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Plant Physiol. (1998) 117: 1217-1225
Control of Meristem Development by CLAVATA1 Receptor Kinase and
Kinase-Associated Protein
Phosphatase Interactions1
Julie M. Stone2, 3,
Amy E. Trotochaud2,
John
C. Walker, and
Steven E. Clark*
Division of Biological Sciences, University of Missouri, Columbia,
Missouri 65211-7400 (J.M.S., J.C.W.); and Department of Biology,
University of Michigan, Ann Arbor, Michigan 48109-1048 (A.E.T.,
S.E.C.)
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ABSTRACT |
The
CLAVATA1 (CLV1) gene encodes a putative
receptor kinase required for the proper balance between cell
proliferation and differentiation in Arabidopsis shoot and flower
meristems. Impaired CLV1 signaling results in masses of
undifferentiated cells at the shoot and floral meristems. Although many
putative receptor kinases have been identified in plants, the mechanism
of signal transduction mediated by plant receptor-like kinases is
largely unknown. One potential effector of receptor kinase signaling is kinase-associated protein phosphatase (KAPP), a protein that
binds to multiple plant receptor-like kinases in a
phosphorylation-dependent manner. To examine a possible role for KAPP
in CLV1-dependent plant development, the interaction of CLV1 and KAPP
was investigated in vitro and in vivo. KAPP binds directly to
autophosphorylated CLV1 in vitro and co-immunoprecipitates with CLV1 in
plant extracts derived from meristematic tissue. Reduction of
KAPP transcript accumulation in an intermediate
clv1 mutant suppresses the mutant phenotype, and the
degree of suppression is inversely correlated with KAPP
mRNA levels. These data suggest that KAPP functions as a negative
regulator of CLV1 signaling in plant development. This may represent a
general model for the interaction of KAPP with receptor
kinases.
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INTRODUCTION |
The shoot meristem in higher plants is the source for all
aboveground organs and tissues (Steeves and Sussex, 1989 ). To
continuously generate new organs, the shoot meristem maintains a
population of undifferentiated cells at the center of the meristem
while directing appropriately positioned progeny cells toward
differentiation. Identical processes occur during organ formation at
the flower meristem (Steeves and Sussex, 1989; Weigel and Clark, 1996).
Proper balance between cell proliferation and differentiation requires the Arabidopsis CLAVATA1 (CLV1) gene.
clv1 mutants accumulate undifferentiated cells at both the
shoot and flower meristems, leading to disrupted organ placement,
enlarged stems, and additional organs generated on the larger
clv1 flower meristem (Leyser and Furner, 1992; Clark et al.,
1993, 1995). CLV1 has been postulated to either inhibit
proliferation of undifferentiated cells at the meristem or promote the
transition of these cells toward differentiation. CLV1
encodes a putative receptor kinase (Clark et al., 1997) with a
predicted extracellular domain composed of 21 tandem LRRs similar to
those in mammalian glycoprotein hormone receptors (Jiang et al., 1995),
a hydrophobic membrane-spanning domain, and a predicted intracellular
domain containing all of the conserved residues found among Ser/Thr
protein kinases (Hanks and Hunter, 1995).
A number of RLKs have been identified in plants. The plant RLKs are
predicted to have Ser/Thr specificity (Braun and Walker, 1996), in
contrast to the majority of animal RTKs, with which RLKs share
structural similarity. The functions of plant RLKs are largely unknown;
however, LRR-RLKs have been implicated in developmental processes
(Torii et al., 1996; Clark et al., 1997), disease resistance (Song et
al., 1995), and hormone response (Li and Chory, 1997).
A LRR-RLK very similar to CLV1 but of unknown function, RLK5, was used
to isolate an interacting protein that was named KAPP (Stone et al.,
1994). KAPP has three functional domains: an N-terminal type I signal
anchor, a KI domain, and a type 2C protein phosphatase catalytic
region. Interaction between KAPP and RLK5 is mediated by the KI domain
and is dependent on phosphorylation (Stone et al., 1994). KAPP
interacts in vitro with a growing subset of plant RLKs (Braun et al.,
1997), but the in vivo relevance of KAPP-RLK interactions is unclear.
Because of the similarity of CLV1 with these KAPP-interacting RLKs, we
hypothesized that KAPP might also interact with CLV1.
In this report we demonstrate that KAPP and CLV1 interact directly
using recombinant fusion proteins and co-immunoprecipitation from plant
extracts. Furthermore, the availability of mutants impaired in CLV1
signaling permits us to determine whether KAPP participates in the CLV1
signal transduction pathway. Analysis of transgenic clv1
mutant plants with altered levels of KAPP mRNA demonstrated that the
clv1 mutant phenotype can be rescued by reducing KAPP mRNA
levels. These data support a model for KAPP's functioning as a
negative regulator of CLV1 signaling. In an independent study using
alternative approaches to examine KAPP-CLV1 interactions, Williams et
al. (1997) came to a similar conclusion. This work provides
complementary yet distinct evidence for the negative regulation of CLV1
signal transduction by KAPP.
