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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: marten@botanik.uni-wuerzburg.de

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]

29

Figure 2

60

50

40

30

20

10

0

conc

entr

atio

n [a

t %]

Mg P S Cl K Ca

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)