SCHIZORIZA controls an asymmetric cell division and restricts epidermal identity in the Arabidopsis root

In plants, meristems are populations of stem cells from which the body of the plant subsequently develops. Cells within the primary shoot meristem give rise to different organs such as leaves, stem and the floral organs. The primary root meristem, however, elaborates a single organ, the root, which has a stereotyped radial pattern of tissues. In Arabidopsis this radial pattern is simple. Root cells are organised as concentric rings of lateral root cap, epidermis, ground tissue (cortex and endodermis), and a pericycle surrounding a central stele (Dolan et al., 1993) (see Fig. 4A). This cell pattern is first laid down during embryogenesis and is maintained by regular divisions of stem cells (initials) in the meristem of the developing root. These initials surround four central cells (quiescent centre) that divide infrequently. There are four sets of initials: the lateral root cap/epidermal initials, the cortical/endodermal initials, the columella initials and the pericycle/vascular initials (Dolan et al., 1993). Initials divide in a stereotypical pattern to give rise to the cells in each of the tissue layers (see Fig. 4A). For example, the cortex/endodermis initial divides anticlinally (new wall perpendicular to the root surface) to generate a new initial and a daughter cell. The daughter cell divides periclinally (new wall parallel to the surface of the root) and asymmetrically to generate a small inner cell which is the daughter cell of the endodermis and a larger outer cell that will develop as cortex. Initials divide slowly, adding to the population of rapidly dividing cells in the meristem. After division, cells elongate before they acquire their mature size and shape in the differentiation zone. Once the radial organisation of tissues is established in the root, the tangential differentiation of cell types occurs in the epidermis. The mature epidermis is composed of cells bearing hairs that develop from meristematic trichoblasts, and non hair-bearing epidermal cells which develop from atrichoblasts.

The identification of mutants with defects in radial pattern has provided a basis for understanding the mechanism of radial patterning in the root. Plants homozygous for the scarecrow (scr) and short root (shr) mutations have defects in the division of the cortex/endodermis initial daughter cell resulting in the formation of a single layer of ground tissue instead of two (Benfey et al., 1993; Scheres et al., 1995). SCR is essential for cell division, but not differentiation, of the ground tissue (Scheres et al., 1995; Di Laurenzio et al., 1996). In contrast, SHR plays a role in both cell division and the differentiation of the endodermis (Benfey et al., 1993; Helariutta et al., 2000). SCR and SHR are members of the GRAS family of transcription factors (Di Laurenzio et al., 1996; Helariutta et al., 2000). SCR is expressed in the quiescent centre, the cortex/endodermis initial and the endodermis (Di Laurenzio et al., 1996; Wysocka-Diller et al., 2000). SHR is expressed in the stele (pericycle and vascular cylinder) and has been shown to act in a non cell-autonomous manner (Helariutta et al., 2000). Genetic analysis has shown that SCR and SHR act in the same pathway since shr is epistatic to scr and SHR is required for SCR transcription.

Here we describe a mutant called schizoriza (scz) which was identified in a screen for genes involved in the development of the root epidermis. Plants homozygous for the scz mutation develop supernumerary ground tissue layers and root hairs emerge from subepidermal cells. The extra periclinal divisions that occur in scz mutants are suppressed by both scr and shr mutations indicating that SHR, SCR and SCZ act in the same pathway in the regulation of cell division in the root meristem. Because scz scr and scz shr double mutants do form root hairs in the ground tissue it suggests that the mis-specification of subepidermal cell fate by SCZ is independent of SHR and SCR.

Seeds were surface sterilised in 5% sodium hypochlorite and sown on half strength Murashige and Skoog (Duchefa, Haarlem, The Netherlands) medium (pH 5.8), containing 1% sucrose and 0.8% phytagel. The plants were stratified for 2 days and grown in continuous light conditions at an angle of 45°.

Col and Col-0 plants were used to pollinate homozygous scz mutant plants (ecotype Ler). An F₂ population was generated. Individual F₂scz mutants were selected for mapping. DNA isolated from the mutants was analysed using CAPS and SSLP markers (Klimyuk et al., 1993; Konieczny and Ausubel, 1993; Bell and Ecker, 1994).