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MATERIALS AND METHODS |
Recombinant Proteins and Antibody Production
MBP fusions were produced using a modified version of pMalcRI (New
England Biolabs), pMalK, and GST fusions were made with a modified
version of pGEX-2T (Pharmacia), GTK. Both vectors were modified to
contain a protein kinase A recognition site at the junction to allow
32P labeling of the proteins. For in
vitro-binding studies, the protein kinase catalytic domain of CLV1
(amino acids 665-980; accession no. U96879) was subcloned into pMalK.
Site-directed mutagenesis to produce a single amino acid substitution
at the conserved Lys required for phosphotransfer (Lys-720 to
Glu-720; K720E) was made using a PCR-based mutagenesis
strategy. Oligonucleotide primers were
5 -TAGACGTCGCGATTAACCGACTCGTTGGCCGT-3 and
5-ACGGCCAACGAGTCGGTTAATCGCGACGTCTA-3.
For antibody production, oligonucleotides primers AntiB
(5-ATGAATTCGGAGTGGTTTTGTTGGAGT-3) and KinC1
(5-ATCTAGATTCAGAACGCGATCAAGTT-3) were used to amplify a
315-bp fragment of the CLV1 C terminus encoding a 10.9-kD
polypeptide, which was subcloned into pMalK. This region of CLV1 was
predicted to generate antibodies specific to CLV1, because only CLV1 is
detected on low-stringency Southern-blot analysis using this fragment
as a hybridization probe (Clark et al., 1997). Primer 5D2
(5-GGGAATTCCTGGAAAAGGATCGA-3) and a universal primer were used to
amplify KAPP (amino acids 162-581), which was subcloned into pMalcRI.
KAPP antibodies were affinity purified on a column with immobilized GST
fusion to the KI domain (KAPP amino acids 99-337; accession no.
U09505), which was the same construct used for the protein probe for in
vitro-binding studies. Recombinant MBP and GST fusion proteins were
expressed in Escherichia coli and purified by affinity
chromatography on amylose-agarose resin or glutathione-agarose resin,
respectively, essentially as described previously (Horn and Walker,
1994). Protein concentration was determined by the Bradford method
(Bradford, 1976).
The antigen for CLV1 antibody production was purified as a MBP
fusion and subjected to 15% SDS-PAGE (Laemmli, 1970) before injection into rabbits by Cocalico Biologicals, Inc. (Reamstown, PA).
The KAPP antibodies were generated in rabbits by the University of
Missouri Animal Care Facility using MBP-KAPP (5D2) antigen directly
after purification. CLV1 preimmune and immune sera were used directly
for immobilization (see "Immunoprecipitations"), whereas the KAPP
antisera were first subjected to affinity purification against GST-KID
(Koff et al., 1992).
Autophosphorylation
For autophosphorylation experiments, 1 µg of affinity-purified
recombinant fusion protein was incubated with
[ -32P]ATP in kinase buffer (50 mM Hepes, pH 7.4, 10 mM
MgCl2, 10 mM MnCl2, 1 mM DTT, and 10 µM cold ATP) for 1 h at room temperature. Reaction
products were separated by 10% SDS-PAGE (Laemmli, 1970), dried, and
exposed to film.
PAA Analysis
The PAA content of autophosphorylated MBP-CLV1CAT was analyzed
essentially as described previously (Boyle et al., 1991). Samples were
acid hydrolyzed in 6 N HCl (Pierce) for 1 h at
110°C, repeatedly lyophilized to remove the HCl, resuspended in pH
1.9 electrophoresis buffer containing PAA standards, and applied to TLC
plates (Merck, Darmstadt, Germany). Samples were electrophoresed at 1.5 kV for 30 min in pH 1.9 buffer (0.22% formic acid, 0.78% acetic acid) in the first dimension, followed by electrophoresis in pH 3.5 buffer
(0.5% acetic acid, 0.05% pyridine) at 1.3 kV for 25 min in the second
dimension using a TLE system (model HTLE 7000, C.B.S. Scientific Co.,
Del Mar, CA). PAA standards corresponding to phospho-Ser, phospho-Thr,
and phospho-Tyr (Sigma) were visualized by spraying the plates with
0.25% ninhydrin in acetone. Plates were exposed to imaging plates
(Bas-IIIS, Fuji Photo Film Co., Tokyo, Japan) to detect
32P.
Two-Dimensional TLE/TLC
Tryptic phosphopeptides were analyzed by two-dimensional TLE/TLC
essentially as described previously (Boyle et al., 1991). Autophosphorylated and trypsin-treated samples applied to TLC plates
were separated by electrophoresis for 40 min at pH 1.9. The
second-dimension separation was achieved by ascending chromatography in
phosphochromatography buffer (37.5% n-butanol, 25%
pyridine, 7.5% acetic acid) for 16 h, followed by exposure to
film.
