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First published online July 9, 2004; 10.1104/pp.104.044354 Plant Physiology 135:1324-1335 (2004) © 2004 American Society of Plant Biologists Cloning and Functional Characterization of a Phospholipid:Diacylglycerol Acyltransferase from Arabidopsis1 Department of Crop Science, Swedish University of Agricultural Sciences, S–230 53 Alnarp, Sweden (A.S.C., A.B., S.S.); Department of Plant Biology and Forest Genetics, Uppsala Genetic Centre, Swedish University of Agricultural Sciences, S–750 07 Uppsala, Sweden (U.S.); Scandinavian Biotechnology Research (ScanBi) AB, S–230 53 Alnarp, Sweden (M.L., A.D.); Institute of Biology, University of Podlasie, 08–110, Siedlce, Poland (W.B); and College of Life Science, Hubei University, Wuhan, 430062, P.R. China (B.H.)
A new pathway for triacylglycerol biosynthesis involving a phospholipid:diacylglycerol acyltransferase (PDAT) was recently described (Dahlqvist A, Stahl U, Lenman M, Banas A, Lee M, Sandager L, Ronne H, Stymne S, [2000] Proc Natl Acad Sci USA 97: 6487–6492). The LRO1 gene that encodes the PDAT was identified in yeast (Saccharomyces cerevisiae) and shown to have homology with animal lecithin:cholesterol acyltransferase. A search of the Arabidopsis genome database identified the protein encoded by the At5g13640 gene as the closest homolog to the yeast PDAT (28% amino acid identity). The cDNA of At5g13640 (AtPDAT gene) was overexpressed in Arabidopsis behind the cauliflower mosaic virus promoter. Microsomal preparations of roots and leaves from overexpressers had PDAT activities that correlated with expression levels of the gene, thus demonstrating that this gene encoded PDAT (AtPDAT). The AtPDAT utilized different phospholipids as acyl donor and accepted acyl groups ranging from C10 to C22. The rate of activity was highly dependent on acyl composition with highest activities for acyl groups containing several double bonds, epoxy, or hydroxy groups. The enzyme utilized both sn-positions of phosphatidylcholine but had a 3-fold preference for the sn-2 position. The fatty acid and lipid composition as well as the amounts of lipids per fresh weight in Arabidopsis plants overexpressing AtPDAT were not significantly different from the wild type. Microsomal preparations of roots from a T-DNA insertion mutant in the AtPDAT gene had barely detectable capacity to transfer acyl groups from phospholipids to added diacylglycerols. However, these microsomes were still able to carry out triacylglycerol synthesis by a diacylglycerol:diacylglycerol acyltransferase reaction at the same rate as microsomal preparations from wild type.
The acylation of diacylglycerols (DAGs) by acyl-CoAs catalyzed by diacylglycerol acyltransferase (DGAT) enzymes was until recently regarded as the only enzymatic step involved in triacylglycerol (TAG) biosynthesis. During the last 5 years three unrelated DGAT genes, the DGAT1, DGAT2, and a bifunctional DGAT/wax synthase, have been identified from different organisms (Cases et al., 1998  ; Lardizabal et al., 2001; Kalscheuer and Steinbuchel, 2003). Recently, a novel enzyme reaction for TAG biosynthesis involving the transacylation of acyl groups from phospholipids to DAGs was described in yeast (Saccharomyces cerevisiae) and plants (Dahlqvist et al., 2000). The LRO1 gene in yeast was shown to code for such an enzyme (phospholipid:diacylglycerol acyltransferase or PDAT; Dahlqvist et al., 2000; Oelkers et al., 2000). The LRO1 has homology to the well-studied enzyme lecithin:cholesterol acyltransferase (LCAT), which catalyzes sterol ester synthesis in blood plasma.
PDAT activity with high specificity for ricinoleoyl groups was demonstrated in microsomal preparations from developing castor bean (Ricinus communis) seeds (Dahlqvist et al., 2000
In yeast, the DGAT and PDAT pathways appear to have different importance for TAG synthesis under different growth phases. The PDAT-pathway is the most important for TAG synthesis during active cell division (Oelkers et al., 2000 In this paper, we show that there are six Arabidopsis gene sequences with homology to both the yeast PDAT (ScPDAT) and human LCAT (HsLCAT) sequences in the Arabidopsis genome database. The closest homolog to ScPDAT encodes the first identified plant PDAT enzyme (AtPDAT). The expression levels of the AtPDAT gene and its closest homolog in various tissues from wild-type Arabidopsis are discussed. Further, substrate specificities of the AtPDAT and the effect on lipid and fatty acid composition of overexpressing the gene in Arabidopsis are reported. It is also shown that an AtPDAT T-DNA insertion mutant has much lower (if any) PDAT activity in membrane fractions prepared from roots but still retains the same capacity as the wild type to synthesize TAG by transacylation between two DAG molecules.
