Download Possible Involvement of Phosphoinositide-Ca

Plant CellPhysiol. 41(4): 399-407 (2000)
JSPP © 2000
Possible Involvement of Phosphoinositide-Ca2+ Signaling in the Regulation
of tf-Amylase Expression and Germination of Rice Seed {Oryza sativa L.)
Mohammad A. Kashem1, Kimiko Itoh 2 , Satoru Iwabuchi1, Hidetaka Hori' and Toshiaki Mitsui 1 ' 3
1
2
Laboratories of Molecular Life Sciences, Graduate School of Science and Technology, Niigata University, Niigata, 950-2181 Japan
Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University, Niigata, 950-2181 Japan
We have studied the effects of neomycin, a potent inhibitor of inositol phospholipid-specific phospholipase C
(PLC), on the germination of rice seed and the gibberellin-induced expression of a-amylase in the aleurone layer
and the scutellar tissues. It was shown that, in the absence
of exogenous Ca 2+ , neomycin markedly reduced the germination speed and seedling growth of rice seeds and inhibited the gibberellin-induced expression of a-amylase in
both secretory tissues. In addition, neomycin decreased the
formation of inositol 1,4,5-trisphosphate (IP3) in the gibberellin-treated aleurone layer and the scutellar tissues.
However, the inhibitory effects on the germination speed
and the expression of a-amylase activity were overcome by
supplementation of Ca 2+ . In addition, gibberellin elevated
the level of IP3, and ABA prevented the gibberellin-induced formation of IP3, although ABA alone did not alter
the IP3 level. The expression of a membrane-bound PLC
molecule in rice aleurone layer was shown to be induced by
gibberellin, and the gibberellin-induced expression of PLC
was markedly delayed by treatment with ABA. These results strongly suggest that the phosphoinositide-Ca2+ signal transduction pathway may play an important role in
the gibberellin-induced expression of a-amylase molecules
closely related to the germination processes of rice seed.
Key words: a-Amylase isoform 1-1 — Ca 2+ — Gibberellin
— Inositol 1,4,5-trisphosphate — Neomycin — Rice seed.
The breakdown of reserve starch in the storage organ
of cereal seed is an essential step for seed germination and
subsequent seedling growth. a-Amylase (EC 3.2.1.1) plays
the most important role in breaking down these starch
molecules. The a-amylase molecules synthesized in the
scutellar tissues and the aleurone layer have been shown to
be subsequently secreted into the starchy endosperm. It is
Abbreviations: GA3, gibberellic acid; GUS, yff-glucuronidase;
IP 3) inositol 1,4,5-trisphosphate; MS, Murashige-Skoog; PBS,
phosphate-buffered saline; PEG, polyethylene glycol; PI, phosphatidylinositol; PIP 2 , phosphatidylinositol-4,5-bisphosphate; PLC,
inositol phospholipid-specific phospholipase C; SD, standard deviation; (+)-trifluoro-ABA, ( + )-8',8',8'-trifluoroabscisic acid.
3
Corresponding author: E-mail, [email protected];
Fax, +81-25-262-6641.
399
widely accepted that the expression of a-amylase is regulated by phytohormones, such as gibberellin and ABA and
by metabolic sugars at both the transcriptional and posttranscriptional steps (Mitsui and Itoh 1997, Ritchie and
Gilroy 1998a). Accumulated evidence has suggested that
Ca 2+ and calmodulin (Gilroy and Jones 1992, Bush 1996,
Schuurink et al. 1996), G protein (Jones et al. 1998), protein phosphorylation (Lue and Lee 1994, Kuo et al. 1996,
Ritchie and Gilroy 1998b), cyclic GMP (Penson et al.
1996), and IP 3 (Murthy et al. 1989, Chen et al. 1997) are
involved at early stages of gibberellin signal transduction.
Recently, it has been reported that the hormonal and metabolic regulation of a-amylase expression appears to proceed in an intricate manner (Perata et al. 1997, Kashem et
al. 1998), and that metabolic sugars regulate the protein
synthesis and posttranslational secretory processes of aamylase molecules, which are mediated by controlling the
Ca 2+ uptake (Mitsui et al. 1999a, b).
In mammalian cells, inositol phospholipid turnover
has been established as an important step in signal transduction across the plasma membrane via second messengers. Breakdown of phosphatidylinositols in response to
external stimuli is catalyzed by inositol phospholipid-specific phospholipase C (PLC). The enzyme cleaves phosphatidylinositol-4,5-bisphosphate (PIP2) to yield IP 3 and
diacylglycerol, which are well known as second messenger
molecules. IP 3 promotes the release of Ca 2+ from internal
stores to the cytoplasm in both animal and plant cells
(Alexandre et al. 1990, Berridge 1993). Calcium ion is
thought to be essential for the expression of enzymatic
activity of cereal seed a-amylase, because a-amylase is
a Ca2+-containing metalloenzyme, and its expression and
secretion require Ca 2+ (Bush et al. 1989, Kadziola et al.
1998, Bethke et al. 1997, Mitsui and Itoh 1997).
Previously, we have identified, purified, and characterized soluble and membrane-bound PLC in rice suspension-cultured cells (Yotsushima et al. 1993), suggesting that
a phosphoinositide signaling system may exist in the cells.
It has been reported that GA3 stimulated the formation of
IP 3 in barley and rice aleurone layer (Murthy et al. 1989,
Chen et al. 1997) and that dehydration, salt stress and ABA
induced the accumulation of PLC1 mRNA in Arabidopsis
thaliana (Hirayama et al. 1995). However, Ritchie and
Gilroy (1998c) have reported that ABA activated phospholipase D to produce phosphatidic acid, which is then
400
IP3-Ca2+ signaling in germinating rice seeds
involved in triggering the subsequent ABA responses of the
barley aleurone protoplast, and that the inositol phosphate
metabolism did not alter gibberellin and ABA responses in
the aleurone cell.
