Download BAM Receptors Regulate Stem Cell Specification and Organ

Copyright Ó 2008 by the Genetics Society of America
DOI: 10.1534/genetics.108.091108
BAM Receptors Regulate Stem Cell Specification and Organ Development
Through Complex Interactions With CLAVATA Signaling
Brody J. DeYoung1 and Steven E. Clark2
Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109-1048
Manuscript received May 6, 2008
Accepted for publication August 10, 2008
ABSTRACT
The CLAVATA1 (CLV1) receptor kinase regulates stem cell specification at shoot and flower meristems of
Arabidopsis. Most clv1 alleles are dominant negative, and clv1 null alleles are weak in phenotype, suggesting
additional receptors functioning in parallel. We have identified two such parallel receptors, BAM1 and
BAM2. We show that the weak nature of the phenotype of clv1 null alleles is dependent on BAM activity, with
bam clv mutants exhibiting severe defects in stem cell specification. Furthermore, BAM activity in the
meristem depends on CLV2, which is required in part for CLV1 function. In addition, clv1 mutants enhance
many of the Bam organ phenotypes, indicating that, contrary to current understanding, CLV1 function is
not specific to the meristem. CLV3 encodes a small, secreted peptide that acts as the ligand for CLV1.
Mutations in clv3 lead to increased stem cell accumulation. Surprisingly, bam1 and bam2 mutants suppress
the phenotype of clv3 mutants. We speculate that in addition to redundant function in the meristem center,
BAM1 and BAM2 act to sequester CLV3-like ligands in the meristem flanks.
T
HE vast majority of tissues and organs of the adult
plant are formed postembryonically. To achieve
continuous organ formation plants rely on the activity
of stem cells located at meristems (Byrne et al. 2003;
Carles and Fletcher 2003; Gross-Hardt and Laux
2003). The shoot apical meristem, which produces the
aerial portion of the plant, is composed of a central pool
of stem cells surrounded by descendant cells that are
directed toward differentiation. Division of stem cells
results in daughter cells that maintain the central stem
cell population as well as daughter cells that are localized
more peripherally, where they perceive positional cues
that drive their differentiation.
Stem cell specification and organogenesis at the Arabidopsis shoot apical meristem is regulated by a receptorkinase signaling system that includes the CLAVATA1
(CLV1), CLV2, and CLV3 gene products. CLV1 encodes
a receptor-kinase protein with 21 leucine-rich repeats
(LRRs) in its predicted extracellular domain, a single
pass transmembrane domain, and a cytoplasmic serine/
threonine kinase domain (Clark et al. 1997). CLV2
encodes a protein structurally similar to CLV1, however
CLV2 has a very small predicted cytoplasmic domain
with no known signaling motifs ( Jeong et al. 1999).
CLV1 accumulation is at least in part dependent on the
presence of CLV2 ( Jeong et al. 1999). Furthermore,
1
Present address: Department of Biology, Indiana University, Bloomington,
IN 47405-7107.
2
Corresponding author: Department of Molecular, Cellular and Developmental Biology, University of Michigan, 830 N. University, Ann
Arbor, MI 48109-1048. E-mail: [email protected]
Genetics 180: 895–904 (October 2008)
CLV1 and CLV2 are hypothesized to form a heterodimer
on the basis of migration in both gel chromatography
and nonreducing protein gel blots (Trotochaud et al.
1999).
Several lines of evidence suggest that CLV3 is the
ligand for CLV1. CLV3 is both secreted and appears to be
subjected to proteolytic maturation, releasing a conserved polypetide, termed the CLV3/ESR-related (CLE)
domain, from the C terminus that is capable of altering
plant development when added exogenously (Fiers
et al. 2005; Ito et al. 2006; Kondo et al. 2006; Ni and
Clark 2006). Null mutations in CLV3 are largely epistatic to mutations in CLV1 and CLV2, while CLV3
overexpression phenotypes are dependent on CLV1,
suggesting that CLV3 functions upstream of CLV1
(Clark et al. 1995; Brand et al. 2000). In addition,
CLV3 is required for the formation of a large molecular
mass complex containing CLV1 (Trotochaud et al.
1999). CLV1 acts to limit the diffusion of CLV3 protein,
suggesting that CLV1 binds to CLV3, sequestering it
(Lenhard and Laux 2003). Recent work has shown that
the CLE peptide is capable of binding to the CLV1
extracellular domain (Ogawa et al. 2008). Taken together these data suggest a model in which CLV1 and
CLV2 form a receptor complex that is capable of activating specific signal transduction pathways upon perception of the CLE peptide derived from CLV3.
CLV1, CLV2, and CLV3 function in a common genetic
pathway to regulate the expression domain of the stem
cell-promoting transcription factor, WUSCHEL (WUS)
(Mayer et al. 1998; Brand et al. 2000; Schoof et al.