Identification of the Arabidopsis PDAT Gene
We have previously identified the ScPDAT gene by the homology to HsLCAT (Dahlqvist et al., 2000
The two closest homologs to ScPDAT in Arabidopsis, At5g13640 (AtPDAT) and At3g44830, were further aligned to the ScPDAT and the HsLCAT sequences using the CLUSTALW algorithm (Fig. 2). In this alignment HsLCAT is shown with its signal peptide resulting in +24 in the numbering of the amino acids compared to what is given by Peelman et al. (1998) . The two Arabidopsis sequences are 57% identical to each other, 28% and 26% to the ScPDAT, and 16% and 18% to the HsLCAT, respectively. As previously reported (McCartney et al., 2004) both Arabidopsis PDAT-like proteins have a C-terminal aromatic amino acid rich endoplasmic reticulum (ER) retrieval motif. All three PDAT and PDAT-like sequences are predicted to have a short N-terminal cytoplasmic tail, a transmembrane-spanning region (Krogh et al., 2001), and the rest of the protein localized on the lumen side of the ER.
There is a considerable amount of information regarding the sequence/function relationship of the animal LCAT. Residues S205, D369, and H401 in HsLCAT have, by site directed mutagenesis, shown to be the catalytic residues involved in the phospholipase A reaction of the enzyme (Peelman et al., 1998 ). These three residues are all conserved in ScPDAT, AtPDAT, and At3g44830. The conserved Ser in HsLCAT as well as in ScPDAT is part of a conserved lipase motif with the consensus sequence GHSXG (Schrag and Cygler, 1997). The two Arabidopsis sequences differ from the lipase motif since the first Gly is replaced with a Pro. F127 in HsLCAT is one of the two suggested oxyanion hole residues that contributes to the stabilization of the intermediate oxyanion during the enzymatic reaction. F127 is in a region similar to F249 in ScPDAT but the Phe are more distantly positioned in the two Arabidopsis sequences (Fig. 2). While the second oxyanion hole residue is a Leu residue in HsLCAT as well as in most other animal LCATs, it is replaced by a Met in the aligned sequences from the two Arabidopsis proteins, ScPDAT (Fig. 2) and in the chicken LCAT (Peelman et al., 1998). Wang and co-workers (1997) have shown that residue E173 in HsLCAT influences the substrate specificity of the enzyme. A Glu to Ala mutation at residue 173 in the HsLCAT changes the sn-2 acyl specificity from linoleate (18:2) to arachidonate (20:4). The mutation is suggested to influence the size of the cavity of the enzyme and facilitate the binding of the more bulky 20:4. None of the PDAT-like sequences has a Glu aligning with this position but instead has Ala or Ser, which have smaller side chains.
