Plant Cell Physiol. 41(11): 1243–1250 (2000)
JSPP © 2000
Involvement of Calcium-Dependent Protein Kinase in Rice (Oryza sativa L.)
Lamina Inclination Caused by Brassinolide
Guangxiao Yang and Setsuko Komatsu 1
Department of Molecular Genetics, National Institute of Agrobiological Resources, Tsukuba, 305-8602 Japan
;
Promotive effect of brassinolide (BL) on green lamina
inclination was concentration-dependent when excised rice
(Oryza sativa L.) lamina was floated on BL solution under
continuous light conditions. Protein kinase inhibitor staurosporine and Ca2+ channel blocker LaCl3 could completely,
while Ca2+ chelator EGTA could partially inhibit the lamina inclination caused by BL. Two protein kinases with
apparent molecular masses of 45 and 54 kDa were detected
using an in-gel kinase assay with histone III-S as a substrate. In particular, the changes in 45 kDa protein kinase
activity correlated with lamina inclination caused by BL.
The 45 kDa kinase activity was inhibited by Ca2+ chelator
EGTA, protein kinase inhibitor, staurosporine and calmodulin antagonist W-7. Therefore, this 45 kDa protein kinase
was identified as a Ca2+-dependent protein kinase (CDPK).
Patterns of 2-dimensional PAGE after in vitro phosphorylation of crude extracts showed that the phosphorylation of
56 and 41 kDa proteins, which was Ca2+ -dependent, was
strongly increased by BL treatment. These results suggested that CDPK and Ca2+-dependent protein phosphorylation are involved in BL-induced rice lamina inclination.
Key words: Brassinolide — CDPK — Protein kinase —
Protein phosphorylation — Rice (Oryza sativa L.) — Lamina
inclination.
nism by which BRs regulate the growth and development of
plants, it is necessary to identify components of the BR signal
transduction pathway. Protein kinase and protein phosphorylation have been shown to play an important role in the response
to various plant hormones. For example, auxin treatment increased the Ca2+ -dependent protein kinase (CDPK) gene expression in mungbean cuttings (Botella et al. 1996). GA3 has
been shown to affect CDPK activity in rice (Abo-El-Saad and
Wu 1995). Protein phosphorylation and dephosphorylation are
required for the induction of ACC oxidase by ethylene (Kwak
and Lee 1997). In pea, ABA-mediated dehydrin gene expression was found to be dependent on protein phosphorylation and
dephosphorylation (Hey et al. 1997). Changes in protein kinase
activities and protein phosphorylations were observed in membrane fractions from rice seeds treated with ABA (Komatsu et
al. 1997). Thus, we supposed that protein kinases and protein
phosphorylation are involved in the BR signal transduction
pathway. We initiated studies on BR signal transduction employing a highly sensitive and BR-specific rice (Oryza sativa
L.) lamina inclination bioassay. Brassinolide (BL) induced rice
lamina inclination and its relation to the changes in protein kinase activity and protein phosphorylation were investigated.
Here, we present our results, which indicate that a 45 kDa
CDPK and Ca2+-dependent protein phosphorylation are involved in BL-induced rice lamina inclination.
Abbreviations: BL, brassinolide; BR, brassinosteroid; CDPK,
Ca2+-dependent protein kinase.
Introduction
Brassinosteroids (BRs) are a group of naturally occurring
plant steroids with structural similarities to insect and animal
steroid hormones (Mandava 1988). Exogenous application of
BRs to plant tissues at nanomolar to micromolar concentrations evokes cell elongation, proliferation, differentiation, organ bending and also affects a number of other physiological
processes (Sasse 1997). Recent molecular genetic studies of
BR action have revealed that these compounds regulate gene
expression and are essential for normal plant growth and development (Arteca et al. 1988, Li et al. 1996, Szekeres et al. 1996,
Zurek and Clouse 1994). However, to understand the mecha1
Materials and Methods
Plant material and lamina inclination assay
Rice (Oryza sativa L. cv. Nipponbare) was grown under white
fluorescent light (about 600 mol m–2 s–1, 12 h light period d–1) at
25C and 75% relative humidity in a growth chamber. The second leaf
lamina segments were prepared from 1-week-old seedlings according
to the rice lamina inclination test as described (Wada et al. 1981). Leaf
lamina segments were floated on 10 ml distilled water in 6015 mm
Petri dishes containing BL alone, or a combination of BL and various
chemicals as indicated in the text.
