Full text of DOI:10.1078/0176-1617-00326

J. Plant Physiol. 158. 1117–1123 (2001)
Urban & Fischer Verlag
http://www.urbanfischer.de/journals/jpp

2
+
The requirements for Ca , protein phosphorylation and concurrent
protein synthesis for zeatin signaling of acidic chitinase transcript
accumulation in Cucumis sativus L.
Sonali P. Barwe, Muthukrishnan Sathiyabama, Chelliah Jayabaskaran*
Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India
Received January 12, 2001 · Accepted May 21, 2001
Summary
We have previously reported that both Ca2 + and staurosporine-sensitive protein kinase(s) are
involved in the cytokinin zeatin induction of cucumber chitinase activity and its protein content
(Barwe et al. 2001). To further characterize signal transduction events involved in this cytokinin induc-
tion of chitinase gene expression, Northern hybridizations of total RNAs prepared from excised, dark-
grown cucumber cotyledons treated with cytokinins and/or various agonists and antagonists of signal
transduction components, were carried out using a cucumber acidic chitinase (CACHT) cDNA probe
(Metraux et al. 1989). CACHT mRNA increased by approximately 5- to 6-fold in response to exoge-
nous zeatin (Z), zeatin riboside (ZR), and benzyladenine (BA) treatment, but failed to accumulate in
response to kinetin (K). Among the cytokinins tested, Z was most effective. The Z-induced accumula-
tion of CACHT mRNA was inhibited by a plasma membrane Ca2 + channel blocker verapamil. Treat-
ment of cotyledons with exogenous CaCl2 and calcium ionophore A23187 in the presence and
absence of cytokinin enhanced CACHT mRNA accumulation. These two observations suggest the
participation of extracellular calcium in signaling Z-induction. Furthermore, the presence of stauro-
sporine (an inhibitor of protein kinase) in Z treatment reduced CACHT mRNA, suggesting the involve-
ment of phosphorylation of one or more cellular proteins. In addition, we provide evidence that the
Z-induction of CACHT mRNA is blocked by protein synthesis inhibitor cycloheximide treatment.
Taken together, these results suggest that Ca2 + influx from extracellular space, protein phosphoryla-
tion, and concurrent protein synthesis events participate in cytokinin signaling during Z-induced
CACHT transcript accumulation.
Key words: Ca2 + – chitinase-cytokinin signaling – Cucumis sativus L. – protein phosphorylation –
transcript level – zeatin
Abbreviations: BA benzyladenine. – CACHT cucumber acidic chitinase. – CHX cycloheximide. –
EGTA ethylene glycol. – bis (2-amino-ethylether) tetraacetic acid. – K kinetin. – NaF sodium fluoride. –
ST staurosporine. – Z zeatin. – ZR zeatin riboside
*
E-mail corresponding author: cjb@biochem.iisc.ernet.in
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176-1617/01/158/09 –1117 $ 15.00/0

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Sonali P. Barwe, Muthukrishnan Sathiyabama, Chelliah Jayabaskaran
β-expansin gene (Crowell 1994, Downes and Crowell 1998)
and Sesbania rostrata early nodulin gene SrEnod 2 (Schmul-
ling et al. 1997).
Introduction
Cytokinins are of interest in view of their possible regulatory
roles in several stages of plant development. They induce cell
division, cell expansion in cotyledon, chloroplast and etio-
plast development, suppression of apical dominance and
senescence, and differentiation of in vitro cultured cells (Leth-
am and Palni 1983, Evans 1984). There is a considerable
amount of evidence to show that treatment of intact plants or
excised organs with exogenous cytokinins stimulates the
activity of several enzymes and/or their respective mRNAs.
These include ribulose-1,5-biosphophate carboxylase/oxy-
genase (Flores and Tobin 1986), PEP carboxylase (Suzuki et al.