2000). Mutations in CLV1, CLV2, or CLV3 result in an
896
B. J. DeYoung and S. E. Clark
increased WUS expression domain and a larger meristem with extra stem cells (Clark et al. 1993, 1995; Kayes
and Clark 1998; Brand et al. 2000; Schoof et al. 2000),
while mutations in WUS result in reduction or elimination of the meristem (Laux et al. 1996). The related
phosphatases POLTERGEIST (POL) and PLL1 are
signaling intermediates acting downstream of CLV1,
CLV2, and CLV3. POL/PLL1 act to maintain WUS
expression within the meristem, while CLV signaling
represses POL/PLL1 activity (Yu et al. 2000, 2003; Song
and Clark 2005; Song et al. 2006). The remaining
genetic and biochemical mechanisms that link CLV1
activation and WUS expression are largely unknown.
Several lines of evidence suggest the presence of
additional receptors that function in parallel with CLV1.
Most of the clv1 mutant alleles identified in mutagenic
screens are dominant-negative missense alleles that
exhibit intermediate-to-strong phenotypes, while clv1
null alleles exhibit quite weak phenotypes by comparison (Diévart et al. 2003). In addition, clv3 null alleles
exhibit strong meristem phenotypes and are epistatic to
both null and dominant-negative alleles of clv1, demonstrating that CLV3 retains some function in the
absence of CLV1 (Clark et al. 1995). Candidates for
CLV1-redundant receptors can be found among the
related BAM receptor kinases (BAM1, BAM2, and
BAM3) (DeYoung et al. 2006). CLV1 and the BAM genes
form a monophyletic group of Arabidopsis genes
encoding related receptor kinases, although the duplication giving rise to the BAM and CLV1 clades predates
the split between monocots and dicots (DeYoung et al.
2006).
Despite the related sequences of the these receptors,
several functional differences exist between CLV1 and
BAM receptors (DeYoung et al. 2006). While CLV1
promotes stem cell differentiation, BAM receptors are
required for stem cell maintenance. In bam1 bam2
double mutants, a reduction in meristem size is observed, while in bam1 bam2 bam3 triple mutants a
frequent incidence of premature shoot and flower
meristem termination is observed. A second key difference lies in the specificity of the receptors. clv1 mutants
only exhibit phenotypes at the shoot and flower meristem, while all three BAM receptors regulate numerous
aspects of Arabidopsis development, from leaf vascular
patterning to the proper development of all floral
organs, especially the anthers and ovules (Clark et al.
1993; DeYoung et al. 2006; Hord et al. 2006). Despite
these differences, CLV1 and BAM receptors appear to
have retained significant similarities in protein function,
because broadly expressed CLV1 can fully rescue bam
mutants while meristem-expressed BAM receptors can
partially rescue clv1 mutants (DeYoung et al. 2006).
Thus, on a genetic level, the differences between CLV1
and BAM function appear to be largely driven by
expression differences. Consistent with this, BAM1 and
BAM2 are expressed throughout the plant and exhibit
relatively high levels of expression on the meristem
flanks, while CLV1 is expressed primarily in the meristem center (Clark 1997; DeYoung et al. 2006).
To understand the relationships between CLV1,
CLV2, and BAM receptors, especially within the meristem where they play apparently complimentary roles,
we sought to assess the genetic interactions of BAM1 and
BAM2 with mutant alleles of CLV1, CLV2, and CLV3. We
show that bam and clv mutants display complex geneand allele-specific interactions. bam mutants suppress
clv3 phenotypes, have no effect on clv2 phenotypes, and
enhance clv1 null phenotypes. Our evidence suggests
that BAM proteins function similarly to CLV1 on a
biochemical level and, in fact, function in parallel with
CLV1 in the meristem center, but carry out separate
roles elsewhere within the plant. We propose that one
of these roles is to sequester CLE peptide ligands on
the flanks of the meristem, creating a buffer around the
meristem that prevents these ligands from upsetting the
delicate balance necessary for stem cell maintenance.
MATERIALS AND METHODS
Plant growth (Yu et al. 2003), SEM analysis (Diévart et al.
2003), and phloroglucinol staining (Prigge et al. 2005) were
performed as previously described unless otherwise noted.
Genetic analysis: clv1-1 bam1-1, clv1-4 bam1-1, clv1-7 bam1-1,
clv1-11 bam1-1, clv2-1 bam1-1, clv3-2 bam1-1, clv1-1 bam2-1, clv1-4
bam2-1, clv1-7 bam2-1, clv1-11 bam2-1, clv2-1 bam2-1, clv3-2 bam21, clv1-7 bam1-3, clv1-11 bam1-3, clv1-7 bam2-3, and clv1-11 bam23 double-mutant combinations were generated by crossing
plants homozygous for the bam1-1, bam2-1, bam1-3, or bam2-3
allele to plants homozygous for the respective clv1, clv2, or clv3
mutant allele. Individual F2 progeny were screened on the
basis of phenotype to identify individuals homozygous for the
respective clv1, clv2, or clv3 mutant alleles and, in the cases of
bam1-1 and bam1-3, to identify lines homozygous for the er-1
allele. No segregation distortion was observed that might
suggest clv homozygous mutants were not segregating in a 3:1
ratio. These plants were subsequently screened using a PCR
strategy to identify individuals homozygous for bam1-1, bam2-1,
bam1-3, or bam2-3, respectively, as previously described
(DeYoung et al. 2006). In some cases, progeny from F2 plants
heterozygous for bam1-1, bam2-1, bam1-3, or bam2-3 were
screened in the F3 generation to identify lines homozygous
for bam1-1, bam2-1, bam1-3, or bam2-3, respectively.