It has also been reported that neomycin, a positively
charged aminoglycoside, forms electroneutral complexes
with PIP2 and thereby blocks the binding of PIP 2 to PLC
(Chen and Boss 1991, Legendre et al. 1993). However,
uncharged aminoglycosides, such as gentamicin exhibit
only weak affinity for PIP 2 . The aminoglycoside antibiotics
are known to inhibit protein synthesis on bacterial ribosomes. Recently, these antibiotics have been reported to
inhibit lysosomal sphingomyelinase and phospholipase Al
in mammalian cells (Montenez et al. 1994).
In the present study, we carefully examined the effect
of neomycin on the germination of rice seeds and the aamylase expression in the secretory tissues. We propose a
possible role for phosphoinositide-Ca2+ signaling in the
gibberellin-inducible expression of a-amylase molecules
and the germination of rice seed.
Materials and Methods
Chemicals—Gibberellic acid (GA3), (±)-ABA and neomycin
(fradiomycin) sulfate were purchased from Wako (Osaka, Japan).
Polyethylene glycol (PEG) 4000 and dextran T-500 were from
Sigma (St. Louis, MO, U.S.A.) and Pharmacia (Uppsala,
Sweden), respectively. PI, L-a-[myoinositol-2-3H(N)] (37-185
GBqmmol" 1 ) and PIP 2) L-a-[myoinositol-2-3H(N)] (37-185 GBq
mmol"1) were from American Radio labeled Chemicals (St. Louis,
MO, U.S.A.). ( + )-ABA was separated from the racemic mixture,
and (+)-8',8',8-trifluoroabscisic acid ((+)-trifluoro-ABA) was
synthesized as described previously (Todoroki et al. 1995).
Plant materials—Rice seeds (Oryza sativa L. cv. Nipponkai
and Kinuhikari line-K43) were hulled and mechanically cut into
two parts. The embryoless and embryo-containing half-seeds were
sterilized in 1% sodium hypochlorite for 15 and 2 min, respectively, and thoroughly rinsed in sterile water. Scutellar tissues
were dissected from the embryo-containing half-seeds soaked with
sterile water for 24 h at 30°C in darkness.
The procedure for rice cell culture (Oryza sativa L. cv. Nipponkai) was identical to the method described previously (Mitsui
et al. 1999a). About 2 g of rice cells were grown in 120 ml of
Murashige-Skoog (MS) medium containing 3% (w/v) sucrose, 2
mg liter"1 2,4-dichlorophenoxyacetic acid, and 5 mg liter ' thiamine-hydrochloric acid at 28°C in darkness. Established suspension-cultured cells were subcultured at 7-day intervals.
All the procedures were performed under aseptic conditions.
Further details of incubation are described in each figure legend.
Preparation of rice soluble and microsomal membrane fractions—The treated rice embryoless half-seeds and scutellar tissues
were homogenized with buffer A [50 mM glycylglycine-sodium
hydroxide (pH 7.5), 1 mM EDTA, 3 mM magnesium chloride, 0.5
M mannitol] and centrifuged at 12,000 x g for 15 min at 4°C. The
supernatant was further centrifuged at 100,000 xg for 1 h at 4°C,
and the pellet was resuspended with buffer A. The supernatant
and pellet were used as soluble and microsomal membrane fractions, respectively.
Preparation of rice plasma membranes and PLC assays—
Rice plasma membranes were isolated by a slightly modified
PEG/dextran aqueous two-phase method (Yoshida et al. 1986).
Rice suspension-cultured cells were homogenized with buffer 1 [50
mM Tris-hydrochloric acid (pH 7.5), 1 mM EDTA, 0.5 M mannitol] and centrifuged at 12,000 xg for 15 min at 4°C. The supernatant was centrifuged at 100,000xg for 1 h at 4°C, and the
pellet was suspended with buffer 2 [10 mM potassium phosphate
(pH7.8), 30mM sodium chloride, 0.5 M mannitol]. PEG and
dextran dissolved in buffer 2 were added to the suspension to give
a 5.8% (w/w) concentration of each polymer. The suspension was
centrifuged at 800xg for 15 min to facilitate phase settling. The
upper phase was collected, diluted with 3 volumes of 20 mM
Tris-hydrochloric acid (pH 7.5), and centrifuged at 100,000xg
for 1 h at 4°C to precipitate plasma membranes. The plasma
membrane fraction contained the PLC activities toward both PI
and PIP 2 (Yotsushima et al. 1993). The assay mixture for PLC
consisted of 0.1 mM radioactive substrate (92.5 MBqmmol" 1 ;
[3H] PI or [3H] PIP 2 ), 50 mM Tris-maleate-sodium hydroxide (pH
6.5), 0.08% (w/v) sodium deoxycholate, 0.5 mM calcium chloride, 1 mM ethylene glycol-bis-(^aminoethylether)-A',N,iV',./Vtetraacetic acid, and enzyme in a final volume of 0.1 ml. The enzyme reaction was started by adding substrate and was incubated
for 30 min at 37°C. It was terminated with the addition of 0.1 ml
of 4 M hydrochloric acid/0.5% (w/v) calcium chloride and 0.6 ml
of chloroform : methanol ( 2 : 1 , v/v) and immediately cooled on
ice. The mixture was vortexed and then centrifuged at 2,000 x g
for 10 min to separate aqueous and organic phases. The radioactivity in the upper aqueous phase was determined by liquid scintillation counting (Aloka LSC-1000, Tokyo, Japan).
Other enzyme and protein assays—a-Amylase activity was
measured as described previously by Kashem et al. (1998). One
enzyme unit was defined according to the procedure described by
Okamoto and Akazawa (1978).