Chemicals
BL, IAA, GA3, staurosporine, ethylene glycol-bis (-aminoethyl
ether)-N,N,N,N-tetraacetic acid (EGTA), LaCl3 and CoCl2 were purchased from Wako (Osaka). N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), N-(6-aminohexyl)-5-chloro-2-naphthalenesulfonamide (W-5), 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7)
and N-(2-[methylamino]ethyl)-5-isoquinolinylsulfonamide (H-8) were
products of Seikagaku Kogyo (Tokyo).
Corresponding author: E-mail, [email protected]; Fax, +81-298-38-7408; Phone, +81-298-38-7446.
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CDPK in brassinolide-induced lamina inclination
Preparation of the protein extract
The following procedures were carried out at 4C. A portion of
leaf lamina segments (100 mg) was homogenized in a mortar with a
pestle in 200 l extraction buffer containing 50 mM Tris-HCl (pH 7.4),
150 mM NaCl, 1% (v/v) Triton X-100, 1% (w/v) sodium deoxycholate, 1 mM EGTA, 5 M sodium vanadate and 1 mM phenylmethylsulfonyl fluoride (PMSF). The homogenate was centrifuged at 15,000g
for 5 min and the supernatant was used as the protein extract.
Preparation of subcellular fraction
The following procedures were carried out at 4C. Leaf lamina
segments (100 mg) were homogenized in a mortar with a pestle in
200 l homogenization buffer containing 20 mM Tris-HCl (pH 7.5),
0.25 M sucrose, 10 mM EGTA, 1 mM dithiothreitol (DTT) and 1 mM
PMSF. The homogenates were centrifuged at 3,300g for 5 min. The
supernatants were centrifuged at 100,000g for 15 min and the cytosolic fraction was obtained by collecting the supernatant. The pellet
was resuspended in 100 l homogenization buffer and washed by centrifugation at 100,000g for 15 min. The pellet was resuspended in
40 l membrane solubilizing buffer containing 1% Triton X-100,
20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 50 mM 2-mercaptoethanol
and solubilized for 30 min on ice. The membrane fraction was obtained from the supernatant after centrifugation at 100,000g for 7 min
(Komatsu and Hirano 1992).
In-gel kinase assay
Protein extracts (20 g) were separated by 15% SDS-polyacrylamide gel containing 2 mg ml–1 histone III-S (Sigma, St. Louis, MO,
U.S.A.) as substrate in the separating gel. After electrophoresis, SDS
was removed by washing the gel with a buffer containing 50 mM TrisHCl (pH 8.0) and 20% 2-propanol for 1 h, and then washed with buffer A containing 50 mM Tris-HCl (pH 8.0) and 5 mM 2-mercaptoethanol for 1 h. The separated proteins were denaturated in buffer A containing 6 M guanidine-HCl for 1 h and renatured in buffer A
containing 0.04% (w/v) Tween-40 at 4C for 12 h. The gels were
equilibrated in 40 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 2 mM DTT
and 0.2 mM CaCl2 for 30 min at room temperature. The reaction was
initiated by addition of 5 M [-32P]ATP (110 TBq mmol–1, Amersham, Buckinghamshire, U.K.) and incubated at 30C for 30 min. The
reaction was stopped by extensive gel washing with 5% (w/v) trichloroacetic acid containing 1% (w/v) potassium PPi until background radioactivity decreased. The gels were stained with Coomassie Brilliant
Blue R-250 (CBB), destained, dried and exposed to X-ray film
(Kondak, Rochester, NY, U.S.A.) at –80C for 3 d (Komatsu et al.
1996).
In vitro protein phosphorylation
5 l protein extracts (40 g) were incubated in 25 l reaction
mixture containing 20 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 39 M
[-32P]ATP (110 TBq mmol–1). The reaction mixture was incubated for
10 min at 30C and terminated by cooling to 0C. After in vitro protein phosphorylation, the sample was added to a lysis buffer containing
8 M urea, 2% Triton X-100, 2% ampholine (Phamarcia, Solna, Sweden), 10% polyvinylpyrrolidone and subjected to 2D-PAGE (O’Farrel
1975). Proteins were separated in the first dimension by isoelectric focusing and in the second dimension by 15% SDS-PAGE. The gels
were stained with CBB, destained, dried and exposed on X-ray film at
–80C for 2 d (Komatsu and Hirano 1993).
