Plant Physiol. (1999) 120: 559-570
Expression of a Gibberellin-Induced Leucine-Rich Repeat
Receptor-Like Protein Kinase in Deepwater Rice and Its Interaction with
Kinase-Associated Protein Phosphatase1
Esther van der Knaap2,
Wen-Yuan Song3,
De-Ling Ruan,
Margret Sauter4,
Pamela C. Ronald, and
Hans Kende*
Michigan State University-Department of Energy Plant Research
Laboratory, Michigan State University, East Lansing, Michigan
48824-1312 (E.v.d.K., M.S., H.K.); and Department of Plant
Pathology, University of California, Davis, California 95616-8680
(W.-Y.S., D.-L.R., P.C.R.)
 |
ABSTRACT |
We
identified in deepwater rice (Oryza sativa L.) a gene
encoding a leucine-rich repeat receptor-like transmembrane protein kinase, OsTMK
(O.
sativa
transmembrane kinase). The
transcript levels of OsTMK increased in the rice
internode in response to gibberellin. Expression of OsTMK
was especially high in regions undergoing cell division and elongation.
The kinase domain of OsTMK was enzymatically active,
autophosphorylating on serine and threonine residues. A cDNA encoding a
rice ortholog of a kinase-associated type 2C protein phosphatase
(OsKAPP) was cloned. KAPPs are putative downstream
components in kinase-mediated signal transduction pathways. The kinase
interaction domain of OsKAPP was phosphorylated in vitro by the kinase
domain of OsTMK. RNA gel-blot analysis indicated that the expression of
OsTMK and OsKAPP was similar in different
tissues of the rice plant. In protein-binding assays, OsKAPP interacted
with a receptor-like protein kinase, RLK5 of Arabidopsis, but not with
the protein kinase domains of the rice and maize receptor-like
protein kinases Xa21 and ZmPK1, respectively.
| |
INTRODUCTION |
The successful existence of all plants depends on their ability to
coordinate complex developmental changes and to sense and respond to
fluctuations in their environment. A stimulus is perceived by the cell,
a signal is generated and transduced, and a biochemical response is
elicited. Deepwater or floating rices belong to a group of cultivars
whose survival is based on their capacity for rapid internodal
elongation when they become submerged during flooding in the rainy
season. Under field conditions, growth rates of up to 25 cm/d have been
reported, resulting in plants that are up to 6 m long (Catling,
1992
). The signal for accelerated growth is an increase in the internal
ethylene concentration (Raskin and Kende, 1984a), which, via a decrease
in ABA levels (Hoffmann-Benning and Kende, 1992), enhances the
responsiveness of the internode to GA (Raskin and Kende, 1984b).
Whereas ethylene and ABA are intermediates in signaling the change in
the environment, the growth response is ultimately elicited by
GA.
In several plant species, the putative components of the GA
signal transduction pathway have been identified by genetic and biochemical approaches (Swain and Olszewski, 1996; Bethke et al., 1997;
Jones et al., 1998; Ritchie and Gilroy, 1998; Steber et al., 1998).
GAMYB, a transcription factor in the cereal aleurone system, has been
isolated and appears to mediate GA-induced expression of a high-pI
-amylase gene (Gubler et al., 1995). In addition to GAMYB, two
putative transcription factors with high sequence similarity to each
other, GAI (Peng et al., 1997) and RGA (Silverstone et al., 1998), were
identified in genetic screens for GA signal transduction mutants. It
has been proposed that these putative transcription factors function as
negative regulators of GA signal transduction. Another negative
regulator of GA signal transduction is encoded by the SPY
gene. The SPY protein is thought to posttranslationally modify target
proteins of the GA-signaling pathway (Jacobsen et al., 1996; Peng et
al., 1997; Silverstone et al., 1998). To our knowledge, despite this
progress in the identification of GA signal transduction components,
the other potential elements of GA transduction (e.g. specific protein
kinases, phosphatases, channel proteins, or heterotrimeric G-proteins)
have not yet been found, nor has the GA receptor been identified.
However, circumstantial evidence has pointed to the plasma membrane as
the site of GA perception (Hooley et al., 1991; Gilroy and Jones,
1994).
Many signals are initially perceived by transmembrane receptors, a
large number of which function by activation of an
intrinsic protein kinase domain. In recent years, many plant RLKs have
been identified. Whereas the majority of animal RLKs autophosphorylate on Tyr residues, the majority of plant RLKs autophosphorylate on Ser
and/or Thr residues (Braun and Walker, 1996). A petunia RLK, PRK1,
exhibits dual specificity, phosphorylating on Tyr as well as Ser
residues (Mu et al., 1994). Another class of plant RLKs shows sequence
similarity to members of the prokaryotic two-component signal
transduction systems, which act as His kinases. This group includes the
ethylene receptors (Chang et al., 1993; Schaller and Bleecker, 1995;
Wilkinson et al., 1995) and CKI1, whose protein product is
involved in cytokinin signaling (Kakimoto, 1996). RLKs with predicted
or demonstrated substrate specificity for Ser and Thr residues can be
classified according to the amino acid composition of their putative
extracellular domains. The two largest subclasses of RLKs are those
whose extracellular domain contains LRRs and those whose extracellular
domain shows sequence similarity to the S-locus
glycoprotein. The extracellular domains of several other plant RLKs are
unique.
We are interested in the mechanism by which GA promotes internodal
elongation in deepwater rice (Oryza sativa; Kende et al., 1998). In a search for GA-regulated transcripts, we identified the
LRR-RLK OsTMK (O.
sativa
transmembrane kinase). The
expression of OsTMK increased during GA treatment of rice stem sections, indicating a role for this gene in plant growth. A
potential downstream signal transduction component of RLKs is KAPP,
originally identified in Arabidopsis by its in vitro interaction with
RLK5, an LRR-RLK (Stone et al., 1994). OsKAPP, a rice
ortholog of the Arabidopsis KAPP, was cloned, and the interaction
between KID of OsKAPP and the kinase domains of the LRR-RLKs OsTMK,
RLK5, and Xa21 of rice (Song et al., 1995) and the
S-locus-like RLK, ZmPK1, of maize (Zhang and Walker, 1993)
were investigated.
| |
MATERIALS AND METHODS |
Plant Material
Seeds of deepwater rice (Oryza sativa L. cv Pin Gaew
56) were obtained from the International Rice Research Institute (Los Baños, Philippines). Plants were grown as described by
Stünzi and Kende (1989). Twenty-centimeter-long stem sections
containing the growing internode were excised and treated with 50 µM GA3 (Raskin and Kende,
1984a, 1984b). Incubation was allowed to proceed for various periods,
after which the different regions of the internode were excised, frozen
immediately, and stored at 
80°C until use.
Identification of OsTMK and OsKAPP
Clones
The derived amino acid sequence of a partial 913-bp cDNA
(OsKIN), which was isolated during a library screen for
cyclin genes, turned out to have similarity to protein kinases. A
305-bp fragment from the 5
end of OsKIN was used to screen
an intercalary-meristem-specific, unamplified cDNA library, and a
full-length clone, OsTMK, was isolated. The phage insert was
inserted into the NotI site of the pBluescript SK()
phagemid (Stratagene), and the DNA sequence was determined as described
previously (Van der Knaap et al., 1996). The sequences were aligned
using the Sequencher program (version 3.0, Gene Codes Corp., Ann Arbor,
MI).