In Vitro-Binding Assays
The GST-KID fusion was expressed in E. coli, purified,
and labeled with [-32P]ATP as described
previously (Stone, 1997). Purified recombinant fusion proteins (1 µg)
were separated on 10% SDS-PAGE gels and transferred to PVDF membranes
(Harlow and Lane, 1988). PVDF membranes were blocked for 4 h at
4°C in 25 mM Hepes, pH 7.5, 5 mM
MgCl2, 1 mM KCl, and 5% nonfat dry
milk, and then incubated overnight with approximately 2.5 × 105 cpm mL 1
32P-labeled GST-KID in B buffer (25 mM Hepes, pH 7.5, 7.5 mM KCl, 0.1 mM EDTA, 2.5 mM MgCl2,
and 1% nonfat dry milk). Filters were washed three times in B buffer
for 10 min, dried, and exposed to radiographic film.
Immunoprecipitations
CLV1 UM174 immune and preimmune sera (200 µL each) were dialyzed
(10,000 Mr cutoff) against 0.1 M NaHCO3 and 0.5 M NaCl.
Protein concentration was determined by
A280. Dialyzed preimmune and immune sera
were cross-linked to cyanogen bromide-activated Sepharose 4 Fast-Flow
beads (Pharmacia) according to the manufacturer's recommendations and
stored in TSA solution (0.01 M Tris-Cl, pH 8.0, 0.14 M NaCl, and 0.025% NaN3) at
4°C.
Cauliflower (Brassica oleracea) meristem tissue (50 g) was
ground in a Waring prechilled blender with 100 mL of 50 mM
Hepes, pH 7.4, 10 mM EDTA, 0.1% Triton X-100, 0.1 mM PMSF, 5 µg mL1 aprotinin, 10 µg mL1 chymostatin, and 1 µg
mL1 leupeptin. The extract was centrifuged
(3000g for 10 min at 4°C) repeatedly until all flocculate
was removed from the supernatant, which was stored at  20°C.
Cauliflower extract (50 mL) was passed through a 0.45 µM
filter and incubated with approximately 5 mL of swelled (1 g dry weight) preimmune coupled beads at 4°C for 2 h. The reaction was spun at 1000g for 1 min at 4°C. One-half of the
supernatant was then incubated with immune beads and the other half
with fresh preimmune beads for 2 h at 4°C. Both reactions were
centrifuged at 1000g for 1 min. Beads were washed once each
in wash buffer (10 mM Tris-Cl, pH 8.0, 140 mM
NaCl, 0.025% NaN3, 0.5% Triton X-100, and 0.5%
SDS) and Tris/Triton/NaCl (50 mM Tris-Cl, 0.1% Triton
X-100, and 0.5 M NaCl) buffer, pH 8.0 and 9.0. The beads were eluted twice with 5 mL of triethanolamine solution (50 mM triethanolamine, pH approximately 11.5, 0.1% Triton
X-100, and 150 mM NaCl) into tubes containing 0.2 volume of
1 M Tris-Cl, pH 6.7.
Eluates were concentrated to approximately 100 µL in Centricon 3000 columns. Preimmune and immune eluates (30 µL) were separated by
12.5% SDS-PAGE (Laemmli, 1970), and transferred to nitrocellulose with
a semidry transfer cell (Bio-Rad) for 20 min at 12 V (Harlow and Lane,
1988). Nitrocellulose filters were blocked overnight in 2.5% dry milk
in PBST (1× PBS and 0.5% Tween 20) and washed three times in PBST.
Filters were incubated with a 1:500 dilution of affinity-purified M12
KAPP antibody or CLV1 UM174 antibody for 2 h. Each filter was
washed three times in PBST, incubated for 1 h with a 1:30,000
dilution of goat anti-rabbit IgG horseradish peroxidase conjugate
(Bio-Rad), again washed three times in PBST, and then incubated for 1 min in 2 mL of oxidizing reagent plus 2 mL of luminol reagent from a
western chemiluminescent kit (Renaissance, NEN-DuPont). Filters were
then exposed to film for 1 to 2 min.
Transgenic Plant Generation
A construct consisting of the cauliflower mosaic virus 35S
promoter fused to the KAPP cDNA in the sense orientation with a nopaline synthase 3 terminator was subcloned into the binary vector
pGA482 (An et al., 1985). Transformation of Agrobacterium tumefaciens strain GV3101 was accomplished by electroporation and
confirmed by Southern-blot analysis. Arabidopsis (Landsberg erecta clv1-1 and clv1-6) plants were transformed
with A. tumefaciens by the vacuum-infiltration method, and
transformants were selected by sowing the resulting seeds on
kanamycin-containing plates (Bechtold et al., 1993). Several
independent lines were examined for a suppression of the
clv1 phenotype.
