1 Department of Cell and Developmental Biology, John Innes Centre, Colney, Norwich NR4 7UH, UK
2 Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA
* Present address: The Sainsbury Laboratory, Norwich Research Park, Norwich NR4 7UH, UK
Author for correspondence (e-mail: Panagiota.Mylona{at}bbsrc.ac.uk)
Accepted 20 June 2002
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SUMMARY |
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Key words: Root, Radial pattern, Epidermis, Root hair, schizoriza, Arabidopsis thaliana
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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Mapping the schizoriza mutation
Col and Col-0 plants were used to pollinate homozygous scz mutant plants (ecotype Ler). An F2 population was generated. Individual F2 scz 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
).
Confocal microscopy
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.
Crosses with marker lines
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.
Number of plants used for the analysis
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.
Semi-quantitative RT-PCR analysis
Total RNA was isolated from Ler and scz 3-day-old roots using an RNeasyTM 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.
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RESULTS |
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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).
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Epidermal patterning genes act in the hair-forming subepidermal layer of scz mutants
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).
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SCZ is required during cell division in the meristem
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).
An epidermis-specific enhancer trap is expressed in the ground tissue of scz mutant meristem
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.
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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 expression in scz mutants
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.
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DISCUSSION |
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SCZ is required for suppression of epidermal fate in the ground tissue
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.
SCZ represses periclinal divisions in the ground tissue
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).
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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.
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ACKNOWLEDGMENTS |
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