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Journal of Experimental Botany, Vol. 48, No. 314, pp. 1623-1630, September 1997
Journal of
Experimental
Botany
Transient light-induced changes in ion channel and
proton pump activities in the plasma membrane of
tobacco mesophyll protoplasts
Margaretha Blom-Zandstra1, Hans Koot, Joke van Hattum and Sake A. Vogelzang
DLO-Research Institute for Agrobiology and Soil Fertility (AB-DLO), PO Box 14, 6700 AA Wageningen,
The Netherlands
Received 20 November 1996; Accepted 14 May 1997
Abstract
Rapid, transient changes of the membrane potential
upon light transitions are generally observed in microelectrode studies. In a patch-clamp study similar
responses to light transitions were found in current
clamp. Corresponding with the changes of membrane
potential, light-induced current changes in voltage
clamp were observed. This paper evaluates the
involvement of outward rectifying conductances and
plasma membrane bound H'-ATPases (proton pump)
to these light responses in mesophyll protoplasts of
Nicotiana tabacum L. The contribution of K + -channels
to these responses, could be minimized by variation
of the holding potential or addition of the K + -channel
blocker verapamil. It was concluded that light transitions modulate both proton pump and K + -channel
activity. Effects of light on membrane current were
not observed in root cells and chlorophyll-deficient
cells, suggesting that the response requires photosynthetic activity. However, blockers of photosystems I
and II did not affect current changes.
Key words: Light,
tobacco, whole cell.
patch-clamp,
plasma
membrane,
Introduction
Under dark conditions, ion transport in plants strongly
differs from that in light. Thus a transition from light to
dark (LD) and dark to light (DL) has a significant effect
on the dynamics of ion transport processes both on a
whole plant and a cellular level. The nature of the initial
response to light transitions is likely to provide important
information about the factors that are involved in changes
of ion transport. Changes of the free running membrane
potential (E^ of photosynthetic plant cells upon light
transitions, have been reported since studies with microelectrodes were started (reviewed by Jeschke, 1976;
Findlay and Hope, 1976). At least two types of responses
upon DL transitions have been identified, differing in
time-scale and direction: a rapid, transient depolarization
of 10-50 ms (Bulychev et al., 1972; Vredenberg, 1974)
and a slow hyperpolarization of 5-20 min (Spanswick,
1972, 1974). Transition to dark causes similar responses
in the opposite direction. Spalding and Cosgrove (1992)
showed, with microelectrode measurements, that lightinduced, transient depolarizations of the plasma membrane result from inactivation of the plasma membranebound H+-ATPase (proton pump). Apart from an effect
on pump activity, light also affects voltage-gated channel
activity: Single channel data from patch-clamp studies
(Spalding et al., 1992) indicated that the activity of
plasma membrane-bound K+-channels increased upon
DL transitions.
A patch-clamp study is presented here in which transient responses of the plasma membrane upon LD and DL
transitions are shown in isolated mesophyll protoplasts
from tobacco leaves. Tobacco protoplasts were used
because ion selectivity, kinetics and pharmacology of the
1
To whom correspondence should be addressed. Fax: +31 317 423 110. E-mail: m.blom-zandstraeab.dlo.nl.
Abbreviations: BTP, 1,3-bis[tris{hydroxyrnethyl)-rnethylamino]propane; CC, current clamp; DL, dark to light; Ea, chloride equilibrium potential; £H,
proton equilibrium potential; EK, potassium equilibrium potential; gm, membrane conductance; gK/wc. potassium conductance; la, chloride current; /H,
proton current; IKOK:, potassium outward rectifying current; lm, membrane current; I/V current-voltage; IRC, inward rectifying current; K4BAPTA,
potassium-1,2-bis(2-aminophenoxy)ethane-N,rV,N',A/l-tetra-acetic-acid; LD, light to dark; ORC, outward rectifying current; VC, voltage clamp; Vm,
membrane potential; Vr>onc, reversal potential of the potassium outward rectifying currents; WC, whole cell.
© Oxford University Press 1997
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Blom-Zandstra et al.
plasma membrane K + -channels have been thoroughly
characterized (Van Duijn, 1993; Van Duijn et al., 1993;
Thomine et al., 1994).
