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University of ZurichZurich Open Repository and Archive
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Year: 2009
The fou2 mutation in the major vacuolar cation channel TPC1confers tolerance to inhibitory luminal calcium
Beyhl, D; Hörtensteiner, S; Martinoia, E; Farmer, EE; Fromm, J; Marten, I; Hedrich, R
Beyhl, D; Hörtensteiner, S; Martinoia, E; Farmer, EE; Fromm, J; Marten, I; Hedrich, R (2009). The fou2 mutationin the major vacuolar cation channel TPC1 confers tolerance to inhibitory luminal calcium. The Plant Journal,58(5):715-723.Postprint available at:http://www.zora.uzh.ch
Posted at the Zurich Open Repository and Archive, University of Zurich.http://www.zora.uzh.ch
Originally published at:The Plant Journal 2009, 58(5):715-723.
Beyhl, D; Hörtensteiner, S; Martinoia, E; Farmer, EE; Fromm, J; Marten, I; Hedrich, R (2009). The fou2 mutationin the major vacuolar cation channel TPC1 confers tolerance to inhibitory luminal calcium. The Plant Journal,58(5):715-723.Postprint available at:http://www.zora.uzh.ch
Posted at the Zurich Open Repository and Archive, University of Zurich.http://www.zora.uzh.ch
Originally published at:The Plant Journal 2009, 58(5):715-723.
The fou2 mutation in the major vacuolar cation channel TPC1confers tolerance to inhibitory luminal calcium
Abstract
Summary The SV channel encoded by the TPC1 gene represents a Ca(2+)- and voltage-dependentvacuolar cation channel. Point mutation D454N within TPC1, named fou2 for fatty acid oxygenationupregulated 2, results in increased synthesis of the stress hormone jasmonate. As wounding causesCa(2+) signals and cytosolic Ca(2+) is required for SV channel function, we here studied theCa(2+)-dependent properties of this major vacuolar cation channel with Arabidopsis thaliana mesophyllvacuoles. In patch clamp measurements, wild-type and fou2 SV channels did not exhibit differences incytosolic Ca(2+) sensitivity and Ca(2+) impermeability. K(+) fluxes through wild-type TPC1 werereduced or even completely faded away when vacuolar Ca(2+) reached the 0.1-mm level. The fou2protein under these conditions, however, remained active. Thus, D454N seems to be part of a luminalCa(2+) recognition site. Thereby the SV channel mutant gains tolerance towards elevated luminalCa(2+). A three-fold higher vacuolar Ca/K ratio in the fou2 mutant relative to wild-type plants seems toindicate that fou2 can accumulate higher levels of vacuolar Ca(2+) before SV channel activity vanishesand K(+) homeostasis is impaired. In response to wounding fou2 plants might thus elicit strongvacuole-derived cytosolic Ca(2+) signals resulting in overproduction of jasmonate.
The fou2 mutation in the major vacuolar cation channel TPC1
confers tolerance to inhibitory luminal calcium
Running title: Luminal Ca2+-tolerant fou2 SV channels
Beyhl1, D., Hörtensteiner2, S., Martinoia2, E., Farmer3, E. E., Fromm, J.4, Marten1, I.
and Hedrich1, R.
1 University of Würzburg, Department of Molecular Plant Physiology and Biophysics,
Julius-von-Sachs - Institute, Julius -von-Sachs-Platz 2, 97082 Würzburg, Germany
2 University of Zürich, Institute of Plant Biology, CH-8008 Zürich, Switzerland
3 University of Lausanne, Plant Molecular Biology, Biophore, CH1015 Lausanne,
Switzerland
4 University of Hamburg, Center for Wood Sciences, Leuschnerstr. 91, 21031 Hamburg,
Germany
Corresponding author: Irene Marten, Julius-von-Sachs-Platz 2, 97082 Würzburg,
Germany, Phone: +49 (0)931/888-6118, Fax: +49 (0)931/888-6158,
E-mail: [email protected]
Word Count: In total 6705 words
2
Abstract (199 words)
The SV channel encoded by the TPC1 gene represents a Ca2+- and voltage-dependent
vacuolar cation channel. Point mutation D454N within TPC1, named fou2 for fatty acid
oxygenation upregulated 2, results in increased synthesis of the stress hormone jasmonate.
Since wounding causes Ca2+ signals and cytosolic Ca2+ is required for SV channel
function, we here studied the Ca2+-dependent properties of this major vacuolar cation
channel with Arabidopsis thaliana mesophyll vacuoles. In patch clamp measurements,
wild type and fou2 SV channels did not exhibit differences in cytosolic Ca2+ sensitivity and
Ca2+ impermeability. K+ fluxes through wild type TPC1 were reduced or even completely
faded away when vacuolar Ca2+ reached the 0.1 mM level. The fou2 protein under these
conditions, however, remained active. Thus, D454N seems to be part of a luminal Ca2+
recognition site. Thereby the SV channel mutant gains tolerance towards elevated luminal
Ca2+. A three-fold higher vacuolar Ca/K ratio in the fou2 mutant relative to wild type
plants seems to indicate that fou2 can accumulate higher levels of vacuolar Ca2+ before SV
channel activity vanishes and K+ homeostasis is impaired. In response to wounding fou2
plants might thus elicit strong vacuole -derived cytosolic Ca2+ signals resulting in
overproduction of jasmonate.