Results
Effect of BL on rice lamina inclination
Laminae, excised from 1-week-old seedlings, were treat-
Fig. 1 Effect of phytohormones on lamina inclination. (A) Laminae,
excised from 1-week-old seedlings grown in 12 h light/12 h dark
growth chamber at 25C, were floated on the distilled water containing BL (0.001, 0.01, 0.1, 1, 10 M), IAA (1, 10 M) and GA3 (1,
10 M) respectively, and incubated under continuous light at 25C for
48 h. (B) The time course of lamina inclination treated with 10 M
BL. The inclination angle between the lamina and its leaf sheath was
measured using a circular protractor. The mean of three experimentsSE are shown.
ed with BL under continuous light at 25C for 48 h. The extent
of lamina inclination caused by different concentrations of BL
is shown in Fig. 1. BL could promote green lamina inclination
and the promotion was concentration-dependent. A measurable promotive effect occurred at 1 nM concentration, and there
was a 78% increase in lamina inclination at 1 M BL as compared to the water control. For comparison, the effects of IAA
and GA3 on lamina inclination were also examined. Even at
CDPK in brassinolide-induced lamina inclination
1245
the water control. It is believed that La3+ competes externally
with Ca2+ for plasma membrane Ca2+ channels (Tester 1990)
but evidence shows that at high concentrations, it also affects
intracellular channels (Knight et al. 1996). Thus, Ca2+ from
both extracellular and intracellular sources may be involved in
BL signal transduction. Then Ca2+ was artificially increased in
cytoplasm without addition of BL, to see whether it can cause
lamina bending. After 48 h treatment with 5 mM CaCl2 and
5 M Ca2+ ionophore A23187, no significant difference of lamina inclination was observed between the water control and the
treatment (data not shown), which indicates that an increase in
Ca2+ in cytoplasm alone is not sufficient to induce lamina inclination.
Fig. 2 Effects of a protein kinase inhibitor, Ca2+ chelator and Ca2+
channel blocker upon lamina inclination caused by BL. Together with
10 M BL, was added 10 M staurosporine (ST), 5 mM LaCl3 (LA) or
5 mM EGTA. The inclination angle was measured 48 h after each
treatment. The mean of three experimentsSE are shown.
10 M neither IAA nor GA3 caused significant lamina inclination (Fig. 1A). Thus, the rice lamina inclination bioassay is
highly sensitive and BL-specific under this experimental condition. To better understand the kinetics of BL action on rice lamina inclination, a time-course experiment was conducted, treating lamina segment with 10 M BL for up to 48 h. BL required
6 h to give a measurable effect on lamina inclination, pronounced inclination occurred at later times, and constant inclination continued until 48 h after BL treatment (Fig. 1B).
Effects of staurosporine, EGTA and lanthanum chloride on
lamina inclination caused by BL
To assess the possible involvement of Ca2+ and protein kinase in lamina inclination caused by BL, the effects of protein
kinase inhibitor staurosporine, Ca2+ chelator EGTA and Ca2+
channel blocker LaCl3 upon lamina inclination caused by BL
were examined. Ten M staurosporine, 5 mM LaCl3 and 5 mM
EGTA were added with 10 M BL at the same time. Inclination angles were measured 48 h after each treatment. As shown
in Fig. 2, 10 M staurosporine completely inhibited the increase in lamina inclination caused by BL, indicating the involvement of protein kinase in BL-induced lamina inclination.