So far, changes in pump and channel activities upon
light transitions have been studied separately. In this
study, the involvement of both K + -channels and proton
fluxes in the transient light responses in the same cell was
evaluated. Patch-clamp experiments in a whole cell (WC)
configuration, provide the opportunity to study light
responses in different ways: In current clamp (CC) the
effect of light on £ m can be studied while in voltage clamp
(VC) both membrane current (7m) and conductance (gm)
can be measured simultaneously. Moreover, the contribution of K + -channels to / m upon light transitions can be
minimized (1) by clamping the membrane potential (Km)
at a potential where these channels are deactivated or (2)
by clamping Vm at the reversal potential (VT) of these
channels. In both situations the potassium current (/K.ORC)
is zero although the potassium conductance (£K.ORC) m a v
not be zero at Vz.
The involvement of the K + -channel is also evaluated
by the addition of verapamil, effective as a blocker of
K +-selective channels in tobacco protoplasts (Thomine
et al., 1994). Addition of verapamil strongly reduces the
contribution of K + -channels to 7m andg m , independent
of Vm.
Materials and methods
Plant material
Seeds of Nicotiana tabacum L., cv. rustica America, were sown
in Perlite and kept at 20 °C for 2 weeks. Further growth
occurred in a growth chamber at the same temperature, RH =
70% and a photoperiod of 12 h (150 ^mol cm" 2 s" 1 ). Onemonth-old seedlings were transferred to a hydroponic system
based on a Steiner solution (1968).
Control experiments were performed with sweet pepper,
ArabidopsLs, couch grass, and Alstroemeria. The growth conditions of sweet pepper {Capsicum annuum L.) were the same as
those for tobacco. Arabidopsis thaliana L. was kindly donated
by the Department of Genetics from the Wageningen
Agricultural University. The growth conditions are described
by Koorneef et al. (1991). Couch grass (Elytrigia repens L.)
was grown as described by De Ruiter et al. (1990). Cut flower
stalks of alstroemeria (Alstroemeria pelegrina L. cv. Westland)
were obtained from a commercial nursery at a developmental
stage as described in detail by Jordi et al. (1993).
0.009 ^ M ) adjusted with mannitol to an osmolarity of 485
mOsmol kg" 1 . Asymmetrical bath solution: 10 mM MES-TRIS
(pH 5.5), 10 mM K G (concentration depended on the desired
equilibrium potential), 2 mM MgCl2, 2 mM CaCl 2 , adjusted
with mannitol to an osmolarity of 510 mOsmolkg" 1 .
Symmetrical bath solution: 10 mM MES-TRIS (pH 7.2),
150 mM KC1 (equal to the K + -concentration of the pipette
solution, resulting in an equilibrium potential for potassium of
nearly 0 mV), 2 mM MgCl2, 2 mM CaCl 2 , adjusted with
mannitol to an osmolarity of 510 mOsmol kg" 1 .
Patch-clamp experiments
Electrodes were prepared from borosilicate glass (GC150-15,
Clark Electromedical Instruments, Reading, UK.). Pipettes were
made with a two-stage puller (List-medical 3P-A, Darmstadt,
Germany), fire-polished (Zeiss 1D03 and List-medical CP-Z10I)
and coated with Sylgard (Dow-Corning, Midland, MI). The
pipette resistance ranged from 5-10 M£>, depending on the
geometry and experimental solutions.
Dishes with protoplasts were mounted in an inverted
microscope (Nikon-TMD). A piezo manipulator (Luigs and
Neumann GmbH, Ratingen, Germany) was used to move the
pipettes in the micrometre range. Conventional patch-clamp
techniques were applied according to Hamill et al. (1981) in
whole-cell (WC) configurations, performed by suction and
monitored by an Axopatch 200 patch-clamp amplifier (Axon
Instruments, USA). The sealing process was monitored on
a digital storage oscilloscope (Hewlett Packard 54501A
100 MHz). Voltage and current data were transferred via a
CED1401 A/D-converter which was controlled by CED patchclamp software (Cambridge, UK). Data were sampled at
2-10kHz and analysed using (Turbo-Pascal) CED compatible
software (ECOPATCH, Vogelzang and Prins, 1992). Light and
dark responses of plasma membranes were measured by
switching the light source (Philips 12 V, 100 W) of the
microscope (Nikon-TMD) on and off. Photon flux density was
about 100 ^mol m " 2 s" 1 .
