The TAG1 locus of Arabidopsis encodes for a diacylglycerol acyltransferase

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Abstract

Diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) is a membrane enzyme that drives the final step in the formation of oils using diacylglycerol (DAG) and acyl-CoA to yield triacylglycerol (TAG). We identified a putative plant DGAT gene (TRIACYLGLYCEROL1: TAG1) and demonstrated its function by the cloning of two mutated alleles, designated AS11 (tag1-1) and ABX45 (tag1-2). One allele, AS11, has been previously characterised at the biochemical level. Mutant seeds contained less oil with a modified fatty acid profile and have reduced germination rates compared to wild-type controls. The TAG1 cDNA encodes for a 520-aa protein that possesses multiple putative transmembrane domains and shows 70 % similarity to a human DGAT cDNA.

Introduction

Triacylglycerols (TAG) represent the most efficient storage form of energy for eukaryotic cells. In plants, TAGs are deposited during seed and fruit development. Oil seed content of different plant species varies from 1–2 % to over 50 % of dry weight and the physiological and biochemical bases of such differences are not well-understood [16]. The structure of fatty acids esterified to TAGs has also been found to be remarkably diverse throughout the plant kingdom. However, eight fatty acids alone contribute to greater than 90 % of the world oil production, as the others are found in non-crop species [13]. This fact represents a major limitation for edible or industrial uses of oils. Recently, the isolation of genes involved in oil biosynthesis has offered exciting opportunities for redesigning plant oil metabolism towards, (a) production of healthier edible oils [5]; (b) creation of new industrial feedstock or chemical products [6], [14], [22]; in addition to (c) increasing yield [24]. For instance, expression of a yeast sn-2-acyltransferase, encoding for a protein exhibiting lysophosphatidic acid acyltransferase activity in Arabidopsis thaliana, has been shown to increase seed oil content from 8 to 48 % [24]. There is also an interest in increasing the amount of very long chain fatty acids (VLCFA), such as 22:1, in rapeseed to provide an industrial feedstock [15], [21]. A similar interest exists for manipulating the level of desaturation of edible oils [22].

Diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) is a membrane bound enzyme, thought to be located in the endoplasmic reticulum [10], [19]. DGAT drives the final and only committed step in the formation of oils by catalysing the acylation of the sn-3 position of the sn-1,2-diacylglycerol (DAG) to yield triacylglycerol (TAG). DAG can be derived from the Kennedy pathway by stepwise acylation of glycerol-3-phosphate [9], from hydrolysis of TAGs or from membrane phospholipids by acyl exchange or PC-DAG inter-conversion [20]. DGAT has been generally assumed to have a broad specificity for fatty acids and might control the flux of the pathway of triglyceride accumulation [16], [20], [23]. Increased DGAT abundance in maturing oilseeds could lead to increased seed oil content and changes in oil quality [8], [24]; hence, the identification of the gene encoding for the DGAT represents an important step towards improvement of plant oilseed production.

Finally, changes in lipid fatty acids have been associated with lower germination properties [12] and dramatic alteration in plant morphology [11]. The availability of the DGAT gene would offer the means to investigate the biochemical and physiological consequences of modifying the expression of this enzyme in seed or in the whole plant.

Recently, a chemically induced mutant of A. thaliana affected in DGAT activity was characterised [8]. This seed lipid mutant, AS11, not only had reduced oil content and DGAT activity throughout seed development, but 18:3 fatty acid levels doubled, whereas those of 18:1 and 20:1 were reduced; furthermore, several enzymes of lipid metabolism had altered activity. Hence, the phenotype observed in the AS11 mutant might be due to a lesion in the DGAT gene itself or in a gene encoding for a regulatory factor acting directly or indirectly on the DGAT activity. The phenotype was shown to result from a single-locus mutation, on a gene designated TAG1, located on chromosome II, between the two markers sti and cp2 [8].

Here, we report the isolation and characterisation of a new tag1-allele, designated ABX45, and the identification of the corresponding DGAT gene.

Section snippets

Isolation and characterisation of a new mutant, ABX45, affected in DGAT activity

To identify the genetic loci involved in seed lipid accumulation in higher plants, we visually screened the T2 progeny of 17 000 T-DNA lines from the Versailles T-DNA insertion mutant collection [1]. The assumption was that a mutation in a key maturation process, such as storage material accumulation, would lead to wrinkled, incompletely filled seeds [4]. Among fifty lines isolated following this criteria, one mutant, namely ABX45, showed this phenotype (figure 1). Moreover, ABX45 oil

Discussion

Based on an exhaustive biochemical study of the AS11 mutant, Katavic et al. [8] concluded that the AS11 mutant was affected in DGAT activity. Both, AS11 and ABX45 alleles used in this paper showed a lower seed oil content although 18:3 fatty acid levels doubled and 18:1, 20:1, 22:1 fatty acid levels were reduced. Several enzymes of lipid metabolism have shown altered activities in the AS11 mutant [8]. This phenotype is consistent with a reduction in DGAT activity leading to a lower oil content

Plant materials

Arabidopsis thaliana mutants AS11 and ABX45, as well as their corresponding wild-type ecotypes Columbia (Col) and Wassilewskija (WS), respectively, were grown in soil under 16 h light in the greenhouse (15 °C night–20 °C day). The AS11 ethylmethanesulphonate mutant (Col ecotype) was obtained from L. Kunst [8]. The ABX45 (WS ecotype) was isolated from T-DNA insertion lines previously described [1].

Fatty acid and lipid analyses

Total lipid extracts were prepared from seed, and individual lipids were separated on

Note added in proof

During the submission of this paper, Hobbs et al. [7] reported the cloning of the same A. thaliana cDNA (EMBL accession number AJ131831) and demonstrated the DGAT activity of the recombinant protein produced in mammalian cells.

Acknowledgements

We thank Dr L. Kunst for kindly providing the AS11 mutant seeds and Dr G. Pelletier for providing T-DNA insertion lines. We also thank Murielle Boisson and Anne-Marie Jaunet for the scanning electron microscopy. The additional technical assistance of V. Tanty is gratefully acknowledged. Finally, we thank Dr A.M. Lescure for critical reading of the manuscript and her constant support and Dr H. North for corrections. This work was partially supported by a grant from the EC (Fair

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