EGTA binds extracellular Ca2+ and makes it unable to enter the
cytoplasm. Five mM EGTA inhibited 60% of the inclination increased by BL, while 5 mM LaCl3 not only completely nullified the effect of BL on the lamina inclination, but also had
very strong inhibition on the basal bending that had occurred in
Changes in kinase activity as affected by BL treatment
In order to identify specific kinase activities affected by
BL treatment, in-gel kinase assay was performed. In this experiment, cytosolic and membrane fractions, prepared from lamina treated with 10 M BL for 48 h, were separated by SDSpolyacrylamide gel containing histone III-S as a substrate. [32
P]ATP was added to visualize phosphorylation. The 17, 45
and 54 kDa proteins in cytosol, 45 and 54 kDa proteins in
membrane fraction, respectively, had kinase activities (Fig. 3A,
B). Activities of 17 and 45 kDa kinase in the cytosol fraction
from BL-treated lamina were greatly increased compared with
that of the water control, while there was no obvious change in
the kinase activity between control and BL treatment in the
membrane fraction (Fig. 3A, B). To examine the changes in the
kinase activities in detail during BL-induced lamina inclination, laminae were collected after treatment with 10 M BL for
1, 3, 6, 12, 24 and 48 h, respectively. The activity of 45 kDa kinase in the cytosolic fraction appeared to increase 1 h after BL
treatment and this increase became more pronounced after 12 h
(Fig. 3C). The increase in activity of 17 kDa kinase occurred
much later. An obvious difference was observed 12 h after BL
treatment (Fig. 3C). In contrast, there was no significant
change in kinase activities in the membrane fraction during BL
treatment (Fig. 3D). This shows that the changes in the kinase
activity, especially the 45 kDa kinase in the cytosolic fraction,
are in good agreement with the lamina inclination caused by
BL treatment.
Characterization of the 45 kDa kinase as a CDPK
In order to characterize the kinase affected by BL, Ca2+dependency of histone III-S phosphorylation by cytosol fraction from laminae treated with BL was tested. The activities of
the 17, 45 and 54 kDa kinases in the cytosol fraction from BLtreated laminae were observed in the presence of Ca2+ using
histone III-S as a substrate. The activities of 45 and 54 kDa kinases was completely inhibited by EGTA, while no obvious influence on the 17 kDa kinase activity was observed (Fig. 4A, 1
and 2). Also, only the 17 kDa kinase activity could be detected
in the presence of EGTA when using MBP as a substrate (Fig.
4A, 3). In the absence of substrate in the gel, the three kinase
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CDPK in brassinolide-induced lamina inclination
Fig. 3 In-gel kinase assay to detect the changes in kinase activity in lamina affected by BL. Laminae were floated on the distilled water containing 10 M of BL or distilled water alone as a control. Cytosolic (A) and membrane (B) fractions were prepared from lamina treated with BL for
48 h. Time course changes in kinase activities were measured (C, D). Proteins were separated by 15% SDS-polyacrylamide containing 2 mg ml–1
histone III-S as a substrate. The in-gel kinase assay was performed in the presence of 0.2 mM CaCl2.
activities could be still observed in the presence Ca2+, but the
45 and 54 kDa kinases had lower activity (Fig. 4A, 4). This
was obviously due to their autophosphorylation.
Further analysis on the in vitro effects of some protein kinase inhibitors and calmodulin antagonist on these kinase activities in the presence of Ca2+ was carried out (Fig. 4B). Staurosporine, a general protein kinase inhibitor, at 10 mM
concentration dramatically inhibited the 45 kDa kinase activity. H-7, an inhibitor of protein kinase C (Hidaka et al. 1984), at
100 mM concentration partially blocked the 45 kDa kinase activity. H-8, an inhibitor of cyclic nucleotide-dependent protein
kinase, showed much stronger inhibition on the 45 kDa kinase
activity at the same concentration. In the presence of W-7, a
potent calmodulin antagonist, the 45 kDa kinase activity was
CDPK in brassinolide-induced lamina inclination
1247
Fig. 4 Characterization of protein kinase affected by BL. Cytosolic fraction was prepared from lamina treated with 10 M BL. (A) Histone III-S
(1 and 2) and myelin basic protein (3) were used as a substrates for the in-gel kinase assay. No substrate was added to detect kinase autophosphorylation in the gel (A 4). The reaction mixture contained either 0.2 mM CaCl2 or 4 mM EGTA as indicated. (B) In vitro effects of Ca2+, EGTA, protein kinase inhibitor and calmodulin antagonist upon protein kinase activity. To each reaction mixture, was added 4 mM EGTA, 1 M
staurosporine (ST), 100 M H-7, 100 M H-8, 100 M W-5 or 100 M W-7. All reaction mixtures contained 0.2 mM CaCl2 except those for
EGTA and the water control. Histone III-S was used as a substrate for the in-gel kinase assay.
strongly inhibited, while W-5, a close structural analog of W-7,
but less effective, had a smaller inhibitory effect than W-7
when applied at the same concentration. From the above results, it can be concluded that the 45 kDa kinase is a CDPK.