This study presents an effort to elucidate the mechanism of
cytokinin-induced gene activation and includes the identifica-
tion of the components of cytokinin signal transduction. We
have earlier shown by TaqMan (Quantitative RT-PCR) analy-
ses (Rajesh 2000) that cytokinin treatment of dark-grown, ex-
cised cucumber cotyledons induced the expression of the
genes encoding a calcium-dependent protein kinase (CuCD-
PK3) and the FtsZ plastid division protein. We have also de-
monstrated the induction of chitinase activity by exogenous
cytokinin in dark-grown, excised cucumber cotyledons and
the involvement of Ca2 + and staurosporine-sensitive protein
kinase in cytokinin-induced stimulation of chitinase activity
and its protein accumulation (Barwe et al. 2001). In this paper
we have investigated the effects of cytokinins on CACHT tran-
script accumulation, and possible second messengers and
signaling components involved in the cytokinin-mediated in-
duction of CACHT transcript accumulation. The results de-
monstrate that CACHT transcript accumulation is enhanced
by cytokinin zeatin, and that this mechanism requires Ca2
influx from extracellular space, protein phosphorylation, and
ongoing protein synthesis.
1
994), protein kinase (Sano and Youssefian 1994), nitrate
reductase (Dilworth and Kende 1974, Lu et al. 1990), chitinase
Memelink et al. 1987), chalcone synthase, chalcone isomer-
(
ase, dihydrofolate reductase, and phenylalanine ammonia
lyase (Deikman and Hammer 1995). These studies demon-
strate that although gene expression is enhanced by cytoki-
nin, the molecular mechanisms controlling the expression of
these genes appear to be different for different enzymes. For
instance, the expression of cytokinin-inducible genes encod-
ing PEP carboxylase, nitrate reductase, chalcone synthase,
and dihydrofolate reductase is regulated at the level of tran-
scription (Dilworth and Kende 1974, Lu et al. 1990, Suzuki et
al. 1994, Deikman and Hammer 1995), whereas the expres-
sion of genes encoding the small subunit of ribulose-1,5-
bisphosphate carboxylase/oxygenase, chalcone isomerase
and phenylalanine ammonia lyase is regulated at the post-
transcriptional level (Flores and Tobin 1986, Deikman and
Hammer 1995). Even with this present knowledge of the role
of these hormones in the enhancement of expression of a var-
iety of genes, very little is known about how cytokinin signals
are perceived and the components required for transducing
them.
+
Materials and Methods
Chemicals and materials
The various chemicals and other materials used in this study were ob-
tained from commercial sources as follows: acrylamide, benzylade-
nine, calcium chloride, cycloheximide, Ca2 + ionophore A23187, EGTA,
kinetin, staurosporine, Tris base, zeatin, and zeatin riboside from
Sigma Chemical Co., USA; nylon membranes from Amersham Phar-
macia Biotech, USA, cucumber (Cucumis sativus L. cv. Poinsette)
seeds from National Seed Corporation, Bangalore, India. Cucumber
acidic chitinase cDNA clone was a kind gift from Dr. Metraux, Ciba-
Geigy, Agricultural Centre, Switzerland. [α32-P]-dCTP was obtained
from New England Nuclear, USA. All other chemicals used were of an-
alytical grade.
There have been few reports on the possible involvement
of the two-component regulatory system, typically consisting
of a sensor histidine kinase and a response regulator in the
signal transduction of cytokinins (Kakimoto 1996, Schmulling
et al. 1997, Kakimoto 1998, Sakakibara et al. 1999). Sakaki-
bara et al. (1998) isolated a cytokinin-inducible gene, ZmCip 1,
encoding a homologue of a response-regulator of a bacterial
two-component signaling system. The authors showed that
the steady-state level of the ZmCip 1 transcript was markedly
enhanced by cytokinin treatment of the excised maize leaves.
In Arabidosis thaliana, CKI1, a gene encoding homologues
domains of His-protein kinase and response regulator, was
isolated using activation tagging, and overexpression of this
gene induced shoot development from callus in the absence
of exogenous cytokinins, suggesting that the two-component
system is involved in cytokinin signal transduction (Kakimoto
Plant material and excised cotyledon treatments
Cucumber (Cucumis sativus L. var. Poinsette) seeds were germinated,
and grown in the dark at 28 ˚C, as previously described (Barwe et al.
2001). After 7 days, cotyledons were excised under dim green light,
transferred to small Petri dishes, and pre-incubated in distilled water
for 24 h in the dark to deplete endogenous cytokinins. Treatments of
the cotyledons with cytokinin (BA, Z, ZR, or K) and/or with one of the
other chemicals, such as staurosporine, sodium fluoride, CaCl2, cal-
cium ionophore A23187, calcium antagonists (EGTA, verapamil),
MgCl2, or cycloheximide were carried out as described previously
1
996). Further, a role for protein phosphorylation/dephos-
(Barwe et al. 2001). The treated as well as water-treated (control) coty-
phorylation has been suggested on the basis of studies using
inhibitors of protein kinases and protein phosphatases in the
expression of cytokinin inducible genes such as soybean
ledons were collected, rinsed with distilled water, and surface blotted
carefully with filter paper. Cotyledon samples were frozen in liquid ni-
trogen and stored at –80 ˚C until extraction of total RNA.

