Plant Physiol. (1999) 120: 913-922
A Plastidial Lysophosphatidic Acid Acyltransferase from Oilseed
Rape1
Fabienne Bourgis2,
Jean-Claude Kader,
Pierre Barret,
Michel Renard,
David Robinson,
Colin Robinson,
Michel Delseny, and
Thomas J. Roscoe*
Laboratoire Physiologie Cellulaire et Moléculaire,
Université Pierre et Marie Curie, Centre National de la Recherche
Scientifique Unite Mixte de Recherche 7632, Tour 53, 4 Place Jussieu,
75252 Paris, France (F.B., J.-C.K.); Institut National de la
Recherche Agronomique Station d'Amélioration des Plantes B.P.
29, 35650 Le Rheu, France (P.B., M.R.); Department of Biological
Sciences University of Warwick, Coventry CV1 4BA United Kingdom (D.R.,
C.R.); and Laboratoire Physiologie et Biologie Moléculaire des
Plantes, Centre National de la Recherche Scientifique Unite Mixte de
Recherche 5545, Université de Perpignan 52 Avenue de Villeneuve,
66860 Perpignan cedex, France (M.D., T.J.R.)
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ABSTRACT |
The
biosynthesis of phosphatidic acid, a key intermediate in the
biosynthesis of lipids, is controlled by lysophosphatidic acid (LPA, or
1-acyl-glycerol-3-P) acyltransferase (LPAAT, EC 2.3.1.51). We have
isolated a cDNA encoding a novel LPAAT by functional complementation of
the Escherichia coli mutant plsC with an
immature embryo cDNA library of oilseed rape (Brassica
napus). Transformation of the acyltransferase-deficient
E. coli strain JC201 with the cDNA sequence BAT2
alleviated the temperature-sensitive phenotype of the
plsC mutant and conferred a palmitoyl-coenzyme
A-preferring acyltransferase activity to membrane fractions. The BAT2
cDNA encoded a protein of 351 amino acids with a predicted molecular mass of 38 kD and an isoelectric point of 9.7. Chloroplast-import experiments showed processing of a BAT2 precursor protein to a mature
protein of approximately 32 kD, which was localized in the membrane
fraction. BAT2 is encoded by a minimum of two genes that may be
expressed ubiquitously. These data are consistent with the identity of
BAT2 as the plastidial enzyme of the prokaryotic glycerol-3-P pathway
that uses a palmitoyl-ACP to produce phosphatidic acid with a
prokaryotic-type acyl composition. The homologies between the deduced
protein sequence of BAT2 with prokaryotic and eukaryotic microsomal LAP
acytransferases suggest that seed microsomal forms may have evolved
from the plastidial enzyme.
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INTRODUCTION |
Phosphatidic acid is a key intermediate in the biosynthesis of
phospho- and glycerolipids, essential components of all cellular membranes and of triacylglycerols. In most plant species, palmitoyl-ACP and oleoyl-ACP are the predominant products of de novo fatty acid biosynthesis in the chloroplast (Ohlrogge et al., 1993
). These fatty
acids may enter the prokaryotic pathway of lipid biosynthesis by
transfer of the acyl group from ACP to glycerol-3-P, which is mediated
by a stromal glycerol-3-P acyltransferase to form LPA or to position
sn-2 of glycerol-3-P mediated by LPAAT (EC 2.3.1.51) to form phosphatidic acid. The phosphatidic acid produced by
the prokaryotic pathway of the chloroplast is then used to produce
phosphatidylglycerol (for review, see Ohlrogge and Browse, 1995) or is
dephosphorylated to diacylglycerol, from which the glycodiacylglycerols
characteristic of the thylakoid membrane are derived (for review, see
Douce and Joyard, 1990).
Alternatively, the elongation of fatty acids may be terminated by the
action of an acyl-ACP thioesterase, which hydrolyzes the acyl-ACP to
release a free fatty acid, which then leaves the plastid. The fatty
acid in the form of an acyl-CoA thioester may participate in the
synthesis of glycerolipids via the eukaryotic pathway located at the
ER. The phosphatidic acid of the cytoplasmic pathway is used to produce
the phospholipids characteristic of extrachloroplastic lipids. The
phosphatidylcholine produced by the eukaryotic pathway is a substrate
for desaturation, after which the diacylglycerol moiety may be returned
to the chloroplast. The contribution of each pathway to the synthesis
of chloroplast lipids differs among species, and the relative amounts
of hexadecatrienoic acid and linolenate present in the leaf
galactolipids are an indication of the relative flux through each
pathway (for review, see Browse and Somerville,
1991).
