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First published online September 20, 2007; 10.1104/pp.107.106278 Plant Physiology 145:974-984 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
Biosynthesis of Phytosterol Esters: Identification of a Sterol O-Acyltransferase in Arabidopsis1,[OA]Plant Biotechnology Institute, National Research Council Canada, Saskatoon, Saskatchewan, Canada S7N 0W9
Fatty acyl esters of phytosterols are a major form of sterol conjugates distributed in many parts of plants. In this study we report an Arabidopsis (Arabidopsis thaliana) gene, AtSAT1 (At3g51970), which encodes for a novel sterol O-acyltransferase. When expressed in yeast (Saccharomyces cerevisiae), AtSAT1 mediated production of sterol esters enriched with lanosterol. Enzyme property assessment using cell-free lysate of yeast expressing AtSAT1 suggested the enzyme preferred cycloartenol as acyl acceptor and saturated fatty acyl-Coenyzme A as acyl donor. Taking a transgenic approach, we showed that Arabidopsis seeds overexpressing AtSAT1 accumulated fatty acyl esters of cycloartenol, accompanied by substantial decreases in ester content of campesterol and  -sitosterol. Furthermore, fatty acid components of sterol esters from the transgenic lines were enriched with saturated and long-chain fatty acids. The enhanced AtSAT1 expression resulted in decreased level of free sterols, but the total sterol content in the transgenic seeds increased by up to 60% compared to that in wild type. We conclude that AtSAT1 mediates phytosterol ester biosynthesis, alternative to the route previously described for phospholipid:sterol acyltransferase, and provides the molecular basis for modification of phytosterol ester level in seeds.
Plant sterols, known generally as phytosterols, are integral components of the membrane lipid bilayer (Demel and De Kruyff, 1976  ; Schuler et al., 1991). Unlike animal systems in which cholesterol is most often the lone final product of sterol synthesis, each plant species has its own characteristic distribution of phytosterols, with the three most common phytosterols in nature being -sitosterol, campesterol, and stigmasterol (Benveniste, 1986, 2004). In addition to free sterol form, phytosterols are also found in the form of conjugates, particularly fatty acyl sterol esters (SEs). SEs can be found in lipid bodies in the cytoplasm and are present at a substantial level in seeds. In canola (Brassica napus), for example, phytosterols constitute about 0.5% of seed oil (Sabir et al., 2003), 35% of which is in the form of SEs (Harker et al., 2003). The biochemical process of sterol acylation is believed to play a role in maintaining the free sterol content of cell membranes at their physiological levels (Schaller, 2004).
The effectiveness of phytosterols as a dietary component to lower serum cholesterol level in humans has been well documented in medical research for more than half a century (Moghadasian et al., 1997
Despite the ubiquitous presence of phytosterol esters in plant cells, the metabolic process of sterol acylation remains poorly understood. Sterol esterification is suggested to take place using several acyl donors including phospholipid, diacylglycerol, or triacylglycerol (Zimowski and Wojciechowski, 1981a
Cloning of AtSAT1 from Arabidopsis
Taking a yeast functional complementation strategy (Bach and Benveniste, 1997
The yeast mutant SCY059 produces only a residual amount of SE because the two principle sterol O-acyltransferase genes, ARE1 and ARE2, were interrupted (Yang et al., 1996
To verify the identity of the product, the HPLC fraction was collected and subjected to alkaline hydrolysis (saponification). The saponification extract was derivatized with N,O-bis(trimethysily)-trifluoroacetamide + 1% trimethylchlorosilane (BSTFA + 1% TMCS) and detected as trimethylsilyl (TMS) derivatives by gas chromatography (GC)-mass spectrometry (MS). GC profile revealed four major peaks from the sterol fraction (Fig. 2A ). All the GC fractions were identified by searching the National Institute of Standards and Technology (NIST) 2.0 mass spectra library. The first peak, also present in the control strain, corresponded to squalene. The other three peaks displayed mass spectra identical to that of ergosterol, lanosterol, and 4,4-dimethyl-8,24-cholestadienol (also known as 4,4-dimethylzymosterol), respectively (Fig. 2A). The five peaks from the fatty acid fraction were found to be 14:0, 16:1, 16:0, 18:1, and 18:0 fatty acids (Fig. 2B). These results confirmed that expression of At3g51970 conferred SCY059 an ability to produce SE and At3g51970 was then tentatively designated as AtSAT1.
