subunit would be
Plant Physiol. Vol. 122, 2000
present in excess. Our data substantiate the suggestion of
Shintani et al. (1997) that the BCase subunit accumulates in
excess, and explains why a substantial decrease in the
accumulation of the BCase subunit is required to bring
about even a conditional phenotype in transgenic tobacco.
Our data indicate that during silique development,
ACCase expression is regulated by mechanism(s) that coordinately affect the steady-state concentrations of each of the
ACCase mRNAs. Reporter transgene analyses of the CAC2
(Bao et al. 1997) and CAC1 (Choi et al., 1997; J.-K. Choi and
B. Nikolau, unpublished data) promoters implicate transcriptional regulation as an important mechanism that establishes the changing patterns in the accumulation of these
mRNAs. Maintaining transcriptional coordination would require communication between the three nuclear ACCase
subunit genes (CAC1, CAC2, and CAC3) and the plastidic
subunit gene (accD). This communication, at least between
the three nuclear genes, could be mediated by common
trans-acting factor(s) that interact directly or indirectly with
each of the three nuclear ACCase genes and coordinately
regulate their transcription.
If there is a direct interaction between this hypothesized
coordinating factor(s) and each nuclear ACCase gene, a
binding site for the coordinating factor(s) would occur on
all three nuclear ACCase genes and may be recognizable as
a conserved nucleotide sequence motif. Indeed, the promoters of the CAC1 (Ke et al., 1997), CAC2 (Bao et al., 1997;
Sun et al., 1997), and CAC3 genes have several potential
transcriptional regulatory motifs in common. These include: multiple E-boxes, which are important for light- and
seed-specific expression (Stalberg et al., 1996); Gt1 consensus sequences, which are involved in light regulation
(Zhou, 1999); and a motif that is common to promoters of
napin genes of B. napus and binds nuclear protein(s), but
has no established function (Ericson et al., 1991). Detailed
molecular studies are required to functionally identify
whether one or more of these motifs provide binding sites
for coordinating regulatory factor(s). That each ACCase
subunit in Arabidopsis is probably coded by a single-copy
gene provides a major advantage in using this species for
investigating coordinating mechanisms of ACCase expression.
Mechanisms to explain the coordination between the
nuclear (CAC1, CAC2, and CAC3) and the plastidic (accD)
ACCase subunit genes involve additional complexities associated with two distinct subcellular genomes and two
distinct subcellular compartments. The interplay between
nuclear- and plastid-coded genetic information generates
the wide variety of plastids that occur in different organs
and tissues (e.g. chloroplasts, amyloplasts, etioplasts,
chromoplasts, and oilseed plastids). Parallel to these developmentally induced changes in plastid morphology and
function are concomitant changes in macromolecular composition. Most studies of plastid development have focused
on chloroplasts, in particular using photosynthetic genes as
molecular tools (Leon et al., 1998; Pyke, 1999). Because the
heteromeric ACCase accumulates in many plastid types,
studies of its biogenesis can provide the means for elucidating the mechanisms of nuclear-plastid interactions that
control the development of a variety of plastids. Specifi-
Coordinate Acetyl-Coenzyme A Carboxylase Gene Expression
cally, ACCase can be useful in understanding the development of seed plastids and the mechanisms that influence
the partitioning of carbon into starch and oil in seeds.
Received July 6, 1999; accepted December 17, 1999.