Three- to 7-day-old seedlings were stained with 0.1 mg/ml propidium iodide solution for 5-15 minutes. Propidium iodide-stained roots were imaged with an MRC1024 Biorad confocal microscope using 568 nm excitation line and a YHS filter block or a Leica TC5 SP confocal microscope using the 568 nm excitation and 498-551 nm emission lines. The 488 nm excitation and 580-700 nm emission lines on the Leica microscope were used to image GFP expression in the enhancer trap lines. Images were processed using NIH Image (http://rsb.info.hih.gov/nih-image) and assembled using Adobe Photoshop 5.

Several GFP enhancer trap lines from the Haseloff collection were crossed into the scz background. J0481 is expressed in all epidermal cells from the elongation and differentiation zones and the lateral root cap cells (lrc). J0672 and J2092 are expressed in all epidermal cells from the elongation and differentiation zones at 3 days after germination, and by 5 days they are only expressed in atrichoblasts. These lines are also expressed in the lrc. J0571 is expressed in the quiescent centre, the cortex/endodermis initial and daughter cells, and all the cells of the ground tissue in the meristem, elongation zone, and differentiation zone. J3611 is expressed in endodermal cells in the differentiation zone and J2672 is expressed in the endodermis from the endodermal daughter cell into the elongation and differentiation zones. J2672 is also expressed in lateral root cap cells. J2931 and Q2393 are expressed in the lateral root cap, epidermis and cortex. N9173 is expressed in the epidermis/lateral root cap daughter cells, in the lateral root cap cells, and in all the epidermal cells of the meristem. In the elongation zone it is expressed in the atrichoblasts of the epidermis. In the differentiation zone, it is expressed in the atrichoblasts and the expression spreads to all cells of the ground tissue, the pericycle and the vasculature.

Fifty scz mutant roots were analysed with confocal microscopy at 3 days, 4 days and 5 days after germination. Thirty wild-type plants and 30 scz GFP-expressing plants were examined using confocal microscopy at 3 days, 5 days and 7 days after germination (10 plants at each time point) for each GFP enhancer trap line. Several hundred plants from each double mutant combination were plated and observed with a stereomicroscope. Thirty plants from each double mutant combination were analysed further with confocal microscopy. 25 scz GL2::GUS plants were stained for GUS activity. Six scz GL2::GUS plants embedded in Technovit, were sectioned to look for GUS activity in subepidermal positioned cells.

Total RNA was isolated from Ler and scz 3-day-old roots using an RNeasy™ Plant Mini Kit (Qiagen). Genomic contamination was removed by DNase treatment. 3 μg of RNA was reverse transcribed with oligo(dT) using murine reverse transcriptase and used as templates for PCR amplification. The gene specific primers SCR5: 5′-GGAATTTACGCGGCTTTGCCTTCACGGTGGATG-3′ and SCR3: 5′-TACAAATCTTCCTAAGAAAGAACCAGCGTGGCT-3′, were used to amplify SCR. These primers produce a fragment of 564 bp when cDNA is used as a template and a fragment of 680 bp when genomic DNA is the template. Primers EF1 5′-GCTCTATGGAAGTTCGACC-3′ and EF2 5′-GGTGTGGCAATCGAGAACTGGG-3′ were used to amplify the Arabidopsis elongation factor 1-alpha 4 (Liboz et al., 1989), for a control of equal amounts of cDNA used in the PCR reactions. These primers produce a fragment of 811 bp when cDNA is used as template and a fragment of 912 bp when genomic DNA is used. PCR reactions were run for 22, 25, 28, 31 and 34 cycles for SCR and 18, 22 and 25 for EF1.

A population of Ac/Ds transposon tagged lines was visually screened to isolate root mutants with defects in epidermal development. The screen identified a mutant that developed hairier roots than wild-type plants. The mutant was named schizoriza (scz). Apart from a decrease in shoot stature the mutant displayed no other obvious aberrant phenotype. The scz mutation segregates as a single recessive Mendellian factor (χ²=1.05), and maps to chromosome 1, between CAPS marker GAPB and SSLP marker nF19K23. Genetic analysis indicates that the transposon in this line and the scz mutation are unlinked.

