Knock-out mutants from anEn-1mutagenizedArabidopsis thalianapopulation generate phenylpropanoid biosynthesis phenotypes

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Abstract
A collection of 8,000Arabidopsis thalianaplants carrying 48,000 insertions of the maize transposable elementEn-1has been generated. This population was used for reverse genetic analyses to identify insertions in individual gene loci. By using a PCR-based screening protocol, insertions were found in 55 genes.En-1showed no preference for transcribed or untranscribed regions nor for a particular orientation relative to the gene of interest. In several cases,En-1was inserted within a few kilobases upstream or downstream of the gene.En-1was mobilized from such positions into the respective gene to cause gene disruption. Knock-out alleles of genes involved in flavonoid biosynthesis were generated. One mutant line contained anEn-1insertion in the flavonol synthase gene (FLS) and showed drastically reduced levels of kaempferol. Allelism tests with other lines containingEn-1insertions in the flavanone 3-hydroxylase gene (F3H) demonstrated thatTRANSPARENT TESTA 6(TT6) encodes flavanone 3-hydroxylase. Thef3handflsnull mutants complete the set ofA. thalianalines defective in early steps of the flavonoid pathway. These experiments demonstrate the efficiency of the screening method and gene disruption strategy used for assigning functions to genes defined only by sequence.