The important role that acyltransferases, and the LPAATs in particular,
play in regulating lipid acyl composition is mediated through their
substrate specificities (for review, see Frentzen, 1993). The
plastidial LPAAT almost exclusively utilizes palmitoyl-ACP to produce a
phosphatidic acid containing a saturated group at position
sn-2, which is characteristic of lipids
synthesized by the prokaryotic pathway. In contrast, the cytoplasmic
LPAAT prefers linoleoyl to oleoyl groups; it discriminates strongly
against saturated C16 and C18 acyl groups and further differs from the plastidial enzyme in that an unsaturated acceptor LPA is preferred. Thus, the phosphatidic acid produced by the eukaryotic pathway is
highly enriched in C18:1 at position sn-2. In
cytoplasmic LPAAT, additional seed-specific isozymes exist whose
substrate specificity varies among species (Sun et al., 1988). These
acyltransferases are of interest for engineering of seed oil
composition.
Membrane-bound LPAATs have not been purified sufficiently to allow
molecular cloning, except that of coconut (Davies et al., 1995).
However, the complementation of the acyltransferase-deficient mutant of
Escherichia coli (Coleman, 1990) using libraries derived from immature embryos of meadowfoam (Brown et al., 1995; Hanke et al.,
1995) and from maize endosperm (Brown et al., 1994) has facilitated the
isolation of cDNAs encoding LPAATs.
We are engaged in studies of structure and function among
plant LPAATs and their role in determining lipid acyl composition. To
this end we have isolated a novel LPAAT by functional complementation in an acyltransferase-deficient E. coli mutant. We describe
the characterization of an oilseed rape (Brassica napus)
cDNA encoding a prokaryotic-type LPAAT and provide evidence identifying
this protein as the enzyme controlling phosphatidic acid biosynthesis in the plastid. We also compare the structural similarities among the
plastidial, microsomal, and prokaryotic proteins and discuss evolutionary relationships among plant LPAATs.
The nucleotide sequence corresponding to the BAT2 cDNA reported in this
article has been entered in the database under accession no. A111161.
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MATERIALS AND METHODS |
Plant Material
The progeny of a cross between the varieties B002 (<1% erucic
acid) and Hokkaido (>50% erucic acid) of oilseed rape (Brassica napus) described in Barret et al. (1998) were used for the
construction of cDNA libraries and the isolation of the BAT2
gene. For the mapping studies we used the double-haploid population,
which was derived from the cross between the varieties
Darmor-bzh (<1% erucic acid) and Yudal (>50% erucic
acid) and was used for the construction of a genetic map of B. napus (Foisset et al., 1996).
Isolation of B. napus cDNAs by Heterologous
Complementation
The protocol used for complementation was based on that of
Delauney and Verma (1990). The construction of an immature embryo cDNA
library of B. napus in the vector Lambda ZAP II (Stratagene) as described in Barret et al. (1998). An aliquot representative of the
primary library was subjected to in vivo excision using the ExAssist
system (Stratagene). Approximately 2 × 106
colonies were recovered after plasmid rescue and pooled for plasmid DNA
preparation. The Escherichia coli mutant JC201 was used in the complementation experiments and has the following genotype: plsC thr-1 ara-14
d(gal-attl)-99 his G4 rpsL136 xyl-5
mtl-1 lacY1 tsx-78 eda-50 rfbD1
thi-1 (Coleman, 1990). E. coli JC201 was
transformed via an electroporation protocol for bacteria based on that
described by Ausubel et al. (1992).
Transformant colonies growing on Luria-Bertani agar containing 100 µg
mL
1 ampicillin and 1 mM IPTG after incubation
at 42°C were collected and plasmid DNA was isolated. An aliquot of
this pooled DNA was used to retransform E. coli JC201 cells,
and the procedure was repeated with pooled DNA from this second
transformation. After three rounds of transformation, plasmid DNA was
isolated from individual clones, which apparently corrected the
temperature-sensitive phenotype. The ability of the individual clones
to complement the temperature-sensitive phenotype of E. coli
JC201 was tested by re-transformation with plasmid DNA isolated from
individual cDNA clones, followed by incubation at 42°C. Plasmid DNA
was isolated from individual transformant clones and sequenced to
confirm identity with the original DNA.
DNA Sequencing and Analysis
Sequences were determined using cycle sequencing (DyeDeoxy
Terminator, Applied Biosystems) on double-stranded DNA templates with a
sequencer (model 373A, Applied Biosystems). Each strand was sequenced
using oligonucleotide primers.
Southern and Northern Hybridization
DNA was extracted from young green leaf tissue according to the
protocol of Doyle and Doyle (1990). A series of reactions containing 15 µg of DNA was digested with the appropriate restriction enzyme, and
the resulting fragments were separated on a 0.75% agarose gel and
transferred to nylon membrane (Hybond N+,
Amersham). A 632-bp probe contained within the coding sequence was
generated by PCR using the following primers: AT2.1, 5
-CAA GAA CTG TGA
CTG TGA GAT CGG-3; and AT2.2, 5-CGT TAT TGG CAC CAC TGG AAC TCC-3.
The resultant amplimer was radiolabeled by random priming (Rediprime,
Amersham). Hybridization and washing at low and high stringency were
according to the manufacturer's protocol.
