1991 RESEARCH ARTICLE INTRODUCTION Studies on the regulation of cell fate and function on the plant epidermis continue to provide important insights into how plant cells are organized, how patterning develops, and how developmental and biochemical pathways interact. Trichome initiation in the model plant Arabidopsis thaliana has been an important model for understanding cell fate and patterning. Trichomes (leaf hairs) are large branched single cells that initiate and develop on young leaves in a regular spacing pattern (Larkin et al., 1997; Marks, 1997; Hulskamp and Schnittger, 1998; Hulskamp et al., 1999). Trichome patterning is not random or dependent on other cell types or position on the leaf, but is thought to be generated de novo by intercellular communication (Larkin et al., 1996; Schnittger et al., 1999). The model assumes that inhibitors, which are activated by self-enhanced activators, can move between cells to mediate competition between equivalent cells, resulting in the pattern formation (Larkin et al., 2003; Pesch and Hulskamp, 2004). Years of genetic and molecular studies have enabled the identification of components of this trichome patterning machinery. Three classes of interacting regulators [including the R2R3-MYB transcription factor GLABRA1 (GL1) (Oppenheimer et al., 1991), the basic helix-loop-helix (bHLH) proteins GLABRA3 (GL3) and ENHANCER OF GLABRA3 (EGL3) (Payne et al., 2000; Zhang et al., 2003), and the WD40 repeat protein TRANSPARENT TESTA GLABRA1 (TTG1) (Walker et al., 1999)] are postulated to form a combinatorial regulatory complex. Evidence comes from yeast two- hybrid studies showing that TTG1 and GL1 physically interact with GL3/EGL3 but not with each other (Payne et al., 2000; Zhang et al., 2003). GLABRA2 (GL2) is a direct target of GL3 and EGL3 (Morohashi et al., 2007) and TRANSPARENT TESTA GLABRA2 (TTG2) is directly regulated by GL1 (Ishida et al., 2007). This activation is believed to be through the formation of TTG1-GL3- GL1 and TTG1-EGL3-GL1 (TTG1-bHLH-GL1) regulatory complexes (Szymanski et al., 1998), thereby regulating trichome cell fate. GL2, a homeodomain (HD-Zip), and TTG2, a WRKY transcription factor, are required for normal trichome development (Rerie et al., 1994; Johnson et al., 2002). Some levels of GL2 overexpression can result in trichome clusters, indicating that this HD-Zip may function in the regulation of trichome spacing (Ohashi et al., 2002). To date, a group of at least four homologous single MYB proteins [TRIPTYCHON (TRY) (Schellmann et al., 2002), CAPRICE (CPC) (Wada et al., 1997), and ENHANCER OF TRY and CPC1 and 2 (ETC1 and 2) (Kirik et al., 2004a; Kirik et al., 2004b)] have been identified as negative regulators of trichome initiation and patterning. The try cpc double and the try cpc etc1 triple mutants (Kirik et al., 2004a; Schellmann et al., 2002) display a greatly enhanced ‘clustered-trichome’ phenotype, indicating that lateral inhibition is disrupted. These inhibitory proteins contain no recognizable transcription activation domain. Therefore, they could work as negative transcriptional regulators. Protein interaction analysis in yeast has suggested that TRY or CPC would interrupt the functionality of the ‘activating’ TTG1-bHLH-GL1 complex by competitive interaction with the bHLH (Esch et al., 2003; Zhang et al., 2003). Additionally, the individual members of this inhibitory protein family may function differently. There is evidence that TRY might be more important in short-range inhibition, while CPC and particularly ETC1 may be important for long-range inhibition (Schellmann et al., 2002; Kirik et al., 2004a). As described above, the identification of these positive and negative trichome regulators has laid an excellent foundation for understanding trichome patterning. However, a large amount of the The TTG1-bHLH-MYB complex controls trichome cell fate and patterning through direct targeting of regulatory loci Mingzhe Zhao 1 , Kengo Morohashi 2 , Greg Hatlestad 1 , Erich Grotewold 2 and Alan Lloyd 1, * A network of three classes of proteins consisting of bHLH and MYB transcription factors, and a WD40 repeat protein, TRANSPARENT TESTA GLABRA1 (TTG1), act in concert to activate trichome initiation and patterning. Using YFP-TTG1 translational fusions, we show that TTG1 is expressed ubiquitously in Arabidopsis leaves and is preferentially localized in the nuclei of trichomes at all developmental stages. Using a conditional transgenic allele, we demonstrate that TTG1 directly targets the same genes as the bHLH protein GLABRA3 (GL3). In vivo binding of the R2R3-MYB protein GLABRA1 (GL1) to the promoters of GLABRA2 (GL2), TRANSPARENT TESTA GLABRA2(TTG2), CAPRICE (CPC) and ENHANCER OF TRIPTYCHON AND CAPRICE1 (ETC1) establishes that these genes are major transcriptional targets for the TTG1-bHLH-MYB regulatory complex. By co-precipitation, we confirm that TTG1 associates with GL3 and GL1 in vivo, forming a complex. The loss of TTG1 and GL1 through mutation, affects the subcellular distribution of GL3. Using particle bombardment, we show that TTG1, GL3, GL1 and the homeodomain protein GL2 do not move between adjacent epidermal cells, while the R3-MYB, CPC, does move to neighboring cells. These data support a model for the TTG1 complex directly regulating activators and repressors and the movement of repressors to affect trichome patterning on the Arabidopsis leaf. KEY WORDS: Epidermis, Pattern formation, Trichome, Gene regulation, Cell differentiation, Leaf, Arabidopsis thaliana, Cell fate, Transcription, TTG1 Development 135, 1991-1999 (2008) doi:10.1242/dev.016873 1 Section of Molecular Cell and Developmental Biology and The Institute for Cellular and Molecular Biology, The University of Texas at Austin, 2500 Speedway, Austin, TX 78712, USA. 2 Department of Plant Cellular and Molecular Biology and Plant Biotechnology Center, The Ohio State University, Columbus, OH 43210, USA. *Author for correspondence (e-mail: lloyd@uts.cc.utexas.edu) Accepted 4 April 2008 DEVELOPMENT