You are on page 1of 17

Dissecting blue light signal transduction

pathway in leaf epidermis using a


pharmacological approach
Branka D.ivanovi, Lana I.Shabala,
Theo J.M.Elzenga & Sergey N.Shabala

Planta
An International Journal of Plant
Biology
ISSN 0032-0935
Planta
DOI 10.1007/s00425-015-2316-2

1 23

Your article is protected by copyright and


all rights are held exclusively by SpringerVerlag Berlin Heidelberg. This e-offprint is
for personal use only and shall not be selfarchived in electronic repositories. If you wish
to self-archive your article, please use the
accepted manuscript version for posting on
your own website. You may further deposit
the accepted manuscript version in any
repository, provided it is only made publicly
available 12 months after official publication
or later and provided acknowledgement is
given to the original source of publication
and a link is inserted to the published article
on Springer's website. The link must be
accompanied by the following text: "The final
publication is available at link.springer.com.

1 23

Author's personal copy


Planta
DOI 10.1007/s00425-015-2316-2

ORIGINAL ARTICLE

Dissecting blue light signal transduction pathway in leaf epidermis


using a pharmacological approach
Branka D. Zivanovic1,2
Sergey N. Shabala1

Lana I. Shabala1 Theo J. M. Elzenga3

Received: 1 February 2015 / Accepted: 28 April 2015


Springer-Verlag Berlin Heidelberg 2015

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.

Keywords Broad bean  Inhibitors  Ion fluxes 


Microelectrodes
Abbreviations
BL
Blue light
CaM
Calmodulin
CRY
Cryptochrome
DPI
Diphenyleneiodonium
ErB
Erythrocin B
EY
Eosin yellow
HCFIII Hexacyanoferrate (III)
MIFE
Microelectrode ion flux estimation
NM
Neomicyn
PHOT
Phototropin
RR
Ruthenium red
TG
Thapsigargin
TFP
Trifluoperazine
TMB-8 8-(N,N-diethylamino) octyl-3,4,5trimethoxybenzoate
W7
N-(6-aminohexyl)-5-chloro-1naphthalenesulfonamide

Introduction
& Branka D. Zivanovic
vunduk@imsi.bg.ac.rs
1

School of Land and Food, University of Tasmania,


Private Bag 54, Hobart, TAS 7001, Australia

Institute for Multidisciplinary Research, University of


Belgrade, Kneza Viseslava 1, Belgrade, Serbia

Ecophysiology of Plants, University of Groningen,


Nijenborgh 7, Groningen, The Netherlands

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

Author's personal copy


Planta

photoreceptors. Blue light responses are not restricted to a


single cell type in plants. For instance, blue light controls
extension growth of epidermal cells in leaf and stem,
stomatal aperture in guard cells and chloroplast position in
mesophyll cells. Both the expression of BL photoreceptors
and their mode of signalling appear to be highly tissue
specific. While in hypocotyls BL inhibits cell expansion
growth (Cosgrove 1994), this process is enhanced by BL in
leaf epidermis (Staal et al. 1994). The major difference
comes from signal transduction pathways to downstream
targets. It is believed that BL perception in hypocotyls is
mediated by CRY1 photoreceptors (Ahmad and Cashmore
1993). These photoreceptors subsequently activate Clchannels, resulting in plasma membrane depolarization
(Spalding and Cosgrove 1992). This depolarization activates a certain class of outward-rectifying K? channels
causing massive K? efflux. The water efflux follows suit,
and the cell volume decreases. The process is rather different in epidermis, where BL perception increases the rate
of H? efflux (Staal et al. 1994), resulting in acidification of
the apoplast, acid-induced cell wall loosening and solute
accumulation for turgor maintenance (Stiles and Van
Volkenburgh 2002).
Despite the broad spectrum of physiological responses,
it appears that the downstream targets of BL signalling are
limited to a set of key transport proteins mediating movements of osmotically active inorganic ions (mainly K? and
Cl-) across the plasma membrane (Spalding 2000). However, despite many years of research, signal transduction
pathways between BL photoreceptors and plasma membrane effectors (downstream targets) have largely remained
elusive. Numerous second messengers were suggested including various kinases and phospholipases (Shimazaki
et al. 1992; Kaufman 1993). BL-induced increase in cytosolic free Ca2? is also widely reported (Kinoshita et al.
1995; Baum et al. 1999).Voltage gating via regulation of
the plasma membrane H?-ATPase (Takemiya et al. 2006)
and involvement of a redox system (Long and Jenkins
1998; Baum et al. 1999) are also widely discussed,
although many details remain unclear.
One of the major hurdles in studying signal transduction
pathways from BL receptors to membrane effectors is the
lack of convenient tools allowing in planta study of activity
of specific plasma membrane transporters, which have
sufficient temporal resolution to study the rapid kinetics of
BL responses and are capable of doing this under physiologically relevant conditions. The MIFE technique for noninvasive microelectrode ion flux measurements (Shabala
et al. 1997, 2012) is one of these tools. The technique
allows in situ measurements of net ion fluxes across a
membrane with high spatial (\2 lm) and temporal (5 s)
resolution (Shabala et al. 2012). Earlier, we have successfully applied the MIFE technique to study ion fluxes

123

associated with phototropism (Babourina et al. 2002) and


the localization of BL receptors (Babourina et al. 2003) in
plants, the association between light-induced transient ion
fluxes and leaf growth and photosynthesis (Shabala and
Newman 1999; Zivanovic et al. 2005; Shabala and Hariadi
2005), and their spectral and dose dependency (Zivanovic
et al. 2007). In the current study, we use the MIFE technique in combination with a pharmacological approach to
dissect BL-induced signal transduction pathways in broad
bean leaf epidermis. Data presented in the current work
suggests that redox signalling and, specifically, plasma
membrane NADPH oxidase, and coupled Ca2? and K?
fluxes, play an essential role in BL signal transduction.