clv1-1 bam1-1 bam2-1, clv1-4 bam1-1 bam2-1, clv1-7 bam1-1
bam2-1, clv1-11 bam1-1 bam2-1, clv2-1 bam1-1 bzm2-1, and clv3-2
bam1-1 bam2-1 triple-mutant combinations were generated by
crossing clv bam1 double-mutant plants to the respective clv
bam2 mutant plants. For example, clv1-1 bam1-1 bam2-1 plants
were generated by crossing clv1-1 bam1-1 plants to clv1-1 bam2-1
and screening the F2 progeny of this cross to identify plants
homozygous for both bam1-1 and bam2-1. Because the bam1-1
bam2-1 allele combination causes infertility, the bam1 bam2
clv1-1 plants used for subsequent analysis were identified from
segregating progeny of sibling plants heterozygous at one
BAM locus and homozygous mutant at the other, as above.
Floral phenotype measurement: The mean number of
carpels per flower was determined by averaging the number
of carpels per flower from the first 10 flowers of each plant.
Image analysis: Photographic images were captured with a
Nikon Coolpix 995 digital camera. Brightness and contrast of
Analysis of CLV-Related Receptors
some photographic and SEM images were adjusted with
Adobe PHOTOSHOP CS.
Accession numbers: Sequence data from this article can be
found in the EMBL/GenBank data libraries under accession
nos. Q9SYQ8 (AT1G75820) for CLV1, AAF02655
(AT1G65380) for CLV2, AAD27620 (AT2G27250) for CLV3,
NM_125967 (AT5G65700) for BAM1, and NM_114827
(AT3G49670) for BAM2. All bam alleles used in this study
have been provided to the Arabidopsis Biological Resource
Center
(http://www.biosci.ohio-state.edu/pcmb/Facilities/
abrc/abrchome.htm).
RESULTS
clv3 phenotypes are suppressed by bam1 bam2
mutations: Our previous genetic studies have suggested
that BAM and CLV1 proteins have retained significant
biochemical redundancy, although the developmental
role of BAM1 and BAM2 is apparently antagonistic to
that of CLV1 in the meristem (DeYoung et al. 2006). We
therefore undertook experiments to characterize the
role of BAM receptors in the absence of specific CLV
pathway components in an attempt to clarify the role of
the BAM receptors within meristem development. In
particular, it is critical to investigate the relationship(s)
between BAM and CLV3, because the expression of
these genes in adjacent cell populations raises the
possibility that CLV3 acts as a ligand for the BAM
receptors on the flanks of the meristem.
To this end, bam1-1 and bam2-1 single and double
mutants were generated in the clv3-2 null mutant
background. To quantify the relative effect of these
mutations on the flower meristem, we took advantage of
previous observations that floral organ number, especially carpel number, reflects the relative size of the
flower meristem and is a sensitive method of detecting
change in flower meristem size (Clark et al. 1993; Song
and Clark 2005). When carpel number per flower was
assessed in bam1 clv3 and bam2 clv3 plants, we observed a
modest, but statistically significant reduction when
compared to clv3 single-mutant plants (Table 1). bam1
mutations lead to a larger absolute reduction in carpel
number in the clv3-2 mutant background than bam2.
bam1 bam2 clv3 triple mutants exhibited more obvious
suppression of the Clv phenotype, with 4.3 carpels
per flower compared to clv3-2 single mutants, which
have 6.7 carpels per flower (Table 1). The suppression
of clv3 by bam mutations is consistent with effects of
multiple bam mutants to reduce meristem size (DeYoung
et al. 2006).
clv2 phenotypes are unaltered by bam1 bam2: CLV2
function is necessary for normal CLV1 function ( Jeong
et al. 1999). Because CLV2 expression can be detected
broadly within the meristem, as well as outside of the
meristem, and because clv2 mutants exhibit several
phenotypes during organ development, we have hypothesized that CLV2 interacts with multiple receptor-like
kinases in addition to CLV1 (Kayes and Clark 1998;
897
TABLE 1
Phenotypic interactions of bam1 and bam2 mutations with
clv3 and clv2 mutations
Genotype
Lerd
bam2-1d
bam2-3d
bam1-1d
bam1-3d
bam1-1 bam2-1d
bam1-3 bam2-3d
clv3-2
clv3-2 bam2-1
clv3-2 bam1-1
clv3-2 bam1-1 bam2-1
clv2-1
clv2-1 bam2-1
clv2-1 bam1-1
clv2-1 bam1-1 bam2-1
Mean
carpels/flower
SEa
nb
2.00
2.00
2.00
2.00
2.00
2.00
2.00
6.66
5.96
5.82
4.32
3.98
3.65
3.34
3.89
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.081
0.057
0.061
0.066
0.083
0.044
0.057
0.065
100
100
100
100
100
100
100
100
300
300
140
100
200
200
100
Pc
,0.001
,0.001
,0.001
,0.001
,0.001
0.394
a
Standard error.
