Seed-specific over-expression of an Arabidopsis cDNA encoding a diacylglycerol acyltransferase enhances seed oil content and seed weight

Abstract

We recently reported the cloning and characterization of an Arabidopsis (ecotype Columbia) diacylglycerol acyltransferase cDNA (Zou et al., 1999) and found that in Arabidopsis mutant line AS11, an ethyl methanesulfonate-induced mutation at a locus on chromosome II designated as Tag1 consists of a 147-bp insertion in the DNA, which results in a repeat of the 81-bp exon 2 in the Tag1 cDNA. This insertion mutation is correlated with an altered seed fatty acid composition, reduced diacylglycerol acyltransferase (DGAT; EC 2.3.1.20) activity, reduced seed triacylglycerol content, and delayed seed development in the AS11 mutant. The effect of the insertion mutation on microsomal acyl-coenzyme A-dependent DGAT is examined with respect to DGAT activity and its substrate specificity in the AS11 mutant relative to wild type. We demonstrate that transformation of mutant AS11 with a single copy of the wild-type Tag1 DGAT cDNA can complement the fatty acid and reduced oil phenotype of mutant AS11. More importantly, we show for the first time that seed-specific over-expression of the DGAT cDNA in wild-type Arabidopsis enhances oil deposition and average seed weight, which are correlated with DGAT transcript levels. The DGAT activity in developing seed of transgenic lines was enhanced by 10% to 70%. Thus, the current study confirms the important role of DGAT in regulating the quantity of seed triacylglycerols and the sink size in developing seeds.

Figure 1

Comparisons of 18:1-CoA-dependent DGAT activity (values expressed as pmol min⁻¹ mg⁻¹ protein) in microsomes prepared from developing seeds at the mid-green stage of embryo development in wild-type and DGAT mutant AS11 lines of Arabidopsis with the seed oil content in WT and AS11 mature seed (values for oil content expressed as percentage of dry weight). In single label experiments, 18 μm ¹⁴C-oleoyl-CoA (10 nCi nmol ⁻¹) was the acyl donor in the absence of exogenous diolein (*18:1-CoA alone) or in the presence of exogenous 200 μm unlabeled sn-1,2 diolein (*18:1-CoA + DAG). In the double label experiment, ³H-labeled oleoyl-CoA was used at a radiospecific activity of 50 nCi nmol ⁻¹ and a final concentration of 40 μm with ¹⁴C-labeled sn-1,2 diolein provided at a specific activity of 2 nCi nmol⁻¹ and a final concentration of 200 μm (*18:1-CoA + *DAG).

Figure 2

Comparison of the acyl-CoA preference of the DGAT in WT and AS11 developing seed (mid-green stage). Seeds of WT and AS11 Arabidopsis were harvested, and homogenates were prepared and DGAT activity measured as described by Katavic et al. (1995). In the comparative specificity studies, the DGAT activity was assayed in the presence of 18 μm [1-¹⁴C]-labeled 18:1-CoA, 18:2-CoA, or 20:1-CoA, supplied individually, and 200 μm unlabeled sn-1,2-diolein. In the comparative selectivity study equal concentrations (18 μm) of [1-¹⁴C]-labeled 18:2-CoA and 20:1-CoA were supplied simultaneously to the seed homogenates in the presence of 200 μm unlabeled sn-1,2-diolein. Separation and measurement of the relative proportions of the radiolabeled TAG products (18:1/18:1/¹⁴C-18:1; 18:1/18:1/¹⁴C-18:2 or 18:1/18:1/¹⁴C-20:1) was conducted by reverse-phase radio-HPLC as described by Weselake et al. (1991).

Figure 3

Complementation of the AS11 DGAT mutation with the WT cDNA leads to a restoration of WT seed oil composition and content. A, Transformation of Arabidopsis mutant line AS11 with single copies of the DGAT cDNA under the control of a napin promoter, leads to a restoration of the WT fatty acid composition in the seed oil of the transformant lines 3-3, 3-4, 9-1, 14-2, and 19-5. Fatty acid composition (% [w/w]) was determined on the seed oil extracted from Arabidopsis WT pSE and AS11 pSE (empty plasmid) control transformants, non-transformed (nt) WT and n-t AS11 controls, and T₃ seeds of napin:DGAT transgenic lines. B, Seed oil content of napin:DGAT T₃ transgenic AS11 mutant seed lines containing a single insertion of the DGAT cDNA. Oil content is expressed as percentage of seed dry weight for pSE in WT (empty plasmid) and n-t WT controls (stippled bars), for pSE in AS11 (empty plasmid) and n-t AS11 controls (black bars), and napin:DGAT AS11 transgenic lines 3-3, 3-4, 9-1,14-2, and 19-5 containing a single copy of the DGAT cDNA (gray bars). se bars are indicated (n = 3–5 replicate analyses performed on seed lots from each line with 100–200 seeds analyzed/replicate).

