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The involvement of calmodulin and protein kinases in
the upstream of cytosolic and nucleic calcium signaling
induced by hypoosmotic shock in tobacco cells
Hieu Nguyen, François Bouteau, Christian Mazars, Masaki Kuse, Tomonori
Kawano
To cite this version:
Hieu Nguyen, François Bouteau, Christian Mazars, Masaki Kuse, Tomonori Kawano. The involvement of calmodulin and protein kinases in the upstream of cytosolic and nucleic calcium signaling induced by hypoosmotic shock in tobacco cells. Plant Signaling and Behavior, Taylor & Francis, 2018, pp.1-7. �10.1080/15592324.2018.1494467�. �hal-02361110�
Running title: Regulatory factors of Hypo-OS-induced calcium ion
1
2
The involvement of calmodulin and protein kinases in the upstream of cytosolic and
3
nucleic calcium signaling induced by hypoosmotic shock in tobacco cells
4
5
Hieu T. H. Nguyen1, François Bouteau2,3,4, Christian Mazars5, Masaki Kuse6 and
6
Tomonori Kawano1,3,4,7,†
7
8
1
Laboratory of Chemical Biology and Bioengineering, Faculty and Graduate School of 9
Environmental Engineering, The University of Kitakyushu, Kitakyushu 808-0135, Japan 10
2
Université Paris Diderot, Sorbonne Paris Cité, Laboratoire Interdisciplinaire des Energies de 11
Demain, Paris, France 12
3
University of Florence LINV Kitakyushu Research Center (LINV@Kitakyushu), 13
Kitakyushu, Japan 14
4
International Photosynthesis Industrialization Research Center, The University of 15
Kitakyushu, Kitakyushu 808-0135, Japan. 16
5
Laboratoire de Recherches en Sciences Végétales, Université de Toulouse UPS, CNRS 17
UMR5546, Castanet-Tolosan, France 18
6
Laboratory of Natural Products Chemistry, Graduate School of Agricultural Science, Kobe 19
University, Kobe 657-8501, Japan 1
7
Univ. Paris-Diderot, Sorbonne Paris Cité, Paris Interdisciplinary Energy Research Institute 2
(PIERI), Paris, France 3
†
To whom correspondence should be addressed. Tel: +81-93-695-3207 E-mail: 4
kawanotom@kitakyu-u.ac.jp 5
Abstract
1
Changes in Ca2+concentrations in cytosol ([Ca2+]C) or nucleus ([Ca2+]N) may play some vital
2
roles in plants under hypoosmotic shock (Hypo-OS). Here, we observed that Hypo-OS 3
induces biphasic increases in [Ca2+]Cand [Ca2+]Nin two tobacco cell lines (BY-2) expressing
4
apoaequorin either in the cytosol or in the nucleus. Both [Ca2+]Cand [Ca2+]Nwere sensitively
5
modulated by the inhibitors of calmodulin and protein kinases, supporting the view that 6
calmodulin suppresses the 1stpeaks and protein kinases enhance the 2nd peaks in [Ca2+]Cand
7
[Ca2+]N. Data also suggested that the 1st and 2nd events depend on the internal and
8
extracellular Ca2+sources, respectively. 9
10
Key words:
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Calmodulin; cytosolic calcium signaling; hypoosmotic shock; nucleic calcium signaling; 12
protein kinases; tobacco cells 13
Introduction
1
Plants must respond to water stress during normal growth and development.1-3Calcium, a 2
ubiquitous second messenger in eukaryotes,4,5plays a vital role in plant signaling pathways in 3
response to water stress, including hypoosmotic shock (Hypo-OS).6,7 Hypo-OS-induced 4
transient changes in cytosolic and nucleic Ca2+ concentrations ([Ca2+]C and [Ca2+]N) have
5
been documented in tobacco cells and the change in [Ca2+]Nis distinct and independent from
6
the events in [Ca2+]C.6,7Hypo-OS-induced Ca2+signaling is also known as one of the reliable
7
models for studying the involvement of mechano-sensitive calcium channels in plant cells.8,9 8
While Hypo-OS-responsive [Ca2+]C increase shows a biphasic signature,6 Hypo-OS
9