The expression of yS-glucuronidase (GUS) activity in rice
seeds (Oryza sativa L. cv. Kinuhikari, line K43) transformed with
the 5' flanking region from - 2 3 2 to +31 of RAmylA/GUS fusion gene (Itoh et al. 1995) was determined according to the
method described by Jefferson (1987). The tissues were hofhogenized with GUS extraction buffer consisting of 50 mM phosphate buffer (pH 7.0), 10 mM dithiothreitol, 1 mM EDTA, 0.1%
(w/v) sodium lauryl sarcosine, and 0.1% (w/v) Triton X-100, and
centrifuged at 15,000 x g for 20 min. The supernatant was used as
the crude enzyme extract.
Protein content was estimated by dye binding procedure of
Bradford (1976) with y-globulin as a standard.
IP3 contents—The embryoless half-seeds, the scutellar tissues, and the suspension-cultured cells were ground in liquid nitrogen into fine powders. An ice-cold 250 /A of distilled water and
100 /A of 10% perchloric acid were added to each tissue sample.
After mixing, the suspension was kept for 30 min on ice and than
centrifuged at 2,000xg for 10min at 4°C. IP 3 contents in the
supernatant were determined according to the manufacturer's
protocol (IP3 [3H]assay system, Amersham, Little Chalfont,
Buckinghamshire, England).
Preparation of antibodies and immunoblotting—The preparation procedure for antibodies against a-amylase isoforms and
their specificity were described previously (Mitsui et al. 1996). To
prepare specific antibodies against plant PLC, the synthesized
oligopeptide for a consensus sequence (RIYPKGTRVDSSNY)
among the PLC enzymes was injected into rabbits at two week
intervals. The first injection of 1 mg of peptide was mixed with
complete Freund's adjuvant. The three subsequent injections (1
mg for each injection) were mixed with incomplete Freund's ad-
IP3-Ca2+ signaling in germinating rice seeds
juvant.
The procedure for immunoblotting was identical to that in
the previous report (Kashem et al. 1998).
Isolation of RNA and northern blotting—Seven g of rice
embryoless half-seeds for each treatment were frozen and ground
in liquid nitrogen. The powdered sample was subjected to RNA
isolation according to the procedure described previously (Mitsui
et al. 1999a).
The isolated RNA (20 fig) was loaded onto 1.4% (w/v)
agarose-formaldehyde gels and blotted onto Hybond N + membrane according to the manufacturer's protocol (Amersham). The
nylon membrane blotted with RNA was incubated with prehybridization solution consisting of 5 x SSPE [0.9 M sodium
chloride, 50 mM sodium phosphate, 5 mM EDTA (pH 7.7)], 5 x
Denhardt's solution, 0.5% SDS and 0.02% (w/v) denatured
salmon sperm DNA for 1 h at 65 °C and then hybridized with
[33P]-labeled DNA probes in the prehybridization solution for 16
h at 65°C. The a-amylase 1-1 (RAmylA) gene-specific DNA (Sheu
et al. 1996) was synthesized by Hokkaido System Science Co.
(Sapporo, Hokkaido, Japan), and the rice actin 1 DNA was amplified by PCR with 5-CATGCTATCCCTCGTCTCGACCT-3'
and 5-CGCACTTCATGATGGAGTTGTATG-3' primers. The
template was a gift from Drs. Hirai and Nakazono (The University Qf Tokyo). The DNA probes were labeled with [a-33P]dCTP
according to the manufacturer's protocol (Amersham). After hybridization, the membrane was washed twice with 2 x SSPE and
0.1% SDS at room temperature and finally washed with 1 x SSPE
and 0.1% SDS for 15 min at 65°C. The air dried membrane was
subjected to autoradiography using a radioisotope imaging
analyzer (BAS-5000, Fuji Film, Tokyo, Japan).
Results
We first determined the specificity of neomycin toward
the enzyme activity of rice PLC. As shown in Figure la,
neomycin markedly inhibited the PIP 2 -PLC activity associated with rice plasma membranes at more than 50 JIM but
did not affect on the PLC activity toward phosphatidylinositol (PI) even at 5 mM. The effect of neomycin on the
release of IP 3 from PIP 2 in vivo was examined employing a
model experimental system with suspension-cultured cells
derived from the rice seed embryo (Oryza sativa L. cv.
Nipponkai). When neomycin was added to the rice cell
culture under Ca2+-free conditions, IP 3 content in the cells
was reduced in the same pattern as the dose response for
enzyme inhibition (Fig. lb). These results indicate that the
neomycin treatment decreased the IP 3 level in rice cells
through the inhibition of PIP 2 -PLC activity.
When rice seeds were incubated in a buffered solution
containing different concentrations of neomycin (0 to 5
mM), a significant inhibition of the elongation of root and
shoot tissues was observed (Fig. 2, 3b), and the germination speed was markedly delayed (Fig. 3a) in the presence
of 0.5 mM neomycin. However, supplementation with 10
mM calcium chloride partially removed the inhibitory
effect of neomycin on seedling growth (Fig. 2, 3b), and the
neomycin effect on germination completely disappeared
with Ca 2+ (Fig. 3a).
401
0.05
0.1
0.2
5
Neomycin (mM)
0.05
0.1
1
Neomycin (mM)
Fig. 1 Effects of neomycin on PLC activity (a) and IP 3 formation (b) in rice cells, (a) Activities of rice plasma membrane-bound
PLC toward PI and PIP 2 were determined in the presence of
different concentrations of neomycin (0 to 10 mM). One hundred
percent activity (2//molmm~ 3 (mgprotein)" 1 ) was set for each
substrate in the absence of neomycin. (b) Rice cells (100 mg) cultured for 3 d in the MS media without 3 mM calcium chloride
were incubated in the sucrose-depleted and Ca2+-free MS media (1
ml) containing neomycin (0 to 0.2 mM) at 30°C for 18 h. The
treated cells were subjected to the IP 3 assay described in the text.
The amount of IP 3 in the absence of neomycin was normalized to
100%. The absolute 100% value was 82pmol (g of cells)"1. The
data present the averages of duplicate experiments.