Photochemical reaction of electrodes to light
Photochemical currents of the Ag/AgCl electrodes in both
pipette and bath solution upon light induction were measured
after transfer of the pipette into the bath medium. At high light
intensity (3800fimol m~ 2 s" 1 ) current changes (/, in pA) could
be described by single exponential curves (/= Imnx (1 — exp(— t/r))
with TbghI on = 5-10s and Tbght ofr=8—15 s) and occurred in the
opposite direction compared with responses of green cells. The
contribution of the photochemical currents to shifts in electrochemical potential was small (<;4 mV). During experiments
with protoplasts electrodes were minimally exposed to light
(less than lOO^jnol m" 2 s" 1 ).
Protoplast isolation
Protoplasts (diameter: 25 ± 7 (im) were isolated without osmotic
shock and centrifugation steps, as described by Blom-Zandstra
et al. (1995). The following solutions were used: wash medium.
10 mM MES-KOH (pH 5.5), 10 mM sucrose, 500 mM mannitol, 2 m M MgCl 2 , 2 m M CaCl 2 . Enzyme medium: l%cellulase
(R10 Onozuka), 0.2% macerozyme (R10 Onozuka) in wash
medium. Pipette solution: 1 mM HEPES-BTP (pH 7.2),
150 mM KC1 (concentration depended on the desired equilibrium potential), 2 mM MgCI 2 , 5 mM MgATP, 2 mM
K 4 BAPTA, 0.1 mM CaCl 2 (calculated free Ca-concentration:
Addition of blockers or elicitors
When indicated in the text, the following chemicals were added
to the bath solution: 10/u.M verapamil, 0.1 mM paraquat,
0.02 mM or 1 mM NaCN, 10 pM fusicoccin (kindly supplied
by Dr AH de Boer, University of Amsterdam, the Netherlands).
The chemicals were stored in methanol. The final concentration
of methanol in the bath solution was 0.1%. Addition of 0.1%
methanol to the bath solution did not affect channel activity or
light responses. When indicated, 5 ^M DCMU was added to
the pipette interior.
Light modulates channel and pump
Results
Electrogenic characteristics of isolated mesophyll
protoplasts from tobacco leaves
Mesophyll protoplasts were isolated from tobacco leaves
and used for patch-clamp measurements in the whole
cell configuration. Figure 1 shows typical recordings of
activation of outward K + -currents (ORCs) in the plasma
membrane upon depolarizing pulse potentials ranging
from —90 mV to 50 mV. Deactivation of the K + -currents
can be seen in the tail currents upon stepping back to the
holding potential (—140 mV). The equilibrium potential
(EK) was —60 mV in this experiment. From the steadystate //K-relationship (see inset) and the tail currents it
can be seen that the ORC activated at potentials close to
and more positive than EK. At potentials more negative
than EK, the ORC did not contribute to the steady-state
membrane current (see inset). Changing £ K to 0 mV
resulted in a shift of the activation potential proportional
to the shift of EK (as shown for ORCs in Plcmtago media
L. root cortex protoplasts, Vogelzang and Prins, 1995).
The tail currents reversed at EK (i.e. —54 mV in Fig.l).
This indicates that the ORC is selective for K + . In most
experiments (n = 40) only ORCs could be detected. In
four cases also inward rectifying currents (IRCs) were
observed. The ORC currents activated with a sigmoidal
time-course. Half-time values of these currents were voltage-dependent and decreased from 2 s to 0.2 s when pulse
potentials increased from —50 mV to +50 mV. The
single exponential deactivation currents were also voltage-dependent, but slower than the activation currents.
1625
Their half-time values varied from 0.8 s to 2.5 s for pulse
potentials from —140 mV to 0 mV.