Zeatin modulation of chitinase transcript accumulation
1119
Total RNA isolation and Northern blot analysis
Total RNA was isolated from frozen cucumber cotyledons according
to the method described by Verwoerd et al. (1989). The RNA was sub-
jected to electrophoresis on agarose gels that contained formalde-
hyde, and blotted onto nylon membranes (Sambrook et al. 1989). The
blots were probed with a cucumber acidic chitinase cDNA (Metraux
et al. 1989) that had been labeled by random-primed labelling with
[
α32-P]-dCTP (Feinberg and Vogelstein 1984). The blots were placed
in hybridization bottles, subjected to pre-hybridization in hurch buffer
containing 0.5 mol/L sodium phosphate buffer, pH 7.2, 10 mmol/L
EDTA, and 7 % (w/v) SDS for 3 h at 65 ˚C in a rotating hybridization
oven, and subsequently hybridized to the probe in the same condi-
tion. After hybridization, the membranes were washed once with
2
×SSC and 0.5 % SDS at room temperature for 15 min, and then twice
with 0.1 × SSC and 0.5 % SDS at 65 ˚C for 30 min. They were then ex-
posed to X-ray film with an intensifying screen at –70 ˚C. Relative lev-
els of RNA were determined by densitometry scanning of the autora-
diogram using the TotalLab Analysis software from Phoretix.
Results
The effects of cytokinins on cucumber acidic chitinase
(CACHT) transcript accumulation
In order to determine if cytokinin treatment influences CACHT
transcript level by increasing the rate of transcriptional initia-
tion and/or by altering some post transcriptional event, total
RNA was isolated from cotyledons treated with various cytoki-
nins and Northern blot analysis was performed. The Northern
blot hybridization and CACHT transcript quantitation data are
shown in Figures 1A and C. Hybridization with the 32P-labeled
CACHT cDNA probe revealed a major transcript of 1.0 kb cor-
responding to the size of the CACHT mRNA (Metraux et al.
Figure 1. Effects of various cytokinins on the accumulation of CACHT
transcripts (A) and 18S rRNA (B). Total RNA was isolated from ex-
cised, dark-grown cucumber cotyledons 48 h after treatment with wa-
ter (control), benzyladenine (BA), zeatin riboside (ZR), zeatin (Z), and
kinetin (K). All tested cytokinins were used at a concentration of
1
989). The level of CACHT transcript increased by approx-
imately 5- to 6-fold following BA, ZR, and Z treatment. BA and
ZR were equally effective while Z appeared to be a slightly
stronger effector among other cytokinins. K was totally inef-
fective and did not affect the level of the transcript. We also
examined the same blot by hybridization with an 18S rDNA
probe. As anticipated the level of 18S rRNA detected with its
probe did not change upon treatment with cytokinins (Fig.
1
00 µmol/L. Ten micrograms of total RNA from each sample were sep-
arated electrophoretically in each lane and analysed by Northern hy-
bridization with 32P-labeled CACHT cDNA and 18S rRNA gene pro-
bes (see Materials and Methods). The Northern blot analysis was re-
peated twice, with similar results. C. Quantifications of CACHT tran-
scripts. The relative levels of CACHT transcript were measured by
densitometric scanning of autoradiograms. The level of CACHT tran-
script present in the water-treated (control) cotyledons was set as 1.
Values are the means and SD from two separate experiments for each
treatment.
1
B). This indicates that the observed effects of cytokinins in
the accumulation of CACHT transcripts were specific.