Keywords: TPC1 – fou2 mutant – Ca2+ – pH – vacuole
3
Introduction (667 words)
Following the application of the patch clamp technique to higher plant plasma membranes
and vacuoles, ion transport properties of both membranes could be studied individually
(Schroeder et al. , 1984; Hedrich et al., 1986; Hedrich and Schroeder, 1989 for review).
Initial studies on isolated barley vacuoles identified a voltage -dependent cation channel
and a proton pump as major conductances of the vacuolar membrane (Hedrich et al. ,
1986). Using the same electrophysiological approach both vacuolar transporters were
identified in almost all plants and cell types (Hedrich et al. , 1988, Pottosin and
Schönknecht, 2007 for review). The vacuolar cation channel, characterized by its slow
activation in response to voltage changes by Hedrich and Neher (1987), was named SV for
Slow Vacuolar channel. Furthermore, this study recognized the Ca2+-dependent activation
of the SV channel. In line with a Ca2+-modulated channel, voltage activation of SV channel
is facilitated in the presence of elevated cytosolic Ca2+ concentrations. A rise of the
cytosolic Ca2+ level has a dual effect on SV channel activity, an increase in the maximal
number of open SV channels at high positive potentials and a shift of the voltage
dependence to less positive potentials (Schulz-Lessdorf and Hedrich, 1995; Pottosin et al.,
1997; Pottosin and Schönknecht, 2007 for review).
The SV channel has a similar permeability for K+ and Na+ (Coyaud et al., 1987; Amodeo
et al., 1994; Pottosin et al., 2001; Ranf et al., 2008). When these cations are present on the
cytosolic side of the vacuolar membrane at about 10 mM and higher concentrations, K+
and Na+ ions enter the vacuole (Ivashikina and Hedrich, 2005). While this reflects a
physiological condition, Ca2+ transport into the lumen of this organelle was shown when
the vacuole was challenged with Ca2+ concentrations exceeding 50000 times that occurring
in vivo in the cytosol (Ward and Schroeder, 1994). When present in the vacuolar lumen,
the SV channel of Arabidopsis suspension-cultured cells mediates the uptake and release of
the major monovalent cation K+. This process is driven by the voltage - and concentration
4
gradient (Ivashikina and Hedrich, 2005). In contrast Na+ and Ca2+ are not released from the
vacuole into the cytosol via this channel. At millimolar concentrations Na+ and Ca2+ even
block the release of K +.
In search for plant Ca2+ channels, a single copy gene with partial homology to a voltage-
dependent Ca2+ channel called two pore channel (TPC) 1 was identified by data base
mining (Ishibashi et al., 2000). The Ca2+ channel signature conserved among species
(Zagotta, 2006), however, is missing in TPC1 (Furuichi et al., 2001). The channel protein
fused to GFP was localized in the central vacuole of Arabidopsis (Peiter et al. , 2005).
Elegant patch clamp studies on a TPC1 -loss-of-function mutant, tpc1-2, enabled the
authors to unequivocally demonstrate that TPC1 encodes the SV channel. In line with
previous findings showing that the SV channel does not very likely catalyze vacuolar Ca2+
release into the cytosol (Pottosin et al., 1997; Ivashikina and Hedrich, 2005), recently Ranf
et al. (2008) could demonstrate that Ca2+ signaling of tpc1-2 is indistinguishable from wild
type. In contrast to tpc1-2 , a point mutation in TPC1 named fou2 showed a pronounced
phenotype (Bonaventure et al., 2007a, b). fou2 for fatty acid oxygenation upregulated 2
was identified in a screen for mutants in wounding-induced jasmonate pr oduction. fou2
channel activation appeared to be shifted in its voltage -dependence to more negative
membrane potentials resulting in faster activation kinetics compared to the wild type SV
channel. The role of this gain-of-function SV channel activity in wounding/jasmonate
signaling is obscure. Also, it is not known how an amino acid residue in the SV channel
that is predicted to face the vacuolar lumen is able to shift the voltage gate of the SV
channel. Here we show comparative patch clamp studies on Arabidopsis mesophyll
vacuoles isolated from wild type and fou2 mutant plants. While both the responses to
changes in the cytosolic Ca2+ level and the Ca2+ impermeability of the fou2 SV channels
remained wild type-like, the SV channel mutation altered sensitivity to luminal Ca2+ levels.
5
Results (1574 words)
fou2 represents a hyperactive version of the SV channel
Previous studies have shown that the SV channel from barley mesophyll vacuoles under
symmetrical 100 mM potassium functions as a Ca2+-dependent outward-rectifying cation
channel (Hedrich et al., 1986). Similar results were obtained when the whole-vacuole
patch clamp configuration was established on mesophyll vacuoles of Arabidopsis thaliana.