10mMCaCI 2 :
0 mM CaCb:
0.5
1.0
25
Neomycin (mM)
5.0
Fig. 2 Effect of neomycin on germination of rice seeds. Rice
seeds were incubated in 10 mM acetate buffer (pH 5.2) containing
different concentrations of neomycin (0 to 5 mM) in presence or
absence of 10 mM calcium chloride for 6 d at 30°C in the dark.
IP3-Ca2+ signaling in germinating rice seeds
402
In order to determine the effects of neomycin on the
gibberellin-inducible a-amylase expression in aleurone layers of rice seeds, the embryoless half-seeds were incubated
with GA3 and different concentrations of neomycin (0 to 5
mM). The GA3-induced a-amylase activities were markedly
inhibited by neomycin at more than 1 mM (Fig. 3c). Interestingly, the inhibitory effect of neomycin on the expression of a-amylase activity was found to be overcome
by the presence of 10 mM calcium chloride (Fig. 3c). It has
been shown that a-amylase 1-1 (isoform A and B), encoded
by RAmylA, is the dominant isoform in germinating rice
seeds (Mitsui and Itoh 1997). We examined the effects of
neomycin on the GA3-induced formation of a-amylase 1-1
molecules by employing immunodetection with anti-AB
(a-amylase 1-1) antibodies. As can be seen in Figure 4a and
c, the effect of neomycin on the a-amylase 1-1 formation
and the suppression of this effect by exogenous Ca 2+ were
similar to those on the activity of a-amylase. We also dear Aleurone Layer
neomycin (mM):
0.5 ixM GA3
10mMCaCl2
0 0.5 1.0 Z5 5.0 0 0.5 1.0 2.5 5.0
b: Scutellar Tissues
Neomycin (mM)
Neomycin (mM)
Fig. 3 Effects of neomycin on germination speed, seedling
growth, and gibberellin-inducible a-amylase expression in aleurone and scutellar tissues of rice seeds, (a, b) Seeds were incubated
in 10 mM acetate buffer (pH 5.2) with different concentrations of
neomycin in the presence or absence of 10 mM calcium chloride
for 6 d at 30°C in the dark, and the speed of germination (a) and
dry weight of seedlings (b) were determined. The speed of germination was calculated using the following formula: Speed of germination =ZGn/Dn, where Gn is number of seeds germinated on
day n and Dn is days from initial sowing (Maguire 1962). The
values were the average of three experiments (16 seeds for one
experiment), and the standard deviations (SD) did not exceed 3%.
(c, d) Embryoless half-seeds (10 segments) of normal (Nipponkai)
and transgenic rice (Kinuhikari, line K43) were incubated in the
treatment solutions (0.75 ml) consisting of 10 mM acetate buffer
(pH 5.2), 0.5 piM GA3, 0 to 5 mM neomycin with or without 10
mM calcium chloride for 4 d at 30°C, and then incubation media
and seed extracts were subjected to the a-amylase (c) and GUS (d)
assays, (e, f) Scutella (10 segments) dissected from normal and
transgenic rice seeds were incubated in the treatment solutions (0.1
ml) consisting of 10 mM acetate buffer (pH 5.2) and 0 to 5 mM
neomycin with or without 10 mM calcium chloride for 24 h at
30°C. Incubation media and tissue extracts were subjected to the
a-amylase (e) and GUS (f) assays, (c-f) Data are the means + SD
(« = 3). The germination speed, dry weight, a-amylase, and GUS
activities in each experimental set without neomycin were normalized to 100%.
neomycin (mM):
10mMCaCl2
N»omyein (mM)
0
0.5 1.0 2.5 5.0 0 0.5 1.0 2.5 5.0
+
Naomycln (mM)
Fig. 4 Effects of neomycin on formation of a-amylase 1-1
molecules in the GA3-treated aleurone layer (a, c) and the scutellar
tissues (b, d). The extracts obtained from the experiments of
Fig. 3c and 3e, 5 and 20 fig proteins, respectively, were subjected
to SDS-PAGE, followed by immunoblotting with anti-AB (aamylase 1-1) antibodies, (c, d) Quantitative results by densitometric analysis. The amounts of a-amylase 1-1 molecules in each
experimental set without neomycin were normalized to 100%.
IP3-Ca2+ signaling in germinating rice seeds
u-Amytase 1-1
(RAmylA)
Actin 1
EtBr
neomycin (mM):
0
0 0.5 1.0 2 5 5.0 0
10mMCaCl2
+
0 0.51.0 2 5 5.0
+
+
+
+
+
Fig. 5 Effect of neomycin on formation of a-amylase 1-1 mRNA
in the GA3-treated aleurone layers. Total RNA (20 jug) extracted
from each embryoless half-seed sample treated under different
conditions as indicated was subjected to agarose gel electrophoresis, followed by northern blotting with the a-amylase 1-1
(RAmylA) and the actin 1 gene-specific DNA probes.
termined the effect of neomycin on the GA3-induced formation of a-amylase 1-1 mRNA in the presence or absence
of Ca 2+ . Neomycin reduced the GA3-induced mRNA formation in the absence of Ca 2+ , and the inhibitory effect of
neomycin was weakly suppressed by the addition of Ca 2+
(Fig. 5). To further elucidate the effects of neomycin on
403
the GA3-induced a-amylase expression in the aleurone layers, we tested the effect of the drug on the expression of
the RAmylA/GUS fusion gene in embryoless half-seeds
of transgenic rice. As shown in Figure 3d, the level of
GA3-induced GUS activities in the aleurone layers was
effectively decreased by neomycin; under the Ca2+-supplemented condition, the neomycin effect was partially
neutralized.