There are no alternating files of root hair and non-root hair cells in the epidermis of scz mutants (Fig. 1B) as there are in wild type (Fig. 1A). In addition, some of the hairs emerged from cells in the subepidermal location and grew out between the cells of the epidermis (arrows in Fig. 1B).

Fig. 2A is a median longitudinal section through a 3-day-old root of a plant homozygous for the scz mutation. The image shows two root hair cells. Transverse sections through the root, at positions indicated by arrows, show a root hair that originates from a cell in the subepidermal location (asterisks in Fig. 2B). In 3-day-old plants approximately 60% of the hairs originate from the layer immediately underneath the epidermis (subepidermal layer) but not every cell in this layer produces a hair. By 10 days, almost all cells in the subepidermal layer have developed hairs.

To determine if subepidermal cells display other epidermis-specific traits, we examined the expression of the GFP-enhancer trap line J0481 in the root hair region of scz mutant roots. This enhancer trap is expressed in all the epidermal cells of the elongation and differentiation zones in wild-type roots (Fig. 2D). In contrast the enhancer trap line is expressed in the epidermis and the underlying layers of 3- to 7-day-old scz roots (Fig. 2C) and every subepidermal root hair-forming cell expresses the marker. The GL2::GUS promoter fusion is preferentially expressed in epidermal atrichoblasts (cells that will develop into non hair-bearing epidermal cells) in wild type (Masucci et al., 1996). GL2::GUS is expressed in some subepidermal cells in the scz mutant (data not shown), further supporting the notion that the subepidermal layer exhibits epidermal traits. We examined the expression of a ground tissue-specific marker (J0571) and two endodermis-specific markers (J3611 and J2672) in the scz mutants. These enhancer traps are not expressed in the hair-forming subepidermal cells of scz mutants (data not shown), suggesting that the cells display epidermal but not ground tissue traits.

TRANSPARENT TESTA GLABRA (TTG), and GLABRA2 (GL2) repress hair cell identity in the epidermis (Galway et al., 1994; Masucci et al., 1996; Di Cristina et al., 1996). CAPRICE (CPC), in contrast, is a positive regulator of hair formation in the epidermis (Wada et al., 1997). To determine if TTG, GL2 and CPC are active in the scz subepidermal layer, double mutants with ttg, gl2 and cpc were generated. The phenotypes of scz, scz gl2, scz ttg and scz cpc mutants are shown in Fig. 3. scz gl2 and scz ttg double mutants (Fig. 3B,C upper panels, respectively) have more root hairs than scz single mutant (Fig. 3A upper panel). In both double mutants almost all epidermal cells form root hairs. In addition, most if not all cells situated in a subepidermal position form root hairs, as shown in 3-day-old scz gl2 and scz ttg roots (Fig. 3B,C lower panels, respectively), compared with scz mutants where fewer cells of the subepidermal layer form root hairs (Fig. 3A lower panel). scz cpc double mutants develop fewer root hairs than scz single mutants (compare Fig. 3D to A upper panel). Of the few root hairs that form in the scz cpc double mutant, some originate from subepidermal cells (Fig. 3D lower panel).

In conclusion, ttg and gl2 enhance root hair formation in the subepidermal layer of scz, while cpc decreases the number of hairs. This indicates that TTG, GL2 and CPC act in the subepidermal layer of scz in the same way as they act in the epidermis of the scz mutant and the wild type, supporting the view that the subepidermal layer of the scz mutant has epidermal characteristics.

Meristems of 3-day-old scz primary roots were analysed and compared to wild-type (Ler) meristems. The cellular organisation of the scz mutant meristem is defective – the planes of division are abnormal compared to wild type and the organisation of the cells next to the presumptive quiescent centre (initial cells) is altered. Consequently it is often difficult to precisely define the position of the epidermis/lateral root cap initials and daughter cells (asterisks, Fig. 4C). During the development of the ground tissue in wild type the cortex/endodermis initial divides anticlinally to generate a new initial and a daughter cell. This daughter in turn undergoes an asymmetric periclinal division to generate an outer cell, which gives rise to cortical cells, and an inner cell, which gives rise to the file of endodermal cells (arrowhead, Fig. 4B). In scz mutants a number of anticlinal divisions may take place before the periclinal division occurs (arrowhead, Fig. 4C). More than one periclinal division may occur in any lineage, here defined as the descendants of a single cortical endodermal initial, which results in an increase in the number of cell layers (arrows, Fig. 4C).