Upon voltage stimulation (+70 mV pulses) of wild type SV channel, outward currents
appeared about 5-6 min right after gaining access to the vacuole lumen during equilibration
of the vacuole sap with patch pipette solution containing 0.1 mM Ca2+. Steady-state
activation was reached after about 12 min in the whole -vacuole mode (Figure 1). Under the
same condition with fou2 vacuoles, depolarizing voltage steps elicited outward SV channel
currents already in the range of 0.5-2 min (Figure 1). These results indicate that a vacuolar
SV channel inhibitor present in the lumen seems to be `washed out´ upon equilibration
with the patch pipette solution (Maathuis and Prins, 1991). Two possibilities may explain
why a potential inhibitor has only a marginal effect in the fou2 mutant: (i) the TPC1
inhibitor concentration is lower in fou2 vacuoles and/or (ii) the sensitivity of the SV
channels towards the inhibitor is reduced in fou2 . The latter situation could be gained by a
point mutation on the luminal side of the channel protein. To distinguish between the two
possibilities we examined the quantities of various elements in mesophyll vacuoles by
energy dispersive X-ray (EDX) analysis (Figure 2). In wild type and mutant plants the
vacuolar Mg, P, S and Cl content was similar while K/Ca ratios appeared quite different.
fou2 mesophyll vacuoles contained significantly less K but more Ca than wild type
vacuoles. Since wild type Arabidopsis plants and the fou2 mutant express TPC1 RNA to a
similar extent (Bonaventure et al., 2007a), the reduced K level in fou2 might be related to
the properties of the mutant form of the SV channel.
6
With symmetrical K+ concentrations on both sides of the vacuolar membrane, fou2 SV
channels activated about 30 mV more negative than wild type channels in whole vacuolar
studies (Figure 3a, cf. Bonaventure et al. , 2007a). Thus, fou2 SV channels conduct inward
K+ currents, in other words K+ release from the vacuole into the cytosol, already at low
negative voltages. When in the presence of 1 mM cytoplasmic Ca2+ the potassium gradient
is directed out of the vacuolar lumen (high [K+]lumen / low [K+]cytosol), wild type channels
conduct both inward and outward potassium currents (Ivashikina and Hedrich, 2005).
Under these asymmetric conditions we found fou2 to activate at about 40 mV less
depolarized voltages than wild type SV channels (Figure 3b). As a consequence, in the
presence of 30 mM K+ in the cytosol and 150 mM in the vacuole fou2 SV channels at -5
mV mediate about five times higher inward currents as the wild type SV channels (Figure
3b). When cytosolic K+ was replaced by 15 mM Ca2+, no currents were elicited by voltage
stimulation in wild type and fou2 mesophyll vacuoles (Figure 3c). This shows that in the
presence of Ca2+ as the sole cytosolic cation, wild type and fou2 SV channels of A.
thaliana mesophyll cell vacuoles neither conduct K+ into the cytoplasm nor Ca2+ into the
vacuolar lumen. Based on these observations, it is unlikely that elevated vacuolar Ca2+
levels in non-stressed fou2 mutants (Figure 2) as well as the predicted wounding-induced
rise in cytosolic Ca2+ concentration (Chico et al. 2002; Fisahn et al., 2004; Dombrowski et
al., 2007) are related to a change in Ca2+ permeability of the SV channel. This, however,
does not exclude the possibility that the fou2 mutation additionally gained altered Ca2+-
dependent properties of the SV channel.
fou2 is activated by cytosolic Ca2+ and tolerates elevated vacuolar Ca2+ levels.
SV channels are characterized by pronounced Ca2+ sensitivity (Hedrich and Neher, 1987;
Allen and Sanders, 1996; see for review Pott osin and Schönknecht, 2007). Our studies on
fou2 shown in Figure 3 were performed with 1 mM cytosolic Ca2+, a level at which this
7
channel is maximally stimulated. To test whether the fou2 mutation alters the sensitivity of
the SV channel towards cytosolic Ca2+ changes, fou2 and wild type SV channels were
challenged with 200, 300, 500 and 1000 µM cytosolic Ca2+ (Figure 4). Under these
conditions, the voltage-dependence of the relative channel open-probability (Figure 4b, c)
was determined by tail-current experiments (Figure 4a). With decreasing cytosolic Ca2+
concentrations the half -activation voltage V1/2 of fou2 and wild type channels linearly
shifted towards more positive voltages (Figure 4b, c). For instance a voltage difference of
∆V1/2 = 46.7 mV was determined for the activation curves with 0.3 and 0.5 mM cytosolic
Ca2+. Hence, fou2 and wild type SV channel exhibit a similar sensitivity towards
regulatory cytosolic Ca2+. Given the fact that the luminal calcium concentration feeds back
on the transport ca pacity and direction of cation fluxes through the wild type SV channel
(see for review Pottosin and Schönknecht, 2007), we examined on the single channel level
whether the vacuolar Ca2+ concentration affects the channel activity of fou2 as well. For
this we exposed excised patches with the vacuolar membrane side facing 0, 100 and 1000
µM Ca2+ under symmetrical K+ and H+ concentrations and measured single channel
activities in the voltage range from -50 to -30 mV. In line with the macroscopic current
recordings the single channel activity in wild type and fou2 plants declined with negative-
going membrane voltages. At voltages positive from -30 mV the channel activities were
too high to resolve single channel events. Upon rising luminal Ca2+ from 0 to 100 or 1000
µM, the wild type SV channel activity almost completely disappeared at -50 to -30 mV - a
range where single channel analysis was feasible. However, fou2 responded much less
dramatically to increasing luminal Ca2+ levels (Figure 5). In contrast to wild type channels,
fou2 still maintained a pronounced channel activity in the presence of 100 and even 1000
µM luminal Ca2+. The fact that the wild type SV channels exhibited a quasi-steady-state
inhibition at both Ca2+ levels (100, 1000 µM) points to an increased tolerance of the SV
channel mutant to elevated luminal Ca2+ concentrations. Thus, the fou2 point mutation
8
seems to alter the luminal Ca2+ affinity of the SV channel underlying the Ca2+-mediated
inhibition of the SV channel.
