Expression in yeast and tobacco of plant cDNAs encoding acyl CoA:diacylglycerol acyltransferase

Eur. J. Biochem. 267, 85±96 (2000) q FEBS 2000

Expression in yeast and tobacco of plant cDNAs encodingacyl CoA:diacylglycerol acyltransferase

Pierrette Bouvier-NaveÂ 1, Pierre Benveniste1, Peter Oelkers2, Stephen L. Sturley2 and Hubert Schaller1

1Institut de Biologie MoleÂculaire des Plantes, Strasbourg, France; 2Institute of Human Nutrition, Columbia University College of Physicians

and Surgeons, New York, USA

During the course of a search for cDNAs encoding plant sterol acyltransferases, an expressed sequence tag clone

presenting substantial identity with yeast and animal acyl CoA:cholesterol acyltransferases was used to screen

cDNA libraries from Arabidopsis and tobacco. This resulted in the isolation of two full-length cDNAs encoding

proteins of 520 and 532 amino acids, respectively. Attempts to complement the yeast double-mutant are1 are2

defective in acyl CoA:cholesterol acyltransferase were unsuccessful, showing that neither gene encodes acyl

CoA:cholesterol acyltransferase. Their deduced amino acid sequences were then shown to have 40 and 38%

identity, respectively, with a murine acyl CoA:diacylglycerol acyltransferase and their expression in are1 are2 or

wild-type yeast resulted in a strong increase in the incorporation of oleyl CoA into triacylglycerols. Incorporation

was 2±3 times higher in microsomes from yeast transformed with these plant cDNAs than in yeast transformed

with the void vector, clearly showing that these cDNAs encode acyl CoA:diacylglycerol acyltransferases.

Moreover, during the preparation of microsomes from the Arabidopsis DGAT-transformed yeast, a floating layer

was observed on top of the 100 000 g supernatant. This fraction was enriched in triacylglycerols and exhibited

strong acyl CoA:diacylglycerol acyltransferase activity, whereas almost no activity was detected in the

corresponding clear fraction from the control yeast. Thanks to the use of this active fraction and

dihexanoylglycerol as a substrate, the de novo synthesis of 1,2-dihexanoyl 3-oleyl glycerol by AtDGAT could

be demonstrated. Transformation of tobacco with AtDGAT was also performed. Analysis of 19 primary

transformants allowed detection, in several individuals, of a marked increase (up to seven times) of

triacylglycerol content which correlated with the AtDGAT mRNA expression. Furthermore, light-microscopy

observations of leaf epidermis cells, stained with a lipid-specific dye, showed the presence of lipid droplets in the

cells of triacylglycerol-overproducer plants, thus illustrating the potential application of acyl CoA:diacylglycerol

acyltransferase-transformed plants.

Keywords: expression in yeast; floating lipid layer; plant diacylglycerol acyltransferase; triacylglycerol synthesis;

transgenic tobacco.

Acyl CoA:diacylglycerol acyltransferase (DGAT; EC 2.3.1.20)is a membrane-bound enzyme that catalyzes the last and onlycommitted step in triacylglycerol synthesis, by using adiacylglycerol and a fatty acyl CoA as its substrates [1,2]. Inanimals, triacylglycerol synthesis is involved in numerousprocesses such as regulation of plasma triacylglycerol concen-tration, fat storage in adipocytes and milk production [3]. Inyeast, triacylglycerols and steryl esters coaccumulate undercertain physiological conditions in lipid particles (or lipidbodies), which constitute a fatty acid and sterol storage pool

[4,5]. In plants, triacylglycerol synthesis is mainly involved inthe generation of seed oils [6] but triacylglycerols are alsostored in fruits, petals and mature pollen grains [7] where theymight be important as a source of fatty acid for the rapid growthof the pollen tube [8].

DGAT-catalyzed esterification of diacylglycerol was pro-posed to be an important step in the control of planttriacylglycerol synthesis [9,10] and cloning of the DGATgene would allow us to ascertain this role. In recent months twocDNA clones have been reported, the deduced proteins ofwhich showed identity with known acyl CoA:cholesterolacyltransferases (ACATs). One called ACAT-related geneproduct (ARGP) was isolated from Homo sapiens [11] andanother from Mus musculus [12]. Actually both clones wereshown not to code for an ACAT. The H. sapiens cDNAencoding ARGP did not complement a yeast are1 are2 double-mutant defective in the two ACATs (ARE1 and ARE2) presentin wild-type yeast. Moreover, the murine cDNA, whenexpressed in H5 insect cells, did not produce any ACATactivity but was unambiguously shown to encode a DGAT [12].More recently an Arabidopsis thaliana cDNA presentingsignificant identity with murine DGAT was cloned by Hobbset al. [13]. This cDNA was expressed in insect cell cultures andthe microsomal preparation of these cells catalyzed the

Correspondence to P. Bouvier-NaveÂ, Institut de Biologie MoleÂculaire des

Plantes, DeÂpartement de Biologie Cellulaire et MoleÂculaire, Institut de

Botanique, 28 rue Goethe, F-67083 Strasbourg cedex, France.

Fax: + 33 3 8835 8484, E-mail: [email protected]

Abbreviations: ACAT, acyl CoA:cholesterol acyltransferase; ARGP,

ACAT-related gene product; DGAT, diacylglycerol acyltransferase; EREE,

ethylene responsive enhancer element; EST, expressed sequence tag; PGK,

phosphoglycerate kinase; p.f.u., plaque-forming units.

Enzymes: acyl CoA:cholesterol acyl transferase (EC 2.3.1.26); acyl

CoA:diacylglycerol acyltransferase (EC 2.3.1.20).

Note: a web page is available at http://www.ibmp.u-strasbg.fr/

(Received 30 July 1999, revised 1 October 1999, accepted

22 October 1999)

86 P. Bouvier-NaveÂ et al. (Eur. J. Biochem. 267) q FEBS 2000

synthesis of [14C] triacylglycerol from [14C]dioleylglycerol andoleyl-CoA thus demonstrating its function. In addition, theseauthors showed that the cDNA AtDGAT is expressed moststrongly in developing embryos and petals [13]. During thecourse of a search for an ACAT-like plant cDNA, we happenedto isolate, independently and using a different procedure, thesame AtDGAT, as well as its homolog from Nicotiana tabacum.Both cDNAs were expressed in yeast where we couldaccumulate and thoroughly characterize the product of theenzymatic reaction. Transgenic tobaccos expressing AtDGATunder the control of a constitutive promoter were generated inorder to prove the function in planta. A strong increase in thetriacylglycerol content of young growing leaves was shown tobe correlated with the appearance of lipid droplets in the cells.

E X P E R I M E N T A L P R O C E D U R E S

Strains, media and culture conditions

Escherichia coli. XL1blue recA-[recA1, lac-, endA1, gyrA96,thi, hsdR17, SupE44, relA1 (F 0proAB, lac1q, lacZ DM15,Tn10)].

Saccharomyces cerevisiae. Two strains of common geneticbackground (can 1±100, his 3±11, 15, leu 2-3, 112, trp 1-1,ura 3-1) were used: SCY059 (MATa, met14DHpaI-SalI,are1DNA::HIS3, are2D ::LEU2) and the corresponding wild-type SCY062 (MATa, ade2±1).

Yeast strains transformed with plasmid pYeDP60, harboringeither no insert or the plant cDNA under study, were alwaysgrown at the same time. For sterol or triacylglycerol analysis,culture conditions were 3 days at 30 8C on minimal medium[6.7 g´L21 yeast nitrogen base (Difco), 20 g´L21 galactose]containing suitable supplements (50 mg´mL21 each), i.e. Ade,Trp and Met for SCY059 (are1 are2) and His, Trp and Leu forSCY062 (wild-type). The cultures were then centrifuged andfreeze-dried.

For subcellular fractionation, these transformed strains weregrown for 3 days at 30 8C in minimum medium containing20 g´L21 glucose and the suitable supplements. The cultureswere then centrifuged under sterile conditions, and cells wereresuspended in a complete medium [10 g´L21 yeast extract(Difco), 10 g´L21 bactopeptone (Difco), 20 g´L21 galactose]and grown overnight at 30 8C. This two-step procedure resultedin a high cell mass, suitable for microsome preparation.