AtSAT1 Is Structurally Related to Acyl-CoA Sterol Acyltransferases of Yeast and Animal Origins
AtSAT1 is predicted to be an integral membrane protein with eight transmembrane domains, and has a putative signal peptide with a cleavage site at the 19th amino acid from the N terminus, which was proposed to direct nascent proteins into the secretory pathway, including the endoplasm reticulum (Nielsen et al., 1997
Heterologous Expression of AtSAT1 in Yeast Produces Mainly Lanosterol Esters
We estimated SE content of the yeast strains by quantifying sterols released through alkaline hydrolysis. As reported previously (Yang et al., 1996 Considering SCY059 still retained a low level of SE synthesis capacity, there was the possibility of AtSAT1 being a facilitator, enhancing the residual SE synthesis activity in the mutant strain. If this were true, SCY059 harboring AtSAT1 would likely produce SE with a profile of sterol or fatty acid moiety similar to that of the vector-alone transformant. This was experimentally proven untrue. As shown in Figure 4A , the molar composition of the fatty acid species saponified from the SE collected from the vector-only strain and the SEs produced by SCY059/AtSAT1 were different. Likewise, the sterol composition of the SE also displayed major differences due to the expression of AtSAT1 (Fig. 4B). The residual SE in SCY059/empty vector consisted of mainly ergosterol. On the other hand, expression of AtSAT1 resulted in production of SE containing chiefly lanosterol, which contributed to 88.1% of total sterol moiety. Thus, AtSAT1 appeared to preferentially acylate lanosterol when expressed in yeast.
Substrate Preference of AtSAT1 In light of substrate preferences of ACAT-related enzymes reported to date, we examined fatty acyl donor preferences of AtSAT1 using cell-free lysate of yeast with various fatty acyl-CoAs. Lipids extracted from in vitro reactions were separated by TLC and the incorporation of [3H]lanosterol into SE was measured. Because the cell-free lysate was expected to contain certain amount of acyl-CoA, reactions without added acyl-CoA were set as blank controls for enzyme activity calculation. As shown in Figure 5A , AtSAT1 had a substrate preference in the order of 16:0 > 18:0 > 16:1 > 18:1. 18:2 was found to negatively affect sterol O-acyltransfease activity.
We used stigmasterol, -sitosterol, and cycloartenol as well as two sterols of yeast origin, lanosterol and ergosterol, to assess [14C]16:0-CoA acylation. Assessment of sterol substrate preferences entailed a methyl--cyclodextrin wash to extract free sterols from the preparations (Rodal et al., 1999), due to the concern that endogenous sterols may interfere with substrate specificity assessment. However, there apparently was still a substantial amount of endogenous sterols remaining, because the background value in the absence of added sterol substrate was nonetheless high. We also attempted acetone extraction of the membrane fractions to reduce the endogenous sterol levels. Unfortunately the acetone treatment essentially eliminated the enzyme activity (data not shown). This led us to use reactions without exogenous sterol additions as control experiments. Under our assay conditions, the yeast strain harboring empty vector displayed a reduced 16:0 acylation activity in the presence of exogenous sterol. Addition of ergosterol to the yeast lysate expressing AtSAT1 also gave rise to a negative value on SE formation. Such a negative effect of exogenous sterol addition on sterol O-acyltransferase activity was previously reported for animal ACAT (Tavani et al., 1982; Yang et al., 1997) and the Arabidopsis AtPSAT1 assays (Bana et al., 2005). Despite these results, we could consistently detect SE synthesis activity with most of the sterol substrates when AtSAT1 was expressed. The highest activity was found with cycloartenol, followed by -sitosterol, lanosterol, and stigmasterol (Fig. 5B).