To isolate a KAPP ortholog, a rice cDNA library (Song et
al., 1995) was screened under low-stringency conditions (Walker, 1993)
using a 0.7-kb EcoRI fragment corresponding to the maize KID
(Braun et al., 1997) as a probe. To isolate a full-length cDNA of rice
KAPP, a second rice cDNA library (W.-Y. Song and P.C.
Ronald, unpublished data) was screened under high-stringency conditions
(Wang et al., 1996) with the partial cDNA clone that had been isolated
in the first screen. The two cDNAs were sequenced using a
cycle-sequencing kit (Sequitherm, Epicentre Technologies, Madison, WI)
and an automated sequencer (model 4000L, LI-COR, Lincoln, NE).
RNA Gel-Blot Analysis
Twenty micrograms of total RNA, isolated according to the method
of Puissant and Houdebine (1990), was separated electrophoretically in
a 1.2% (v/v) formaldehyde-agarose gel (Ausubel et al., 1987) and
transferred to a Hybond-N membrane (Amersham). DNA fragments containing
the inserts of RL5, E37, and OsKIN
were isolated from agarose gels by digestion with 
-agarase (New
England Biolabs). RL5 encodes the 5S rRNA-binding
protein (Kim and Wu, 1993), and E37 is a truncated cDNA
corresponding to a chloroplast inner membrane protein (Van der Knaap
and Kende, 1995). RL5 and E37 are constitutively expressed transcripts and served as loading controls. Fifty nanograms of template DNA was labeled in the presence of
[-32P]dCTP (3000 Ci
mmol
1) using a random-primer labeling kit
(Boehringer Mannheim). RNA gel blots were hybridized and washed as
previously described (Van der Knaap et al., 1997). For gel-blot
analysis of OsKAPP expression, an RNA probe was prepared
from the region encoding KID in the presence of
[-32P]UTP. Blots were prehybridized and
hybridized overnight in 3× SSPE, 10× Denhardt's solution, 0.5%
(w/v) SDS, 50 µg mL1 denatured salmon-sperm
DNA, and 50% formamide at 65°C. Blots were washed twice for 5 min
each time in 2× SSC and 0.5% (w/v) SDS at 65°C and then washed
twice in 0.1× SSC and 0.5% (w/v) SDS at 65°C for 30 min each. The
radioactivity on blots was quantified with a PhosphorImager (Molecular
Dynamics, Sunnyvale, CA).
DNA Gel-Blot Analysis
Rice genomic DNA was isolated from a CsCl gradient according to
the method of Ausubel et al. (1987). Four micrograms of genomic DNA was
digested with the appropriate restriction enzyme, and the DNA fragments
were separated on a 0.8% (w/v) agarose gel for 20 h at 30 V. The
gel was treated following standard protocols (Ausubel et al., 1987),
and the DNA was transferred to a Hybond-N+ membrane
(Amersham). OsKIN was used to prepare a probe by
random-primer labeling in the presence of
[-32P]dCTP. Blots were prehybridized and
hybridized overnight in 6× SSC, 5× Denhardt's solution, and 1%
(w/v) SDS at 65°C. Nonspecific hybridization was removed by stringent
washes in 0.1× SSC and 0.1% (w/v) SDS at 65°C.
Overexpression and Purification of the Fusion Proteins
To facilitate the cloning of the kinase domain of OsTMK
in-frame with MBP, a restriction enzyme site was introduced via PCR. The primers used were 5-ATGGAATTCTCAATTCAAGTCCTC-3 and the
reverse primer present in pBluescript SK(). The product was amplified from a plasmid containing the full-length clone of OsTMK
with Pwo polymerase (Boehringer Mannheim). The PCR product was digested with EcoRI and HindIII and inserted directionally
in the same sites of pMAL-cRI (New England Biolabs). The
construct created, pMBP-OsTKD, encoded MBP fused to residues
596 to 962 of OsTMK. pMBP-OsTKD was sequenced over the
primer region to verify that the proper fusion construct had been
obtained and was introduced into the Escherichia coli host
ER2508 (New England Biolabs). The cells were grown in rich medium
(containing, per liter: 10 g of Bactotryptone, 5 g of yeast
extract, 5 g of NaCl, and 2 g of Glc) supplemented with 1 mM MnCl2 and 50 µg
mL1 carbenicillin at 37°C until an
A600 of 0.6 was reached.
The cells were induced by the addition of 50 µM isopropyl
-D-thiogalactoside for 2 h at room temperature and
harvested by centrifugation. The bacterial pellet was resuspended in
lysis buffer (10 mM Tris-HCl, pH 7.3, 150 mM
NaCl, 1 mM DTT, 0.1% [v/v] Tween 20, and 1 mM PMSF) and stored at 20°C overnight. The cells were
lysed with a cup sonicator at a 30% duty cycle, a probe setting of 4 to 5, or with a French press. MBP and MBP-OsTKD fusion protein (83 kD)
were allowed to bind to amylose resin (New England Biolabs) for 20 min,
and the resin was washed several times with lysis buffer. The final
wash was performed in lysis buffer without Tween 20, and the two
proteins were eluted with 10 mM maltose and 10 mM Tris-HCl, pH 7.0.
KID of rice KAPP (KID defined according to Stone et al., 1994) was
amplified using the primers 5-GTGATTCTGGCAGGACTTATCCTGC-3 and
5-AACACCCTGCAGAGGGCACTGGC-3 and inserted into the pGEX-2T-derived expression vector (Pharmacia). The construct created,
pGST-OsKID, contained the region encoding the rice KID fused
in-frame to GST. Both GST and GST-OsKID contained a protein kinase A
recognition site. Overexpression of GST-OsKID (55 kD) and GST was
performed essentially as described for MBP-OsTKD, except that
MnCl2 was omitted from the rich medium. After the
cells were lysed, the proteins were purified by affinity binding to
glutathione agarose resin (Sigma). After several washes, the proteins
were eluted with 10 mM GSH in 10 mM Tris-HCl, pH 7.0. Protein concentrations were
determined by comparison of band intensity against known standards on a
Coomassie Blue-stained polyacrylamide gel. The fusion proteins used in
the filter binding assay shown in Figure 6 were overexpressed and
purified according to the method of Horn and Walker (1994). The
GST-Xa21CAT fusion protein contained the catalytic domain of Xa21 fused
in-frame to GST (W.-Y. Song and P.C. Ronald, unpublished data). The
MBP-RLK5CAT and GST-PK1CAT fusion proteins contained the catalytic
domains of RLK5 and ZmPK1 fused in-frame to MBP or GST, respectively
(Stone et al., 1994; Braun et al., 1997).
View larger version (61K):
[in this window]
[in a new window]
| Figure 6.