Northern-Blot Analysis
RNA was isolated using the RNeasy Plant Mini Kit (Qiagen,
Chatsworth, CA) from 100 mg of inflorescence tissue. Concentration was
determined by A260, and 10 µg of RNA from
each sample was separated on a 1.0% agarose-formaldehyde gel (Brown
and Mackey, 1993). RNA was transferred to Hybond-N membranes overnight
using 10× SSC. The membrane was baked in a vacuum oven at 80°C for
2 h and incubated for 2 h at 37°C in 5× SSPE, 50%
formamide, 0.5% SDS, and 5× Denhardt's reagent. KAPP
cDNA was used as a template for a PCR reaction that amplified a 715-bp
fragment from oligonucleotide primers 5-GGGATTTGCAGAGACCA-3 and
5-CTTTGTTGTTGT TCCCA-3, used as a template in a random-primed
labeling reaction in the presence of
[ -32P]dCTP. Unincorporated nucleotides were
removed using a nucleotide-removal kit (Qiagen). 18S rRNA probe was
generated by random-primed labeling of a DNA fragment corresponding to
the 18S rRNA sequence (accession no. X02623). The membrane was
hybridized overnight at 37°C, washed once in 5× SSPE and 0.5% SDS
at 37°C for 30 min, washed twice in 2× SSPE at 37°C for 30 min
each, and exposed in a molecule-imaging system (model GS363, Bio-Rad).
The amount of signal in each band was determined using the Molecular
Analyst program (Bio-Rad).
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RESULTS |
CLV1 and KAPP Interact in Vitro
We first established that CLV1 encodes a functional
protein kinase. The CLV1 protein kinase catalytic domain, expressed as a recombinant fusion protein (MBP-CLV1) in E. coli, was
capable of autophosphorylation when incubated with
[-32P]ATP. Site-directed mutagenesis of a
conserved active-site residue (K720E) abolished this activity,
indicating that incorporation of 32P requires an
active kinase domain (Fig. 1A). PAA
analysis demonstrated that autophosphorylation occurred exclusively on
Ser residues (Fig. 1B), and analysis of CLV1 phosphopeptides generated
by trypsin digestion indicated that multiple sites were phosphorylated
(Fig. 1C).
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| Figure 1.
CLV1 encoded an active
protein kinase that autophosphorylates on multiple Ser residues. A,
MBP fusions to CLV1CAT and CLV1CAT(K720E), which contains a point
mutation at the conserved Lys required for phosphotransfer, were
expressed in E. coli. Recombinant MBP-CLV1CAT is capable
of incorporation of 32P when incubated with
[-32P]ATP, indicating that it autophosphorylates
(lanes 2), whereas the mutated version, MBP-CLV1CAT(K720E), is inactive
(lanes 1). On the left is a Coomassie blue-stained SDS-PAGE gel and on
the right the corresponding autoradiogram. Sizes of molecular mass
standards in kilodaltons are indicated on the left. B,
Autophosphorylation of MBP-CLV1CAT occurred on Ser residues.
Autophosphorylated MBP-CLV1CAT was excised from an SDS-PAGE gel,
hydrolyzed to individual amino acids in 6 N HCl,
separated by two-dimensional TLE, and exposed to film. Positions of PAA
standards (PSer, PThr, and PTyr) are indicated. C, Autophosphorylation
of MBP-CLV1CAT occurred at multiple sites. Autophosphorylated
MBP-CLV1CAT was excised from an SDS-PAGE gel, treated with trypsin,
separated by two-dimensional TLE/TLC, and exposed to film. Tryptic
peptides were applied to a cellulose plate (+) and resolved by
electrophoresis in pH-1.9 buffer, followed by ascending
chromatography.
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To determine whether CLV1 interacts with the KI domain of KAPP, active
and inactive CLV1 fusion proteins were transferred to a PVDF membrane
and incubated with 32P-labeled, KI-containing
fusion protein. Interaction was observed only between the KI domain and
the active, phosphorylated CLV1 kinase domain (Fig.
2). These results are consistent with the phosphorylation-dependent interaction observed with other RLKs (Stone
et al., 1994; Braun et al., 1997).
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| Figure 2.
The KI domain of KAPP interacted with the
autophosphorylated form of CLV1. Affinity-purified recombinant fusion
proteins were subjected to SDS-PAGE, electrophoretically transferred to
PVDF, and probed with 32P-labeled fusion protein containing
the KI domain. Molecular mass markers are indicated on the left in
kilodaltons. On the left is a gel stained with Coomassie blue and on
the right the corresponding autoradiogram after probing with GST-KI.
The KI domain was capable of interacting with the catalytic domain of
CLV1 (MBP-CLV1CAT) (lanes 2); however, the KI domain could not bind to
the mutated form of CLV1 (MBP-CLV1CAT[K720E]) (lanes 1), which was
incapable of autophosphorylation. Control blots probed with
32P-labeled GST showed no binding.
