The FATTY ACID ELONGATION1 (FAE1) gene of Arabidopsis is required for the synthesis of very long chain fatty acids in the seed. The product of the FAE1 gene is presumed to be a condensing enzyme that extends the chain length of fatty acids from C18 to C20 and C22. We report here the cloning of FAE1 by directed transposon tagging with the maize element Activator (Ac). An unstable fae1 mutant was isolated in a line carrying Ac linked to the FAE1 locus on chromosome 4. Cosegregation and reversion analyses established that the new mutant was tagged by Ac. A DNA fragment flanking Ac was cloned by inverse polymerase chain reaction and used to isolate FAE1 genomic clones and a cDNA clone from a library made from immature siliques. The predicted amino acid sequence of the FAE1 protein shares homology with those of other condensing enzymes (chalcone synthase, stilbene synthases, and beta-ketoacyl-acyl carrier protein synthase III), supporting the notion that FAE1 is the structural gene for a synthase or condensing enzyme. FAE1 is expressed in developing seed, but not in leaves, as expected from the effect of the fae1 mutation on the fatty acid compositions of those tissues.

We have investigated the pattern of transposition of an intact, 4.6-kbAc element inArabidopsis thaliana. Because the trans-acting transposition function (transposase) ofAc is not fully penetrant in Arabidopsis, it is not possible to use it as a diagnostic feature to scoreAc genetically, as has been done in maize and tobacco. Instead, the presence or absence of a transposedAc (trAc) was monitored by Southern blots. Germinal transpositions from the marker SPT::Ac were selected using a streptomycin germination assay and scored for the presence of atrAc. Segregation of thetrAc element and the SPT donor locus was scored in the F2 progeny of the germinal revertants, and the recombination fraction between thetrAc element and SPT was estimated by the method of maximum likelihood. We have found that, as in maize and tobacco, receptor sites fortrAcs in Arabidopsis tend to be linked to theAc donor locus.

The effect of Ac copy number on the frequency and timing of germinal transposition in tobacco was investigated using the streptomycin phosphotransferase gene (SPT) as an excision marker. The activity of one and two copies of the element was compared by selecting heterozygous and homozygous progeny of transformants carrying single SPT::Ac inserts. It was observed that increasing gene copy not only increases the transposition frequency, but also occasionally alters the timing of transposition such that earlier events are obtained. The result is that some homozygous plants generate multiple streptomycin resistant progeny carrying the same transposed Ac (trAc) element. We have also investigated the effect of modification of the sequence in the region around 82 bp downstream of the polyadenylation site and 177 bp from the 3' end of the element on germinal excision frequencies. Alteration of three bases to create a Bgl II site at this location caused a minor decrease in germinal excision events, but insertion of four bases to create a Cla I site caused a 10-fold decrease in the transposition activity of the Ac element.

We have investigated the germinal and somatic activity of the maize Activator (Ac) element in Arabidopsis with the objective of developing an efficient transposon-based system for gene isolation in that plant. Transposition activity was assayed with a chimeric marker that consists of the cauliflower mosaic virus 35S promoter and a bacterial streptomycin phosphotransferase gene (SPT). Somatic activity was detected in seedlings germinated on plates containing streptomycin as green-resistant sectors against a background of white-sensitive cells. Germinal excisions resulted in fully green seedlings. The transposition frequency was extremely low when a single copy of the transposon was present, but appeared to increase with an increase in Ac copy number. Plants that were selected as variegated produced an increased number of green progeny. The methylation state of the Ac elements in lines with either low or high levels of excision was assessed by restriction analysis. No difference was found between these lines, indicating that the degree of methylation did not contribute to the level of Ac activity. Germinal excision events were analyzed molecularly and shown to carry reinserted transposons in about 50% of the cases. In several instances, streptomycin-resistant siblings carried the same transposed Ac element, indicating that excision had occurred prior to meiosis in the parent. We discuss parameters that need to be considered to optimize the use of Ac as a transposon tag in Arabidopsis.

The strategy to be followed in a transposon tagging experiment will be determined largely by the transposition pattern of the transposon in question. With a view to utilizing the maize element Activator (Ac) as a transposon tag in heterologous systems, we investigated the pattern of Ac transposition from six different loci in transgenic tobacco. We isolated germinal revertants from plants carrying mutable alleles of the antibiotic-resistant gene streptomycin phosphotransferase (SPT) and mapped the location of the transposed Ac (trAc) elements relative to the donor SPT gene. A comparison of the distributions of trAcs among the six loci revealed that, although the receptor sites for trAcs tend to be linked to the donor locus, the pattern of Ac transposition in tobacco displays surprising locus-to-locus variation. Some trAc distributions showed the same tight clustering around the donor locus previously seen in maize, whereas others were more dispersed. The possible meaning of these findings and their implication for transposon tagging in heterologous systems are discussed.