Because phosphatidylcholine (PC) has been previously implicated as an acyl donor for SE synthesis (Bana
Based on transcript profile obtained from Genevestigator at https://www.genevestigator.ethz.ch/, AtSAT1 is expressed in all tissues examined, but particularly abundant in the elongation zone of roots followed by developing microspore and germinating seedlings. Previous published works (Ting et al., 1998
To further confirm that AtSAT1 is involved in SE biosynthesis in plants, we cloned AtSAT1 cDNA into a plant expression vector under the control of a seed-specific napin promoter (Josefsson et al., 1987
Seeds harvested from four wild-type plants and 12 transgenic plant (T1) lines, raised simultaneously in the same growth chamber, were selected for sterol analysis. AtSAT1 overexpression led to increases in SE by more than 2-fold and total sterol content by 64% in some transgenic lines (Table I ). Overexpression of AtSAT1 in seed, as exemplified by one transgenic line shown in Fig. 7 , also drastically altered the SE-sterol profile. In wild-type seeds, the two major SE species, -sitosterol ester and campesterol ester, accounted for 80.4% and 17.5% of total SE, respectively. Cycloartenol ester was detected as a minor component at a distant third with 2.1%. Strikingly, the AtSAT1 transgenic seeds produced cycloartenol ester as the most prominent component, representing up to an average of 64.1% of total SE. On the other hand, the esters of -sitosterol and campesterol were reduced to averages of 25.2% and 5.2%, respectively. We also found that 24-methylene cycloartenol was increased from nondetectable level in wild-type seeds to 5.6% in the transgenic line seeds. The enhanced SE overproduction also caused reduction of free sterol from 58.8% in wild type to 28.3% of total sterols in transgenic plants.
We further analyzed the profile of the fatty acid component of the SEs in seeds overexpressing AtSAT1 (Table II ). In comparison to wild-type seeds, there was a drastic increase in the molar percentage of 16:0, from less than 15% to approaching 30%. Significant increases were also apparent in the composition of very long-chain fatty acids 20:0 and 20:1. The overall changes in fatty acid component of the SEs in the transgenic lines can be summarized as saturated and long-chain fatty acids replacing unsaturated fatty acids in wild-type seeds, particularly 18:2 and 18:3. The fatty acid composition of SE in AtSAT1-overexpressed seeds was consistent with the in vitro fatty-CoA specificity results.
In this study we identified AtSAT1 as a sterol O-acyltransferase from Arabidopsis that belongs to the MBOAT family. Our conclusion on the functional identity of AtSAT1 was based on the following lines of evidence: (1) heterologous expression of AtSAT1 enabled a yeast SE-deficient mutant SCY059 to synthesize a considerable amount of SE; (2) AtSAT1 mediated the synthesis of SE with sterol and fatty acid profiles distinctively different from that of the trace amount of SE synthesized in the yeast mutant strain; and (3) transgenic overexpression of AtSAT1 in Arabidopsis substantially increased SE content in seeds. Furthermore, since we did not find detectable levels of TAG when expressing AtSAT1 in the are1are2lro1dga1 quadruple yeast mutant, it appears that AtSAT1 could not acylate diacylglycerol for TAG biosynthesis.
Expression of AtSAT1 in yeast strain SCY059 resulted in the accumulation of SE consisting mainly of lanosterol, rather than the most abundant sterol being ergosterol, the end product of the sterol biosynthesis pathway in yeast (Zweytick et al., 2000 Data obtained from transgenic plants were generally in line with results of enzyme property assessment performed through heterologous expression of AtSAT1 in yeast. Consistent with the high enzyme activity detected with cycloartenol in our in vitro assays, enhanced AtSAT1 expression drastically advanced accumulation of cycloartenol esters in Arabidopsis seeds. Likewise, for the fatty acid components of the SEs, we observed increases in the molar ratio of saturated fatty acids, including 16:0, 18:0, and 20:0. Elevated sterol acylation with the long-chain monounsaturated eicosenoic acid was also evident. On the other hand, the polyunsaturated fatty acids were proportionally reduced.
