The Maize CRINKLY4 Receptor Kinase Controls a CellAutonomous Differentiation Response1
Philip W. Becraft*, Suk-Hoon Kang, and Sang-Gon Suh
Zoology and Genetics/Agronomy Departments, 2116 Molecular Biology Building, Iowa State University,
Ames, Iowa 50011 (P.W.B.); and Department of Horticulture, Yeungnam University, Daedong 214–1,
Kyongsan 712–749, Republic of Korea (S.-H.K., S.-G.S.)
The maize (Zea mays) CRINKLY4 (cr4) gene encodes a receptor-like kinase that controls a variety of cell differentiation
responses, particularly in the leaf epidermis and in the aleurone of the endosperm. In situ hybridization indicated that the
cr4 transcript is present throughout the shoot apical meristem and young leaf primordia. A genetic mosaic analysis was
conducted to test whether CR4 signal transduction directly regulated the cellular processes associated with differentiation
or whether differentiation was controlled through the production of a secondary signal. Genetic mosaics were created using
␥-rays to induce chromosome breakage in a cr4/Cr4⫹ heterozygote. The mutant cr4 allele was marked with the albino
mutation, Oy-700. Breakage and loss of the chromosome arm carrying the wild-type alleles created a sector of albino, cr4
mutant tissue in an otherwise normal leaf. Analysis of such sectors indicated that cr4 functions cell autonomously to regulate
cell morphogenesis, implying that CR4 signal transduction regulates cell differentiation through strictly intracellular
functions and not the production of secondary intercellular signals. However, several sectors altered cell patterning in
wild-type tissue adjacent to the sectors, suggesting that cr4 mutant cells are defective in the production of other lateral
signals.
Cell interactions are important in the development
of all multicellular organisms. Proper cell patterning
and coordinated cell differentiation necessitate communication among cells. Receptor protein kinases
mediate cellular responses to many extracellular
stimuli, including developmental signals and several
receptor-like kinases (RLKs) are known to be important for plant development (Becraft, 1998).
The maize (Zea mays) CRINKLY4 (CR4) RLK mediates cellular differentiation responses in tissues of
the shoot and endosperm (Becraft et al., 1996; Jin et
al., 2000). Kernels mutant for cr4 often display mosaic
aleurone, where portions of the endosperm fail to
differentiate aleurone. Cells on the surface of the
endosperm have attributes of starchy endosperm
cells indicating that cell fate was not properly specified in these cells. This suggests that CR4 may function in the perception of positional cues that specify
aleurone cell fate throughout endosperm development (Becraft and Asuncion-Crabb, 2000). In leaves
and other organs of the shoot, the most pronounced
mutant effects are on the epidermis (Becraft et al.,
1
This research was supported by the U.S. National Science
Foundation (grant no. IBN–9604426) and by the Basic Research
Program of the Korea Science and Engineering Foundation (grant
no. 2000 –1–22100 – 001–3). This is journal paper no. J–19277 of the
Iowa Agriculture and Home Economics Experiment Station
(Ames, IA; project no. 3379; supported by Hatch Act and State of
Iowa funds).
* Corresponding author; e-mail becraft@iastate.edu; fax
515–294 – 6755.
Article, publication date, and citation information can be found
at www.plantphysiol.org/cgi/doi/10.1104/pp.010299.
486
1996; Jin et al., 2000). Epidermal cells are abnormally
large, irregularly shaped, and cell pattern is disorganized. Abnormal proliferation is evident in localized
regions and sometimes leaves form graft-like fusions.
Defects in cell differentiation are also evident internally in strong mutant phenotypes. This array of
differentiation defects indicates that CR4 regulates a
diverse set of cellular functions during development,
analogous to the function of growth factor receptors
in animals. Growth factor receptors are receptor protein kinases. Binding of the growth factor to the
extracellular domain activates the cytoplasmic kinase
domain, triggering a phosphorylation cascade that
leads to a change in cell activity.
When studying the developmental function of a
gene, it is of interest to determine whether it functions cell autonomously. Nonautonomy would indicate either that the gene of interest encodes a product
that can be transmitted from cell to cell, or that it
regulates the production of such a product. Testing
whether a gene product functions cell autonomously
is accomplished by a genetic mosaic analysis: the
analysis of individuals composed of both mutant and
wild-type cells. If the mutant and wild-type cellular
phenotypes correspond strictly to the cellular genotypes, the gene acts cell autonomously. If genetically
wild-type cells are able to rescue the phenotype of
neighboring mutant cells (or vice versa, depending
on the nature of the gene product and the mutation),
the gene acts nonautonomously.
In the case of receptors, although the direct response to receptor activation is intracellular, the phenotypic consequences of growth factor reception are
Plant Physiology, October 2001, Vol. 127, pp. 486–496, www.plantphysiol.org © 2001 American Society of Plant Biologists
The CRINKLY4 Receptor Kinase Acts Cell Autonomously
often non-cell-autonomous. Such an example is the
specification of dorsal cell fate in the Drosophila melanogaster embryo. The oocyte is surrounded by a layer
of maternal cells called follicle cells. The D. melanogaster sp. epidermal growth factor receptor, a receptor Tyr kinase, is expressed in the follicle cells and is
activated in the dorsal region by a signal from the
oocyte (Schüpbach, 1987; Price et al., 1989; NeumanSilberberg and Schüpbach, 1993). The activated receptor promotes dorsal follicle cell fate, which entails
the repression of another signal back to the embryo.
If not repressed, this second signal specifies ventral
cell fate in the embryo (Stein et al., 1991; Stein and
Nüsslein-Volhard, 1992). Thus, specification of dorsal cell fate in the embryo requires the activation of a
receptor kinase located in the maternal follicle cells.