To determine if the mis-specification of the ground tissue (cortex/endodermis) occurs during early development, in the meristem, the expression of the GFP enhancer trap line N9173 was examined in scz mutant roots. In wild type, this enhancer trap is expressed after division of the daughter cells of the lateral root cap and the epidermal initial (arrow, Fig. 5A) and expression is maintained in all the cells of the epidermis and the lateral root cap (Fig. 5A). In scz roots, the enhancer trap is expressed in these cell types but is also expressed at low levels in cells of the extra layer that forms as a result of the supernumerary periclinal division in the ground tissue. The expression is weaker than in the epidermis/lateral root cap and it occurs in clusters of cells (arrowheads, Fig. 5B,C). This suggests that the subepidermal layers of the scz roots exhibit epidermal characteristics in the meristem as well as in the mature zone where hairs develop (as shown above). Furthermore the expression of the enhancer trap is displaced upwards (away from the root tip) in scz mutants compared to wild type (arrow, Fig. 5B). This suggests that SCZ may be required for position-dependent development along the apical basal axis of the root.

Since an epidermis-specific enhancer trap is mis-expressed in the meristem of the scz roots, we predicted that the expression of ground tissue-specific genes would be defective in the subepidermal layers of scz roots. In wild type, the J0571 enhancer trap is expressed in the quiescent centre, the cortex/endodermis initials, cortex/endodermis daughter cells and throughout the cortex and endodermis (Fig. 6A). The expression pattern of this enhancer trap is altered in the scz meristem. In regions of the mutant root where additional ground tissue layers develop the enhancer trap is expressed in the two layers of the ground tissue next to the vascular cylinder. Furthermore the enhancer trap is not expressed in the presumptive quiescent centre, or in the ground tissue initials. Along the apical basal axis the expression is first observed after the first periclinal divisions have taken place (asterisk, Fig. 6B), i.e. it is displaced upwards along the root as is the case for the epidermal enhancer trap (N9173). Expression in the meristem is discontinuous (arrow, Fig. 6B). Occasionally, there are clusters of a few cells in the extra ground tissue layer, below the epidermis, that express the enhancer trap (arrowhead, Fig. 6B). Hence, very few cells of the supernumerary ground tissue layers express the ground tissue specific marker. This is consistent with the majority of the cells in the subepidermal layer being epidermal in identity. The mis-specification of the subepidermal layer as epidermis is mostly evident in the elongation/differentiation zone.

SCARECROW (SCR) and SHORT ROOT (SHR) are required for the development of the ground tissue and scr and shr mutant roots develop a single layer of ground tissue instead of two (Benfey et al., 1993; Scheres et al., 1995). To determine if SCZ is involved in the same process as SHR and SCR, double mutant plants for scz and either scr or shr were constructed. Both scz scr and scz shr have fewer cell layers than the scz single mutant (Fig. 7A,B respectively), indicating that scr and shr suppress the extra periclinal divisions occurring in the scz ground tissue. Occasionally, periclinal divisions are observed in scz scr ground tissue (arrow, Fig. 7A). Such divisions lead to the formation of a partial double ground layer as has been already reported in the scr single mutant (Wysocka-Diller et al., 2000).

scz scr and scz shr double mutants have a single layered epidermis and ground tissue, and the ground tissue forms root hairs (Fig. 7C,D and E respectively) indicating that scr and shr do not suppress subepidermal hair cell development. This suggests that the supernumerary periclinal cell divisions are not required for the mis-specification of cells in the ground tissue.

In conclusion, both scr and shr suppress the extra periclinal divisions that take place in scz mutants indicating that these genes may act in the same pathway. Nevertheless root hairs are formed in the single cell layer ground tissue of the double mutants, indicating that the mis-specification of the ground tissue of scz mutants is not a consequence of the formation of extra ground tissue layers.

