Professional Documents
Culture Documents
Planta
An International Journal of Plant
Biology
ISSN 0032-0935
Planta
DOI 10.1007/s00425-015-2316-2
1 23
1 23
ORIGINAL ARTICLE
Abstract
Main conclusion Blue light signalling pathway in
broad bean leaf epidermal cells includes key membrane
transporters: plasma- and endomembrane channels and
pumps of H1, Ca21 and K1 ions, and plasma membrane redox system.
Blue light signalling pathway in epidermal tissue isolated
from the abaxial side of fully developed Vicia faba leaves
was dissected by measuring the effect of inhibitors of second
messengers on net K?, Ca2? and H? fluxes using non-invasive ion-selective microelectrodes (the MIFE system).
Switching the blue light onoff caused transient changes of
the ion fluxes. The effects of seven groups of inhibitors were
tested in this study: CaM antagonists, ATPase inhibitors,
Ca2? anatagonists or chelators, agents affecting IP3 formation, redox system inhibitors, inhibitors of endomembrane
Ca2? transport systems and an inhibitor of plasma membrane Ca2?-permeable channels. Most of the inhibitors had a
significant effect on steady-state (basal) net fluxes, as well as
on the magnitude of the transient ion flux responses to blue
light fluctuations. The data presented in this study suggest
that redox signalling and, specifically, plasma membrane
NADPH oxidase and coupled Ca2? and K? fluxes play an
essential role in blue light signal transduction.
Introduction
& Branka D. Zivanovic
vunduk@imsi.bg.ac.rs
1
Higher plants measure quantity, quality, direction and periodicity of incident light via a collection of specific photoreceptors, belonging to several classes of photoreceptors
that perceive different wavelengths of light (Gyula et al.
2003; Jenkins 2009). Two of thesephototropins and
cryptochromesdetect incident light from the UV-A/blue
region of the spectrum (Cashmore et al. 1999; Kimura and
Kagawa 2006) and are classified as blue light (BL)
123
123
(a)
-6
H+
5.751
-7
-8
5.743
-9
5.739
5.735
-10
(b)
pH units
5.747
the measuring chamber and the microelectrodes were adjusted under dim green microscope light. The electrodes
were placed 40 lm above the epidermal tissue surface with
their tips aligned and separated by 34 lm. During measurements, electrodes were moved by a computer-controlled stepper motor at a frequency of 0.1 Hz in a squarewave manner between 40 and 60 lm from the tissue surface. Net ion fluxes were calculated from recorded potential differences at these positions, assuming cylindrical
diffusion geometry (Shabala et al. 1997; Shabala and
Newman 1999; Newman 2001). The epidermal strips were
illuminated with blue light (30 lmol m-2 s-1) from a
150 W quartz halogen lamp (Phillips, Type 6423) with a
flexible light guide (KL 1500 LCD, Schott, Mainz, Germany) and blue light filter (BG37, 481 nm, Schott). Light
intensity was measured by a Li-Cor quantum photometer
(LI-250 Light meter, Lincoln, NE, USA).The epidermal
strips were exposed to several light/dark cycles (5/5 min
light/dark) until they obtained stable light-induced transient
changes in ion fluxes before the addition of an inhibitor
(Fig. 1). The choice of the 5/5 min cycle was driven by the
fact that this period was close to the frequency of the
natural (self-sustained) oscillations in membrane-transport
activity in plant cells (see Shabala et al. 2006 for comprehensive review and modelling). As a result, although the
entire transient response takes typically between 30 and
40 min (Shabala and Newman 1999), the strongest response (the peak value) is achieved at around 5 min.
Hence, using 5/5 min cycle was the most optimal, as it
came at a resonant frequency and allowed us to maximize
the magnitude of response (hence, the resolution of the
method).