Materials and methods


Plant material
Broad bean (Vicia faba L. cv Oswald; Hollander Imports,
Hobart, Australia) plants were grown from seeds in standard potting mix (70 % composted pine bark, 20 % coarse
sand and 10 % sphagnum peat, pH 6.0) supplemented with
fertilizer mix (1.8 kg m-3 Limil, 1.8 kg m-3 dolomite,
6.0 kg m-3 Osmocote Plus and 0.5 kg m-3 ferrous sulphate) in 2 L pots in temperature-controlled glasshouse
facilities at the University of Tasmania (26/19 C mean
day/night temperatures; 16-h day length; 5570 % relative
humidity; natural sunlight) as previously described (Shabala 2000; Percey et al. 2014). Four seeds per pot were
sown and thinned to two per pot when the plants reached
the two-leaf stage. Plants were watered daily with tap water
and used for measurements after 23 weeks.
Broad bean epidermal strips for MIFE
measurements
The newest fully developed leaves were excised from 15to 20-day-old broad bean plants. The abaxial epidermis was
peeled off by means of fine forceps (Eye-Instruments, Albert Heiss H3376, Tuttlingen, Germany). Isolated epidermal strips were left floating on aerated experimental
solution (0.1 mM KCl, 0.1 mM NaCl, 0.1 mM MgCl2,
0.05 mM CaCl2, 0.05 mM NH4Cl, 10 mM sucrose; pH
approximately 5.3, non-buffered) for 1 h before starting the
measurements to avoid wounding effects and to stabilize
ion fluxes. The isolated epidermal strips were rolled around
a glass holder with the mesophyll-facing side on the outside, exposed to the experimental solution.
The epidermal strip attached to its glass holder was
placed in the measuring chamber filled with measuring
solution containing 0.1 mM KCl, 0.1 mM CaCl2 and
10 mM sucrose, pH 5.3 (non-buffered). The positions of

Author's personal copy


Planta

Ion flux measurements


Net fluxes of K?, Ca2? and H? were measured using the
MIFE technique (University of Tasmania, Hobart, Australia) for non-invasive microelectrode ion flux measurements. All details of fabrication and calibration of ionselective microelectrodes are available in our previous
publications (Shabala and Newman 1999; Shabala and
Shabala 2002; Babourina et al. 2003). In brief, glass microelectrodes were pulled from non-filamentous glass
capillaries (GC150-10, Clark Electrochemical instruments,
Pangbourne, UK), salinized with tributylchlorosilane
(Fluka Chemicals 90796), backfilled with solutions

(a)

-6

H+

5.751

-7

-8
5.743

-9

5.739

5.735

-10

(b)

pH units

5.747

Net fluxes (nmol m-2 s-1)

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)

Fig. 1 Representative tracings of BL-induced transient changes in


net fluxes of H? (a), Ca2? (b) and K? (c) ions (in nmol m-2 s-1)
(open circle) and pH units (filled circle) recorded from broad bean
(Vicia faba L.) leaf epidermis in the absence of inhibitors. Epidermal
tissue was exposed to several light-on/off (L/D, 5 min/5 min) cycles
before the onset of inhibitory treatment. White and black bars indicate
5 min period of light and dark, respectively

(15 mM NaCl, 40 mM KH2PO4, adjusted to pH 6.0 by


NaOH for H?; 0.5 mM CaCl2 for Ca2?), and filled with
appropriate ion-selective cocktail (Fluka: K?: 60031;
Ca2?: 21048; H?: 95297). Electrodes were then calibrated
in a set of pH, K? or Ca2? standards and used for
measurements.
Inhibitors
The effects of seven groups of the inhibitors were tested in
this study (Table 1) [(1) CaM (calmodulin) antagonists: W7,
N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide; and
TFP, trifluoperazine; (2) inhibitors of P2B-type ATPase: EY,
eosin yellow; and ErB, erythrocin B; (3) Ca2? antagonists or

123

Author's personal copy


Planta

Net H+ flux (nmol m-2 s-1)

20 M DPI
6

amplitudes of the light-induced transients recorded from


different epidermal strips were averaged before and after
treatment. The effect of pharmacological treatment was
expressed as the difference in net basal fluxes and lightinduced amplitudes before (control) and after treatment.
Data are presented as mean SE and tested for significance by using Students t test. Mean values of basal
fluxes were simply tested for difference between control
and treated samples, while the magnitude of the flux response to the light before and after treatment was presented
as a relative value (% of control) which was also tested for
significant difference.

BL on

4
2
0
-2

BL off
-4

6.05

pH units

5.95
5.85

Results

5.75

Blue light-induced transients of the ion fluxes


5.65
10

20

30

40

50

60

70

80

90

Time (min)

Fig. 2 Experimental protocol illustrated by a typical example of


recording of net H? flux and pH from broad bean leaf epidermis. Leaf
epidermis was immobilized in the measuring chamber and exposed to
a rhythmical blue light treatment (30 lmol m-2 s-1; ON/OFF 5 min/
5 min cycle). After five or six cycles (5060 min later), a metabolic
inhibitor was added to the bath solution (20 lM DPI in this particular
example) and fluxes were recorded for another 6080 min. Black and
white bars indicate 5 min period of dark and light, respectively

chelators: TMB-8, 8-(N,N-diethylamino) octyl-3,4,5trimethoxybenzoate; and EGTA, ethylene glycol tetraacetic


acid; (4) agents affecting IP3 formation: NM, neomycin; and
LiCl; (5) redox system inhibitors: DPI, diphenyleneiodonium; and HCFIII, hexacyanoferrate (III); (6) inhibitors of
endomembrane Ca2? transport systems: TG, thapsigargin;
and RR, ruthenium red; and (7) inhibitor of plasma membrane Ca2?-permeable channels: LaCl3]. The stock solutions
of the inhibitors were prepared in distilled water, except DPI
and TG which were dissolved in DMSO and ethanol, respectively. Potential effects of all inhibitors on ion-selective
microelectrode characteristics and pH of the experimental
solution were tested separately. Ion-selective microelectrodes were calibrated in the experimental solution first and
then in the presence of the inhibitor after the experiment.
Data processing and analysis
Initial calculations of ion fluxes were performed after the
measurements and data were processed in the spreadsheet
software Excel (Microsoft). The ion fluxes obtained from
individual epidermal strips were averaged. There were two
groups of ion flux values analysed, i.e. steady-state (net
basal fluxes) values and the amplitudes of the blue lightinduced transients of ion fluxes. Basal fluxes and the

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

Author's personal copy


Planta
Table 1 Details on metabolic inhibitors (CaM antagonists, inhibitors of P2B type ATPase, Ca2? antagonists, Ca2? chelators, agents affecting IP3
formation, redox inhibitors, inhibitors of endomembrane Ca2?transport systems) and channel blockers used in this study
Abbreviation