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CLV1 and KAPP Interact in Vivo
To demonstrate that this interaction occurs in plant cells,
polyclonal antibodies were generated to portions of KAPP and CLV1 expressed as recombinant proteins. Specificity of the CLV1 antibodies was assessed by comparing the signals on immunoblots of extracts from
wild-type (Landsberg erecta) and clv1-6 plants.
No immunoreactive polypeptides within the range of detection were
expected in extracts from clv1-6 plants, because the
mutation in this allele is predicted to result in a truncated protein
lacking the C-terminal protein kinase domain (Clark et al., 1997) to
which the CLV1 antisera was directed. Although equivalent amounts of
protein were analyzed (Fig. 3A), no
signal was present in extracts from clv1-6 plants, whereas a
band corresponding to the predicted molecular mass of CLV1 (105 kD) was
detected in wild-type plant extracts (Fig. 3B).
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| Figure 3.
KAPP and CLV1 associated in vivo. Antibodies
raised to recombinant KAPP and CLV1 were used to co-immunoprecipitate
the proteins from cauliflower meristem extracts. A and B, Demonstration
of the specificity of CLV1 antibodies. Extracts (25 µg
lane1) from mutant (clv1-6) and wild-type
(Landsberg erecta [Ler]) Arabidopsis inflorescence
tissue, including mature flowers, cauline leaves, and some stem tissue,
were resolved by SDS-PAGE. A, Coomassie blue-stained gel. B, Results of
chemiluminescence detection of immunoreactive polypeptides after
incubation with CLV1 antisera. C, Proteins extracted from cauliflower
(Caul.) and Arabidopsis (Arab.) were subjected to immunodetection using
affinity-purified KAPP antibodies. D and E, Cauliflower meristematic
tissue extracts were incubated with immobilized preimmune (PI) or
immune (I) antisera against CLV1. Immunocomplexes were eluted, resolved
by SDS-PAGE, and immunodetected with immune antisera against CLV1 (D)
or KAPP (E). The positions of molecular mass standards are indicated on
the left of each panel in kilodaltons.
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Because of the limited range of expression of CLV1 (Clark et
al., 1997) and the small size of Arabidopsis meristems,
co-immunoprecipitation experiments were performed with extracts from
cauliflower meristematic tissue. Affinity-purified KAPP antibodies
recognized a single polypeptide of the predicted molecular mass (65 kD)
in extracts from both Arabidopsis and cauliflower (Fig. 3C). When
cauliflower extracts were incubated with immobilized serum, CLV1 immune
serum, but not preimmune serum, precipitated an immune complex
containing a 105-kD polypeptide detected by CLV1 antiserum (Fig. 3D)
and a 65-kD protein recognized by the KAPP antibodies (Fig. 3E). These data demonstrate that KAPP and CLV1 associate in vivo as well as in
vitro.
CLV1 and KAPP Participate in Control of Meristem Development
To determine a possible in vivo significance of KAPP-CLV1
interactions, we sought to alter KAPP levels in the
partial-loss-of-function clv1-1 and clv1-6 mutant
plants in which CLV1 signaling is limited. clv1-1 and
clv1-6 plants were transformed with the complete KAPP cDNA
under control of the strong, constitutive 35S cauliflower mosaic virus
promoter (Bevan et al., 1985). Several independent lines exhibited
varying degrees of suppression of the clv1 phenotype. One
family of clv1-1 primary transformants displaying heritable phenotypes was further characterized to determine a relationship between the phenotypes and the level of KAPP mRNA. In subsequent generations, individuals with a range of phenotypes were observed and
classified into three phenotypic categories: a normal clv1-1 phenotype, a partially suppressed phenotype, and a completely suppressed phenotype (Fig. 4A).
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| Figure 4.
Suppression of the clv1-1 phenotype
correlated with a reduction in KAPP mRNA levels. A, Comparison of
typical siliques from a wild-type plant (Landsberg
erecta [Ler]) and three clv1-1 plants
transformed with a 35S KAPP construct exhibiting varying degrees of
suppression of the clv1 phenotype: complete, partial,
and none. B, Analysis of RNA isolated from the corresponding transgenic
plants shown in A. RNA was isolated from inflorescence tissue,
separated in a formaldehyde-containing agarose gel, blotted to Hybond-N
membranes, and hybridized with a 32P-labeled KAPP cDNA
probe. The membrane was also probed with 18S rRNA as a loading control.
On the left is an autoradiogram of the RNA blots and on the right a
graph showing the relative levels of KAPP transcript determined by
quantitation of the blots. KAPP mRNA levels are expressed as a
percentage of that in plants exhibiting no suppression. C, Partially
(Partial) and completely (Complete) suppressed 35S KAPP plants
accumulated reduced levels of KAPP mRNA compared with untransformed
wild-type plants (WT). D, KAPP mRNA levels were equivalent in wild-type
(WT) and clv1-1 inflorescence tissues. On the left is an
autoradiogram of RNA blots hybridized with KAPP cDNA and 18S rRNA as a
loading control and on the right a graph showing the relative levels of
KAPP mRNA determined by quantitation of the blots. RNA analysis was
repeated with similar results.