The inhibitor of appropriate concentration was added to
the experimental chamber during a dark cycle, and light/dark transients of ion fluxes in the presence of the inhibitor
were continuously recorded for several cycles. For clarity
reasons, the spurious peaks in the recording during the
addition of the inhibitor are omitted from the analysis
(Fig. 2).
10
15
20
25
15
20
25
15
20
25
15
Ca2+
10
-5
-10
0
(c)
10
-5
K+
-15
-25
-35
-45
0
10
Time (min)
123
20 M DPI
6
BL on
4
2
0
-2
BL off
-4
6.05
pH units
5.95
5.85
Results
5.75
20
30
40
50
60
70
80
90
Time (min)
123
Net basal H? ion fluxes recorded from different leaf samples were rather heterogeneous showing either net uptake
(influx) or efflux, while for Ca2? and K? ions influx and
efflux were observed in most cases, respectively (Figs. 3, 5,
6, 7, 8, 9). The blue light induced transient changes of H?,
Ca2? and K? ions close (25 lm) to the surface of the
epidermal strips. BL caused bimodal changes (efflux-influx) of H? fluxes, while Ca2? and K? fluxes displayed a
transient influx upon BL illumination (Fig. 1). Switching
the light off caused a change in all ion fluxes opposite of
the transients that were induced by light-on. After obtaining stable BL-induced transients in ion fluxes, the inhibitors were added to the experimental solution during a
dark period and the ion fluxes were recorded for at least
three more BL-on/-off cycles. Such experimental protocol
for the application of all inhibitors used in this study is
illustrated in Fig. 2 by showing an example of one treatment with 20 lm DPI. Most of the inhibitors induced a
significant effect on steady-state (basal) net fluxes (Fig. 3),
as well as on the magnitude of ion flux responses to blue
light-on/light-off changes (Fig. 4).
Effect of CaM antagonists
The application of CaM antagonists (W7 and TFP) aimed
at testing the involvement of CaM in BL-induced activation of proton pump and coupled K? and Ca2? ion channels, which is expected for the leaf epidermis upon BL
illumination. Moreover, the inhibition of CaM by these
CaM antagonists was previously reported to stimulate
proton pumping outside of the guard cell (Shimazaki et al.
1992). It was expected that CaM antagonists-induced increase H? pumping outside of the epidermis should stimulate K? and Ca2? uptake. Indeed, both 200 lM W7 and
100 lM TFP caused a significant (P \ 0.05) shift towards
Full name
Mode of action
Concentration
References
W7
N-(6-aminohexyl)-5-chloro-1naphthalenesulfonamide
CaM antagonist
200 lM
Li et al. (2004)
TFP
Trifluoperazine
CaM antagonist
100 lM
EY
Eosin yellow
0.5 lM
ErB
Erythrocin B
4 lM
TMB-8
8-(N,N-diethylamino)octyl-3,4,5trimethoxybenzoate
Intracellular Ca2?
antagonist
50 lM
EGTA
NM
Ca2? chelator
Decreases formation of
IP3
2 mM
10 lM
LiCl
LiCl
5 mM
DPI
Diphenyleneiodonium
20 lM
HCFIII
Hexacyanoferrate (III)
Electron acceptor
0.5 mM
TG
Thapsigargin
Endomembrane Ca2?
ATPase inhibitor
5 lM
RR
Ruthenium red
Endomembrane Ca2?
channel inhibitor
1 lM
La3?
LaCl3
500 lM
increased net basal Ca2? and K? uptake (Fig. 3b, c). Basal
H? fluxes were also significantly affected and shifted towards a net H? efflux (Fig. 3a). The addition of CaM antagonists to the bath also potentiated the magnitude of BLinduced ion flux responses significantly (Fig. 4), with
three- to fivefold higher responses compared to pre-treated
tissue (Fig. 5). Adding TFP to the bath proved to have a
major detrimental effect on H?-specific electrodes. H? flux
data in response to TFP are therefore unavailable in Figs. 3
and 4.