Total RNA was extracted from various tissues by a protocol based on the
method of Kay et al. (1987). Total RNA (20 µg) or 1 µg of
poly(A+) RNA was fractionated on 1.2% agarose
gels containing formaldehyde and transferred to nylon membrane (Hybond
N, Amersham). The probe and the hybridization were as for the genomic
Southern analysis. The filter was washed at 65°C in 0.125× SSC and
0.1% SDS and autoradiographed with intensifying screens at 
80°C.
The filters were subsequently stripped and reprobed with a 18S RNA
probe to compare the relative amounts of RNA in each lane.
Chloroplast Import Studies
Plasmid DNA isolated from clone BAT2 was used to generate the
precursor form of BAT2 by in vitro transcription using T3 polymerase, after which capped transcripts were translated in a wheat germ lysate
in the presence of (35S)-Met. The translation
product was incubated with intact, isolated pea chloroplasts, and the
organelles were treated with thermolysin and then fractionated as
described in Robinson (1993).
Assay of LPAAT Activity
E. coli JC201 cells were transformed in pBluescript SK
vector (Stratagene) with the BAT2 sequence and in the vector only. Cells were sedimented at 3000g from a culture grown at
30°C to an optical density of 0.6, induced by the addition of IPTG to 1 mM final concentration, and grown for 3 h
at 30°C. The cells were fractionated into periplasmic, cytoplasmic,
and membrane fractions according to the protocol of Henderson and
Macpherson (1986). The membrane pellet fraction was resuspended with 50 mM Tris-HCl, pH 8.0, 2 mM
MgCl2, and 2 mM DTT, and
stored at 80°C.
The assay was comprised of 50 mM Tris-HCl, pH 8, 1 mM MgCl2, 1 mM
dithiotheitol, 55 µM 1-oleoyl-LPA, 10 µM
[1-14C] acyl-CoA, and 15 µg of the membrane
fraction and was incubated for 5 min at 30°C. This was essentially
the protocol of Cao et al. (1990), as was the TLC analysis of products.
The phosphatidic acid spot was visualized by autoradiography and
collected for scintillation counting.
Mapping of the BAT2 Genes
The DNA insert contained within cDNA clone BAT2 was amplified by
PCR and purified according to protocols described in Barret et al.
(1998) using the following primers: BnAT2.4 = 5-GTC ATC GGT TGG
GCT ATG-3 and BnAT2.5 = 5-CCG ATC TCA CAG TCA CAG-3.
RFLP analysis was performed as described in Sharpe et al. (1995). The
BAT2/DraI markers were scored for each genotype, and linkage
analysis was performed using Mapmaker (Lander et al., 1987) and
Mapmaker QTL software, as described in Jourdren et al. (1996).
| |
RESULTS |
Heterologous Complementation of E. coli plsC
A population of plasmid clones excised from and representative of
an immature embryo cDNA library of B. napus was transformed into the E. coli strain JC201 to identify clones that were
capable of complementing the deficiency of LPAAT activity that is
characteristic of this mutant (Coleman, 1990). This strain is unable to
grow at 42°C but grows well at 30°C. Thus, the selection was based on the restoration of growth at the nonpermissive temperature in the
presence of IPTG and ampicillin. We performed three successive rounds
of transformation with pooled DNA obtained from colonies growing at the
previous transformation step, with the expectation that each round of
transformation would enrich the population with authentic complementing
clones and eliminate revertants that apparently complement after
acquisition of antibiotic resistance.
At the third round of transformation, approximately 1.5 × 106 colonies per microgram of DNA were obtained.
An aliquot of this pooled DNA was propagated in E. coli
XL-1-Blue (Stratagene) cells, and plasmid DNA was isolated from
individual transformant colonies. When 100 clones were screened for
insert size by PCR, approximately 85% contained an insert of 1.2 kb
that shared homology with known LPAAT sequences. Four of these clones
were sequenced at each extremity, and were found to be identical; these
clones were designated BAT2 (Brassica
acyltransferase 2).
A final transformation experiment was performed with plasmid DNAs of
individual BAT2 clones. Plasmid DNA (50 ng) of one of the 1.2-kb clones
was used to transform E. coli JC201 via heat shock, which
resulted in the growth at 42°C of a minimum of 10,000 colonies in the
presence of 1 mM IPTG and 100 µg
mL1 ampicillin. In contrast, 125 transformant
colonies were present after transformation of E. coli JC201
with the vector pBluescript. We conclude that the expression of the
1.2-kb insert present in BAT2 resulted in the abolition of the
temperature sensitivity of E. coli JC201 and restored growth
at 42°C.
Sequence Analysis of a BAT2 cDNA Clone
The BAT2 cDNA (accession no. A111161) was composed of a 1155-bp
sequence preceeding a poly(A+) tail of 18 bp.
