Leucoanthocyanidin Dioxygenase in Arabidopsis thaliana: characterization of mutant alleles and regulation by MYB-BHLH-TTG1 transcription factor complexes

In Arabidopsis thaliana, most mutants impaired in flavonoid accumulation were identified through screens for altered seed pigmentation. Mutations in more than 20 loci have been described that can result in a transparent testa (tt) or tannin deficient seed (tds) phenotype. For some of these mutants it is still unclear if they represent additional loci or if they are allelic to known mutations. In this study, we found that tt17 is allelic to tt11 and tds4 and identified a point mutation in tt17 that affects the gene encoding Leucoanthocyanidin Dioxygenase (LDOX). The mutation results in replacement of a cysteine close to the active site of the enzyme by the hydrophobic amino acid tyrosine. Effects of this mutation on protein structure and activity are discussed in the context of LDOX sequences from various genotypes. Regulation of the LDOX promoter was analyzed and found to be directly controlled by different MYB-BHLH-TTG1 transcription factor complexes containing the BHLH factors EGL3 and TT8. Experiments with single and double loss-of-function mutants identified EGL3 and TT8 as necessary regulators of anthocyanin accumulation in developing A. thaliana seedlings.