The GA3 treatment increased the content of IP 3 in
embryoless half-seeds from 37 to 200pmol per half-seed
(Table 1). To evaluate role of ABA on the GA3-induced
IP 3 formation in the aleurone layer, the embryoless halfseeds were incubated in different mixtures of GA3 and
ABA. As shown in Table 1, the GA3-induced IP 3 formation was significantly reduced by 10 /uM ABA; however,
ABA alone, even ( + )-trifluoro-ABA, which is known to be
a highly potent and long-lasting analog of ABA (Kashem et
al. 1998), scarcely increased the IP 3 level. These results
strongly suggest that GA3 and ABA coordinate the level of
IP 3 in the aleurone layer of rice seed. When 0.5 mM neomycin was added to the GA3-treated half-seeds, the IP 3
level decreased from 200 to 90pmol per half-seed. Five
mM neomycin completely inhibited the GA3 induced formation of IP 3 to the basal level observed in GA3-untreated
tissues (Table 1). Supplementation with 10 mM calcium
Table 1 Effects of neomycin and ABA on GA3-induced IP 3 formation in embryoless
half-seeds of rice"
IP 3 (pmol half-seed"1)
Treatments
none
0.5 JUM GA3 + 0mM neomycin
0.5 JUM GA3 + 0.5 mM neomycin
0.5 JUM GA3 + 1.0 mM neomycin
0.5 /uM GA3 + 2.5 mM neomycin
0.5 JUM GA3 + 5.0mM neomycin
10 mM CaCl2
0.5 JUM GA3 + 0 mM neomycin +10 mM CaCl2
0.5 pM GA3 + 0.5 mM neomycin+10 mM CaCl2
0.5 /uM GA3 + 1.0mM neomycin +10 mM CaCl2
0.5 JUM GA 3 +2.5 mM neomycin+10 mM CaCl2
0.5 fM GA3 + 5.OmM neomycin +10 mM CaCl2
0.5/M GA3 + 10^M (±)-ABA
0.5 fM GA3 + 10//M (+)-ABA
0.5//M GA3 + 10JUM ( + )-Trifluoro-ABA
10,uM (+)-ABA
\0JUM(+)-Trifluoro-
AB A
37± 6b
200 ±30
90 ±14
63± 9
53± 9
38± 7
18± 1
81±21
31± 5
27± 5
23 ± 4
16± 2
97±18
93 ±15
89±11
55±10
47± 6
54±10
" Rice embryoless half-seeds were incubated in 10 mM acetate buffer (pH5.2) with different supplementations for 18 h at 30°C in the dark. The treated seeds were extracted and subjected to the
IP3 assay.
* Data are the mean±SD (« = 3).
IP3-Ca2+ signaling in germinating rice seeds
404
Table 2 Effect of neomycin on IP 3 formation in scutellar
tissues of rice seedsa
IP 3 (pmol scutellum x)
Treatments
2.9±0.44*
4.4±0.67
2.7±0.41
2.3±0.35
2.0±0.30
1.7±0.27
no incubation
0 mM neomycin
0.5 mM neomycin
1.0 mM neomycin
2.5 mM neomycin
5.0 mM neomycin
0
Scutella tissues dissected from rice seeds soaked with distilled
water for 24 h at 30°C in the dark were incubated in 10 mM acetate buffer (pH 5.2) with different concentrations of neomycin for
6 h at 30°C. The treated scutellar tissues were extracted and subjected to the IP 3 assay.
* Data are the mean±SD (n = 3).
chloride decreased the IP 3 level regardless of the presence
or absence of GA3 and neomycin (Table 1).
The a-amylase expression in rice scutellar tissues has
been reported to be regulated by endogenous gibberellin
synthesized in the tissues (Sugimoto et al. 1998). To determine the neomycin effects on a-amylase expression in the
scutellar tissues, the dissected scutella were incubated with
different concentrations of neomycin in the presence or
absence of 10 mM calcium chloride without phytohormone. As shown in Figure 3e, the expression of a-amylase
activity was markedly inhibited by neomycin at more than
0.5 mM, and the inhibitory effect increased depending on
the dose of drug. In the presence of external Ca 2+ , the
neomycin effect was clearly disappeared (Fig. 3e). The
effect of neomycin on the a-amylase 1-1 formation and the
neutralizing effect of exogenous Ca 2+ were similar to those
on the activity of a-amylase (Fig. 4b, d). Neomycin also
exhibited a significant inhibitory effect on the RAmylA/
GUS expression in the scutellar tissues; this effect was also
weakly inhibited by the presence of external Ca 2+ (Fig. 3d).
Estimations of the levels of IP 3 in the scutellar tissues
treated with or without neomycin indicated that 0.5 mM
neomycin treatment strongly prevented the production of
IP 3 (Table 2). The inhibitory effects of neomycin on the
tissues of rice seeds were relatively weaker than those on
the rice cell culture. This discrepancy might have resulted
b: Aleurone
a*. Scutellum
soluble
kD
66 —
45—
36 —
29—
12
29 —
kD
3 4 5
6
7
membrane
66 —
45—
36—
29—
12
3 4 5 6 7 8
V VV>
V
*
Fig. 6 Immunodetection of PLC isoform molecules in rice scutellar and aleurone tissues, (a) Scutellar tissues were dissected from rice
seeds soaked with distilled water for 2 d at 30°C in the dark, (b) Embryoless half-seeds were incubated in 10 mM acetate buffer (pH 5.2)
with 0.5 fjM GA3 alone (lane 1-4), 0.5 /uM GA3 and 10/M (±)-ABA (lane 6-8), or without phytohormones (lane 5) for 0 to 6 d at 30°C.
The soluble (15 fig) and microsomal membrane (12 jug) fractions prepared from both tissues were subjected to SDS-PAGE, followed by
immunoblotting with anti-PLC antibodies. Arrows represent the membrane-bound and soluble PLC isoforms.
IP3-Ca2+ signaling in germinating rice seeds
from differences in drug permeability and Ca 2+ content
between the tissues and the suspension-cultured cells.