In der genetic mosaics, it is the genotype of the follicle
cells, not the oocyte or embryo, that determines
whether the dorsal-ventral axis of the embryo is
properly specified (Schüpbach, 1987).
To better understand the role of CR4 signaling in
coordinating cell differentiation in maize leaves, we
examined the cellular expression pattern of cr4 by in
situ hybridization and performed a genetic mosaic
analysis. The results indicate that CR4 functions cell
autonomously and is required in both the mesophyll
and epidermis. Wild-type tissues bordering mutant
sectors occasionally showed altered cell patterns,
suggesting that lateral signals were disrupted in mutant cells.
RESULTS
Figure 1. Expression pattern of the cr4 transcript in the vegetative
shoot apical meristem (SAM) and leaf primordia. A, In situ hybridization of a digoxigenin (DIG)-labeled cr4 probe to a longitudinal
section of wild-type W22 inbred. B, In situ hybridization of cr4 in a
transverse section of a wild-type W22 inbred. C, Magnified view of
the wild-type cr4 hybridization pattern shown in the boxed region of
B. D, cr4 expression in a transverse section of a cr4-651 mutant. E, In
situ hybridization of cr4 in a transverse section of a cr4-60 mutant.
Size bars: A and C, 200 ␮m; B, D, and E, 500 ␮m.
The cr4 Gene Is Expressed throughout Developing
Leaf Tissues
In situ hybridization was performed to examine the
cellular pattern of cr4 gene expression. Previous analysis demonstrated that cr4 expression was highest in
developing tissues of the shoot, decreasing with tissue maturation (Jin et al., 2000). As shown in Figure
1, cr4 is expressed throughout the vegetative shoot
apical meristem and young leaf primordia. The cr4
transcript is distributed evenly in cells of the apical
dome and the several youngest leaf primordia. As
cells begin to mature, as evidenced by increased vacuolation, the signal intensity diminishes. This is first
evident in the midrib region of the plastochron 3 leaf
and progresses toward the margins. In plastochron 5,
signal levels remain high in the leaf margins, vascular traces, and epidermises, whereas the signal is
weaker in the mesophyll. By plastochron 6, only the
epidermises show consistently strong signal with the
parenchyma cells of some vascular traces also retaining high levels. Expression in the bundle sheaths was
similar to the mesophyll. In the stem, strong signal
becomes localized to the vasculature and epidermis
Plant Physiol. Vol. 127, 2001
within four nodes of the apex, with weaker signal
throughout the ground tissues.
Two cr4 mutants were examined. The cr4-60 allele
contains a Mu transposon in the region coding for the
extracellular domain and shows no detectable transcript on RNA gel blots probed with 3⬘ regions of the
cr4 gene (data not shown). Consistent with this, no
detectable signal was produced from in situ hybridization to cr4-60 tissues (Fig. 1E), confirming that the
probe was specific to the cr4 transcript. The cr4-651
allele contains a point mutation producing a premature stop codon in subdomain IX of the protein kinase domain. This mutation is predicted to eliminate
kinase function and to delete the carboxy terminal
domain from the protein but shows near-normal
transcript levels on RNA gel blots (data not shown).
In this mutant, the tissue-specific diminution of cr4
transcript levels seen in wild type was less evident.
The transcript levels remained relatively high and
evenly distributed at least through plastochron 6,
diminishing with no preferential expression in the
epidermis thereafter (Fig. 1D).
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Becraft et al.
Generation of cr4 Mutant Sectors
Previous analyses indicated that cr4 mutants could
show defects in both the epidermis and the mesophyll, with the epidermis showing more pronounced
effects. (Becraft et al., 1996; Jin et al., 2000). These
studies showed that cr4 is required for the normal
development of both tissues but left open the possibility that the site of action for cr4 was in one tissue
and that differentiation of the other was controlled
by secondary signaling. A genetic mosaic analysis
was conducted to test this possibility. The strategy
for generating cr4 mutant sectors is depicted in Figure 2. Seedlings heterozygous for cr4 linked to the
chlorophyll deficient marker, oy, were treated with
␥-rays to induce chromosome breakage. Breakage
and loss of the chromosome 10S arm carrying the
wild-type alleles uncovers the recessive cr4 allele and
the oy marker, resulting in sectors of cr4 mutant
tissue, marked by chlorophyll deficiency, in an otherwise normal plant. Wild-type cells can be identified
with fluorescence microscopy by the red fluorescence
of chlorophyll, which is absent in albino mutant cells
Figure 2. Generation of cr4 genetic mosaics. A, Seedlings heterozygous for cr4 linked to the Oy-700 mutant allele were ␥-irradiated to
induce chromosome breaks. Breakage and loss of the chromosome
10S arm carrying the wild-type alleles uncovers the mutant cr4 allele
and the Oy-700 marker. Hemizygous Oy-700 cells are albino; thus,
clones of cells derived from such an event form a hemizygous cr4
mutant sector marked by the lack of chlorophyll. B, An example of a
marked mutant sector.
488
(Fig. 3). The epidermal genotype is inferred from the
guard cells, the only epidermal cells to contain
chloroplasts.
Experiments were conducted using two different
oy alleles, oy and Oy-700. Sectors that were hemizygous for the recessive oy mutation are yellow because
of a defect in chlorophyll biosynthesis; however, by
fluorescence microscopy, mutant cells were difficult
to discern because the chloroplasts showed only subtle changes in fluorescence color. Figure 3A shows an
example of a sector containing mutant mesophyll
and epidermis marked by the recessive oy allele. To
avoid ambiguity, these sectors were not considered
further and the experiment was repeated using the
dominant Oy-700 allele as a marker. In the heterozygous state, Oy-700 confers a yellow-green phenotype
but in the homozygous or hemizygous state, Oy-700
produces an albino phenotype that was readily distinguishable by fluorescence microscopy (Fig. 3,
B–F).