Ca2? flux responses (Figs. 4b, 6), while at the same time
induced a doubling of the K? flux (Figs. 4c, 6). These data
argue in favour of the proposed model given in Fig. 10. No
significant effects of either 0.5 lM EY or 4 lM ErB on the
basal net Ca2? fluxes were found (Fig. 3b), while net K?
efflux was slightly enhanced by ErB (Fig. 3c). Also, no clear
trends were noticed for basal H? fluxes and BL-induced H?
flux responses (Figs. 3a, 4a, 6).
To explore the involvement of calcium in blue light signalling in the epidermis, Ca2? chelators and antagonists
were employed in this study. It was expected that decrease
in intracellular Ca2? concentration would promote proton
pumping outside the cell and activate KIR/KOR channels
due to voltage change on plasma membrane in the presence
of these inhibitors. Intracellular Ca2? antagonist 50 lM
TMB and Ca2? chelator 2 mM EGTA both resulted in
increased net basal H? efflux (Fig. 3a) and reduced net
Ca2? uptake (Fig. 3b), which is in the line with our expectation. Significant (P \ 0.05) effects on net K? basal
fluxes were also detected (Fig. 3c). When epidermal strips
were subjected to cyclic BL fluctuation, pre-treatment with
TMB and EGTA significantly reduced the magnitude of
Ca2? flux responses, but led to up to threefold increase in
123
(a)
350
RR
TG
HCFIII
La3+
Fig. 3 Steady-state (basal) net fluxes (in nmol m-2 s-1) of H? (a),
Ca2? (b) and K? (c) ions measured 60 min after the addition of a
pharmacological agents (for details, see Table 1). Positive and
negative values of ion fluxes correspond to influx and efflux,
respectively. Mean SE (n = 512). Control bars refer to the basal
flux values before addition of a pharmacological agent. Treatments
labelled with asterisks are significantly different from controls at
P \ 0.05
RR
TG
DPI
NM
TMB
EGTA
EY
ErB
W7
TFP
LiCl
NM
EGTA
EY
ErB
TFP
TMB
*
DPI
*
NM
TMB
EY
ErB
-100
EGTA
LiCl
-50
TFP
RR
TG
HCFIII
NM
* *
W7
W7
Control
100
123
K+ flux
200
K+ flux
*
TG
DPI
300
50
* * *
HCFIII
500
400
100
(c)
200
* * * *
600
(c)
150
*
EGTA
*
LiCl
EGTA
TMB
EY
ErB
W7
TFP
Control
NM
-50
* *
La3+
RR
HCFIII
* *
TG
Ca2+ flux
TMB
Ca2+ flux
100
* *
EY
150
700
600
500
400
300
200
100
0
ErB
200
(b)
TFP
(b)
- 50
W7
La3+
TG
RR
DPI
HCFIII
NM
LiCl
TMB
EGTA
ErB
EY
W7
TFP
Control
-20
50
* *
50
-15
HCFIII
150
DPI
DPI
* *
LiCl
-5
-10
250
La3+
H+ flux
La3+
10
RR
flux
450
LiCl
H+
15
La3+
(a)
After treatment
-2
-3
4
3
-8
H+
-4
-16
-5
-6
-24
0
10
15
20
15
550
10
450
350
10
15
20
30
20
Ca2+
10
250
0
10
15
20
10
15
20
-10
500
-15
300
60
-20
100
40
-25
-100
-30
-300
80
K+
20
10
15
20
0
C
0
10
15
20
Time (min)
Before treatment
After treatment
1.5
1
H+
0.5
0
C
0
0
10
15
20
10
15
20
45
35
40
30
35
25
30
20
25
15
0
10
15
0
C
0
20
30
10
-20
-10
-40
-30
-60
Ca2+
10
15
40
30
20
K+
10
0
C
-50
0
10
15
20
20
-80
0
10
15
20
Time (min)
123
0.1
After treatment
-5
-6
-0.1
-7
6
5
4
3
2
1
0
H+
-8
-0.2
0
10
15
C
0
20
10
10
10
15
20
5
4
Ca2+
3
2
0
C
-5
-5
0
10
15
20
-70
-20
-90
10
15
20
40
30
K+
20
-40
10
-110
0
-60
0
10
15
20 -130
C
0
10
15
20
Time (min)
Before treatment
-3
After treatment
-3
-5
4
3
-5
H+
2
-7
-7
-9
-9
1
0
10
15
20
C
0
10
15
29
14
22
15
-7
1
5
10
15
20
10
15
20
-30
-30
-40
-40
30
-50
-50
20
-60
-60
-70
-70
Ca2+
-14
0
20
40
K+
10
0
10
15
20
10
15
20
Time (min)
123
Discussion
Ion transporters involved in BL signalling in bean
epidermis
Since the guard cells form only a small fraction of overall
membrane area in leaf epidermis, and the BL-induced H?