Plasmid for yeast transformation

The plasmid pYeDP60 [14] was used to transform yeast strains.This plasmid contains an E. coli replication origin, a yeast2 mm plasmid replication origin, an E. coli ampicillin-resistance gene and the yeast genes URA3 and ADE2. Itutilizes an expression cassette including a galactose-induciblehybrid promoter and a phosphoglycerate kinase (PGK)terminator. Gene expression is driven by the upstreamactivating sequence of the yeast GAL10 and CYC4 genes.

Cloning of a cDNA encoding DGAT from A. thaliana

An EST cDNA clone (ABRC DNA Stock Center, Columbia,OM 43210-1002) showing significant identity with ACATs wassequenced. This 849 bp EST fragment was used to screen acDNA library CD4-15 [15] supplied by the ABRC DNA StockCenter. In total, 500 000 plaque-forming units (p.f.u.) werescreened with the EST clone labeled with 32P after randompriming leading to 12 cDNA clones of different lengths. Amongthem 10 were larger than 1.5 kb. When cut with EcoRI these

clones showed a digestion pattern suggesting that theypossess an identical ORF but differ by the respective lengthof the 5 0-UTR and 3 0-UTR. Two of them were sequenced, thefirst (3D11) contained 1845 bp with an ORF of 1560 bp flankedby 5 0-UTR and 3 0-UTR of, respectively, 90 and 195 bp. Thesecond (3D71) contained 2062 bp with an identical ORF flankedby 5 0-UTR and 3 0-UTR of, respectively, 173 and 429 bp.

Cloning of a cDNA encoding DGAT from N. tabacum

A cDNA library from a 3-week-old N. tabacum cv. Xanthi lineLAB 1-4 calli derived from leaf protoplasts [16] was screenedwith the Arabidopsis EST clone of 849 bp. First, 250 000 p.f.u.were screened with an ORF from the Arabidopsis cDNA 3D11leading to two clones (1A111 and 2A113). One of them(2A113) was sequenced and shown to contain 1354 bp and itsdeduced amino acid sequence (317 amino acids) displayed astrong (. 70%) identity with the Arabidopsis DGAT deducedprotein sequence. This clone, called NtDGAT2, was likelyuncomplete. After digestion with EcoRI and HindIII, a 1-kbfragment was randomly labeled with [32P]dCTP and used toscreen again the tobacco library (250 000 p.f.u.), seven clonesof different lengths were isolated. NtDGAT2 was again foundamong them and was shown to be identical to the cDNA foundpreviously. Another clone presented a length (2099 bp)comparable with that of the Arabidopsis DGAT. Aftersequencing it was shown to encode a polypeptide of 532amino acids presenting 68% identity with Arabidopsis DGATand 86% identity with NtDGAT2. This cDNA was calledNtDGAT1.

Reformatting and cloning DGAT cDNAs into the yeastexpression vector pYeDP60

Deletion of the 5 0-UTR and 3 0-UTR of the DGAT cDNAs wasperformed by PCR amplification using specific primers.

Arabidopsis thaliana DGAT. Specific primers were designed tointroduce a BamHI restriction site immediatly upstream of theinitiation codon and a KpnI site immediatly downstream of thestop codon. Direct primer: 5 0-atatatggatccATGGCGATTTTG-GATTCTGC-3 0. Reverse primer: 5 0-tatataggtaccTCATGA-CATCGATCCTTTTCG-3 0. The DGAT was amplified using25 thermal cycles (1 min 93 8C, 2 min 56 8C, 3 min 72 8C)with Pyrococcus furiosus (Pfu) DNA polymerase under thestandard conditions. The PCR product was digested withBamHI and KpnI and subsequently cloned into Bluescript togive At DGAT-pSK. The BamHI, KpnI insert in pSK wasextracted and subcloned into BamHI, KpnI of pYeDP60 leadingto AtDGAT±pYeDP60.

A. thaliana FLAG DGAT. In order to evaluate the amount ofArabidopsis DGAT expressed in yeast, an N-terminal FLAGepitope (MDYKDDDDK, epitope underlined) was fused to theDGAT protein. For this purpose a DNA molecule containing at5 0-end the appropriated nucleotide sequence was synthesizedby PCR. Direct primer: atatatggatccATGGACTACAAGGAC-GACGATGACAAGGCGATTTTGGATTCTGCTGG. The reverseprimer is the one used above to amplify the At DGAT. The PCRproduct was digested with BamHI and KpnI and subsequentlycloned into pYeDP60 to give AtFLAGDGAT±pYeDP60.

N. tabacum DGAT1. Direct primer: atatatggatccATGGTGAT-CATGGAATTGCC. Reverse primer tatataggtaccTTAACGTG-CACTGCTTTTCT. The tobacco DGAT1 was amplified using

q FEBS 2000 Plant cDNAs encoding diacylglycerol acyltransferase (Eur. J. Biochem. 267) 87

25 thermal cycles (1 min at 93 8C, 2 min at 56 8C, 3 min at72 8C) with Pfu DNA polymerase under the standardconditions. The PCR product was digested with BamHI andKpnI and subsequently cloned into pYeDP60 to giveNtDGAT1±pYeDP60.

Caenorhabditis elegans DGAT. cDNA EST yk 453 a2 waskindly donated by Y. Kohara (Mishima, Japan). After sequen-cing, this clone perfectly matches a processed gene (Z75526)arising from the systematic sequencing of the genome of thisnematode. The ORF was amplified by PCR under standardconditions (see above) using the following primers. Directprimer: atatatggatccATGCAAATGCGTCAACAAACG. Reverseprimer: tatataggtaccTCAAATACCAACGGTTTGG. The PCRproduct was digested with BamHI and KpnI and subcloned intoBamHI, KpnI of pYEDP60 leading to CeDGAT±pYeDP60.

Nucleotide sequence determination

Sequencing of cDNAs AtDGAT, AtFLAGDGAT, NtDGAT1 andCeDGAT, as well as their ORF derivatives made by PCR, wasperformed using an automatic sequencer Perkin-Elmer model373 with T3, T7 primers, specific oligonucleotide sequencebelonging to the sequenced DNA and a modified Taqpolymerase capable of incorporating fluorescent dNTP. Com-plete sequencing of both DNA strands was performed. cDNAswere in pSK or in pYeDP60.

Transformation of yeast

Transformation was performed according to Schiestl and Gietz[17] with some modifications. A fresh yeast culture (initialabsorbance = 0.2) was grown in complete medium YPG[10 g´L21 yeast extract (Difco), 10 g´L21 bactopeptone(Difco), 20 g´L21 glucose] for 5 h. The cells were collected,washed twice with water and then with 1.5 mL of a 0.1 mlithium acetate solution in Tris/EDTA buffer (1 mm EDTA,10 mm Tris/HCl, pH 7.5) and finally resuspended in 200 mL ofthe same solution. Salmon sperm was added as a DNA carrier(100 mg from a 10 mg´mL21 solution in Tris/EDTA) aftersonication (10 s) and boiling (20 min) to the plasmid DNA(1 mg). Competent yeast cells (50±80 mL) and 50 mL of40% poly(ethylene glycol), 0.1 m lithium acetate solution inTris/EDTA (pH 7.5) were added. The mixture was incubatedfor 30 min at 30 8C followed by 15 min at 42 8C. Aftercentrifugation, the cells were resuspended in YPG (1 mL),incubated 1 h at 30 8C, collected and then plated (with 100 mLwater) on minimum medium containing suitable supplements(50 mg´mL21 each).

Plant expression vectors

A cDNA 3D11 encoding the ORF of At DGAT was subclonedinto the BamHI±KpnI sites of a pBD 121 binary vectorderivative engineered as described by Husselstein et al. [18].In particular, the vector contained between the left and rightT-DNA borders a kanamycin resistance gene and a multiplecloning site flanked in the 5 0-region by a duplicated 35Spromoter described by Kay et al. [19] and by a nopalinesynthase terminator at the 3 0-end. The At DGAT constructand the corresponding void plasmid were transferred fromE. coli XL1 Blue into Agrobacterium tumefaciens LB4404[20] by triparental mating with the helper plasmid pRK2013 [21].

