Cloning and functional characterization of a phospholipid:diacylglycerol acyltransferase from Arabidopsis - PubMed (original) (raw)
Cloning and functional characterization of a phospholipid:diacylglycerol acyltransferase from Arabidopsis
Ulf Ståhl et al. Plant Physiol. 2004 Jul.
Abstract
A new pathway for triacylglycerol biosynthesis involving a phospholipid:diacylglycerol acyltransferase (PDAT) was recently described (Dahlqvist A, Stahl U, Lenman M, Banas A, Lee M, Sandager L, Ronne H, Stymne S, [2000] Proc Natl Acad Sci USA 97: 6487-6492). The LRO1 gene that encodes the PDAT was identified in yeast (Saccharomyces cerevisiae) and shown to have homology with animal lecithin:cholesterol acyltransferase. A search of the Arabidopsis genome database identified the protein encoded by the At5g13640 gene as the closest homolog to the yeast PDAT (28% amino acid identity). The cDNA of At5g13640 (AtPDAT gene) was overexpressed in Arabidopsis behind the cauliflower mosaic virus promoter. Microsomal preparations of roots and leaves from overexpressers had PDAT activities that correlated with expression levels of the gene, thus demonstrating that this gene encoded PDAT (AtPDAT). The AtPDAT utilized different phospholipids as acyl donor and accepted acyl groups ranging from C10 to C22. The rate of activity was highly dependent on acyl composition with highest activities for acyl groups containing several double bonds, epoxy, or hydroxy groups. The enzyme utilized both sn-positions of phosphatidylcholine but had a 3-fold preference for the sn-2 position. The fatty acid and lipid composition as well as the amounts of lipids per fresh weight in Arabidopsis plants overexpressing AtPDAT were not significantly different from the wild type. Microsomal preparations of roots from a T-DNA insertion mutant in the AtPDAT gene had barely detectable capacity to transfer acyl groups from phospholipids to added diacylglycerols. However, these microsomes were still able to carry out triacylglycerol synthesis by a diacylglycerol:diacylglycerol acyltransferase reaction at the same rate as microsomal preparations from wild type.
Figures
Figure 1.
Evolutionary dendrogram showing the six Arabidopsis PDAT/LCAT homologs together with the HsLCAT, HsPLA2, and the ScPDAT. The dendrogram was calculated from aligned protein sequences using the CLUSTALX multiple alignment program (Thompson et al., 1997) with default settings. Pair wise alignment scores were computed, and an unrooted tree was obtained from these scores by using the neighbor-joining method (Saitou and Nei, 1987) with correction for multiple substitutions and exclusion of gapped positions.
Figure 2.
Alignment of the AtPDAT, At3g44830, HsLCAT, and ScPDAT sequences (accession nos. AAK96619, NP_190069, AAR03499, and P40345). Sequences were aligned by using the CLUSTALW algorithm at the PBIL web site (
http://npsa-pbil.ibcp.fr/cgi-bin/npsa\_automat.pl?page=npsa\_clustalw.html
) with default settings. Identical residues in all four sequences are highlighted with white letters on black background and similarities in at least three sequences are highlighted with black letters on light gray background. The signal peptide in the HsLCAT sequence is outlined as SP and the predicted membrane spanning regions in the three PDAT like sequences are marked with dark gray background. The three active site residues of HsLCAT, S205, D369, and H401, are indicated with a star above, and the two oxyanion hole residues of HsLCAT, F127 and L206, are indicated with a black circle above. The four Cys residues in HsLCAT which are involved in forming two disulfide bridges are indicated with arrows and numbers. _N_-glycosylations sites in HsLCAT and potential _N_-glycosylation sites (Asn-Gly-Ser) in the other proteins are encircled. The HsLCAT lid-structure (interfacial recognition site) spanning residue 74 to 98 is outlined as Lid. The C-terminal ER retrieval motif in AtPDAT and At3g44830 is outlined and the aromatic amino acids underlined. Note that HsLCAT is shown with its signal peptide resulting in +24 in the numbering of the amino acids compared to what is given by Peelman et al. (1998).
Figure 3.
PDAT activity in microsomal preparations of Arabidopsis transformants expressing the AtPDAT gene under the control of the 35S promoter. A, PDAT activity in microsomal preparations of roots from control plants (transformed with empty vector) and three different transgenic lines expressing the AtPDAT under the control of the 35S promoter and (Top) northern-blot analysis of total root RNA from corresponding plants. PDAT activity was measured with _sn_-1-oleoyl(18:1)-_sn_-2-[14C]ricinoleoyl-PC and unlabeled di-18:1-DAG as substrates. B, PDAT activity as a function of DAG concentrations in microsomal preparations of roots from AtPDAT overexpresser (line 1-3b-44). The PDAT activity was measured with _sn_-1-18:1-_sn_-2-[14C]18:3-PE and with di-18:1-DAG added in amounts indicated in the figure.
Figure 4.
Autoradiogram of neutral lipid fraction separated on TLC after incubation of microsomal preparations of roots from different Arabidopsis lines with _sn_-1-18:1-_sn_-2-[14C]18:2-PE or _sn_-1-18:1-_sn_-2 -[14C]18:2-DAG in the presence of vernoloyl-DAG. A, Incubations with microsomal preparations from AtPDAT overexpresser (line 1-1-6) and control plants (transformed with empty vector). B, Incubation with microsomal preparations from AtPDAT T-DNA insertion mutant (mutant) and the null segregant (null) to this mutant. The figures given correspond to percentage radioactivity in the TAG areas of total radioactivity in the lane. Amount of substrates used in each assay: _sn_-1-18:1-_sn_-2-[14C]18:2-PE: Figure A, 2.5 nmol; Figure B, 5 nmol; _sn_-1-18:1-_sn_-2 -[14C]18:2-DAG: Figure A, 1.5 nmol; Figure B, 6 nmol; vernoloyl-DAG: Figure A, 1.5 nmol; Figure B, 6 nmol. TAG, triacylglycerol; 1-epoxy-TAG, TAG with one vernoloyl moiety; FFA, free fatty acids; DAG, diacylglycerol; MAG, monoacylglycerol; PL, polar lipids.
Figure 5.
Positional specificity of AtPDAT. PDAT activity was measured in microsomal preparations from leaves of Arabidopsis control plants (transformed with empty vector) and an AtPDAT overexpresser (line 1-1-6) with 5 nmol of either _sn_-1-[14C]18:1-_sn_-2-18:1-PC or _sn_-1-18:1-_sn_-2-[14C]18:1-PC.
Figure 6.
Acyl (A) and lipid (B) specificity of the AtPDAT. The PDAT activity was measured in microsomal preparation of leaves from AtPDAT overexpresser (line 1-1-6) with _sn_-2 labeled substrates. Labeled 18:0-PC, 20:4-PC, 22:1-PC, 10:0-PC, and 10:0-PE had 16:0 at position _sn_-1, whereas all other substrates had 18:1 in the _sn_-1 position. The results are presented as relative amounts of radioactive TAG synthesized compared to assays with _sn_-2-[14C]18:1-PC. Crep-PC, crepenynoyl-PC; Vern-PC, vernoloyl-PC; Ric-PC, ricinoleoyl-PC; Ric, ricinoleoyl.
Figure 7.
RT-PCR analysis of the expression of the genes encoding AtPDAT and At3g44830. One microgram of total RNA from developing seeds (S), roots (R), flower (F), and leaves (L) were used for cDNA synthesis. Transcripts shown are obtained after 35 cycles using cDNA as template. ACTIN2 was used as a control.
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