Position-dependent cell fate determination and pattern formation are unique aspects of the development of plant structures. The establishment of single-celled leaf hairs (trichomes) from pluripotent epidermal (protodermal) cells in Arabidopsis thaliana provides a powerful system to determine the genetic networks and positional signals involved in cell fate determination (Szymanski et al., 2000; Larkin et al., 2003; Schellmann and Hulskamp, 2005; Serna and Martin, 2006). Over 30 genes controlling various aspects of trichome initiation, spacing, size and morphology have been cloned (Schellmann and Hulskamp, 2005).
Trichome initiation is regulated by the combinatorial action of the R2R3-MYB GLABRA1 (GL1) or AtMYB23 together with the bHLH GLABRA3 (GL3) or ENHANCER OF GLABRA3 (EGL3) transcription factors (Oppenheimer et al., 1991; Payne et al., 2000; Kirik et al., 2001; Esch et al., 2003; Schiefelbein, 2003; Zhang et al., 2003; Kirik et al., 2005). While gl1 mutants are mostly glabrous, mutations in gl3 have only a modest effect, primarily affecting branching, DNA endoreduplication and trichoblast size (Hulskamp et al., 1994; Payne et al., 2000). In contrast, egl3 plants have no obvious trichome defect, but gl3 egl3 double mutants show a complete glabrous phenotype (Zhang et al., 2003). Expression of both GL3 and EGL3 is low in the developing epidermis of young leaves, increases in initiating and young trichomes and drops in mature trichomes and pavement cells of mature leaves (Zhang et al., 2003). This is similar to GL1 and consistent with their participation in the selection of protodermal cells to the trichome pathway (Larkin et al., 1993). (Larkin et al., 1993). In addition to GL1 and GL3/EGL3, trichome initiation also requires the presence of TRANSPARENT TESTA GLABRA1 (TTG1), a WD-repeat containing protein (Walker et al., 1999) that physically interacts with GL3 and EGL3 (Zhang et al., 2003).
Figure 1: Induction of trichome formation by GL3. GL3-induced trichome formation in gl3 egl3 pGL3::GL3-GR seedlings treated with DEX. The transgenic plants were mock treated for a week (A) or treated with DEX for 4 hours (B) or 24 hours (C). Alternatively, plants were continuously grown for a week in media containing DEX (D).
The control of trichome patterning is likely to occur by a self-organizing system (Larkin et al., 1996). This involves lateral inhibition with feedback regulation for the initiation of trichome formation (Schellmann et al., 2002). At least four partially redundant single-repeat R3 MYB proteins CAPRICE (CPC) (Wada et al., 1997), TRIPTYCHON (TRY) (Hulskamp et al., 1994) and ENHANCER OF TRY and CPC1 and 2 (ETC1 and 2) (Kirik et al., 2004) play central roles in this lateral inhibition, by targeting specific components of the MYB/bHLH/TTG regulatory complex, making it non-functional (Schellmann et al., 2002). The expression of the TRY, CPC and ETC inhibitory proteins in trichome initials and in mature trichomes (Schellmann et al., 2002; Kirik et al., 2004) poses the problem of how the MYB/bHLH/TTG complex can function to promote a trichome. To explain this conundrum, models have been proposed in which the expression of the inhibitors is tightly controlled by the activatory complex, and which involve the ability of the inhibitory proteins to move to adjacent cells, resulting in, at first equivalent cells, competing by mutual lateral inhibition for the ability to accumulate sufficient quantity of the MYB/bHLH/TTG complex to trigger trichome initiation (Schellmann et al., 2002; Schiefelbein, 2003).
Figure 2: Transcriptional regulatory interactions controlling trichome development. GL3, GL1 and TTG1 are positive regulators of trichome froamtion. Protein complexes are indicated by circles. Orange and red lines describe the inter-cellular movement of the small single MYB-repeat proteins, CPC and ETC1. The cell on the left has entered the trichome pathway while the cell on the right will remain a pavement cell. The figure was drawn using Biotapestry.
We have shown that trichome initiation is triggered within 4 hours of the induction of the GL3 bHLH transcription factor. Within this developmental window, GL3 binds to the promoters of at least three genes previously implicated in the development and patterning of trichomes (GL2, CPC and ETC1) and activates their transcription. The in vivo binding of GL3 to the promoters of these genes requires the presence of the R2R3-MYB factor GL1, supporting a model in which a GL3-GL1 complex is part of the trichome initiation enhanceosome. In contrast, GL3 is recruited to its own promoter in a GL1-independent manner, and this results in decreased GL3 expression, suggesting the presence of a GL3 negative auto-regulatory loop. In support of genetic analyses indicating that EGL3 is partially redundant with GL3, we show that EGL3 shares some direct targets with GL3. However, our results suggest that GL3 and EGL3 work independently of each other. Since we have shown GL3 functions as both activator and repressor, it is intriguing to investigate genome-wide functions of GL3. We performed ChIP-chip experiments with transgenic plants expressing epitope-tagged GL3 proteins. The research is underway.