Figure 4

Northern analysis of TAG1 gene expression in non-transformed Arabidopsis WT and AS11 lines, a pSE (empty plasmid) AS11 control transformant, as well as napin:DGAT AS11 T₃ transgenic lines 3-3, 9-1,14-2, and 19-5, each containing a single copy of the DGAT cDNA. Total RNA was extracted from siliques containing mid-green (G) developing seeds. The TAG1 DNA probe was ³²P-labeled by random priming.

Figure 5

Transformation of WT Arabidopsis with the DGAT cDNA under the control of a napin promoter leads to a higher seed oil content. Homozygous T₃ napin:DGAT lines were sampled in triplicate, each sample consisting of 100 to 200 seeds/sample, accurately counted and weighed. For the plasmid only pSE in WT transgenic controls and nt-WT and nt-AS11controls, 10 individual transgenic plants were sampled and individual control seed lots similarly analyzed. Each error bar indicates se. Seed oil content (oil as a percentage of mature seed weight) is shown for Arabidopsis T₃ seeds of pSE (empty plasmid) control WT transgenics (stippled bars show representative oil content ranges; solid black bar is the average of oil contents in 10 independent pSE in WT plasmid only controls), nt-WT controls (white bar), nt-AS11 controls (checkered bar), and homozygous napin:DGAT transgenic lines with multiple inserts 2-2, 2-5, 9-2, 9-5, and 16-1(hatched bars), and lines with single inserts 10-4, 21-1, 21-6, 23-5, 23-6, 24-1, and 24-3 (gray bars).

Figure 6

Introduction of the napin:DGAT cDNA results in increases in the average 1,000-seed weight (expressed as milligrams of weight/1,000 seeds) of Arabidopsis T₃ seeds. Homozygous T₃ napin:DGAT lines were sampled in triplicate, each sample consisting of 100 to 200 seeds/sample accurately counted and weighed. For the plasmid only pSE controls, 10 individual transgenic plants were sampled and individual control seed lots similarly analyzed. Each error bar indicates se. Data are presented from pSE in WT (empty plasmid) control transgenics (solid black bar), nt-WT control lines (white bar), nt-AS11 control lines (checkered bar), homozygous napin:DGAT transgenic lines with multiple inserts 2-2, 2-5, 9-2, 9-5, and 16-1(hatched bars), and single insert lines 10-4, 21-1, 21-6, 23-5, 23-6, 24-1, and 24-3 (gray bars).

Figure 7

Heritability of the high oil trait from the pooled segregating T₂ generation to the average values for the corresponding selected homozygous T₃ progeny, shows a strong linear correlation. ▪, Intercepts for the pSE in WT control generations; ●, intercepts for the napin:DGAT generations.

Figure 8

Number of seeds per plant in Arabidopsis n-t WT controls (n = 6; white bar), six individual pSE in WT T₃ transgenic controls and their average (black bars), versus homozygous napin:DGAT T₃ transgenic lines with multiple inserts (2-2, 2-5, 9-2, 9-5, and 16-1, hatched bars), and single insert lines 10-4, 21-1, 21-6, 23-5, 23-6, 24-1, and 24-3 (gray bars).

Figure 9

A, Northern analysis of TAG1 gene expression in pooled developing T₃ seed samples from Arabidopsis pSE (empty plasmid) WT control transformants, as well as developing progeny from parent transgenic lines 2, 9, 10, 16, 21, 23, and 24, transformed with the napin:DGAT construct. Total RNA was extracted from siliques containing mid-green developing seeds. The TAG1 DNA probe was ³²P-labeled by random priming. B, Correlation of relative DGAT transcript level (♦) with oil content (percentage of mature seed weight); pSE control (black bar) or napin:DGAT multiple insert (hatched bars) and single insert (gray bars) transgenics. Values for transgenics represent the average from the corresponding homozygous T₃ progeny as reported in Figure 5. C, Correlation of relative DGAT transcript level (♦) with average seed weight (expressed as milligram/1,000 seeds); pSE control (black bar) or napin:DGAT multiple insert (hatched bars) and single insert (gray bars) transgenics. Values for transgenics represent the average from the corresponding homozygous T₃ progeny as reported in Figure 6.

Figure 10

Alignment of DGAT sequences (and GenBank accession nos.) from Arabidopsis (AJ238008 and AJ131831), B. napus (AF155224), Nicotiana tabacum (AF129003), mouse (AF078752), human (AF059202), and Caenorhabditis elegans (Z75526) in the region covering the insertion repeat found in mutant AS11. The conserved R and E residues, indicated by an asterisk, are proposed to constitute key residues at the active site. A proposed “box I” type of motif, which is conserved in all DGAT sequences reported thus far (and analogous to the motif found in other acyl-CoA acyltransferases) is underlined.