Plant transformation

Tobacco (N. tabacum L. var Xanthi) was transformed viaAgrobacterium tumefaciens according to a modification [22] ofthe method reported by Horsch et al. [23]. In brief, leaf pieceswere cocultivated with an overnight A. tumefaciens culture onMurashige and Skoog medium supplemented with glucose(30 g´L21), ANA (0.1 mg´L21) and 6-benzylaminopurine(1 mg´L21) for 2 days and then transferred onto the samemedium supplemented with kanamycin (100 mg´L21) as theplant-selective agent and cefotaxime (500 mg´L21) to preventfurther bacterial growth. Regenerated shoots were rooted onMurashige and Skoog medium with a half-reduced concentra-tion of NH4NO3 and supplemented with sucrose (30 g´L21),kanamycin (200 mg´L21) and cefotaxime (200 mg´L21). Invitro cultures were grown under a 16-h light period at 24 8Cand an 8-h dark period at 20 8C. Primary transformants weresubcultured as T1 vitro-plantlets every 7±8 weeks in order togenerate leaf material in sufficient quantities for analyticalprocesses. Primary transformants were then transferred to thegreenhouse and grown under standard conditions.

RNA gel blot analysis

Total RNAs from leaf material of 6-week-old in vitro-grownprimary transformants were extracted according to the methoddescribed by Goodall et al. [24]. Northern analysis was carriedout by separating 10 mg RNA samples on formaldehyde gel asdescribed previously [25] and blotting them onto nylonfilters (Hybond-N+, Amersham). Randomly primed (Strata-gene) 32P-labeled probes polymerized from the 3D11 cDNAwere hybridized to the filters as recommended by themanufacturer. Filters were then washed in 0.2� NaCl/Citcontaining 0.1% SDS at 65 8C before autoradiography. Filterswere further washed in 0.1% SDS at 70 8C for 3 h and thenhybridized to randomly primed 32P-labeled probes polymerizedfrom a 18S rRNA as an internal standard.

Lipid analysis

Lipids were extracted from freeze-dried yeast cells or tobaccoleaves by immersion in methylene chloride/methanol (2 : 1)and heating under reflux for 1 h. The extract was submitted toTLC in methylene chloride into sterol esters (Rf = 0.8±0.7),triacylglycerols (Rf = 0.5±0.4) and free sterols (Rf = 0.3±0.1)using cholesteryl palmitate, trioleylglycerol, lanosterol andcholesterol as references.

Sterol esters were hydrolyzed by heating under reflux for 1 hwith methanolic KOH (6%). The sterols were then extractedwith hexane. Both free sterols and sterols corresponding tosterol esters were acetylated according to Schmitt andBenveniste [26]. Sterol acetates were purified by TLC inmethylene chloride, then quantified and identified by GC on aDB-1 capillary column (according to their relative retentiontime to the internal standard cholesterol), as describedprevioulsy [22].

Triacylglycerols purified by TLC from the lipid extract ofeither yeast or transgenic tobacco leaves were quantified usingthe colorimetric assay Perichrom Triglycerides GPO-PAP fromRoche-Diagnostics (Meylan, France), according to McGowanet al. [27].

GC-MS was performed on a computerized gas-chromato-graph mass spectrometer (Fison MD500) equipped with anon-column injector and a capillary column (30 m � 0.25 mminternal diameter) coated with DB5 (J&W Scientific). The


different fragments obtained are designated by the ratio m/z andtheir relative intensity.

Lipid-specific staining of cells

Leaf epidermis was stained in a satured Sudan IV (dye no.26106 according to the color index) solution in 70% (v/v)ethanol. Lipid droplets appear as orange spherical granules onlight-microscopy.

Subcellular fractionation

Yeasts were grown for 3 days in 100 mL glucose minimummedium followed by 16 h in 200 mL galactose completemedium (see above). The harvested cells were then disrupted asdescribed previously [28] except that KCl was omitted from thewashing buffer and BSA (1%) was added to the disruptionmedium. The homogenate was centrifuged for 10 min at12 000 g and the supernatant for 60 min at 100 000 g. Thelipid layer floating at the top of the 100 000 g supernatant fromthe plant DGAT-transformed yeasts was carefully withdrawn aswas the corresponding volume (4±6 mL) from the voidplasmid-transformed yeast. The microsomal pellet was resus-pended (2±5 mg protein´mL21) in 0.1 m Tris/HCl pH 7containing 20% (v/v) glycerol. In some experiments (Fig. 3A,C) microsomes and the floating lipid layer were washed bydilution in Tris/HCl buffer and recentrifugation. Coarse orwashed fractions were kept at 280 8C for months withoutsignificant loss of activity.

Proteins were quantified using the Bio-Rad protein assayaccording to Bradford [29]. Western blots of microsomes (6 mgprotein) or the floating lipid layer (5 mL) from AtFLAGDGAT-transformed yeast were achieved after SDS/PAGE, electro-transfer to a nylon membrane (Hybond C, Amersham) andimmunoblotting with the anti-FLAG M2 mAb from IBI/Kodak.

Enzymatic assays

ACAT assays were performed according to Billheimer et al.[30] using either [14C]cholesterol or [14C]oleyl CoA.

DGAT was assayed according to Cases et al. [12] with somemodifications. Diacylglycerol (either dioleylglycerol or dihex-anoylglycerol, 400 mm) was resuspended in 0.1 m Tris/HClbuffer pH 7 containing 20% glycerol, by vigorous shaking. Theenzymatic preparation, either microsomes (50 mg protein) or100 000 g supernatant top fraction (40 mL) was then added. Inthe control assays, proteins were omitted. After the addition of[14C]oleyl CoA (20 mm, 200 000 c.p.m.) the reaction mixture(100 mL) was incubated for 10 min at 30 8C. Under theseconditions the substrates were shown to be saturating, thereaction was linear with protein and time and the reaction yieldwas kept below 10%. The reaction was stopped by adding amixture of methylene chloride (400 mL) and methanol(100 mL) containing trioleylglycerol (100 mg) and cholesterylpalmitate (100 mg) as carriers. After further addition of water(300 mL) and methylene chloride (600 mL), the organic phasewas withdrawn and the water phase was extracted twice morewith methylene chloride. The lipid extract was separated byTLC with trioleylglycerol as standard and the radioactivetriacylglycerol bands (Rf 0.5±0.4 for incubations with orwithout dioleylglycerol, Rf 0.3±0.2 for those with dihexanoyl-glycerol) were detected with an automatic TLC-linear analyzer(Berthold), and scrapped off the plate for counting.

A large-scale, high-yield DGAT assay was set up for GC andGC-MS. The incubation mixture (2 mL) had the same

composition as described above except that unlabeled oleylCoA (40 mm) was used and a longer incubation time (2 h) wasapplied. After purification, the triacylglycerol formed fromdihexanoylglycerol was quantified by GC using the samecolumn and temperature program as for steryl acetates. GC-MSanalysis yielded the following spectrum: 552 (M+; 0.1), 534(M+-H2O; 0.2), 438 (5), 437 (M+-hexanoyloxy; 5), 393(oleyl + 128; 3), 363 (2), 340 (2), 339 (oleyl + 74; 2), 337(2), 283 (oleic acid + 1; 5), 271 (M+-oleyloxy; 49), 265 (oleyl;6), 264 (10), 257 (3), 227 (hexanoyl + 128; 10), 173(hexanoyl + 74; 21), 99 (hexanoyl; 100). These characteristictriacylglycerol fragmentation ions, as described by Barberet al. [31] and Christie [32], correspond unambiguously to1,2-dihexanoyl 3-oleyl glycerol.

R E S U LT S

Cloning of a cDNA encoding DGAT from A. thaliana

In order to isolate a full-length cDNA clone encoding anArabidopsis enzyme able to acylate plant sterols, we startedfrom an EST clone (GenBank accession number AA042298).After sequencing, this EST cDNA was shown to contain 849 bpand the deduced protein sequence had 30 and 25% identity withH. sapiens [33] and S. cerevisiae [34] ACATs, respectively.This EST clone was used as a probe to screen an A. thalianacDNA library, as described above. After screening500.000 p.f.u., 12 cDNA clones of different lengths wereisolated. Among them, 10 had a length compatible with those(< 2 kb) reported for mammalian and baker's yeast ACATcDNAs. Two (3D11 and 3D71) were sequenced and contained1845 and 2162 bp, respectively. They differed only by therespective length of their 5 0-UTR and 3 0-UTR. The 5 0-UTR and3 0-UTR of 3D71 were 173 and 429 bp. Both were shown toencode a polypeptide of 520 amino acids (58 947 Da)presenting 25% identity with both H. sapiens and baker'syeast ACATs and 40% identity with an ARGP from H. sapiens[11] and a DGAT from Mus musculus [12] (Table 1). It wasidentical to that isolated by Hobbs [13].

