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Figure 8.4 Speculative model for GNOM-mediated auxin canalization. (A) Wild type (top) and strong gnom allele (bottom) heart stage embryos. Apical (a), central (c) and basal (b) regions of the embryos and the successive relating areas in the mature seedling are indicated. Auxin flow from source to sink is indicated by black arrows. (B) Relation between PIN1 localization (grey stubs) and auxin-response gradient (grey filling) in lateral root primordium development. Gradual reorientation of the individual transport polarities of cells by auxin canalization are indicated by arrows. Grey stubs indicate the cell walls to which the respective PIN1 label is thought to belong. (C) Presumptive critical step during lateral root formation for the canalization of auxin flow. Stage II primordium immediately after division (left) with two daughter cells displaying opposite polarities. Gradual, GNOM-dependent, relocalization of the efflux carriers are possibly guided by weak polarizing cues from adjacent cells; this supplies more auxin to the inner layer, which as a result imposes its auxin transport on the outer layer. Arrows indicate the direction of the auxin flux; auxin efflux carrier PIN1 in grey; GN, GNOM-positive endosomes involved in recycling auxin carriers. Adapted from Geldner et al. (2004).

gnomP5

Figure 8.4 Speculative model for GNOM-mediated auxin canalization. (A) Wild type (top) and strong gnom allele (bottom) heart stage embryos. Apical (a), central (c) and basal (b) regions of the embryos and the successive relating areas in the mature seedling are indicated. Auxin flow from source to sink is indicated by black arrows. (B) Relation between PIN1 localization (grey stubs) and auxin-response gradient (grey filling) in lateral root primordium development. Gradual reorientation of the individual transport polarities of cells by auxin canalization are indicated by arrows. Grey stubs indicate the cell walls to which the respective PIN1 label is thought to belong. (C) Presumptive critical step during lateral root formation for the canalization of auxin flow. Stage II primordium immediately after division (left) with two daughter cells displaying opposite polarities. Gradual, GNOM-dependent, relocalization of the efflux carriers are possibly guided by weak polarizing cues from adjacent cells; this supplies more auxin to the inner layer, which as a result imposes its auxin transport on the outer layer. Arrows indicate the direction of the auxin flux; auxin efflux carrier PIN1 in grey; GN, GNOM-positive endosomes involved in recycling auxin carriers. Adapted from Geldner et al. (2004).

mutation, caprice (cpc), affects root epidermis cell specification in a different manner. Rather than causing ectopic root hair cells, the cpc mutant produces fewer hairs than normal root cells. This implies that CPC is a positive regulator of the root hair cell fate (Wada et al, 1997).

All these genes encode putative transcriptional regulators. WER codes for a MYB-related putative transcriptional regulator that is mainly expressed in the N cells. Yeast two-hybrid assays showed that WER is able to interact with a bHLH-type protein to control epidermal cell patterning during Arabidopsis root development (Lee & Schiefelbein, 1999).

The GL2 gene encodes a homeodomain-containing protein (Rerie et al., 1994). It is preferentially expressed in the N cells (Masucci et al., 1996) already early during embryogenesis (Lin & Schiefelbein, 2001; Costa & Dolan, 2003). It is first expressed in the future epidermis in the heart stage embryo and its expression is progressively restricted to those cells that will acquire an N identity at the transition between torpedo and mature stage (Costa & Dolan, 2003).

CPC encodes a small protein with a MYB-like DNA-binding domain, but it does not have a typical transcriptional activation domain (Wada et al., 1997). It is preferentially expressed in the differentiating N cells (Wada et al., 1997, 2002). CPC has been suggested either to work together with the TTG gene product or in an independent pathway that controls the number of root hairs upstream of GL2 in the developmental pathway of root hair formation. (Wada et al., 1997). Interestingly, CPC protein is able to move to the hair-forming cells and repress gene expression (Wada et al., 2002). These studies indicate that transcriptional feedback loops between the WER, CPC and GL2 genes help to establish position-dependent epidermal patterning (Lee & Schiefelbein, 2002; Costa & Dolan, 2003).

Various gene expression studies have shown that WER positively regulates GL2 expression in the N cells (Lee & Schiefelbein, 1999) and is thus required to specify the positional information for GL2 expression (Hung et al., 1998; Lin & Schiefelbein, 2001). The WER gene is also required for positive regulation of CPC transcription in the developing N cells, and CPC acts as a negative regulator of GL2 (Wada et al., 1997; Schellmann et al., 2002; Wada et al., 2002), WER and itself in the developing H cells (Lee & Schiefelbein, 2002).

Based on GL2 promoter-reporter gene expression studies, it was proposed that the epidermal cell patterning mechanism in the root initiates during the early heart stage and that it occurs before the establishment of a functional meristem (Lin & Schiefelbein, 2001). Lee and Schiefelbein (2002) have proposed a simple model for the origin of the epidermal cell pattern. In this model, the specification of an H or N cells relies on the relative activity of two competing transcription factors, WER and CPC. In heart stage embryos, all epidermal cells express WER and GL2 and, in the absence of positional cues, use CPC to inhibit their neighbours from expressing these genes. In the growing root the pattern is established by positional cues from the underlying tissue that break the symmetry of the inhibition and cause greater WER transcription in the cells overlying a single cortical cell (Fig. 8.5). This leads to a high level of GL2 and CPC expression and to the N cell fate. In the alternate cell position, the CPC protein produced by the developing N cell is proposed to accumulate by virtue of its cell-to-cell trafficking and it represses GL2, WER and CPC expression, permitting H cell differentiation to proceed (Lee & Schiefelbein, 2002).

