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The Plant Cell, Vol. 12, 1307–1318, August 2000, www.plantcell.org © 2000 American Society of Plant Physiologists
Molecular Analysis of the SCARECROW Gene in Maize
Reveals a Common Basis for Radial Patterning in
Diverse Meristems
Jun Lim,a Yrjo Helariutta,a,b,1 Chelsea D. Specht,a Jee Jung,a Lynne Sims,c Wesley B. Bruce,c Scott Diehn,c
and Philip N. Benfey a,2
a Department
of Biology, New York University, New York, New York 10003
B. and Dorothy Cullman Program for Molecular Systematic Studies, New York Botanical Garden, Bronx, New York
10458-5126
c Pioneer Hi-Bred International, Inc., Johnston, Iowa 50131
b Lewis
Maize and Arabidopsis root apical meristems differ in several aspects of their radial organization and ontogeny. Despite the large evolutionary distance and differences in root radial patterning, analysis of the putative maize ortholog of
the Arabidopsis patterning gene SCARECROW (SCR) revealed expression localized to the endodermis, which is similar
to its expression in Arabidopsis. Expression in maize extends through the quiescent center, a population of mitotically
inactive cells formerly thought to be undifferentiated and to lack radial pattern information. Zea mays SCARECROW
(ZmSCR), the putative maize SCR ortholog, was used as a molecular marker to investigate radial patterning during regeneration of the root tip after either whole or partial excision. Analysis of the dynamic expression pattern of ZmSCR as
well as other markers indicates the involvement of positional information as a primary determinant in regeneration of
the root radial pattern.
INTRODUCTION
In contrast to animal development, plant development exhibits an open growth system in which ontogenetic processes (growth and organogenesis) continue throughout
the life of the organism. These processes are mediated by
a population of stem cells located at the shoot and root
apical meristems of the plant. Within the root apical meristem (RAM) of higher plants is a group of mitotically inactive cells termed the quiescent center (QC). The QC has
been described as consisting entirely of undifferentiated
cells (Barlow, 1976). Initial cells, the divisions of which give
rise to the different root cell types, are generally thought to
be located immediately adjacent to the QC (Clowes, 1954,
1956).
Root meristems have been classified as open or closed
based on the anatomy of the root tip. In open meristem
roots, there is no discrete boundary between the root
proper and the root cap, making it difficult to trace cell files
1 Current address: Plant Molecular Biology Laboratory, University of
Helsinki, P.O. Box 56, FIN-00014, Finland.
2 To whom correspondence should be addressed. E-mail philip.benfey
@nyu.edu; fax 212-995-4204.
to distinct initials. In closed meristem roots, convergence of
cell files at the root apex makes it straightforward to identify
putative initials in median longitudinal sections (Clowes,
1981).
Both Arabidopsis and maize have closed root meristems.
Common to the radial organization of root tissues in the two
species is a single epidermal layer as well as a single layer of
endodermis that surrounds the stele (pericycle and vascular
cylinder). There are, however, important differences in the
radial pattern and ontogeny between maize and Arabidopsis
roots. First, maize roots have a large number of ground tissue cell files compared with two ground tissue cell files (cortex and endodermis) in Arabidopsis. Second, in Arabidopsis,
cortex and endodermis share an initial, as do epidermis and
root cap, whereas in maize the epidermis has its own initials,
which are independent of those of the root cap (Esau, 1965).
Third, the QC in maize consists of 1000 to 1500 cells, compared with only four cells in Arabidopsis (Feldman, 1984;
Dolan et al., 1993). Finally, the ways in which secondary roots
are formed are strikingly different. The root system of maize,
typical of grasses, consists of distinct types of roots, which
are formed at different developmental stages and locations
during plant development. Primary roots as well as seminal
lateral roots are formed during embryogenesis, whereas
crown roots and brace/prop roots arise from stem tissue
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during postembryonic development (Feldman, 1994; Hetz
et al., 1996; Hochholdinger and Feix, 1998; Kiesselbach,
1999). In contrast, Arabidopsis secondary roots normally are
formed postembryonically from the primary root (Malamy
and Benfey, 1997).