Number of flowers evaluated.
c
P-value calculated using Student’s t-test comparing clv
bam1, clv bam2, or clv bam1 bam2 to the respective clv single
mutant.
d
Data from DeYoung et al. (2006).
b
Jeong et al. 1999). Considering these observations, CLV2
could conceivably act as a partner for the BAM receptors.
To test this idea, bam1-1 and bam2-1 single and double
mutants were combined with the clv2-1 null mutation.
While bam1 clv2 and bam2 clv2 exhibited a modest but
statistically significant suppression in carpel number
compared to clv2 single mutants, the mean carpel
number of bam1 bam clv2 triple-mutant flowers was not
significantly different from that of clv2-1 alone (Table
1). Thus, clv2 is unaltered bam1 bam2 double mutants, in
direct contrast to clv3. This raises the possibility that
CLV2 may be necessary for both CLV1 and BAM
function, which would imply that the Clv2 phenotype
is the combined result of the loss of both the meristeminhibiting CLV1 pathway and the meristem-promoting
BAM pathway.
Synergistic meristem interactions of bam1, bam2, and
clv1 null alleles: To determine the net effect of removing
both BAM and CLV1 receptors from the meristem,
bam1-1 and bam2-1 mutations were combined with clv1-7
or clv1-11 mutations. clv1-11 is a null allele containing a
T-DNA insertion in the coding sequence for the LRR
domain, while clv1-7 contains a nonsense mutation that
results in truncation of a large portion of the kinase
domain and appears phenotypically similar to clv1 null
alleles (Diévart et al. 2003). Unexpectedly, bam1 and
bam2 alleles significantly enhanced the flower meristem
defects in both the clv1-11 and clv1-7 backgrounds
(Table 2). To test if these results could be attributed to
linked mutations in the bam1-1 or bam2-1 background,
898
B. J. DeYoung and S. E. Clark
TABLE 2
Phenotypic interactions of bam1 and bam2 mutations with null
and dominant-negative clv1 alleles
Genotype
clv1-7
clv1-7 bam2-1
clv1-7 bam2-3
clv1-7 bam1-1
clv1-7 bam1-3
clv1-7 bam1-1 bam2-1
clv1-11
clv1-11 bam2-1
clv1-11 bam2-3
clv1-11 bam1-1
clv1-11 bam1-3
clv1-11 bam1-1 bam2-1
clv1-1
clv1-1 bam2-1
clv1-1 bam1-1
clv1-1 bam1-1 bam2-1
clv1-4
clv1-4 bam2-1
clv1-4 bam1-1
clv1-4 bam1-1 bam2-1
Mean
carpels/flower
SEa
nb
3.09
3.80
3.35
4.62
4.34
7.44
3.60
4.40
4.26
5.18
5.44
8.65
4.32
4.29
4.37
7.35
5.45
6.67
6.41
8.97
0.047
0.037
0.054
0.069
0.080
0.116
0.042
0.074
0.045
0.072
0.087
0.198
0.051
0.056
0.050
0.185
0.077
0.057
0.076
0.200
300
200
200
300
230
100
200
100
200
200
179
40
100
200
200
60
130
300
200
30
Pc
,0.001
,0.001
,0.001
,0.001
,0.001
,0.001
,0.001
,0.001
,0.001
,0.001
0.693
0.483
,0.001
,0.001
,0.001
,0.001
a
Standard error.
Number of flowers evaluated.
c
P-value calculated using Student’s t-test comparing clv1
bam1, clv1 bam2, or clv1 bam1 bam2 to the respective clv1 single
mutant.
b
we repeated these experiments with the bam1-3 and
bam2-3 alleles. Again we observed a significant enhancement
of the meristem defect (Table 2). For all combinations,
bam1 alleles resulted in a stronger enhancement than
bam2 alleles. Interestingly, bam1 caused a stronger
suppression of clv3 than bam2 (Table 1).
To assess the effect of removing all three receptor
kinases on meristem development, bam1-1 bam2-1 clv111 and bam1-1 bam2-1 clv1-7 triple-mutant plants were
generated. In both cases the bam1 bam2 clv1 triple
mutants exhibited rather extreme phenotypes, with
the mean number of carpels per flower .8.6 for the
clv1-11 combination (Table 2).
To determine if these unexpected interactions were
specific to the flower meristem, we examined shoot
apical meristem size and organization in clv1, bam1 clv1,
bam2 clv1, and bam1 bam2 clv1 triple-mutant plants. In
each case, bam mutations enhanced the shoot meristem
defects of clv1-7 and clv1-11, in accordance with the
flower meristem enhancement. Namely, bam1 clv1 were
more enhanced compared to clv1 than bam2 clv1, and
the bam1 bam2 clv1 triple mutant exhibited extreme
shoot meristem defects (Figure 1). Indeed, the bam1
bam2 clv1 triple mutants exhibited novel phenotypes
that are discussed below.
These data are not consistent with a model in which
BAM genes simply function in a separate pathway
antagonistic to the CLV pathway. The observation that
BAM genes are required for the weak phenotype of clv1
null alleles suggests that the BAM and CLV loci interact
in a complex manner and that BAM and CLV1 genes
may act in a functionally redundant manner in the
center of the meristem.