Since protein kinase inhibitors used had no obvious influence
on the 17 kDa kinase activity, it should not be a protein kinase.
Its molecular mass and nature detected by the in-gel kinase as-
say were the same as previously reported and it was found to
be nucleoside diphosphate kinase (NDP kinase) using antiNDP kinase antibody (Hamada et al. 1999). NDP kinase from
various origins is auto-phosphorylated at the histidine residue,
and the phosphoric group can be transferred to histone III-S
(Moisyadi et al. 1994).
1248
CDPK in brassinolide-induced lamina inclination
2 corresponding to 56 and 41 kDa respectively, were observed
only in BL-treated lamina but not in the water control (Fig. 6A,
B). In the presence of 0.2 mM CaCl2 in the reaction mixture,
these two phosphoproteins appeared only weakly in the control compared with that in the BL-treated lamina (Fig. 6C, D).
In addition, another two phosphoproteins marked as 3 and 4
corresponding to about 17 kDa and 10 kDa were more intensified by Ca2+ in the BL-treated lamina than in the control (Fig.
6C, D). Most of the protein phosphorylation was strongly inhibited in the presence of 4 mM EGTA in the reaction mixture
(Fig. 6E, F).
Discussion
Fig. 5 Effects of BL, IAA and CoCl2 on lamina inclination and on
protein kinase activities. Two hundred M CoCl2 was added either
alone or with 10 M BL or with 10 M BL and 10 M IAA. The ingel kinase assay was performed in the presence of 0.2 mM CaCl2 using
histone III-S as a substrate. The inclination angle was measured 48 h
after each treatment. The mean of three experimentsSE are shown. 1,
water; 2, BL; 3, CoCl2; 4, BL + IAA; 5, BL + CoCl2; 6, BL + IAA +
CoCl2.
Influences of interaction of BL with IAA and ethylene on lamina
inclination and CDPK activity
A synergistic interaction between 10 mM BL and 10 mM
IAA was observed in the present experiment (Fig. 5A). CoCl2,
an inhibitor of ACC oxidase, not only completely inhibited the
increase in lamina inclination caused by BL, but also showed
some inhibitory effect on the basal bending of the water control
when applied at a concentration of 200 mM. On the other hand,
CoCl2 partially inhibited the bending caused by BL and IAA cotreatment (Fig. 5A). An in-gel kinase assay showed that among
all treatments, the activity of the 45 kDa CDPK was the highest
in the lamina co-treated with BL and IAA, which showed a
good correlation with the degree of lamina inclination (Fig. 5B).
CoCl2 inhibited lamina inclination, and the activity of the
45 kDa CDPK as well. These results confirmed the involvement of CDPK in BL-induced lamina inclination and also indicated a role for ethylene in BL signaling in lamina inclination.
In vitro protein phosphorylation affected by BL
In order to assess changes in protein phosphorylation occurring as a result of BL treatment of excised lamina, protein
extracts were phosphorylated and the labeled phosphoproteins
were separated by 2D-PAGE. Phosphoproteins marked as 1 and
In the present study, the rice lamina inclination bioassay
system was adopted to explore BR signal transduction. The results suggested the involvement of protein kinase, in particular
CDPK and its catalyzed protein phosphorylation in the signal
transduction pathway of BRs.
Lamina inclination, which resembles the epinasty phenomenon caused by ethylene, is the result of the greater cell expansion of adaxial cells relative to the adorsal cell in the joint
region (Takeno and Pharis 1982, Cao and Chen 1995). A
change in cell wall extensibility or loosening is necessary for
cell expansion (Campbell and Braam 1999). Xyloglucan endotransglycosylase, a cell wall-loosening enzyme has been
shown to be up-regulated by BL (Uozu et al. 2000) in rice. Although the molecular basis for cell wall modifications is in a
large part unknown, it is possible that BL and its induced ethylene might work in sequence or together, resulting in lamina inclination.
Ca2+ functions as a second messenger in a variety of plant
responses. In order to show the involvement of Ca2+ in a given
process, Ca2+ chelator and Ca2+ channel blocker have been widely used in plants to block the Ca2+ -mediated processes (Hepler
and Wayne 1985, Kwak and Lee 1997, Katou et al. 1999). Treatments with Ca2+ chelator EGTA partially, while Ca2+ channel
blocker LaCl3 completely inhibited lamina inclination caused by
BL, suggesting that Ca2+ influx or probably Ca2+ release from internal sources may play a role in the BL signaling.