To determine if the anatomical differences in radial patterning between maize and Arabidopsis reflect underlying differences in expression of genes responsible for radial
patterning, we isolated the putative ortholog of the Arabidopsis SCARECROW (SCR) gene. In Arabidopsis scr mutants, one of the two ground tissue layers is missing, and the
remaining layer has differentiated attributes of both cortex
and endodermis, suggesting that the primary role of SCR
is to regulate the asymmetric division of the cortex/endodermis initials (Di Laurenzio et al., 1996). The SCR gene encodes a transcription factor of the GRAS gene family (Pysh
et al., 1999), and its expression in Arabidopsis roots is localized to the cortex/endodermis initials. After division of the
daughter cells, expression is restricted to the endodermal
cell file, making it an informative marker for radial patterning
(Di Laurenzio et al., 1996).
Here, we report that expression of the putative maize
ortholog Zea mays SCARECROW (ZmSCR) is localized to the
endodermal cell layer, suggesting that SCR plays a similar
role in conserved pathways involved in regulation of the radial
patterning of the diverse RAMs in these two species. Expression extends through the QC, providing evidence for the existence of tissue-specific pattern information within the QC.
Microsurgical removal of the QC and the root cap in maize
initiates a regeneration process that ultimately leads to the
reformation of an apparently normal root tip (Feldman,
1976). An analysis of carrot somatic embryo regeneration
showed that the replacement of excised material proceeded
by sequentially replacing the missing tissues. The final result
was regeneration along the apical–basal axis with the same
order and polarity of tissues as that found in intact controls
(Schiavone and Racusen, 1991). Exclusively on the basis of
histological characteristics, Schiavone and Racusen concluded that the regeneration process proceeded “with no
appreciable discontinuity of structure, directly from cells at
or near the site of surgery” (1991). Similar conclusions had
been drawn from the anatomical analysis of regeneration in
the maize root tip (Feldman, 1976).
We sought to study the patterning process by disturbing
the normal radial pattern and observing molecular markers
during the regeneration process. Regeneration of the maize
root tip requires not only reformation of a QC but also restoration of a root cap, production of convergent cell files for
the root body, separation of the root cap and the root body,
and assignment of proper cell identities throughout the regenerated portion of the root. To address how radial patterning occurs during root tip regeneration, we examined
ZmSCR expression and other markers after removing a portion of either the apical–basal axis or the radial axis. In both
cases, our results indicate a dynamic process involving a cell
respecification that is dependent on positional information.
RESULTS
Isolation and Characterization of ZmSCR
In a search for homology to the deduced amino acid sequence of Arabidopsis SCR, we identified several expressed
sequence tags (ESTs) in maize for which the predicted gene
products were highly similar to SCR. On the basis of sequence comparison among the maize ESTs (data not
shown), we used a partial EST sequence (CNDAH53) with
the greatest identity (77%) to SCR as a probe to isolate a
full-length genomic clone of ZmSCR. As shown in Figure 1,
the ZmSCR gene encodes a putative protein of 668 amino
acids, which is ⵑ60% identical to SCR. Although the N-terminal regions of ZmSCR and SCR are divergent, ZmSCR retains the homopolymeric repeats of proline, serine,
threonine, alanine, and glutamine characteristic of SCR and
shares ⵑ34% identity with SCR (Figures 1A and 1B). The
similarity between the two coding regions extends throughout their entire length (Figure 1B). In addition, the positions
of the exon/intron boundaries are conserved in the two
genes (Figures 1A and 1B). Among the maize ESTs, the
highest identity is that of ZmSCR to SCR, compared with
the other known members of this family (Pysh et al., 1999).
This is consistent with the hypothesis that ZmSCR is the
ortholog of SCR.
Characterization of the Expression Pattern of ZmSCR in
Maize Roots
To determine whether differences in the radial organization
of root tissues in Arabidopsis and maize reflect differences
in expression of genes that regulate radial patterning, we
analyzed the RNA expression pattern of ZmSCR. In spite of
their evolutionary distance and the substantial differences in
root architecture between maize and Arabidopsis, their SCR
expression patterns were remarkably similar. In the maize
primary root, ZmSCR was found to be expressed in the single layer of the endodermis (Figures 2A to 2C).
In addition, ZmSCR expression was consistently observed to extend in a single file of cells through the QC (Figures 2A and 2B). Observations of serial sections confirmed
that this expression pattern was indeed found within the region defined as the QC (data not shown).