Previous biochemical studies suggested that there are several potential acyl donors for SE biosynthesis in plants (Garcia and Mudd, 1978a
SE has been long proposed to be the storage and transport form of sterols for their intracellular movement (Kemp et al., 1967
Incorporating phytosterols into the diet may be an effective approach to lower total and LDL cholesterol levels in humans, but free phytosterols are difficult to incorporate into commercial foods because of their low solubility. Fortunately, phytosterol esters can be dissolved in vegetable oil at a concentration 10 times higher than that of the free phytosterols. In this study, we found that overexpression of AtSAT1 could lead to enhanced total sterol content, primarily due to the increase in cycloartenol ester level. Cycloartenol is particularly enriched in oryzanol derived from rice (Oryza sativa) bran oil (Rukmini and Raghuram, 1991 ). It has been suggested that cycloartenol is the effective ingredient in rice bran oil for lowering plasma non-HDL cholesterol level (Wilson et al., 2007). We believe that the engineering outcome of the current research is likely to be repeated in crops species, particularly canola, offering a novel genetic engineering target for enhanced phytosterol content in vegetable oil. We must note, although the increase in seed oil SE content was significant, increase in total sterol content was still modest. Further studies on the regulation of phytosterol biosynthesis are clearly required to improve phytosterol content in seeds.
TOPA TA Cloning and Yeast Complementation
RT-PCR of the open reading frames of Arabidopsis (Arabidopsis thaliana) MBOAT family genes At1g12460, At1g34490, At1g34500, At1g34520, At1g57600, At3g08930, At3g51970, At5g01460, At5g55320, At5g55330, At5g55340, At5g55350, At5g55360, At5g55370, and At5g55380 was performed with primer pairs designed based on sequences of gene annotation available on The Arabidopsis Information Resource Web site. The primer pair for At3g51970 cDNA was 5'-CCATGGCGAGTTTCATCAAGGCAT-3' (forward primer) and 5'-GGCAGGGTTAAAAAAGATATGCGGTCAGT-3' (reverse primer). The cDNA was cloned into vector pYES2.1 using pYES2.1 TOPO TA cloning kit according to the manufacturer's protocol (Invitrogen), and subsequently introduced into yeast (Saccharomyces cerevisiae) strain SCY059 (MAT
Single colonies of vector-only or AtSAT1 transformed yeast were inoculated in 10 mL of synthetic complete medium with Leu, His, and Ura omitted, and grown overnight at 28°C. Twenty-milliliters of fresh medium was then added and the cultures were further maintained overnight. Neutral lipid fraction was extracted following an established protocol (Folch et al., 1957
SEs were saponified for 2 h in 1 mL of 7.5% methanolic KOH at 80°C. The released sterol was extracted twice with hexane and dried under N2 stream. The aqueous layer was neutralized with HCl and extracted twice with hexane to obtain free fatty acids. The fatty acid fraction was derivatized with BSTFA containing 1% TMCS and sterol derivatives were obtained in a mixture of BSTFA and pyridine (1:1, v/v) at room temperature for 1 h, and separated on a Hewlett-Packard 6890 N GC with a capillary column DB-5 (Hewlett-Packard HP5, 35 m length x 0.25 mm diameter x 0.25 µm thickness). GC-MS analysis was accomplished using an Agilent 5973 mass selective detector coupled to an Agilent 6890 N gas chromatograph. The mass selective detector was run under standard electron impact conditions (70 eV), scanning an effective mass-to-charge ratio range of 40 to 700 at 2.26 scans/s. The mass spectra were compared with entries in the NIST mass-spectral database, version 2.0.
After HPLC separation of neutral lipid, SE fraction was collected, dried under N2 stream, and saponified in 7.5% KOH in 95% methanol. Separation and quantification of sterol species was performed on a Hewlett-Packard 6890 series GC with a capillary column DB-5 and flame ionization detector. The peaks of each sterol species were compared to the cholesterol (internal standard) peak to determine the amount of each sterol present. Fatty acid methyl esters were separated on GC with DB23 column. The quantification was performed in triplicate with three different batches of yeast cultures. The content of SE was expressed as micromoles of sterol moiety saponified from SE on a dry yeast cell weight basis.