Mutant Sectors Display a Typical cr4 Phenotype
The phenotypes displayed by mutant sectors
showed all the attributes associated with cr4 mutants
(Becraft et al., 1996; Jin et al., 2000). Cells were abnormally expanded, irregularly shaped, and disorganized (Figs. 2–4). Crenulations of neighboring epidermal cells may not match up or interlock (Fig. 4E) and
the cell wall corrugations visible in Figure 4C probably correspond to the ridges noted in transmission
electron microscopy (Jin et al., 2000). Regions of overabundant cell proliferation were evident on some
sectors (Figs. 2B and 3E). The epidermis showed the
most striking effects of cr4 loss (Fig. 3, A,B, and E). As
shown in Table I, roughly one-half the sectors with a
mutant phenotype only showed discernable defects
in the epidermis. Of the sectors that included all cell
layers, none showed mesophyll defects that did not
also show epidermal defects.
The cr4 mutant epidermis also appears more prone
to over-proliferation than the mesophyll. The arrows
in Figure 3E denote cell walls that appear continuous
with the inner epidermal walls of the adjacent wildtype tissues. This suggests that the cells to the outside
of these walls are derived from the proliferation of
epidermal cells. The proliferation appeared much
more extensive in the epidermis than the mesophyll.
The phenotypic variability of cr4 mutants was evident in this study, both among and within sectors.
Some sectors showed phenotypes that would have
likely been lethal had the entire shoot been so effected, whereas almost one-third of the sectors that
carried the hemizygous Oy-700 marker showed no
discernible defects. Variability along the length of a
single sector was common and one sector that extended for three nodes showed a strong mutant phenotype in two leaves but appeared normal in the
third leaf. Figure 4, D through F, shows examples of
Plant Physiol. Vol. 127, 2001
The CRINKLY4 Receptor Kinase Acts Cell Autonomously
sectors that show phenotypic variation across the
width of the sector, with some regions showing a
strong phenotype and other areas with a weak phenotype. Each of these sectors was mutant throughout
the thickness of the leaf, so the strong and mild
regions did not correlate with different constellations
of cell layer genotypes.
Cr4 Functions Cell Autonomously in Both
Mesophyll and Epidermis
Figure 3. Fluorescence micrographs of mutant sectors. A, Sector
marked by the recessive oy allele, encompassing both the epidermis
and mesophyll. The epidermis showed more dramatic morphological
effects than the mesophyll. The oy marker was difficult to score
unambiguously by fluorescence microscopy. B, The region delimited
by the arrows had a mutant abaxial epidermis but normal mesophyll
and adaxial epidermis. The guard cell just inside the right arrow
lacked chlorophyll. The mutant phenotype was restricted to the
epidermis, showing that the Cr4⫹ allele is required autonomously in
the epidermis. The leaf in this region was distorted, highlighting the
importance of the epidermis in determining leaf morphology. The
mutant mesophyll to the left of the major vascular bundle was
distinctly thinner than the wild-type mesophyll to the right, showing
the requirement for Cr4⫹ in mesophyll development. C, Sector with
wild-type epidermis and mutant mesophyll. The mutant mesophyll
cells were smaller than normal, causing reduced leaf thickness in
the sector and showing that Cr4⫹ is required autonomously in the
mesophyll. The wild-type adaxial epidermis was indicated by the
red-fluorescing guard cell just behind the plane of focus (arrow).
Plant Physiol. Vol. 127, 2001
Mutant sectors displayed a cr4 mutant phenotype,
regardless of sector width, and the phenotype was
confined to mutant cells. Control sectors in plants
that were heterozygous for the Oy-700 marker but
homozygous wild type for Cr4 showed no morphological defects, indicating that the effects were not
caused by hemizygosity for chromosome 10S or Oy700. The results from sectors that did not include all
tissue layers of the leaf further indicated that cr4
function is required autonomously in both epidermal
and mesophyll tissues. Figure 3B shows an example
of a sector where mutant epidermis overlies wildtype mesophyll. The epidermal cells show mutant
morphology, indicating that the underlying mesophyll cells are not able to rescue their phenotype.
Although the mutant phenotypes of mesophyll
cells are not typically as dramatic as epidermal cells
(Fig. 3A), several examples clearly showed that cr4
function is also required in the mesophyll. Figure 3C
shows a sector that had wild-type epidermis on both
surfaces but the mesophyll was mutant throughout.
The sectored area shows a noticeably reduced leaf
thickness indicating that the mesophyll cell development was not completely normal in the absence of cr4
function, even though the overlying epidermis was
wild type. Figure 3D shows an example where the
mutant sector encompassed only the adaxial one-half
of the mesophyll, and the mutant cells were smaller
than normal despite their juxtaposition to normal
epidermal cells. Thus, wild-type epidermal cells are
not able to confer a normal phenotype on mutant
mesophyll cells.
Our results were also consistent with a cell autonomous function of cr4 within the epidermal layer or
the mesophyll, although this point cannot be stated
definitively. The sector borders in the epidermis had
sharp borders, with one cell appearing normal and
D, Sector that was mutant through just the adaxial portion of the
mesophyll. The mutant cells were smaller than normal, indicating
that wild-type mesophyll cells are not capable of rescuing the mutant
cells. Red fluorescence of guard cells in adjacent sections showed
both epidermises were wild type. E, Sector showing cell proliferation.