In maize, distinct root types are formed at specific times
and places during development. We characterized the temporal and spatial expression patterns of ZmSCR in lateral,
crown, and seminal lateral roots. Expression of ZmSCR in
the lateral root appeared similar to that observed in Arabidopsis (Malamy and Benfey, 1997). It was expressed in early
stages of lateral root primordium formation (Figure 2D), becoming restricted to the endodermal cell lineage at later
stages (Figure 2E). Crown roots, representing the predominant root type of the mature plant, are formed at consecu-
Radial Patterning in the Maize Root
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Figure 1. Sequence Comparison of the Predicted Gene Products of ZmSCR and SCR.
(A) Alignment of the deduced amino acid sequences of ZmSCR and SCR. Identical amino acid residues are indicated by asterisks, and gaps are
introduced by dashes. Motifs are designated as previously described (Pysh et al., 1999). The arrowhead indicates the position of an intron for
which the location is conserved between the two genes. GenBank accession numbers of the proteins are AF263457 (ZmSCR) and U62798
(SCR).
(B) Schematic structure of ZmSCR and SCR with functional motifs. Boxes represent coding and thin lines represent noncoding regions. The
names of each motif and the percentage of conserved amino acid residues are indicated.
tive early nodes of the stem, beginning with the coleoptilar
nodes (Feldman, 1994; Hetz et al., 1996; Hochholdinger and
Feix, 1998; Kiesselbach, 1999). ZmSCR expression was restricted to the endodermal cell lineage in both early and later
stages of crown root development (Figures 2F and 2G). As
in the primary root, expression of ZmSCR in the seminal lateral root was found only in the endodermal layer (Figure 2H).
These results suggest that ZmSCR is utilized in common developmental mechanisms that give rise to all root types in
maize.
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Figure 2. Expression of ZmSCR in the Maize Root System.
(A) to (C) Primary roots. ZmSCR mRNA was localized in the single endodermal layer (arrowheads), extending through the QC. RC, root cap.
(D) and (E) Early stages in lateral root primordium formation. Expression was found only in an internal layer and later became restricted to the
endodermal cell lineage.
(F) and (G) ZmSCR expression in the crown root primordium at the coleoptilar node before (F) and after (G) emergence.
(H) Expression in the seminal root emerged from the scutellar node.
(I) Cross-section of the primary root probed with a sense probe.
Bars in (A) to (I) ⫽ 50 ␮m.
Radial Patterning in the Maize Root
Analysis of Expression of ZmSCR during Root
Tip Regeneration
After excision of the maize QC and root cap, the damaged
root is capable of regenerating an intact root tip with a new
QC (Feldman, 1976). To begin to understand how radial patterning occurs during regeneration of the maize root tip, we
made two types of excisions and used ZmSCR expression
as a marker for radial patterning. The first excision involved
removal of the entire root tip, including the root cap and QC,
and addressed the question of regeneration of a missing
portion of the apical–basal axis. The second involved a longitudinal excision resulting in removal of approximately half
of the root cap and QC and addressed how regeneration occurs upon removal of a portion of the radial axis.
After excision of the root cap and QC, we analyzed the
expression pattern of ZmSCR by RNA in situ hybridization at
different times during the regeneration process. Analysis of
⬎50 regenerating roots at each sampling point indicated
that the timing and morphological aspects of the regeneration process were very similar to those previously reported
(Feldman, 1976). Controls for the effects of excision revealed that ZmSCR expression was found exclusively in the
endodermal tissue after removal of the root cap alone (Figure 3A) and appeared immediately after root cap and QC excision (Figure 3B).
From the reported analysis of carrot embryonic regeneration (Schiavone and Racusen, 1991), we expected new endodermal cells expressing ZmSCR to be formed directly
below the existing endodermal cell file. As shown in Figures
3C to 3E, the expression of ZmSCR 24 hr after root cap and
QC excision was not consistent with this hypothesis. At this
stage, replacement of excised tissues appeared to occur
through divisions of the exposed cells at the cut surface. In
addition to ZmSCR expression in the endodermal cell file of
the unexcised portion of the root, expression was observed
consistently in the newly formed cells at the base of the endodermal cell file. These ZmSCR-expressing cells were frequently located internally with respect to the endodermal
cell file, directly below the stele cell files (arrows in Figures
3C and 3D). Moreover, ZmSCR mRNA was also found in
isolated cells located between the endodermal cell files (arrowheads in Figures 3C to 3E). Examination of serial transverse and longitudinal sections indicated that these internal
cells were not directly adjacent to any other cells expressing
ZmSCR mRNA (data not shown). The lack of expression in
cells below this region indicates that ZmSCR is activated
only after the initial proliferation phase.