KID interacts with the kinase domain of
Arabidopsis RLK5 but does not interact with the kinase domains of Xa21
and ZmPK1. A, Coomassie Blue-stained SDS-PAGE gel. Lane 1, GST-Xa21CAT;
lane 2, MBP-RLK5CAT; lane 3, GST-PK1CAT; lane 4, GST; and lane 5, MBP.
B, Corresponding autoradiogram probed with the 32P-labeled
rice GST-OsKID. The strongly interacting protein (80.5 kD) corresponds
to the full-length MBP-RLK5CAT, whereas the weaker, smaller protein may
be a degraded form of MBP-RLK5CAT.
|
|
Filter Binding Assay
MBP, GST, GST-Xa21CAT, MBP-RLK5CAT, and GST-PK1CAT were separated
on a 7.5% SDS-PAGE gel and electrophoretically transferred to PVDF
membranes. The GST-OsKID fusion protein was labeled with P at the protein kinase A recognition site
at the junction of the fusion using bovine heart-muscle kinase (Sigma)
and allowed to bind to the proteins on the membrane (Stone et al.,
1994). The filter was then subjected to autoradiography.
Auto- and Transphosphorylation Assays and Phosphoamino Acid
Analysis
Purified MBP-OsTKD, in the presence or absence of GST-OsKID, was
incubated in 15 µL of 50 mM Tris-HCl, pH 7.3, containing 10 µCi [
-32P]ATP (6000 Ci
mmol1), 20 µM nonradioactive ATP,
1 mM DTT, and 10 mM
MnCl2. The reaction was allowed to proceed for
the appropriate time at room temperature or at 30°C and then was
stopped by the addition of 35 µL of ice-cold 10% (v/v) TCA. The
radiolabeled proteins were collected by centrifugation, washed with 50 µL of ice-cold 10% (v/v) TCA, and resuspended in 4 µL of 1 M Tris base and 5 µL of 2× SDS sample buffer (Bio-Rad). The resuspended proteins were directly loaded onto a 10% or 12% (w/v)
polyacrylamide gel and electrophoretically separated with a constant
current of 15 mA. The gel was stained with Coomassie Blue to verify
equal loading, dried, and radiographic film was exposed to the gel at
room temperature. The radioactivity was quantified with a
PhosphorImager.
For phosphoamino acid analysis, the autophosphorylated MBP-OsTKD was
eluted from the polyacrylamide gel overnight in 50 mM NH4HCO3. The sample was
precipitated with TCA and hydrolyzed with acid in 6 M HCl
at 110°C for 1 h. The HCl was evaporated, and the pellet was
resuspended in electrophoresis buffer (2.2% [v/v] formic acid and
7.8% [v/v] acetic acid, pH 1.9) containing phosphoamino acid
standards and applied to a cellulose TLC plate (Merck, Rahway, NJ) as
described by Boyle et al. (1991). Samples were subjected to
electrophoresis at 1.5 kV for 20 min in pH 1.9 buffer in the first
dimension and then in pH 3.5 buffer (5% [v/v] acetic acid and 0.5%
[v/v] pyridine) at 1.3 kV for 16 min in the second dimension. Phosphoamino acid standards were visualized by spraying the plate with
0.25% (w/v) ninhydrin in acetone and heating it at 65°C for 30 min.
Radiographic film was exposed to the TLC plate for 2 d at
80°C.
| |
RESULTS |
OsTMK Encodes an RLK with High Sequence
Similarity to Two LRR-RLKs from Arabidopsis
A screen of the rice cDNA library with the partial clone
OsKIN resulted in the isolation of three independent inserts
of approximately 3100 bp, a size similar to that expected from RNA
gel-blot analysis, indicating that a full-length clone had been
isolated. When the longest insert was sequenced, no in-frame
stop codon was observed upstream of the putative start Met. However, an
otherwise identical rice expressed sequence tag (D41598) was found to
have a five-nucleotide extension with an in-frame stop codon at the 5
end. As shown in Figure 1A, the putative
signal sequence is followed by an extracellular LRR region containing
nine potential N-glycosylation sites (consensus N-X-S/T).
The first 10 LRRs are flanked by two Cys residues spaced 11 and 8 amino
acids apart. One Cys pair, spaced 8 amino acids apart, was found
at the N-terminal side of the last three LRRs.
View larger version (94K):
[in this window]
[in a new window]
| Figure 1.
Amino acid sequence comparisons between OsTMK and
other LRR-RLKs. A, Alignment of OsTMK, TMK1, and AC000103 using Clustal
W. Amino acids identical to those in OsTMK are indicated by a period,
and gaps in the alignment are denoted with dashes. The putative signal
sequence and transmembrane region are single-underlined. The LRR region
is boxed in gray, and the three Cys pairs are double-underlined. The
Roman numerals refer to the 12 subdomains found in protein kinases. The
conserved residues in the kinase domains (Hanks and Quinn, 1991) are
boxed in black. B, Alignment of the 11 complete and two incomplete LRRs
in OsTMK, TMK1, and AC000103. The LRR of all three proteins are grouped
as indicated by the horizontal bar and the number behind the repeat.
The gap between LRR10 and LRR11 is not shown. Amino acid residues with
identity to the consensus are boxed in gray. "x" in the consensus
sequence indicates any amino acid; "a" indicates an aliphatic amino
acid. C, Alignment of the Cys pairs present in the N terminus of the
LRRs (top) and the alignment of the Cys pairs present in the C terminus
of the LRRs (bottom). Amino acid residues with identity to the
consensus are boxed in gray. "x" in the consensus sequence
indicates any amino acid. References are: OsTMK, Van der Knaap et al.,
1996 (accession no. Y07748); TMK1, Chang et al., 1992; AC000103, same
as for genomic sequence of Arabidopsis BAC (accession no. F21J9); Xa21,
Song et al., 1995; BRI1, Li and Chory, 1997; CLV1, Clark et al., 1997;
ER, Torii et al., 1995; RLK5, Walker, 1993; PRK1, Mu et al.,
1994; DcSERK, Schmidt et al., 1997; RPK1, Hong et al., 1997. The Cys
pair in RPK1 is located between the first and second LRR.
|
|
LRRs are believed to play a role in protein-protein interaction, and
several LRR domains are flanked by Cys clusters (Kobe and Deisenhofer,
1994). The extracellular domain is followed by a putative transmembrane
region and the C-terminal intracellular portion of the protein contains
the 12 characteristic kinase subdomains (Hanks and Quinn, 1991).
Database searches with the BLAST program (Altschul et al., 1990)
indicated amino acid sequence similarity to RLKs from plants. Because
the highest similarity was to TMK1 from Arabidopsis (Chang et al.,
1992), we named the rice gene OsTMK. A high level of
similarity was also found to an Arabidopsis open reading frame (locus
2213607 on BAC F21J9; accession no. AC000103).
The spacing of the LRRs and the presence of the Cys pairs is conserved
in all three proteins. The alignment of the LRRs in the three proteins
is shown in Figure 1B. The consensus amino acids shown below the
alignment are commonly found in other plant proteins containing LRRs
(Li and Chory, 1997), except for the presence of S/T and S at positions
5 and 6, respectively. The alignment of the sequences surrounding the
two Cys residues located at either the N-terminal or the C-terminal
side of the LRRs in several different LRR-RLKs is shown in Figure 1C.