In-gel kinase assay has been proved useful to detect kinases as well as to study their roles in several physiological processes of plants (Abo-El-Saad and Wu 1995, Komatsu et al.
1996, Karibe and Komatsu 1998, Katou et al. 1999). Using an
in-gel kinase assay, we found that a 45 kDa kinase activity in
cytosol fraction from BL-treated lamina increased, which coincided with the lamina inclination. This 45 kDa kinase was further characterized as a CDPK. This indicated that CDPK is involved in lamina inclination caused by BL. Plant CDPKs have
been found in cytosol (Putnam-Evans et al. 1990) or associated with chromatin (Robert and Harmon 1993), membrane
(Abo-El-Saad and Wu 1995) and the cytoskeleton (PutnamEvans et al. 1989). This multiple subcellular localization of
CDPKs also suggested that they have multiple functions. For
CDPK in brassinolide-induced lamina inclination
1249
Fig. 6 In vitro effect of Ca2+ upon protein phosphorylation in lamina joint treated with BL. 2D-PAGE was carried out after in vitro phosphorylation of crude protein extracts from lamina treated with distilled water as the control (A, C, E) and with 10 mM BL (B, D, F) for 48 h. The reaction
mixture contained either 0.2 mM CaCl2 (C, D), 4 mM EGTA (E, F), or with water alone (A, B).
example, the activity of a rice seed membrane CDPK is increased by gibberellin (Abo-El-Saad and Wu 1995). A CDPK
has been partially purified from rice leaves, and found to phosphorylate three endogenous proteins as detected by in vitro
phosphorylation on 2D-PAGE (Karibe et al. 1996). Two rice
cDNAs (OSCPK 2 and OSCPK 11) encoding for putative
CDPK have been cloned and characterized. ABA treatment inhibited the elongation of rice coleoptile and OSCPK 11 mRNA
expression, which suggests the possible role that CDPKs play
in ABA signaling (Breviario et al. 1995). The present results
suggested that the 45 kDa CDPK in the cytosol fraction plays
an important role in BL signaling in rice lamina inclination.
The activity of the 45 kDa CDPK from lamina treated
with BL and IAA was higher than that of the treatments with
BL alone. In the present study, CoCl2, an inhibitor of ACC oxidase, inhibited BL- and BL applied together with IAA-induced
lamina inclination and it also inhibited the activity of the
45 kDa CDPK increased by either BL alone or in combination
with IAA. These results not only confirmed the involvement of
CDPK, but also indicated a possible role for ethylene in BL
signaling in rice lamina inclination.
In Azuki bean, BL alone or in combination with auxin, induced elongation of epicotyls, which related with the increase
in the percentage of transversely oriented microtubules and
maintenance of a transverse orientation requires protein phosphorylation (Mayumi and Shibaoka 1996). Recently, some BRinsensitive mutants of Arabidopsis, which turned out to be alleles of a single locus, BRI1, have been identified. BRI1 gene en-
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CDPK in brassinolide-induced lamina inclination
codes a leucine-rich repeat receptor-like kinase (Li and Chory
1997). BRI1 might function as a cell surface receptor that tranduces the BR signal to the cytoplasm through protein phosphorylation (Schumacher and Chory 2000). Ca2+-dependent protein
phosphorylation was shown to occur in the crude extract of
lamina. The phosphorylation of proteins, especially 56 and
41 kDa proteins, was strongly increased by BL treatment, and
was Ca2+ -dependent. These phosphoproteins may be the endogenous substrates of the CDPK affected by BL. Although
their nature remains to be clarified, these phosphoproteins may
be involved in the BL signaling in the rice lamina inclination.
References
Abo-El-Saad, M. and Wu, R. (1995) A rice membrane calcium-dependent protein kinase is induced by gibberellin. Plant Physiol. 108: 787–793.
Arteca, R.N., Bachman, J.M. and Mandava, B.N. (1988) Effects of indole-3-acetic acid and brassinosteroid on ethylene biosynthesis in etiolated mung bean
hypocotyls segment. J. Plant Physiol. 133: 430–435.