The brightly fluorescing cell walls designated by the arrows were
continuous with the inner epidermal walls in the normal part of the
leaf, suggesting that the proliferated cells were epidermally derived.
F, Control sector containing the Oy-700 marker but which had a
wild-type Cr4⫹ allele, demonstrating that hemizygosity for chromosome 10S or Oy-700 have no effect on cell morphology.
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Becraft et al.
Figure 4. Scanning electron microscope (SEM) images of sectors. A through C, Mutant sector showing a sharp delineation
between the normal and mutant phenotypes. The area in the white box is enlarged in B, whereas the area within the black
box is enlarged in C. The black arrowheads denote the first file of mutant cells with normal cells to the left and the mutant
sector to the right. The sector boundary in B is offset by several cell files. The mutant cells in B show several different
morphologies, including nearly spherical (e.g. to the right of the arrowhead) to irregularly shaped with scalloping caused by
noninterlocking crenulations (white arrow). Some cells in C show additional defects including cell wall corrugations and
several cells are collapsed. D, Sector where part of the sector showed a strong mutant phenotype, whereas some showed
a mild phenotype. The arrowheads identify the genotypic sector boundary, with mutant cells to the left and normal on the
right. The far left part of the sector showed a typical strong mutant phenotype, whereas the right-hand part of the sector
appears nearly normal. The mild versus strong phenotypes did not correlate with the mesophyll genotype because both areas
contained mutant underlying cells. E, The sector shows a gradation from severely mutant to wild type (on the right). The
exact sector boundary is not readily distinguishable. F, Another sector showing two distinct severities of the mutant
phenotype. There is an abrupt change from strong to mild at the cleft (white arrow) but a gradual change from mild to wild
type (somewhere between the black arrows). The severity of the epidermal phenotype did not correlate with the mesophyll
genotype. Size bars: A ⫽ 500 ␮m; B, E, and F ⫽ 100 ␮m; C ⫽ 50 ␮m; and D ⫽ 200 ␮m.
the adjacent cell showing a mutant phenotype (Fig.
4A-C). However, in the epidermis, only the stomatal
guard cells contain chlorophyll and can therefore be
scored for genotype. No sectors were observed with
a marked stomatal file directly adjacent to a wildtype stomatal file. Sectors were observed with a cell
file containing marked guard cells and showing the
cr4 mutant phenotype adjacent to a cell file with a
normal phenotype, giving the appearance of cell autonomy. However, it is formally possible that the cell
file with the normal phenotype could have been genetically mutant and been phenotypically rescued by
nonautonomous functions in neighboring wild-type
490
cells. Thus, although the sectors had the appearance
of cell autonomy within the epidermis, this cannot be
proved because of limitations in the marker system.
Within the mesophyll, mutant cells were readily
identified by the albino marker but the cr4 mutant
phenotype is difficult to recognize because the effect
on cell morphology is subtle. Nonetheless, the results
suggest that CR4 is likely to act cell autonomously
within the mesophyll as well. Twelve sectors were
observed that contained mutant cells through only
part of the thickness of the leaf and yet still showed
a mutant phenotype. Figure 3D shows a sector where
the adaxial side of the mesophyll was mutant and the
Plant Physiol. Vol. 127, 2001
The CRINKLY4 Receptor Kinase Acts Cell Autonomously
Table I. Summary of the sectors in the major genotypic and phenotypic classes
Genotype
Phenotype
Ep.a
Mes.b
cr4 ep.
cr4 mes.
cr4 ep. and mes.
Normal
cr4
cr4
⫹
n.a.
Controlsd
Oy/–
⫹
cr4
⫹
cr4
cr4/⫹c
43
15
0
0
0
0
21
12
39
1
0
0
38
–
20
7
0
0
0
0
0
0
12
4
Oy/–
Oy/–
a
Epidermis (genotype ascertained by guard cell chlorophyll fluob
c
rescence).
Mesophyll.
cr4/⫹ represents sectors where the
mesophyll was mutant partway through the thickness of the leaf and
d
wild type the rest of the way.
Sectors mutant only for the
Oy-700 marker but not cr4.
abaxial side was wild type. The mutant cells show a
clearly decreased cell size, indicating that a partially
wild-type mesophyll is not sufficient to confer a normal phenotype through the entire thickness of the
leaf. Thus, if there are any non-cell-autonomous effects of CR4 on mesophyll cell phenotype, they occur
over a very short range.
One impetus for this study was the possibility that
defects noted in mesophyll tissues of cr4 mutants
could have been secondary effects caused by physical
stresses imposed by the deformed epidermis (Becraft
et al., 1996). Although it is now clear that CR4 has a
direct function in mesophyll development, it is also
clear that a deformed epidermis resulting from a cr4
mutation can have indirect effects on the morphology
of mesophyll cells. Figure 3B shows an example
where one-half the sector is mutant in only the adaxial epidermis and yet the whole leaf is bent in that
region. In addition, as shown in Table I, one individual that was mutant only in the epidermis showed
cellular defects in the subtending wild-type epidermis. Therefore, in cr4 mutant plants, the cellular abnormalities observed in the mesophyll likely arise
from a combination of direct and indirect effects.
(Fig. 5A), whereas one had no effect. Therefore, either
our hypothesis that cr4 mutant cells do not acquire
epidermal identity is incorrect, or B is not a marker of
epidermal identity per se.
There was variability for the enhanced anthocyanin
accumulation within sectors. One sector extended for
six nodes and entered five leaf blades. The sector
showed elevated pigmentation throughout the entire
length in the culm and sheaths but only showed this
effect in one of the five leaf blades. Another showed
the effect in all but one of the five leaf blades it
entered, whereas a third had enhanced anthocyanin
in two of three leaf blades.