Cloning of a cDNA encoding DGAT from N. tabacum andcomparison of its deduced protein sequence with those ofother acyltransferases

A cDNA library from a 3-week-old N. tabacum cv. Xanthi lineLAB 1-4 calli derived from leaf protoplasts [16] was screenedusing the A. thaliana EST clone. As described above, sevencDNA clones of different lengths were isolated. Among themone had a length (2099 bp) comparable with that of theArabidopsis DGAT. It was shown to encode a polypeptide of532 amino acids presenting 68% identity with ArabidopsisDGAT (Table 1). Strikingly, the first 100 amino acids from theN-terminal sequence show a low identity (19%) with thecorresponding Arabidopsis sequence. After amino acid 135, theidentity increases abruptly and becomes . 70%. The KyteDoolittle hydrophobicity profile is very similar to that of theArabidopsis DGAT with a hydrophilic moiety of 120 aminoacids and 10 hydrophobic domains [13]. As in the ArabidopsisDGAT, and at exactly the same place, four leucines (L230,L237, L244, L251), are regularly spaced out by six variableamino acids forming a typical leucine zipper. This cDNA clonewas called Nt DGAT1. Another cDNA clone had a length of1354 bp. Its deduced amino acid sequence (317 amino acids)was much shorter than that of NtDGAT1 but had a high identity(86%) with it. This second cDNA (NtDGAT2) was of course


incomplete but its presence in the library showed that there areat least two genes encoding DGAT in tobacco.

Sequence comparison of the DGATs from Arabidopsis andtobacco with the two human ACATs [33], murine DGAT [12],the putative human DGAT called ARGP [11] and an unknowncDNA from C. elegans (putative DGAT) is shown in Fig. 1.Remarkably, all these sequences present the same global patternand are composed of a strongly hydrophilic moiety of < 100amino acids (in blue) followed by 10 hydrophobic domains (inred and boxed) which may correspond, in the case of DGATs,to nine membrane helix spanning domains as confirmed by thetransmembrane predict program (http://ulrec3.unil.ch/software/TMPRED-form.html). In addition all these cDNAs have severalinvariant domains in the second moiety of the deduced protein.A remarkably conserved domain (AELLCFGDREFYKDWW)is situated between amino acids 382 and 400 of the AtDGAT.This sequence is present in a hydrophilic domain situatedbetween two supposed transmembrane helices. It is common toboth DGATs and ACATs. Also worthy of interest is thepresence in all DGATs and ACATs of an invariant serine(Ser254 in AtDGAT) which has been shown to be essential forACAT activity [37]. However, the DGAT sequences can easilybe distinguished from the ACAT sequences. The five DGATsequences possess a remarkable motif consisting of fourconsecutive positively charged amino acids (four Arg in fourof five DGATs, three Arg and one Lys in AtDGAT). This clusteris absent from all ACATs. Several other motives are alsorestricted to the five DGATs, one example is a sequenceIERVLKL starting at Ileu352 of AtDGAT.

Organization of the gene encoding A. thaliana DGAT

In recent months a genomic sequence originated fromchromosome II sequencing appeared in GenBank (AC005917)and was designated as `putative acyl CoA:cholesterol acyl-transferase'. According to databases (gene prediction programgrail), this sequence was processed into a polypeptide (441amino acids) matching partially the protein (520 amino acids)deduced from the AtDGAT cDNA. Using the GTX and XAGrule defining introns boundaries for splicing, we reconstitutedfrom a 5051-bp genomic sequence corresponding to thepolypeptide of 441 amino acids, a cDNA perfectly matchingour AtDGAT cDNA. According to this study the gene encodingthe Arabidopsis DGAT was formed by 16 exons separated by 15introns (Fig. 2). The sequence (817 bp) situated upstream ofthe ATG was submitted to the place signal scan program(http://www.dna.affrc.go.jp/htdoc/PLACE/WAIS.html) to identify

cis-acting regulatory DNA elements possibly intervening in theregulation of DGAT expression. Interestingly an ethylene-responsive element (ATTTCAAA) [36] was shown to bepresent 719 bp upstream of the ATG. This important findingis discussed below.

If one now considers the sequence downstream of the ATG, itappears that the first exon (130 amino acids) correspondsexactly to the hydrophilic moiety of the protein. The secondexon fits precisely with the first hydrophobic trans-membranesegment.

Expression of AtDGAT in a yeast mutant deficient in ACATactivity

Because this cDNA was first expected to encode an ACAT-likeprotein, it was expressed in the yeast double-mutant are1 are2under control of the GAL10-CYC1 promoter from the vectorpYeDP60. Because in this mutant the two ACAT genes havebeen knocked out, sterol ester biosynthesis is absent [33] andmicrosomes from this yeast lack ACAT activity [32]. Sterolester and free sterol contents were determined for AtDGAT-transformed are1 are2 and the void plasmid-transformed yeast.Both were devoid of sterol esters and had similar sterol profile(data not shown). Microsomes were prepared from both yeastsand ACAT activity was assayed. It was undetectable underconditions in which microsomes from the corresponding wild-type yeast, transformed or not with AtDGAT, displaidsignificant activity (160 pmol sterol esters per mg protein permin). Taken together, these results clearly showed that cDNAAtDGAT does not encode an ACAT.

Meanwhile, Cases et al. [12] described a mouse cDNAencoding a DGAT, with which AtDGAT shared 40% identity.Therefore, the DGAT activities of AtDGAT-transformed are1are2 and void plasmid-transformed are1 are2 were measuredand compared. Microsomes from both yeasts were incubatedwith [14C]oleyl CoA in the absence or presence of dioleyl-glycerol or dihexanoyl-glycerol (Fig. 3A). The results clearlyshow that although microsomes from the void plasmid-transformed yeast display a significant DGAT activity due tothe yeast enzyme, microsomes from the AtDGAT-transformedyeast show a higher (more than double) activity, correspondingto the expression of both the yeast and the plant genes. Theseresults further show that yeast and plant DGAT activities aremeasurable without added diacylglycerols, presumably becausediacylglycerols are present in the enzymatic preparations orproduced during the course of the incubation. The addition ofdioleylglycerol or dihexanoylglycerol significantly increases

Table 1. Comparison of the deduced amino acid sequence of A. thaliana DGAT with those of other acyltransferases showing % identity. The names

and accession numbers are those in Fig. 1, except ScACAT1 and 2, which are the Saccharomyces cerevisiae sequences (P25268 and U51790, respectively)

and CeACAT, which is the Caenorhabditis elegans sequence (Z68131).

AtDGAT NtDGAT HsARGP MmDGAT CeDGAT HsACAT1 HsACAT2 ScACAT1 ScACAT2 CeACAT

AtDGAT 100

NtDGAT 68 100

HsAGRP 40 38 100

MmDGAT 39 38 86 100

CeDGAT 36 37 47 47 100

HsACAT1 25 25 26 27 25 100

HsACAT2 24 26 25 26 25 49 100

ScACAT1 26 26 21 23 24 23 25 100

ScACAT2 26 27 23 24 26 28 24 49 100

CeACAT 20 19 24 24 21 34 31 23 25 100


both yeast and plant activities. Expression of AtDGAT in thecorresponding wild-type yeast gave identical results (data notshown). Thus expression of cDNA AtDGAT in yeast produces a2±3 times increase of DGAT activity in the microsomalfraction.

Expression of AtDGAT, AtFLAGDGAT and NtDGAT1 inwild-type yeast

The wild-type yeast was transformed with AtDGAT, a FLAGepitope-tagged version of AtDGAT called AtFLAGDGAT, aN. tabacum homolog of AtDGAT, called NtDGAT1, or the void

plasmid. The triacylglycerol content of these strains wasdetermined. The three DGAT-transformed yeasts had signifi-cantly higher triacylglycerol contents than the void plasmid-transformed yeast (25±80 versus 9 ^ 3 nmol triacylglycerolper mg dry weight).