LITERATURE CITED
Alban C, Baldet P, Douce R (1994) Localization and characterization of two structurally different forms of acetyl-CoA carboxylase in young pea leaves, of which one is sensitive to aryloxyphenoxyproprionate herbicides. Biochem J 300: 557–565
Alban C, Julien J, Job D, Douce R (1995) Isolation and characterization of biotin carboxylase from pea chloroplasts. Plant
Physiol 109: 927–935
Banhegyi G, Csala M, Mandl J, Burchell A, Burchell B, Marcolongo P, Fulceri R, Benedetti A (1996) Fatty acyl-CoA esters
and the permeability of rat liver microsomal vesicles. Biochem J
320: 343–344
Bao X, Shorrosh BS, Ohlrogge JB (1997) Isolation and characterization of an Arabidopsis biotin carboxylase gene and its promoter. Plant Mol Biol 35: 539–550
Benjamin AM, Murthy CR, Quastel JH (1983) Calcium-dependent
release of acetyl-coenzyme A from liver mitochondria. Can
J Physiol Pharmacol 61: 154–158
Bowman J (1994) Arabidopsis. An Atlas of Morphology and Development. Springer-Verlag, New York
Brown JW (1989) A catalogue of splice junction and putative
branch point sequences from plant introns. Nucleic Acids Res
14: 9549–9559
Caffrey JJ, Choi J-K, Wurtele ES, Nikolau BJ (1998) Tissue distribution of acetyl-CoA carboxylases in leaves of leek (Allium
porrum L.). J Plant Physiol 153: 265–269
Choi J-K, Ke J, McKean AL, Weaver LM, Wen T-N, Sun J, Diez
T, Yu F, Guan X, Wurtele ES, Nikolau BJ (1997) Molecular
biology of biotin-containing enzymes required in lipid metabolism. In JP Williams, MU Khan, NW Lem, eds, Physiology,
Biochemistry and Molecular Biology of Plant Lipids. Kluwer
Academic Publishers, Dordrecht, The Netherlands, pp 363–367
Choi J-K, Yu F, Wurtele ES, Nikolau BJ (1995) Molecular cloning
and characterization of the cDNA coding for the biotincontaining subunit of the chloroplastic acetyl-coenzyme A carboxylase. Plant Physiol 109: 619–625
Conn EE (1981) The Biochemistry of Plants: A Comprehensive
Treatise. Vol 7, Secondary Plant Metabolism. Academic Press,
New York
Eastwell K, Stumpf PK (1983) Regulation of plant acetyl-CoA
carboxylase by adenylate nucleotides. Plant Physiol 72: 50–55
Egli MA, Gengenbach BG, Gronwald JW, Somers DA, Wyse DL
(1993) Characterization of maize acetyl-coenzyme A carboxylase. Plant Physiol 101: 499–506
Elborough KM, Winz R, Deka RK, Markham JE, White AJ,
Rawsthone SR, Slabas AR (1996) Biotin carboxyl carrier protein
and carboxyltransferase subunits of the multi-subunit form of
acetyl-CoA carboxylase from Brassica napus: cloning and analysis of expression during oilseed rape embryogenesis. Biochem J
315: 103–112
Ericson ML, Muren E, Gustavsson H-O, Josefsson L-G, Rask L
(1991) Analysis of the promoter region of napin genes from
Brassica napus demonstrates binding of nuclear protein in vitro
to a conserved sequence motif. Eur J Biochem 197: 741–746
Ghislain M, Frankard V, Vandenbossche D, Matthews BF, Jacobs M (1994) Molecular analysis of the aspartate kinasehomoserine dehydrogenase gene from Arabidopsis thaliana. Plant
Mol Biol 24: 835–851
Gornicki P, Faris J, King I, Podkowinski J, Gill B, Haselkorn R
(1997) Plastid-localized acetyl-CoA carboxylase of bread wheat
is encoded by a single gene on each of the three ancestral
chromosome sets. Proc Natl Acad Sci USA 94: 14179–14184
1069
Gornicki P, Scappino LA, Haselkorn R (1993) Genes for two
subunits of acetyl coenzyme A carboxylase of Anabaena sp. strain
PCC7120: biotin carboxylase and biotin carboxyl carrier protein.
J Bacteriol 175: 5268–5272
Guan X, Diez T, Prasad KT, Nikolau BJ, Wurtele ES (1999).