reportedly induces a single-phased slow increase in [Ca2+]N,7implying that nucleus possesses
10
a distinct [Ca2+]Nsignaling pathway as reported in a number of reports.5,7,10-12
11
The elevation of [Ca2+]Cis decoded and transmitted to downstream responses, by several
12
types of Ca2+ sensor proteins containing a high-affinity Ca2+-binding motif, such as 13
calmodulin (CaM), calmodulin-like protein, calcium-dependent protein kinase (CDPK) and 14
calcineurin B-like protein.5,13,14The identification of CaM and CDPK in nucleus provided the 15
evidences that CaM and CDPK have a pivotal role in the decoding of Ca2+signaling in both 16
cytosol and nucleus.5 Currently, a number of studies have focused on the downstream of 17
Hypo-OS-induced [Ca2+]Csignaling. Early studies by Takahashi et al. (1997b) and Cazale et
18
al. (1999) focusing on [Ca2+]C suggested that Hypo-OS-induced [Ca2+]C elevation is
transduced to the protein kinase cascades in tobacco cells through the activation of 50-, 75-1
and 80-kDa protein kinases as well as MAPK kinase.15,16 A novel type of Calmodulin 2
(CaM)-binding protein (AtCaMBP25) was reported to function as a negative regulator of 3
osmotic stress responses in Arabidopsis thaliana.17 4
However, Ca2+ sensors are not only responsible in the downstream but also in the 5
upstream of intracellular Ca2+signaling. Accumulating evidences show that CaM and protein 6
kinases actively mediate the elevation of intracellular Ca2+ concentrations in response to 7
extracellular stimuli.18-22 In order to clarify the involvement of CaM and protein kinases in 8
the upstream of Hypo-OS-induced [Ca2+]Cand [Ca2+]Nelevations, effects of pharmacological
9
inhibitors on these events were examined in the present study, by employing two tobacco cell 10
lines (BY-2) expressing aequorin either in the cytosol or in the nucleus. 11
12
Results
13
Hypo-OS-induced biphasic responses in [Ca2+]Cand [Ca2+]Nelevations 14
By using the native coelenterazine and a synthetic F-coelenterazine for reconstituting 15
aequorin in the cytosol and nucleus, respectively, aequorin luminescence showed that 16
Hypo-OS can induce the biphasic increase in [Ca2+] in both cellular compartments in tobacco 17
cells (Fig. 1a,b and Fig. 2). It should be noted that the first minor peak (1stpeak) of [Ca2+]Cis
18
occasionally unobservable as shown by the size of error bars in Fig. 1a. Notably, the peaking 19
times in [Ca2+]C and [Ca2+]N were almost identical, as the 1st peaks of [Ca2+]C and [Ca2+]N
1
were attained within initial 30 s after Hypo-OS was given, and the peaking times for the 2
second but major increase (2ndpeak) in [Ca2+]Cand [Ca2+]Ncommonly ranged at c.a. 85 ± 15
3
s. 4
5
Effect of Trifluoperazine (CaM inhibitor) on Hypo-OS-induced [Ca2+]Cand [Ca2+]N 6
Trifluoperazine (TFP), a CaM antagonist known to bind directly to CaM,23 was used to 7
examine the involvement of CaM in the regulation of [Ca2+]C and [Ca2+]Nsignatures upon
8
Hypo-OS. Tobacco cells were incubated with different concentrations (50, 100, 200 and 300 9
µM) of TFP for 5 min prior to Hypo-OS treatment. Under the influence of TFP, the patterns 10
of [Ca2+]Cand [Ca2+]Ninduced by Hypo-OS were dose-dependently altered (Fig. 1a-d). While
11
the initial peaks of [Ca2+]Cand [Ca2+]Nwere significantly enhanced by high concentration of
12
TFP, the 2nd peaks of [Ca2+]C and [Ca2+]N were less influenced by the lower range of TFP
13
concentrations and partially inhibited by high concentration of TFP (Fig. 1a-d). Amplitudes of 14
TFP-dependent enhancement in aequorin luminescence were 1300 % (13-fold) and 120 % in 15
the 1stpeaks of [Ca2+]Cand [Ca2+]N, respectively (in the presence of 300 µM TFP, Fig. 1e).