Translation of the cDNA sequence revealed the presence of a single ORF
in the 5 to 3 direction of the cDNA, 351 amino acids
(Mr 38,748), and a pI of 9.73. There
were no in-frame stop codons present before the ORF, so it is not
certain that the cDNA sequence isolated is complete. The first in-frame
Met codon is positioned at nucleotides 24 to 26. The sequence
surrounding this initiation codon does not correspond to the consensus
for plants (Lutcke et al., 1987), so translation in the plant
may not initiate from this codon. However, the BAT2 cDNA sequence, together with the EcoRI adaptor sequence, is in frame with
and was probably expressed as a fusion protein with the
-galactosidase of the pBluescript vector. We used the technique of
PCR walking (Devic et al., 1997) to determine the genomic sequence
corresponding to the region 5 to the cDNA coding sequence. This
technique revealed the continuation of the ORF in the same reading
frame as the BAT2 sequence and suggested that the BAT2 coding sequence
may contain an additional eight amino acid residues to the nearest
upstream AUG codon. Therefore, the BAT2 sequence may code for a protein of 359 amino acids with a Mr of 39,606 D. We conclude that the 1155-nt sequence of the pBAT2 clone contains
sufficient information for the translation of a fusion protein in
E. coli JC201 that eliminates the temperature-sensitive
phenotype.
The deduced amino acid sequence of the ORF present in BAT2 was used to
identify homologous sequences via an advanced BLASTX2 search (Gish and
Slates, 1993). The BAT2 protein was found to share significant
homologies with LPAAT sequences of eukaryotic and prokaryotic species
(Fig. 1). The highest scores were
obtained with a LPAAT of yeast, the SLC1 gene product
(Nagiec et al., 1993), which revealed 32% identity and 51% similarity
over a 204-amino acid overlap. A similar degree of homology was evident
with the three sequences identified as microsomal LPAATs expressed in
seeds of meadowfoam (Brown et al., 1995; Hanke et al., 1995; Lassner et
al., 1995), which revealed 30% identity and 54% similarity within a
182-residue overlap, and with the coconut endosperm LPAAT (Knutzon et
al., 1995), which revealed 31% identity and 47% similarity over 229 residues. Over a shorter sequence of 143 amino acids the BAT2 sequence
shared a similar degree of homology (30% identity and 55% similarity
with a putative ORF identified within the genome of
Synechocystis sp. (accession no. P74498). Significant scores were obtained with bacterial LPAATs: The homology between the BAT2
protein and that of E. coli plsC was 31% identity and 50% similarity but within a shorter overlap of 115 residues. Absent from
the BLAST alignments were the putative LPAAT sequences pMAT1 (Brown et
al., 1994) and its B. napus homolog pBAT1 (accession no.
Z49860).
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| Figure 1.
Comparison of the partial deduced amino acid
sequences of B. napus BAT2 protein with LPAATs from
meadowfoam (Limnanthes; accession no. Q42870), coconut (Cocos;
accession no. Q42670), E. coli (accession no. P26647),
yeast (Saccharomyces cerevisiae; accession no. P33333),
and Synecocystis (Synechocys; accession no. P74498).
Outlined boxes indicate identity; shaded boxes indicate conservative
differences and unshaded boxes nonconservative differences.
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A comparison of the deduced amino acid sequence of BAT2 with each of
the previously identified LPAAT sequences via the BLAST alignments
suggested that the BAT2 protein was longer at the amino terminus by
approximately 90 residues. This sequence is characterized by a paucity
of acidic residues but is rich in Ser and enriched Ala and Val, and
contains basic residues, all of which are characteristics of a
chloroplast-targeting sequence (Keegstra et al., 1989). Sequence analysis by the PSORT program (Nakai and Horton, 1999) predicted that
the BAT2 protein possessed an N-terminal chloroplast-targeting sequence
together with an intrachloroplast sorting signal, so the BAT2 was
predicted to be a thylakoid protein. The hydropathy profile of the
deduced BAT2 protein sequence (Fig. 2)
revealed the presence of several hydrophobic regions, three of which
were predicted by the TOPRED2 program to be transmembrane
domains. These putative membrane-spanning domains are located between
residues 121 and 141, 217 and 237, and 282 and 302 with respect to the most N-terminal residue deduced from the cDNA sequence of BAT2.
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| Figure 2.
Hydropathy analysis of the BAT2 protein.
Hydropathy profiles were determined using the deduced amino acid
sequence of B. napus BAT2 using the Kyte-Doolittle
algorithm. Horizontal numbers indicate amino acid positions in the
protein sequence, and positive numbers on the vertical scale indicate a
hydrophobic region. Three potential transmembrane domains are indicated
by horizontal bars.