Transient light responses
In the WC configuration, LD and DL transitions were
applied in both voltage clamp (VC) and current clamp
(CC). Measurements in CC always showed that DL
transitions induced rapid depolarization, while LD transitions caused hyperpolarization of the plasma membrane
(Fig. 2). In VC, DL transitions always induced a transient
rapid decrease in current, while LD transitions caused a
transient increase in current (Fig. 3, upper panel). The
steady-state 7/K-curves (measured > 5 m i n after light
transitions) were similar in light and dark (data not
shown). In addition, the activation and deactivation time
constants did not differ between light and dark.
Apparently, in WC measurements light transitions
induced only transient changes (<5min) of both membrane current in VC and membrane potential in CC.
While the transient responses to LD and DL transitions
were reproducible, the long-term changes (> 1 min) were
not.
To evaluate the role of ion channels in the transient
light responses further, the effect of LD and DL transitions on membrane conductance (#„,) was studied.
Membrane conductances were determined in VC by
application of small voltage pulses, which were repeated
every second and were superimposed on the holding
potential during the LD and DL transitions. To prevent
activation or deactivation of the channels, these pulses
were small (3-10 mV) and short (50-100 ms) compared
to the activation and deactivation halftime values. An
example of an applied pulse and the corresponding / pulK
is shown in the inset of Fig. 3. The direction of the
300
E
LU
-80
Fig. 1. Whole cell recordings (upper panel) of a tobacco leaf mesophyll
protoplast in asymmetrical solutions. Outward rectifying currents were
activated upon depolarizing pipette potentials. Pulses (see lower panel)
were applied ranging from —90 to 50 mV with 10 mV steps.
Deactivation tail currents appeared when the voltage was returned to
the holding potential of —140 mV. A leak subtraction of 4 GQ has
been applied to all tracks. The inset displays the corresponding whole
cell current voltage curve measured at the end of each applied
pulse (9.5 s).
-100
1
2
3
time (min)
Fig. 2. Whole cell recordings in current clamp in asymmetrical solutions
Light- ( • ) and dark- ( • ) induced changes of the free running
membrane potential (£„,) of tobacco mesophyll protoplasts
1626
Blom-Zandstra et al.
time (min)
Fig. 3. Whole cell recordings in voltage clamp in asymmetrical solutions
Light- ( • ) and dark- ( • ) induced current changes of the plasma
membrane of tobacco mesophyll protoplasts (upper panel) and
membrane conductance (lower panel). The holding potential was —50
mV. The membrane conductance was calculated (explanation: see text)
from the change in membrane current upon application of —10 mV
for a period of 0.1 s (inset). The values on the v-axis of the inset apply
to both the current (pA) and voltage (mV).
applied Vpu\K (upward or downward) did not affect the
amplitude of Ipulx (data not shown). The current changes
( V I K ) w e r e usec * t o calculate gm by:
6m
'pulie/ ' pulte
(Note: gm is denned as the sum of different conductances,
i.e. (/) the voltage and time-independent, non-selective
conductances usually described as the leak conductance,
comprising the seal and membrane bilayer conductance;
(II) the voltage and time-dependent ion channel conductances and pump conductances.)
Light transitions caused transient changes of gm (Fig. 3,
lower panel). These changes ofg m corresponded well to
the light-induced changes of/ m (Fig. 3, upper panel). As
no channels were activated or deactivated by Vpu\K (see
above), changes in / m or gm could not result from Vpu\x.
Therefore, variations of both 7m andg m (Fig. 3) were
caused by changes in the light conditions only.
the K + conductance (gK.oRc>0)> but the contribution of
the ORCs to current changes is small.
Apart from changes in channel activities, changes of
the pump activity may also be involved in the light effect.
To evaluate the involvement of the pump in light
responses, conditions were created in which changes of
7K ORC will not contribute to changes of / m . This can be
achieved by (I) changing the holding potential with
respect to the activation and reversal potential of the
ORC or by (II) changing the conditions of the bath and
pipette solutions from asymmetrical to symmetrical.