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To determine if the changes in the clv1-1 phenotype were
caused by changes in KAPP transcript levels, RNA was collected from inflorescence tissue of plants from each phenotypic class. Compared with the normal clv1-1 plants by RNA-blot analysis using 18S
rRNA as an RNA-loading control standard, the partially suppressed
plants exhibited a 70% reduction in KAPP transcript accumulation,
whereas the completely suppressed plants exhibited a 94% reduction
(Fig. 4B). Direct comparison of KAPP mRNA accumulation in untransformed wild-type plants and several partially suppressed and completely suppressed isolates from the single transformed clv1-1 line
indicated that KAPP levels were significantly reduced in the suppressed isolates (Fig. 4C). To confirm that the KAPP mRNA level was not altered
by the clv1 mutation, comparison of KAPP transcript
accumulation in untransformed wild-type and clv1-1 plants
revealed that KAPP was expressed equally in these plants (Fig. 4D).
Thus, the differences in KAPP transcript accumulation were not caused
by differences in the amount of meristematic tissue present, but appear
to have been the result of sense suppression of the endogenous KAPP
gene.
Progeny exhibiting suppression of the clv1 phenotype were
found to have variable kanamycin resistance. We observed that wild-type plants transformed with CLV1 under the control of the 35S
promoter that exhibited sense suppression also had reduced frequency of kanamycin resistance that correlated with the level of sense
suppression based on the mutant phenotype. This was perhaps the result
of simultaneous suppression of the transgene and the npt II
gene that provides kanamycin resistance (A.E. Trotochaud and S.E.
Clark, unpublished results). Therefore, kanamycin resistance was
compared for the different phenotypic classes. Analysis of
kanamycin-resistance rates, which may reflect sense suppression of
KAPP, correlated with the level of suppression of the clv1
phenotype (Table I).
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Table I.
Kanamycin resistance of control clv1-1 plants and
those exhibiting a range of suppression of the clv1 phenotype after
transformation with a cauliflower mosaic virus 35S promoter::KAPP cDNA
construct
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DISCUSSION |
CLV1 encodes a receptor kinase required for maintenance
of the size of shoot and inflorescence meristems in Arabidopsis. The similarity of CLV1 to other receptor kinases shown to interact with
KAPP (Stone et al., 1994; Braun et al., 1997) motivated us to
investigate whether KAPP might be involved in CLV1 signal transduction. The availability of clv1 mutants with an easily observable
phenotype (Leyser and Furner, 1992; Clark et al., 1993, 1997) permits
direct assessment of the in vivo significance of the KAPP-CLV1
interaction.
We demonstrate that KAPP and CLV1 interact in vitro by binding KAPP to
immobilized CLV1 recombinant protein. Moreover, the observed
interaction is dependent on a functional protein kinase domain, i.e.
the KI domain of KAPP fails to bind to an inactive mutant version of
CLV1. This is consistent with other previously observed interactions
between KAPP and other protein kinases that have been demonstrated to
be phosphorylation dependent (Stone et al., 1994; Braun et al., 1997).
Using these assay conditions, KAPP has previously been shown to
interact with several RLKs in vitro, but it exhibits some specificity
and does not interact with several phosphorylated RLKs (Braun et al.,
1997; J.M. Stone and J.C. Walker, unpublished data). This in vitro
interaction is direct and not dependent on other factors, because the
KAPP and CLV1 recombinant proteins were purified before analysis.
Although our findings are consistent with separate observations of KAPP and CLV1 interaction using in vitro-translated KAPP and CLV1 (Williams et al., 1997), the association in vitro is not a guarantee of in vivo
interaction. For example, MADS-box-containing proteins appear
very promiscuous in their binding to other MADS-box proteins, co-precipitating with proteins in vitro that they are unlikely to
interact with in vivo (Riechmann et al., 1996).
To overcome the difficulty in interpreting in vitro associations, we
sought to determine if CLV1 and KAPP associate in vivo. Because
CLV1 is expressed in a small number of cells of the shoot meristem, biochemical analysis of CLV1 from Arabidopsis is less than
ideal. Instead, we generated extracts from cauliflower heads, which are
composed almost entirely of reiterative meristems and are therefore
likely to be an excellent system in which to study CLV1 signaling.