The Cys residues are often spaced seven amino acids apart; for OsTMK,
TMK1, and AC000103, they are usually eight amino acids apart.
The percentage of amino acid identity, including the putative signal
sequences and the transmembrane regions, between several LRR-RLKs is
shown in Table I. Whereas the amino acid
identity among OsTMK, TMK1, and AC000103 ranges from 49% to 59%, the identity among all other LRR-RLKs is approximately 25%. The kinase domain of OsTMK is 79% identical to that of TMK1 and 63% identical to
that of AC000103. The LRR domain of OsTMK is 49% identical to that of
TMK1 and 40% to that of AC000103. This indicates that OsTMK, TMK1, and
AC000103 belong to a subgroup within the larger LRR-RLK family.
View this table:
[in this window]
[in a new window]
|
Table I.
Amino acid identity between LRR-RLKs
The numbers indicate overall percentages of identity between the pairs,
based on pairwise comparisons using ALIGN. The last column shows the
number of LLRs found in the presumed extracellular domain of the
kinases.
|
|
Changes in Transcript Levels of OsTMK in
Response to GA and the Gene Copy Number of OsTMK
in Rice
We were interested in determining whether OsTMK plays a
role in GA-mediated internodal growth. In rice stem sections, GA
enhances the rate of cell production in the intercalary meristem
at the base of the growing internode and also results in a 3- to 4-fold increase in the final size of cells in the elongation zone (Raskin and
Kende, 1984b; Sauter and Kende, 1992). In deepwater rice internodes, several genes have been identified whose transcript levels increase in
response to GA (Kende et al., 1998). Results of gel-blot analyses of
RNA isolated from different regions of GA-treated internodes are shown
in Figure 2. The top panels in Figure 2,
A to C, represent blots hybridized to OsTMK; the bottom
panels show hybridization of the same blots to RL5, a cDNA
corresponding to a gene whose expression did not change significantly
in the intercalary meristem (Fig. 2A) and in the growing zone (Fig. 2B)
of the internode during treatment with GA. The signals were quantified
with a PhosphorImager and normalized for equal loading with the signals
for RL5. OsTMK transcript levels increased more
than 3-fold in the region of the intercalary meristem and the lower
elongation zone after 2 to 3 h of treatment with GA and reached a
9-fold increase after 15 h (Fig. 2A). OsTMK transcript
levels also increased in the elongation zone (Fig. 2B). In the
differentiation zone (Fig. 2C), the increase in OsTMK
expression was accompanied by an increase in RL5 mRNA. The
expression of OsTMK was very low in the oldest part of the
internode and did not change during GA treatment (Fig. 9 and data not
shown). The transcript levels for OsTMK did not increase in
control stem sections (data not shown). These results indicate that the
mRNA levels of OsTMK increase in growing tissues of the
internode in response to GA.
View larger version (81K):
[in this window]
[in a new window]
| Figure 2.
Expression of OsTMK in GA-treated
stem sections. Stem sections were incubated in 50 µM
GA3 for the times indicated above the lanes. Shown are gel
blots containing RNA (20 µg per lane) isolated from the 0- to 5-mm
region above the second-highest node, which includes the intercalary
meristem and the basal part of the elongation zone (A), 5 to 10 mm
above the second highest node, which includes the elongation zone (B),
and 10 to 20 mm above the second highest node, which includes the upper
part of the elongation zone and the differentiation zone (C). All blots
were hybridized to RL5, a constitutively expressed gene
encoding a 5S rRNA-binding protein.
|
|
View larger version (34K):
[in this window]
[in a new window]
| Figure 9.
Tissue-specific expression of OsTMK
and OsKAPP in rice. N2, Second highest node; N1, highest
node containing the shoot apex; L2b, basal 2 cm of second youngest leaf
blade; L2s, basal 2 cm of second youngest leaf sheath; L1, youngest
leaf; Co, coleoptile 3 d after germination; Ro, root 3 d
after germination; 0-3, internodal region 0 to 3 mm above N2
containing the intercalary meristem; 3-8, internodal region 3 to 8 mm
above N2 containing the elongation zone; 8-18, internodal region 8 to
18 mm above N2 containing the upper part of the elongation zone and the
differentiation zone; old, oldest part of the internode. Top,
Hybridization signals with OsKIN, which corresponds to
the kinase domain of OsTMK, as a probe; middle,
hybridization signals with OsKAPP; bottom, hybridization
signals with E37, a truncated cDNA corresponding to a
constitutively expressed chloroplast inner membrane protein.
|
|
High-stringency DNA gel-blot analysis of the region encoding the kinase
domain as a probe showed, with the exception of the HindIII
digest, only one band in each lane (Fig.
3). The additional faint band seen in the
lane containing the HindIII digest was expected because the
insert used as probe had an internal HindIII restriction
site. The size of the smallest band was 2 kb, which eliminated the
possibility that the rice genome contains two linked copies of
OsTMK. The presence of a single band in each digest on the
DNA gel blot indicates that OsTMK is represented by a single gene in the rice genome.
View larger version (32K):
[in this window]
[in a new window]
| Figure 3.
DNA gel-blot analysis of OsTMK in
rice. A blot containing genomic DNA (5 µg per lane), digested with
either BamHI (B), EcoRI (E),
HindIII (H), or PstI (P) was probed with
random-primer-labeled OsKIN, which corresponds to the
kinase domain of OsTMK. M, Molecular size markers (in kb).
|
|
Phosphorylation Assays with the Kinase Domain of OsTMK
To examine whether OsTMK constituted an active kinase and to
determine which amino acid residues were phosphorylated, the kinase
domain, OsTKD, was inserted in-frame into a cDNA encoding MBP and was
overexpressed in E. coli. The resulting fusion
protein, MBP-OsTKD, was purified to homogeneity and possessed
autophosphorylating activity (Fig. 4A).
To determine substrate specificity, the autophosphorylated fusion
protein was eluted from the polyacrylamide gel and hydrolyzed with HCl;
the hydrolysate was analyzed by electrophoresis (Fig. 4B).
32P-labeled spots corresponding to the positions
of phospho-Thr and phospho-Ser, but not to that of phospho-Tyr, were
detected. This indicates that OsTMK, like TMK1, is a Ser/Thr protein
kinase. Most RLKs undergo intermolecular phosphorylation (second order with respect to enzyme concentration; Horn and Walker, 1994). An
intramolecular phosphorylation mechanism, which is a first-order reaction, would result in a linear increase of phosphorylation as a
function of enzyme concentration. Because the relationship between the
rate of autophosphorylation and the MBP-OsTKD concentration was not
linear but approached the curve expected for a second-order reaction,
MBP-OsTKD appears to phosphorylate itself via an intermolecular, higher-order reaction mechanism (Fig. 4C).
View larger version (33K):
[in this window]
[in a new window]
| Figure 4.