Lipases, as well as HsLCAT, contain a so-called lid domain which is closed by a disulfide bridge. The lid structure occurs between the two Cys, C74 and C98, at the N- and C- terminal parts of the segment in HsLCAT (Fig. 2). This 20 to 25 amino acid long, highly mobile element covers the hydrophobic active site of the enzymes. These lids have highly variable sequences in lipases and are able to form an amphipatic helix interacting with the lipid interface. It has been suggested that they destabilize the bilayer and facilitate both the binding of the hydrophobic substrate and its diffusion into the active site cavity of the enzyme (Peelman et al., 1999
In order to investigate whether the AtPDAT gene encodes an enzyme with PDAT activity and to further characterize the potential PDAT activity, the cDNA coding for AtPDAT was overexpressed in Arabidopsis using the constitutive cauliflower mosaic virus promoter (35S). The AtPDAT coding sequence was obtained from a cDNA clone, p5D6T7P, of the AIMS database. The clone was sequenced and found, by comparison to the GenBank genomic sequence (AB006704), to contain an insertion of one base that introduces a frame shift in the open reading frame. This extra base was deleted through site directed mutagenesis. To test whether the transformed Arabidopsis plants expressed the AtPDAT gene, RNA was extracted from T2 plants transformed with empty vector (control) or AtPDAT. Since the T2 seedlings were segregating for the inserted gene, seeds were germinated on agar plates containing kanamycin to eliminate nontransgenic segregating individuals. T2 seedlings from Arabidopsis transformed with empty vector and T2 seedlings from three independent transgenic lines containing AtPDAT were then grown in liquid culture and RNA was prepared from leaves and roots for northern blotting. Expression of AtPDAT gene was not detectable by northern blots in either leaves or roots of Arabidopsis transformed with empty vector but expression was clearly detectable in both roots (Fig. 3A) and leaves (data not shown) in all three 35S-AtPDAT transgenic lines. AtPDAT was most highly expressed in the transgenic line 1-1-6, while lines 1-3b-44 and 1-2-13 gave bands with approximately 70% and 25%, respectively, of the radioactivity of the 1-1-6 hybridizing band when measured by electronic autoradiography (Fig. 3A).
PDAT Activities in Microsomal Preparations PDAT activity was determined in microsomal preparations of leaves and roots of T2 plants of Arabidopsis transformed with empty vector (control) and from the three independent 35S-AtPDAT lines. Plants used for microsomal preparation were grown under the same conditions as plants used for detection of AtPDAT mRNA by northern blotting. PDAT assays were performed with microsomal preparations from roots with phosphatidylcholine (PC) containing [14C]labeled ricinoleate in the sn-2 position and di-oleoyl-DAG as substrates. The amount of synthesized [14C]labeled 1-ricinoleoyl-TAG correlated positively with the expression levels of the AtPDAT gene in the plant material with membrane preparations from the highest expresser yielding about 10 times more radioactive TAG than the control (Fig. 3A). It was further shown that addition of DAG stimulated the production, indicating limiting amounts of endogenous DAG in the microsomes (Fig. 3B).
Since some radioactivity also was found in DAG after the incubation, we performed an experiment to elucidate if the synthesis of radioactive TAG could have occurred by a DAG/DAG transacylase that was previously postulated to occur in plants (Stobart et al., 1997
Since LCATs have been shown to have phospholipase activity (Aron et al., 1978 ) we investigated if AtPDAT also had this activity. We could not observe any consistent difference in the amount of radioactive free fatty acids between assays with microsomal fractions from wild type and overexpresser, whether or not exogenous DAG was added (data not shown). However, the phospholipase activities varied much more than PDAT activities from one microsomal preparation to another.
In order to investigate the positional specificity of the AtPDAT, microsomal preparations from leaves of overexpresser and control plants were incubated with either the sn-1-[14C]labeled or sn-2-[14C]labeled di-18:1-PC. The activity of the sn-1 substrate was one-third of the activity of the sn-2 substrate, demonstrating a clear preference of the enzyme for the sn-2 position (Fig. 5). That the activity with the sn-1 labeled substrate was catalyzed by the AtPDAT is evident by comparing the assays with those from control microsomes, where activity was below detection limit. When checked with phospholipase A2, the sn-1 labeled substrate had, at the most, 10% of the label in the sn-2 position (see "Materials and Methods"). Consequently the major part of the radioactive TAG must have been derived from sn-1 position of PC in assays with the sn-1 labeled substrate.
Acyl and Lipid Specificity of the AtPDAT The acyl specificity of the AtPDAT for different acyl groups at position sn-2 of PC was investigated in microsomal preparations from the overexpresser (Fig. 6A). The enzyme activity was highly dependent on the nature of the acyl group with stearic acid (18:0) and erucic acid (22:1) giving the lowest activities and with ricinoleoyl group yielding 23-fold higher activity. Increased number of double bonds greatly increased activity as did the introduction of functional groups such as epoxy or hydroxy groups. However, replacement of a double bond with an acetylenic bond decreased the activity. Caproyl(10:0)-PC was utilized much better than 18:0-PC and somewhat better than 18:1-PC. When phosphatidic acid (PA), PC, and PE with [14C]labeled 18:1 in the sn-2 position were compared as substrates, the enzyme showed a 5.6-fold and 1.7-fold higher activity with PE than with PA and PC, respectively (Fig. 6B). The relative activities of the enzyme for the different phospholipids appeared to be similar if phospholipids with 10:0 or ricinoleoyl groups (ricinoleoyl-PE was not tested) were used as substrates (Fig. 6B).