Role of Ca2+ in Z-induced CACHT transcript
accumulation
however, no further increase was observed in the CaCl2 plus
Z-treated cotyledons. This observation clearly indicates that
an increase in the intracellular Ca2 + level might be involved in
the observed zeatin stimulation of CACHT transcript accumu-
lation. To confirm the involvement of Ca2 + in CACHT tran-
script accumulation, the cotyledons were treated with a spe-
cific calcium chelator, EGTA, and the Ca2 +-induced CACHT
transcript accumulation was examined. Chelation of extracel-
lular Ca2 + with EGTA inhibited the Ca2 +-induced expression
of CACHT mRNA to a level similar to that of the water-treated
We investigated the role of Ca2 + in the accumulation of
CACHT transcript in general, and zeatin-induced accumula-
tion of CACHT transcript in particular, using the CACHT cDNA
probe. Northern blot was carried out using total RNA isolated
from cotyledons treated with agents that influence intracellu-
lar Ca2 + concentrations. The results of the Northern analysis
are presented in Figures 2 A and B. Interestingly, the CACHT
transcripts increased upon either CaCl2 or zeatin treatment;

1
120
Sonali P. Barwe, Muthukrishnan Sathiyabama, Chelliah Jayabaskaran
Role of protein phosphorylation in Z-induced CACHT
transcript accumulation
To determine whether protein phosphorylation/dephosphory-
lation is involved in Z-induced CACHT transcript accumula-
tion, the cotyledons were treated with zeatin in the presence
or absence of inhibitors of protein kinase and protein phos-
phatase, and the Z-induced CACHT transcript accumulation
was examined by Northern analysis. As shown in Figures 3 A
and B, staurosporine strongly inhibited Z-induced CACHT
transcript accumulation, whereas NaF treatment showed no
change. Water-treated (control) cotyledons indicated the bas-
al level of CACHT transcript accumulation, which was not
affected by treatment with dimethylsulfoxide (DMSO), the sol-
vent used for dissolving staurosporine. The former result indi-
cates that protein kinase activity is involved in cytokinin regu-
lation of CACHT transcript level.
Figure 2. Effects of Ca2 +, EGTA, Ca2 + ionophore (Ca2 +I), and Ca2 +
channel blocker verapamil (V) on uninduced and Z-induced accumu-
lation of CACHT transcripts. Total RNA was isolated from excised,
dark-grown cucumber cotyledons 48 h after treatment with water
(
control), 100 µmol/L zeatin (Z), 5 mmol/L calcium chloride (CaCl2),
mmol/L calcium chloride plus 100 µmol/L zeatin (CaCl2 +Z), 5 mmol/L
EGTA (EGTA), 10 µmol/L verapamil (V), 10 µmol/L verapamil plus
5
1
1
00 µmol/L zeatin (V+Z), 100 µmol/L calcium ionophore (Ca2 +I), and
00 µmol/L calcium ionophore plus 100 µmol/L zeatin (Ca2 +I+Z).
Northern blot analysis was carried out as described in the legend to
Figure 1. The rRNA detected with ethidium bromide was indicated as
the loading control. B. Densitometric quantitation of CACHT transcript
levels. Other explanations are as described in the legend to Figure
1
C.
(
control) cotyledons. To further test the role of extracellular
Figure 3. Effects of inhibitors of protein kinase (staurosporine, ST) and
protein phosphatase (sodium fluoride, NaF) on uninduced and Z-in-
duced accumulation of CACHT transcripts. Total RNA was isolated
from excised, dark-grown cucumber cotyledons 48 h after treatment
with water (control), 100 µmol/L zeatin (Z), 0.1 % DMSO, 10 nmol/L
staurosporine (ST), 10 nmol/L staurosporine plus 100 µmol/L zeatin
2
+
Ca
in the Z-induced CACHT transcript accumulation, the
2
+
plasma membrane Ca
Ca2 + ionophore A23187 were used. Treatment of cotyledons
channel blocker, verapamil, and
with the Ca2 + ionophore reproduced the regulatory effect of
Ca2 + on Z-induced accumulation of CACHT transcript. Vera-
pamil treatment inhibited the Z-induced CACHT transcript ac-
cumulation. These results indicate that Z-induced CACHT
transcript accumulation is due to the elevations in cellular
Ca2 + concentrations as a result of Ca2 + uptake from the ex-
tracellular space through plasma-membrane channels.
(ST+Z), 1mmol/L sodium fluoride (NaF), 1mmol/L sodium fluoride plus
1
00 µmol/L zeatin (NaF+Z). Northern blot analysis was carried out as
described in the legend to Figure 1. The rRNA detected with ethidium
bromide was indicated as the loading control. B. Densitometric quan-
tification of CACHT transcript levels. Other explanations are as de-
scribed in the legend to Figure 1C.