Luminal protons and Ca2+ ions differentially affect the activity and K + transport capacity of
fou2 and wild type SV channel
Vacuolar proton pumps can modulate the acidity of the vacuolar lumen (see for review
Martinoia et al. , 2007) and luminal pH changes were shown to alter SV c hannel properties
(Schulz-Lessdorf and Hedrich, 1995; Pottosin et al., 1997). Thereby, Ca2+ apparently
competes with H+ for the same binding sites. Removal of vacuolar Ca2+ at neutral pH
results in a dramatic negative shift of the voltage-dependent SV channel activation that is
opposite to the effect of reduced cytoplasmic Ca2+ levels (Pottosin et al. , 1997, 2004). To
test whether protons affect the luminal Ca2+ sensitivity of the SV channel too and thereby
rendering this transporter less sensitive to blocking Ca2+ ions, we studied the vacuolar Ca2+
response of the SV channel to vacuolar acidification. When a 100-fold proton gradient (pH
7.5cyt and 5.5vac) was generated across the vacuolar membrane in the absence of luminal
Ca2+, the wild type single -channel activity was much lower (Figure 6) than under
symmetrical pH 7.5 conditions (Figure 5). An increase in the luminal Ca2+ concentration
from 0 to 100 µM at acidic pH (Figure 6b) affects the wild type SV channel activity less
than at symmetric pH conditions (Figure 5). A further rise to 1000 µM luminal Ca2+ almost
completely blocked the wild type channel. In comparison to neutral pH (Figure 5b), fou2
single -channel activity appeared to be almost similar in the absence of luminal Ca2+ but of
altered sensitivity towards vacuolar Ca2+ loads at acid vacuolar pH (Figure 6b). A rise from
0 up to 1000 µM luminal Ca2+ only slightly reduced the channel activity in fou2 (Figure
6b). In the absence of luminal Ca2+, acidification of the vacuole decreased SV channel
activity in wild type but only marginally in fou2. This observation points to a mutation-
related pK change of a critical luminal site of the TPC1 protein. To further characterize
9
this, we decreased the luminal pH stepwise from pH 7.5 to 4.5 in the absence of luminal
Ca2+ and presence of 1 mM cytosolic Ca2+ (Figure 7). The single channel conductance was
in the order of 35-40 pS at symmetrical pH 7.5 for both wild type and mutant SV channels
(Figure 7) and increased upon pH decrease. At pH 5.5 and 4.5, the unitary conductance
reached about 80 pS, a transport capacity which is twice as high as at neutral pH. Similar
single -channel open probabilities in fou 2 plants in the absence of luminal Ca2+ at luminal
pH 5.5 and 7.5 (cf. Figures 5b and 6b) is indicative for an effect of pr otons on the unitary
conductance of SV channels in fou2 only (Figure 7). However, under same experimental
conditions both the open probability (Figures 5b, 6b) and unitary conductance (Figure 7) of
SV channels in wild type responded to pH changes in an opposite manner. While the open
probability of wild type SV channels decreased with increasing proton concentration, the
unitary conductance increased. Thus, the mutation in fou2 does not seem to affect
protonation of the `conductance -site´ but alters the pK of the site associated with the open
probability.
10
Discussion (1171 words)
fou2 mimics Ca2+ activation of wild type SV channels
As shown by Bonaventure et al. (2007a), under symmetrical K+ conditions on both
vacuolar membrane sides fou2 SV channels activate at more negative potentials and shuttle
more K+ into the cytosol than wild type SV channels (Figure 3a). The presence of inward-
directed K+ gradients across the vacuolar membrane caused a negative shift in the voltage
threshold for activation of the wild type channel (Figure 3a, b; see also Ivashikina and
Hedrich, 2005). In fou2 the latter process was comparable but associated with higher
inward K+ fluxes compared to wild type TPC1 (Figure 3a, b). Under related physiological
conditions the fou2 mutant seems to gain its phenotype most likely from the increased
capacity for K+ release from the vacuole into the cytosol resulting in a reduced K level in
fou2 vacuoles (Figure 2). The impaired potassium homeostasis in the mutant is likely
related to the voltage -dependent activation of fou2 SV channels at less negative voltages
(Figure 3a, b) as well as to its reduced sensitivity towards increased luminal Ca2+ levels
(Figures 5, 6) maintaining more SV channels open in fou2 even at elevated luminal Ca2+
levels. Interestingly, the transcript profile of fou2 plants resembles the K+ starvation
transcriptome (Armengaud et al., 2004; Bonaventure et al., 2007b). While the expression
of the K+ transporters did not change under K+ deficiency, the transcript level of the
vacuolar Ca2+/H+ antiporter CAX3 was up-regulated (Armengaud et al. 2004). The latter
could contribute to an increase in the vacuolar Ca2+ content (as observed with fou2, Figure
2) because CAX3 promotes Ca2+ entry into the vacuole . In tomato, wound-induced
cytosolic Ca2+ signals have been observed (Moyen et al., 1998) leading to wound-response
gene activation (Dombrowsky and Bergey, 2007). Considering the elevated vacuolar Ca2+
content in fou2, a more pronounced signal-induced rise in the cytosolic Ca2+ level might
occur upon wounding. As one possible consequence increased lipoxygenase (LOX) gene or
11
enzyme activity could explain the rise in transcript numbers (e.g. 25-fold for LOX2) and
LOX activity observed with the fou2 mutant (Bonaventure et al., 2007a).