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Subcellular Localization of the pBAT2 Protein
To establish whether the protein encoded by the cDNA clone pBAT2
may be located in the plastid, we tested the ability of isolated chloroplasts to import and process the putative precursor protein. The
primary translation product was synthesized by in vitro transcription of the cDNA, followed by translation in a wheat germ cell-free system
in the presence of radiolabeled Met. Figure
3 shows that this procedure generated a
precursor protein of approximately 40 kD. Incubation of this
polypeptide with intact pea chloroplasts resulted in the import and
processing of the precursor protein to a mature protein of 32 kD, which
was resistant to proteinase, indicating localization in the
chloroplast. The preprotein present during the incubation with
chloroplasts and evident in Figure 3, lane C, was sensitive to
proteolysis, indicating that these molecules are associated with the
external surface of the chloroplast, and are perhaps bound to the
import receptors. Fractionation of the chloroplasts after the uptake
incubation revealed the mature BAT2 protein (migrating with an apparent
molecular mass greater than 32 kD in the presence of unlabeled
thylakoid proteins) to be localized almost exclusively in the membrane
fraction. We conclude from these data that the BAT2 protein is
synthesized with a cleavable signal that is capable of targeting the
preprotein to plastids and allowing compartmentalization of the mature
protein into a membrane fraction.
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| Figure 3.
Import of BAT2 protein into isolated chloroplasts.
35S-Labeled proteins derived from an in vitro
transcription-translation programmed by the BAT2 cDNA (lane Tr) were
incubated with intact chlorplasts (lane C), treated with thermolysin
(lane C+), and fractionated into stromal (lane S) and membrane (lane M)
fractions. preBAT2, Preprotein; BAT2, mature protein. On the right are
the apparent molecular masses in kD.
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Mapping of the B. napus BAT2 Genes
The BAT2 gene was positioned as a molecular marker on a B. napus genetic map (Foisset et al., 1996) using the BAT2 cDNA as a
RFLP probe. Hybridization of the probe to Southern blots of the
parental varieties Darmor-bzh and Yudal digested with
DraI revealed at least two loci: AT2.1 (dominant) and AT2.2
(co-dominant). Examination of 90 progeny plants indicated that marker
AT2.1 was positioned at the extremity of linkage group DY12, and AT2.2
was located at the extremity of group DY4 (Fig.
4A). A RFLP marker, 4NB12a, was located
14.3 cM from AT2.2, indicating either that DY4 and DY12 are homeologous
chromosomes or that a fragment of DNA is duplicated between the two
linkage groups. Using a cleaved amplified polymorphic sequences
approach (Konieczny and Ausubel, 1993), a major band of 2.1 kb and
several smaller bands were obtained after PCR, which gave rise to two
markers. One 1.7-kb fragment, BnAT2a, was dominant and positioned on
the DY19 linkage group. The second fragment was 1.2 kb and codominant.
Digestion of the 2.1-kb fragment by HaeIII revealed a third
marker, BnAT2b, which was positioned in the middle of the DY3 linkage
group (Fig. 4A) 53 cM from locus L2, one of two loci controlling seed
linolenic acid content. The marker BnAT2c was positioned on the DY12
group within 3.3 cM of the RFLP marker AT2.1 using 40 plants. The
distance of 3.3 cM was obtained with a single recombinant plant between the two markers.
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| Figure 4.
A, Localization of BAT2 markers on a genetic map
of B. napus: AT2.1 and AT2.2 were mapped by RFLP; BnAT2
(a, b, and c) were mapped by CAPS. B, Southern hybridization of
B. napus BAT2 genes. Pooled, genomic DNA isolated from
20 HEAR segregant plants was digested with the enzymes
DraI, EcoRI, and EcoRV,
resolved on an agarose gel, and transferred to a nylon membrane. A
truncated BAT2 coding sequence probe of 632 bp was hybridized, and the
filter was washed to a stringency of 0.125× SSC and 0.1% SDS at
60°C and autoradiographed. Arrows indicate position of weakly
hybridizing fragment.
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Southern hybridization performed to confirm the number of genes
encoding BAT2 revealed the presence of two strongly hybridizing fragments from each restriction digest of B. napus DNA. In
addition, weakly hybridizing fragments were detected with the DNA
digested with the enzymes DraI and EcoRI, as
indicated by arrows in Figure 4B. Using the BAT2 sequence as a probe,
we have subsequently isolated a highly homologous (97.2% amino acid
identity) yet distinct sequence, BAT2.3. The heterogeneity between BAT2
and BAT2.3 cDNAs may be a consequence of the allotetraploid nature of
B. napus and may reflect divergence between the parental
BAT2 gene copy present in each donor A and C genome. This
observation is consistent with the putative identification of pairs of
homoeologous loci located in DY4 and DY12 linkage groups, a
frequent feature of RFLP mapping in this amphidiploid species. The two
strong hybridization signals detected on the Southern blot may
correspond to the two cDNAs. The detection of additional, weakly
hybridizing fragments may indicate the existence of structurally
related genes. Thus, B. napus BAT2 protein is encoded by a
minimum of two homologous genes and may be present in a small
multigenic family of four members.