Both soluble and membrane-bound PLCs have been
purified from rice suspension-cultured cells, and the molecular sizes of soluble and membrane-bound PLC have
been estimated to be 55 and 40 kDa, respectively (Yotsushima et al. 1993). Employing polyclonal antibodies raised
against a peptide sequence highly conserved in plant PLC,
the expression of soluble and membrane-bound PLC molecules in germinating rice seed was examined. Both 55 and
40 kDa PLCs were detected in the scutellar tissues (Fig. 6a).
Fifty-five kDa PLC was also detected in the soluble fraction of embryoless half-seeds, and the expression of 55 kDa
PLC was stimulated by the GA3 treatment (Fig. 6b). Furthermore, a membrane-bound PLC (38 kDa) was found to
be expressed only in the GA3-treated tissues, and ABA
markedly delayed the GA3-induced expression of membrane-bound PLC (Fig. 6b).
Discussion
PLC is a key effector in most receptor-mediated signal
transduction pathways, a large family of closely related
enzymes that reversibly associate with membranes to carry
out hydrolysis of membrane resident phosphoinositide
substrates (Williams and Katan 1996). IP 3 , derived from
the hydrolysis of PIP 2 by PLC, binds to its specific receptor, which is an IP3-induced Ca2+-releasing channel. Recent molecular cloning studies have revealed that there are
at least three types of IP 3 receptor from distinct genes
in mammalian cells (Mikoshiba 1997, Yoshida and Imai
1997). Previously, we have succeeded in identifying, purifying and characterizing both soluble and membranebound PLC from rice suspension-cultured cells (Yotsushima et al. 1993). Recently, cDNA clones of PLC have
been isolated from Arabidopsis thaliana (Hirayama et al.
1995), soybean (Shi et al. 1995), and potato (Kopka et al.
1998). Muir and Sanders (1997), by using immunodetection, have suggested that the animal type I IP 3 receptor
exists in microsomal membranes prepared from cauliflower. Furthermore, several experimental results suggest that
IP3 may also play a role in elevating [Ca2+]j in higher plant
cells (Cote and Crain 1994, Franklin-Tong et al. 1996, Kim
et al. 1996, Scanlon et al. 1996, Muir and Sanders 1997).
In the present investigation, we determined the effects
of neomycin, a potent inhibitor of PIP 2 -PLC, on the
germination and the gibberellin-inducible expression of aamylase molecules in rice seeds. In the absence of exogenous Ca 2+ , neomycin markedly reduced the germination
speed and seedling growth of rice seeds, and inhibited
the GA3-induced expression of a-amylase molecules in the
aleurone layer and the scutellar tissues (Fig. 2-5). In addition, neomycin decreased the level of IP 3 in the GA3treated aleurone layer and the scutellar tissues (Table 1,2).
405
Importantly, the inhibitory effects on the germination
speed and the expression of a-amylase activity were found
to be overcome by supplementation with Ca2+ (Fig. 3a, c,
e). The expression of a-amylase 1-1 molecules (isoform A
and B), which are the dominant a-amylase isoforms in
germinating rice seeds, was also recovered by Ca 2+ regardless of the presence of neomycin (Fig. 4). It has been
reported that aminoglycoside antibiotics induce lysosomal
phospholipidosis in mammalian cells, associated with a
decrease in the activities of lysosomal sphingomyelinase
and phospholipase Al (Montenez et al. 1994). Therefore,
it is possible that the inhibitory effect of neomycin was
caused by PLC-independent pathways. We tested the effect
of gentamicin, which has a structure similar to neomycin
but only weak affinity for PIP 2 , on the GA3-induced expression of a-amylase activity in aleurone and found that
the inhibitory effect of gentamicin was weaker compared
with that of neomycin and that this effect was not overcome by supplementation with Ca 2+ (data not shown).
These results strongly suggest that the phosphoinositideCa 2+ signal transduction pathway plays an important role
in the gibberellin-inducible expression of a-amylase molecules closely related to the germination processes of rice
seed.
Neomycin also inhibited the GA3-induced transcription of a-amylase 1-1 gene (RAmylA) and the seedling
growth of rice seed; these inhibitory effects were partially
suppressed by adding exogenous Ca 2+ (Fig. 2, 3b, 3d, 3f,
5). These results appear to indicate that the phosphoinositide-Ca2+ signaling might be involved in the GA3-induced
gene expression; however, some part of the inhibition by
neomycin involves PLC-independent pathways.
The results presented in Table 1 indicate that GA3
elevated the level of IP 3 , ABA prevented the GA3-induced
formation of IP 3 , and ABA alone did not increase the
IP 3 level. The expression of PLC molecules in rice aleurone
layer was induced by GA3, and the GA3-induced expression
of PLC was markedly delayed by the treatment with ABA
(Fig. 6b). These results support the hypothesis that gibberellin and ABA do alter the inositol phospholipid turnover in germinating rice seeds.
It has been reported that the activity of rice PIP2-PLC
in vitro was Ca2+-dependent (Yotsushima et al. 1993).
However, when Ca 2 " was added to the incubation medium
for rice aleurone layer, the level of IP 3 in the tissue was
significantly reduced (Table 1). We think this is an understandable phenomenon, because the Ca 2+ homeostasis in
cytosol should be strictly maintained in plant cells (Bush
1995). At present, details of the control mechanism for
IP3 formation and degradation in the presence of external
Ca 2+ are unknown.
Molecular weights of plant PLC, which are isolated
from their cDNA clones, are calculated to be 64,000 to
69,000 (Hirayama et al. 1995, Shi et al. 1995, Kopka et al.