Autophosphorylation of MBP-OsTKD. A, Purified
MBP-OsTKD (500 ng) was allowed to autophosphorylate for 20 min at room
temperature. Right, Autoradiogram of the Coomassie Blue-stained gel
shown on the left. M, Molecular mass markers (in kD). B,
Electrophoretic analysis of phosphoamino acids autophosphorylated by
MBP-OsTKD. pS, Phospho-Ser; pT, phospho-Thr; pY, phospho-Tyr. C,
Semilogarithmic plot of the rate of phosphorylation as a function of
MBP-OsTKD concentration. The amount of MBP-OsTKD used ranged from 30 to
810 nM (38 ng to 1 µg per 15-µL reaction). The
phosphorylation reaction was performed at 30°C for 20 min.
|
|
Identification of OsKAPP
A potential downstream component of several RLKs in plants is
KAPP. Arabidopsis and maize KAPP have been shown to interact with TMK1
via KID (Braun et al., 1997). For this reason, we identified the rice
ortholog of KAPP and determined its interaction with plant RLKs.
OsKAPP contains an open reading frame encoding 585 amino
acids (Fig. 5). Sequence comparison
indicated that OsKAPP has a high level of overall similarity to maize
KAPP (81.3% similarity, 77.1% identity, Fig. 5) and Arabidopsis KAPP
(54.3% similarity, 45.4% identity, Fig. 5). Like the Arabidopsis and
maize KAPP, the predicted rice protein consists of three domains: a
potential N-terminal membrane anchor, a centrally located KID, and a
carboxy-terminal type 2C protein phosphatase. The N-terminal anchor
shares relatively low similarity to the corresponding regions of the
Arabidopsis (42.9% similarity, 25.0% identity) and maize KAPP (73.1%
similarity, 69.2% identity). In contrast, KID and the protein
phosphatase domain of OsKAPP are more related to those of Arabidopsis
(KID: 53.5% similarity, 46.9% identity; phosphatase: 68.3%
similarity, 57.9% identity) and maize KAPP (KID: 83.3% similarity,
80.0% identity; phosphatase: 87.5% similarity, 83.2% identity). The
protein phosphatase domain of OsKAPP carries the 11 structurally
conserved motifs of type 2C protein phosphatases, which is a strong
indication for protein phosphatase activity (Bork et al., 1996; Fig.
5).
View larger version (54K):
[in this window]
[in a new window]
| Figure 5.
Alignment of KAPPs from rice (OsKAPP), maize
(ZmKAPP), and Arabidopsis (AtKAPP). Amino acids identical to those in
OsKAPP are indicated by periods, and gaps in the alignment with dashes.
The 11 structurally conserved motifs of type 2C protein phosphatases
are underlined and numbered along the top. Accession numbers are as
follows: OsKAPP, AF075603l; ZmKAPP, U81960; and AtKAPP, U09505.
|
|
Filter Binding Assays of OsKID to RLKs and the Phosphorylation of
OsKID by MBP-OsTKD
To determine the protein-binding properties of rice KID, filter
binding assays were performed as described for Arabidopsis and maize
KID (Stone et al., 1994; Braun et al., 1997). OsKID was expressed in
E. coli as a GST fusion protein and
phosphorylated with 32P at the protein kinase A
site. The probe was allowed to interact with the recombinant kinase
domains MBP-RLK5CAT, GST-PK1CAT, and GST-Xa21CAT. The results of the
protein-protein interaction assays showed that GST-OsKID interacted
with MBP-RLK5CAT but not with GST-PK1CAT (Fig.
6), as has also been found for the
Arabidopsis and maize KID (Braun et al., 1997). Furthermore, the
labeled GST-OsKID did not bind to GST, MBP, or GST-Xa21CAT. These
results indicate that OsKID interacts with RLK5, an LRR-RLK from
Arabidopsis, but not with Xa21, an LRR-RLK from rice.
Arabidopsis and maize KID have been shown to interact with TMK1 (Braun
et al., 1997). We wanted to determine whether rice KID also interacted
with OsTMK and whether it was a substrate for phosphorylation by OsTMK.
As shown in Figure 7, MBP-OsTKD phosphorylated GST-OsKID but not GST alone. GST and GST-OsKID did not
autophosphorylate or phosphorylate each other. This indicates that
GST-OsKID is a substrate for MBP-OsTKD and that these proteins interact
in vitro.
View larger version (25K):
[in this window]
[in a new window]
| Figure 7.
Phosphorylation of GST-OsKID. Left, Coomassie
Blue-stained gel. The reaction mixture contained 1.5 µg of GST, 0.5 µg of GST-OsKID, and/or 0.5 µg of MBP-OsTKD, as indicated above the
lanes. The reaction was allowed to proceed for 20 min at room
temperature. The lower bands are proteolytic degradation products.
Right, Autoradiogram of the same gel.
|
|
Because MBP-OsTKD and GST-OsKID are both substrates for
phosphorylation, the level of phosphorylation was determined as a function of protein concentration. Increasing amounts of MBP-OsTKD led
to an increase in autophosphorylation of MBP-OsTKD and an increase in
phosphorylation of GST-OsKID (Fig. 8A).
However, the level of autophosphorylation was greatly increased by
decreasing amounts of GST-OsKID. Autophosphorylation is inhibited by
increasing amounts of inactive kinase (Horn and Walker, 1994;
Williams et al., 1997). To investigate whether inhibition of in vitro
autophosphorylation of MBP-OsTKD was specific to its substrate, a
phosphorylation assay with MBP-OsTKD and GST-OsKID in the presence of
excess GST-OsKID, GST, or BSA was performed (Fig. 8B). Phosphorylation
of MBP-OsTKD and GST-OsKID were both reduced at higher GST-OsKID
concentrations (Fig. 8B, compare lanes 1 and 4). Inhibition of
MBP-OsTKD activity was also observed in the presence of BSA or GST
(Fig. 8B, lanes 2 and 3, respectively), indicating that in vitro
inhibition of OsTMK activity is not specific to its substrate and
that the kinase assay is sensitive to protein concentration.
View larger version (46K):
[in this window]
[in a new window]
| Figure 8.
Phosphorylation activity of MBP-OsTKD. A,
Phosphorylation assay in the presence of different amounts of fusion
proteins as indicated above the lanes. The reaction mixtures were
incubated at room temperature for 20 min. B, Inhibition of MBP-OsTKD
activity as shown by a phosphorylation assay with 500 ng of MBP-OsTKD
and 500 ng of GST-OsKID. Lane 1, No additions; lane 2, 3 µg of GST
added; lane 3, 2.5 µg of BSA added; lane 4, additional 750 ng of
GST-OsKID added. The reaction mixtures were incubated at 30°C
for 20 min. Left, Coomassie Blue-stained gel; right, corresponding
autoradiogram.
|
|
Expression of OsTMK and OsKAPP in Rice
KAPP was shown to interact in vitro with several RLKs (Braun et
al., 1997). To determine whether OsTMK and OsKAPP may interact in vivo,
the expression patterns of these genes were investigated by RNA
gel-blot analysis. High transcript levels of OsTMK were detected in many tissues of rice, particularly in regions containing dividing and elongating cells (Fig. 9).