Fatty Acid and Lipid Composition and Content in AtPDAT Overexpresser Fatty acid composition and lipid content of 17-d-old seedlings of the highest AtPDAT overexpresser were compared with control (wild-type) seedlings (Table I). No significant differences were found in total acyl composition or in the amount of polar lipids, TAG or other neutral lipids. It should be noted that the amount of TAG was very low (0.6%–0.8% of all acyl groups) in these seedlings. The amount of acyl groups per fresh weight (FW) was also identical between the overexpresser and the wild type. In order to investigate if there were any differences between individual polar lipids in the overexpressor compared to wild type, we analyzed the relative distribution and acyl composition of the major polar lipids in leaves and roots from plants grown for 14 d in liquid culture. We found no significant differences between overexpresser and wild type in any major polar lipid classes or in the lyso-PC (data not shown). The amount of lyso-PC was very low in roots and leaf tissues of both wild type and overexpressor (0.3%–0.4% of total acyl groups).
We also analyzed the fatty acid composition and oil content per FW and per seed of the T3 seeds from all three overexpressers and did not find any significant differences compared to the wild type (data not shown). It should be noted that northern blots showed that the developing T3 seeds of the highest overexpresser had an expression level of the AtPDAT that was about 30% of that of leaves from the T2 plants as judged on northern blots by electronic autoradiography (data not shown). The expression level of AtPDAT in developing seeds from wild type was below detection limit on our northern blots (data not shown).
Since the expression of the AtPDAT gene was below detection limit in wild-type Arabidopsis in our northern blots, we examined the expression of AtPDAT and its closest homolog At3g44830 by semi-quantitative RT-PCR. We detected expression of AtPDAT at similar levels in leaves, roots, flowers and developing seeds (Fig. 7). At3g44830 was clearly expressed in developing seeds but only at a very low levels in leaves and flowers while no transcript was detected in roots.
Catalytic Activities of PDAT and LCAT-Like Proteins
The gene encoding ScPDAT from yeast was previously cloned based on the assumption that the catalytic similarities between LCAT and PDAT would also be reflected in sequence similarities (Dahlqvist et al., 2000
Reasonable amino acid sequence similarities exist between the two Arabidopsis PDAT-like sequences, the ScPDAT and the HsLCAT sequences throughout the proteins. Putative active site residues, Cys involved in disulfide bridges and putative glycosylation sites are all relatively conserved in the PDAT-like sequences. The HsLCAT lid domain is suggested to interact and bind to the lipid substrates, but there is no homology to this domain in the three PDAT-like sequences (Fig. 2). The PDAT-like proteins might not need an interfacial recognition sequence since they are predicted to have an N-terminal located membrane-spanning region (Fig. 2) and would by this be anchored to the membrane and thus be in close contact with the substrates. The three PDAT-like proteins are predicted to have small cytosolic N-terminal tails before the predicted membrane spanning regions and with the rest of the proteins facing the lumen side of the ER. The Arabidopsis proteins have an aromatic amino acid rich stretch in the C-terminal end that recently have been shown to act as a ER retrieval motif (McCartney et al., 2004
Assays with microsomal preparations of leaves and roots from AtPDAT overexpressers and control plants (empty plasmid) showed that the PDAT activity correlated positively with expression levels. The activity was 10-fold higher in the membranes from the highest overexpresser than in the control. The evolutionarily related LCAT, that normally catalyzes the esterification of cholesterol, can also esterify alcohols, DAGs, lyso-phospholipids as well as catalyze hydrolysis of phospholipids (Czarnecka and Yokoyama, 1993
The positional specificity of the AtPDAT was tested using radioactive di-18:1-PC labeled with [14C]18:1 in either sn-1 or sn-2 position. The enzyme was shown to have a preference for sn-2 position but utilized the sn-1 position at about one-third of the rate of the sn-2 labeled substrate. When corresponding assays were performed with yeast PDAT it showed a 7-fold preference for the sn-2 position (Dahlqvist et al., 2000