Divergent interactions of clv1 dominant-negative
alleles: The apparent functional overlap of the BAM
and CLV1 receptors evident from the bam clv1 combinations could occur either within the same cells or
within separate cells. Overlap occurring within separate
cells would suggest that BAM acts in a complicated
fashion on the meristem periphery to regulate meristem
development. Overlap within the same cells would
suggest that although BAM expression is low at the
center of the meristem (DeYounget al. 2006), functional
BAM receptors are still present and acting redundantly
with CLV1 in these cells. To distinguish between these
possibilities, we combined bam1 and bam2 mutations
with the clv1 dominant-negative alleles clv1-1 and clv1-4.
These alleles contain missense mutations in the kinase
domain and LRR domain, respectively, and result in
phenotypes significantly more severe than clv1 null
alleles (Clark et al. 1997; Diévart et al. 2003). We have
hypothesized that clv1 dominant-negative alleles block
the action of receptor kinases with functional overlap.
If the overlapping function of BAM receptors with
CLV1 occurs in separate cells, we would expect similar
interactions of bam mutants with clv1 null and dominant-negative alleles. If the functional overlap occurs
within the same cells, clv1 dominant-negative alleles
might partially block BAM1 and/or BAM2 function.
When the bam clv1-1 double mutants were assayed, we
observed that neither the bam1 clv1-1 nor the bam2 clv1-1
double mutants exhibited a stronger phenotype than
clv1-1 alone (Table 2). This indicates that the genetic
interactions of bam1 and bam2 with clv1-1 are different
from those with the clv1 null alleles. When determining
the genetic interactions with clv1-4, we observed a
different result: both bam1 and bam2 were capable of
enhancing the clv1-4 allele. Thus, the nature of the
dominant-negative effect in the kinase domain missense
allele (clv1-1) may be different from the LRR domain
missense allele (clv1-4). The LRR domain dominantnegative alleles of clv1 are invariably more severe phenotypically than the kinase domain dominant-negative
alleles (Diévart et al. 2003), providing additional evidence of functional differences in dominant-negative
action between LRR and kinase mutants.
Neither clv1-1 nor clv1-4 exhibit the most severe clv
mutant phenotypes, and CLV3 still functions in these
mutant backgrounds (Clark et al. 1995). Consistent
with this, the bam1 bam2 clv1-1 and the bam1 bam2 clv1-4
triple-mutant plants were enhanced compared to the
corresponding clv1 single mutant, and displayed phenotypes similar to bam1 bam2 clv1 null triple mutants
(Table 2).
Analysis of CLV-Related Receptors
899
Figure 1.—bam1 and
bam2 enhance meristem enlargement by clv1 and function synergistically with clv1
resulting in novel phenotypes. Scanning electron micrographs of Ler (A), clv1-11
(B), clv1-11 bam2-3 (C), clv111 bam1-3 (D), clv1-7 (E),
clv1-7 bam2-3 (F), clv1-7
bam1-3 (G and H), clv1-11
bam1-1 bam2-1 (I and J),
clv1-7 bam1-1 bam2-1 (K–N).
Older flowers were removed
for clarity. H, L, and N correspond to the boxed regions
in G, K, and M, respectively.
Differentiated cells surrounded by undifferentiated meristem cells are
indicated with an arrow (H
and L). Lack of flower primordia is indicated by an
arrow (N). A, B, E, and
F are shown at the same
magnification. Bars: 50 mm
(A, B, E, F, H, and L); 100
mm (C, D, and N); 250 mm
( J); and 500 mm (G, I, K,
and M).
Novel phenotypes in bam1 bam2 clv triple mutants:
The shoot apical meristems of bam1 bam2 clv1 plants
exhibited unique phenotypes not seen in any clv single
mutant. These meristems occasionally lost organogenesis in portions of the meristem periphery (Figure 1, M
and N). Furthermore, bam1 bam2 clv1 shoot apical
meristems, and to a lesser degree, clv1 bam1 shoot apical
meristems, developed patches of differentiated cells (in
one case, complete leaves) across the normally continuous field of undifferentiated meristem cells (Figure 1,
G, H, and J–L). Finally, bam1-1 bam2-1 clv1-11 and bam1-1
bam2-1 clv1-7 flowers developed an enormous indeterminate meristem in the center of each flower (Figure
2Q).
In addition to the synergistic interactions observed in
the shoot meristem, bam1-1 bam2-1 clv1-11 and bam1-1
bam2-1 clv1-7 triple mutants displayed a number of novel
phenotypes outside of the meristem (Figure 2). bam1-1
bam2-1 clv1-11 and bam1-1 bam2-1 clv1-7 plants were
much smaller than wild-type and bam1-1 bam2-1 plants,
even when they were fully grown (Figure 2, E, F, and N;
data not shown). bam1-1 bam2-1 clv1-11 and bam1-1 bam21 clv1-7 plants had very small leaves and the number of
rosette leaves was increased. The stems of the primary
inflorescence of clv1-11 bam1-1 bam2-1 and clv1-7 bam1-1
bam2-1 plants were also extremely thick (Figure 2N; see
below). The sepals, petals, and stamens of bam1-1 bam2-1
clv1-11 and bam1-1 bam2-1 clv1-7 flowers were sometimes
filamentous and generally looked less developed than
those of bam1 bam2 flowers (Figure 2M; data not shown).