Changing the holding potential: In this experiment, protoplasts were selected without IRC activity and a holding
potential of —120 mV (60 mV more negative than the
reversal potential of the ORC (KrORc)) w a s applied. Thus,
outward rectifying channels were fully inactivated and
V.ORC = 0 pA (see inset Fig. 1). So, without IRC and
ORC activity, potassium channels do not contribute to
the current. At this holding potential, DL and LD transitions still resulted in changes of 7m (Fig. 4), whileg m was
not significantly affected by the transitions. These current
changes may result from changes in channel activities of
Cl" and Ca2 + , or a passive proton conductance. Changes
of proton pump activity may also be involved in the
light effect.
Changing bath and pipette solutions: To eliminate the
effects of Cl~ and passive proton conductances to lightinduced changes of Im, the light-responses with symmetrical solutions were studied. Here, the equilibrium potentials of protons, free potassium and free chloride ions are
close to zero (Ea = - 0 . 8 mV; EK = - 1 . 2 mV, £ H = °
mV). Consequently, at a holding potential of 0 mV,
ar)
AC.ORO ^ci d / H are zero. Therefore, potassium, chloride
or passive proton conductances will not contribute to
light-induced current changes at this potential. In VC,
DL transitions still induced a transient rapid decrease in
Changing the contribution of the ORC in the WC
configuration
Changes of Im and gm upon LD and DL transitions, as
shown in Fig. 3, may result from changes in the activities
of different channels, i.e. K + , Cl~, Ca 2+ or a passive H +
conductance. Of these, the K + channel is the most
dominant one. The holding potential (—50 mV) in this
experiment was slightly positive to KrORC (—60 mV). At
this potential, it is expected that the ORCs contribute to
~*
'.—r
time (min)
Fig. 4. Whole cell recordings in voltage clamp in asymmetrical solutions.
Light- (C) and dark- ( • ) induced current changes of the plasma
membrane of tobacco mesophyll protoplasts (upper panel) and
membrane conductance (lower panel). The holding potential was
- 1 2 0 mV.
Light modulates channel and pump
Im, while LD transitions caused a transient increase in /m
(Fig. 5). Concomitantly, DL and LD transitions also
caused transient changes ofgm. The results agreed with
the data measured in the asymmetrical solutions shown
in Fig. 3. However, the amplitude of the changes in gm
was smaller. In this experiment, a proton pump and a
calcium channel are the only potential sources for current
changes.
From the experiments it was concluded that apart from
channel activity the proton pump may substantially contribute to light-induced changes of the membrane current.
Different channels that are hard to distinguish from each
other may be responsible for the light-induced changes
ofgm. The potassium ORC may be the most important
source: when the ORC is inactivated (Fig. 4) changes
of gm have faded.
1627
400 -
-120 mV
+Verapamil
Evaluation of the involvement of the proton pump
To verify the contribution of the proton pump to lightinduced current changes of the plasma membrane, the
proton pump activity was strongly increased by the
addition of fusicoccin (Marre, 1979), and the ORC and,
if present, the calcium channels, were inactivated by the
addition of 10 pM verapamil (Thomine et al., 1994). The
inhibition of the ORC by addition of this chemical was
very effective: as a result the steady-state outward rectifying currents were strongly blocked (Fig. 6). Similar to
the experiment in which the ORC was inactivated (Fig. 4),
addition of verapamil inhibited the effect of LD and
DL transitions on changes ofgm (n = 5), while lightinduced current changes could still be detected (Fig. 7).
Apparently, the proton pump contributes significantly to
the light response, although a contribution of chloride
fluxes cannot be excluded.
Fig. 6. Whole cell recordings of a fusicoccin-treated tobacco leaf
mesophyll protoplast in asymmetrical solutions before and after
addition of verapamil. Outward rectifying currents were activated upon
depolarizing pipette potentials. Pulses were applied ranging from —100
to 60 or 80 mV (as indicated in the figure) with 10 mV steps.
Deactivation tail currents appeared when the voltage was returned to
the holding potential of —120 mV. A leak subtraction of 5 GQ has
been applied to all tracks. The inset displays the corresponding whole
cell current voltage curves, measured at the end of each applied pulse
(6 s), before (—) and after (+) addition of verapamil.
