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 G LABRA 2 (GL2), TRANSPARENT TESTA GLABRA 2 ( TTG 2), 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.

The specification of the hair and non-hair cells in the Arabidopsis root epidermis provides a useful model for the study of cell fate determination in plants. A network of putative transcriptional regulators, including the related bHLH proteins GLABRA3 (GL3) and ENHANCER OF GLABRA3 (EGL3), is known to influence the patterning of these cell types. Here, we analyze the expression and regulation of GL3 and EGL3 during root epidermis development. Although they are thought to act in both the hair and non-hair cell types, we surprisingly found that GL3 and EGL3 gene expression and RNA accumulation occurs preferentially in the developing hair cells. By analyzing the expression of GL3::GUS and EGL3::GUS reporter fusions in various mutant and overexpression lines, we discovered that the expression of both genes is negatively regulated by WER, GL3 and EGL3 in the developing non-hair cells,and positively regulated by the CPC and TRY proteins in the developing hair cells. Further, the analysis of a GL3-YFP translational fusion, expressed under the GL3 promoter, indicates that the GL3 protein moves from the hair cells to the non-hair cells. These results suggest that GL3/EGL3 accumulation in the N cells is dependent on specification of the hair cell fate, which itself is known to be influenced (via CPC-mediated lateral inhibition) by the non-hair cells. This bi-directional signaling mechanism defines a new regulatory circuit of intercellular communication to specify the epidermal cell types.

The position-dependent specification of the hair and non-hair cell types in the Arabidopsis root epidermis provides a simple model for the study of cell fate determination in plants. Several putative transcriptional regulators are known to influence this cell fate decision. Indirect evidence from studies with the maize R gene has been used to suggest that a bHLH transcription factor also participates in this process. We show that two Arabidopsis genes encoding bHLH proteins, GLABRA3 ( GL3 ) and ENHANCER OF GLABRA3 ( EGL3 ), act in a partially redundant manner to specify root epidermal cell fates. Plants homozygous for mutations in both genes fail to specify the non-hair cell type,whereas plants overexpressing either gene produce ectopic non-hair cells. We also find that these genes are required for appropriate transcription of the non-hair specification gene GL2 and the hair cell specification gene CPC , showing that GL3 and EGL3 influence both epidermal cell fates. Furthermore, we show that these bHLH proteins require a functional WER MYB protein for their action, and they physically interact with WER and CPC in the yeast two-hybrid assay. These results suggest a model in which GL3 and EGL3 act together with WER in the N cell position to promote the non-hair cell fate, whereas they interact with the incomplete MYB protein CPC in the H position, which blocks the non-hair pathway and leads to the hair cell fate.

GLABRA3 ( GL3 ) encodes a bHLH protein that interacts with the WD repeat protein, TTG1. GL3 overexpression suppresses the trichome defect of the pleiotropic ttg1 mutations. However, single gl3 mutations only affect the trichome pathway with a modest trichome number reduction. A novel unlinked bHLH-encoding locus is described here, ENHANCER OF GLABRA3 ( EGL3 ). When mutated, egl3 gives totally glabrous plants only in the gl3 mutant background. The double bHLH mutant, gl3 egl3 , has a pleiotropic phenotype like ttg1 having defective anthocyanin production, seed coat mucilage production, and position-dependent root hair spacing. Furthermore, the triple bHLH mutant, gl3 egl3 tt8 , phenocopies the ttg1 mutation. Yeast two-hybrid and plant overexpression studies show that EGL3, like GL3,interacts with TTG1, the myb proteins GL1, PAP1 and 2, CPC and TRY, and it will form heterodimers with GL3. These results suggest a combinatorial model for TTG1-dependent pathway regulation by this trio of partially functionally redundant bHLH proteins.

Silencing of homeotic gene expression requires the function of cis -regulatory elements known as Polycomb Response Elements (PREs). The MCP silencer element of the Drosophila homeotic gene Abdominal-B has been shown to behave as a PRE and to be required for silencing throughout development. Using deletion analysis and reporter gene assays, we defined a 138 bp sequence within the MCP silencer that is sufficient for silencing of a reporter gene in the imaginal discs. Within the MCP138 fragment, there are four binding sites for the Pleiohomeotic protein (PHO) and two binding sites for the GAGA factor (GAF), encoded by the Trithorax-like gene. PHO and the GAF proteins bind to these sites in vitro. Mutational analysis of PHO and GAF binding sequences indicate that these sites are necessary for silencing in vivo. Moreover, silencing by MCP138 depends on the function of the Trithorax-like gene, and on the function of the PcG genes, including pleiohomeotic . Deletion and mutational analyses show that, individually, either PHO or GAF binding sites retain only weak silencing activity. However, when both PHO and GAF binding sites are present, they achieve strong silencing. We present a model in which robust silencing is achieved by sequential and facilitated binding of PHO and GAF.