SCR is required for the execution of the periclinal cell division of the cortex/endodermis initial daughter cell, generating the cortex and endodermis cell files (Di Laurenzio et al., 1996). Since shr and scr suppress the development of additional cell layers in scz mutants, it is possible that SCZ regulates the transcription of SCR directly and that the extra periclinal divisions occurring in scz mutants are the result of the deregulation of SCR expression. To test this hypothesis mRNA was isolated from roots of 3-day-old scz and wild-type plants. The amount of SCR mRNA was determined by semi-quantitative RT-PCR. As it is shown in Fig. 8 (upper panel), there are the same amounts SCR mRNA in scz mutants and in wild type. As a control for equal amounts of cDNA used for amplification (Fig. 8 lower panel), we used the Arabidopsis elongation factor 1-alpha 4 (Liboz et al., 1989). Since scz mutation does not affect the steady state levels of SCR mRNA, it is unlikely that SCZ regulates SCR transcription.

To determine when SCZ is first active during the life of the plant, the organisation of cells in scz embryos was compared to wild type at different developmental stages. At the early heart stage scz and wild-type embryos were indistinguishable – cell layers and initials were laid down around the quiescent centre as in wild-type embryos (data not shown). Differences in cell division patterns between wild type and scz mutant were first seen during the transition from heart to torpedo stage when the promeristem is formed (Fig. 9A). The periclinal division of the presumptive cortex/endodermis initial daughter cell takes place earlier in the scz mutant than in wild type (arrow, Fig. 9A). This suggests that SCZ activity is required between the heart and the torpedo stages of embryogenesis.

At the walking stick stage in scz mutant embryos the pattern of cell divisions in the ground tissue is altered. A periclinal division occurs at one cell distance from the presumptive central cells (arrowhead, Fig. 9B) resulting in the formation of two files. A second periclinal division takes place in the cells of the inner file. These combined divisions result in the formation of three ground tissue cell layers in the scz embryo instead of the two that normally develop in wild type (cortex and endodermis). The ectopic periclinal divisions resulting in the formation of an extra cell layer were observed in all scz mutant embryos examined (n=8). These data indicate that the control of the position and number of periclinal divisions of the cortex/endodermis initial daughter cell is defective in scz mutants. This results in the formation of supernumerary cell layers in developing embryos.

We have shown that SCZ is required for the establishment of the radial organisation of tissues in the root. The development of subepidermal root hair cells in the scz mutant indicates that SCZ represses epidermal identity in the ground tissue of wild-type roots. Furthermore the development of additional layers in the ground tissue of the scz mutant root indicates that SCZ also functions to repress periclinal divisions in the ground tissue of wild-type roots. Taken together these data suggest that SCZ plays a pivotal role in the development and maintenance of ground tissue in the root.

Evidence presented here indicates that SCZ is required to repress epidermal identity in subepidermal layers of the root, i.e. to restrict epidermal fate to the outer layer of the mature root. The subepidermal layer in scz mutants is transformed into an epidermis as revealed by the formation of root hairs and the expression of molecular markers in these cells. We examined the behaviour of 3 independent GFP enhancer trap lines expressed in the epidermis in the scz background (J0481, J0672 and J2092). All lines showed expression in the subepidermal layer(s). In addition, the GL2 gene, which is normally only expressed in the atrichoblasts and non hair cells of the epidermis, is expressed in cells of the subepidermal layer of the scz mutant. Further support that the scz subepidermal layer displays characteristics of epidermis comes from double mutants in which scz is combined with other mutations affecting the development of the epidermis. The TTG, GL2 and CPC genes active in the specification of cell fate in the epidermis are also active in the subepidermal layer of scz mutant roots, supporting the view that the subepidermal layer in scz mutants is epidermal in character. While the subepidermal cells of scz roots are located where cortical cells are situated in a wild-type root, we found no evidence, using molecular markers, that these cells exhibit cortical identity (J0571). One possibility is that SCZ represses the expression of genes required for epidermal identity and promotes the expression of those required for cortical cell development in the ground tissue. In addition, some of the markers expressed in cortex or endodermis used for the analysis showed no expression in the mutant background, indicating that the genes are downstream of the SCZ pathway (J2931, Q2393, J3611 and J2672). Alternatively the lack of expression might be a result of ground tissue mis-specification.