Mutant Sectors Disrupt Spatial Patterns of
Adjacent Cells
Two types of sectors were observed that suggested
that cr4 mutant cells were defective in communicating spatial cues with neighboring cells. The first type
of sector produced a high density of macrohair
trichomes along the sector border. Figure 6, A and B
shows an example of such a sector on the abaxial
surface of a leaf and Figure 6, C through E shows an
example on the adaxial surface. The abaxial surface
does not normally produce macrohairs (Becraft,
1999). The hairs appeared to be produced by wildtype cells adjacent to mutant cells along the sector
border. Three examples of hairy sector boundaries
were observed, two in the abaxial and one in the
adaxial leaf surface. The sectors included all cell layers in every case.
Two sectors were observed that showed ligule displacement from one side to the other (Fig. 5B). No
ligule or auricle structures differentiated within the
cr4 mutant sector. Plants with strong mutant phenotypes often show disrupted ligular regions with sporadic or discontinuous ligule formation (not shown).
Mutant Sectors Show Enhanced
Anthocyanin Pigmentation
We hypothesized that CR4 was involved in promoting epidermal cell fate. As such, we predicted
that cr4 mutant epidermal cells might show reduced
expression of an epidermal marker. Anthocyanins
accumulate preferentially in epidermal cells of maize
lines carrying dominant alleles of the B gene (Poethig
et al., 1986); thus, we expected that cr4 mutant sectors
would express less anthocyanin than wild-type tissues. To test this, three families were generated for
the mosaic analysis in a B, pl background. pl makes
anthocyanin accumulation sunlight dependent (Cone
et al., 1993). Five sectors were generated in these lines
and contrary to our prediction, four of the mutant
sectors showed elevated anthocyanin accumulation
Plant Physiol. Vol. 127, 2001
Figure 5. Leaves with mutant sectors showing unusual properties. A,
Sector showing elevated anthocyanin accumulation in the cr4 mutant cells. B, Sector showing a displaced ligule. The ligule and
auricles do not align normally across the sector; the ligule is displaced basally on the marginal side of the sector relative to the
midrib side.
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Becraft et al.
Figure 6. SEM images of sectors with hairy borders. A and B, cr4
mutant sector that had hairy borders on the abaxial side of the leaf.
The mutant cells within the sector show an extremely severe phenotype. The trichomes appear to originate from normal cells bordering
the mutant sector. The boxed area in A is magnified in B. C through
E, Hairy sector on the adaxial surface of the leaf. In this case,
trichomes appear to originate from both normal and mutant cells
along the sector boundary and within the sector. The boxed area in
C is enlarged in D. E, Area within the mutant sector showing clusters
of misshapen, fused, and incompletely formed trichomes. Size bars:
A and C, 500 ␮m; and B,D, and E, 100 ␮m.
One sector occurred in a tiller and extended for six
leaves. The sector was about 7 mm wide and located
adjacent to the midrib in the blade of the first leaf.
The lamina on the outside portion of the leaf was
deleted (not shown). This suggests that the sector
disrupted the ability of the leaf to coordinate the
development of tissues from one side of the sector to
the other. The sector was mutant in all cell layers and
showed a cr4 mutant phenotype in every leaf. The
other leaves in this sector were shaped normally and
this effect was not seen with any other sector. Thus,
although the cr4 mutant phenotype per se (i.e. abnormal cell morphology) appears cell autonomous, there
are non-cell-autonomous effects on the development
of neighboring wild-type cells.
DISCUSSION
The Cellular Expression Pattern of cr4
Expression of the cr4 gene was observed evenly
throughout the apical dome and early leaf primordia.
492
By plastochron 3, the hybridization signal in the mesophyll and bundle sheaths declined in the midrib
region and decreased progressively toward the margins as maturation, as evidenced by cell vacuolation,
proceeded. Signal remained prominent in the epidermis near the leaf base through plastochron 7. We
believe this pattern to be indicative of gene expression and not merely a consequence of vacuolation
because in the cr4-651 mutant, there is no such preferential signal either in the epidermis or in the vascular traces. This expression pattern is also consistent
with a role of CR4 in directing cell differentiation
with a preferential requirement in the epidermis (Becraft et al., 1996; Jin et al., 2000).
In the cr4-651 mutant, which contains a point mutation but produces a strong mutant phenotype, transcript levels remained nearly uniform in primordial
leaf tissues through plastochron 6. This suggests that
expression of the cr4 gene might be regulated by a
feedback mechanism. The wild-type expression pattern suggests that cr4 expression is regulated by, or at
least correlates with, the maturation state of the cell.
One possibility is that as cells differentiate and mature, under the direction of CR4 signaling, the level
of cr4 expression declines. In the mutant with defective CR4 signaling, differentiation may be impeded,
allowing prolonged expression of the cr4 transcript.
A delayed rate of maturation was suggested by a
developmental analysis of cr4 mutants (Jin et al.,
2000). Another possibility is that the expression of the
cr4 transcript could be directly regulated as a target
of the CR4 signal transduction system. These possibilities await further examination.
The Developmental Function of CR4
Analysis of cr4 mutant sectors showed that the
function of cr4 is required throughout the tissues of
the shoot, and that the phenotype appeared to be cell
autonomous. That is, wild-type cells could not rescue
the phenotype of neighboring mutant cells. Thus, the
differentiation response does not appear to be controlled by a secondary signal produced in response to
CR4 signal transduction in neighboring cells. The fact
that the mutant phenotype was confined to mutant
cells also indicates that CR4 signaling does not negatively regulate a non-cell-autonomous signal that
inhibits normal differentiation.