Zeatin modulation of chitinase transcript accumulation
1121
Requirement of protein synthesis in the Z-induced
CACHT transcript accumulation
Discussion
To investigate whether the enhancement of CACHT transcript
accumulation by zeatin was dependent on concurrent protein
synthesis, the effect of cycloheximide, a protein synthesis in-
hibitor, on the basal as well as Z-induced CACHT transcript
level was studied. Excised, dark-grown cucumber cotyledons
were treated with 100 µmol/L cycloheximide in the presence
or absence of 100 µmol/L Z, after which RNA was isolated,
and CACHT transcript levels were examined by Northern blot
hybridization. As shown in Figures 4 A and B, cycloheximide
treatment completely inhibited the Z-induced accumulation of
CACHT transcript. The decrease in the basal level of CACHT
transcript after treatment with cycloheximide was not as pro-
nounced as the decrease in the level of Z-induced CACHT
transcript. This data suggests that ongoing protein synthesis
is required for the cytokinin-stimulated increased level of
CACHT transcript accumulation.
Chitinase has been shown to be expressed constitutively in
cucumber (Majean et al. 1990, Barwe et al. 2001), and was in-
duced by elicitors, wounding, pathogens, salicylic acid, and
phytohormones, such as abscisic acid, ethylene, and cytoki-
nins (Mauch et al. 1988, Siegrist and Kauss 1990, Zhang and
Punja 1994, Yu et al. 1998, Petruzzelli et al. 1999, Barwe et al.
2
001). A cDNA encoding an acidic chitinase has been iso-
lated and characterized from cucumber: its gene has been
shown to be present as a single copy gene in the cucumber
genome, and its protein was found to have no significant ho-
mology with either the basic chitinase isolated from bean or
tobacco or the chitinase isolated from Serratia marcescens
(Metraux et al. 1989). We were able to use this cDNA as
probe to determine effects of cytokinins and/or various ago-
nists and antagonists of signal transduction as part of a
study to characterize cytokinin regulation of CACHT gene ex-
pression. In this paper we present evidence that there is an
approximately 5- to 6-fold induction in CACHT mRNA in re-
sponse to three of the four cytokinins tested (zeatin, zeatin-
riboside, and benzyladenine). The cytokinin-induced en-
hancement of the CACHT transcript appears to be dependent
of influx of extracellular Ca2 +, protein phosphorylation and
concurrent protein synthesis.
The primary action of cytokinins has been associated with
enhanced calcium ion influx and the action of calmodulin
(
Hepler and Wayne 1985, Poovaiah and Reddy 1987). A rise
2
+
in intracellular Ca was shown to mediate cytokinin-induced
bud formation in Funaria by the use of calcium antagonists
such as EGTA, La3 +, and TMB and Ca2 + channel blockers,
such as verapamil (Saunders and Hepler 1983). We used a
Ca2 + chelator EGTA, Ca2 + channel blocker verapamil and
calcium ionophore, to investigate the role of Ca2 + in our sys-
tem. Both EGTA and verapamil blocked the basal and/or the
cytokinin-induced accumulation of CACHT transcript. Be-
cause EGTA and verapamil are unlikely to enter the cells and
verapamil blocks the Ca2 + channels localized in the plasma
membrane (Graziana et al. 1988, Knight et al. 1992), our data
indicates that the influx of Ca2 + from the extracellular space
via the Ca2 + channels localized in the plasma membrane is
required for the transduction of the cytokinin signal, and that
the cytokinin may influence the activity of the Ca2 + channels.
This conclusion is supported by the fact that increasing intra-
cellular Ca2 + levels by enhancing uptake from the extracellu-
lar space using a Ca2 + ionophore, reproduced the effects of
exogenous cytokinin or CaCl2 treatment on accumulation of
CACHT transcripts. Nevertheless, our study does not rule out
the possibility of mobilization of calcium from intracellular
compartments. Ca2 + signaling often coordinates parallel and/
or sequential use of different sources of Ca2 + and different
Ca2 + channels in different subcellular locations. It was dem-
onstrated in tobacco cells that hypo-osmotic shock stimulates
Ca2 + fluxes in a sequential manner, deriving first from external
Figure 4. Effect of cycloheximide on uninduced and Z-induced ac-
cumulation of CACHT transcripts. Total RNA was isolated from ex-
cised, dark-grown cucumber cotyledons 48 h after treatment with wa-
ter (control), 100 µmol/L zeatin (Z), 100 µmol/L cycloheximide (CHX)
and 100 µmol/L cycloheximide plus 100 µmol/L zeatin (CHX+Z). North-
ern blot analysis was carried out as described in legend to Figure 1.