16
17
Effect of Staurosporine (protein kinase inhibitor) on Hypo-OS-induced [Ca2+]Cand [Ca2+]N 18
Staurosporine, an inhibitor of broad range of serine/threonine protein kinases,24 was used 19
for pre-incubation of cells for 5 min prior to Hypo-OS application. High (10 µM) and low (1 1
µM) concentrations of staurosporine, were used to assess the impact of protein kinases on the 2
elevations of both [Ca2+]Cand [Ca2+]N(Fig. 2). As shown in Fig. 2, staurosporine specifically
3
lowered the 2ndpeaks of [Ca2+]Cand [Ca2+]Nwhich are induced by Hypo-OS, without altering
4
the extent of the 1st peak. While low dose (1 µM) of staurosporine partially suppressed the 5
increase of 2nd peaks in both [Ca2+]Cand [Ca2+]N, high dose (10 µM) completely abolished
6
the 2ndpeaks in both [Ca2+]Cand [Ca2+]N(Fig. 2). These results suggested the involvement of
7
protein kinase(s) in induction of the major late phase of [Ca2+]C and [Ca2+]N in response to
8
Hypo-OS. 9
10
Effect of K252a (protein kinase inhibitor) on Hypo-OS-induced [Ca2+]Cand [Ca2+]N 11
Another popular protein kinase inhibitor, K252a, was used to confirm the action of 12
protein kinases in the regulation of [Ca2+]C and [Ca2+]N upon Hypo-OS. Analogous to
13
staurosporine, K252a is known to target serine/threonine protein kinases while it is also 14
expected to inhibit the activity of receptor-type tyrosine kinases within the Trk family.25 The 15
influence of K252a was shown in Fig. 2. Lower dose (10 µM) of K252a interfered with the 16
increase in 2ndpeaks in [Ca2+]Cand [Ca2+]Nwithout influencing the 1stpeaks (Fig. 2). In the
17
presence of high dose (50 µM) of K252a, Hypo-OS-responsive [Ca2+]C was completely
18
blocked and the increase in [Ca2+]Nwas partially lowered (Fig. 2). These data support for the
role of protein kinases in regulation of [Ca2+]Cand [Ca2+]Nupon Hypo-OS.