Botella, J.R., Arteca, J.M., Somodevilla, M. and Arteca, R.N. (1996) Calciumdependent protein kinase expression in response to physical and chemical
stimuli in mungbean (Vigna radiata). Plant Mol. Biol. 30: 1129–1137.
Breviario, D., Morello, L. and Giani, S. (1995) Molecular cloning of two novel
rice cDNA sequences encoding putative calcium-dependent protein kinases.
Plant Mol. Biol. 27: 953–967.
Campbell, P. and Braam, J. (1999) Xyloglucan endotransglycosylases: diversity
of genes, enzymes and potential wall-modifying functions. Trends Plant. 4:
361–366.
Cao, H. and Chen, S. (1995) Brassinosteroid-induced rice lamina joint inclination and its relation to indole-3 acetic acid and ethylene. Plant Growth Regul.
16: 189–196.
Clouse, S.D. (1996) Molecular genetic studies confirm the role of brassinosteroids in plant growth and development. Plant J. 10: 1–8.
Estruch, J.J., Kadwell, S., Merlin, E. and Crossland, L. (1994) Cloning and characterization of a maize pollen-specific calcium-dependent calmodulin-independent protein kinase. Proc. Natl. Acad. Sci. USA 91: 8837–8841.
Hamada, T., Hasunuma, K. and Komatsu, S. (1999) Phosphorylation of proteins
in the stem section of etiolated rice seedling irradiated with red light. Biol.
Pharm. Bull. 22: 122–126.
Hepler, P.K. and Wayne, R.O. (1985) Calcium and plant development. Annu.
Rev. Plant Physiol. 36: 397–439.
Hey, S.J., Balcon, A., Burnett, E. and Neill, S.J. (1997) Abscisic acid signal
transduction in epidermal cells of Pisum sativum L. Argentenum: both dehydrin mRNA accumulation and stomatal responses require protein phosphorylation and dephosphorylation. Planta 202: 85–92.
Hidaka, K., Inagaki, M., Kawamoto, S. and Sasaki, Y. (1984) Isoquinolinesulfonamides; novel and potent inhibitor of cyclic nucleotide dependent protein
kinase and protein C. Biochemistry 23: 5036–5041.
Hubbard, M.J. and Cohen, P. (1993) On target with a new mechanism for the
regulation of protein phosphorylation. Trends Biochem. Sci. 18: 172–177.
Karibe, H. and Komatsu, S. (1998) The involvement of Ca2+-dependent protein
kinase in the regeneration of rice cultured suspension cells. Biol. Pharm. Bull.
21: 163–166.
Karibe, H., Komatsu, S. and Hirano, H. (1996) Partial purification and characterization of a calcium-dependent protein kinase in rice leaves. Phytochemistry 41: 1459–1464.
Katou, S., Senda, K., Yoshioka, H., Doke, N. and Kawakita, K. (1999) A 51 kDa
protein kinase of potato activated with hyphal wall components from Phytophthora infestans. Plant Cell Physiol. 40: 825–831.
Knight, H., Trewavas, A.J. and Knight, M.R. (1996) Cold calcium signaling in
Arabidopsis involves two cellular pool and a change in calcium signature
after acclimation. Plant Cell 8: 489–503.
Komatsu, S. and Hirano, H. (1992) 80 kDa mouse sperm protein as a substrate
of protein kinase C. Chem. Pharm. Bull. 40: 2780–2782.
Komatsu, S. and Hirano, H. (1993) Protein kinase activity and protein phosphorylation in rice (Oryza sativa L.). Plant Sci. 94: 127–137.
Komatsu, S., Karibe, H. and Matsuda, T. (1997) Effect of abscisic acid on phosphatidylserine-sensitive calcium dependent protein kinase activity and protein phosphorylation in rice. Biosci. Biotech. Biochem. 61: 418–423.
Komatsu, S., Karibe, H., Xia, B. and Hirano, H. (1996) Phosphatidylserine-sensitive calcium dependent protein kinase in rice embryo. Phytochemistry 42:
21–27.
Kwak, S.H. and Lee, S.H. (1997) Requirements for Ca2+, protein phosphorylation, and dephosphorylation for ethylene signal transduction in Pisum sativa
L. Plant Cell Physiol. 38: 1142–1149.
Li, J. and Chory, J. (1997) A putative leucine-rich repeat receptor kinase
involved in brassinosteroid signal transduction. Cell 90: 929–938.