The SV channel seems to present a primary voltage-dependent cation channel with its
voltage gate modulated by Ca2+, luminal pH and transmembrane K+ gradient. Recent
studies on red beet vacuoles showed that changes in luminal pH at different luminal Ca2+
levels did not affect the activation potential of SV channels (Pérez et al., 2008).
Accordingly, the observed decrease in the open probability (Po) of wild type SV channels
upon luminal acid loads in the absence of Ca2+ (Figures 5, 6) might be related to a change
in voltage sensitivity rather than voltage dependence. This pH effect on Po is gone in fou2
(Figures 5, 6), suggesting that aspartate at residue 454 of the SV channel protein might be
involved in pH control of the single channel activity upon its voltage sensitivity. In line
with a Ca2+-activated cation channel, voltage changes triggered SV channel-mediated K+
currents in the presence of elevated cytosolic Ca2+ only (Ward and Schroeder, 1994; Peiter
et al., 2005). Thereby cytosolic Ca2+ seems to shift the voltage threshold for SV channel
activation into the range of physiological vacuolar potentials (cf. Hedrich and Neher,
1987). A similar effect of Ca2+ on the voltage range of activity has been described for maxi
K channels in neurons (Marty, 1981). In the absence of cytosolic Ca2+, SV channels seem
to activate at extreme positive voltages, a condition which is not tolerated by isolated
vacuoles under patch clamp conditions. Elevation of the cytosolic Ca2+ level resulted in a
similar shift in the voltage -dependent activation of wild type and fou2 SV channels (Figure
4b, c), indicating that the SV channel mutation does not affect the cytosolic Ca2+
dependence of the channel. When challenged with increasing Ca2+ concentrations on the
luminal side, SV channel activity ceased in wild type, but not in fou2 (Figures 5, 6).
Inhibition of K+ currents through the SV channel occurred at luminal Ca2+ concentrations
similar in range to activating cytosolic Ca2+ concentrations. In contrast to the action of the
12
divalent cation at the cytosolic side, an increase in the vacuolar Ca2+ level, therefore, albeit
increasing the driving force for Ca2+ release, results in SV channel closure.
SV channel/TPC1 in mesophyll cells is neither a Ca2+ channel nor capable of mediating
CICR
Replacing physiological 100 mM K+ by 5 mM Ca2+ on the cytoplasmic side of the vacuolar
membrane in the presence of 50 mM luminal Ca2+, the Vicia faba guard cell SV channel
was shown to mediate cation currents into the vacuole lumen (Ward and Schroeder, 1994).
From the K+ and Ca2+ gradients (Nernst potentials) and reversal of tail currents, a Ca2+/K+
permeability of about 3:1 was calculated for the V. faba guard cell SV channel (Ward and
Schroeder, 1994). Related experiments with non-physiologically high cytosolic Ca2+ loads
(15 mM Ca2+) with isolated vacuoles from cultured Arabidopsis cells showed SV channels
mediating Ca2+ currents into the vacuole (Ivashikina and Hedrich, 2005). However, under
similar conditions neither the SV channels of mesophyll vacuoles from Arabidopsis
thaliana wild type nor from fou2 mutant plants (Bonaventure et al., 2007a, b) conducted
Ca2+ influx into the vacuole (Figure 3c). Under physiological Ca2+ concentrations (10-1000
nM cytosol / 200-2000 µM vacuole) (Bethke and Jones, 1994; Felle, 1988; Pérez et al. ,
2008) together with K + (symmetrical 100 mM; cf. Pérez et al., 2008) SV channel-mediated
Ca2+ fluxes across the vacuolar membrane have not been observed. Thus the hypothesized
capability of the SV channel to mediate CICR (Ca2+-Induced Ca2+ Release; Ward and
Schroeder, 1994), however, seems rather unlikely because at luminal Ca2+ concentrations
under which this channel type is active, Ca2+ release into the cytosol through SV channels
would not result in a pronounced Ca2+ signal anymore. This prediction is further supported
by (i) recent non-invasive Ca2+ flux measurements (Pérez et al., 2008) and (ii) the fact that
in the SV-channel-loss-of-function mutant tpc1-2 and in TPC1 overexpressing plants Ca2+
13
signals in response to the entire set of stimuli known to trigger a rise in cytosolic Ca2+
concentration are wild type-like (Ranf et al., 2008).
Conclusion
The gene expression profile of tpc1-2 when compared to wild type is reminiscent of
potassium starvation (Bonaventure et al., 2007b). Together with the above-mentioned
vacuolar K+ and Ca2+ content and properties of fou2 and wild type SV channel, this
channel type at least in mesophyll cells seems to play a role in the control of vacuolar
membrane potential and potassium homeostasis rather than Ca2+ release from this major
plant organelle. Our data showed that fou2 plants could accumulate higher levels of
vacuolar Ca2+ than wild type before SV channels are blocked and potassium homeostasis is
impaired. SV channels in fou2 activate in response to small changes in membrane potential
already. It is thus very likely that compared to wild type fou2 gains more pronounced
jasmonate biosynthesis from wounding-induced membrane polarization and in turn
elevated vacuolar Ca2+ release - mediated by transporters other than TPC1 - driven by
steeper gradients of this regulatory cation across the vacuolar membrane.
