Regulation of arabidopsis trichome patterning and anthocyanin biosynthesis by the TTG1-bHLH-MYB complex

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 in Arabidopsis. These proteins also regulate the flavonoid-based pigment biosynthetic pathway in almost all higher plants including Arabidopsis. Using TTG1-YFP translational fusions, I show that TTG1 is expressed ubiquitously in Arabidopsis leaves and is preferentially localized in the nuclei of trichomes at all developmental stages. Using conditional transgenic alleles I demonstrate that TTG1 directly regulates the same genes as GL3. In vivo binding of GL3, GL1 and TTG1 to the promoters of GL2, TTG2, CPC and ETC1 establishes that these genes are major transcriptional targets for the TTG1-bHLH-MYB regulatory complex. By co-precipitation, I confirm that TTG1 interacts with the GL3 (bHLH) and GL1 (Myb) proteins in vivo, forming a complex. The loss of members of the TTG1 complex through mutation, affects the subcellular distribution of other complex members. Using particle bombardment, I show that TTG1, GL3, GL1 and GL2 do not move between adjacent epidermal cells while 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. In addition, I also show that GL3 is recruited to its own promoter in a GL1-independent manner, which results in decreased GL3 expression, suggesting the presence of a GL3 negative auto-regulatory loop. Expression studies using GL3-GR (GL3-glucocorticoid receptor) and TTG1-GR fusions reveal direct regulation of the late anthocyanin biosynthetic genes, but not of early biosynthetic genes. Taken together, our results provide insights on the molecular mechanisms by which the combinatorial TTG1-bHLH-MYB regulatory complexes activate and repress both developmental and biosynthetic pathways in Arabidopsis.