AtSAT1 expression induction in yeast cells was performed according to manufacturer's protocol for pYES2.1 TOPO TA expression kit (Invitrogen). The cells, resuspended in 1 mL cell wall-breaking buffer, were shaken vigorously in the presence of acid-washed glass beads. The resultant homogenate was centrifuged at 1,500g for 5 min at 4°C. The supernatant was removed, aliquoted, and stored at –76°C. For sterol substrate specificity determination, the supernatant was washed twice with 80 mM methyl-
L-
A cDNA of AtSAT1 was PCR amplified with primers FP-XbaI (5'-CAAGAATCTAGAATGGCGAGTTTCATCAAGGCA-3') and RP-KpnI (5'-GATCACTCAAGTTACCACACACGGCAGGGTTA-3'), and ligated into the XbaI and KpnI sites of pSE129 vector. The AtSAT1 sequence and the construct integrity were verified by sequencing. The resulting gene expression cassette in the transformation vector contained a canola (Brassica napus) seed-specific napin promoter and Agrobacterium NOS terminator flanking the 5' and 3' ends of the AtSAT1 cDNA, respectively. A single colony of Agrobacterium (GV3101) carrying napin:AtSAT1 transformation vector was cultured in Luria-Bertani medium containing 50 mg mL–1 kanamycin and 25 mg mL–1 gentamycin. Plant transformation was performed through vacuum infiltration. Selection of T0 transgenic seeds was carried on media containing one-half Murashige and Skoog Gamborg medium, 0.8% (w/v) phytagar, 3% (w/v) Suc, 50 mg mL–1 kanamycin, and 50 mg mL–1 timentin.
To quantify transcript levels, total RNA was extracted from siliques of wild-type and transgenic T2 plants using Qiagen NEasy Plant Mini kit. One microgram of DNase I-treated RNA was used as template for cDNA synthesis (Invitrogen superscript First-Strand Synthesis kit). qRT-PCR was performed on Applied Biosystem StepOne Real-Time PCR system in triplicates. The
Six to 8 mg of mature seeds from wild-type and AtSAT1 transgenic lines were suspended in 2 mL of chloroform:methanol (2:1, v/v) containing 20 µg cholesterol and 20 µg cholesteryl myristate as internal standards. The suspension was probe sonicated, washed with 0.9% NaCl, and then vigorously vortexed. The bottom chloroform layer was transferred into a new solvent-washed tube. The aqueous layer was extracted twice with 2 mL of chloroform:methanol (2:1, v/v), and the organic layers were combined and dried down under N2 stream. The residue was dissolved in 200 µL hexane. SE and free sterol were separated and collected through HPLC. The SE fraction was saponified in 2 mL 7.5% KCl in 95% methanol and the resultant free sterols were repeatedly extracted with hexane. Sterol-TMS derivatives were qualitatively and quantitatively analyzed following the same method for yeast sterol analysis. Campesterol, Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number NM_115056.
We thank Dr. Stephen L. Sturley for providing the yeast strain SCY059 and Dr. Sten Stymne for providing the quadruple disrupted yeast strain devoid of TAG synthesis. We thank Mike Giblin and Darwin Reed for technical guidance in HPLC separation of SEs, and Ms. Kim Bryce, project manager for the National Research Council Canada-Crops for Enhanced Human Health Program. Helpful discussion with Drs. Sten Stymne, Pat Covello, and Mark Smith are acknowledged. This is National Research Council Canada publication number 48407. Received July 25, 2007; accepted September 6, 2007; published September 20, 2007.
1 This work was supported by the National Research Council Canada-Crops for Enhanced Human Health Program and a Natural Science and Engineering Research Council of Canada grant (grant no. NSERC RGPIN 327217–06 to J.Z.). Q.C. is a recipient of the Post-Doctoral Fellowship for Visiting Government Laboratories from the Natural Science and Engineering Research Council of Canada. 
2 Present address: Department of Biology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E2. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Jitao Zou (jitao.zou{at}nrc-cnrc.gc.ca).
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.107.106278 * Corresponding author; e-mail jitao.zou{at}nrc-cnrc.gc.ca.
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, his 3-11,15 leu 2-3, 112 trp 1-1, ura 3-1, kan 1-100, ade2 met14
are1::HIS3 are2::LEU2), kindly provided by Dr. Stephen Sturley (Columbia University), and a quadruple mutant strain (are1 are2 lro1 dgat1) from Dr. Sten Stymne (Swedish University of Agricultural Sciences). 