Using this system we were able to demonstrate that KAPP and CLV1
co-immunoprecipitate, suggesting that KAPP and CLV1 are associated in
vivo, and providing a strong argument for the significance of the in
vitro studies that we and others (Williams et al., 1997) have
performed. Although co-immunoprecipitation is not evidence of direct
interaction, and it is quite likely that other proteins may be
associated with the KAPP/CLV1 immunocomplex, in vitro experiments
suggest that the interaction is direct. Identification of other
proteins in the KAPP/CLV1 signaling complex might be facilitated by the
cauliflower extract system. It will be interesting to determine whether
KAPP is constitutively associated with CLV1, or if it only does so in
response to CLV1 activation. The fact that the CLV1 antibody, which we
demonstrated is specific for CLV1, and the affinity-purified KAPP
antibody each recognize a protein from cauliflower extracts of the
appropriate mass indicates that these proteins are sufficiently
conserved in the closely related Arabidopsis and Brassica
genera. Furthermore, homologs have also been reported in more divergent
plants, such as maize (Braun et al., 1997; Clark et al., 1997),
suggesting that CLV1 and KAPP signal transduction pathways may be
common to higher plants.
The CLV1 signaling pathway is an excellent system in which to study
KAPP participation in receptor kinase-mediated signal transduction,
because multiple mutant alleles of clv1 have been isolated
that exhibit phenotypes ranging from barely detectable (clv1-7) to possibly null (clv1-4) (Clark et al.,
1993). In the weak and intermediate mutant alleles (such as
clv1-1), CLV1 signaling is rate limiting, meaning that any
changes in activity should be translated into an enhanced or suppressed
mutant phenotype. This is evident by the fact that normally recessive
alleles such as clv3-1 and stm-1 become dominant
in a clv1-1 background (Clark et al., 1995, 1996). Thus, in
a clv1-1 mutant, CLV1 signaling is intrinsically different
from other receptor-kinase signaling pathways, in that in other systems
significant alterations in the level of signaling will not necessarily
lead to changes in phenotype. For example, mutations in or absence of
other putative receptor kinases involved in disease resistance and
developmental control are recessive (Song et al., 1995; Torii et al.,
1996), indicating that a 50% reduction in the amount of receptors
likely has no effect on phenotype. This was the rationale for using
clv1 mutant backgrounds for generating transgenic plants
over- and underexpressing KAPP. We hypothesized that if KAPP is
involved in multiple signaling pathways, over- or underexpression of
KAPP at levels sufficient to induce clv1 mutant
phenotypes might also lead to defects in other signaling pathways,
which could result in gamete inviability.
Transgenic experiments were performed in plants carrying the
intermediate clv1-1 and clv1-6 alleles.
Theoretically, if KAPP is involved in CLV1 signal transduction, a
change in KAPP levels in a genetic background in which signal from CLV1
is rate-limiting should lead to an alteration of the clv1
phenotype. For example, if KAPP positively regulates CLV1 signaling,
reducing KAPP in clv1 plants should enhance the mutant
phenotype. If, on the other hand, KAPP negatively regulates CLV1
signaling, reducing KAPP should suppress the clv1 phenotype.
Plants were transformed with a construct containing the entire
KAPP cDNA driven by the strong cauliflower mosaic virus 35S promoter. Phenotypes of transformed plants were moderately stable. For
example, the progeny of partially suppressed plants were generally partially suppressed, although the full range of phenotypes was observed. In subsequent generations of the suppressed plants and/or in
F2 and F3 generations of
outcrosses to wild-type plants, a number of phenotypes were
consistently observed at low frequency. These included reduced
fertility, the absence of cauline leaves, terminated shoot meristems,
shortened and/or absent root hairs, and early flowering. Because KAPP
interacts with several kinase domains in vitro (Braun et al., 1997) and
may therefore act as a common intermediate in multiple signaling
cascades, these additional phenotypes were not unexpected.
Rather than overexpression of KAPP, we found that KAPP mRNA
levels were reduced in the lines exhibiting partial to complete suppression of the clv1 mutant phenotype. Furthermore, the
degree of suppression of the phenotype was inversely correlated with KAPP mRNA levels, lending greater support for interacting roles of CLV1
and KAPP in meristem development.
A reduction in expression of endogenous KAPP in plants harboring a
sense construct may be explained by sense suppression or gene silencing
caused by the introduction of homologous DNA sequences, which is a
frequently reported phenomenon in plants (Meyer and Saedler, 1996).
Low-stringency RNA and DNA analyses suggest that no other genes closely
related to KAPP are present in the Arabidopsis genome (data not shown).
Therefore, the observed suppression should be restricted to KAPP.
Our observations suggest that KAPP most likely acts as a negative
regulator of the CLV1 signaling pathway, because the suppression of the
clv1 phenotype correlated with reduced levels of KAPP
transcript. CLV1 is predicted to either promote differentiation and
organ formation or repress cell division in the meristematic regions; KAPP would therefore function to promote cell division or suppress differentiation. One scenario that is consistent with our observations is that KAPP acts to attenuate the signal through the CLV1 pathway. In
wild-type plants CLV1 signaling is appropriately maintained and normal
meristems are generated. In clv1 mutant plants CLV1 signaling is impaired, leading to an increased pool of undifferentiated cells. By reducing the level of an attenuator, KAPP, a normal meristem
is produced. This model predicts that overexpression of KAPP in
wild-type plants would lead to a phenocopy of the clv1 mutant phenotype by promoting cell division or suppressing
differentiation in the meristems.