Although it is not surprising that a receptor would
function cell autonomously, there are known examples where one signaling system regulates another,
leading to a non-cell-autonomous phenotype. For example, activation of the D. melanogaster EGF receptor
(a Tyr receptor kinase), in dorsal follicle cells represses another signaling system that would otherwise specify ventral fate in the embryo (Nilson and
Schüpbach, 1999). Thus, loss of Der function in the
follicle cells leads to ventralization of the embryo.
The mutual regulation of the Wingless and HedgePlant Physiol. Vol. 127, 2001
The CRINKLY4 Receptor Kinase Acts Cell Autonomously
hog signaling systems in segment polarity formation
provides another example (DiNardo et al., 1994). In
plants, CLAVATA1 (CLV1) provides an example of
apparently non-cell-autonomous receptor kinase
function. CLV1 regulates the relative rates of cell
proliferation and differentiation throughout the
shoot apical meristem, including the surface tunica
layers, even though CLV1 RNA is only detected in
internal corpus regions of the meristem (Clark et al.,
1997; Fletcher et al., 1999; Brand et al., 2000). Thus, if
the in situ hybridization results accurately depict the
full extent of CLV1 expression, the CLV1 RLK regulates meristem proliferation non-cell autonomously.
A genetic mosaic analysis would be required to test
this definitively. In addition to regulating a second
signaling system, non-cell-autonomous action could
occur if the receptor protein were trafficked between
cells as has been demonstrated for several plant transcription factors, including KNOTTED (Lucas et al.,
1995).
Although CR4 appears to function cell autonomously in both epidermal and mesophyll development, this analysis demonstrated that a cr4 mutant
epidermis can have secondary effects on the morphology of mesophyll cells. Several sectors that contained a mutant epidermis but genetically wild-type
mesophyll showed deformations in mesophyll tissue.
These likely were caused by physical stresses imposed by the malformed epidermal cells. The mesophyll defects seen in the leaves of cr4 mutant plants
are therefore due to a combination of direct and
indirect effects. This also supports a proposed role
for the epidermis in controlling leaf morphogenesis
(Dale, 1988). Additional deformations in cr4 mutant
leaves probably result from stresses caused by leaf
adherence (Becraft et al., 1996), where growth cannot
be accommodated by leaves sliding past one another.
No leaf adherence was observed in this study, suggesting that mutant and normal epidermal cells are
not capable of forming these unions. Adherence appears to require contact between mutant epidermal
cells.
This analysis reaffirmed the preferential function
of cr4 in the epidermis. Of the 82 sectors that included both epidermis and mesophyll and that
showed a mutant phenotype, 43 showed phenotypic
defects only in the epidermis (Table I). Several sectors produced wart-like cell masses. In most cases,
the tissue in these masses was too disorganized to
determine the cellular origin but in several, the outgrowth could be ascribed to the overproliferation of
epidermal cells (Fig. 3E).
Sixty-five sectors were obtained that displayed the
Oy-700 marker but that did not show a cr4 mutant
phenotype. Several factors probably contributed to
this. The cr4 and oy loci are located 10 cM apart;
therefore, some of these sectors could have arisen in
recombinant individuals that no longer had the cr4
mutant allele linked to the Oy-700 marker. A more
Plant Physiol. Vol. 127, 2001
significant factor is likely to be the variability inherent in the phenotype of cr4 mutants (Jin et al., 2000).
Within a mutant leaf, it is not uncommon to find
areas that appear relatively normal. The cr4-R allele
used in this analysis is a moderate-strength allele,
caused by insertion of a Mu transposon, and is prone
to this type of variation. That we observed large
sectors showing no mutant phenotype and very narrow sectors that showed a strong phenotype argues
against nonautonomous rescue of mutant cells by
neighboring wild-type cells as the explanation for the
normal phenotyped sectors. Furthermore, one sector
that extended for three leaves showed a mutant phenotype in two leaves but looked normal in the third.
This particular sector showed ligule displacement in
one leaf.
cr4 Mutant Cells Are Defective in Lateral Signaling
Although the cr4 mutant phenotype per se appeared to be cell autonomous, several mutant sectors
caused perturbations in patterning outside the sectors. Two sectors caused ligule displacement. A similar phenomenon was seen with liguleless1 (lg1) mutant sectors and it was concluded that the lg1 gene
product was required to propagate an inductive signal that organizes the ligular region of maize leaves
(Becraft et al., 1990; Becraft and Freeling, 1991). Thus,
it appears that cr4 mutant sectors also occasionally
disrupt this signal. One possibility is that CR4 is
directly involved in propagating this signal. Another
possibility, which we prefer, is that ligule displacement could be an indirect effect of the cr4 mutation
disrupting the differentiation of the cellular machinery needed to propagate this signal. We favor this
possibility because of the widespread cellular defects
seen in cr4 mutants and because of the rarity of this
occurrence.
Several sectors were lined along the borders with
macrohair trichomes. Two of these sectors occurred
on the abaxial face of the leaf where macrohairs do
not normally form. The hairs appeared to originate
from the wild-type cells bordering the mutant sector.
Therefore, the mutant sectors either induced the
neighboring cells to form hairs, or failed to inhibit
macrohair formation. The phenotype is reminiscent
of the phenotype of D. melanogaster mosaic for components of the notch signaling pathway. Notch signaling is important for the lateral inhibition that establishes the spacing pattern of neural precursors
within the equipotent cells of the neural ectoderm.
Disruption of the Notch signaling pathway causes
the entire neurogenic ectoderm to adapt neural precursor cell fates at the expense of epidermal cells.