The AtPDAT enzyme showed a broad acceptance for acyl groups of different chain lengths but had a strong preference for acyl groups with multiple double bonds or a functional group such as a hydroxy or epoxy group. In general, the AtPDAT accepted acyl groups ranging from C10 to C22 and with the activity of the enzyme negatively correlating to the melting point of the acyl group except for the hydroxy and epoxy fatty acids. PDAT activity was originally identified in microsomal preparations from developing seeds of castor bean (Ricinus communis) and Crepis palaestina plants, which accumulate high levels of hydroxy (ricinoleoyl) and epoxy (vernoloyl) acyl groups, respectively, in their seed TAGs (Dahlqvist et al., 2000
At this stage we can only speculate about the physiological relevance of the AtPDAT. The PDAT reaction catalyzes a breakdown of the major membrane lipids (PC and PE), thus forming lysophospholipids, which indicate that PDAT could be involved in various signal transduction pathways. LPC and LPE are products from the PDAT as well as of phospholipase A2 activities but, unlike the lipase reaction, no free fatty acids are formed. Lysophospholipids have been reported to stimulate a number of different activities in the cell (Palmgren and Sommarin, 1989 The identification of the PDAT gene in plants and its functional expression as well as the identification of a plant PDAT T-DNA insertion mutant, as described in this work, open the possibility to experimentally address PDAT's potential involvement in storage lipid synthesis and other metabolic processes in plants. Our experiments with the AtPDAT mutant have also revealed the presence of a third type of enzyme reaction, a DAG:DAG transacylation, involved in TAG biosynthesis in plant tissues. This reaction is carried out by an enzyme(s) other than AtPDAT and thus, adds another number to the growing list of transacylases found in plants.
Plant Material Arabidopsis (ecotype Columbia-0) plants were grown in peat-based soil media in a growth chamber with 70% humidity and 16 h light (200 µmol radiation m–2 s–1) at 20°C and 8 h dark at 18°C regime. Six-week-old plants, with inflorescence cut back once, were used for transformation. Plant material for gene expression and enzymatic studies were obtained by germinating Arabidopsis seeds in petri dishes containing one-third (1.4 g/L) Murashige and Skoog medium, 1% Suc in absence (nontransgenic) or presence of kanamycin (50 µg/mL) for 10 d. The seedlings were then transferred into beakers with liquid medium containing one-half Murashige and Skoog medium and 1% (w/w) Suc and grown for additional 27 d at 23°C under constant light (80 µmol radiation m–2 s–1) and gentle shaking (80 rpm).
A putative AtPDAT insertion mutation line, SALK_032261 (Alonso et al., 2003
The cDNA clone, p5D6T7P, coding for a putative Arabidopsis PDAT (AtPDAT) was obtained from the AIMS database. The clone was sequenced and found, by comparison to the GenBank genomic sequence, to contain an insertion of one base at position 302 counting from the A in the start codon, which caused a frame shift in the open reading frame. This extra base was deleted through site directed mutagenesis and in the same mutagenesis a SmaI site was created by a single and silent base change of a thymine to a guanine at nucleotide number 326. The mutagenesis was performed in two consecutive PCR reactions run with the AIMS clone, pD6T7P, as DNA template and with Taq DNA polymerase (Sigma, St. Louis). In the first PCR reaction two 45-bp overlapping DNA fragments, 326 and 1,751 bp long, were amplified using the primers (5'-GGAATTCCATGCCCCTTATTCATCGG-3' and 5'-CGCCTTAAGACCTTCTTTTTTGAGCTTAACCCCGGGCGGGTCAGG-3') for the N-terminal short fragment and the primers (5'-CCTGACCCGCCCGGGGTTAAGCTCAAAAAAGAAGGTCTTAAGGCG-3' and 5'-GCTCTAGATCACAGCTTCAGGTCAATA-3') for the C-terminal long fragment. The primers introduced an EcoRI site just before the start codon and an XbaI site right after the stop codon of the cDNA. The two PCR fragments were gel purified and equal amounts of each band, together with the end primers and Taq DNA polymerase, were used for the second PCR reaction. The amplified PCR fragment from the second reaction was gel purified and cloned into the plasmid pUni/V5-HIS-TOPO (Invitrogen, Carlsbad, CA) thus generating the plasmid pUS56. The insert in pUS56 was verified by sequencing.