flux was stimulated by calmodulin antagonists (Fig. 4)
which is opposite to reported responses from V. faba guard
cells (Shimazaki et al. 1992), it is likely that the fluxes
measured in this work are predominantly associated with
the basement cells and, given the leaf age, could be part of
the BL-related growth response. As growth of the epidermal
After treatment
Before treatment
3
-1
-1
-3
-3
H+
1
0
Fig. 9 Effect of
diphenyleneiodonium (DPI; a
known NADPH oxidase
inhibitor) on blue light-induced
ion flux responses measured
from broad bean epidermis.
Typical flux recordings are
shown for each ion (H?, Ca2?,
and K?) before and after adding
20 lM to the bath solution. The
right column shows the mean
peak magnitudes (in nmol m-2
s-1) before (control; C) and
after treatment (T). Mean SE
(n = 15)
10
15
15
10
10
-5
-5
-10
-10
-15
0
10
15
C T
0
20
20
10
15
20
12
8
Ca2+
4
0
C
0
10
15
20
-10
-10
15
-20
-20
10
-30
-30
20
K+
5
0
-40
-40
0
10
15
20
10
15
20
Time (min)
123
K+
H2O2
O 2-
DPI, HCF
NSCC
e-
O2
Ca2+
Voltage
HACC
H+
Ca2+
NADPH
La3+
La3+
ATP
TMB-8, EGTA
EY, ErB
Ca2+
LiCl
ATP
K+
CaM
W7,TFP
IP3
ATP
TG
NM
BL
Ca2+
RR
KIR
K+
KOR
Ca2+
123
H1-ATPase
In our working hypothesis, activation of the proton pump is
essential to acidify the cell wall to increase extensibility
and generate the driving force for K? influx to maintain an
osmotic difference between cytoplasm and apoplast and
thus maintain turgor. Modulation of BL has resulted in
significant fluctuation in net H? fluxes (all figures). The
fact that in most cases background H? was outwardly directed (net efflux; Fig. 3) and that BL-induced changes in
H? fluxes were suppressed by vanadate (Shabala and
Hariadi 2005; Percey et al. 2014) explicitly suggests involvement of plasma membrane H?-ATPase. BL-induced
activation of H?-ATPase has been reported before, both for
stomata guard cells (Schwartz et al. 1991; Taylor and
Assmann 2001) and leaf epidermis (Staal et al. 1994).