14
Experimental procedures (743 words)
Isolation of mesophyll vacuoles
Arabidopsis thaliana ecotype Columbia (Col-0) and fou2 mutant (Bonaventure et al. ,
2007a) were grown on soil in a growth chamber at a 8/16 h day/night regime, 22/16°C
day/night temperature and a light intensity of 800 lx. For daily enzymatical protoplast
isolation fully developed young rosette leaves of 3-5 week-old plants were used. After
removing the lower epidermis, the leaves were incubated in 0.5% (w/v) cellulase Onozuka
R-10 (Serva, Heidelberg, Germany), 0.05% (w/v) pectolyase Y23 (Seishin Corp., Tokyo,
Japan), 0.5% (w/v) macerozyme R10 (Serva, Heidelberg, Germany), 1% (w/v) bovine
serum albumine (Sigma-Aldrich), 1 mM CaCl2, 10 mM Hepes/Tris (pH 7.4) for 45 min at
23°C and 80 rpm on a rotary shaker. The enzyme solution was adjusted to an osmolality of
400 mosmol kg-1 with sorbitol. Released protoplasts were filtered through a 50-µm nylon
mesh and washed with 400 mM sorbitol and 1 mM CaCl2. After centrifugation at 60 x g
and 4°C for 6 min, the enriched protoplasts were stored on ice until aliquots were used for
vacuole isolation and patch clamp experiments. Upon exposure to hypotonic medium (10
mM EGTA, 10 mM Hepes/Tris pH 7.4 adjusted to 200 mosmol kg-1 with D-sorbitol)
protoplasts bursted and spontaneously released vacuoles.
Electrophysiology
Patch clamp experiments on mesophyll vacuoles were performed in the whole -vacuole and
excised patch configuration essentially as described by Schulz-Lessdorf and Hedrich
(1995) and Ivashikina and Hedrich (2005). Patch pipettes were prepared from Kimax-51
glass capillaries (Kimble products, Vineland, NY, USA) and were covered from the inside
with sigmacoat (Sigma-Aldrich Chemie GmbH, Steinheim, Germany). Close to the tip the
outside of the patch pipette was coated with silicone (Sylgard 184 silicone elastomer kit;
15
Dow Corning GmbH, Wiesbaden, Germany). Pipette resistance was about 5 to 6 MO for
single channel recordings, whereas the pipette resistance for whole vacuole experiments
was about 3 MO in solutions with 100 mM cytosolic potassium and 2 MO in solutions
with 150 mM cytosolic potassium. Macroscopic and single channel recordings were
performed with an EPC-7 patch clamp amplifier (HEKA Lambrecht, Germany) choosing a
data acquisition rate of 500 µs and 50 µs, respectively. The macroscopic currents were
low-pass filtered at 5 kHz and the single channel currents at 1 kHz. Data were digitized by
an ITC-16 interface (Instrutech Corp., Elmont, NY, USA), stored on a Maxdata computer
and analysed using different software programs such as Pulse and PulseFit (HEKA
Elektronik, Lambrecht, Germany), IGORPro (Wave Metrics Inc., Lake Oswego, OR,
USA) and TAC V3.0 (Bruxton Coporation, Seattle, USA). The current recordings were
performed according to the convention for electrical measurements on endomembranes
(Bertl et al., 1992) . The vacuolar membrane was clamped to voltages V as indicated in the
respective figure legends. To allow comparison of macroscopic current magnitudes among
different vacuoles, the current densities (Iss/Cm) were determined upon dividing the
macroscopic ionic current through the whole -vacuolar membrane capacitance Cm of the
individual vacuole as shown in the figures. The relative voltage-dependent open-
probability shown as G(V) curves in Figure 4 was determined from tail current
experiments in the whole vacuolar mode. The derived conductance values G were plotted
against the voltages V, fitted with a Boltzmann distribution using a fixed number of
apparent gating charges (z = 1.6) and normalized with respect to the maximal conductance
(G/Gmax). The half-maximal activation voltage V1/2 was derived from the Boltzmann fit
and represents the voltage at which 50% of the maximal conductance is reached. Single
channel amplitudes (i) were determined from single channel recordings by using the
software program TAC V3.0, and the single channel activity expressed as the single-
channel open probability (Po) was estimated as described by Bertl and Slayman (1990).
16
Patch clamp solutions
Bath and pipette solutions were composed of 2 mM DTT, varied KCl/CaCl2 concentrations
and set to an osmolality of 400 mosmol kg-1 with D-sorbitol. Vacuolar side media
additionally contained 2 mM MgCl2. pH values were adjusted to pH 7.5 or pH 6.5 with 10
mM Hepes/Tris, to pH 5.5 with 10 mM Mes/Tris, and to pH 4.5 with 2 mM citrate/Tris.
The free Ca2+ concentrations for the pipette and bath mediums were calculated with
webmaxc standard (http://www.stanford.edu/~cpatton/webmaxc/webmaxcS.htm). Details
about the composition of the solutions are given in the figure legends.
EDX analysis
Leaves of 7-8 week-old wild type and fou2 plants were quickly frozen to about -175°C in
liquid nitrogen. Cross sections were examined by scanning electron microscopy (SEM, S-
520 Hitachi) equipped with an energy dispersive X-ray device (EDX eumex Si(Li)-
detector, EUMEX GV, Mainz, Germany).
Acknowledgments
The work was funded by the Deutsche Forschungsgemeinschaft to RH (SFB 487). We
thank Petra Dietrich for helpful discussion.