Discovery and characterization of geranoyl-CoA carboxylase in
plants. Archives Biochem Biophys 362: 12–21
Guchhait RB, Polakis SE, Dimaroth P, Stoll E, Moss J, Lane MD
(1974) Acetyl coenzyme A carboxylase system of Escherichia coli.
J Biol Chem 249: 6633–6645
Hablacher M, Ivessa AS, Paltauf F, Kohlwein SD (1993) AcetylCoA carboxylase from yeast is an essential enzyme and is regulated by factors that control phospholipid metabolism. J Biol
Chem 268: 10946–10952
Higo K, Ugawa Y, Iwamoto M, Higo H (1998) PLACE: a database
of plant cis-regulatory DNA elements. Nucleic Acids Res 26:
358–359
Hirsch S, Soll J (1995) Import of a new chloroplast inner envelope
protein is greatly stimulated by potassium phosphate. Plant Mol
Biol 27: 1173–1181
Hunaiti AR, Kolattukudy PE (1982) Isolation and characterization
of an acyl-coenzyme A carboxylase from an erythromycinproducing Streptomyces erythreus. Arch Biochem Biophys 216:
362–371
Hunter SC, Ohlrogge JB (1998) Regulation of spinach chloroplast
acetyl-CoA carboxylase. Arch Biochem Biophys 359: 170–178
Jacobson BS, Stumpf PK (1972) Fat metabolism in higher plants:
LV. Acetate uptake and accumulation by class I and class II
chloroplasts from Spinacia oleracea. Arch Biochem Biophys 153:
656–663
Kannangara CG, Stumpf PK (1972) Fat metabolism in higher
plants: LIV. Prokaryotic type acetyl-CoA carboxylase in spinach
chloroplasts. Arch Biochem Biophys 152: 83–91
Ke J (1997) Expression of genes encoding acetyl-coA carboxylase,
biotin synthase, and acetyl-CoA generating enzymes in Arabidopsis thaliana. PhD thesis. Iowa State University, Ames
Ke J, Choi J-K, Smith M, Horner HT, Nikolau BJ, Wurtele ES
(1997) Structure of the CAC1 gene and in situ characterization of
its expression: the Arabidopsis thaliana gene coding for the biotincontaining subunit of the plastidic acetyl-coenzyme A carboxylase. Plant Physiol 113: 357–365
Keegstra K, Olsen LJ, Theg SM (1989) Chloroplastic precursors
and their transport across the envelope membranes. Plant Mol
Biol 40: 471–501
Kieber JJ, Rothenberg M, Roman G, Feldmann KA, Ecker JR
(1993) CTR1, a negative regulator of the ethylene response pathway in Arabidopsis, encodes a member of the raf family of
protein kinases. Cell 72: 427–441
Kim J-S, Raines RT (1993) Ribonuclease S-peptide as a carrier in
fusion proteins. Protein Sci 2: 348–356
Kondo H, Shiratsuchi K, Yoshimoto T, Masuda T, Kitazono A,
Tsuru D, Anai M, Sekiguchi M, Tanabe T (1991) Acetyl-CoA
carboxylase from Escherichia coli: gene organization and nucleotide sequence of the biotin carboxylase subunit. Proc Natl Acad
Sci USA 88: 9730–9733
Konishi T, Sasaki Y (1994) Compartmentalization of two forms of
acetyl-CoA carboxylase in plants and the origin of their tolerance toward herbicides. Proc Natl Acad Sci USA 91: 3598–3601
Konishi T, Shinohara K, Yamada K, Sasaki Y (1996) Acetyl-CoA
carboxylase in higher plants: most plants other than gramineae
have both the prokaryotic and eukaryotic forms of this enzyme.