The rRNA detected with ethidium bromide was indicated as the load-
ing control. B. Densitometric quantification of CACHT transcript lev-
els. Other explanations are as described in the legend to Figure 1C.

1
122
Sonali P. Barwe, Muthukrishnan Sathiyabama, Chelliah Jayabaskaran
and then internal Ca2 + stores, and that these fluxes are me-
diated by Ca2 + channels (Cessna et al. 1998).
tor protein factor or some rapidly turned over protein involved
in the control of mRNA stability. The data shown here indi-
cates that concurrent protein synthesis is necessary for the
cytokinin-enhanced accumulation of CACHT transcript. Treat-
ment of cotyledons with cytokinin in the presence of protein
synthesis inhibitor cycloheximide inhibited cytokinin-en-
hanced CACHT transcript accumulation (Fig. 4). Silver et al.
(1996) reported the inhibition of cytokinin-induced SrEnod 2
mRNA accumulation by cycloheximide, and proposed that
concurrent protein synthesis is required for cytokinin enhance-
ment of SrEnodZ mRNA accumulation. Since the cytokinin ef-
fect is dependent upon concurrent protein synthesis, it is pos-
sible that cytokinin induces synthesis and phosphorylation of
some rapidly turned over protein, which, in turn, may ultima-
tely cause the accumulation of CACHT transcript. It remains
to be determined whether the cytokinin-induced accumula-
tion of the chitinase transcript is solely caused by enhanced
transcription, or is also partly due to post-transcriptional pro-
cesses and/or stabilization of mRNA.
The observation that the inhibitory effect of protein kinase
inhibitor staurosporine on cytokinin-induced CACHT tran-
script accumulation (Fig. 3) showed that a staurosporine-sen-
sitive protein kinase(s) plays an important role in the trans-
duction of the cytokinin signal, resulting in accumulation of
the CACHT transcript. Thus, it is likely that the staurosporine-
sensitive protein kinase(s) may participate in cytokinin-
induced protein phosphorylation and, in turn, that the cytoki-
nin-induced protein phosphorylation may ultimately cause the
accumulation of CACHT transcript. Our results suggest the
involvement of either Ca2 +-dependent protein kinase(s) or
Ca-CaM (calmodulin)-dependent protein kinase(s) in the cy-
tokinin response. Thus, it is possible that the influx of Ca2 +
from the extracellular space by cytokinin regulates a complex
network of a protein kinase cascade in regend to CACHT
gene expression. Crowell (1994) has shown that in soybean,
the Cim1 mRNA accumulation is stimulated by staurosporine
in the absence of cytokinin, by okadaic acid, an inhibitor of
protein phosphatases, and is inhibited in the presence of cy-
tokinin. The authors suggest that dephosphorylation of one or
more cellular proteins is involved in cytokinin regulation of
Cim1 abundance. Moreover, it has been recently reported
that cytokinin-induced Cim1 stability is blocked by the protein
phosphatase inhibitor okadaic acid, and the involvement of
protein phosphatase activity has been suggested for cytoki-
Acknowledgements. This work was supported by a grant (SP/SO/
A49/98) from the Department of Science and Technology, Govern-
ment of India. S.P.B. was supported by a fellowship from CSIR, New
Delhi, India. We thank Mr. K. K. Pramod for help in the preparation of
the manuscript.
nin-induced mRNA stability, and subsequent accumulation of References
Cim1 in soybean cells (Downes and Crowell 1998).
Barwe SP, Sathiyabama M, Jayabaskaran C (2001) Induction of chitin-
In another study, okadaic acid treatment of cultured to-
bacco cells has been shown to slightly stimulate the accumu-
lation of msr1 mRNA in the absence of auxin or cytokinin,
whereas staurosporine treatment blocked cytokinin- and aux-
in-induced msr1 mRNA accumulation (Dominov et al. 1992).
Furthermore, in the tobacco cell cultures desensitized with
auxin, okadaic acid treatment was shown to result in a strong
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gene expression, and argue that the protein dephosphoryla-
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staurosporine-sensitive protein kinase is involved in the cyto-
kinin-signaling pathway for the enhancement of CACHT tran-
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specifically increases wheat protein kinase wpk4 mRNA ac-
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