Lower levels of expression were found in the basal part of the second
youngest leaf blade and in the oldest part of the internode; these
tissues show reduced growth or no growth, respectively. The expression
of OsKAPP showed a similar trend (Fig. 9). Whereas the
transcript levels of OsTMK clearly increased during GA
treatment of stem sections (Fig. 2), the GA-induced accumulation of
OsKAPP mRNA was small, reaching only a 3-fold increase after
24 h (data not shown).
| |
DISCUSSION |
Amino Acid Sequence Comparisons of OsTMK with TMK1 and Other
LRR-RLKs
We identified an LRR-RLK that was highly expressed in growing
regions of deepwater rice. The gene encoding this kinase was named
OsTMK for several reasons: (a) it encodes a putative TMK; (b) its highest amino acid sequence identity is to TMK1 from
Arabidopsis (Chang et al., 1992); and (c) the expression pattern of
OsTMK resembles that of TMK1 in Arabidopsis
(Chang et al., 1992). DNA gel-blot analysis indicated that
TMK1 is a unique gene in Arabidopsis. However, a related
gene (AC000103) showing 53.3% amino acid identity to TMK1 was
identified in the Arabidopsis genome sequence database. DNA gel-blot
analysis of rice genomic DNA and the region corresponding to the OsTMK
kinase domain as a probe also indicated the presence of only one gene
in the rice genome. However, a rice expressed sequence tag (D39936)
with high sequence similarity to part of the LRR domain of OsTMK was
identified recently. Therefore, it is possible that homologs of
OsTMK exist in rice as well.
Arabidopsis AC000103 and TMK1 are likely to be
evolutionarily related. The position of a unique intron (84 nucleotides in both TMK1 and AC000103) is conserved between
the first and the second nucleotide of the codon corresponding to Val
at position 766 in TMK1, immediately following kinase
subdomain VIII (Chang et al., 1992). For OsTMK, the intron
position(s) has not been determined. However, the lane containing
HindIII-digested genomic DNA was expected to show a band of
1.7 kb based on the cDNA sequence. Instead, the band detected was 2 kb,
indicating the presence of an intron in this fragment that spans the
region containing the intron in AC000103 and TMK1.
Based on amino acid sequence similarity, TMK1 is more related to OsTMK
than to AC000103. Several gaps exist in the sequence alignment of TMK1
and AC000103. In Arabidopsis, TMK1 is found in the membrane fraction
and is glycosylated in vivo (Schaller and Bleecker, 1993). Six of the
nine potential glycosylation sites in the presumed extracellular domain
are conserved between OsTMK and TMK1. Between AC000103 and OsTMK or
TMK1, only one and two of the nine potential glycosylation sites,
respectively, are conserved. Furthermore, phylogenetic analysis using
the Clustal W method (Thompson et al., 1994) with the PAM250
residue weight table showed that TMK1 and OsTMK are more related to
each other than to AC000103 (data not shown). The high sequence
conservation and similar expression pattern between two RLKs from a
monocot and a dicot species indicate an important role and functional relatedness for this gene.
Potential Role of OsTMK in GA-Mediated
Growth
Treatment with GA increased the transcript level of
OsTMK in deepwater rice internodes. The expression of this
gene was particularly high in all regions undergoing cell division and
elongation and low in the nongrowing region of the internode. This
suggests a role for this gene in plant growth. Recently, BRI1, an
LRR-RLK that plays a role in brassinosteroid signaling, was identified (Li and Chory, 1997). Because both GA and brassinosteroids are terpenoids, the intriguing possibility arises that an LRR-RLK such as
OsTMK may also be involved in GA signal transduction, as is the case in
brassinosteroid signaling.
Hormones and external stimuli regulate the transcript levels of several
genes encoding RLKs in plants. The mRNA encoding SFR, an
S-locus-like RLK of cauliflower, accumulates after wounding and bacterial infection (Pastuglia et al., 1997). Similarly, the transcript levels of RPK1, which codes for an LRR-RLK in
Arabidopsis, increases after ABA treatment and in response to a variety
of environmental stresses (Hong et al., 1997). Inducible expression of
some receptor kinase genes has been observed in animals as well. The
epidermal and platelet-derived growth factors cause an increase in the
transcript levels of their respective receptors (Clark et al., 1985;
Ericksson et al., 1991). Ligand-induced changes in receptor transcript
levels have also been observed in plants. The mRNA levels for the
putative ethylene receptors NR in tomato (Wilkinson et al., 1995) and
ERS1, ERS2, and ETR2 in Arabidopsis (Hua et al., 1998) increase in
response to ethylene. Therefore, there is precedence for ligands
changing the transcript level of their respective receptors. To
elucidate the function of OsTMK, it will be necessary to determine
whether this RLK is indeed involved in mediating growth and, if so,
whether it is a component of the GA transduction pathway. If the latter
turns out to be the case, it will be interesting to find out whether GA
by itself or GA with an accessory protein can bind to OsTMK and, as a
ligand, induce the accumulation of OsTMK mRNA.
Phosphorylation Characteristics of MBP-OsTKD and the Potential in
Vivo Role of OsKAPP
OsTMK is an active protein kinase that autophosphorylates
primarily on Thr residues and to a lesser extent on Ser residues, as
has been shown for TMK1 (Chang et al., 1992). The autophosphorylation mechanism of MBP-OsTKD is complex and appears to occur via a
higher-order reaction mechanism. Complex autophosphorylation was found
for RLK5 as well (Horn and Walker, 1994). Phosphorylation of the
inactive RLK5 kinase domain by active RLK5 kinase showed that RLK5 uses primarily an intermolecular mechanism. In animals, it is known that
inactive receptor monomers are in equilibrium with active receptor
dimers and that ligand binding stabilizes the active dimeric form
(Ullrich and Schlessinger, 1990). Dimerization (or higher-order
oligomerization) is responsible for activation of the intrinsic protein
kinase activity and for autophosphorylation; both processes are
mediated by an intermolecular mechanism (Lemmon and Schlessinger,
1994).
One candidate for a signal transduction component downstream of several
RLKs is KAPP. We isolated a rice ortholog of KAPP using a
heterologous cloning approach. Similar to Arabidopsis (Stone et al.,
1994) and maize (Braun et al., 1997) KAPP, the rice gene
encodes a putative carboxy-terminal 2C type protein phosphatase with a
potential N-terminal membrane anchor and a centrally located KID.
Because the phosphatase domain of OsKAPP carries the 11 structurally
conserved motifs of type 2C protein phosphatases, it is likely to be an
active phosphatase. Previous studies have shown that Arabidopsis and
maize KIDs interact in vitro with the protein kinase domain of several
RLKs, including RLK5 and TMK1, but not with ZmPK1 (Braun et al., 1997).
Similarly, rice KID interacts with the kinase domain of RLK5 and OsTMK
but not with the kinase domain of ZmPK1 and Xa21. These results suggest that KAPP-mediated signaling is conserved across dicot and monocot plant species. To date, the in vivo role of these interactions is
unknown. In the CLV1-signaling pathway, KAPP appears to interact with
this LRR-RLK in vivo (Williams et al., 1997; Stone et al., 1998).