At 48 hr postexcision (PE), cell files appeared to descend
in nearly vertical lines from the excised surface, as shown in
Figures 3F to 3H. ZmSCR expression was found in a plate of
cells (seen as a band of expression in median longitudinal
sections) that linked the base of the endodermal cell files.
Observation of roots with different edge configurations indicated that the expression pattern of ZmSCR appeared to mirror the configuration of the edge of the root (Figures 3F to 3H).
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At 72 hr PE, the anatomical pattern appeared to be similar
to that of roots with an open meristem, in which there is no
distinct boundary between the root cap and the rest of the
root (Figures 3I and 3J). Although there was no obvious convergence of cell files at the root tip at this stage, the ZmSCR
expression pattern resembled that found in an unsevered
root (Figure 3I). This pattern was formed by expression in
progressively more interior cell files (arrow in Figure 3J). In
other words, the expression appeared to “jump” laterally to
adjacent cell files. At this time, convergence of the cell files
appeared to be partially restored in some roots, suggesting
that the transition is gradual (Figure 3K).
Between 72 and 96 hr PE, there was an anatomical shift
resembling the adoption of a closed meristem, in which cell
files became convergent at the tip (Figure 3L). By 96 hr, an
intact root tip had been regenerated. Expression of ZmSCR
was localized to a single file of cells extending through the
newly formed QC in a manner similar to that seen in the normal root (Figure 3M).
Analysis of ZmSCR Expression during Regeneration of
Excised Half Root Tips
Our analysis of ZmSCR expression during regeneration
along the apical–basal axis suggests that ZmSCR responds
to positional cues rather than lineage restrictions. Expression of ZmSCR in the central region of the regenerating root
tip at 24 hr PE raised the possibility that this marks the center of the newly forming root. Maintenance of radially symmetric ZmSCR expression in the endodermal cell files of the
unexcised portion of the root suggests that this expression
may provide important radial patterning information during
the regeneration process.
To test these hypotheses, we excised half of the QC and
root cap. After excision of half of a root tip, two roots with
reduced diameters were regenerated within 3 to 4 days (Figure 4).
At 24 hr PE, ZmSCR RNA was expressed in the center
and in the endodermal cell file in each side of the root in a
pattern similar to that seen in whole root tip regeneration at
the same time (Figure 4C). At 36 hr PE, the central domain of
ZmSCR expression disappeared, and both half roots began
to regenerate separately (Figures 4D and 4E). ZmSCR was
expressed in each half internal to the preexisting endodermal cell file (arrows in Figures 4D and 4E). We observed no
expression of ZmSCR in isolated cells in the region that
would become the center of the two “half” roots.
By 48 hr PE, ZmSCR expression from each of the two regenerating roots became contiguous, as shown in Figure
4F. At this stage, anatomical analysis indicated little increase in the diameter of each half root. In the unexcised
half root, ZmSCR expression was found on one side in the
existing endodermal cell file and on the other side in a mirror
image pattern formed by expression in cells that were previously part of the stele (Figure 4G).
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Figure 3. Localization of ZmSCR Transcript during Whole Root Tip Regeneration.
(A) After excision of the root cap alone.
(B) Immediately after root cap and QC excision. Removal of neither the root cap (A) nor the root cap and QC (B) appeared to alter the expression
pattern.
(C) to (E) 24 hr PE. Arrows indicate the ZmSCR-expressing cells internal to the endodermal cell file, and arrowheads indicate central cells expressing ZmSCR.
(F) to (H) 48 hr PE. The ZmSCR expression pattern appears to mimic the configuration of the cut edge of the root.
(I) to (K) 72 hr PE. Arrow in (J) indicates a shift of expression from the endodermis to the adjacent cell file. (K) A root with partial restoration of
the radial organization into converging cell files.
(L) 72 to 96 hr PE. A new boundary between the root proper and the root cap was formed (arrowhead).
(M) 96 hr PE. Anatomical and expression patterns were fully restored.
Bars in (A) to (M) ⫽ 50 ␮m.