1
2
Discussion
3
F-aequorin reconstituted from F-coelenterazine possesses the higher Ca2+-sensitivity than 4
native one,26 therefore, the use of F-coelenterazine might be an appropriate choice for 5
monitoring even small change in low-[Ca2+] compartments in plant cells, such as nucleus.27 6
In the present study, the use of F-coelenterazine for monitoring the [Ca2+]Nsignature has
7
revealed that Hypo-OS can induce the biphasic increase in [Ca2+]N. Note that the resolution of
8
two peaks in [Ca2+]N would be lost with native aequorin.7 In contrast, native aequorin with
9
lesser Ca2+-sensitivity (compared to F-aequorin) was shown to be a good choice for detecting 10
a larger change in [Ca2+]C.6,7,28,29 Here, biphasic increase in [Ca2+]Cwas also observed with
11
native aequorin. These data suggest the hypoosmotically induced responses in [Ca2+]C and
12
[Ca2+]Nare likely caused by the same manner. Thus, the use of appropriate aequorin is
13
recommended for the monitoring the [Ca2+] signatures in different compartments of plant 14
cells. 15
The formation of Ca2+/CaM complex reportedly inhibits the 1,4,5-triphosphate receptor 16
(IP3R), a calcium-release channel located on the membrane of the intracellular Ca2+ stores
17
such as endoplasmic reticulum (ER), by interacting with the N-terminal ligand-binding 18
domain of IP3R.19,22 Action of TFP effectively liberating the IP3R from the inhibitory action
of Ca2+/CaM complex was recently described in animal cells.22Since TFP binds to CaM, the 1
Ca2+/CaM complex formation is no-longer formed, thus, allowing the release of Ca2+ from 2
internal stores through IP3R. By analogy, the result with TFP is possibly suggesting that the
3
major source of free Ca2+ for the 1stpeak of [Ca2+]C is from the internal Ca2+ stores, most
4
likely ER. Our result is consistent with a result presented by Goddard et al. (2000), which 5
suggests the positive role for IP3in the hypoosmotically induced Ca2+release in the nucleus
6
regions of marine alga, Fucus.30 7
Our data showed that the 1stpeak of [Ca2+]Ndose-dependently increases with TFP, but the
8
original source of Ca2+ is not yet confirmed. The fact that the initial peaks of [Ca2+]C and
9
[Ca2+]Ncoincide may be suggesting that both events in two distinct compartments could be
10
reflecting the same phenomenon. Assuming that the release of free Ca2+ from ER which 11
occupies the boundary between cytosolic and nucleic spaces were induced under Hypo-OS, 12
transient increases in [Ca2+] in both side of two compartments facing ER should be observed. 13
Our view that the initially synchronized peaks of [Ca2+]Cand [Ca2+]Nare caused by the same
14
mechanism, is consistent with the action of TFP which is a likely activator of 15
Ca2+/CaM-dependently silenced IP3R on ER. Currently, the presence of IP3R on nuclear
16
membrane has been identified in animal cells,31-33 but this information has not been 17
elucidated in plant cells. Xiong et al. (2004) have reported that nuclei isolated from tobacco 18
BY-2 cells maintaining intact [Ca2+]C elevating activity upon mechanical stimulus showed
sensitivity to two class of chemicals targeting the IP3-gated and TRP (transient receptor
1
potential) channels, suggesting that BY-2 cells possess the putative ligand-gated channels and 2
homologues of TRP channels located on the nuclear membrane.11 3
Endogenous protein kinase A and CDPKs are two protein kinases potentially responsible 4
for phosphorylation of aquaporins,34,35 enhanced movement of water through plasmas 5
membrane in plant cells by maintaining the aquaporin pores at open state.36-38Upon Hypo-OS, 6
the activation of aquaporin channels allows the movement of bulk water into intracellular 7
space and thus causes the mechanical stretching of plasma membrane, followed by the influx 8
of Ca2+ from extracellular through mechano-sensitive calcium channels. According to earlier 9