Mutant sectors of delta, a ligand for the Notch receptor, contain dense clusters of misshapen sensory bristles (Heitzler and Simpson, 1991). Unlike the situation observed here, the extra bristles were confined to
the mutant sector; normal cells rescued the mutant
493
Becraft et al.
cells along the sector boundary but mutant sectors
had no effect on neighboring wild-type cells. Lateral
inhibition is thought to be important for generating
the spacing pattern of Arabidopsis trichomes (Larkin
et al., 1996; Schnittger et al., 1999).
The maize leaf has at least two types of macrohairs.
On the adaxial surface, associated with bulliform
rows, are macrohairs that possess a multicellular
base. On the leaf margin, macrohairs have no multicellular base. The hairs lining the sectors did not have
a multicellular base. One intriguing idea is that the
cells bordering the sector cannot sense the abnormally differentiated cr4 mutant cells. They would
only detect cells on one side and thus perceive their
position as at the leaf margin. This is speculative
because the sector borders did not form fiber bundles
like those found at true margins. In any case, it is
clear that lateral signaling is important for specifying
epidermal cell patterns and that this signaling is disrupted in the cr4 mutant tissue. Disruption of this
signaling on one side of normal epidermal cells has
the potential to cause an abnormally high proportion
of epidermal cells to adapt a trichome cell fate. These
results suggest that the disrupted cell patterning seen
in mutant leaves might be an indirect consequence of
the cr4 mutation rather than a direct function of CR4
signal transduction (Becraft et al., 1996; Jin et al.,
2000).
Enhanced Anthocyanin Accumulation in cr4 Sectors
An interesting effect was observed when cr4 mutant sectors were generated in plants carrying a B
allele that confers anthocyanin accumulation to
leaves. The mutant sectors showed elevated levels of
anthocyanin (Fig. 5A). This was the opposite of the
expected result; we hypothesized that cr4 interferes
with epidermal differentiation and because anthocyanin accumulates preferentially in the epidermis of B
plants, we expected to see reduced pigmentation in
cr4 sectors. Either our hypothesis was incorrect or B
expression does not strictly mark epidermal cell
identity.
The enhanced anthocyanin accumulation could result from several factors. First, anthocyanins typically accumulate in the vacuole (Marrs et al., 1995).
Epidermal cells of cr4 mutants have altered vesicle
trafficking, leading to or resulting from disturbed
vacuole formation (Jin et al., 2000). It is not clear
whether vacuoles are missing or are present as many
small bodies instead of a single large central vacuole.
If vacuoles were completely absent, then the cytosolic
anthocyanin would be expected to be oxidized and
have a brownish color similar to bronze2 mutants
(Marrs et al., 1995). The light responsiveness of anthocyanin accumulation in a pl genetic background
provides another possible explanation. It has not
been reported what light wavelengths pl responds to
but because anthocyanins are often cited as being UV
494
protectants, it would make sense that they might
accumulate in response to UV light. The cuticle normally forms barrier to UV and the cuticle is partially
disrupted in cr4 mutant epidermal cells. Therefore,
cr4 mutant cells could receive greater UV exposure,
which might increase anthocyanin accumulation.
It was not possible to conduct a genetic mosaic
analysis of Cr4 function in the endosperm. There are
no suitable endosperm markers linked to the cr4
locus and the incomplete penetrance of cr4 in the
endosperm results in sporadic phenotypes (Becraft et
al., 1996; Jin et al., 2000). Therefore, genetic mosaic
experiments would be uninterpretable. Whether the
developmental function of CR4 is similar in endosperm and leaf cells will be determined when targets of CR4 signaling are determined in the two
tissues.
MATERIALS AND METHODS
In Situ Hybridization
Shoot apices and young leaf primordia were fixed in 2%
(v/v) formaldehyde, 5% (v/v) glacial acetic acid, and 45%
(v/v) ethanol under vacuum for 2 h, stored for 2 d at 4°C,
dehydrated with a graded ethanol/tertiary butyl alcohol
series, and embedded in Paraplast Plus (Fisher, Pittsburgh). Eight-micrometer sections were floated on poly-lLys-coated glass slides (Sigma, St. Louis) flooded with 1%
(v/v) formaldehyde in water, drained, and dried overnight
at 43°C. Sections were dewaxed in xylene, transferred to
100% (v/v) ethanol, and rehydrated in a graded ethanolwater series. Sections were then treated for 30 min at 37°C
with proteinase K (Sigma) at 100 ␮g mL⫺1 in 0.01 m Tris
and 0.005 m EDTA (pH 8.0)
RNA probes for in situ hybridization were labeled with
DIG-11-rUTP using a nucleic acid labeling kit (Boehringer,
Indianapolis). The plasmid cr4c-XE520, containing the
unique carboxy terminal domain and the 3⬘-untranslated
region of the cr4 cDNA inserted into pBluescript KS (Stratagene, La Jolla, CA), was linearized with XbaI or EcoRI and
1 ␮g used as a template to synthesize DIG-labeled RNA
using T3 (antisense probe) or T7 (sense probe) polymerase,
respectively. The RNA probes were subjected to mild alkali
hydrolysis by heating at 60°C for 40 min in 100 mm carbonate buffer, and 4% of each labeling reaction was used as
a probe in 40 ␮L of hybridization buffer (Ingham et al.,
1985).