Radial Patterning in the Maize Root
After 60 hr PE, the two roots were very similar in appearance. ZmSCR expression on the internal side of each root
had an even more pronounced jagged quality, caused by
“skipping cell files,” than was seen in regeneration of the
whole root tip (Figure 4H), indicating that ZmSCR expression is, at minimum, a marker that prepatterns the anatomical restoration of the root radial pattern. The expression
pattern formed a loop at the point at which the two roots
emerged (Figure 4I).
At later stages, twin primary roots became indistinguishable in shape, except that they were thinner than the unexcised primary root (data not shown).
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Thus, UDP-GDH can serve as a positional marker for external cell layers during root tip regeneration.
Unlike starch staining, expression of UDP-GDH was detectable in the outer layer of cells at the root tip as early as
24 hr PE (Figure 5H). At 48 hr PE, UDP-GDH expression was
found in a broader band in the edge of the root, where the
new root cap was formed (Figure 5I). Thus, the expression
pattern of UDP-GDH indicates that the properties of the
outer cell layers appear to differ from those of the rest of the
root tip, even at the earliest stages of regeneration.
DISCUSSION
Expression of Differentiation Markers during
Root Regeneration
An interesting observation from the analysis of carrot somatic embryo regeneration was that the initial phases of regeneration involved formation of a “regeneration dome,”
which did not have the histological attributes of an apical
meristem and for which the cellular identity was left unassigned (Schiavone and Racusen, 1991). Because no ZmSCR
expression was found in cells at the very tip of the regenerating root, we reasoned that these cells might form a regeneration dome.
To determine the differentiation status of cells at the tip of
the regenerating root, we applied histochemical staining for
starch granules in amyloplasts as a marker of differentiated
root cap cells. In an unsevered root, starch staining was observed in the root cap but was excluded from root cap initials (Figure 5A). Immediately after root tip excision (Figure
5B) and at 24 hr PE (Figure 5C), there was no detectable
starch staining.
We first detected starch staining 48 hr after removal of the
root tip (Figure 5D). At this stage, there was still no clear histological separation between the root cap and the rest of the
regenerating root. The starch-stained root cap became even
more evident at 72 hr PE, by which time the root had regained a relatively normal shape (Figure 5E). However, the
size of the newly regenerated root cap did not appear to
match that of the unexcised root, and there was still no anatomical separation between the root cap and the root body.
At 96 hr PE, the amputated root was completely regenerated, and starch staining was indistinguishable from that of
the undissected root (Figure 5F). Analysis of starch staining
indicated that cells at the tip of the regenerating root were
already differentiated as root cap cells by 48 hr PE.
To further examine the differentiation status of the cells at
the tip of the root during the earliest stages of root tip regeneration, we analyzed expression of the uridine diphosphate
glucose dehydrogenase (UDP-GDH) gene. Our expression
analysis of UDP-GDH in undissected roots revealed expression throughout the root tip, with the highest expression in
the epidermis of the meristematic zone and root cap cells
just internal to the mucilage-secreting cells (Figure 5G).
Tissue-Specific Expression of the Putative Maize SCR
Ortholog (ZmSCR)
Two criteria for claiming orthology between genes of different species are (1) sequence similarity when compared with
other gene family members and (2) similarity of expression
patterns. ZmSCR shares extensive sequence similarity with
Arabidopsis SCR at the deduced amino acid level. Comparison of the known maize ESTs with the other known members of the Arabidopsis GRAS gene family, which includes
SCR (Pysh et al., 1999), indicates that ZmSCR is most
closely related to SCR. Conservation of intron placement
provides further evidence for orthology.
The expression pattern of ZmSCR is remarkably similar to
that of Arabidopsis SCR, providing strong evidence that it is
the likely maize ortholog of SCR. The pattern also indicates
that despite differences in root radial organization, this aspect of radial patterning is highly conserved. The conserved
expression pattern, combined with the sequence similarity,
also raises the strong possibility of conserved function.
Further evidence that ZmSCR may function in a manner
similar to that of SCR is its expression in all root types of
maize. Isolation of mutants defective in unique maize root
types implies that some aspects of their development
are under different genetic controls (Hetz et al., 1996;
Hochholdinger and Feix, 1998). To determine whether radial
patterning is likely to be a conserved process in different
maize root types, we examined ZmSCR expression in lateral, crown, and seminal lateral roots. As seen in the primary
root and in secondary roots, ZmSCR was found exclusively
in the single endodermal layer, indicating that seemingly
very different roots share similar developmental pathways.