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

Liu et al. (2005)

EY

Eosin yellow

Inhibitor of P2B type


ATPase

0.5 lM

De Michelis et al. (1993)

ErB

Erythrocin B

Inhibitor of P2B type


ATPase

4 lM

Cocucci and Marre (1986)

TMB-8

8-(N,N-diethylamino)octyl-3,4,5trimethoxybenzoate

Intracellular Ca2?
antagonist

50 lM

Mayer et al. (1997), Schumaker and Sze


(1987), Alexandre et al. (1990)

EGTA
NM

Ethylene glycol tetraacetic acid


Neomycin

Ca2? chelator
Decreases formation of
IP3

2 mM
10 lM

Vitrac et al. (2000)


Mayer et al. (1997), Toyoda et al. (1992)

LiCl

LiCl

Increases IP3 levels

5 mM

Monreal et al. (2007)

DPI

Diphenyleneiodonium

NADPH oxidase inhibitor

20 lM

HCFIII

Hexacyanoferrate (III)

Electron acceptor

0.5 mM

Khokon et al. (2011), Morre (2002)


Grabov and Bottger (1994)

TG

Thapsigargin

Endomembrane Ca2?
ATPase inhibitor

5 lM

Pang et al. (2007), Ordenes et al. (2002)

RR

Ruthenium red

Endomembrane Ca2?
channel inhibitor

1 lM

Muir et al. (1997)

La3?

LaCl3

Plasma membrane Ca2?


channel blocker

500 lM

Huang et al. (1994)

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).

Effect of inhibitors of P2B-type ATPase

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

The inhibitors of P2B-type ATPase (EY and ErB) have been


previously shown to affect Ca2? pumping out of the plant
cells (Romani et al. 2004; Beffagna et al. 2005; Nemchinov
et al. 2008), so we tested their potential effect on broad bean
epidermal tissue. We supposed that the inhibition of Ca2?
ATPase by P2B type ATPase inhibitors could result in accumulation of positive ions in the cell (depolarization of the
membrane potential), which should stimulate opening of K?
channels (KOR) enabling the efflux of K? ions from the cell
and subsequently return membrane potential to hyperpolarized level. When epidermal strips were subjected to cyclic
BL fluctuation, pre-treatment with these compounds indeed
induced a twofold (P \ 0.05) reduction in magnitude of

Effect of Ca21 antagonists and chelators

123

Author's personal copy


Planta

(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

the magnitude of H? and K? flux responses (Fig. 4a, c, 7).


Thus, data presented in this study confirmed the involvement of intracellular Ca2? concentration in BL signal
transduction pathway.
Effect of agents affecting IP3 formation
It has been previously reported that the enzymes and
metabolites of the phosphoinositide cycle are present in
plants (Stevenson et al. 2000). Plant vacuoles were suggested as a major IP3-regulated intracellular Ca2? store. In
this study we tested the effects of the two compounds affecting IP3 formation: neomycin (NM) and LiCl, to explore

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

Magnitude of response (% control)

50

* *

50

-15

HCFIII

150

DPI

DPI

* *

LiCl

-5
-10

Net basal fluxes (nmol m-2 s-1)

250

La3+

H+ flux

La3+

10

RR

flux

450

LiCl

H+

15

La3+

(a)

Fig. 4 Effect of metabolic inhibitors (for details, see Table 1) on the


magnitude of flux responses (% control) for H? (a), Ca2? (b) and K?
(c) measured in response to blue light on/off cycles. Mean SE
(n = 825 values from at least four biological specimens). Treatments labelled with asterisks are significantly different from controls
at P \ 0.05

the existence of IP3-gated endomembrane Ca2? channels.


Neomycin (NM) decreases IP3 formation via inhibition of
enzyme phospholipase C (Mayer et al. 1997), while LiCl
increases cytosolic IP3 levels (Monreal et al. 2007). NM
(10 lM) caused non-significant shift in basal H? towards
net influx and non-significant effect on K? fluxes, while the
Ca2? net efflux was increased significantly (P \ 0.05)
(Fig. 3). Moreover, the use of NM reduced the magnitude
of BL-induced Ca2? flux responses to about 50 % of that in
control (Fig. 4b), while the effects on H? and K? fluxes
were not significant (Fig. 4a, c). Opposite effects were
observed for 5 mM LiCl. In the latter case, responses of
both Ca2? and K? fluxes to BL were substantially potentiated (about twofold; Figs. 4b, c, 8), and basal H? flux was
shifted to towards net efflux (Fig. 3a). Although the effects
of NM and LiCl on Ca2? ions were observed in this work,
our data did not show clearly the existence of IP3 gated
Ca2? channel responsible for regulation of intracellular
Ca2? concentration.

Author's personal copy


Planta
Before treatment

After treatment

-2

-3

4
3

-8

H+

-4

-16

-5

Net ion fluxes (nmol m-2 s-1)

Fig. 5 Effect of CaM


antagonist W7 on blue lightinduced ion flux responses
measured from broad bean
epidermis. Typical flux
recordings are shown for each
ion (H?, Ca2? and K?) before
and after 200 lM W7 adding to
the bath solution. Different
scales for Y axis are given for
fluxes recorded before and
after treatment to make
visible kinetics of control fluxes
of all ions measured. The right
column shows mean peak
magnitudes (in nmol m-2 s-1)
before (control; C) and after
treatment (T). Mean SE
(n = 16)

-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

Net ion fluxes (nmol m-2 s-1)

Fig. 6 Effect of Erythrosin B


(an inhibitor of P2B-type Ca2?ATPase) 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 4 lM
ErB adding 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 = 20)

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)

Effect of redox system inhibitors


Reduction of impermeable artificial electron acceptors,
such as hexacyanoferrate III (HCFIII), has been reported
on several intact plants since the early 1980s (Luthje et al.
1997; Berczi and Mu`ller 2000). Depolarization of the

plasma membrane and increase in apoplastic acidification


were also observed upon application of redox inhibitors.
The application of the two redox system inhibitors on
broad epidermis in this study, 20 lM DPI and 0.5 mM
HCFIII, did reduce the net basal Ca2? uptake and decreased basal K? leak (Fig. 3b, c), but significantly