DGAT activity was measured in microsomes from yeasttransformed with either AtDGAT, AtFLAGDGAT or NtDGAT1in the presence of [14C]oleyl CoA and dioleyl-glycerol assubstrates and shown to be significantly higher than that of thevoid plasmid-transformed yeast (20±24 versus 6 nmol triacyl-glycerol formed per mg protein per h). Taken together, theseresults obtained with the wild-type yeast confirm those obtained

Fig. 1. Sequence alignment of various putative or demonstrated diacylglycerol or acyl CoA cholesterol acyltransferases. DGAT, acyl

CoA:diacylglycerol acyltransferase; ACAT, acyl CoA:cholesterol acyltransferase; ARGP, ACAT-related gene product. Alignments were performed using

the pileup program of the GCG package run with default parameters and the seqvu program. AtDGAT is the A. thaliana sequence (GenBank accession

number AF051849). NtDGAT1 is the N. tabacum sequence (GenBank accession number AF129003). MmDGAT is the M. musculus sequence

(GenBank accession number AF078752). HsARGP is the H. sapiens sequence (GenBank accession number AF059202). CeDGAT is the C. elegans

sequence (GenBank accession number Z75526). HsACAT1 is the H. sapiens sequence (GenBank accession number L21934). HsACAT2 is the

H. sapiens sequence (GenBank accession number AF059203). Hydrophilic domains are in blue. Ten hydrophobic segments (red) are boxed. Signs are

placed under important amino acids (e.g. the Ser254 of the Arabidopsis cDNA) or highly conserved motives such as the invariant

FGDREFYRDWWN segment. Clusters of arginines present in DGATs are underlined. Leucines involved in a putative leucine zipper are circled.


for AtDGAT with the mutant are1 are2, indicate that the FLAGepitope-tagged version of AtDGAT produces a functionalprotein and clearly show that NtDGAT1 encodes a DGAT.

The microsomal fractions from AtDGAT-transformed andAtFLAGDGAT-transformed yeast cells, were submitted toSDS/PAGE followed by a Western blot analysis usingcommercial anti-FLAG serum (Fig. 3B). The blot revealed atagged protein with a molecular mass (54 kDa) close to that(59 kDa) calculated for AtDGAT according to its sequence.

Expression of AtDGAT in the floating lipid layerfrom the transformed yeast: identification of theenzymatic product

In the course of the preparation of microsomes from plantDGAT-transformed yeasts, we consistently observed theformation of a cloudy floating layer on top of the 100 000 gsupernatants from the AtDGAT-tranformed, AtFLAGDGAT-transformed or, to a lesser extent, NtDGAT1-transformedyeasts, whereas the supernatant from the void plasmid-transformed yeast remained clear. These floating layers andthe corresponding region of the 100 000 g supernatant fromthe void plasmid-transformed yeast were taken from thecentrifugation tubes for analysis.

Extraction of lipids and purification of triacylglycerols byTLC revealed that the floating layer from the AtDGAT-transformed yeast had a much higher triacylglycerol mass(assessed by I2 visualization) than the 100 000 g supernatanttop fraction from the void plasmid-transformed yeast (data notshown). Furthermore, the floating lipid layers from yeasttransformed with AtDGAT, AtFLAGDGAT or NtDGAT1 wereshown to display much higher DGAT activities than the100 000 g supernatant top from the void plasmid-transformedyeast (5, 4 and 1 versus 0.1 nmol´mL21´h21). The presence ofplant DGAT in the floating lipid layer from AtFLAGDGAT-transformed yeast was confirmed by SDS/PAGE followed byWestern blot analysis (Fig. 3B).

The DGAT activities of the 100 000 g supernatant topfractions from yeast transformed with AtDGAT or the voidplasmid were studied further. The activities were measured with[14C]oleyl CoA in the absence or presence of dioleylglycerol ordihexanoylglycerol (Fig. 3C). The 100 000 g supernatant topfrom the control yeast is almost devoid of activity, whereas thefloating lipid layer from the AtDGAT-transformed yeastcontains significant activity, 200±600 times higher than thatof the control. Hence the floating lipid layer obtained fromAtDGAT-transformed yeast exhibits a DGAT activity almostexclusively due to the plant gene. Because of the presence ofBSA in the 100 000 g supernatant, specific activities could notbe determined for this fraction. Its total DGAT activityrepresented one half to one sixth, according to the experiment,that of the corresponding microsomal fraction.

As observed with the microsomal preparations (Fig. 3A) theDGAT activity of the floating lipid layer is measurable without

added diacylglycerols, probably for the same reasons. However,the addition of dioleylglycerol or dihexanoylglycerol signifi-cantly increases the activity of the plant enzyme expressed inyeast (Fig. 3C). Dihexanoylglycerol was again a good substrateand, because of its low molecular mass, the resultingtriacylglycerol (Rf = 0.2) could be easily separated from the

Fig. 3. DGAT enzymatic activities in subfractions of yeast expressing

AtDGAT. Yeasts were transformed with B the void pYeDP60 plasmid, B

AtDGAT in pYeDP60, or AtFLAGDGAT in pYeDP60. (A) DGAT activities

(nmol triacylglycerol per mg protein per h) in washed microsomes from

void plasmid-transformed or AtDGAT-transformed are1 are2 mutant. (B)

Expression of the FLAG-tagged DGAT. Microsomes (6 mg protein) from

wild-type yeast transformed with (1) AtFLAGDGAT or (2) AtDGAT and

100 000 g supernatant top fractions (5 mL) from wild-type yeast trans-

formed with (3) AtFLAGDGAT or (4) AtDGAT were resolved by SDS/PAGE.

Proteins were submitted to immunoblotting with an anti-FLAG serum.

(C) DGAT activities (nmol triacylglycerol´mL21´h21) in twice-washed

100 000 g supernatant top fractions from void plasmid-transformed or

At DGAT-transformed wild-type yeast. DOG, dioleylglycerol; DHG,

dihexanoylglycerol.

Fig. 2. Organization of the gene encoding the diacylglycerol acyltransferase from A. thaliana. The part of the gene corresponding to the coding region

(3020 bp) contains 16 exons and 15 introns. Exons are shown by continuous heavy lines, introns are shown by continuous thin lines. Parts of the gene

upstream and downstream of the coding region are shown by broken lines. The numbers under the exons and above the introns are in bp. The motif

ATTTCAAA at 2719 bp is a putative EREE. The arrow indicates the translation start point determined by Hobbs et al. [13]. S,E,A: SacI, EcoRI, AseI.

Letters b and e mark the 5 0-end and the 3 0-end of the longest cDNA (3D71).


usual long-chain fatty acid triacylglycerols (Rf = 0.4) usingTLC. This latter TLC band corresponds to unlabeled pre-existing triacylglycerols together with labeled triacylglycerolsformed during the incubation from endogenous diacylglycerols.In order to identify unambiguously the product of the reactioncatalyzed by the A. thaliana DGAT, we scaled up (20 times)the usual incubation mixture with dihexanoylglycerol andthe floating lipid layer from AtDGAT-transformed yeast,lengthened the incubation time (to 2 h) and could thus purify20 mg of the short-chain fatty acid triacylglycerol. GC-MSanalysis of this pure product clearly confirmed its structure as1,2-dihexanoyl 3-oleyl glycerol (see MS in Experimentalprocedures). It must be noted that the same high-scale, long-time incubation with the 100 000 g supernatant top fractionfrom the control yeast yielded 0.5 mg of this low molecularmass triacylglycerol. Under these conditions, the floating lipidlayer from the AtDGAT-transformed yeast produced 40 times

more 1,2-dihexanoyl 3-oleyl glycerol, which definitely provesthe function of the protein encoded by AtDGAT.

Expression of AtDGAT in N. tabacum

AtDGAT cDNA encoding the ORF of the gene was introducedinto N. tabacum under the control of a strong constitutivepromoter (tandem CaMV 35S). Nineteen primary transformantsgrown in vitro on kanamycin-supplemented Murashige andSkoog medium were analyzed and compared with wild-typetobacco. The upper leaves from 6-week-old plants wereseparated into two batches: one for triacylglycerol dosage andthe other for RNA gel blots.

The results shown in Fig. 4 clearly show that, in all cases, theexpression of AtDGAT in tobacco is associated with asignificant increase in the triacylglycerol content. In the bestcase, this increase was up to seven times the mean value

Fig. 4. Expression of At DGAT in N. tabacum.

Lanes 1±19, primary transformants; lane 20,

wild-type tobacco. (A) Triacylglycerol content.

Triacylglycerols were extracted from young

leaves, purified by TLC and quantified using a

colorimetric assay. The column in lane 20

corresponds to the mean of three different

wild-type plants (21 ^ 5 nmol triacylglycerol

per mg dry weight). (B) Northern analysis: total

leaf RNA from primary transformants (around

10 mg) was hybridized with a probe derived

from the Arabidopsis DGAT. The arrow denotes

the DGAT 1.5 kb transcript. (C) 18S rRNA

hybridization of the same blot is shown to

illustrate the relative amount of RNA loaded

and transferred.