Plant Cell Physiol 37: 117–122
Kozaki A, Sasaki Y (1999) Light-dependent changes in redox
status of the plastidic acetyl-CoA carboxylase and its regulatory
component. Biochem J 339: 541–546
Kyhse-Andersen J (1984) Electroblotting of multiple gels: a simple
apparatus without buffer tank for rapid transfer of proteins from
polyacrylamide to nitrocellulose. J Biochem Biophys Methods
10: 203–209
Laemmli UK (1970) Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 227: 680–685
Lane MC, Moss J, Polakis SE (1974) Acetyl coenzyme A carboxylase. Curr Top Cell Regul 8: 139–195
1070
Ke et al.
Leon P, Arroyo A, Mackenzie S (1998) Nuclear control of plastid
and mitochondrial development. Annu Rev Plant Physiol Mol
Biol 49: 453–480
Li S-J, Cronan JE (1992a) The genes encoding the two carboxyltransferase subunits of Escherichia coli acetyl-CoA carboxylase.
J Biol Chem 267: 16841–16847
Li S-J, Cronan JE Jr (1992b) The gene encoding the biotin carboxylase subunit of E. coli acetyl-CoA carboxylase. J Biol Chem 267:
855–863
Li S-J, Cronan JE Jr (1992c) The genes encoding the two carboxyltransferase subunits of E. coli acetyl-CoA carboxylase. J Biol
Chem 267: 16841–16847
Li S-J, Cronan JE Jr (1992d) Putative zinc finger protein encoded
by a conserved chloroplast gene is very likely a subunit of a
biotin-dependent carboxylase. Plant Mol Biol 20: 759–761
Lopez-Casillas F, Bai D-H, Luo X, Kong I-S, Hermodson MA,
Kim K-H (1988) Structure of the coding sequence and primary
amino acid sequence of acetyl-coenzyme A carboxylase. Proc
Natl Acad Sci USA 85: 5784–5788
Mansfield SG, Briarty LG (1991) Early embryogenesis in Arabidopsis thaliana II. The developing embryo. Can J Bot 69: 461–476
Mansfield SG, Briarty LG (1992) Cotyledon cell development in
Arabidopsis thaliana during reserve deposition. Can J Bot 70:
151–164
Meurer J, Berger A, Westhoff P (1996) A nuclear mutant of
Arabidopsis with impaired stability on distinct transcripts of the
plastid psbB, psbD/C, ndhH, and ndhC operons. Plant Cell 8:
1193–1207
Nakai K, Horton P (1999) PSORT: a program for detecting sorting
signals in proteins and predicting their subcellular localization.
Trends Biochem Sci 24: 34–36
Nikolau BJ, Wurtele ES, Stumpf PK (1984) Tissue distribution of
acetyl-coenzyme A carboxylase in leaves. Plant Physiol 75:
895–901
Ohyama K, Fukuzawa H, Kohchi T, Shirai H, Sano T, Sano S,
Umesono K, Shiki Y, Takeuchi M, Chang Z, Aota S-I, Inokuchi
H, Ozeki H (1986) Chloroplast gene organization deduced from
complete sequence of liverwort Marchantia polymorpha chloroplast DNA. Nature 322: 572–574
Olszewski NE, Martin FB, Ausubel FM (1988) Specialized binary
vector for plant transformation: expression of the Arabidopsis
thaliana AHAS gene in Nicotiana tabacum. Nucleic Acids Res 16:
10765–10782
Post-Beittenmiller D, Jaworski JG, Ohlrogge JB (1991) In vivo
pools of free and acylated acyl carrier proteins in spinach. J Biol
Chem 266: 1858–1863
Post-Beittenmiller D, Roughan G, Ohlrogge JB (1992) Regulation
of plant fatty acid biosynthesis. Plant Physiol 100: 923–930
Pyke KA (1999) Plastid division and development. Plant Cell 11:
549–556
Raynal M, Guilleminot J, Gueguen C, Cooke R, Delseny M,
Gruber V (1999) Structure, organization and expression of two
closely related novel Lea (late-embryogenesis-abundant) genes
in Arabidopsis thaliana. Plant Mol Biol 40: 153–165
Reverdatto S, Beilinson V, Nielsen NC (1999) A multisubunit
acetyl coenzyme A carboxylase from soybean. Plant Physiol 119:
961–978
Roesler K, Shintani D, Savage L, Boddupalli S, Ohlrogge J (1997)
Targeting of the Arabidopsis homomeric acetyl-coenzyme A
carboxylase to plastids of rapeseed. Plant Physiol 113: 75–81
Roesler KR, Shorrosh BS, Ohlrogge JB (1994) Structure and expression of an Arabidopsis acetyl-coenzyme A carboxylase gene.