Li, J., Nagpal, P., Vitart, V., McMorris, T.C. and Chory, J. (1996) A role for
brassinosteroid in light-dependent development of Arabidopsis. Science 272:
398–401.
Mandava, N.B. (1988) Plant growth-promoting brassinosteroids. Annu. Rev.
Plant Physiol. Plant Mol. Biol. 39: 23–52.
Mayumi, K. and Shibaoka, H. (1996) The cyclic reorientation of cortical microtubules on walls with a crossed polylamellate structure: effects of plant hormones and an inhibitor of protein kinases on the progression of the cycles.
Protoplasma 195: 112–122.
Mizoguchi, T., Gotoh, Y., Nishida, E., Yamaguchi-Shinozaki, K., Hayashida, N.,
Iwasaki, H., Kamada, H. and Shinozaki, K. (1994) Characterization of two
cDNA that encode MAP kinase homologue in Arabidopsis thaliana and analysis of possible role of auxin in activating such kinase activity in cultured
cell. Plant J. 5: 111–122.
Moisyadi, S., Dharmasiri, S., Harringron, H.M. and Lukas, T.J. (1994) Characterization of a low molecular mass autophosphorylation protein in culture
sugarcane cell and its identification as a nucleoside diphosphate kinase. Plant
Physiol. 104: 1401–1409.
O’Farrel, P.F. (1975) High resolution two-dimensional electrophoresis of protein. J. Biol. Chem. 250: 4007–4021.
Putnam-Evans, C., Harmon, A.C., Palevitz, B.A., Fechheimer, M. and Cormier,
M.J. (1989) Calcium-dependent protein kinase is localized with F-actin in
plant cells. Cell Motil. Cytoskel. 12: 12–22.
Putnam-Evans, C.L., Harmon, A.C., Cormier, M.J. (1990) Purification and
characterization of a novel calcium-dependent protein kinase from soybean
Biochemistry. 29: 2488–2495.
Reddy, A.S.N., Chengappa, S. and Poovaiah, B.W. (1987) Auxin-regulated
changes in protein phosphorylation in pea epicotyl segments. Biochem. Biophys. Res. Commun. 144: 944–950.
Robert, D.M. and Harmon, A.C. (1993) Calcium-modulated protein: Targets of
intracellular calcium signals in higher plants. Annu. Rev. Plant Physiol. Plant
Mol. Biol. 43: 375–414.
Sasse, J.M. (1997) Recent progress in brassinosteroid research. Physiol. Plant.
100: 696–701.
Schumacher, K. and Chory, J. (2000) Brassinosteroid signal transduction: still
casting the actors. Curr. Opin. Plant Biol. 3: 79–84.
Sheen, J. (1996) Ca2+-dependent protein kinases and stress signal transduction in
plants. Science 274: 1900–1902.
Szekeres, M., Nemeth, K., Koncz-Kalman, Z., Mathur, J., Kauschmann, A.,
Altaman, T., Redei, G.P., Nagy, F., Schell, J. and Koncz, C. (1996) Brassinosteroid rescue the deficiency of CYP90, a cytochrome P450, controlling cell
elongation and de-etiolation in Arabidopsis. Cell 85: 171–182.
Takeno, K. and Pharis, R.P. (1982) Brassinosteroid-induced bending of leaf lamina of dwarf rice seedlings: an auxin-mediated phenomenon. Plant Cell Physiol. 23: 1275–1281.
Tester, M. (1990) Plant ion channel: whole-cell and single-channel studies. New
Phytol. 114: 305–340.
Uozu, S., Tanaka-Ueguchi, M., Kitano, H., Hattori, K. and Matsuoka, M. (2000)
Characterization of XET-related genes of rice. Plant Physiol. 122: 853–859.
Wada, K., Marumo, S., Ikekawa, N., Morisaki, M. and Mori, K. (1981) Brassinolide and homobrassinolide promotion of lamina inclination of rice seedlings. Plant Cell Physiol. 22: 323–325.
Zurek, D.M. and Clouse, S.D. (1994) Molecular cloning and characterization of
a brassinosteroid-regulated gene from elongating soybean epicotyls. Plant
Physiol. 104: 161–170.
(Received April 4, 2000; Accepted August 21, 2000)