Recently, a different model for the establishment of the root epidermal pattern has been proposed based on more detailed examination of GL2, WER and CPC gene expression during embryogenesis (Costa & Dolan, 2003). According to this model the development of cell patterning in the root epidermis is also initiated

Figure 8.5 Model of root epidermal patterning in the growing root. Positional cues from underlying cells generate a bias in WER expression either because of inhibition of WER in H cells or because of activation of WER in N cells. This bias is enhanced by increased CPC levels, which carry an inhibitory signal from N cells to H cells. Dark grey tint corresponds to cell files that will produce non-hair cells (N cells). Light grey tint corresponds to cell files that will produce hair cells (H cells). Adapted from Larkin etal. (2003).

Figure 8.5 Model of root epidermal patterning in the growing root. Positional cues from underlying cells generate a bias in WER expression either because of inhibition of WER in H cells or because of activation of WER in N cells. This bias is enhanced by increased CPC levels, which carry an inhibitory signal from N cells to H cells. Dark grey tint corresponds to cell files that will produce non-hair cells (N cells). Light grey tint corresponds to cell files that will produce hair cells (H cells). Adapted from Larkin etal. (2003).

in the heart stage embryo, but it is not completed until the embryo reaches the mature stage. The model is based on the complex regulatory interactions between WER, CPC and GL2 that occur during the formation of epidermal pattern in the embryo. GL2 is first expressed in the heart stage embryo in a subset of cells in the protoderm and WER positively regulates its expression. Then, by the torpedo stage, GL2 expression has spread to all cells in the future epidermis. WER and CPC expression is then detectable and WER promotes GL2 expression throughout the epidermis. CPC is in turn required for the preferential accumulation of GL2 transcript in future N cells, perhaps by negatively regulating GL2 transcription in H cells position. In the mature embryo, GL2 negatively regulates WER transcription whereas WER positively regulates CPC expression from the torpedo to mature stages. These events result in GL2 being expressed at high levels in the future N cells and being absent from the future H cells in the mature embryo. The pattern of GL2 expression is then maintained in the root of the seedling and accounts for the pattern of H cells and N cells in the root epidermis, where GL2 negatively regulates hair formation in cells located in the N position (Costa & Dolan, 2003).

In addition to WER, GL2 and CPC, yet other genetic loci have been implicated in the root hair patterning. The TTG gene encodes a small WD40 repeat protein (Walker et al., 1999). It has been proposed to mediate protein-protein interactions (Galway et al., 1994) and to be involved in signal transduction to downstream transcription factors (Walker et al., 1999). Expression of the maize R gene (a transcription factor involved in anthocyanin biosynthetic pathway) under the CaMV 35S promoter complemented the ttg1 mutant phenotype, suggesting that TTG1 might regulate an R-like gene in Arabidopsis to adopt a hairless cell fate (Lloyd et al., 1992; Galway et al., 1994). Indeed, yeast two-hybrid assays indicate that GLABRA3 (GL3), an R homologous bHLH protein expressed in shoot epidermal cells in Arabidopsis, interacts with TTG (Payne et al., 2000). Thus, TTG may act as a general regulator of epidermal cell patterning. However, its precise role in root epidermis remains to be elucidated.

In addition, the TRY (TRIPTHYCON) gene encodes a CPC-homologous MYB-related transcription factor that lacks a recognizable activation domain. TRY has been shown to function as a negative regulator of trichome development in the shoot but it is also expressed in roots, suggesting that it has a role in root hair patterning. Indeed, over-expression of TRY results in formation of extra root hairs. Furthermore, various expression studies with TRY have shown that TRY levels are controlled by TRY and CPC, and that TRY is likely to be expressed in N cells (Schellmann et al., 2002).

Although TTG and TRY involvement in root epidermal pattern formation has not been rigorously examined, several studies indicate that they have a role in this process. The emerging picture is that TTG and an as yet unknown bHLH-related transcription factor, together with WER, begin to act an early stage in embryonic development to positively regulate the expression of GL2 (and perhaps other as yet unidentified genes) in a cell position-dependent manner to specify the N cell type (Hung et al., 1998; Lin & Schiefelbein, 2001). Furthermore, it is likely that TRY, in addition to CPC, is also involved in the lateral suppression of GL2 expression in root hair files (Schellmann et al., 2002). The control of epidermal patterning in aerial tissue and its overlap with the system described in the root is explored further in Chapter 9, this volume.

8.4.4 Patterning of ground tissue

As discussed above, pattern formation in the Arabidopsis root is the result of highly asymmetric cell divisions and subsequent cell specification events. One of the best characterized examples is the radial pattern formation of ground tissue that originates from a set of stem cells ('initials') that undergo asymmetric cell divisions to give rise to the youngest cells of the endodermis and cortex cell lineages (Figs. 8.7A and 8.7B). Subsequently, cells in both lineages undergo differential expansion resulting in cell types with distinct morphologies and differentiated features. Two loci, SHORT-ROOT (SHR) and SCARECROW (SCR), controlling ground tissue patterning during root development have been identified in Arabidopsis (Benfey et al., 1993; Scheres et al., 1995). Mutations in these loci result in a single ground tissue layer, indicating

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