works, staurosporine and K252a, which are the protein kinase inhibitors by acting at the 10
ATP-binding site or the catalytic domain of protein kinases,24,39 may indirectly block the 11
aquaporin water channels through inhibition of protein kinase A and CDPKs. The actions of 12
staurosporine and K252a inhibiting only the 2nd peaks of [Ca2+]C and [Ca2+]N, strongly
13
suggested that the 2nd peaks is resulted from water entry-dependently stimulated Ca2+ 14
movement through mechano-sensitive calcium channels on the plasma membrane. Therefore, 15
free Ca2+must be of extracellular origin. This view is supported by early studies showing that 16
[Ca2+]Celevation induced by Hypo-OS could be inhibited by rare-earth metal ions, chiefly
17
Gd3+, a known inhibitor of stretch-activated Ca2+ channels.7,40 Here, we propose the 18
hypothesis on the different Ca2+sources responsible for the increases in [Ca2+]Cfor the 1stand
the 2ndpeaks to be attributed to the internal and extracellular sources, respectively. 1
Some studies suggested that ROS could be a factor triggering the elevation of [Ca2+] in 2
plant cells.29,41-43Indeed, one of the earliest events in response to Hypo-OS is the production 3
of reactive oxygen species (ROS) which is mediated by NADPH oxidases.44,45 Hence, ROS 4
might participate in the upstream of Hypo-OS-induced Ca2+elevation and therefore the role 5
for ROS must be elucidated in the future studies. 6
Lastly, we summarized the knowledge on the early stage of Hypo-OS response leading to 7
Ca2+ signaling (Fig. 3). The increases in [Ca2+]C and [Ca2+]N in response to Hypo-OS are
8
negatively and positively regulated by CaM and protein kinases, respectively. The likely 9
targets of CaM and protein kinases are the 1stpeak and 2nd peak, respectively. Although 10
[Ca2+]Cand [Ca2+]N likely share the same regulatory mechanism, the presence of CaM and
11
protein kinases in nucleus suggest that nucleus may possess its own regulatory mechanism. 12
13
Materials and Methods
14
Transgenic tobacco cell lines – Tobacco cells (Nicotiana tabacum L. cv. Bright Yellow-2, 15
BY-2), which express apoaequorin protein specifically either in the cytosol or in the nucleus 16
were cultured in Murashige-Skoog (MS) liquid medium (pH 5.8) containing 0.2 µg ml-1 of 17
2,4-dichlorophenoxy acetic acid at 28 ± 1○C on a rotary shaker (130 rpm) in darkness.6,46 18
Cells were subcultured with weekly intervals with 3% - 6% of inoculating culture at the 19
exponential stage. These 7 d-old cultured cells were collected by filtration and re-suspended 1
in fresh medium with a 25% packed cell volume prior to incubation with coelenterazines. 2
3
Chemicals – Native coelenterazine and F-coelenterazine were chemically synthesized by the 4
group of Prof. M. Isobe and Prof. Masaki Kuse, Nagoya University.47 TFP, staurosporine, 5
K252a and other chemicals were purchased from Sigma-Aldrich, Japan (Tokyo). 6
7
Monitoring of [Ca2+]C and [Ca2+]N by native and F-coelenterazines – Tobacco cells 8
expressing apoaequorin either in the cytosol or in the nucleus were incubated with 0.5 µM of 9
either native coelenterazine or F-coelenterazine, respectively, in darkness for 8 h to 10
reconstitute the chemiluminescence-active aequorin protein. Aequorin chemiluminescence 11
reflecting the increases in [Ca2+]C or [Ca2+]N induced by a given stimulus (∆112 mOsm of
12
Hypo-OS) was measured by using a luminometer (Luminescencer-PSN, model AB-2200-R, 13
ATTO, Japan) and expressed as relative luminescent units (rlu). 14
15
Hypo-osmotic stress treatments – To treat the cells with Hypo-OS, a known volume of pure 16
water was injected by a syringe onto differently conditioned cells in glass tubes set inside the 17
luminometer. The increase in the volume of the added water directly contributes to the 18
increase in the extent of Hypo-OS, which is proportional to the difference in osmolarity 19
(ΔmOsm).