K1 transport systems
For the inward-directed K? fluxes, several candidates are
included in our model. TEA-sensitive K? channels play a
key role in BL-induced cell expansion growth (Stiles and
123
Thus, it can be assumed that BL-increased H? pump activity has hyperpolarized plasma membrane leading to
opening of the HACC channel and KIR channels to uptake
Ca2? and K? ions from apoplast to cytosol, respectively
(Fig. 10). Moreover, increased [Ca2?]cyt should activate
CaM protein to indirectly inhibit (via some unknown Y
component) H? pump, which is antagonized by CaM inhibitors (Fig. 10). This suggests that CaM acts either as a
negative feedback regulator of 14-3-3 protein binding to
H?-ATPase, or the observed regulation is indirect and may
be related to altered ionic conditions in cell cytosol. It is
known that K? may act as an intrinsic uncoupler of the
proton pump by binding to its site involving Asp-617 in the
cytoplasmic domain and inducing dephosphorylation of the
reaction cycle intermediate by a mechanism involving Glu184 in the conserved TGES motif of the pump phosphatase
domain (Buch-Pedersen et al. 2006). H?-ATPase is also
known to be sensitive to [Ca2?]cyt (Kinoshita et al. 1995).
Thus, stomatal opening is promoted by activation of H?ATPase due to lowered [Ca2?]cyt, whereas the increase in
[Ca2?]cyt reduces the activity of H?-ATPase leading to
stomatal closure. We show here that activation of increased
cytosolic Ca2? inhibits modulation of BL-induced Ca2?
flux responses by TFP or W7 may have a domino effect on
intracellular K? and Ca2? concentrations and, this, indirectly regulate H?-ATPase activity.
BL-induced [Ca2?]cyt elevation
BL-induced [Ca2?]cyt elevation has always been considered to be central to BL signalling, both in guard cells
(Shimazaki et al. 1992) and leaf epidermis (Elzenga et al.
1997). Early pharmacological studies have suggested that
CaM- and Ca2?/CaM-dependent myosin light chain kinases (MLCK) are the components of the signal transduction pathway in BL-dependent proton pumping in guard
cells (Shimazaki et al. 1992). In this study, the evidence for
[Ca2?]cyt involvement is from the observed modulation of
BL responses by EGTA (Ca2? chelator), W7 and TFP
(CaM antagonists) (Fig. 4). Intracellular Ca2? storage (and,
hence, endomembrane Ca2? transport systems) appears to
be central to BL signal transduction. Baum et al. (1999)
have suggested that Ca2? may regulate the activity of the
nuclear pore controlling movement of constitutively photomorphogenic repressor protein 1 (COP1) between the
cytosol and the nucleus. Photo 2 was implicated in Ca2?
release from intracellular stores in Arabidopsis leaves
(Baum et al. 1999; Babourina et al. 2002; Harada et al.
2003).
One of the possible candidates for the pathway for Ca2?
release from the intracellular stores may be IP3-gated
Ca2?-permeable channels (Allen et al. 1995). At the same
time, some other researchers have questioned the existence
123
epidermal tissue of the broad bean plant. Taking into account the fact that the specificity of inhibitors is not absolute, we proposed a tentative model for blue light
signalling in broad bean epidermal cells. Data presented
suggest the involvement of various plasma membrane/endomembrane ion channels and pumps in blue light signalling, i.e. plasma membrane K? channels (KIR, KOR),
Ca2? channel (HACC), non-selective cation channels
(NSCC), endomembrane Ca2? channel, plasma membrane
pumps as P-type H?-ATPase, Ca2?-ATPase, endomembrane Ca2?-ATPase, and plasma membrane redox system
(NADPH oxidase). It is also reasonable to include in the
model blue light photoreceptors such as cryptochromes and
phototropins. Additional electrophysiological and biochemical experimentation are required to confirm the involvement of all these components given in the
hypothetical model of blue light signalling in broad bean
epidermal tissue.
Author contribution statement SS and BZ designed the
experiments and wrote the manuscript. BZ performed
MIFE measurements and analysed data. LS contributed to
methodological aspects of this study and critically assessed
the data. TE critically read the manuscript and contributed
to data interpretation.
Acknowledgments This work was supported by the Australian
Research Council Discovery grant to Prof Sergey Shabala and partly
by Grant No. 173040 from the Serbian Ministry of Education and
Science.
Conflict of interest
of interest.
References
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