BAT2 Is Ubiquitously Expressed
The pattern of B. napus BAT2 gene expression was
determined to gain insight into its function. Northern hybridization
was performed with total RNA isolated from root and stem tissues and with poly(A+) RNA isolated from leaf, flower, and
embryos at 28 d after pollination (Fig.
5). A probe corresponding to 632 bp of
the coding sequence was found to hybridize to at least one class of
transcript of approximately 1.3 kb in each tissue examined. Apparent
differences in the BAT2 transcript level among leaf, flower, and embryo
were associated with a degree of contamination of the leaf and embryo poly(A+) RNA preparations by total RNA, as
revealed by the use of the 18S rRNA probe as a quantitative control. We
concluded that the BAT2 gene is expressed in a ubiquitous manner in all
tissues, including nonphotosynthetic tissues where plastids are
present.
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| Figure 5.
Northern hybridization of B. napus
BAT2 expression. Total RNA was extracted from root and
stem and poly(A+) RNA from leaf, flower, and immature
embryos isolated at 28 d after pollination. The RNAs were resolved
on formamide agarose gels, blotted to a nylon membrane, and hybridized
with a 632-bp probe corresponding to the truncated BAT2 coding sequence
cDNA insert and subsequently with an rRNA probe. The filters were
washed to a stringency of 0.125× SSC and 0.1% SDS at 60°C and
autoradiographed.
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The BAT2 Protein Possesses LPAAT Activity
To confirm that the presence of the BAT2 cDNA sequence confers
LPAAT activity, membranes were isolated from the
acyltransferase-deficient E. coli mutant JC201 by expressing
sequences present on the plasmid vector and from the pBAT2 cDNA, which
were then assayed for the incorporation of acyl groups into
phosphatidic acid using [1-14C]oleoyl-CoA or
[1-14C]palmitoyl-CoA. Despite a preference for
acyl-ACP thioesters, their physiological substrates, plastidial
acyltransferases are capable of utilizing acyl-CoA thioesters for the
synthesis of phosphatidic acid (Frentzen et al., 1983). LPAAT activity
was determined in the presence of 55 µM
1-oleoyl-LPA, and varying concentrations of palmitoyl-CoA or
oleoyl-CoA. The LPAAT activities of membrane preparations from E. coli JC201 transformants as a function of acyl-CoA concentration
are shown in Figure 6.
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| Figure 6.
LPAAT activities in E. coli
membrane preparations. E. coli JC201 cells were
transformed in pBluescript SK vector (Stratagene) with the BAT2
sequence and in vector only. Membranes were obtained from cells induced
by IPTG addition to cultures grown at 30°C. The membranes were
assayed in the presence of 50 mM Tris-HCl, pH 8, 1 mM MgCl2, 55 µM LPA, 1 mM DTT, and varying concentrations of
[1-14C]palmitoyl-CoA or [1-14C]oleoyl-oA,
and the products of the reaction were resolved on TLC. The phosphatidic
acid spot was visualized by autoradiography and collected for
scintillation counting. Values are the means obtained with two
individual membrane preparations. The values obtained with E. coli JC201/pBSK for palmitoyl-CoA and oleoyl-CoA were similar
and are labeled as acyl-CoA.
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The rate of formation of phosphatidic acid in the presence of membranes
isolated from E. coli JC201 cells transformed with a vector
containing the B. napus cDNA BAT2 was approximately 120-fold greater with [1-14C]palmitoyl-CoA and
approximately 42-fold greater with
[1-14C]oleoyl-CoA as substrates, compared with
membranes isolated from E. coli JC201 cells transformed with
the vector at acyl-CoA concentrations greater than 30 µM. Furthermore, the rate of formation of
phosphatidic acid associated with the presence of the BAT2 cDNA
sequence was significantly greater with
[1-14C]palmitoyl-CoA (approximately 4.7-fold
greater at saturating substrate concentrations) than with
[1-14C]oleoyl-CoA. At 10 µM acyl-CoA the rate of formation of
phosphatidic acid was approximately 2.2-fold greater with
[1-14C]palmitoyl-CoA than with
[1-14C]oleoyl-CoA. These data are consistent
with a preference of the enzyme for the palmitoyl-CoA substrate when
1-oleoyl-LPA was the acyl acceptor.
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DISCUSSION |
Heterologous complementation in auxotrophic mutants of E. coli by eukaryotic cDNA expression libraries is a powerful
approach for the isolation of genes not suitable for the conventional
techniques of protein purification, and requires functional
conservation of the mutated E. coli gene and its eukaryotic
counterpart (Delauney and Verma, 1990). Although not all genes are
suitable for this approach, LPAATs involved in the biosynthesis of
triacylglycerols have been successfully isolated by complementation
(Brown et al., 1995; Hanke et al., 1995). By adopting a similar
approach, we have isolated a B. napus cDNA encoding a
plastidial acyltransferase homologous to prokaryotic LPAATs. It is
intriguing that our complementation screen resulted in the isolation of
a plastidial LPAAT isoform only. This indicates, at least under our
conditions of selection, that the plastidial enzyme was the most
efficient in providing a functional substitution for the LPAAT
deficiency, and is consistent with the sequence similarity between the
plastidial and bacterial proteins. The abundance of cDNA does not seem
to have been an important factor, since the BAT2 mRNA was not highly
expressed (as evidenced by northern analysis and the absence of BAT2
homologs in expressed sequence tag databases).