123

Author's personal copy


Planta
Before treatment

0.1

Net ion fluxes (nmol m-2 s-1)

Fig. 7 Effect of EGTA (a


known Ca2? chelator) 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 2 mM EGTA adding
to the bath solution. Different
scale for Y axis is given for H?
fluxes recorded before and
after treatment. The right
column shows mean peak
magnitudes (in nmol m-2 s-1)
before (control; C) and after
treatment (T). Mean SE
(n = 13)

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

Net ion fluxes (nmol m-2 s-1)

Fig. 8 Effect of LiCl (a known


IP3 signalling 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 5 mM LiCl 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 = 20)

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)

increased H? efflux (Fig. 3a), which is in the line with


previously reported data (Menckhoff and Luthje 2004).
The magnitude of BL-induced oscillations in net ion fluxes
was significantly (P \ 0.05) inhibited for all ions (Fig. 4)

123

in epidermal strips pre-treated with these agents, resulting


in a reduction ranging from three to fivefold (Fig. 9). These
data confirmed the involvement of the redox systems in BL
signalling of broad bean epidermis.

Author's personal copy


Planta

Effect of inhibitors of endomembrane Ca21


transport systems

as previously reported on Arabidopsis (Wang et al. 2004).


La3? has been also shown to block both inward-rectifying
(KIR) (Tester and MacRobbie 1990) and outward-rectifying (KOR) (Ketchum and Poole 1991) K? channels. The
application of 500 lM La3? on epidermis has significantly
reduced net basal Ca2? uptake and increased basal H?
efflux (Fig. 3b, a), while the basal K? efflux was reduced
(Fig. 3c). A significant (3 to 4-fold) reduction in the
magnitude of BL-induced oscillations in net fluxes of Ca2?
and K? ions and increased BL-induced oscillations in net
H? fluxes were observed in epidermal strips pre-treated
with La3? (Fig. 4). Thus, the results obtained in the present
work with La3? are consistent with previously reported
data (Tester and MacRobbie 1990; Ketchum and Poole
1991; Wang et al. 2004).

Endomembrane Ca2? transporters regulate cytosolic Ca2?


concentration either passively by Ca2? channels enabling
export Ca2? from internal stores to cytosol or actively by
Ca2? efflux mechanisms (Ca2? ATPase) which rapidly remove the excess of Ca2? from cytosol by sequestering it into
internal stores. The effects of the two inhibitors (thapsigargin, TG, and ruthenium red, RR) of the endomembrane
Ca2? transporters, mostly used in animal systems (Thastrup
et al. 1990; Bae et al. 2003), were also tested on plants
(Pottosin et al. 1999; Chang et al. 2001; Ordenes et al. 2002;
Urbina et al. 2006). In this study, TG (5 lM) and RR (1 lM)
significantly reduced basal Ca2? uptake, but had no impact
on basal K? fluxes (Fig. 3b, c). The net H? efflux was
stimulated by both compounds (Fig. 3a). Both TG and RR
reduced (about twofold; P \ 0.05) the magnitude of BLinduced net Ca2? flux oscillations (Fig. 4b), but had no
significant effect on K? flux (Fig. 4c) and H? flux (Fig. 4a)
responses to BL. These results indicate that endomembrane
Ca2? channel (inhibited by RR) and Ca2? ATPase (inhibited
by TG) might be included in the model on BL signalling in
the broad bean epidermis (Fig. 10).

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

Effect of plasma membrane Ca21-permeable


channels block
La3? ion was added in the form of LaCl3 solution to inhibit
Ca2?-permeable channel activity at the plasma membrane,

After treatment

Before treatment
3

-1

-1

-3

-3

H+

1
0

Net ion fluxes (nmol m-2 s-1)

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

Author's personal copy


Planta

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+

Fig. 10 Tentative model for blue light signalling in broad bean


epidermal cells. HACC hyperpolarization-activated Ca2?-permeable
channel, KIR inward-rectifying hyperpolarization-activated K? channel, KOR outward-rectifying depolarization-activated K? channel,
NSCC non-selective cation channel, R receptor; at least two
unidentified signalling molecules are postulated (X and Y): one

mediates transduction of BL stimulus from receptor to IP3 (factor X in


the model), while another (factor Y in the model) acts as a negative
feedback regulator between CaM and H?-ATPase. The relationship
between IP3 and endomembrane Ca2?-permeable channel was not
confirmed pharmacologically in this model (indicated by question
mark). See text for more details and other abbreviations

basement cells is mainly the result of cell expansion, it is


essential that, firstly, both the vacuole and the cytoplasm
need to accumulate osmolytes to allow water uptake leading
to volume increase and, secondly, that the pH in the cell
wall becomes or remains low, to increase the plasticity and
allow cell increase. For both of them H?-pumping ATPase
needs to be activated and the driving force created for the
uptake of K? (or other small solutes) and the acidification of
the cell wall. In our working hypothesis, BL activates the
H?-pumping ATPase through a signalling cascade that involves an increase in the cytoplasmic calcium concentration
(released from internal stores or coming from outside the
cell), which in turn activates CaM/CDPK, leading to
changes in the activity of K? influx. Since the Ca2?/CaM
signal transduction is a central pathway in signal transduction of many external factors, other elements (redox
reactions) might directly or indirectly interfere with the
coupling between BL perception and ATPase and K?
channel activation. The tentative BL signal transduction
pathways and key membrane transporters involved in BL
signalling are depicted in Fig. 10. The latter include four
channels and two pumps: K?-permeable inward (KIR) and
outward (KOR) rectifying channels and non-selective cation channels (NSCC); Ca2? transporting hyperpolarization-activated cannel (HACC) and P2B-type Ca2?-ATPase;
and H?-translocation ATPase. In addition, endomembrane

Ca2? channel and P2A-type Ca2?-ATPase are included in


the model as a part of the endomembrane systems. The
reasons for this are given below.