However, CLV1 is expressed only in the shoot and floral meristems (Clark et al., 1997). Because of its ubiquitous expression in
the plant and its interaction with several RLKs, KAPP is likely to
function in a number of other signaling pathways. The expression pattern of OsKAPP showed a trend similar to that of
OsTMK, which, along with the in vitro phosphorylation of
GST-OsKID by MBP-OsTDK, may be indicative of in vivo interaction of
these two proteins and a role for OsKAPP in OsTMK-mediated signaling.
| |
FOOTNOTES |
1
This work was supported by grants from the
National Science Foundation (no. IBN9722915 to H.K.), the U.S.
Department of Energy (no. DE-FG02-91ER20021 to H.K.), and the National
Institutes of Health (no. GM47907 to P.C.R.) and by a Visiting
Fellowship for Interlaboratory Research from the Multi-Institutional
Plant Protein Phosphorylation Group (to W.-Y.S.).
2
Present address: Department of Plant Breeding
and Biometry, Cornell University, Ithaca, NY 14853-1673.
3
Present address: Department of Plant Pathology,
University of Florida, Gainesville, FL 32611-0680.
4
Present address: Institut für Allgemeine
Botanik, AMP II, Universität Hamburg, D-22609 Hamburg, Germany.
*
Corresponding author; e-mail hkende{at}pilot.msu.edu; fax
1-517-353-9168.
Received October 28, 1998;
accepted February 25, 1999.
| |
ABBREVIATIONS |
Abbreviations:
GST, glutathione S-transferase.
KAPP, kinase-associated type 2C protein phosphatase.
KID, kinase
interaction domain.
LRR, Leu-rich repeat.
MBP, maltose-binding protein.
RLK, receptor-like kinase.
TKD, transmembrane kinase domain.
TMK, transmembrane kinase.
| |
ACKNOWLEDGMENTS |
We are grateful to Dr. E.A. Maher (University of Wisconsin,
Madison) for help with the initial phosphorylation experiments and for
critically reading the manuscript and to Dr. John Walker (University of
Missouri, Columbia) and Dr. David Braun (University of California,
Berkeley) for helpful discussions and the use of the maize KID probe.
| |
LITERATURE CITED |
Altschul SF,
Gish W,
Miller W,
Myers EW,
Lipman DJ
(1990)
Basic local alignment search tool.
J Mol Biol
215:
403-410
[CrossRef][ISI][Medline]
Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA,
Struhl K (1987) Current Protocols in Molecular Biology. Wiley, New
York
Bethke PC,
Schuurink R,
Jones RL
(1997)
Hormonal signalling in cereal aleurone.
J Exp Bot
48:
1337-1356
Bork P,
Brown MP,
Hegyi H,
Schultz J
(1996)
The protein phosphatase 2C (PP2C) superfamily: detection of bacterial homologues.
Protein Sci
5:
1421-1425
[Abstract]
Boyle WL,
van der Geer P,
Hunter T
(1991)
Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates.
Methods Enzymol
201:
110-149
[ISI][Medline]
Braun DM,
Stone JM,
Walker JC
(1997)
Interaction of the maize and Arabidopsis kinase interaction domains with a subset of receptor-like protein kinases: implications for transmembrane signaling in plants.
Plant J
12:
83-95
[CrossRef][ISI][Medline]
Braun DM,
Walker JC
(1996)
Plant transmembrane receptors: new pieces in the signaling puzzle.
Trends Biochem Sci
21:
70-73
[CrossRef][ISI][Medline]
Catling D (1992) Rice in Deepwater. MacMillan Press, London
Chang C,
Kwok SF,
Bleecker AB,
Meyerowitz EM
(1993)
Arabidopsis ethylene-response gene ETR1: similarity of product to two-component regulators.
Science
262:
539-544
[Abstract/Free Full Text]
Chang C,
Schaller GE,
Patterson SE,
Kwok SF,
Meyerowitz EM,
Bleecker AB
(1992)
The TMK1 gene from Arabidopsis codes for a protein with structural and biochemical characteristics of a receptor protein kinase.
Plant Cell
4:
1263-1271
[Abstract/Free Full Text]
Clark AJL,
Ishii S,
Richert N,
Merlino GT,
Pastan I
(1985)
Epidermal growth factor regulates the expression of its own receptor.
Proc Natl Acad Sci USA
82:
8374-8378
[Abstract/Free Full Text]
Clark SE,
Williams RW,
Meyerowitz EM
(1997)
The CLAVATA1 gene encodes a putative receptor kinase that controls shoot and floral meristem size in Arabidopsis.
Cell
89:
575-585
[CrossRef][ISI][Medline]
Ericksson A,
Nistér M,
Leveen P,
Westermark B,
Heldin C-H,
Claesson-Welsh L
(1991)
Induction of platelet-derived growth factor - and -receptor mRNA and protein by platelet-derived growth factor BB.
J Biol Chem
266:
21138-21144
[Abstract/Free Full Text]
Gilroy S,
Jones RL
(1994)
Perception of gibberellin and abscisic acid at the external face of the plasma membrane of barley (Hordeum vulgare L.) aleurone protoplasts.
Plant Physiol
104:
1185-1192
[Abstract]
Gubler F,
Kalla R,
Roberts JK,
Jacobsen JV
(1995)
Gibberellin-regulated expression of a myb gene in barley aleurone cells: evidence for Myb transactivation of a high-pI alpha-amylase gene promoter.
Plant Cell
7:
1879-1891
[Abstract]
Hanks SK,
Quinn AM
(1991)
Protein kinase catalytic domain sequence database: identification of conserved features of primary structure and classification of family members.
Methods Enzymol
200:
38-62
[ISI][Medline]
Hoffmann-Benning S,
Kende H
(1992)
On the role of abscisic acid and gibberellin in the regulation of growth in rice.
Plant Physiol
99:
1156-1161
[Abstract/Free Full Text]
Hong SW,
Jon JH,
Kwak JM,
Nam HG
(1997)
Identification of a receptor-like protein kinase gene rapidly induced by abscisic acid, dehydration, high salt, and cold treatments in Arabidopsis thaliana.
Plant Physiol
113:
1203-1212
[Abstract]
Hooley R,
Beale MH,
Smith SJ
(1991)
Gibberellin perception at the plasma membrane of Avena fatua aleurone protoplasts.
Planta
183:
274-280
[ISI]
Horn MA,
Walker JC
(1994)
Biochemical properties of the autophosphorylation of RLK5, a receptor-like protein kinase from Arabidopsis thaliana.
Biochim Biophys Acta
1208:
65-74
[CrossRef][Medline]
Hua J,
Sakai H,
Nourizadeh S,
Chen QG,
Bleecker AB,
Ecker JR,
Meyerowitz EM
(1998)
EIN4 and ERS2 are members of the putative ethylene receptor gene family in Arabidopsis.
Plant Cell
10:
1321-1332
[Abstract/Free Full Text]
Jacobsen SE,
Binkowski KA,
Olszewski NE
(1996)
SPINDLY, a tetratricopeptide repeat protein involved in gibberellin signal transduction in Arabidopsis.
