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.

Land plants secrete a layer of wax onto their aerial surfaces that is essential for survival in a terrestrial environment. This wax is composed of long-chain, aliphatic hydrocarbons derived from very-long-chain fatty acids (VLCFAs). Using the Arabidopsis expressed sequence tag database, we have identified a gene, designated CUT1 , that encodes a VLCFA condensing enzyme required for cuticular wax production. Sense suppression of CUT1 in transgenic Arabidopsis plants results in waxless ( eceriferum ) stems and siliques as well as conditional male sterility. Scanning electron microscopy revealed that this was a severe waxless phenotype, because stems of CUT1 -suppressed plants were completely devoid of wax crystals. Furthermore, chemical analyses of waxless plants demonstrated that the stem wax load was reduced to 6 to 7% of wild-type levels. This value is lower than that reported for any of the known eceriferum mutants. The severe waxless phenotype resulted from the downregulation of both the decarbonylation and acyl reduction wax biosynthetic pathways. This result indicates that CUT1 is involved in the production of VLCFA precursors used for the synthesis of all stem wax components in Arabidopsis. In CUT1 -suppressed plants, the C24 chain-length wax components predominate, suggesting that CUT1 is required for elongation of C24 VLCFAs. The unique wax composition of CUT1 -suppressed plants together with the fact that the location of CUT1 on the genetic map did not coincide with any of the known ECERIFERUM loci suggest that we have identified a novel gene involved in wax biosynthesis. CUT1 is currently the only known gene with a clearly established function in wax production. INTRODUCTIONWaxes are major constituents of the cuticle, a hydrophobic barrier covering the aerial portions of land plants. They are embedded within the cuticular matrix (intracuticular waxes) and also form the outermost layer of the cuticle (epicuticular waxes). The chemical and physical properties of waxes determine functions vital for plant life, such as regulation of nonstomatal water loss and protection against UV radiation (Reicosky and Hanover, 1978). Waxes also help plants resist bacterial and fungal pathogens (Jenks et al., 1994) and play a role in plant-insect interactions (Eigenbrode and Espelie, 1995). In addition, waxes found in the tryphine layer of pollen grains are essential for proper pollen-stigma signaling required for fertilization (Preuss et al., 1993).Cuticular waxes are complex mixtures of lipids, and their composition differs widely among plant species as well as among the organs and tissues of a single plant (PostBeittenmiller, 1996). They are composed mainly of longchain aliphatic hydrocarbons derived from saturated verylong-chain fatty acids (VLCFAs; chain length is Ͼ 18 carbons). The chain length of these hydrocarbons is species dependent, but typically they are 26 to 34 carbons long.VLCFAs, the precursors for wax biosynthesis, are formed by a microsomal fatty acid elongation (FAE) system. FAE involves sequential ad...

In characterizing the enzymes involved in the formation of very long-chain fatty acids (VLCFAs) i n the Brassicaceae, we have generated a series of mutants of Arabidopsis thaliana that have reduced VLCFA content. Here we report the characterization of a seed lipid mutant, AS1 1, which, in comparison to wild type (WT), has reduced levels of 20:l and 18:l and accumulates 18:3 as the major fatty acid in triacylglycerols. Proportions of 18:2 remain similar to WT. Cenetic analyses indicate that the fatty acid phenotype is caused by a semidominant mutation in a single nuclear gene, designated TAC1, located on chromosome 2. Biochemical analyses have shown that the AS11 phenotype is not due to a deficiency in the capacity t o elongate 18:l or to an increase in the relative A1 5 or A1 2 desaturase activities. Indeed, the ratio of desaturase/elongase activities measured in vitro is virtually identical in developing WT and AS1 1 seed homogenates. Rather, the fatty acid phenotype of AS1 1 is the result of reduced diacylglycerol acyltransferase activity throughout development, such that triacylglycerol biosynthesis is reduced. This leads to a reduction in 20:l biosynthesis during seed development, leaving more 18:l available for desaturation. Thus, we have demonstrated that changes to triacylglycerol biosynthesis can result i n dramatic changes i n fatty acid composition and, in particular, i n the accumulation of VLCFAs i n seed storage lipids.The fatty acyl composition of seed TAGs determines their physical and chemical properties and, thus, their use in edible oil or industrial applications. TAG composition depends on the interaction of several different groups of enzymes in the lipid biosynthesis pathway. The enzymes of the fatty acid synthase complex in the plastids of developing seeds are responsible for the biosynthesis of fatty acids up to and including oleic acid. Modifying enzymes, such as the extraplastidic A12 and A15 desaturases, elongases, hydroxylases, and epoxidases, yield polyunsaturated, very long-chain, hydroxy-, and epoxy-fatty acids, respectively. Acyltransferases insert specific acyl moieties onto the glyc-

A putative yeast sn-2 acyltransferase gene (SLC7-7), reportedly a variant acyltransferase that suppresses a genetic defect in sphingolipid long-chain base biosynthesis, has been expressed in a yeast SLC deletion strain. The SLC7-7 gene product was shown in vitro to encode an sn-2 acyltransferase capable of acylating sn-1 oleoyl-lysophosphatidic acid, using a range of acyl-COA thioesters, including 181-, 22:l-, and 240-COAS. The SLC7-7 gene was introduced into Arabidopsis and a high erucic acid-containing Brassica napus cv Hero under the control of a constitutive (tandem cauliflower mosaic virus 35s) promoter. The resulting transgenic plants showed substantial increases of 8 to 48% in seed oil content (expressed on the basis of seed dry weight) and increases in both overall proportions and amounts of very-long-chain fatty acids in seed triacylglycerols (TAGs). Furthermore, the proportion of very-long-chain fatty acids found at the sn-2 position of TAGs was increased, and homogenates prepared from developing seeds of transformed plants exhibited elevated lysophosphatidic acid acyltransferase (EC 2.3.1 51) activity. Thus, the yeast sn-2 acyltransferase has been shown to encode a protein that can exhibit lysophosphatidic acid acyltransferase activity and that can be used to change total fatty acid content and composition as well as to alter the stereospecific acyl distribution of fatty acids in seed TAGS.

SummaryA full-length cDNA encoding a putative diacylglycerol acyltransferase 1 (DGAT1, EC seed-specific expression of TmDGAT1 was able to complement the low TAG/unusual fatty acid phenotype of the Arabidopsis AS11 ( DGAT1 ) mutant. Over-expression of TmDGAT1 in wild-type Arabidopsis and high-erucic-acid rapeseed (HEAR) and canola Brassica napus resulted in an increase in oil content (3.5%-10% on a dry weight basis, or a net increase of 11%-30%). Site-directed mutagenesis was conducted on six putative functional regions/ motifs of the TmDGAT1 enzyme. Mutagenesis of a serine residue in a putative SnRK1 target site resulted in a 38%-80% increase in DGAT1 activity, and over-expression of the mutated TmDGAT1 in Arabidopsis resulted in a 20%-50% increase in oil content on a per seed basis.Thus, alteration of this putative serine/threonine protein kinase site can be exploited to enhance DGAT1 activity, and expression of mutated DGAT1 can be used to enhance oil content.