The pUS56 insert, harboring the AtPDAT cDNA, was cut out by an EcoRI/XbaI digestion and ligated into a EcoRI/XbaI digested cloning vector. The cloning vector used was pART7-35S (Gleave, 1992
Arabidopsis plants were transformed with A. tumefaciens GV3101 harboring the binary plasmid pAtPDAT-27 to 35 s (overexpressers) or the pART27 equipped with an empty expression cassette from pART7-35S (control plants) using the floral dip method (Bent and Clough, 1998
Total RNA was isolated from root and leaf tissue of wild-type plants grown on agar plates and from open flowers and developing seeds (mid stage) from soil-grown wild-type plants using Concert Plant RNA reagent (Invitrogen, Paisley, UK) as described by the manufacturer. One microgram of RNA was reverse transcribed at 43°C using Moloney murine leukemia reverse transcriptase and the RevertAid H Minus First Strand cDNA Synthesis kit (Fermentas GmbH, St. Leon-Rot, Germany) with oligo(dT) as primer according to the protocol provided by the supplier. The resulting cDNA was diluted 10-fold and 1 µL used as template for 35 cycles of PCR amplification using Taq DNA Polymerase (Sigma-Aldrich) with the specific oligo-nucleotides for AtPDAT (forward 5'-TCAAAGCTATCGCTGAGTATAAGG-3'; reverse 5'-CGGTAATTTTGTCTCTAACGGATTT-3'), AT3g44830 (forward 5'-CTAAACGTCAAGGAACTGTCAAG-3'; reverse 5'-GTGCCTCCGGTAATTTGG-3'). Amplification of the ACTIN2 control transcript was done using the primers Actin2-1 (5'-TCCCTCAATCTCATCTTCTTCC-3') and Actin2-1 (5'-GACCTGCCTCATCAATCTTCTTCC-3'). In order to avoid amplification of genomic products, one primer in each primer pair was designed to span an exon border. The PCR products were analyzed on ethidium bromide stained agarose gels.
T2 plants and T3 developing seeds, either transformed with empty vector or with pAtPDAT-27-35s, were used for RNA extraction. Total RNA was extracted by a modified phenol extraction method (Sambrook et al., 1989
Radiolabeled ricinoleic (12-hydroxy-9-octadecenoic) and vernolic (12,13-epoxy-9-octadecenoic) acids were synthesized enzymatically from [1-14C]18:1 and [1-14C]18:2, by incubation with microsomal preparations from seeds of R. communis and Euphorbia lagascae, respectively (Bafor et al., 1991
Microsomes from leaves and roots from plants grown in liquid culture were prepared using the procedure described by Stobart and Stymne (1985)
Plant material was homogenized in chloroform:ethanol:0.15 M acetic acid (1:2:0.9) using a Potter Elvehjem homogenizer and extracted into chloroform according to Bligh and Dyer (1959)
Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes, subject to the requisite permission from any third-party owners of all or parts of the material. Obtaining any permission will be the responsibility of the requestor. Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession numbers AAR03499, AY072914, P40345, NM_100282, NM_102512, NM_111202, NM_118106, AAK96619, NP_190069, AAR03499, and AB006704.
We are grateful to Helen Lindgren for technical assistance and Susanne Hjerdin for maintenance of the plants. We thank the Salk Institute Genomic Analysis Laboratory for providing the sequence-indexed Arabidopsis T-DNA insertion mutant and the Nottingham Arabidopsis Stock Centre for providing seeds. Funding for the SIGNAL indexed insertion mutant collection was provided by the National Science Foundation. Received April 9, 2004; returned for revision May 21, 2004; accepted May 22, 2004.
1 This work was supported by the Swedish University of Agricultural Science's strategic research grants (The Biological Factory and AgriFunGen), ScanBi AB, Stiftelsen Svensk Ojeväxtforskning, the Swedish Farmers Foundation for Agricultural Research, Stiftelsen Västsvenska Lantmännen Odal, and the European Commissions (grant no. QLRT1999–00213). 
2 These authors contributed equally to the paper. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.044354. * Corresponding author; e-mail anders.carlsson{at}vv.slu.se; fax 46–40–415519.
Alonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen H, Shinn P, Stevenson DK, Zimmerman J, Barajas P, Cheuk R, et al (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301: 653–657
Aron LJ, Jones S, Fiedling CJ (1978) Human plasma lecithin-cholesterol acyltransferase. Characterization of cofactor-dependent phospholipase activity. J Biol Chem 253: 7220–7226 Bafor M, Smith MA, Jonsson L, Stobart AK, Stymne S (1991) Ricinoleic acid biosynthesis and triacylglycerol assembly in microsomal preparations from developing castor-bean endosperm. Biochem J 280: 507–514 Banas A, Johansson I, Stymne S (1992) Plant microsomal phospholipases exhibit preference for phosphatidylcholine with oxygenated acyl groups. Plant Sci 84: 137–144[CrossRef] Bent FB, Clough SJ (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis. Plant J 16: 735–743[CrossRef][ISI][Medline] Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Med Sci 37: 911–917 Bouvier-Navé P, Noiriel A, Benveniste P (2003) Characterization of a novel phospholipase A1 cDNA from A. thaliana. 1st European Symposium on Plant Lipids, Aachen, Germany, September 10–13, 2003
Cases S, Smith SJ, Zheng YW, Myers HM, Lear SR, Sande E, Novak S, Collins C, Welch CB, Lusis AJ, et al (1998) Identification of a gene encoding an acyl CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis. Proc Natl Acad Sci USA 95: 13018–13023
Czarnecka H, Yokoyama S (1993) Regulation of lecithin-cholesterol acyltransferase reaction by acyl acceptors and demonstration of its "idling" reaction. J Biol Chem 268: 19334–19340
Dahlqvist A, Stahl U, Lenman M, Banas A, Lee M, Sandager L, Ronne H, Stymne S (2000) Phospholipid:diacylglycerol acyltransferase: an enzyme that catalyzes the acyl-CoA-independent formation of triacylglycerol in yeast and plants. Proc Natl Acad Sci USA 97: 6487–6492 Gleave AP (1992) A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome. Plant Mol Biol 20: 1203–1207[CrossRef][ISI][Medline]
Goyal J, Wang K, Liu M, Subbaiah PV (1997) Novel function of lecithin-cholesterol acyltransferase. Hydrolysis of oxidized polar phospholipids generated during lipoprotein oxidation. J Biol Chem 272: 16231–16239
Hiraoka M, Abe A, Shayman JA (2002) Cloning and characterization of a lysosomal phospholipase A(2), 1-O-acylceramide synthase. J Biol Chem 277: 10090–10099 Holsters M, de Waele D, Depicker A, Messens E, van Montagu M, Schell J (1978) Transfection and transformation of Agrobacterium tumefaciens. Mol Gen Genet 163: 181–187[CrossRef][ISI][Medline]
Kalscheuer R, Steinbuchel A (2003) A novel bifunctional wax ester synthase/acyl-CoA: diacylglycerol acyltransferase mediates wax ester and triacylglycerol biosynthesis in Acinetobacter calcoaceticus ADP1. J Biol Chem 278: 8075–8082 Kanda P, Wells MA (1986) Dihexanoylphosphatidylethanolamine: effect of head group charge on rates of alkaline and phospholipase-a2 catalyzed hydrolyzes. Chem Phys Lipids 39: 31–39[Medline] Krogh A, Larsson B, von Heijne G, Sonnhammer ELL (2001) Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 305: 567–580[CrossRef][ISI][Medline]
Lardizabal KD, Mai JT, Wagner NW, Wyrick A, Voelker T, Hawkins DJ (2001) DGAT2 is a new diacylglycerol acyltransferase gene family: purification, cloning, and expression in insect cells of two polypeptides from Mortierella ramanniana with diacylglycerol acyltransferase activity. J Biol Chem 276: 38862–38869
Lee HY, Bahn SC, Kang YM, Lee KH, Kim HJ, Noh EK, Palta JP, Shin JS, Ryu SB (2003) Secretory low molecular weight phospholipase A(2) plays important roles in cell elongation and shoot gravitropism in Arabidopsis. Plant Cell 15: 1990–2002
Lee M, Lenman M, Banas A, Bafor M, Singh S, Schweizer M, Nilsson R, Liljenberg C, Dahlqvist A, Gummeson P-O, et al (1998) Identification of non-heme diiron proteins that catalyze triple bond and epoxy group formation. Science 280: 915–918
MacDonald MH, Mogen BD, Hunt AG (1991) Characterization of the polyadenylation signal from the T-DNA-encoded octopine synthase gene. Nucleic Acids Res 19: 5575–5581