IP3-Ca2+ signaling in germinating rice seeds
406
1998). The expression of PLC1 mRNA in Arabidopsis
thaliana has been shown to be induced by ABA (Hirayama
et al. 1995). We have purified two PLC isoforms from rice
cells that were estimated to be 40 and 55 kDa (Yotsushima
et al. 1993). Indeed, the specific antibodies against a consensus sequence among the plant PLC recognized 38 to 40
kDa membrane-bound and 55 kDa soluble PLC in the
germinating rice seeds (Fig. 6). The anti-PLC antibodies
also recognized 55 kDa soluble and 70 kDa membranebound PLC in soybean seedlings (data not shown). Furthermore, the 40 and 55 kDa PLC were GA3-inducible in
rice aleurone. We infer from these results that there may be
a gibberellin signaling system composed of unique subtypes
of PLC in the aleurone layer of rice seed.
This research was supported by Grant-in-Aid no. 10640628
(to T.M.) from the Ministry of Education, Science, Sports and
Culture, Japan. We thank Drs. A. Hirai and M. Nakazono
(Graduate School of Agricultural and Life Sciences, The University of Tokyo) for gift of rice actin 1 gene.
References
Alexandre, J., Lassalles, J.P. and Kado, R.T. (1990) Opening of Ca2 f
channels in isolated red beet root vacuole membrane by inositol 1,4,5trisphosphate. Nature 343: 567-570.
Berridge, M.J. (1993) Inositol trisphosphate and calcium signalling. Nature 361: 315-325.
Bethke, P.V., Schuurink, R. and Jones, R.L. (1997) Hormonal signalling
in cereal aleurone. J. Exp. Bot. 48: 1337-1356.
Bradford, M.M. (1976) A rapid and sensitive method for the quantitation
of microgram quantities of protein utilizing the principle of protein-dye
binding. Anal Biochem. 72: 248-254.
Bush, D.S. (1995) Calcium regulation in plant cells and its role in signaling.
Annu. Rev. Plant Physiol. Plant Mol. Biol. 46: 95-122.
Bush, D.S. (1996) Effects of gibberellic acid and environmental factors on
cytosolic calcium in wheat aleurone cells. Planta 199: 88-89.
Bush, D.S., Sticher, L., Huystee, R., Wagner, D. and Jones, R.L. (1989)
The calcium requirement for stability and enzymatic activity of two
isoforms of barley aleurone a-amylase. /. Biol. Chem. 264: 1939219398.
Chen, Q. and Boss, W.F. (1991) Neomycin inhibits the phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate stimulation of plasma membrane ATPase activity. Plant Physiol. 96: 340-343.
Chen, X., Chang, M., Wang, B. and Wu, R. (1997) Cloning of a Ca2 ATPase gene and the role of cytosolic Ca 2+ in the gibberellin-dependent
signaling pathway in aleurone cells. Plant J. 11: 363-371.
Cote, G.G. and Crain, R.C. (1994) Why do plants have phosphoinositides?
BioEssays 16: 39-46.
Franklin-Tong, V.E., Drobak, B.K., Allan, A.C., Watkins, P.A.C. and
Trewavas, A.J. (1996) Growth of pollen tubes of Papaver rhoeas is
regulated by a slow-moving calcium wave propagated by inositol 1,4,5trisphosphate. Plant Cell 8: 1305-1321.
Gilroy, S. and Jones, R.L. (1992) Gibberellic acid and abscisic acid coordinately regulate cytoplasmic calcium and secretory activity in barley
aleurone protoplasts. Proc. Natl. Acad. Sci. USA 89: 3591-3595.
Hirayama, T., Ohto, C , Mizoguchi, T. and Shinozaki, K. (1995) A gene
encoding a phosphatidylinositol-specific phospholipase C is induced by
dehydration and salt stress in Arabidopsis thaliana. Proc. Natl. Acad.
Sci. USA 92: 3903-3907.
Itoh, K., Yamaguchi, J., Huang, N., Rodriguez, R. L., Akazawa, T. and
Shimamoto, K. (1995). Developmental and hormonal regulation of rice
a-amylase (RAmylA)-gusA fusion genes in transgenic rice seeds. Plant
Physiol. 107: 25-31.
Jefferson, R.A. (1987) Assaying chimeric genes in plants: The GUS gene
fusion system. Plant Mol. Biol. Rep. 5: 387-405.
Jones, H.D., Smith, S.J., Desikan, R., Plakidou-Dymock, S., Lovegrove,
A. and Hooley, R. (1998) Heterotrimeric G proteins are implicated in
gibberellin induction of a-amylase gene expression in wild oat aleurone.
Plant Cell 10: 245-253.
Kadziola, A., Sogaard, M., Svensson, B. and Haser, R. (1998) Molecular
structure of a barley a-amylase-inhibitor complex: Implications for
starch binding and catalysis. /. Mol. Biol. 239: 104-121.
Kashem, M.A., Hori, H., Itoh, K., Hayakawa, T., Todoroki, Y., Hirai,
N., Ohigashi, H. and Mitsui, T. (1998) Effects of (+)-8',8',8'-trifluoroabscisic acid on a-amylase expression and sugar accumulation in rice
cells. Planta 205: 319-326.
Kim, H.Y., Cote, G.G. and Crain, R.C. (1996) Inositol trisphosphate may
mediate closure of potassium channels by light and darkness in Samanea
saman motor cells. Planta 198: 279-287.
Kopka, J., Pical, C , Gray, J.E. and Muller-Rober, B. (1998) Molecular
and enzymatic characterization of three phosphoinositide-specific phospholipase C isoforms from potato. Plant Physiol. 116: 239-250.
Kuo, A., Cappelluti, S., Cervantes-Cervantes, M., Rodriguez, M. and
Bush, D.S. (1996). Okadaic acid, a protein phosphatase inhibitor,
blocks calcium changes, gene expression, and cell death induced by
gibberellin in wheat aleurone cells. Plant Cell 8: 259-269.
Legendre, L., Yueh, Y.G., Crain, R., Haddock, N., Heinstein, P.F. and
Low, P.S. (1993) Phospholipase C activation during elicitation of the
oxidative burst in cultured plant cells. /. Biol. Chem. 268: 24559-24563.