17
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22
Figure legends (1147 words)
Figure 1. Transient increase in the macroscopic steady-state currents of wild type and fou2
SV channels after establishing the whole vacuolar configuration on mesophyll vacuoles of
Arabidopsis thaliana.
The steady-state currents Iss were normalized to the maximal value and plotted against the
time in the whole vacuolar mode. Closed and open symbols represent wild type and fou2,
respectively. Iss were recorded at the following clamped voltages: +70 mV (open
downward triangles, open circles and closed symbols) and +50 mV (open upward triangles,
open squares). For analysis vacuoles of similar sizes reflected by the measured membrane
capacitance Cm (20-30 pF) were chosen. The experiments were performed under
symmetrical KCl (100 mM) und pH 7.5 conditions. The luminal and cytosolic Ca2+
concentrations were 0.1 and 1 mM, respectively.
Figure 2. Semi-quantitative EDX analysis of ion concentrations in mesophyll vacuoles.
Data were obtained from leaves of four wild type as well as four fou2 plants. Columns
(open = wild type, closed = fou2) show mean values with standard deviations of at least 15
recorded spectra. The scale on the left gives the atomic% of recorded X-ray signals. Note
that differences in the relative K+ and Ca2+ concentration were statistically significant (t-
test, P<0.001).
23
Figure 3. Dependency of the voltage-dependent activation of fou2 and wild type SV
channels on the K+ and Ca2+ gradient.
Macroscopic currents were recorded in the whole -vacuolar configuration either with 150
mM K+ and 1 mM Ca2+ (a), with 30 mM K+ and 1 mM Ca2+ (b), or with 0 mM K+ and 15
mM Ca2+ (c) in the bath medium (cytosol) at symmetrical pH 7.5. The luminal solution
(pipette) contained in (a-c) 150 mM K+ and was nominal Ca2+-free. Representative traces
of the current densities (I/Cm) evoked upon 15-mV steps in the voltage range from -50 mV
to +70 mV are shown in the left (wild type) and middle lane (fou2). Corresponding steady-
state current densities were plotted against the voltages and are shown in the right lane.
Please note the different scaling of the current densities for wild type and fou2. Closed
symbols = wild type; open symbols = fou2. Arrows indicate the respective voltage
thresholds of channel activation. Data points represent the mean, and the errors bars give
the standard error. The respective number of experiments for wild type was n=3 in (a-c),
for fou2 n=9 in (a), n=7 in (b) and n=3 in (c).
24
Figure 4. Effect of cytosolic Ca2+ on the voltage-dependent activation of fou2 and wild
type SV cha nnels.
(a) Representative tail currents shown for wild type and fou2 (framed and enlarged on the
right side) were recorded at -60 mV subsequent to +70 mV as a pre-activating voltage
pulse. The holding voltage was -60 mV. Traces at cytosolic Ca2+ concentrations of 1 and
0.3 mM are shown.
(b) The relative voltage-dependent open-probabilities (G(V) curves) were determined upon
tail currents after SV channels were activated at different pre-pulse voltages in the range of
-100 mV to +100 mV. The experiments w ere performed in the presence of 100 mM KCl at
pH 7.5 in the bath and pipette medium. The pipette medium (vacuole lumen) contained 100
µM Ca2+. Open and closed symbols represent wild type and fou2, respectively, at the
following Ca2+ concentrations: squares = 1 mM; triangles = 0.5 mM and circles = 0.3 mM.
(c) The half-activation voltages (V1/2) determined from the Boltzmann fit of the G(V)
curves were plotted against the respective cytosolic Ca2+ concentration.
Number of experiments in (b, c) were nwild type = 7 and nfou2 = 6 for 1 mM Ca2+, nwild
type, fou2 = 4 for 0.5 mM Ca2+, nwild type, fou2 = 3 for 0.3 mM Ca2+ and nwild type, fou2 = 4 for
0.2 mM Ca2+. Error bars show standard error of the mean.
25
Figure 5. Effect of different luminal Ca2+ concentrations on the wild type and fou2 single
channel activity under symmetrical KCl and pH conditions.
(a) Single channel fluctuations were recorded from excised membrane patches with the
cytoplasmic side of the vacuolar membrane facing the bath medium. The membrane
potential was clamped to -30 mV. C indicates the current baseline where all channels are
closed. O 1,2,3 give the current levels at which 1, 2 or 3 channels were simultaneously open.
(b) Single -channel open probabilities (Po) were determined at -50, -40 and -30 mV from
single channel recordings as illustrated in (a). Since the resolution limit for analysis of wild
type single -channel fluctuations was reached at elevated luminal Ca2+, the respective
single -channel open probabilities were set to 0.
The experiments in (a, b) were performed in the presence of 100 mM KCl and pH 7.5 on
both membrane sides. The cytosolic medium (bath) contained 1 mM CaCl2 while the CaCl2
concentration of the luminal medium (pipette) was altered as indicated. The number of
expe riments was n=3-5, and the error bars show standard deviation.
26
Figure 6. Effect of an acidic vacuolar pH on the luminal Ca2+ dependency of the wild type
and fou2 single channel activity.
Single channel recordings were performed at varied luminal Ca2+ concentrations under
asymmetrical pH conditions (cytosol/bath medium: pH 7.5; vacuolar lumen/pipette
medium: pH 5.5).
(a) Current traces were recorded at different Ca2+ levels from excised membrane patches
with the cytoplasmic side of the vacuolar membrane facing the bath medium. The
membrane potential was clamped to -30 mV.