The BAT2 cDNA Encodes a Plastidial LPAAT
Several lines of evidence confirm that the protein encoded by BAT2
is a LPAAT. A selection based on restoration of growth at 42°C of the
temperature-sensitive, acyltransferase-deficient mutant of E. coli, JC201 (Coleman, 1990), allowed us to isolate a cDNA with a
deduced protein sequence that revealed homologies with sequences known
to possess LPAAT activity (Fig. 1). The strongest similarities were
with the yeast SLC1 gene product (Nagiec et al., 1993) and plant
microsomal LPAATs (Hanke et al., 1995; Knutzon et al., 1995; Lassner et
al., 1995). We therefore conclude that the BAT2 cDNA encodes an
activity that functionally compensates for the LPAAT deficiency, and
thus complements the lesion associated with the temperature-sensitive
phenotype of E. coli JC201. Because of the presence of a
functional chloroplast-targeting sequence, the recovery of a
corresponding genomic sequence from plant DNA, the isolation of pBAT2
from two independently constructed cDNA libraries of different
populations of plants, and the strength of hybridizing signals on
high-stringency genomic Southern blots, we believe that we have cloned
an authentic plant cDNA and not an LPAAT from a contaminating
endophyte, despite the sequence similarities among the BAT2,
prokayotic, and yeast LPAATs.
Like the E. coli, yeast, and plant LPAATs (Lassner et al.,
1995), the hydropathy profile of the deduced BAT2 protein indicates the
presence of a minimum of three transmembrane-spanning domains. Such
structural features of the BAT2 protein are consistent with results of
protein purification confirming the LPAATs to be integral membrane
proteins (Knutson et al., 1995). Protein-import assays (Fig. 3) suggest
that the BAT2 protein may be imported into the chloroplast and
localized in the total chloroplastic membrane fraction. Chloroplast
glycerolipids are synthesized at the membranes of the plastid envelope,
and chloroplastic LPAAT has been purified from inner and outer envelope
membranes of spinach and from the inner envelope of pea chloroplasts
(Douce and Joyard, 1990).
Taken together, these observations are consistent with the BAT2 protein
being the LPAAT that incorporates an acyl group derived from an
acyl-ACP thioester at the sn-2 position of a
LPA-acceptor molecule during glycerolipid biosynthesis in the plastid.
This enzyme, isolated from both C16:3 and C18:3 plants, has been shown to exhibit a nearly exclusive preference for palmitoyl groups (Frentzen
et al., 1983). Consistent with this assignment, membrane fractions
isolated from the acyltransferase-deficient E. coli mutant
JC201 transformed with a plasmid containing the BAT2 sequence were
demonstrated to possess LPAAT activity. Furthermore, the LPAAT activity
was at least 4.7-fold greater with a palmitoyl-CoA substrate than with
a oleoyl-CoA substrate (Fig. 6). The apparently ubiquitous expression
of BAT2 and the presence of BAT2 transcripts in root tissues is
consistent with the autonomy of nonphotosynthetically active tissues
for glycerolipid biosynthesis (Browse and Slack, 1985; Stahl and
Sparace, 1990).
BAT2 encodes the acyltransferase ultimately responsible for the
production of hexadecatrienoic acid, which is present in substantial amounts at position sn-2 of galactolipids
produced by the prokaryotic pathway and is characteristic of C16:3
plants. The availability of a plant deficient in chloroplastic LPAAT
activity would confirm the assignment of the identity of BAT2. Such a
mutant may be expected to have a phenotype similar to that of the
Arabidopsis act1 mutant (Kunst et al., 1988), which is
characterized by a deficiency in chloroplastic glycerol-3-P
acyltransferase activity and results in a loss of the prokayotic
pathway that is compensated for by the increased synthesis of lipids in
the cytoplasmic pathway. The lesion effectively converts the C16:3
plant into a C18:3 plant. To our knowledge, no mutant of the
chloroplastic LPAAT has been identified to date, and other Arabidopsis
mutants with a similar fatty acid composition appear to be allelic to
act1. It is possible that a lesion in chloroplastic LPAAT
may be lethal because of accumulation of phosphatidic acid or depletion
of available plastidial glycerol-3-P.