Fig. 5. Light micrographs (� 850) of petiole or limb epidermis cells after staining in a solution of Sudan IV. (A) Wild-type petiole epidermis; (B) wild-

type limb epidermis; (C) petiole epidermis of triacylglycerol-overproducer line 5; (D) line 5 limb epidermis. triacylglycerol dosage gave the following values

for the samples shown in this figure: wild-type leaf material, 3.1 nmol triacylglycerol per mg dry weight; line 5 leaf material, 11.5 nmol triacylglycerol per

mg dry weight.


measured for wild-type leaf material. The specificity of theexpression was attested to by the fact that control tobacco didnot exhibit any AtDGAT expression, showing that under ourexperimental conditions the AtDGAT probe did not hybridizewith the endogenous NtDGAT RNA and allowed quantificationof transgene expression only.

The primary transformants were then transferred to thegreenhouse and grown under standard conditions. Leaf materialwas collected at the flowering stage (3.5 months old) andsubmitted to triacylglycerol dosage. The results were in fullagreement with those obtained with the corresponding in vitroplantlets, i.e. an accumulation of triacylglycerols up to fivetimes the control value (data not shown). To address thequestion of the intracellular location of the overproducedtriacylglycerols, we stained petiole and limb epidermis peelsfrom the wild-type and transgenic lines in an ethanolic solutionof Sudan IV, a lipid-specific dye. Light-microscopy obser-vations showed the presence of lipid droplets that appeared asorange spheres in the cytoplasm of stained cells from transgenictriacylglycerol-overproducer plants (Fig. 5C,D); control lines,as well as low-triacylglycerol-containing transgenic plants,showed very few (or none) of these lipid droplets (Fig. 5A,B).Taken together these data demonstrate that, as expected,AtDGAT is involved in triacylglycerol synthesis in planta.

D I S C U S S I O N

As shown here cDNA clones encoding DGAT from Arabidopsis(AtDGAT ) and tobacco (NtDGAT ) have been characterized.This was carried out using their functional expression in yeastand in tobacco. At the time when this was performed, a similarstudy on the functional expression of AtDGAT in insect cellswas published [13]. However, our work is original in thefollowing aspects.

Protein sequences deduced from the cDNA clones

As shown in Table 1 the protein sequences deduced from thecDNAs or genes considered here can be divided into twofamilies. The first is composed of proteins having < 35±75%identity with the At DGAT. In this group we find the identifiedDGATs from Arabidopsis, tobacco and mouse, the human clonecalled ARGP, which was shown not to be an ACAT andsuggested to be a DGAT [11], and a cDNA clone fromC. elegans, which can be considered as a putative DGATbecause of its relatively high identity (36%) with that of theArabidopsis DGAT. The second group is composed of proteinshaving < 25% identity with AtDGAT. In that group we find thetwo human ACATs (HsACAT1 and HsACAT2) and the twoyeast ACATS (ScACAT1 and ScACAT2). As shown in thesequence alignments (Fig. 1), divergences between DGATs andACATs are concentrated in the first moiety starting from theN-terminal part of the protein, while the second moiety of theprotein is remarkably conserved among the seven sequences.These strong identities show clearly that ACATs and DGATsare closely related evolutionarily. This is not surprising becauseboth enzymes catalyze the acylation by a fatty acyl CoA esterof strongly hydrophobic alcohols associated with endoplasmicreticulum: diacylglycerol for DGAT and cholesterol for ACAT.The other acyl CoA acyltransferases known to date, glycero-phosphate acyltransferase, lysophosphatidate acyltransferase[38] and vindoline acetyltransferase [39], did not show anysignificant sequence identity suggesting that they are unrelated.

Expression of the Arabidopsis and tobacco DGATs in yeast

Whereas expression of the Arabidopsis cDNA cloned here, inthe double mutant (are1 are2) defective in sterol acyltrans-ferases, did not lead to either complementation of the mutationor detection of any ACAT activity at the microsomal level, astrong increase in the triacylglycerol content of the yeast and inthe DGAT activity of its microsomes were observed. Becauseno yeast mutant defective in triacylglycerol synthesis has beenreported to date [5] we used either are1 are2 or a wild-typeyeast. Fortunately under the conditions used, the in vitroincorporation of [14C]oleyl CoA into triacylglycerols wassignificantly higher in microsomes from yeast transformedwith AtDGAT-pYeDP60 than with the void vector (Fig. 3A).Under these conditions the increment of DGAT activity due tothe expression of the plant cDNA culminated at . 40 nmoltriacylglycerol´h21´mg protein21, a value 102 times higher thanthat (300 pmol triacylglycerol´h21´mg protein21) reported inthe insect expression system [13]. As significant endogenousDGAT activity was observed in control yeast, we tried to deviseconditions to decrease this yeast activity. Such a goal wasachieved using Triton X-100 in the incubation medium. Indeedthe detergent almost completely inhibited the endogenousDGAT activity, but also decreased the plant DGAT activity inthe AtDGAT-transformed yeast by a factor of 2 or moreaccording to the experiments (results not shown).

The best results were obtained after the discovery that thefloating lipid layer appearing during the course of themicrosome preparation from yeast transformed with AtDGATexhibited strong DGAT activity, whereas the correspondingzone of the 100 000 g supernatant from the yeast transformedwith the void vector was almost devoid of activity (Fig. 3C).Such selective expression of AtDGAT in the floating lipidlayer from the transformed yeast allowed us to demonstratethe net synthesis by AtDGAT of a specific triacylglycerol(1,2-dihexanoyl 3-oleyl glycerol), which could be separatedthoroughly from endogenous triacylglycerols by TLC andclearly identified by GC-MS. Finally, the use of a N-terminalFLAG epitope in the cDNA AtDGAT allowed us to show thatthe enzymatic activity measured in transformed yeast can beascribed to the presence of a protein with a molecular mass(54 kDa) compatible with that (59 kDa) deduced from theproteic sequence. As expected, this protein was shown to bepresent both in the microsomal fraction and in the floating lipidlayer (Fig. 3B). Therefore, the yeast system appears to beappropriate for the expression of AtDGAT. It also allowedidentification of a new DGAT from tobacco. The charac-terization of a DGAT from C. elegans is in progress in the sameexpression system.

As mentioned above, DGAT is a membrane-bound enzyme.In plants it is thought to be located in the endoplasmicreticulum [40±42] but its activity has also been suggested in oilbody fractions from Brassica napus [43]. This dual localizationof DGAT was also suggested in the case of yeast [44] anddescribed in an oleaginous fungus [45]. As in animals, the lipidbodies (or oil bodies) of plants and yeast are believed to arisefrom specific microdomains of the endoplasmic reticulummembrane that contain the full set of triacylglycerol bio-synthesis enzymes [7,42,46]. Hence, some of these enzymesmight remain attached to the membrane of the lipid (oil) bodiesafter their budding [7]. Indeed such a dual localization has beendescribed in yeast for some enzymes involved in triacylglycerolsynthesis, glycerol 3-phosphate acyltransferase and 1-acyl-glycerol 3-phosphate acyltransferase [5], as well as enzymes ofsterol biosynthesis, sterol methyl transferase ERG6 [47] and


squalene epoxidase ERG1 [48]. The localization of the plantDGAT in At DGAT-transformed yeast is therefore not surprisingeven thought yeast DGAT is localized mainly in microsomes inour conditions of culture.

Expression of AtDGAT in tobacco

Although the gene AtDGAT was efficiently expressed in yeastto give an enzymatically active protein, this result did not giveany information about the activity of this protein inside theplant. For a complete characterization it was necessary to showthat the gene could be expressed in plants. To this purposetransgenic tobaccos expressing AtDGAT cDNA and over-producing triacylglycerols have been characterized. AlthoughDGAT enzymatic assays were not performed here, it seemsreasonable to assume that triacylglycerol overproductioncorresponds to increased DGAT activity. Relevant to theseconsiderations, an EMS-mutant of Arabidopsis showing acorrelation between lower DGAT activity and poor triacyl-glycerol formation has been reported previously [49].