Tissue-Specific Expression of ZmSCR in the Maize QC
Theories of maize QC function have postulated that the QC
cells are generally undifferentiated and lack patterning information (Barlow, 1976; reviewed in Feldman, 1984). For this
reason, it is remarkable that ZmSCR expression extends in
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Figure 4. Analysis of ZmSCR Expression during Regeneration of Excised Half Root Tips.
(A) Just after excision.
(B) Three to 4 days after excision of a half root tip. Two “half roots” were regenerated separately.
(C) 24 hr PE. ZmSCR was expressed in the center (arrowhead) and in the endodermal cell lineage.
(D) and (E) 36 hr PE. The central expression domain of ZmSCR disappeared. Expression was found near the tip of each root (arrows).
(F) and (G) 48 hr PE. (F) ZmSCR expression from the two roots became contiguous. (G) The longer half root appeared to restore cell files in the
radial axis, and ZmSCR expression prepatterned the anatomical restoration of the root.
(H) and (I) 60 hr PE. The regenerated half roots looked similar but were thinner than the unexcised primary root. Arrowhead identifies the likely
point at which the two roots began to regenerate.
Bars in (C) to (I) ⫽ 100 ␮m.
Radial Patterning in the Maize Root
the single endodermal cell layer through the QC. In Arabidopsis, QC cells play a role in maintaining the adjacent initials in an undifferentiated state (van den Berg et al., 1997).
Moreover, genes have been shown to express in Arabidopsis QC cells, including SCR (Wysocka-Diller et al., 2000).
However, there are distinct differences between the QCs of
the two species. In Arabidopsis, the QC consists of only four
cells, and thus the possibility of internal radial patterning
does not exist. In maize, ⵑ1000 cells make up the QC, and
within the region of the QC exist distinct cell files, indicative
of radial patterning. Thus, the extent of shared functions between these two quiescent cell populations remains to be
determined.
The location and function of initials in the root meristem
have been the subject of debate for ⬎100 years. The expression of ZmSCR in a single file of the endodermal cells
within the QC raises the possibility of an ontogenetic relationship between the ZmSCR-expressing cells within the QC
and those outside of it. This relationship would provide support for a variation on the founder cell theory (Barlow, 1976),
in which the cell files within the QC are the source of potential replacements for the initials directly above them. To test
this hypothesis would require a means of following the fate
of individual cells within the QC.
Radial Patterning during Regeneration
Regeneration in animals has been classified as either epimorphic or morphallactic (Wolpert, 1971). Epimorphosis involves production of new cells and subsequent intercalation
of positional values, whereas morphallaxis involves respecification of cell fates without the production of new cells.
Analysis of the ZmSCR expression pattern after excision of
either the entire root tip or only half of the root tip revealed
that both types of regeneration appear to take place.
After excision of the entire root tip, epimorphic regeneration occurs, with cell divisions causing the outgrowth of a
new tip region. Within this region, cells take on identities dependent on their position. This is consistent with a large
body of data indicating that during plant development, cellular identities are primarily dependent on positional information (van den Berg et al., 1995, 1997). The ZmSCR expression
pattern reveals that the process of filling in the tip region is
more complex than adding cells of like identity to preexisting cell files, as was previously suggested (Schiavone and
Racusen, 1991). The dynamically changing expression pattern of ZmSCR suggests that the regeneration process involves early establishment of the root radial organization by
apparently position-dependent mechanisms.
Longitudinal incisions that split the root meristem of
Ginkgo biloba embryos result in the production of twin primary roots with the normal RAM organization and approximately half the size of the normal root (Ball, 1956). Excision
of half of the maize root tip (root cap and QC) resulted in a
similar regeneration pattern. Anatomical analysis indicated
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that regeneration in the radial axis was primarily (but not exclusively) through morphallaxis. Rather than filling in the
missing half of the root by cell divisions, the existing cells
appeared to be respecified.
Expression of ZmSCR in either “half root” proceeded in a
manner similar to that seen when the entire root tip was excised, with two interesting exceptions: there was no marking
by expression in the central region of the new roots; and
ZmSCR expression in the internal part of the root occurred
in cells that were either formerly part of the stele (for the
longer root) or derived from stele cell files (for the shorter
root), thus producing an apparent mirror image of the endodermal cell file found on the outer edge of both twin roots.