Slides were hybridized with the probes overnight at
50°C. Slides were washed in 2⫻ SSC, 50% (v/v) formamide, 0.2⫻ SSC, 50% (v/v) formamide, and 2⫻ SSC at
60°C for 1 h each, followed by two rinses with 0.5 m NaCl,
10 mm Tris-HCl (pH 7.5), 1 mm EDTA and treated with 20
␮g mL⫺1 RNase A in this buffer at 37°C for 30 min. The
slides were then washed in 2⫻ SSC, 50% (v/v) formamide,
and 0.2⫻ SSC, 50% (v/v) formamide at room temperature
for 1 h each. Immunological detection was as described
(Jackson et al., 1994). The slides were rinsed in 130 mm
NaCl and 10 mm sodium phosphate (pH 7.0). Slides were
incubated with gentle agitation for 45 min in 0.5% (v/v)
Plant Physiol. Vol. 127, 2001
The CRINKLY4 Receptor Kinase Acts Cell Autonomously
blocking agent (Boehringer) in 100 mm Tris-HCl and 150
mm NaCl (pH 7.5), followed by 45 min in 0.5% (v/v)
bovine serum albumin, 0.3% (v/v) Triton X-100, 100 mm
Tris-HCl, and 150 mm NaCl, pH 7.5 (buffer A). This was
followed by 2 h incubation in dilute antibody-conjugate
(1:2,500) in buffer A and four washes of 20 min each in
buffer A without antibody. Slides were briefly washed in
100 mm Tris-HCl, 100 mm NaCl, and 50 mm MgCl2 (pH
9.5), and incubated for 1 to 2 d in 0.34 mg mL⫺1 nitroblue
tetrazolium salt and 0.175 mg mL⫺1 5-bromo-4-chloro-3indoyl phosphate toluidinium salt in 100 mm Tris-HCl, 100
mm NaCl, and 50 mm MgCl2 (pH 9.5). The color reaction
was stopped with 1 mm Tris and 1 mm EDTA (pH 8.0).
formed a single layer. The seed received a dose of 10 gray
over a period of 10 min. Approximately 3,000 test seeds
and 1,000 control seeds were irradiated.
Genetic Mosaic Induction
Scanning Electron Microscopy
Ionizing radiation-induced chromosome breakage was
used to uncover the recessive cr4-R mutant allele in heterozygous individuals. The cr4-R allele was marked with
either oy or Oy-700. The oy locus is located approximately
10 CM proximal to cr4 on chromosome 10S (Stinard, 1992).
The oil yellow (oy) gene is involved in chlorophyll biosynthesis (Mascia and Robertson, 1980; Polacco and Walden,
1987); oy1 is a recessive mutant allele that confers a yellow
phenotype when homozygous or hemizygous. Oy-700 is a
dominant mutant allele that produces a yellow phenotype
when heterozygous and an albino phenotype when homozygous or hemizygous. Philip Stinard (U.S. Department
of Agriculture-Agricultural Research Service Maize Genetics Cooperation Stock Center, Urbana, IL) and Tony Pryor
(Commonwealth Scientific and Industrial Research Organization, Canberra, Australia) provided genetic stocks carrying the oy and Oy-700 markers, respectively. Each mutant
oy allele was linked in coupling to cr4-R (Cr4⫹ Oy⫹/cr4-R
oy or Cr4⫹ Oy⫹/cr4-R Oy-700) and heterozygous seeds
were ␥-irradiated to induce chromosome breakage. Breakage and loss of the chromosome arm carrying the dominant
wild type Cr4⫹ allele generated a sector of hemizygous cr4
mutant cells marked with the chlorophyll deficiency conferred by oy or Oy-700, in otherwise normal plants. As a
control, Cr4⫹ Oy⫹/Cr4⫹ oy and Cr4⫹ Oy⫹/Cr4⫹ Oy-700
seeds were used to generate hemizygous oy or Oy-700
sectors that were not mutant for cr4. Two sets of irradiations were conducted in consecutive years. The 1st year,
seed was imbibed for 24 h at room temperature, placed in
plastic bags, and put in a box that was 26 cm square by 4
cm thick. The box was centered 46 cm from a 137Cs point
source and exposed to gamma rays for 15.5 h. The kernels
received a calculated mean dose of 14 gray with a range of
9.3 to 19 gray, depending on distance from the source and
shielding by other kernels. Approximately 4,500 test seeds
(3,500 marked with oy and 1,000 marked with Oy-700) and
1,500 control seeds were irradiated. The irradiated seeds
were immediately hand planted in the field.
The second year, only the Oy-700 marker was used.
Seeds were imbibed 40 h prior to irradiation and placed in
plastic bags. Seeds were irradiated at the University of
Iowa Radiation Laboratory (Iowa City) with a 137Cs rod
source. Bags were laid flat beneath the source so the seed
Tissue was fixed in 2% (v/v) formaldehyde, 5% (v/v)
glacial acetic acid, and 45% (v/v) ethanol, dehydrated
through an ethanol series, and critical point dried. Samples
were sputter coated with palladium in a Denton sputter
coater and examined with a JEOL 5800LV SEM operating at
10-kV accelerating voltage. Images were digitally recorded.
Plant Physiol. Vol. 127, 2001
Microscopy
Sectors were examined by observing fresh hand sections
with a BX-60 fluorescent microscope (Olympus, Melville,
NY) equipped with a narrow violet filter (excitation 400–
410 nm, dichroic mirror and barrier filter, 455 nm). In
situ-hybridized slides were observed and photographed
with bright-field and DIC optics. All microphotography
was performed with an Olympus PM-20 photography system using Ektachrome 160T film (Kodak, Rochester, NY).
ACKNOWLEDGMENTS
Thanks to Erin Irish (University of Iowa) and the University of Iowa Radiation Laboratory and to the Iowa State
University Environmental Health and Safety Department
(Ames) for assistance with seed irradiations. Philip Stinard
and Tony Pryor provided genetic stocks. Yvonne
Asuncion-Crabb (Iowa State University) provided valuable
technical assistance and the entire Becraft lab provided
helpful discussion and critical reading of the manuscript.
Received March 28, 2001; returned for revision May 25,
2001; accepted June 28, 2001.
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