The accumulation of triacylglycerols in leaf material fromtransgenic DGAT plants was shown to be associated with thepresence of lipid droplets in the cytoplasm of their cells(Fig. 5). Although this does not prove directly that the dropletscontain exclusively the overproduced triacylglycerols andcorrespond to the well-defined plant lipid bodies [7], it ismost likely that they are budding from the endoplasmicreticulum where triacylglycerols are synthetized. Synthesisand storage of triacylglycerols, for example in seeds, have beenwidely documented as a process in which these compoundsaccumulate in the endoplasmic reticulum membrane and budoff as droplets surrounded by a phospholipid monolayer [7]. Wepreviously observed lipid droplets similar to those shown inFig. 5, in the cytoplasm of cells from sterol-overproducer plantlines. Sterols, which are biosynthetized in the endoplasmicreticulum as triacylglycerols are, were shown to accumulate, inthese plant lines, as fatty acid steryl esters [16,50,51].

Triacylglycerol accumulation has already been described inthe case of a plant (Arabidopsis) expressing a yeast gene(encoding lysophosphatidic acid acyltransferase) [52]. Never-theless, the present work constitutes the first report to show anaccumulation of triacylglycerols in a transgenic plant expres-sing a plant gene (DGAT ). This result suggests that DGATactivity would be limiting, at least in tobacco leaves, whichnormally produce small amounts of triacylglycerols. That thesame stimulation of triacylglycerol synthesis that occurs intissues such as tobacco flowers and seeds, but also in fruits andtubers in other species, remains to be shown.

These considerations raise the interesting problem of theregulation of the AtDGAT gene. The DGAT gene correspondingto the cDNA described here was sequenced recently onBAC F27F23 (GenBank accession number AC003058) andBAC F3 P11 (GenBank accession number AC005917) and wasshown to be positioned on chromosome II. The region upstreamof the translation starting point possesses interesting features.As shown recently [13] the transcription starting point issituated at 2225 bp, leading to a leader sequence much longerthan the average in higher plant genes. A similar situation hasbeen observed in the gene encoding D7-sterol-C5-desaturase inArabidopsis [18]. As shown previously [53] sequencescontained within the 5 0-transcribed, UTR may play a key rolein regulation of hydroxymethyl glutaryl CoA reductaseexpression in Arabidopsis, therefore similar studies should beconducted in the DGAT gene. A characteristic ethyleneresponsive enhancer element (EREE) ATTTCAAA has been

found in the promoter of genes activated by ethylene incarnation flower petals [36] and in ripening tomato fruit [54].The same EREE motif has been found in the DGAT gene at2719 bp upstream to ATG. As triacylglycerols have beenshown to accumulate in flower petals and DGAT was shown tobe strongly expressed in Brassica napus flower petals [13], itmay be suggested that the EREE motif would play an importantrole in the regulation of triacylglycerol synthesis.

Finally, it appears from the literature that triacylglycerol andsteryl ester biosynthesis in animals and plants are closelyinterwoven. Doubtless the DGAT cDNAs described hereconstitute potent tools for studying these interactions. However,plant genes encoding the enzyme catalyzing the acylation ofplant sterols have still to be characterized. Whilst a revision ofthis manuscript after it's review was in progress, another paperdescribing A. thaliana DGAT in the mutant line AS11 waspublished [55].

A C K N O W L E D G M E N T S

We are grateful to Dr Y. Kohara (National Institute of Genetics,

Mishima, Japan) who kindly gave the EST cDNA yk453a2 from

C. elegans and to Dr D. Pompon (Center National de la Recherche

Scientifique, Gif sur Yvette) for allowing us to use the plasmid pYeDP60.

We warmly acknowledge the skillful assistance of M. Schmitz for the

transformation and in vitro culture of tobacco, A. Hoeft for the GC-MS,

B. Bastian and L. Thiriet for typing the manuscript. We also wish to

thank T. Husselstein, I. Benveniste and R. Bronner for their advice

on yeast transformation, Western blotting and microscopic observation,

respectively.

R E F E R E N C E S

1. Kennedy, E. (1961) Biosynthesis of complex lipids. Fed. Proc. 20,

934±940.

2. Lehner, R. & Kuksis, A. (1996) Biosynthesis of triacylglycerols. Prog.

Lipid Res. 35, 169±201.

3. Bell, R.M. & Coleman, R.A. (1980) Enzymes of glycerolipid synthesis

in eukaryotes. Annu. Rev. Biochem. 49, 459±487.

4. Leber, R., Zinser, E., Zellnig, G., Paltauf, F. & Daum, G. (1994)

Characterization of lipid particles of the yeast, Saccharomyces

cerevisiae. Yeast 10, 1421±1428.

5. Daum, G., Lees, N.D., Bard, M. & Dickson, R. (1998) Biochemistry,

cell biology and molecular biology of lipids of Saccharomyces

cerevisiae. Yeast 14, 1471±1510.

6. Lassner, M. (1997) Transgenic oilseed crops: a transition from basic

research to product development. Lipid Technol. 9, 5±9.

7. Murphy, D.J. & Vance, J. (1999) Mechanisms of lipid-body formation.

Trends Biol. Sci. 24, 109±115.

8. Piffanelli, P., Ross, J.H.E. & Murphy, D.J. (1997) Intra- and

extracellular lipid composition and associated gene expression

patterns during pollen development in Brassica napus. Plant J. 11,

549±562.

9. Perry, H.J. & Harwood, J.L. (1993) Changes in the lipid content

of developing seeds of Brassica napus. Phytochemistry 32,

1411±1415.

10. Ichihara, K., Takahashi, T. & Fujii, S. (1988) Diacylglycerol

acyltransferase in maturing safflower seed: its influences on the

fatty acid composition and on the rate of triacylglycerol synthesis.

Biochim. Biophys. Acta 958, 125±129.

11. Oelkers, P., Behari, A., Cromley, D., Billheimer, J.T. & Sturley, S.L.

(1998) Characterization of two human genes encoding acyl coenzyme

A: cholesterol acyltransferase-related enzymes. J. Biol. Chem. 273,

26765±26771.

12. Cases, S., Smith, S.J., Zheng, Y.W., Myers, H.M., Lear, S.R.,

Sande, E., Novak, S., Collins, C., Welch, C.B., Lusis, A.J.,

Erickson, S.K. & Farese, R.V. Jr (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.

13. Hobbs, D.H., Lu, C. & Hills, M.J. (1999) Cloning of a cDNA encoding

diacylglycerol acyltransferase from Arabidopsis thaliana and its

functional expression. FEBS Lett. 452, 145±149.

14. Urban, P., Cullin, C. & Pompon, D. (1990) Maximizing the expression

of mammalian cytochrome P450 monooxygenase activities in yeast

cells. Biochimie 72, 463±472.

15. Kieber, J.J., Rothenberg, M., Roman, G., Feldmann, K.A. & Ecker, J.R.

(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.

16. Maillot-Vernier, P., Gondet, L., Schaller, H., Benveniste, P. & Belliard,

G. (1991) Genetic study and further biochemical characterization

of a tobacco mutant that overproduces sterols. Mol. Gen. Genet.

231, 33±40.

17. Schiestl, R.H. & Gietz, R.D. (1989) High efficiency transformation of

intact cells using stranded nucleic acids as carrier. Curr. Genet. 16,

339±346.

18. Husselstein, T., Schaller, H., Gachotte, D. & Benveniste, P. (1999)

D7-sterol-C5-desaturase: molecular characterization and functional

expression of wild type and mutant alleles. Plant Mol. Biol. 39,

891±906.

19. Kay, R., Chan, A., Daly, M. & McPherson, J. (1987) Duplication of

CaMV35S promoter sequences creates a strong enhancer for plant

genes. Science 36, 1299±1302.

20. Hoekema, A., Hirsch, P.R., Hooykaas, P.J.J. & Schilperoort, R.A.

(1983) A binary plant vector strategy based on separation of vir- and

T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature 303,

179±180.

21. Bevan, M. (1984) Binary Agrobacterium vectors for plant transfor-

mation. Nucleic Acids Res. 12, 8711±8721.

22. Schaller, H., Bouvier-NaveÂ, P. & Benveniste, P. (1998) Overexpression

of an Arabidopsis thaliana (L.) Heynh. cDNA encoding a sterol-

C241-methyltransferase in Nicotiana tabacum L. modifies the ratio of

24-methyl cholesterol to sitosterol and is associated with growth

reduction. Plant Physiol. 118, 461±469.

23. Horsch, R.B., Fry, J.E., Hoffmann, N.L., Eichholtz, D., Rogers, S.G. &

Fraley, R.T. (1985) A simple and general method for transferring

genes into plants. Science 227, 1229±1231.