The development of epidermal trichomes in subepidermal tissues has been described in leaves that constitutively express GLABRA1 (GL1) in a triptyphon (try) mutant background (Schnittger et al., 1998; Szymanski and Marks, 1998). Since GL1 is a positive regulator of trichome development, its ubiquitous expression might be expected to predispose cells to trichome development. When misexpressed in the absence of TRY, the negative regulator of trichome development, cells in subepidermal tissues then differentiate as trichomes. Given the dominant, gain-of-function effect of overexpressing GL1 this observation does not indicate that either GL1 or TRY are active in repressing or promoting trichome development in the subepidermal tissues of leaves during normal development. Rather it suggests that the fate of cells can be switched if the appropriate regulatory genes are expressed or repressed. On the contrary our observation that plants homozygous for scz presumptive loss-of-function mutations develop root hairs from the subepidermal layers suggests that SCZ is required to repress epidermal cell differentiation in internal cells.

Plants homozygous for the presumptive loss of function scz mutation develop supernumerary cell layers because of an increased number of periclinal divisions in cells of the ground tissue. Such deviations from the wild-type pattern occur during embryogenesis when the cellular organisation of the future root meristem is being laid down. Defects are also observed during the formation of lateral root meristems, indicating that SCZ is required for the establishment of radial pattern in roots throughout the life of the plant.

In wild type, the cortex/endodermis initial daughter cell undergoes a single periclinal division giving rise to cortical and endodermal daughter cells. Anticlinal divisions in each of these cells result in the formation of groups of cortical and endodermal cells (Fig. 10A). In scz, additional rounds of periclinal cell divisions occur, resulting in the formation of a multi-layered ground tissue. We propose that in scz a periclinal division results in the formation of two cells. The outer cell continues to divide anticlinally generating a new layer. The inner cell may then undergo an additional periclinal division resulting in the formation of two files of cells. This process can be repeated a number of times resulting in the formation of a multi-layered ground tissue (Fig. 10A).

In addition to the cell division defects of scz, the expression of the enhancer traps is also altered, indicating that SCZ may be involved in organising the tissues of the root. A number of enhancer traps, that are expressed in cells in the vicinity of the quiescent centre in wild type, are displaced upwards. This suggests that the scz mutant displays a defect along the apical-basal axis similar to that described for roots exposed to auxin transport inhibitors for extended periods of time (Sabatini et al., 1999). Furthermore, the defective organisation of cells in the root meristem in mutants with either defective auxin responses or auxin transport suggests that perception and transport of auxin are involved in patterning (Sabatini et al., 1999; Friml et al., 2002). The precise role of auxin in the cell division and cell specification defects of scz remains to be tested.

Periclinal division in the cortex/endodermis daughter cells does not occur in shr mutants, while ectopic expression of SHR results in extra periclinal divisions and the consequent development of supernumerary layers (Helariutta et al., 2000). Since the pattern of cell divisions in scz roots resembles those found in plants that overexpress SHR, it is likely that SCZ and SHR play opposite roles: SHR is a positive regulator of the asymmetric cell division and SCZ is a negative regulator. That these genes may act in the same pathway is supported by the observation that the scr and shr mutations suppress the Scz- phenotype of scz mutants.

SHR has been shown to act as a transcriptional activator of SCR. Our data suggest that it is unlikely that SCZ is a transcriptional repressor of SCR, indicating that SCZ represses SCR activity. A plausible explanation for SCZ action in the ground tissue is that it represses SCR cell division activity in the inner daughter cell after the periclinal division of the cortex/endodermis initial daughter cell. The existence of such a repressor is in accordance with the fact that expression of SCR in the endodermis daughter cell and in the endodermal cells does not result in continuous rounds of cell division (Fig. 10B). While the extra periclinal divisions of scz roots are suppressed by shr and scr, the defective development of hair cells from subepidermal layers is not suppressed in scz scr and scz shr double mutants. This indicates that specification of the ground tissue is independent of the initial cell division pattern.

The GAL4-GFP enhancer trap lines were generated by Jim Haseloff, University of Cambridge, and were provided by the Nottingham Arabidopsis Stock Centre. Financial support was received from the EU-funded FORMA network and the BBSRC (UK).