0
-4
-8
-12
0.6
0.4
Mvr^P
0.2
25
5
time (mm)
7.5
Fig. 7. Whole cell recordings in voltage clamp in asymmetrical solutions
measured in the same protoplast as in Fig. 6 after blocking the outward
rectifier by addition of 10^M verapamil. Light- (D) and dark- ( • )
induced current changes of the plasma membrane of tobacco mesophyll
protoplasts (upper panel) and membrane conductance (lower panel).
The holding potential was —60 mV.
2
4
time (min)
Fig. 5. Whole cell recordings in symmetrical solutions in voltage clamp.
Light- ( • ) and dark- ( • ) induced current changes of the plasma
membrane of tobacco mesophyll protoplasts (upper panel) and
membrane conductance (lower panel). The holding potential was 0
mV. Both voltage and current clamp measurements were performed in
the same protoplast.
Origin of the light response
Similar light responses as described above were also
detected in protoplasts from other plant species, e.g.
Capsicum annuum L., Arabidopsis thaliana L., Elytrigia
repens L., and Alstroemeriapelegrina L. (data not shown).
These responses were only found in mesophyll cells.
1628
Blom-Zandstra et al.
Protoplasts from root or cortex cells did not respond to
light transitions, nor did protoplasts from chlorophylldeficient cells from a tissue-cultured tobacco plant. This
strongly indicates that a light-sensitive-complex is
involved in the response. To test the involvement of the
light-harvesting complex (LHC) the effect of a photosynthetic inactive wavelength was examined. For this purpose, a filter (Vitatron, 5l5nm, bandwidth 5 nm) was
placed between the light source and the bath, to obtain
green light with a low light intensity (12^mol m~ 2 s" 1 ).
However, current changes by DL and LD transitions still
occurred (data not shown). While light responses
depended on the presence of chlorophyll, addition of
inhibitors of photosynthesis I and II (DCMU, paraquat,
NaCN) did not alter the light response either.
Discussion
The starting point of this study was to evaluate the initial
response of £ m of the plasma membrane to DL and LD
transitions as shown in microelectrode studies (Bulychev
etcil., 1972; Vredenberg, 1974; Jeschke, 1976). The patchclamp technique was used to study the light responses in
the whole cell configuration. The plasma membrane of
mesophyll protoplasts from tobacco leaves showed electrogenic characteristics (Fig. 1) similar to those described
earlier (Van Duijn, 1993; Van Duijn et al., 1993; Thomine
et al., 1994).
A transient hyperpolarization or depolarization of the
plasma membrane was observed in CC (Fig. 2) after DL
and LD transitions, respectively, in addition to a clear
decrease or increase of membrane current in VC (Fig. 3).
These results agree very well with the microelectrode
studies. Apparently, patch-clamp experiments in the WC
configuration are a feasible approach for the investigation
of transient light effects.
To study the light responses, a novel method was used:
at different values of Vm, light-induced changes of Im
were separated from changes in gm by application of
repetitive voltage pulses during DL and LD transitions.
The results show that / m and gm were not always proportionally affected by light transitions. The effect of these
transitions on changes of Im and gm was strongly influenced by the applied Km and by the activity of the ORC
involved. This indicates that apart from channel activity,
obeying Ohm's law, pump activity may also be involved.
It is assumed that in the domain in which the experiments
were performed, only the pump can behave in a voltageindependent manner as suggested by the model of Blatt
(1991). Ion channels or other transporters show voltageindependency only at their extremes, beyond the voltagerange of these observations. To evaluate the involvement
of pump and/or channel activity the experimental conditions were varied to change the contribution of the ORC
to changes of Im. The results of these experiments presented in Figs 3-5 and Fig. 7 are summarized in Table I.
For every experiment a set of potential sources for current
changes upon light is suggested. When Vm was 10 mV
more positive than Vr ORC, light transitions induced both
changes of / m and gm (Fig. 3). As ORCs are activated at
this potential (shown in Fig. 1) it was concluded that
changes in gm by light transitions are caused by modulation of the ORC activity. So, changes of / m may also
result from modulation of the ORC activity, although its
contribution may be small as Vm is close to EK. Apart
from the ORCs, other channels (Ca 2 + , Cl") may be
involved. The proton pump and the passive proton conductances are also likely candidates. The latter have been
described for different plant cell membranes (Althoff
et al., 1989; Beilby et al., 1993) and may be involved in
the cells also because the equilibrium potential for protons
is more positive than +70 mV. Consequently, there is a
large driving force for protons through H + channels.