24. Goodall, G.J., Wiebauer, K. & Filipowicz, W. (1990) Analysis of pre-

mRNA processing in transfected plant protoplasts. Methods Enzymol.

88, 148±161.

25. Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989) Molecular Cloning: a

Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press,

Cold Spring Harbor, New York, USA.

26. Schmitt, P. & Benveniste, P. (1979) Effect of AY-9944 on sterol

biosynthesis in suspension cultures of bramble cells. Phytochemistry

18, 445±450.

27. McGowan, M.W., Artiss, J.D., Strandbergh, D.R. & Zak, B. (1983) A

peroxidase-coupled method for the colorimetric determination of

serum triglycerides. Clin. Chem. 29, 538±542.

28. Urban, P., Werck-Reichhart, D., Teutsch, H.G., Durst, F., Regnier, S.,

Kazmaier, M. & Pompon, D. (1994) Characterization of recombinant

plant cinnamate 4-hydroxylase produced in yeast. Kinetic and

spectral properties of the major plant P450 of the phenylpropanoid

pathway. Eur. J. Biochem. 222, 843±850.

29. Bradford, M.M. (1976) A rapid and sensitive method for the

quantitation of microgram quantities of protein utilizing the principle

of protein±dye binding. Anal. Biochem. 72, 248±254.

30. Billheimer, J.T., Tavani, D. & Nes, W.R. (1981) Effect of a dispersion

of cholesterol in Triton WR-1339 on acyl CoA: cholesterol

acyltransferase in rat liver microsomes. Anal. Biochem. 111,

331±335.

31. Barber, M., Merren, T.O. & Kelly, W. (1964) The mass spectrometry of

large molecules I. The triglycerides of straight chain fatty acids.

Tetrahedron Lett 18, 1063±1067.

32. Christie, W.W. (1989) Molecular species analysis by GC. In Gas

Chromatography and Lipids. A Practical Guide (Walker, C.W., ed.),

pp. 202±204. The Oily Press, Ayr, UK.

33. Yang, H., Cromley, D., Wang, H., Billheimer, J.T. & Sturley,

S.L. (1997) Functional expression of a cDNA to human

acyl-coenzyme A: cholesterol acyltransferase in yeast. J. Biol. Chem.

272, 3980±3985.

34. Yang, H., Bard, M., Bruner, D.A., Gleeson, A., Deckelbaum, R.J.,

Aljinovic, G., Pohl, T.M., Rothstein, R. & Sturley, S.L. (1996)

Sterol esterification in yeast: a two-gene process. Science 272,

1353±1356.

35. Hanley, B.A. & Schuler, M.A. (1988) Plant intron sequences:

evidence for distinct groups of introns. Nucleic Acids Res. 16,

7159±7176.

36. Itzhaki, H., Maxson, J.M. & Woodson, W.R. (1994) An ethylene-

responsive enhance element is involved in the senescence-related

expression of the carnation glutathion-S-transferase (GST1) gene.

Proc. Natl Acad. Sci. USA 19, 8925±8929.

37. Cao, G., Goldstein, J.L. & Brown, M.S. (1996) Complementation of

mutation in acyl-Coa: cholesterol acyltransferase (ACAT) fails to

restore sterol regulation in ACAT-defective sterol resistant hamster

cells. J. Biol. Chem. 24, 14642±14648.

38. Hanke, C., Wolter, F.P., Coleman, J., Peterek, G. & Frentzen, M. (1995)

A plant acyltransferase involved in triacylglycerol biosynthesis

complements an Escherichia coli sn-1-acylglycerol-3-phosphate

acyltransferase mutant. Eur. J. Biochem. 232, 806±810.

39. St-Pierre, B., Laflamme, P., De Alarco, A.M. & Luca, V. (1998)

The terminal O-acetyltransferase involved in vindoline biosyn-

thesis defines a new class of proteins responsible for coenzyme

A-dependent acyl transfer. Plant J. 14, 703±713.

40. Cao, Y.Z. & Huang, A.H.C. (1986) Diacylglycerol acyltransferase in

maturing oil seeds of maize and other species. Plant Physiol. 82,

813±820.

41. Settlage, S.B., Wilson, R.F. & Kwanyuen, P. (1995) Localization of

diacylglycerol acyltransferase to oil body associated endoplasmic

reticulum. Plant Physiol. Biochem. 33, 399±407.

42. Lacey, D.J. & Hills, M.J. (1996) Heterogeneity of the endoplasmic

reticulum with respect to lipid synthesis in developing seeds of

Brassica napus L. Planta 199, 545±551.

43. Murphy, D.J. & Mukherjee, K.D. (1987) Acyltransferases in sub-

cellular fractions of developing seeds of rape (Brassica napus L.).

Lipids 22, 293±298.

44. Christiansen, K. (1978) Triacylglycerol synthesis in lipid particles from

baker's yeast (Saccharomyces cerevisiae). Biochim. Biophys. Acta

530, 78±90.

45. Kamisaka, Y., Mishra, S. & Nakahara, T. (1997) Purification

and characterization of diacylglycerol acyltransferase from the

lipid body fraction of an oleaginous fungus. J. Biochem. 121,

1107±1114.

46. Lacey, D.J., Beaudoin, F., Dempsey, C.E., Shewry, P.R. & Napier, J.A.

(1999) The accumulation of triacylglycerols within the endoplasmic

reticulum of developing seeds of Helianthus annuus. Plant J. 17,

397±405.

47. Zinser, E., Paltauf. F. & Daum, G. (1993) Sterol composition

of yeast organelle membranes and subcellular distribution of

enzymes involved in sterol metabolism. J. Bacteriol. 175,

2853±2858.

48. Leber, R., Landl, K., Zinser, E., Ahorn, H., SpoÈk, A., Kohlwein, S.D.,

Turnowsky, F. & Daum, G. (1998) Dual localization of squalene

epoxidase, erg1p, in yeast reflects a relationship between the

endoplasmic reticulum and lipid particles. Mol. Biol. Cell. 9,

375±386.

49. Katavic, V., Reed, D.W., Taylor, D.C., Giblin, E.M., Barton,

D.L., Zou, J., MacKenzie, S.L., Covello, P.S. & Kunst, L.

(1995) Alteration of seed fatty acid composition by an

ethylmethanesulfonate-induced mutation in Arabidopsis thaliana

affecting diacylglycerol acyltransferase activity. Plant Physiol. 108,

399±409.

50. Gondet, L., Bronner, R. & Benveniste, P. (1994) Regulation of

sterol content in membranes by subcellular compartmentation of


steryl-esters accumulating in a sterol-overproducing tobacco mutant.

Plant Physiol. 105, 509±518.

51. Schaller, H., Grausem, B., Benveniste, P., Chye, M.L., Tan, C.T., Song,

Y.H. & Chua, N.H. (1995) Expression of the Hevea brasiliensis

(H.B.K.) MuÈll. Arg. 3-hydroxy-3-methylglutaryl-coenzyme A reduc-

tase 1 in tobacco results in sterol overproduction. Plant Physiol. 109,

761±770.

52. Zou, J., Katavic, V., Giblin, E.M., Barton, D.L., MacKenzie, S.L.,

Keller, W.A., Hu, X. & Taylor, D.C. (1997) Modification of seed oil

content and acyl composition in the brassicaceae by expression of a

yeast sn-2 acyltransferase gene. Plant Cell 9, 909±923.

53. Enjuto, M., Lumbreras, V., Marin, C. & Boronat, A. (1995) Expression

of the Arabidopsis HMG2 gene, encoding 3-hydroxy-3-methyl-

glutaryl coenzyme A reductase, is restricted to meristematic and

floral tissues. Plant Cell 7, 517±527.

54. Montgomery, J., Goldman, S., Deikman, J., Margessian, L. & Fisher,

R.L. (1993) Identification of an ethylene-responsive region in the

promoter of a fruit ripening gene. Proc. Natl Acad. Sci. USA 13,

5939±5943.

55. Zou, J., Wei, Y., Jako, C., Kumar, A., Selvaraj, G. & Taylor, D.C.

(1999) The Arabidepsis thaliana TAG1 mutant has a mutation in a

diacylglycerol acyltransferase gene. Plant J. 19, 645±653.

Expression in yeast and tobacco of plant cDNAs encoding acyl CoA:diacylglycerol acyltransferase

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