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

Author's personal copy


Planta

van Volkenburgh 2002). KIR channels are activated by


plasma membrane hyperpolarization caused by BL-induced
activation of H?-ATPase and are responsible for solute
accumulation for turgor maintenance, while KOR channels
are activated by depolarization to drive K? efflux (Stiles
and van Volkenburgh 2002). In our case, increased magnitude of BL-induced response in H? flux was often accompanied by the concurrent increase in the magnitude of
K? flux response (e.g. for W7; TMB; EGTA; Fig. 4a, c)
suggesting that this was the case for broad bean as well.
Addition of depolarization-activated KOR channels is
justified by the fact that net K? efflux was measured for
some treatments (Fig. 3c). In addition, BL-induced K? flux
responses were strongly suppressed by La3?, a known
blocker of K?-permeable NSCC (Figs. 3c, 4c). Hence, the
latter are also considered in the model.

work also confirmed La3? inhibitory effect on K? channels


(Figs. 3c, 4c). At the same time, Ca2? flux responses were
also sensitive to ErB and EY (Fig. 4b), two inhibitors of
P2B-type Ca2?-ATPase located at the plasma membrane
(Axelsen and Palmgren 2001). Thus, the latter is also added
to the model (Fig. 10). The two known major types of Ca2?
efflux systems, Ca2?-pumps and Ca2?/H? antiporters, are
essential for restoring the basal levels of cytosolic free
calcium at normal physiological condition (Ca2?-ATPase)
or under extreme stress (Ca2?/H? antiporters), intracellular
calcium concentration ([Ca2?]cyt) (Sze et al. 2000; White
and Broadley 2003) and, together with Ca2? influx channels are responsible for stimulus-induced calcium signatures in plant cells (Bose et al. 2011) required for growth
and development.
Endomembrane systems

Ca21 transport systems


The cellular signal transduction system responsible for the
coupling of excitation of the BL photoreceptor and the
activation of H?-ATPase is hypothesized to be the Ca2?/
CaM system. Any treatment that will increase the cytoplasmic Ca2? concentration (release form internal stores or
entry from the apoplast) is assumed to activate the H?ATPase, while inhibition of CaM is expected to reduce the
H? efflux. Ca2? plays an important role in plant growth,
development, and signal transduction. Ca2? uptake into
cytosol is a thermodynamically passive process mediated
by a range of Ca2?-permeable channels, while the removal
of Ca2? from the cytosol and return to endomembrane
compartments is against an electrochemical potential gradient for Ca2? and is therefore an energy-dependent process driven by ATP-dependent Ca2? pumps (reviewed in
Medvedev 2005). Furthermore, plants have two types of
Ca2? efflux transporters, ATP-dependent Ca2? pumps (Sze
et al. 2000; Boursiac and Harper 2007) and Ca2?/H? antiporters (exchangers) that utilize the proton motive force
(Shigaki and Hirschi 2006). Ca2?-permeable plasma
membrane channels can be separated into voltage-gated
(e.g. HACC, or hyperpolarization-activated Ca2? channels;
Miedema et al. 2001 and references therein) and weakly
voltage dependent (NSCC, or non-selective cation channels; Demidchik and Maathuis 2007). Both of them are
known to be sensitive to trivalent cations such as La3? and
Gd3? (Demidchik et al. 2002; Hetherington and Brownlee
2004) and in our case BL-induced fluctuations of Ca2? flux
were significantly reduced by La3? (Fig. 4b), suggesting
the involvement of such NSCC and/or HACC (Fig. 10). It
cannot be ignored that La3? apart from inhibiting Ca2?
channels (Wang et al. 2004) has been reported to block
both KIR and KOR K? channels (Tester and MacRobbie
1990; Ketchum and Poole 1991). The results shown in this

Net ion flux response were significantly modulated by RR,


a known inhibitor of endomembrane Ca2? channels, as
well as by TG, an inhibitor of P2A-type Ca2?-ATPase
(endomembrane-located) suggesting involvement of both
these transport systems in cytosolic Ca2? signalling and
homeostasis (Figs. 3b, 4b, 10).
BL signalling to downstream targets
Regulation of H?-ATPase
The mechanism by which the perception of BL is transduced into the pump activation is far from being clear
(Assmann et al. 1985). It was suggested for guard cells that
BL receptor PHOT1 contains in addition to the lightsensing LOV domain(s) a serine/threonine protein kinase
domain which enables activation of PHOT1 by autophosphorylation upon BL illumination, resulting in the binding
of 14-3-3 proteins to the H?-ATPase. Thus, in guard cells
BL activates the H?-ATPase via phosphorylation of the C
terminus with subsequent binding of 14-3-3 protein to the
H?-ATPase (Palmgren 2001; Kinoshita et al. 2003). By
contrast, in leaf pulvina BL perception by phototropin results in dephosphorylation of the phosphorylated Thr in the
C terminus of H?-ATPase and the subsequent dissociation
of 14-3-3 protein (Gaxiola et al. 2007). The mechanism of
activation (phosphorylation)/deactivation (dephosphorylation) of H?-ATPase is not clear. Our data reported here
suggests that CaM may be an important component of the
signal transduction pathway from BL receptor to H?ATPase. Indeed, several-fold increase in BL-induced ion
flux fluctuations were observed in epidermal strips treated
with two CaM antagonists, W7 and TFP (Figs. 4, 5). CaM
antagonists increased H? efflux (stimulate H? pumping
outside) and increased Ca2? and K? influxes (Fig. 3).