In a recent report, weak, clv1-like phenotypes were produced
upon transformation of KAPP driven under the 35S promoter (Williams et
al., 1997). However, there was no demonstration that KAPP levels actually increased in the transgenic plants, leaving these results open
to a number of interpretations. clv1-Like phenotypes could conceivably result from down-regulation of CLV1, CLV2, or CLV3, as well
as positive up-regulation of STM. Cytokinin application has also
been shown to result in clv1-like phenotypes (in addition to
other effects on organ identity and development) (Venglat and Sawhney,
1996). In the experiments described here, a weak clv1 mutant
was used as the starting material for transgenic work. Therefore, as
discussed above, CLV1 signaling is fundamentally different from other
signaling pathways in these plants. Specifically, a phenotypic change
should result from any minor alteration in the level of CLV1 signaling,
whereas other pathways are likely to be insensitive to minor changes in
the level of signaling. The partially suppressed plants retained 30%
of the normal level of KAPP transcript accumulation. This was
apparently sufficient for other pathways to behave in a manner
phenotypically similar to that of the wild type, while at the same time
causing a significant alteration in the clv1-1 phenotype.
The in vitro and in vivo interaction between CLV1 and KAPP is
reminiscent of interactions between RTKs and PAA-binding SH2-PTPs. Tyr
phosphorylation of RTKs induced by ligand binding recruits SH2-PTPs and
other SH2-containing proteins to form an activated receptor complex for
signaling. Some SH2-PTPs have been shown to act downstream of multiple
RTKs (Perkins et al., 1996; Su et al., 1996), which may also be the
case for KAPP. SH2-PTPs have been demonstrated to act as positive
regulators, negative regulators, and attenuators of RTK signaling.
These different roles can be attributed to the fact that SH2-PTPs can
act as substrates for RTKs (Vogel et al., 1993), dephosphorylate the
RTK for desensitization (Klinghoffer and Kazlauskas, 1995; Tomic et
al., 1995), serve as adaptor molecules (Kharitonenkov et al., 1995), or
dephosphorylate downstream signaling components (Herbst et al., 1996;
Kharitonenkov et al., 1997). Unlike the RTK/SH2-PTP paradigm, however,
CLV1 autophosphorylates on Ser residues (Fig. 1B) and KAPP is a
phospho-Ser/phospho-Thr protein phosphatase (Stone et al., 1994).
Although multiple scenarios can be envisioned for the participation of
KAPP and CLV1 in the control of meristem development, the simplest
model to explain our data, KAPP as a negative regulator of CLV1 signal
transduction, is that KAPP could act directly on activated CLV1 to
attenuate or desensitize the receptor kinase. KAPP is capable of
dephosphorylating recombinant CLV1 protein in vitro (Williams et al.,
1997). The work presented here contributes to our understanding of
general mechanisms of receptor kinase-mediated signal transduction and
control of plant development. Future work should elucidate other
components of the CLV1 signal transduction pathway and other pathways
in which KAPP might participate.
| |
FOOTNOTES |
1
This work was supported by the National Science
Foundation (NSF) (grant no. MCB-9417732 to J.C.W.), by the University
of Missouri Food for the 21st Century Program (grant to J.C.W.), by the
Department of Energy (DOE) (grant no. DE-FG02-96ER20227 to S.E.C.), and
by the Triagency DOE/NSF/U.S. Department of Agriculture (Plant Biology Grant to Promote Collaboration in Plant Protein Phosphorylation no.
92-37105-7675).
2
J.M.S. and A.E.T. made equal contributions to
this publication.
3
Present address: Department of Molecular
Biology, Massachusetts General Hospital, Boston, MA 02114.
*
Corresponding author; e-mail clarks{at}umich.edu; fax
1-734-647-0884.
Received February 19, 1998;
accepted April 21, 1998.
| |
ABBREVIATIONS |
Abbreviations:
GST, glutathione S-transferase.
KAPP, kinase-associated protein phosphatase.
KI, kinase interaction.
KID, KI domain.
LRR, Leu-rich repeat.
MBP, maltose-binding protein.
PAA, phosphoamino acid.
PTP, phospho-Tyr phosphatase.
RLK, receptor-like kinase.
RTK, receptor Tyr kinase.
SH2, src
homology 2.
TLC, thin-layer cellulose.
TLE, thin-layer
electrophoresis.
| |
ACKNOWLEDGMENTS |
We acknowledge Tsung-Luo Jinn for preparing the
affinity-purified KAPP antibodies and Hannah Alexander for constructing
the modified pMalK vector. We also thank members of the Clark and Walker laboratories for comments on the manuscript.
| |
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Top
Abstract
Introduction