The fruits of bam1-1 bam2-1 clv1-11 and bam1-1 bam2-1
clv1-7 plants were very short, however the pedicels were
longer than those of wild type (WT) (Figure 2N).
The replacement of valve tissue by replum tissue
(valvelessness) has been previously observed in clv2
mutants (Kayes and Clark 1998). This clv2 phenotype
is enhanced by clv1 and clv3 mutants. Valvelessness was
also strongly enhanced in the bam1 bam2 clv1 triple
mutant, such that every bam1-1 bam2-1 clv1-11 and bam11 bam2-1 clv1-7 flower had missing or incomplete valves
(Table 3). In addition, large sections of the gynoecia of
these plants frequently lacked all valve character. We
also observed large valveless regions that developed very
thin stripes of valve-like tissue (Figure 2, I and J).
None of these synergistic shoot meristem or organ
phenotypes were seen in either bam1 bam2 clv2 plants
or bam1 bam2 clv3 plants (Table 3; Figure 1; data not
shown), indicating a specific role for CLV1 outside the
meristem. This represents a novel range of developmental pathways regulated by CLV1, and prompts
consideration of broad functions for the orthologous
rice FON1 and maize thick tassel dwarf1 (td1), both of
whose expression appears broader than CLV1 (Suzaki
et al. 2004; Bommert et al. 2005).
A defect in internal stem differentiation was observed
in bam1 bam2 combinations with all clv mutations. clv
mutations normally result in increased vascular bundle
number, and rarely vascular bundles located away from
900
B. J. DeYoung and S. E. Clark
Figure 2.—clv1 bam1 bam2 plants exhibit novel
phenotypes in floral meristem and nonmeristem
tissues. Rosettes of Ler (A), clv1-7 (B), clv1-11 (C),
bam1-1 bam2-1 (D), clv1-7 bam1-1 bam2-1 (E), and
clv1-11 bam1-1 bam2-1 plants 14 days after germination. Gynoecia of Ler (G), clv1-7 (H), and
clv1-7 bam1-1 bam2-1 (I and J) plants. Scanning
electron micrographs of clv1-7 (K), bam1-1
bam2-1 (L), and clv1-7 bam1-1 bam2-1 (M) young
flowers are shown. Some sepals were removed
from the clv1-7 (K) and bam1-1 bam2-1 (L) flowers
to reveal the inner organs. No organs were removed from the clv1-7 bam1-1 bam2-1 flower
(M). clv1-7 bam1-1 bam2-1 plant (right, boxed) beside a sibling plant (left) that is either heterozygous or homozygous wild type at both BAM1
and BAM2 loci (N). Close-up of clv1-7 bam1-1
bam2-1 plant (N, inset). Scanning electron micrographs of clv1-7 (O), bam1-1 bam1-2 (P), and clv111 bam1-1 bam1-2 gynoecia are shown. A–F are
shown at the same magnification as are G, H,
and I. J is a close-up view of a clv1-7 bam1-1
bam2-1 gynoecium. Bars, 100 mm (L and M);
250 mm (K); 500 mm (O–Q). Asterisks indicate attachment site of sepals, petals, and stamens. S,
stigmatic tissue; VL, valve-like strips; se, sepal primordium; pe, petal primordium; st, stamen primordium; and ca, carpel primordium.
the periphery of the stem, presumably a result of the
enlarged shoot meristem (L. Doil and S. E. Clark,
unpublished data; Figure 3). In various bam1 bam2 clv
mutant combinations, a more dramatic patterning deTABLE 3
bam1 and bam2 affect valvelessness
Genotype
Ler
clv1-7
clv1-7
clv1-7
clv1-7
clv2-1
clv2-1
clv2-1
clv2-1
bam2-1
bam1-1
bam1-1 bam2-1
bam2-1
bam1-1
bam1-1 bam2-1
% valvelessa
nb
0.0
46.3
22.5
95.0
100.0
86.0
45.5
78.5
80.0
100
300
200
300
100
100
200
200
100
a
Percentage of flowers with at least one missing or incomplete valve. Missing valves were defined as the replacement of
a complete valve in the gynoecium by replum tissue. Incomplete valves were defined as valves that occupied ,75% of the
distance from the receptacle to the stigma.
b
Number of flowers evaluated.
fect was observed. Single, large ectopic lignified cells, as
well as circular rings of smaller lignified cells were
observed in the center of the stem (Figure 3, D–F and
H–J). Furthermore, in stronger clv mutant combinations, large portions of the internal stem were composed
of poorly vacuolated cells with a mixture of lignified and
nonlignified cells (Figure 3, F and I). Stems of plants
with this phenotype would occasionally break open
during growth, revealing a fiber-like consistency to these
abnormally developing stem regions (Figure 3, K and
L). What role defects in meristem development and
what role defects in later stem differentiation play in this
phenotype are unclear.
