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.

SummaryIn Arabidopsis thaliana (ecotype Columbia) mutant line AS11, an EMS-induced mutation at a locus on chromosome II results in a reduced diacylglycerol acyltransferase (DGAT; EC 2.3.1.20) activity, reduced seed triacylglycerol, an altered seed fatty acid composition, and delayed seed development. A mutation has been identi®ed in AS11 in a gene, which we designated as TAG1, that encodes a protein with an amino acid sequence which is similar to a recently reported mammalian DGAT, and, to a lesser extent, to acyl CoA:cholesterol acyltransferases. Molecular analysis revealed that the mutant allele in AS11 has a 147 bp insertion located at the central region of intron 2. At the RNA level, an 81 bp insertion composed entirely of an exon 2 repeat was found in the transcript. While the seed triacylglycerol content is reduced by the lesion in AS11, there is no apparent effect on sterol ester content in the mutant seed. The TAG1 cDNA was over-expressed in yeast, and its activity as a microsomal DGAT con®rmed. Therefore, the TAG1 locus encodes a diacylglycerol acyltransferase, and the insertion mutation in the TAG1 gene in mutant AS11 results in its altered lipid phenotype.

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We have used RNA gel blot analysis to demonstrate the anther-specific expression of three genes in sunflower. Expression of these genes was first detected shortly before flower opening, which occurs sequentially on the sunflower inflorescence, and continues during pollination. In contrast, these genes are not expressed (or only weakly expressed) in a male-sterile line in which anther development aborts. In situ hybridization experiments showed that these genes are only expressed in the single cell layer of the sunflower anther epidermis. In the case of one of these genes, which codes for an abundant mRNA, we report the peptide sequences deduced from the sequence of two similar but non identical cDNAs. These proteins contain a potential signal peptide and are characterized by the presence of a proline-rich region which reads KPSTPAPPPPPP(PP)K. Our results also suggest that several proline-rich proteins of unknown functions are specifically synthesized during the maturation of anthers in sunflower.

The cloning of small GTP-binding proteins from Petunia hybrida was performed using a PCR-based strategy. Degenerate primers were designed from the DTAGQE and FMETSA consensus sequences. Three different cDNAs were amplified. The deduced polypeptide sequences PhPCRGP1 and PhPCRGP2 were homologous to RB11_HUMAN and PhPCRGP3 to RAB1A_HUMAN. Using PhPCRGP3 as a probe, 8 identical clones were selected from a Petunia leaf cDNA library. They all encode the same 22.5 kDa polypeptide, PhRAB1, able to bind GTP in vitro and 72% identical to RAB1A_HUMAN. Hybridizable mRNAs encoding PhRAB1 accumulated preferentially in opened flowers.