123

Author's personal copy


Planta

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

of such ligand-gated channels endomembranes (reviewed


by Pottosin and Schonknecht 2007). In our case, BL-induced flux responses were modulated by LiCl and NM
(Fig. 4b, 8), two chemicals known to modify cytosolic IP3
levels (Monreal et al. 2007), and also by RR (Fig. 4), a
known blocker of IP3-gated Ca2? channels (Pineros and
Tester 1997). However, the question of whether IP3 may
directly modulate such endomembrane Ca2? channels, or
this process is mediated by some other additional signal
component, needs to be resolved in direct patch-clamp
experiments. This is reflected by putting a question mark
between IP3 and Ca2? channels in our model (Fig. 10).
Endomembrane Ca2?-ATPases are also involved in signal
transduction pathway, as BL-induced responses were
modulated by TG, an inhibitor of P2A-type ATPase Ca2?ATPase (endomembrane-located) (Figs. 4b, 10).
Redox signalling component
Redox reactions have long been suggested as the early
steps in BL signalling (Long and Jenkins 1998; Cashmore
et al. 1999). Baum et al. (1999) provided evidence that BL
signal transduction involves changes in redox process;
according to their model, the LOV domains of NPH1 detect
BL modification in its redox state and activate protein kinase. Alternatively, redox changes may modify PM potential stimulating Ca2? uptake via voltage-gated channels
(Baum et al. 1999). UV-B-induced stimulation of NADPH
oxidase activity has been reported elsewhere (Jenkins 2009
and references within). Also, BL-stimulated medium
acidification by guard cells was found to be vanadate insensitive and interpreted as evidence for the operation of
proton-extruding redox system (Raghavendra 1990),
although this view was challenged later by Taylor and
Assmann (2001) who showed that BL-stimulated H? currents in guard cell protoplasts were not affected by either
NADH or NADPH added to pipette.
Our data also provide strong evidence that redox signalling and, specifically, plasma membrane NADPH oxidase, play essential role in BL signal transduction. Both
DPI (NADPH oxidase inhibitor) and HCFIII (electron acceptor) resulted in significant reduction of the magnitude of
BL-induced Ca2? and K? flux oscillations (Figs. 4b, c, 9).
This can be explained by the presence of positive feedback
between NADPH oxidase and Ca2? uptake via HACC
mediated by apoplastic H2O2 production, and by concurrent stimulation of ROS-induced K? leak via NSCC, respectively (Fig. 10). Both effects have been previously
reported in direct patch-clamp experiments on Arabidopsis
roots (e.g. Demidchik et al. 2002). It remains to be shown
that the same channels are present in the bean epidermis.
In this study we used pharmacological approach to
dissect blue light signal transduction pathway in the leaf

Author's personal copy


Planta

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.

The authors declare that they have no conflict

References
Ahmad M, Cashmore AR (1993) HY4 gene of A. thaliana encodes a
protein with characteristics of a blue-light photoreceptor. Nature
366:162166
Alexandre J, Lassalles JP, Kado RT (1990) Opening of Ca2? channels
in isolated red beet root vacuole membrane by inositol 1,4,5trisphosphate. Nature 343:567570
Allen GJ, Muir SR, Sanders D (1995) Release of Ca2? from
individual plant vacuoles by both InsP3 and cyclic ADP-ribose.
Science 268:735737
Assmann SM, Simoncini L, Schroeder JI (1985) Blue light activates
electrogenic ion pumping in guard cell protoplasts of Vicia faba
L. Nature 318:285287
Axelsen KB, Palmgren MG (2001) Inventory of the superfamily of
P-type ion pumps in Arabidopsis. Plant Physiol 126:696706
Babourina O, Newman I, Shabala S (2002) Blue light-induced
kinetics of H? and Ca2? fluxes in etiolated wild-type and
phototropin-mutant Arabidopsis seedlings. Proc Natl Acad Sci
USA 99:24332438
Babourina OK, Newman IA, Shabala SN (2003) Electrophysiological
localization of blue light sensory sites in etiolated dicotyledon
seedlings. Plant, Cell Environ 26:15051514

Bae JH, Park JW, Kwon TK (2003) Ruthenium red, inhibitor of


mitochondrial Ca2? uniporter, inhibits curcumin-induced apoptosis via the prevention of intracellular Ca2? depletion and
cytochrome c release. Biochem Bioph Res Co 303:10731079
Baum G, Long JC, Jenkins GI, Trewavas AJ (1999) Stimulation of the
blue light phototropic receptor NPH1 cause a transient increase
in cytosolic Ca2?. Proc Natl Acad Sci USA 96:1355413559
Beffagna N, Buffoli B, Busi C (2005) Modulation of reactive oxygen
species production during osmotic stress in Arabidopsis thaliana
cultured cells: involvement of the plasma membrane Ca2?ATPase and H?-ATPase. Plant Cell Physiol 46:13261339
Berczi A, Mu`ller IM (2000) Redox enzymes in the plant plasma
membrane and their possible roles. Plant, Cell Environ
23:12871302
Bose J, Babourina O, Rengel Z (2011) Role of magnesium in
alleviation of aluminium toxicity in plants. J Exp Bot
62:22512264
Boursiac Y, Harper JF (2007) The origin and function of calmodulin
regulated Ca2? pumps in plants. J Bioenerg Biomembr
39:409414
Buch-Pedersen MJ, Rudashevskaya EI, Berner TS, Venema K,
Palmgren MG (2006) Potassium as an intrinsic uncoupler of the
plasma membrane H?-ATPase. J Biol Chem 281:3828538292
Cashmore AR, Jarillo JA, Wu YJ, Liu DM (1999) Cryptochromes:
blue light receptors for plants and animals. Science 284:760765
Chang S-C, Cho MH, Kang BG, Kaufman PB (2001) Changes in
starch content in oat (Avena sativa) shoot pulvini during the
gravitropic response. J Exp Bot 52:10291040
Cocucci MC, Marre E (1986) Erythrosin B as an effective inhibitor of
electrogenic H? extrusion. Plant, Cell Environ 9:677679
Cosgrove D (1994) Photomodulation of growth. In: Kendrick RE,
Kronenberg GHM (eds) Photomorphogenesis in plants. Kluwer
Academic Publishers, Dordrecht, pp 631658
De Michelis MI, Carnelli A, Rasi-Caldogno F (1993) The Ca2? pump
of the plasma membrane of Arabidopsis thaliana: characteristics
and sensitivity to fluorescein derivatives. Bot Acta 106:2025
Demidchik V, Maathuis FJM (2007) Physiological roles of nonselective cation channels in plants: from salt stress to signalling and
development. New Phytol 175:387404
Demidchik V, Bowen HC, Maathuis FJM, Sergey N, Shabala SN,
Tester MA, White PJ, Davies JM (2002) Arabidopsis thaliana
root non-selective cation channels mediate calcium uptake and
are involved in growth. Plant J 32:799808
Elzenga TJM, Staal M, Prins HBA (1997) Calcium-calmodulin
signaling is involved in light-induced acidification by epidermal
leaf cells of pea, Pisum sativum L. J Exp Bot 48:20552060
Gaxiola RA, Palmgren MG, Schumacher K (2007) Plant proton
pumps. FEBS Lett 581:22042214
Grabov A, Bottger M (1994) Are redox reactions involved in
regulation of K? channels in the plasma membrane of Limnobium stoloniferum root hairs? Plant Physiol 105:927935
Gyula P, Schafer E, Nagy F (2003) Light perception and signalling in
higher plants. Curr Opin Plant Biol 6:446452
Harada A, Sakai T, Okada K (2003) Phot1 and phot2 mediate blue
light-induced transient increases in cytosolic Ca2? differently in
Arabidopsis leaves. Proc Natl Acad Sci USA 100:85838588
Hetherington AM, Brownlee C (2004) The generation of Ca2? signals
in plants. Annu Rev Plant Biol 55:401427
Huang JW, Grunes DL, Kochian LV (1994) Voltage-dependent Ca2?
influx into right-side-out plasma membrane vesicles isolated
from wheat roots: characterization of a putative Ca2? channel.
Proc Natl Acad Sci USA 91:34733477
Jenkins GI (2009) Signal transduction in responses to UV-B radiation.
Annu Rev Plant Biol 60:407431
Kaufman LS (1993) Transduction of blue-light signals. Plant Physiol
102:333337