Plant Physiol 105: 611–617
Rost B, Sander C (1993) Prediction of protein structure at better
than 70% accuracy. J Mol Biol 232 584–599
Roughan PG (1997) Stromal concentrations of coenzyme A and its
esters are insufficient to account for rates of chloroplast fatty
acid synthesis: evidence for substrate channelling within the
chloroplast fatty acid synthase. Biochem J 327: 267–273
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A
Laboratory Manual, Ed 2. Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, NY
Plant Physiol. Vol. 122, 2000
Sasaki Y, Hakamada K, Suama Y, Nagano Y, Furusawa I, Matsuno R (1993) Chloroplast-encoded protein as a subunit of
acetyl-CoA carboxylase in pea plant. J Biol Chem 268: 25118–
25123
Sasaki Y, Konishi T, Nagano Y (1995) The compartmentation of
acetyl-coenzyme A carboxylase in plants. Plant Physiol 108:
445–449
Sasaki Y, Kozaki A, Hatano M (1997) Link between light and fatty
acid synthesis: thioredoxin-linked reductive activation of plastidic acetyl-CoA carboxylase. Proc Natl Acad Sci USA 94: 11096–
11101
Sauer A, Heise K-P (1984) Regulation of acetyl-coenzyme A carboxylase and acetyl-coenzyme A synthetase in spinach chloroplasts. Z Naturforsch 39c: 268–275
Savage LJ, Ohlrogge JB (1999) Phosphorylation of pea chloroplast
acetyl-CoA carboxylase. Plant J 18: 521–527
Schulte W, Schell J, Topfer R (1994) A gene encoding acetylcoenzyme A carboxylase from Brassica napus. Plant Physiol 106:
793–794
Shinozaki K, Ohme M, Tanaka M, Wakasugi T, Hayashida N,
Matsubayashi T, Zaita N, Chungwongse J, Obakata J,
Yamaguch-Shinozaki K, Ohto C, Torazawa K, Meng B-Y, Sugita
M, Deno H, Kamogashira T, Yamada K, Kusuda J, Takaiwa F,
Kato A, Tohdoh N, Shimada H, Sugiura J (1986) The complete
nucleotide sequence of the tobacco chloroplast genome: its gene
organization and expression. EMBO J 5: 2043–2049
Shintani D, Roesler K, Shorrosh B, Savage L, Ohlrogge J (1997)
Antisense expression and overexpression of biotin carboxylase
in tobacco leaves. Plant Physiol 114: 881–886
Shintani DK, Ohlrogge JB (1995) Feedback inhibition of fatty acid
synthesis in tobacco suspension cells. Plant J 7: 577–587
Shorrosh BS, Dixon RA, Ohlrogge JB (1994) Molecular cloning,
characterization, and elicitation of acetyl-CoA carboxylase from
alfalfa. Proc Natl Acad Sci USA 91: 4323–4327
Shorrosh BS, Roesler KR, Shintani D, van de Loo FL, Ohlrogge
JB (1995) Structural analysis, plastid localization, and expression
of the biotin carboxylase subunit of acetyl-coenzyme A carboxylase from tobacco. Plant Physiol 108: 805–812
Shorrosh BS, Savage LJ, Soll J, Ohlrogge JB (1996) The pea
chloroplast membrane-associated protein, IEP96, is a subunit of
acetyl-CoA carboxylase. Plant J 10: 261–268
Stalberg K, Allerstom M, Ezcurra I, Ablov S, Rask L (1996)
Disruption of an overlapping E-box/Abre motif abolished high
transcription of the napA storage-protein promoter in transgenic