Lue, M.-Y. and Lee, H.-T. (1994) Protein phosphatase inhibitors enhance
the expression of an a-amylase gene, aAmy3, in cultured rice cells.
Biochem. Biophys. Res. Commun. 205: 807-816.
Maguire, J.D. (1962) Speed of germination-aid in selection and evaluation
for seedling emergence and vigor. Crop Sci. 2: 17'6-177.
Mikoshiba, K. (1997) The Ins(l,4,5)P3 receptor. In Signalling by Inositides—a Practical Approach, Edited by Shears, S. pp. 173-193. IRL
Press, Oxford.
Mitsui, T. and Itoh, K. (1997) The a-amylase multigene family. Trends
Plant Sci. 2: 255-261.
Mitsui, T., Loboda, T., Itoh, A. and Ikarashi, T. (1999a) Sugar-controlled
Ca 2+ uptake and a-amylase secretion in cultured cells of rice (Oryza
sativa L.). Plant Cell Physiol. 40: 773-783.
Mitsui, T., Loboda, T., Kamimura, I., Hori, H., Itoh, K. and Mitsunaga,
S. (1999b) Sucrose-controlled transport and turnover of a-amylase in
rice (Oryza sativa L.) cells. Plant Cell Physiol. 40: 884-893.
Mitsui, T., Yamaguchi, J. and Akazawa, T. (1996) Physicochemical and
serological characterization of rice a-amylase isoforms and identification
of their corresponding genes. Plant Physiol. 110: 1395-1404.
Montenez, J.-P., Kishore, B.K., Maldague, P. and Tulkens, P.M. (1994)
Leupeptin and E-64, inhibitors of cysteine proteinase, prevent gentamicin-induced lysosomal phospholipidosis in cultured rat fibroblasts.
Toxicol. Lett. 73: 201-208.
Muir, S.R. and Sanders, D. (1997) Inositiol 1,4,5-trisphosphate-sensitive
Ca 2+ release across nonvacuolar membranes in cauliflower. Plant
Physiol. 114: 1511-1521.
Murthy, P.P.N., Renders, J.M. and Kerane, L.M. (1989) Phosphoinositides in barley aleurone layers and gibberellic acid-induced changes in
metabolism. Plant Physiol. 91: 1266-1269.
Okamoto, K. and Akazawa, T. (1978) Purification of a-amylase and 0amylase from endosperm tissues of germinating rice seeds. Agric. Biol.
Chem. 42: 1379-1384.
Penson, S.P., Schuurink, R . C , Fath, A., Gubler, F., Jacobsen, J.V. and
Jones, R.L. (1996) cGMP is required for gibberellic acid-induced gene
expression in barley aleurone. Plant Cell 8: 2325-2333.
Perata, P., Matsukura, C , Vernieri, P. and Yamaguchi, J. (1997) Sugar
repression of a gibberellin-dependent signaling pathway in barley embryos. Plant Cell 9: 2197-2208.
Ritchie, S. and Gilroy, S. (1998a) Tansley review No. 100. Gibberellins:
regulating genes and germination. New Phytol. 140: 363-383.
Ritchie, S. and Gilroy, S. (1998b) Calcium-dependent protein phosphorylation may mediate the gibberellic acid response in barley aleurone.
Plant Physiol. 116: 765-776.
Ritchie, S. and Gilroy, S. (1998c) Abscisic acid signal transduction in the
IP3-Ca2+ signaling in germinating rice seeds
barley aleurone is mediated by phospholipase D activity. Proc. NatI.
Acad. Sci. USA 95: 2697-2702.
Scanlon, C.H., Martinec, J., Machackova, I., Rolph, C.E. and Lumsden,
P.J. (1996) Identification and preliminary characterisation of a calcium-dependent high-affinity binding site for inositol trisphosphate from
Chenopodium rubrum. Plant Physiol. 110: 867-874.
Schuurink, R.C., Chan, P.V. and Jones, R.L. (1996) Modulation of
calmodulin mRNA and protein levels in barley aleurone. Plant Physiol.
I l l : 371-380.
Shi, J., Gonzales, R.A. and Bhattacharyya, M.K. (1995) Characterization
of a plasma membrane-associated phosphoinositol-specific phospholipase C from soybean. Plant J. 8: 381-390.
Sheu, J.-J., Yu, T.-S., Tong, W.-F. and Yu, S.-M. (1996) Carbohydrate
starvation stimulates differential expression of rice a-amylase genes that
is modulated through complicated transcriptional and posttranscriptional processes. J. Biol. Chem. 271: 26998-27004.
Sugimoto, N., Taketa, G., Nagato, Y. and Yamaguchi, J. (1998) Temporal
and spatial experssion of the a-amylase gene during seed germination in
407
rice and barley. Plant Cell Physiol. 39: 323-333.
Todoroki, Y., Hirai, N. and Koshimizu, K. (1995) 8',8'-difluoro-and
8',8',8-trifluoroabscisic acids as highly potent, long-lasting analogues of
abscisic acid. Phytochemistry 38: 561-568.
Yoshida, S., Kawata, T., Uemura, M. and Niki, T. (1986) Properties of
plasma membrane isolated from chilling-sensitive etiolated seedlings of
Vigna radiata L. Plant Physiol. 80: 152-160.
Yoshida, Y. and Imai, S. (1997) Structure and function of inositol 1,4,5trisphosphate receptor. Jpn. J. Pharmacol. 74: 125-137.
Yotsushima, K., Mitsui, T., Takaoka, T., Hayakawa, T. and Igaue, I.
(1993) Purification and characterization of membrane-bound inositol
phospholipid-specific phospholipase C from suspension-cultured rice
(Oryza sativa L.) cells: identification of a regulatory factor. Plant
Physiol. 102: 165-172.
Williams, R.L. and Katan M. (1996) Structure views of phosphoinositidespecific phospholipase C: signalling the way ahead. Structure 4: 13871394.
(Received September 30, 1999; Accepted January 11, 2000)