Wolpert has hypothesized that in morphallactic regeneration, a boundary is reset, and then a sharper morphogen
gradient is reinterpreted by the remaining cells (Wolpert,
1971). The response to excision of a half root indicates that
if a boundary is being reset, it does not require symmetric
cell files.
The dynamic expression pattern of ZmSCR suggests that
it is more than a marker for presumptive endodermis. Particularly intriguing is the “skipping of cell register” expression
pattern during the later stages of root regeneration, which
results in a “prepattern” of convergent ZmSCR expression
before the convergence of cell files. Cell divisions must be
highly regulated to coordinate the dynamic reorganization
that appears to occur during root tip regeneration. Based on
SCR function in Arabidopsis, we speculate that asymmetric
cell divisions mediated by ZmSCR during this stage of the
regeneration process play an important role in the formation
of the new radial pattern.
Cell Fate and Differentiation during Root
Tip Regeneration
Analysis of regeneration of carrot somatic embryos revealed
the presence of a regeneration dome at the leading edge of
regeneration (Schiavone and Racusen, 1991). During maize
root tip regeneration, we observed cells at the leading edge
that did not express ZmSCR and that appeared to be morphologically less differentiated than the cells above them.
We used histochemical and molecular markers to determine
the differentiation state of these cells. Staining for the presence of starch in amyloplasts was used to identify differentiated root cap cells. Staining was first observed at 48 hr PE,
indicating that cells in the newly formed root tip had differentiated properties of root cap even in the absence of a
boundary between the root cap and the root body.
The lack of starch staining at 24 hr PE left open the possibility of an early stage in which these cells could have undergone dedifferentiation and thus had a meristematic quality.
To test for this, we used the molecular marker UDP-GDH,
which was normally expressed strongly in the epidermis and
outer layers of the root cap. At 24 hr PE, UDP-GDH was
clearly expressed in cells at the edge of the regenerating
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Figure 5. Analysis of Cellular and Molecular Markers during Root Tip Regeneration.
Starch staining is shown in (A) to (F); expression of UDP-GDH is shown in (G) to (I).
(A) Before amputation, starch granules were detected in root cap cells but not in root cap initials (arrow).
(B) Immediately after excision.
(C) 24 hr PE. No starch staining was apparent.
(D) 48 hr PE. Starch staining reappeared.
(E) 72 hr PE.
(F) 96 hr PE. The regenerated root tip was indistinguishable from an undissected root.
(G) Unamputated root probed with the UDP-GDH antisense probe. UDP-GDH was expressed strongly in the epidermis of the meristematic region and in root cap cells just internal to the external mucilage-secreting layer.
(H) 24 hr PE. Expression of UDP-GDH was found in a small band of cells in the tip of the regenerating root.
(I) 48 hr PE. A somewhat broader band of UDP-GDH–expressing cells appeared in the place of the future root cap.
Bars in (A) to (I) ⫽ 50 ␮m.
root tip, indicating that these cells sense the continued presence of positional cues. The combined results from these
two markers make it seem unlikely that these cells perform a
specialized “meristematic function.” Rather, it seems more
likely that the entire regenerating root tip undergoes dynamic respecification and highly ordered cell divisions, resulting in the formation of an organized root tip.
In conclusion, our studies indicate a common molecular
basis for radial patterning of roots from the distantly related
species maize and Arabidopsis. In addition, the QC is patterned at least to some degree, as evidenced by the expression of ZmSCR in a subset of the QC cells. The expression
pattern of ZmSCR during regeneration of the RAM indicates
that positional values are generated in the newly formed tissue independently of cell–cell contacts. Two other markers,
UDP-GDH and starch, are expressed in accordance with
their positions in the newly formed RAM and therefore support this conclusion. Finally, the regeneration of a mirror image of a halved meristem demonstrates that not only can a
radial pattern be regenerated in unpatterned tissue but that
previously patterned tissues can be respecified in accordance with new positional cues probably set by new boundaries. The dynamic and highly specific expression pattern of
the putative maize SCR ortholog suggests that this gene
plays an important role in establishing and maintaining the
correct radial pattern in the RAM.
Radial Patterning in the Maize Root
METHODS
1317
germination. The collected samples were fixed and used for in situ
analysis as described above.