Brassica napus seeds Planta 199: 515–519
Sun J, Ke J, Johnson JL, Nikolau BJ, Wurtele ES (1997) Biochemical and molecular biological characterization of CAC2: the Arabidopsis thaliana gene coding for the biotin carboxylase subunit of
plastidic acetyl-coenzyme A carboxylase. Plant Physiol 115:
1371–1383
Takai T, Yokoyama C, Wada K, Tanabe T (1988) Primary structure of chicken liver acetyl-coenzyme A carboxylase deduced
from cDNA sequence. J Biol Chem 263: 2651–2657
Van Dyke MW, Sirito M, Sawadogo M (1992) Single-step purification of bacterially expressed polypeptides containing an oligohistidine domain. Gene 111: 99–104
Voytas DF, Konieczny A, Cummings MP, Ausubel FM (1990) The
structure, distribution and evolution of the Ta1 retrotransposable element family of Arabidopsis thaliana. Genetics 126: 713–721
Walid A-F, Chirala SS, Wakil SJ (1992) Cloning of the yeast FAS3
gene and primary structure of yeast acetyl-CoA carboxylase.
Proc Natl Acad Sci USA 89: 4534–4538
Wang H-Q, Colbert JT, Wurtele ES (1995a) A molecular marker of
the root cortical ground meristem is present in both root and
coleorhiza of the maize embryo. Am J Bot 82: 1083–1088
Wang X, Wurtele ES, Nikolau BJ (1995b) Regulation of
␤-methylcrotonyl-coenzyme A carboxylase activity by biotinylation of the apoenzyme. Plant Physiol 108: 1133–1139
Weaver LM, Lebrun L, Franklin A, Huang L, Hoffman N, Wurtele ES, Nikolau BJ (1995) 3-Methylcrotonyl-CoA carboxylase of
Arabidopsis thaliana: isolation and characterization of cDNA coding for the biotinylated subunit. Plant Physiol 107: 1013–1014
Coordinate Acetyl-Coenzyme A Carboxylase Gene Expression
Woodbury NW, Roberts LL, Palmer JD, Thompson WF (1988) A
transcription map of the pea chloroplast genome. Curr Genet 14:
75–89
Woods A, Munday MR, Scott J, Yang X, Carlson M, Carling D
(1994) Yeast SNF1 is functionally related to mammalian AMPactivated protein kinase and regulates acetyl-CoA carboxylase
in vivo. J Biol Chem 269: 19509–19515
Wurtele ES, Behal RH, Cui X, Ke J, Johnson JL, Lui F, Nikolau BJ,
Oliver BJ, Schnable PS (1997) In J Sanchex, E Cerdalmedo, E Martinez-Force, eds, Molecular Biology Of AcetylCoA Generation. Advances in Plant Lipids. University of Sevilla
Press, Seville, Spain, pp 54–56
1071
Wurtele ES, Nikolau BJ (1990) Plants contain multiple biotin
enzymes: discovery of 3-methylcrotonyl-CoA carboxylase,
propionyl-CoA carboxylase and pyruvate carboxylase in the
plant kingdom. Arch Biochem Biophys 278: 179–186
Yanai Y, Kawasaki T, Shimada H, Wurtele ES, Nikolau BJ,
Ichikawa N (1995) Genomic organization of 251 kDa acetyl-CoA
carboxylase genes in Arabidopsis: tandem gene organization has
made two differentially expressed isozymes. Plant Cell Physiol
36: 779–787
Zhou D-X (1999) Regulatory mechanism of plant gene transcription by GT-elements and GT-factors. Trends Plant Sci 4:
1360–1385
Download