At Vm values 60 mV more negative than EK, light
transitions induced large changes of Im (Fig. 4). These
were not accompanied by corresponding changes ofg m .
The changes in / m may result from different channels
other than the K + -ORC. If K + -channels were responsible
Table 1. Change of membrane current and conductance as a result of LD and DL transitions at changing ORC activity and the
potential candidates possibly involved
Typical responses
presented in
ORC-activated
Effect of LD and DL transitions
Change of /„
Change of gm
Fig. 3
channels: H \ C r ,
Fig. 4
+
Fig. 5
Fig. 7
Potential source for
current change
—
t
"Involvement of K * channel activity different from ORC and IRC activity cannot be excluded, but seems unlikely.
H + pump
channels: H + . Cl".
H + pump
channels Ca2 +
H + pump
channels: Cl"
H + pump
Light modulates channel and pump
for the current changes, a conductance change of at least
0.34 nS would have been required. Moreover, the whole
cell 7/K-curve in Fig. 1 (inset) suggests that all ORCs
were inactivated at —120 mV. It was concluded that the
ORCs are not involved in the light response at these
potentials. Alternatively, at these values of the membrane
potential the proton pump and a passive proton conductance are likely candidates to cause changes of 7m.
To evaluate the contribution of the passive proton
conductances, symmetrical solutions for bath and pipette
were used to nullify the effects of a proton gradient. At
0 mV (Km near KrORC and VTC]), large positive currents
could be detected (Fig. 5), which can only be caused by
proton pump activity. Again, light transitions induced
changes of Im and gm. At this Vm, potassium, chloride or
passive proton fluxes do not contribute to changes in 7m.
So, transient light-induced current changes should be due
to changes in pump activity, although a small contribution
of calcium fluxes cannot be excluded. At present there is
no explanation for the irreproducibility of the long-term
responses.
Apparently, both channel and pump activities contributed to the light responses. The inhibiting effect ofverapamil (Fig. 6) on light-induced changes of gm (Fig. 7)
confirmed the involvement of the proton pump activity.
The mechanism of the light response in protoplasts is
still unclear. In these experiments only mesophyll cells
responded to light, while root cells and chlorophylldeficient cells did not respond. This suggests a relationship
with the presence of chloroplasts. Several authors
(Mimura and Tazawa, 1986; Hansen et al., 1987; Vanselow
et al., 1989) have suggested that light interception at the
chloroplast is coupled with hyperpolarization or depolarization at the plasma membrane. It has been suggested
that rapid changes of the plasma membrane are coupled
with light-induced proton uptake by the inner thylakoid
space (Vanselow et al., 1989). Hansen et al. (1987)
presented a detailed model which describes the relation
between the light response at the thylakoid membrane
and polarization of the plasma membrane. In this model
cytosolic pH changes play an intermediate role. Spalding
and Goldsmith (1993) demonstrated with single channel
experiments that light-induced K + channel activity at the
plasma membrane depends on the availability of ATP.
This suggests that the photosynthetic activity in photosystems I and II initiates a cascade of responses which results
in current changes at the plasma membrane. Indeed, in
these experiments the responses to light transitions only
occurred in protoplasts that contained chloroplasts.
Surprisingly, inhibitors of photosystems 1 and II did not
block the light responses. Moreover, when green light of
low intensity was used, normal LD and DL responses
were detected. A possible explanation for this phenomenon is that only a minimum of the photosynthetic
1629
activity which remained after addition of the inhibitors,
may be required to trigger the light response.
Although the origin of the transient light responses at
the plasma membrane was not revealed, the existence of
light responses in which both proton pump and channel
activity are involved was shown clearly. It may be concluded that the whole cell configuration is a powerful
tool to study the effect of light transitions on pump and
channel activity simultaneously.
Acknowledgements
We are very grateful to Dr JPFG Helsper and Dr BW Veen for
the critical reading of the manuscript.
References
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