1
2
Monitoring of osmolarity – To measure the osmolarity of the culture medium before and after 3
the addition of the water to the known volumes of cell suspensions, the extracellular liquid 4
was sampled and filtered through a nylon mesh (pore size, 10 µm). Then, the resultant liquid 5
samples were used for measurement of the final osmolality (mOsm of solute/kg of solvent, 6
mOsm/kg) by using an osmometer (model Advanced InstrumentsTM 3250 single-sample 7
osmometer, Thermo Fisher Scientific Inc., USA), with support by Japan Food Research 8
Laboratories. 9
10
Inhibitor treatment – Cells were incubated with TFP (50, 100, 200 and 300 µM), 11
staurosporine (1 and 10 µM) and K252a (10 and 50 µM) for 5 min prior to Hypo-OS 12
treatment. 13
14
Statistical analysis – All measurements described with mean± S.D were from at least three 15
replicates. Statistical significance was determined using one-way ANOVA and Tukey’s test, 16
and Student’s t-test (as indicated in the figure captions), with values of P < 0.05 were 17
considered for statistical significance. 18
Acknowledgments
1
TK was supported by a grant of the Regional Innovation Cluster Program implemented by the 2
Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. H.T.H.N. is 3
supported by a scholarship from MEXT. 4
This work was supported by a grant of the Regional Innovation Cluster Program from the 5
Ministry of Education, Culture, Sports, Science and Technology of Japan. 6
7
Conflict of interest
8
The authors declare no conflict of interest. 9
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Figure legends
1
Figure 1. Effect of CaM inhibitor (TFP) on the Hypo-OS-induced [Ca2+]C and [Ca2+]N.
2
Typical traces of aequorin luminescence reflecting the changes in Hypo-OS-induced [Ca2+]C
3
(a) and [Ca2+]N(b) in the presence of TFP are shown. Peak-heights of aequorin luminescence
4
for [Ca2+]C (c) and [Ca2+]N (d) are compared. Effects of TFP concentrations on the yield of
5
aequorin luminescence are shown (e). TFP was incubated with cells for 5 min prior to 6
addition of Hypo-OS (∆112 mOsm). Mean traces of aequorin luminescence from 5 replicates 7
monitored for 300 s are shown. H2O was injected at the initial 30 s of monitoring as indicated
8
by the arrows. Statistical significance (P < 0.05) was calculated using one-way ANOVA and 9
Tukey’s test.
10
11
Figure 2. Effects of protein kinase inhibitors (Staurosporine and K252a) on the
12
Hypo-OS-induced [Ca2+]Cand [Ca2+]N. Hypo-OS-induced [Ca2+]C (left column) and [Ca2+]N
13
(right column) in the presence of staurosporine (1 µM, 10 µM) or K252a (10 µM, 50 µM) 14
were compared. Cells were incubated with inhibitors for 5 min prior to Hypo-OS (∆112 15
mOsm). Mean curves from 5 replicates are shown (error bars at peak tops, S.D.). H2O was
16
injected at the times indicated by the arrows (as in Fig. 1). Statistical significance (*P < 0.01; 17
n.s., not significant) was calculated using Student’s t-test. 18
Figure 3. Schematic illustration of Hypo-OS-induced intracellular Ca2+ elevation pathways. 1
Upon Hypo-OS, cells perceive the Hypo-OS signals through a putative osmosensors and/or 2
PM-localized NADPH oxidases on plasma membrane, followed by the ROS production, the 3
release of IP3, the activation of protein kinases (PKA, CDPKs) and the elevation of Ca2+ in
4
cytosolic and nucleus spaces. NADPH oxidases are likely activated by Hypo-OS, thus rapidly 5
generating ROS. ROS production is one of the earliest events responding to Hypo-OS that 6
possibly stimulating the elevation of the intracellular Ca2+in the cytosol and nuclei. IP3might
7
act on the IP3R to release Ca2+ from ER located between the cytosol and nucleus. As
8
IP3-mediated Ca2+release is spontaneously blocked by the formation of Ca2+/CaM complex,
9
inhibition of CaM by TFP enables the release of Ca2+from ER. Protein kinases are involved 10
in aquaporin activation and allowing the entry of water into the cells. This event is followed 11
by the activation of the mechano-sensitive calcium channels which allows the influx of Ca2+ 12
from extracellular space into cytosol. AP: aquaporin channels, CC: calcium channels, ER: 13
endoplasmic reticulum, IP3: inositol 1,4,5-trisphosphate, IP3R: inositol 1,4,5-trisphosphate
14
receptors, PKs: protein kinases, PM: plasma membrane, ROS: reactive oxygen species. 15
Figure 1.
Figure 2.
1
2 3
Figure 3.
1