Proc Natl Acad Sci USA
93:
9292-9296
[Abstract/Free Full Text]
Jones HD,
Smith SJ,
Desikan R,
Plakidou-Dymock S,
Lovegrove A,
Hooley R
(1998)
Heterotrimeric G-proteins are implicated in gibberellin induction of -amylase gene expression in wild oat aleurone.
Plant Cell
10:
245-253
[Abstract/Free Full Text]
Kakimoto T
(1996)
CKI1, a histidine kinase homolog implicated in cytokinin signal transduction.
Science
274:
982-985
[Abstract/Free Full Text]
Kende H,
van der Knaap E,
Cho H-T
(1998)
Deepwater rice: a model plant to study stem elongation.
Plant Physiol
118:
1105-1110
[Free Full Text]
Kim J-K,
Wu R
(1993)
A rice (Oryza sativa L.) cDNA encodes a protein sequence homologous to the eukaryotic ribosomal 5S RNA-binding protein.
Plant Mol Biol
23:
409-413
[CrossRef][Medline]
Kobe B,
Deisenhofer J
(1994)
The leucine-rich repeat: a versatile binding motif.
Trends Biochem Sci
19:
415-421
[CrossRef][ISI][Medline]
Lemmon MA,
Schlessinger J
(1994)
Regulation of signal transduction and signal diversity by receptor oligomerization.
Trends Biochem Sci
19:
459-463
[CrossRef][ISI][Medline]
Li J,
Chory J
(1997)
A putative leucine-rich repeat receptor kinase involved in brassinosteroid signal transduction.
Cell
90:
929-938
[CrossRef][ISI][Medline]
Mu J-H,
Lee H-S,
Kao T-H
(1994)
Characterization of a pollen-expressed receptor-like kinase gene of Petunia inflata and the activity of its encoded kinase.
Plant Cell
6:
709-721
[Abstract/Free Full Text]
Pastuglia M,
Roby D,
Dumas C,
Cock JM
(1997)
Rapid induction by wounding and bacterial infection of an S gene family receptor-like kinase gene in Brassica oleracea.
Plant Cell
9:
49-60
[Abstract]
Peng J,
Carol P,
Richards DE,
King KE,
Cowling RJ,
Murphy GP,
Harberd NP
(1997)
The Arabidopsis GAI gene defines a signaling pathway that negatively regulates gibberellin responses.
Genes Dev
11:
3194-3205
[Abstract/Free Full Text]
Puissant C,
Houdebine LM
(1990)
An improvement of the single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction.
Biotechniques
8:
148-149
[ISI][Medline]
Raskin I,
Kende H
(1984a)
Regulation of growth in stem sections of deep-water rice.
Planta
160:
66-72
[CrossRef][ISI]
Raskin I,
Kende H
(1984b)
Role of gibberellin in the growth response of submerged deep water rice.
Plant Physiol
76:
947-950
[Abstract/Free Full Text]
Ritchie S,
Gilroy S
(1998)
Calcium-dependent protein phosphorylation may mediate the gibberellic acid response in barley aleurone.
Plant Physiol
116:
765-776
[Abstract/Free Full Text]
Sauter M,
Kende H
(1992)
Gibberellin-induced growth and regulation of the cell division cycle in deepwater rice.
Planta
188:
362-368
[ISI]
Schaller GE,
Bleecker AB
(1993)
Receptor-like kinase activity in membranes of Arabidopsis thaliana.
FEBS Lett
333:
306-310
[CrossRef][Medline]
Schaller GE,
Bleecker AB
(1995)
Ethylene-binding sites generated in yeast expressing the Arabidopsis ETR1 gene.
Science
270:
1809-1811
[Abstract/Free Full Text]
Schmidt EDL,
Guzzo F,
Toonen MAJ,
de Vries SC
(1997)
A leucine-rich repeat containing receptor-like kinase marks somatic plant cells competent to form embryos.
Development
124:
2049-2062
[Abstract]
Silverstone AL,
Ciampaglio CN,
Sun T-P
(1998)
The Arabidopsis RGA gene encodes a transcriptional regulator repressing the gibberellin signal transduction pathway.
Plant Cell
10:
155-169
[Abstract/Free Full Text]
Song W-Y,
Wang G-L,
Chen L-L,
Kim H-S,
Pi L-Y,
Holsten T,
Gardner J,
Wang B,
Zhai W-X,
Zhu L-H,
and others
(1995)
A receptor kinase-like protein encoded by the rice disease resistance gene, Xa21.
Science
270:
1804-1806
[Abstract/Free Full Text]
Steber CM,
Cooney SE,
McCourt P
(1998)
Isolation of the GA-response mutant sly1 as a suppressor of ABI1-1 in Arabidopsis thaliana.
Genetics
149:
509-521
[Abstract/Free Full Text]
Stone JM,
Collinge MA,
Smith RD,
Horn MA,
Walker JC
(1994)
Interaction of a protein phosphatase with an Arabidopsis serine-threonine receptor kinase.
Science
266:
793-795
[Abstract/Free Full Text]
Stone JM,
Trotochaud AE,
Walker JC,
Clark SE
(1998)
Control of meristem development by CLAVATA1 receptor kinase-associated protein phosphatase interactions.
Plant Physiol
117:
1217-1225
[Abstract/Free Full Text]
Stünzi JT,
Kende H
(1989)
Gas composition in the internal air spaces of deepwater rice in relation to growth induced by submergence.
Plant Cell Physiol
30:
49-56
[Abstract/Free Full Text]
Swain SM,
Olszewski NE
(1996)
Genetic analysis of gibberellin signal transduction.
Plant Physiol
112:
11-17
[Medline]
Thompson JD,
Higgins DG,
Gibson TJ
(1994)
CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice.
Nucleic Acids Res
22:
4673-4680
[Abstract/Free Full Text]
Torii KU,
Mitsukawa N,
Oosumi T,
Matsuura Y,
Yokoyama R,
Whittier RF,
Komeda Y
(1996)
The Arabidopsis ERECTA gene encodes a putative receptor protein kinase with extracellular leucine-rich repeats.
Plant Cell
8:
735-746
[Abstract]
Ullrich A,
Schlessinger J
(1990)
Signal transduction by receptors with tyrosine kinase activity.
Cell
61:
203-212
[CrossRef][ISI][Medline]
Van der Knaap E,
Jagoueix S,
Kende H
(1997)
Expression of an ortholog of replication protein A1 (RPA1) is induced by gibberellin in deepwater rice.
Proc Natl Acad Sci USA
94:
9979-9983
[Abstract/Free Full Text]
Van der Knaap E,
Kende H
(1995)
Identification of a gibberellin-induced gene in deepwater rice using differential display of mRNA.
Plant Mol Biol
28:
589-592
[CrossRef][Medline]
Van der Knaap E,
Sauter M,
Wilford R,
Kende H
(1996)
Identification of a gibberellin-induced receptor-like kinase in deepwater rice (PGR96-100).
Plant Physiol
112:
1397-1398
[Medline]
Walker JC
(1993)
Receptor-like protein kinase genes of Arabidopsis thaliana.
Plant J
3:
451-456
[CrossRef][ISI][Medline]
Wang XQ,
Zafian P,
Choudhary M,
Lawto
Top
Abstract
Introduction