Isolation of ZmSCR
Starch Staining
Using the deduced amino acid sequence of the Arabidopsis thaliana
SCR gene, we identified several maize expressed sequence tags
(ESTs) in a maize EST database whose predicted gene products appeared to be highly homologous to SCR. Among the maize ESTs, we
used a 1.3-kb partial cDNA (CNDAH53) with the greatest identity
(77%) to SCR as a probe to screen a B73 inbred maize genomic library. The three clones were isolated, and on the basis of the results
of restriction enzyme analysis, they were overlapping portions of the
same genomic region. Subsequently, a 5-kb SalI fragment from one
of the three clones was subcloned and sequenced. Initial alignment
of the putative gene products of ZmSCR and SCR was performed
using GeneWorks 2.3 (IntelliGenetics, Oxford, UK), and databases
were searched with the BLAST program (Altschul et al., 1997); the results were modified by manual alignment. The appropriate motifs
were designated as previously described (Pysh et al., 1999).
Microsurgical Excision of the Root Tip and in Situ Hybridization
Seeds of maize (Zea mays L. cv Merit; Asgrow Seed Co., Kalamazoo,
MI) were surface-sterilized, imbibed, and germinated in the dark as
described previously (Feldman, 1976). After 48 hr, seedlings with primary roots 2 to 3 cm long were selected and used in all quiescent
center (QC) excision experiments. Either whole root tips or half tips
including the root cap and QC were removed from the primary root
under a dissecting microscope, using a microscalpel. Severed roots
were then returned to the dark. At different times during the regeneration process, several root tips were removed and fixed in 4%
paraformaldehyde in PBS overnight at 4⬚C. Subsequently, the fixed
roots were dehydrated, embedded, and sectioned as previously described (Jackson, 1991; Di Laurenzio et al., 1996). Antisense and
sense riboprobes for ZmSCR and UDP-GDH (gift of G. Lu) were labeled with digoxigenin-11-UTP, using T3 or T7 RNA polymerase
(Boehringer Mannheim) according to the manufacturer’s instructions.
In particular, templates for riboprobes of ZmSCR were derived from
a polymerase chain reaction–amplified 230-bp fragment from the 3⬘
untranslated region (UTR) or a 354-bp PstI fragment corresponding
to the VHIID motif. Even though the 354-bp PstI fragment included a
very conserved region, its expression was identical to that of the
gene-specific 230-bp probe derived from the 3⬘ UTR. Sections were
pretreated, hybridized, washed, and mounted as previously described (Di Laurenzio et al., 1996), with minor modifications. Proteinase K was used at 10 ␮g/mL for 30 min in the section pretreatment.
After the immunological detection reaction, sections were mounted
either with or without dehydration with a graded ethanol series, resulting in purple or dark-brown precipitates, respectively. Photographs were taken with a Leitz MPS52 camera, and images were
processed using Adobe Photoshop 5.0 (Adobe Systems) to create
composite figures.
Tissue Preparation of the Distinct Root Types of Maize
Seminal lateral roots were sampled from 5-day-old seedlings, and
crown roots at the coleoptilar node were collected at 5 or 7 days after
germination. The various regions of the primary root along the axis
were sampled to collect lateral roots at various stages ⵑ6 days after
Root tips at each time point were sampled and fixed in 4% paraformaldehyde in PBS overnight at 4⬚C as described above. The fixed root
tips were stained with IKI solution (2% iodine, 5% potassium iodine,
and 20% chloral hydrate [all w/v]) for 1 min, cleared with chloral hydrate/glycerol/water solution (8:1:2 [w/v/v]) for 2 min, and mounted
on slides with the clearing solution as described previously (Fukaki et
al., 1998). Samples were observed using compound microscopy.
ACKNOWLEDGMENTS
We thank members of the Benfey laboratory for helpful suggestions
and discussions, Guihua Lu for the UDP-GDH clone, and Lewis
Feldman for helpful suggestions on the QC excision experiments. We
are also grateful to David Jackson, Jocelyn Malamy, Philip Newmark,
Alejandro Sanchez, and Ben Scheres for critical reading and comments on the manuscript. Y.H. was supported by a fellowship from
the Academy of Finland. This work was supported by grants to
P.N.B. from the National Institutes of Health (Grant No. RO1GM34778), Pioneer Hi-Bred International Inc., and the Human Frontier Science Program.
Received March 2, 2000; accepted May 19, 2000.
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