Biosynthesis of phytosterol esters: identification of a sterol o-acyltransferase in Arabidopsis

Abstract

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 beta-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.

Figure 1.

TLC and HPLC separation of neutral lipids from SCY059 harboring control vector (vector-only), SCY059 expressing AtSAT1 (AtSAT1), and a parental strain SCY062 (WT). A, Neutral lipids separated through TLC developed in hexane/diethyl ether/acetic acid (40/10/1.5). B, Normal-phase HPLC separation of neutral lipids extracted from SCY059 expressing AtSAT1 (AtSAT1) and SCY059 harboring the vector (vector-only). Arrows denote the novel product found in SCY059 expressing AtSAT1.

Figure 2.

Analysis of TMS derivatives of sterols and fatty acids of SE. A, GC profiles of sterols saponified from SE of yeast strain SCY059 harboring control vector (I) and SCY059 expressing AtSAT1 (II). B, GC profile of TMS derivatives of fatty acids released from SE of AtSAT1 yeast transformant. The TMS derivatives of peaks in the profiles were subsequently subjected to mass spectrum analysis and the mass spectra were found as being identical to: 1, Squalene; 2, ergosterol; 3, lanosterol; 4, 4,4-dimethyl-8,24-cholestadienol; 5, myristic acid (14:0); 6, palmitoleic acid (16:1); 7, palmitic acid (16:0); 8, oleic acid (18:1); and 9, stearic acid (18:0).

Figure 3.

Sequence alignment of AtSAT1 with other reported sterol acyltransferases. Alignment was performed with CLUSTALV from the DNASTAR package run with default multiple alignment parameters (gap opening penalty: 10, gap extension penalty: 10). Accession numbers of proteins are: AtSAT1 (Arabidopsis, AAQ65159); HsACAT1 (Homo sapiens, NP_003092); HsACAT2 (H. sapiens, NP_003569); MmACAT1 (Mus musculus, Q61263); MmACAT2 (M. musculus, NP_666176); RnACAT1 (Rattus norvegicus, O70536); RnACAT2 (R. norvegicus, NP_714950); ScARE1 (yeast, P25628); and ScARE2 (yeast, P53629). Residues boxed in dashed or solid lines were previously reported as conserved functional motifs in ACATs. The black bar on the bottom marks a shared domain, and the arrow indicates the His likely to be an active-site residue (Hofmann, 2000).

Figure 4.

Fatty acid profile and sterol composition of SE in yeast strains. A, Mol % of fatty acid species released from SE of SCY059 expressing AtSAT1 in comparison to that of the vector-only control and the parental strain (WT). B, Profiles of the sterol moieties of SE. The SE was saponified in methanolic-KOH for 2 h at 80°C. The fatty acids were transmethylated with methanolic HCl and free sterols were derivatized with BSTFA (1% TMCS).

Figure 5.

Substrate preference assessment of AtSAT1. A, Fatty acyl-CoA selectivity of AtSAT1. Cell-free lysates were assayed for acylation of [³H]lanosterol. Fatty acyl-CoAs used for the assay were palmitoyl-CoA (16:0), palmitoleoyl-CoA (16:1), stearoyl-CoA (18:0), oleoyl-CoA (18:1), and linoleoyl-CoA (18:2). B, Sterol preference of AtSAT1. Cell-free lysates were assessed for acylation of ergosterol, lanosterol, stigmasterol, β-sitosterol, and cycloartenol in the presence of [¹⁴C]palmitoyal-CoA. Reactions without exogenous sterol (or fatty acyl-CoA) served as control. Enzyme activity was calculated based on the difference of sterol acylation between reactions with added sterol (or fatty acyl-CoAs) substrates and reactions without sterols (or fatty acyl-CoAs). The negative values in some reactions were caused by high background sterol acylation in the control in the absence of exogenous sterol substrates.

Figure 6.

qRT-PCR analysis of transcript levels of AtSAT1. Each sample contained RNA extracted from pooled developing siliques. qRT-PCR was performed using Applied Biosystem StepOne Real-Time PCR system. C_T was automatically determined by software StepOne (version 1.0). AtSAT1 expression levels were calculated through normalization with β-Actin-8 by the formula 2^{−(CTAtSAT1 − CTβ-actin)}. Data represent the mean and sd of three replicates.

Figure 7.

GC profiles of sterols saponified from SE extracted from seeds of wild type (WT) and one representative AtSAT1 overexpression line. Lipids were extracted from seeds with chloroform:methanol (2:1, v/v) and separated though normal-phase HPLC. SE fractions were collected and saponified with 7.5% KOH in 95% methanol. The resulting free sterols were derivatized with BSTFA:pyridine (1:1, v/v). Identification of mass spectra and assignation of GC/MS peaks was carried out by a library search (NIST, version 2.0). 1, Cholesterol (internal standard); 2, campesterol; 3, β-sitosterol; 4, cycloartenol; and 5, 24-methylene cycloartenol.

Figure 8.

A proposed role for AtSAT1 in the sterol biosynthesis pathway. The diagram accentuates the section where the conversion of squalene to sterol end products occurs. Solid lines denote single-step enzymatic reactions and dashed lines represent multiple steps. The open arrows suggest the roles of AtSAT1 in sterol biosynthesis.