RESEARCH PAPER Transparent Testa Glabra 1 (TTG1) and TTG1-like genes in Matthiola incana R. Br. and related Brassicaceae and mutation in the WD-40 motif A. Dressel & V. Hemleben Department of General Genetics, Center of Plant Molecular Biology (ZMBP), Tu ¨ bingen, Germany INTRODUCTION Many processes in eukaryotes are influenced and regu- lated by WD-40 repeat proteins, a large protein family (reviewed in Neer et al. 1994), e.g. the well known Gb subunit of small heterotrimeric G proteins is involved in signal transduction pathways (Li & Roberts 2001). These proteins share a general feature leading to a seven bladder propeller-like structure; in common is the WD-40 repeat structure with subrepeats that are approx. 40 amino acids in length. TTG1, a member of this WD-40 repeat protein family in plants, has been characterized by mutational screening in Arabidopsis thaliana (Koorneef 1990; Larkin et al. 1994; Walker et al. 1999). TTG1 is thought to regulate flavonoid ⁄ anthocyanin biosynthesis (Ramsay & Glover 2005), seed coat development ⁄ pigmentation and trichome formation on leaves (Larkin et al. 1994, 1999; Payne et al. 2000; Szymanski et al. 2000; Ramsay & Glo- ver 2005). A mutation in the TTG1 gene causes defects in all these processes, suggesting a common regulatory path- way (Western et al. 2004; Broun 2005). Homologues were described for several plants, e.g. Petunia (AN11; de Vetten et al. 1997), tetraploid cotton (TTG1 and TTG3; Humph- ries et al. 2005) and maize (PAC1; Selinger & Chandler 1999; Carey et al. 2004). Multiple cellular differentiation pathways are initiated by TTG1 associated in a protein complex with MYB and bHLH transcription factors (Ramsay & Glover 2005). In our collection of isogenic and genetically defined Matthiola incana lines, that were used earlier as a model Keywords Anthocyanin biosynthesis; dihydroflavonol 4-reductase (DFR); GC content; gene duplication; gene evolution. Correspondence Prof. Dr. Vera Hemleben, Department of General Genetics, ZMBP, Center of Plant Molecular Biology, University of Tu ¨ bingen, Auf der Morgenstelle 28, 72076 Tu ¨ bingen, Germany. E-mail: vera.hemleben@zmbp. uni-tuebingen.de Editor J. T. M. Elzenga Received: 24 August 2007; Accepted: 25 March 2008 doi:10.1111/j.1438-8677.2008.00099.x ABSTRACT TTG1 (Transparent Testa Glabra 1), a WD-40 repeat protein, is involved in regulation of flavonoid ⁄ anthocyanin biosynthesis, seed coat (mucilage) development ⁄ pigmentation and trichome formation in leaves. Here, we characterized the TTG1 gene of Matthiola incana wild type (e locus), show- ing 85.3% similarity to TTG1 of A. thaliana on the nucleotide level and 96.2% on the protein level. A white-flowered and glabrous mutant, line 17, of M. incana exhibits one nucleotide change, leading to an amino acid sub- stitution directly in the WD motif (W158R). Correspondingly, the DFR (dihydroflavonol 4-reductase) gene, in which the expression is known to be dependent on TTG1, is not expressed in Matthiola mutant lines 17 (and 19). Comparison of the GC content of the Matthiola TTG1 (54.1%) and Arabid- opsis TTG1 (46.1%) genes revealed a strong difference, mostly obtained by neutral substitutions (C to T transitions). To examine whether this is an ecologically influenced trend, a fragment of TTG1 was characterized from another Matthiola species (M. tricuspidata) and from Malcolmia flexuosa subsp. naxensis from the eastern Mediterranean, near a beach with sandy and salty soils. Both Matthiola species have a higher GC content in the TTG1 gene than Arabidopsis and the closer-related Malcolmia, indicating that the GC content is rather an evolutionary than an ecological signal. A similar WD-40 repeat protein gene (containing no intron in the 3¢ untrans- lated region) with high similarity to the Arabidopsis TTG1-like (AtAN11) gene was found in Matthiola. Plant Biology ISSN 1435-8603 204 Plant Biology 11 (2009) 204–212 ª 2008 German Botanical Society and The Royal Botanical Society of the Netherlands