Two Classes of Higher Plant LPAAT
To date, three LPAAT activities have been characterized, appearing
in the plastidial, mitochondrial, and cytoplasmic compartments of plant
cells (Frentzen, 1993). In addition, cytoplasmic isozymes have been
cloned from seed tissues (Brown et al., 1994; Hanke et al., 1995;
Knutzon et al., 1995; Lassner et al., 1995). The isolation of the
plastidial form confirms that at least two distinct classes of LPAAT
sequences exist within higher plants. Despite the plastidial location
and the difference in substrate preference, the protein encoded by BAT2
shares a strong identity to the microsomal LPAATs of seeds (Brown et
al., 1995; Hanke et al., 1995). The similarity of the plastidial BAT2
sequence to that of a homologous sequence present in the genome of the
blue-green algae Synechocystis sp. suggests a common
progenitor gene and a direct line of descent to the ancestral
chloroplastic gene (and its subsequent transfer to the nucleus).
The structural and functional similarity (evidenced by the
complementation of the plsC lesion) of the plastidial
sequence with that of microsomal LPAATs indicates that a duplication
may have occurred, as well as the recruitment of the plastidial
housekeeping gene for specialization of function in oilseeds. In
this manner, enzymes with a substrate preference for the particular
acyl group present at sn-2 of phosphatidic acid
destined for triacylglycerol synthesis may have evolved. Molecular
phylogenetic analysis using the DARWIN program (Gonnet et al., 1992)
revealed the evolutionary proximity of the BAT2 protein to the
Synechocystis sp. protein and that of the seed microsomal
LPAATs to the prokaryotic LPAATs (data not shown); thus, the
chloroplastic and ER forms involved in glycerolipid biosynthesis may
constitute paralogous sequences. The precedent of the evolutionary
relationships between seed-specific and ubiquitous
acyl-ACP-thioesterases provides support for this hypothesis (Jones et
al., 1995).
The second sequence type, represented by a LPAAT encoded by the BAT1
cDNA of B. napus (accession no. Z49860), a homolog of the
maize MAT1 sequence (Brown et al., 1994), and the meadowfoam LAT1
protein (Brown et al., 1995), shares little identity with the protein
encoded by BAT2. What, then, is the evolutionary relationship and role
of the BAT1-type enzyme? The ubiquitously expressed transcript encoding
the BAT1 protein is not imported into chloroplasts, but is probably
located at the ER (data not shown), and thus may be involved in the
production of phosphatidic acid by the eukaryotic pathway. The low
sequence identity between BAT1 and BAT2 LPAATs of the same species
suggest that these may be analogous sequences. The ubiquitous
microsomal LPAAT of the eukaryotic pathway may have a different
evolutionary origin, since no homologs of BAT1 are known to exist in
the cyanobacterial genome, and molecular phylogenetic analysis reveals
an evolutionary substantial distance from other LPAATs.
Biotechnological Applications for the Plastidial LPAAT
The acyl composition of seed oils is determined by the substrate
specificities of the three microsomal acyltransferases of the Kennedy
pathway, together with the composition of the donor acyl-CoA pool. The
microsomal LPAATs that control the acylation at position
sn-2 show species variation in substrate
specificity. In B. napus, oleoyl-CoA is the preferred
substrate, whereas in contrast, the LPAAT of meadowfoam incorporates
C22:1 at position sn-2 of LPA. Similarly, the
coconut enzyme displays a different specificity, accepting medium-chain
acyl groups. It is suggested that expression of LPAAT with the
appropriate substrate specificities in transgenic B. napus
will alter the stereochemical composition of its seed oil. Transgenic
plants expressing the plastidial enzyme redirected to the ER, together
with a seed acyl-CoA pool enriched with C16:0 groups, may result in
high-palmitoyl oils. The availability of the plastidial sequence is
expected to provide insight into the structure and function
relationships of LPAATs to aid protein engineering of enzymes
possessing desired substrate and kinetic parameters.
| |
FOOTNOTES |
1
This work was supported by a grant from the
French Ministère de la Recherche et de la Technologie (no.
1994G0090) and the Organisation Nationale Interprofessionelle des
Oléagineux, Paris.
2
Present address: Department of Horticultural
Sciences, University of Florida, P.O. Box 110690, Gainesville, FL
32611.
*
Corresponding author; e-mail roscoe{at}univ-perp.fr; fax
33-04-68-66-84-99.
Received December 15, 1998;
accepted April 6, 1999.
| |
ABBREVIATIONS |
Abbreviations:
ACP, acyl-carrier protein.
cM, centimorgan.
LPA, lysophosphatidic acid (1-acyl-glycerol-3-P).
LPAAT, LPA
acyltransferase.
IPTG, isopropyl-1-thio--D-galactopyranoside.
ORF, open reading
frame.
RFLP, restriction fragment length polymorphism.
| |
ACKNOWLEDGMENTS |
We are grateful to Dr. Jack Coleman of the Louisiana State
University School of Medicine (New Orleans) for his generous gift of
E. coli JC201. The authors also thank Dr. Martine Devic for critical appraisal of this manuscript and the Centre National de la
Recherche Scientifique for financial support.
| |
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Abstract
Introduction