DISCUSSION
A model for BAM function within the meristem: We
investigated the genetic interactions of bam null alleles
with various clv mutations. bam1 and bam2 exhibit dramatically different genetic interactions with clv1, clv2,
and clv3, resulting in enhancement, no effect, and suppression, respectively. This indicates that the effects of
BAM1 and BAM2 on stem cell homeostasis are integral
Analysis of CLV-Related Receptors
901
Figure 3.—Synergistic vascular patterning defects clv bam1 bam2 mutants. Cross sections of
wild-type Ler (A), bam1-1 bam2-1 (B), clv1-1 (C),
clv1-1 bam1-1 bam2-1 (D–F), clv3-2 (G), clv3-2
bam1-1 bam2-1 (H), clv2-1 (I), clv2-1 bam1-1
bam2-1 ( J) stems stained with phloroglucinol
(red) to detect lignified tissues. Scanning electron micrographs of clv1-11 bam1-1 bam2-1 mutants (K and L) show stems that split open
during growth to reveal a fiber-like inner structure. Portions of the stem were removed to better
visualize this structure. VB, vascular bundle, SM,
shoot meristem. Arrows indicate large lignified
cells; asterisks indicate rings of smaller lignified
cells.
to the CLV signaling pathway and do not represent a
separate pathway regulating meristem development.
bam1 bam2 mutations significantly suppressed the
stem cell accumulation in clv3-2 flowers, consistent with
a requirement for BAM receptors in stem cell maintenance. In contrast, clv2 was unaltered by bam1 bam2
within the meristem. Given previous data suggesting
that CLV2 is required in part for CLV1 protein accumulation, we propose that BAM1 and BAM2 function
within the meristem is dependent on CLV2. These
results suggest that the clv2 phenotype, which is the
strongest of any receptor null, would result from the
combined reduction of CLV1 and BAM function.
Unexpectedly, when combined with clv1 null alleles,
both bam1 and bam2 strongly enhanced the Clv
phenotype, and the triple mutant exhibited very severe
defects in stem cell homeostasis. Thus bam mutants have
an opposite effect on stem cell accumulation in a clv1
CLV3 background compared to a CLV1 CLV3 or a CLV1
clv3 background, raising the question of how these data
can be reconciled.
A previous report has indicated that CLV1 likely acts
to sequester the ligand CLV3 (Lenhard and Laux
2003). One speculative possibility is that BAM receptors
also act to sequester CLE polypeptide ligand(s) (Figure
4, B–E). We have shown that many CLE-containing
proteins can replace CLV3 function when expressed
within the meristem, and some can act through CLV1
(Ni and Clark 2006). Furthermore, the CLE domain of
these proteins appears to be proteolytically released as
the active signaling molecule, and this domain is well
conserved among Arabidopsis CLE-containing proteins
(Cock and McCormick 2001; DeYoung and Clark
2001; Fiers et al. 2004, 2005; Ito et al. 2006; Kondo et al.
2006). Indeed, multiple CLE domains can bind to the
CLV1 extracellular domain (Ogawa et al. 2008). Over
902
B. J. DeYoung and S. E. Clark
Figure 4.—A model for BAM function through signal transduction and ligand sequestration. (A) In wild-type cell layer
three (L3) meristem cells, CLV1 is expressed at higher levels
than BAM proteins and is the primary transducer of CLE signal.
CLV1/CLV1 and CLV1/BAM complexes are consistent with
the dominant-negative function of many clv1 alleles and their
interaction with bam mutations. In clv1-1 L3 cells, BAM signaling may be blocked by interference of the mutant clv1-1 kinase
domain. Residual signaling may come from a partially active
BAM/clv1-1 heterodimer and/or the presumably small
amount of BAM/BAM homodimers (not shown). In the absence of CLV1 (clv1-null), BAM proteins are available to transduce CLE signal, albeit at a reduced level compared to WTcells.
To simplify the diagram, CLV2 and other components have not
been included. (B) In a wild-type shoot meristem, CLV3 signals
primarily through CLV1, but also through BAM1 and BAM2 to
regulate the WUS expression domain. BAM1 and BAM2 also
function on the flanks of the meristem where they sequester
CLV3-related CLE ligands, creating a buffer around the meristem center. (C) In a clv3 apex, WUS repression is lost, leading
to an expansion of the WUS expression domain and an enlargement of the meristem, while CLE ligands outside of the meristem are sequestered. (D) In a bam1 bam2 apex, CLE ligands
are no longer sequestered, leading to overactivation of CLV1
and reduction in the meristem size, even meristem termination
in bam1 bam2 bam3 triple mutants. (E) In a clv3 bam1 bam2 apex,
loss of CLV3 is partially balanced through activation of CLV1 by
unsequestered CLE ligands resulting in partial WUS expression
control. (F) A model depicting genetic pathways through which
CLV1, BAM1, and BAM2 function.
30 CLE-containing proteins are expressed in a large
number of different tissues, including the meristem
periphery, and evidence indicates that these peptides
can diffuse over many cell diameters (Lenhard and
Laux 2003; Sharma et al. 2003; Fiers et al. 2005). Thus,
insulating the meristem center from CLE peptides
diffusing from outside the meristem may be critical
to maintain the proper feedback between WUS expression and CLV1 signaling (Brand et al. 2000; Schoof
et al. 2000).