(b) Single-channel open probabilities (Po) were calculated at -50, -40 and -30 mV from
single channel recordings as illustrated in (a). Since the resolution limit for analysis of the
wild type single -channel fluctuations was reached at 1 mM Ca2+, the respective single-
channel open probability was set to 0. With the exception of the luminal pH, the solutions
were composed as in Figure 5. The number of experiments was n=3-4, and the error bars
show standard deviation.
27
Figure 7. Luminal pH-induced changes in the single channel conductance γ of fou2 and
wild type SV channels.
(a) Single channel amplitudes were determined and plotted against the respective voltages.
The single channel conductances γ were derived from the slope of a linear regression
describing the data points. Open and closed symbols represent fou2 and wild type,
respectively. Inset: Representative channel fluctuations for fou2 at pH 7.5 (circles) and 4.5
(squares) recorded at -30 mV are shown. C and O1,2,3 have the same meaning as in Figure
5.
(b) Single channel conductances were determined at different vacuolar pH values as
indicated. Open and closed bars represent fou2 and wild type, respectively.
(a, b) Channel fluctuations were recorded from excised membrane patches with the
cytoplasmic side of the vacuolar membrane facing the bath medium. The experiments were
carried out in the presence of 100 mM KCl on both membrane sides. The cytosolic
medium (bath) also contained 1 mM CaCl2 and was adjusted to pH 7.5 while the luminal
medium (pipette) was nominal Ca2+-free. The data points represent the mean of 3-4
individual experiments. Error bars show standard deviation.
28
Figure 1
1.0
0.8
0.6
0.4
0.2
0.01000800600400200
wild typefou2
norm
aliz
ed I s
s
time in whole vacuolar mode [s]
30
Figure 3
150
100
50
-50
8040-40-80
600
400
200
-80 -40 40
V [mV]
V [mV]
I ss/C
m [p
A/p
F]I s
s/Cm
[pA
/pF]
0.05 s
0.05 s
150
pA/p
F30
0 pA
/pF
0.1 s
150
pA/p
F70 mV
70 mV
55
55
40
40
25
10
-20
fou2
-525
-20
-5-35
-50
150
pA/p
F15
0 pA
/pF
150
pA/p
F
0.1 s
0.1 s
0.1 s
70 mV
55
40
70 mV5540
25
10
wild type(a)
(b)
(c)
31
Figure 4
1.0
0.5
G/G
max
-100 -50 50 100V [mV]
100
50
V1/
2 [mV
]
1.00.80.60.40.2
Ca2+cyt [mM]
(a)
(b)
wild type fou2
300 µM
1 mM
300 µM
1 mM
1 mM
300 µM
50 p
A/p
F
100
pA/p
F
0.05 s0.01 s
300 µM
1 mM
(c)
32
Figure 5
(a)
O1
O2
0 mM Ca2 +
0.1 mM Ca2+
1 mM Ca2 +
CC
O1O2
wild type fou2
CC
O1 O2
C
O1O2
O1
C
O1
O3
O3
pH 7.5vac / pH 7.5cyt
1 pA0.1 s
0.4
0.3
0.2
0.1
0.0
-40 mV-50 mVwild typewild typewild type fou2fou2 fou2
open
pro
babi
lity
P o
-30 mV
0.1 mM Ca
2+
1 mM Ca2+
0 mM Ca2+
(b)
0.4
0.3
0.2
0.1
0.0
-40 mV-50 mVwild typewild typewild type fou2fou2 fou2
open
pro
babi
lity
P o
-30 mV
0.1 mM Ca
2+
1 mM Ca2+
0 mM Ca2+
(b)
33
Figure 6
0.3
0.2
0.1
0.0wild typewild typewild type fou2fou2fou2
-30 mV-40 mV-50 mV
open
pro
babi
lity
P o
0.1 mM Ca
2+
1 mM Ca2+
0 mM Ca2+
(b)
0.3
0.2
0.1
0.0wild typewild typewild type fou2fou2fou2
-30 mV-40 mV-50 mV
open
pro
babi
lity
P o
0.1 mM Ca
2+
1 mM Ca2+
0 mM Ca2+
(b)
(a) pH 5.5vac / pH 7.5cyt
wild type fou20 mM Ca
2+
0.1 mM Ca2+
1 mM Ca2+
0.1 s2 pA
C
C
C
C
CC
O1O1
O1
O1O1
O1
O2O2
O2
O2
O3
O3
O4
34
Figure 7
0 mM Ca2+
vac
γ [p
S]
100
80
60
40
20
0pH 7.5 pH 6.5 pH 5.5 pH 4.5
-6
-5
-4
-3
-2
-1-60 -40 -20 20 40 60
0.1 s
2 pAi [pA
]V [mV]
C
C
O1
O1
O2
O2O3
(a) (b)
0 mM Ca2+
vac
γ [p
S]
100
80
60
40
20
0pH 7.5 pH 6.5 pH 5.5 pH 4.5
-6
-5
-4
-3
-2
-1-60 -40 -20 20 40 60
0.1 s
2 pAi [pA
]V [mV]
C
C
O1
O1
O2
O2O3
(a) (b)
-6
-5
-4
-3
-2
-1-60 -40 -20 20 40 60
0.1 s
2 pAi [pA
]V [mV]
C
C
O1
O1
O2
O2O3
(a) (b)