123

Author's personal copy


Planta
Ketchum KA, Poole RJ (1991) Cytosolic calcium regulates a
potassium current in corn (Zea mays) protoplasts. J Membr Biol
119:277288
Khokon MDAR, Okuma E, Hossain MA, Munemasa S, Uraji M,
Nakamura Y, Mori IC, Murata Y (2011) Involvement of
extracellular oxidative burst in salicylic acid-induced stomatal
closure in Arabidopsis. Plant, Cell Environ 34:434443
Kimura M, Kagawa T (2006) Phototropin and light-signaling in
phototropism. Curr Opin Plant Biol 9:503508
Kinoshita T, Nishimura JM, Shimazaki K (1995) Cytosolic concentration of Ca2? regulates the plasma membrane H?-ATPase in
guard cells of Fava bean. Plant Cell 7:13331342
Kinoshita T, Emi T, Tominaga M, Sakamoto K, Shigenaga A, Doi M,
Shimazaki K (2003) Blue-light-and phosphorylation-dependent
binding of a 14-3-3 protein to phototropins in stomatal guard
cells of broad bean. Plant Physiol 133:14531463
Li B, Liu H-T, Sun D-Y, Zhou R-G (2004) Ca2? and calmodulin
modulate DNA-binding activity of maize heat shock transcription factor in vitro. Plant Cell Physiol 45:627634
Liu HT, Sun DY, Zhou RG (2005) Ca2? and AtCaM3 are involved in
the expression of heat shock protein gene in Arabidopsis. Plant,
Cell Environ 28:12761284
Long JC, Jenkins GI (1998) Involvement of plasma membrane redox
activity and calcium homeostasis in the UV-B and UV-A/blue
light induction of gene expression in Arabidopsis. Plant Cell
10:20772086
Luthje S, Doring O, Heuer S, Luthen H, Bottger M (1997)
Oxidoreductases in plant plasma membranes. Biochim Biophys
Acta 1331:81102
Mayer W-E, Hohloch C, Kalkuhl A (1997) Extensor protoplasts of the
Phaseolus pulvinus: light-induced swelling may require extracellular Ca2? influx, dark-induced shrinking inositol 1, 4,
5-trisphosphate induced Ca2? mobilization. J Exp Bot
48:219228
Medvedev SS (2005) Calcium signaling system in plants. Russ J Plant
Physiol 52:249270
Menckhoff M, Luthje S (2004) Transmembrane electron transport in
sealed and NAD(P)H-loaded right-side-out plasma membrane
vesicles isolated from maize (Zea mays L.) roots. J Exp Bot
55:13431349
Miedema H, Bothwell JHF, Brownlee C, Davies JM (2001) Calcium
uptake by plant cellschannels and pumps acting in concert.
Trends Plant Sci 6:514519
Monreal JA, Lopez-Baena FJ, Vidal J, Echevarria C, Garcia-Maurino
S (2007) Effect of LiCl on phosphoenolpyruvate carboxylase
kinase and the phosphorylation of phosphoenolpyruvate carboxylase in leaf disks and leaves of Sorghum vulgare. Planta
225:801812
Morre DJ (2002) Preferential inhibition of the plasma membrane
NADH oxidase (NOX) activity by diphenyleneiodium chloride
with NADPH as donor. Antioxid Redox Signal 4:207212
Muir SR, Bewell MA, Sanders D, Allen GJ (1997) Ligand-gated
Ca2? channels and Ca2? signalling in higher plants. J Exp Bot
48:589597
Nemchinov LG, Shabala L, Shabala S (2008) Calcium efflux as a
component of the hypersensitive response of Nicotiana benthamiana to Pseudomonas syringae. Plant Cell Physiol 49:4046
Newman IA (2001) Ion transport in roots: measurement of fluxes
using ion-selective microelectrodes to characterize transporter
function. Plant, Cell Environ 24:114
Ordenes VR, Reyes FC, Wolff D, Orellana A (2002) A thapsigarginsensitive Ca2? pump is present in the pea golgi apparatus
membrane. Plant Physiol 129:18201828
Palmgren MG (2001) Plant plasma membrane H?-ATPases: powerhouses for nutrient uptake. Annu Rev Plant Physiol Plant Mol
Biol 52:817845