We suggest a model in which one of the functions of
BAM receptors on the meristem periphery is to sequester CLE ligands and insulate the meristem center from
extraneous signals (Figure 4, B–E). In the absence of
BAM receptors (i.e., in bam mutants), ectopic CLE signal
would diffuse to the meristem center. This signal would
not be subjected to the feedback WUS/CLV3 loop that
normally regulates CLV3 expression and could lead to
overactivation of CLV1 and meristem reduction or termination. Furthermore, this ectopic CLE signal should
be able to replace, in part, CLV3 function. Finally,
the impact of this ectopic CLE signal resulting from
bam mutations would only have an impact in a CLV1
background. Thus, the model explains the reduced
meristem size of bam mutants, the bam suppression of
clv3, as well as the requirement of CLV1 for meristem
reduction in bam mutant backgrounds. While consistent with the genetic data, this model is quite speculative. We have no evidence of which CLE peptide might
bind BAM receptors and whether binding alone, as
opposed to binding and signaling, is sufficient for
this proposed BAM activity. One possible test as CLE
expression data become available would be to inactivate candidate CLEs expressed on the meristem flanks
in a bam mutant background. Another possible test
would be to determine if CLE-binding, but nonfunctional BAM receptors expressed specifically on the
meristem flanks could carry out BAM function there.
These experiments are complicated by the exquisite
sensitivity of the meristem center to BAM expression
(see below), and the tendency of nonfunctional receptor kinases to act as dominant-negative isoforms
(Williams et al. 1997; Shpak et al. 2003; Diévart et al.
2003, 2006).
The enhancement of clv1 null alleles by bam mutations could indicate that while BAM receptors are at best
expressed at low levels within the meristem center, they
are redundant with CLV1 there (Figure 4A). clv1
dominant-negative alleles might be so effective in blocking signaling because there would theoretically be much
more CLV1 than BAM receptors present in the meristem center. Redundant BAM/CLV1 function in the
meristem center would explain the observation that
single bam mutations had no effect on the dominantnegative clv1-1 allele. Interestingly, the LRR missense
allele clv1-4 did not display the same interaction with
bam mutants, raising the possibility that its mechanism
Analysis of CLV-Related Receptors
of dominant-negative action may differ from that of the
kinase-domain missense mutants.
CLV1/BAM synergy: Despite previous extensive phenotypic and expression analysis suggesting CLV1 function is meristem specific, genetic analysis with bam
mutants suggested otherwise. bam1 bam2 clv1 triple
mutants exhibited many phenotypes not seen in any
other genetic combination. These included strong
dwarfism, filamentous floral organs, and loss of recognizable valve tissue. None of these phenotypes were seen
in bam1 bam2 clv2 or bam1 bam2 clv3 plants, indicating
that the phenotypes were not indirect consequences of
defects in meristem development. This suggests that
CLV1 plays a role in regulating the development of
many organs in parallel with both BAM1 and BAM2.
In addition, synergistic interactions were seen in the
development of clv1 bam1 bam2 shoot meristems. Here,
we observed differentiated cells, or even organs in the
center of the meristem, and the occasional loss of
organogenesis on the flanks of the meristem. These
phenotypes are reminiscent of the effects of corona
mutants and PLL1 overexpression, both of which
enhance clv mutants, leading to a loss of organogenesis
at the meristem periphery, massive stem cell accumulation, and predicted eventual loss of meristem identity
resulting in differentiation of the entire apex (Green
et al. 2005; Song et al. 2006). Alternatively, the loss of
organogenesis might reflect a function for BAM1 and
BAM2 on the periphery of the meristem, where the
corresponding genes are expressed at relatively high
levels. Determining the precise developmental roles
for BAM1 and BAM2 in the meristem periphery as well
as in the various plant organs is an essential next step
in uncovering the function of these broad-ranging
receptors.
Genetic background effects: One potential complication of our analysis is that the bam1 alleles were
isolated in the Columbia (Col) background, while all
of the clv alleles were isolated or backcrossed into the
Landsberg er (Ler) background (Koornneef et al. 1983;
Clark et al. 1993, 1995; Diévart et al. 2003; DeYoung
et al. 2006). We have shown previously that crosses
between clv alleles in the Ler and Col backgrounds can
have subtle, but measurable effects on the meristem
phenotype (Diévart et al. 2003). Two lines of evidence,
however, indicate that background effects are not the
source of the genetic interactions that we observed.
First, the effects caused by genetic background are
much smaller in magnitude than those observed here.
Second, and most importantly, bam2-1 and bam2-3 alleles
were isolated in the Ler background, and they exhibit
the same type of interactions with each clv allele as
observed for bam1.
This work was supported by a grant from the National Institutes of
Health (R01GM62962) (to S.E.C.). B.J.D. was supported in part by the
National Institutes of Health Cellular Biotechnology Training Program (5 T32 GM08353).
903
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Communicating editor: V. Sundaresan