Download Salinity Effects on the Activity of Plasma Membrane H+ and Ca2+

Annals of Botany 85: 681±686, 2000
doi:10.1006/anbo.2000.1131, available online at http://www.idealibrary.com on
Salinity E€ects on the Activity of Plasma Membrane H‡ and Ca2‡ Transporters
in Bean Leaf Mesophyll: Masking Role of the Cell Wall
S E R G E Y SH A B A L A *{ and IA N N E W M A N {
{School of Agricultural Science, University of Tasmania, Australia and {School of Mathematics and Physics,
University of Tasmania, Australia
Received: 15 November 1999 Returned for revision: 6 January 2000
Accepted: 25 January 2000
Net ¯uxes of H ‡ and Ca2‡ were measured in the mesophyll tissue of broad bean (Vicia faba L.) leaves and in
protoplasts derived from these cells. NaCl at 90 mM enhanced H ‡ extrusion in both protoplasts and tissue, but in
di€erent ways. Proton extrusion was inhibited by vanadate, suggesting the involvement of the plasma membrane H ‡ ATPase in cell responses to salinity. There was virtually no e€ect of NaCl on the net Ca2‡ ¯ux in protoplasts, while in
the tissue a large transient Ca2‡ e‚ux followed the salt treatment. Salt-induced Ca2‡ e‚ux was essentially
independent of external Ca2‡ concentrations in the range 0.1 to 10 mM. Also, Ca2‡ ¯ux responses were `saturated'
above 50 mM NaCl. It is suggested that almost all the measured Ca2‡ ¯ux originates from Na ‡ /Ca2‡ and H ‡ /Ca2‡
ion exchange in the cell wall. This conclusion was supported by the results of modelling cation exchange in the cell
# 2000 Annals of Botany Company
wall.
Key words: Salinity, membrane transporters, wall ion exchange, proton, calcium, Vicia faba.
I N T RO D U C T I O N
There is much controversy over the way high levels of
external NaCl a€ect plasma membrane Ca2‡ and H ‡
transporters. Some authors have reported rapid elevation
of cytosolic free calcium, [Ca2‡ ]cyt , in response to NaCl
treatment (Bittisnich et al., 1989; Lynch et al., 1989;
Okazaki et al., 1996), although others attribute these data
to methodological drawbacks of dye loading (Cramer and
Jones, 1996). The origin of these [Ca2‡ ]cyt changes is not
clear. Both Ca2‡ transport through the plasma membrane
and Ca2‡ release from intracellular stores have been
suggested as possible sources (Cramer et al., 1985, 1987;
Lynch et al., 1987; Rengel, 1992). None of the techniques
used so far has included direct measurements of ¯uxes
through the plasma membrane, so an unambiguous conclusion has not been possible. The problem is further
complicated by the possibility that high external Na ‡ may
exchange with Ca2‡ in the cell wall. This exchange may
confound observations of salinity e€ects on the activity of
plasma membrane Ca2‡ transporters (Zidan et al., 1991).
There are also reports suggesting that NaCl stimulates
plasma membrane H ‡ -ATPase activity (Nakamura et al.,
1992; Ayala et al., 1996; Vera-Estrella et al., 1999), which
therefore may enhance H ‡ extrusion through the plasma
membrane. Thus, as well as the above Na ‡ /Ca2‡ interaction, the extruded H ‡ could exchange with cell wall Ca2‡
(Arif and Newman, 1993). To our knowledge, however,
there have been no reports of direct H ‡ ¯ux measurements
under NaCl application to date. Does NaCl really activate
* For correspondence. Fax 613 62262642, e-mail Sergey.Shabala@
utas.edu.au
0305-7364/00/050681+06 $35.00/00
H ‡ extrusion, and to what extent does Ca2‡ /H ‡ wall
exchange mask plasma membrane H ‡ ¯ux kinetics?
To answer these questions, a comparison was made
between NaCl e€ects on the net ¯uxes of H ‡ and Ca2‡
measured from bean leaf mesophyll and from protoplasts
derived from these cells using our high-resolution noninvasive microelectrode ion ¯ux measurement (MIFE)
technique.