Martinelle M, Holmquist M, Clausen IG, Patkar S, Svendsen A, Hult K (1996) The role of Glu87 and Trp89 in the lid of Humicola lanuginosa lipase. Protein Eng 9: 519–524 McCartney AW, Dyer JM, Dhanoa PK, Kim PK, Andrews DW, McNew JA, Mullen RT (2004) Membrane-bound fatty acid desaturases are inserted co-translationally into the ER and contain different ER retrieval motifs at their carboxy termini. Plant J 37: 156–173[CrossRef][ISI][Medline] Munnik T, Irvine RF, Musgrave A (1998) Phospholipid signalling in plants. Biochim Biophys Acta 1389: 222–272[Medline]
Oelkers P, Cromley D, Padamsee M, Billheimer JT, Sturley SL (2002) The DGA1 gene determines a second triglyceride synthetic pathway in yeast. J Biol Chem 277: 8877–8881
Oelkers P, Tinkelenberg A, Erdeniz N, Cromley D, Billheimer JT, Sturley SL (2000) A lecithin cholesterol acyltransferase-like gene mediates diacylglycerol esterification in yeast. J Biol Chem 275: 15609–15612
Palmgren MG, Sommarin M (1989) Lysophosphatidylcholine stimulates ATP dependent proton accumulation in isolated oat root plasma-membrane vesicles. Plant Physiol 90: 1009–1014 Paul RU, Holk A, Scherer GFE (1998) Fatty acids and lysophospholipids as potential second messengers in auxin action. Rapid activation of phospholipase A(2) activity by auxin in suspension-cultured parsley and soybean cells. Plant J 16: 601–611[CrossRef]
Peelman F, Vanloo B, Perez-Mendez O, Decout A, Verschelde JL, Labeur C, Vinaimont N, Verhee A, Duverger N, Brasseur R, et al (1999) Characterization of functional residues in the interfacial recognition domain of lecithin cholesterol acyltransferase (LCAT). Protein Eng 12: 71–78 Peelman F, Vinaimont N, Verhee A, Vanloo B, Verschelde JL, Labeur C, Seguret-Mace S, Duverger N, Hutchinson G, Vandekerckhove J, et al (1998) A proposed architecture for lecithin cholesterol acyl transferase (LCAT): identification of the catalytic triad and molecular modelling. Protein Sci 7: 587–599[Abstract]
Ryu SB, Karlsson BH, Ozgen M, Palta JP (1997) Inhibition of phospholipase D by lysophosphatidylethanolamine lipid-derived senescence retardant. Proc Natl Acad Sci USA 94: 12717–12721 Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4: 406–425[Abstract] Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual, Ed 2. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY
Sandager L, Gustavsson MH, Stahl U, Dahlqvist A, Wiberg E, Banas A, Lenman M, Ronne H, Stymne S (2002) Storage lipid synthesis is non-essential in yeast. J Biol Chem 277: 6478–6482
Schrag JD, Cygler M (1997) Lipases and the Stahl U, Banas A, Stymne S (1995) Plant microsomal phospholipid acyl hydrolases have selectivities for uncommon fatty-acids. Plant Physiol 107: 953–962[Abstract] Stobart K, Mancha M, Lenman M, Dahlqvist A, Stymne S (1997) Triacylglycerols are synthesised and utilized by transacylation reactions in microsomal preparations of developing safflower (Carthamus tinctorius L) seeds. Planta 203: 58–66[CrossRef] Stobart K, Stymne S (1985) The interconversion of diacylglycerol and phosphatidylcholine during triacylglycerol production in microsomal preparations of developing cotyledons of safflower (Carthamus tinctorius L.). Biochem J 232: 217–221[Medline] Stymne S, Banas A, Carlsson AS, Ståhl U, Lenman M (2003) PDAT and PDAT-like enzymes in plants. NPLC 2003 Meeting, Fallen Leaf Lake, CA, June 4–8, 2003 Subbaiah P, Liu M, Paltauf F (1994) Role of sn-2 acyl group of phosphatidylcholine in determining the positional specificity of lecithin-cholesterol acyltransferase. Biochemistry 33: 13259–13266[CrossRef] |
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ABSTRACT
RESULTS
12desaturase-like enzymes are synthesized from acyl groups linked to phospholipids and then rapidly transferred from the phospholipids to TAG (Bafor et al., 1991
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hydrolase fold. Methods Enzymol 284: 85–107