123

Pang JY, Cuin T, Shabala L, Zhou MX, Mendham N, Shabala S


(2007) Effect of secondary metabolites associated with anaerobic
soil conditions on ion fluxes and electrophysiology in barley
roots. Plant Physiol 145:266276
Percey WJ, Shabala L, Breadmore MC, Guijt RM, Bose J, Shabala S
(2014) Ion transport in broad bean leaf mesophyll under saline
conditions. Planta 240:729743
Pineros M, Tester M (1997) Calcium channels in higher plant cells:
selectivity, regulation and pharmacology. J Exp Bot 48:551577
Pottosin II, Schonknecht G (2007) Vacuolar calcium channels. J Exp
Bot 58:15591569
Pottosin II, Dobrovinskaya OR, Muniz J (1999) Cooperative block of
the plant endomembrane ion channel by Ruthenium Red.
Biophys J 77:19731979
Raghavendra AS (1990) Blue light effects on stomata are mediated by
the guard cell plasma membrane redox system distinct from the
proton translocating ATPase. Plant, Cell Environ 13:105110
Romani G, Bonza MC, Filippini I, Cerana M, Beffagna N,
DeMichelis MI (2004) Involvement of the plasma membrane
Ca2?-ATPase in the short-term response of Arabidopsis thaliana
cultured cells to oligogalacturonides. Plant Biol 6:192200
Schumaker KS, Sze H (1987) Inositol 1,4,5-trisphosphate releases
Ca2? from vacuolar membrane vesicles of oat roots. J Biol Chem
262:39443946
Schwartz A, Illan N, Assmann SM (1991) Vanadate inhibition of
opening in epidermal peels of Commelina communis. Cl- interferes with vanadate uptake. Planta 183:590596
Shabala S (2000) Ionic and osmotic components of salt stress
specifically modulate net ion fluxes from broad bean leaf
mesophyll. Plant, Cell Environ 23:825837
Shabala S, Hariadi Y (2005) Effects of magnesium availability on the
activity of plasma membrane ion transporters and light-induced
responses from broad bean leaf mesophyll. Planta 221:5665
Shabala S, Newman I (1999) Light-induced changes in hydrogen,
calcium, potassium, and chloride ion fluxes and concentrations
from the mesophyll and epidermal tissues of bean leaves.
Understanding the ionic basis of light-induced bioelectrogenesis.
Plant Physiol 119:11151124
Shabala S, Shabala L (2002) Kinetics of net H?, Ca2?, K?, Na?,
NH4?, and Cl- fluxes associated with post-chilling recovery of
plasma membrane transporters in Zea mays leaf and root tissues.
Physiol Plant 114:4756
Shabala SN, Newman IA, Morris J (1997) Oscillations in H? and
Ca2? ion fluxes around the elongation region of corn roots and
effects of external pH. Plant Physiol 113:111118
Shabala S, Shabala L, Gradmann D, Chen Z, Newman I, Mancuso S
(2006) Oscillations in plant membrane transport: model predictions, experimental validation, and physiological implications.
J Exp Bot 57:171184
Shabala S, Cuin TA, Shabala L, Newman I (2012) Quantifying kinetics
of net ion fluxes from plant tissues by non-invasive microelectrode
measuring MIFE technique. In: Shabala S, Cuin TA (eds) Plant salt
tolerance: methods and protocols, vol 913. Springer, New York,
Heidelberg, Dordrecht, London, pp 119134
Shigaki T, Hirschi KD (2006) Diverse functions and molecular
properties emerging for CAX cation/H? exchangers in plants.
Plant Biol 8:419429
Shimazaki K, Kinoshita T, Nishimura M (1992) Involvement of
calmodulin and calmodulin-dependent myosin light chain kinase
in blue light-dependent H? pumping by guard-cell protoplasts
from Vicia faba L. Plant Physiol 99:14161421
Spalding EP (2000) Ion channels and the transduction of light signals.
Plant, Cell Environ 23:665674
Spalding EP, Cosgrove DJ (1992) Mechanism of blue-light-induced
plasma-membrane depolarization in etiolated cucumber hypocotyls. Planta 188:199205

Author's personal copy


Planta
Staal M, Elzenga JTM, Vanelk AG, Prins HBA, VanVolkenburgh E
(1994) Red and blue light-stimulated proton efflux by epidermal
leaf-cells of the Argenteum mutant of Pisum sativum. J Exp Bot
45:12131218
Stevenson JM, Perera IY, Heilmann I, Persson S, Boss WF (2000)
Inositol signaling and plant growth. Trends Plant Sci 5:252258
Stiles KA, Van Volkenburgh E (2002) Light-regulated leaf expansion
in two Populus species: dependence on developmentally
controlled ion transport. J Exp Bot 53:16511657
Sze H, Liang F, Hwang I, Curran AC, Harper JF (2000) Diversity and
regulation of plant Ca2? pumps: insights from expression in
yeast. Annu Rev Plant Physiol Plant Mol Biol 51:433462
Takemiya A, Kinoshita T, Asanuma M, Shimazaki KI (2006) Protein
phosphatase 1 positively regulates stomatal opening in response
to blue light in Vicia faba. Proc Natl Acad Sci USA
103:1354913554
Taylor AR, Assmann SM (2001) Apparent absence of a redox
requirement for blue light activation of pump current in broad
bean guard cells. Plant Physiol 125:329338
Tester M, MacRobbie EAC (1990) Cytoplasmic calcium affects the
gating of potassium channels in the plasma membrane of Chara
corallina: a whole-cell study using calcium-channel effectors.
Planta 180:569581
Thastrup O, Cullen PJ, Brobak BK, Hanley MR, Dawson AP (1990)
Thapsigargin, a tumor promoter, discharges intracellular Ca2?
stores by specific inhibition of the endoplasmic reticulum Ca2?
ATPase. Proc Natl Acad Sci USA 87:24662470

Toyoda K, Shirashi N, Yoshioka H, Yamada T, Ichinose Y, Oku H


(1992) Regulation of polyphosphoinositide metabolism in pea
plasma membranes by elicitor and suppressor from a pea
pathogen, Mycosphaerella pinodes. Plant Cell Physiol
33:445452
Urbina DC, Silva H, Meisel LA (2006) The Ca2? pump inhibitor,
thapsigargin, inhibits root gravitropism in Arabidopsis thaliana.
Biol Res 39:289296
rillon
Vitrac X, Larronde F, Krisa S, Decendit A, Deffeux G, MeA
J-M (2000) Sugar sensing and Ca2?-calmodulin requirement in
Vitis vinifera cells producing anthocyanins. Phytochemistry
53:659665
Wang Y-F, Fan L-M, Zhang W-Z, Zhang W-W, Wu W-H (2004)
Ca2?-permeable channels in the plasma membrane of Arabidopsis pollen are regulated by actin microfilaments. Plant Physiol
136:38923904
White PJ, Broadley MR (2003) Calcium in plants. Ann Bot
92:487511
Zivanovic BD, Pang J, Shabala S (2005) Light-induced transient ion
flux responses from maize leaves and their association with leaf
growth and photosynthesis. Plant, Cell Environ 28:340352
Zivanovic BD, Cuin TA, Shabala S (2007) Spectral and dose
dependence of light-induced ion flux responses from maize
leaves and their involvement in leaf expansion growth. Plant Cell
Physiol 48:598605

123

You might also like