M AT E R I A L S A N D M E T H O D S
Net ¯uxes of H ‡ and Ca2‡ from the mesophyll tissue of
broad bean (Vicia faba L. `Coles Dwarf'; Cresswell's Seeds,
New Norfolk, Australia) leaves and from protoplasts
derived from mesophyll cells were measured non-invasively
using ion-selective vibrating microelectrodes. Plants were
grown essentially as described by Shabala and Newman
(1999). Seven to 10 d old leaves were harvested and the
lower epidermis was peeled o€. Leaf segments, 5 8 mm,
were cut and ¯oated ( peeled-side down) on the experimental solution (unbu€ered 0.1 mM CaCl2 ‡ 1.0 mM KCl)
as described previously (Shabala and Newman, 1999). The
cut segment was mounted in a Perspex holder that provided
gentle bending of the plant tissue (to allow a clear view for
electrode positioning; see Shabala and Newman, 1999 for
details). Flux measurements started 4±5 h after segments
were cut, to avoid wounding e€ects.
The protoplast isolation procedure was modi®ed from
Lin and Ehleringer (1997). About 200 mg of peeled leaf
tissue was incubated in the enzyme medium (1% cellulase,
0.1% macerozyme, 0.1 mM CaCl2 , 1.0 mM KCl, 0.4 M
mannitol) for 2.5 h. The protoplast suspension was ®ltered,
centrifuged at 300 rpm for 6 min, resuspended twice, and
# 2000 Annals of Botany Company
Shabala and NewmanÐSalinity and Cell Wall Exchange
R E S U LT S A N D D I S C U S S I O N
H‡
¯ux kinetics
In both mesophyll cells and protoplasts, 90 mM NaCl
treatment caused an immediate shift in net H ‡ ¯ux towards
e‚ux (Fig. 1A). For protoplasts, this newly established H ‡
¯ux value was relatively stable over the period of measurements, while for the mesophyll tissue there was a drift
towards larger e‚ux, becoming steady after about 50 min.
The magnitude of NaCl-induced H ‡ e‚ux ( per unit surface
area) at the end of the 60 min interval was four±®ve times
larger for mesophyll tissue than for protoplasts (Fig. 1A).
One reason for this di€erence is the di€erent geometry of a
single protoplast surface compared with a tissue surface for
which several cells contribute to the external ¯ux. However,
Net H+ flux (nmol m−2 s−1)
40
0
−40
−80
Protoplast
90 mM
NaCI
−120
A
Tissue
−160
−200
0
5
10 15 20
25 30 35
40 45 50 55 60
Time (min)
100
Net Ca2+ flux (nmol m−2 s−1)
then measured in enzyme-free bathing solution (0.1 mM
CaCl2 , 1.0 mM KCl, 0.4 M mannitol). A similar bath
composition (except mannitol) was used in experiments
with mesophyll tissue. In some experiments, Ca2‡ composition in the bath varied between 0.1 and 10 mM (added as
CaCl2 salt); K ‡ concentration in the bath remained
constant (1.0 mM). NaCl treatment was given as 1M stock
added into the 5 ml chamber and mixed to produce the
required ®nal concentration of NaCl. In experiments with
protoplasts, the bathing solution was replaced by one
additionally containing 90 mM NaCl. The period of time
required for solution changes was omitted from the analysis
and appears as a gap in our ®gures.
Speci®c details of microelectrode ion ¯ux measurements
using the MIFE2 system (University of Tasmania, Hobart,
Australia) have been given previously (Shabala et al., 1997,
1998; Shabala and Newman, 1999). Brie¯y, pulled and
silanized electrode blanks, with external tip diameter of 3±
4 mm, were ®lled with commercially available H ‡ Ð(95297)
or Ca2‡ Ð(21048) ionophore cocktails (Fluka Chemie AG,
Buchs, Switzerland). Back ®lling solutions were 500 mM
CaCl2 for calcium, and 15 mM NaCl plus 40 mM KH2PO4
( pH adjusted to 6.0 using NaOH) for H ‡ . Electrodes were
calibrated in a known set of standards (calcium from 50 to
500 mM; pH from 4.5 to 8) twiceÐwithout NaCl (used to
calculate net ion ¯uxes before salt treatment), and with
90 mM NaCl present in each standard (used to calculate
¯uxes after salt was applied). In both types of standards,
the average electrode slopes were 53±54 mV/pH and
27±28 mV/pCa. If a range of NaCl concentrations was
used (Fig. 3), an appropriate correction was made to
compensate for the changed Ca2‡ activity due to the
changed ionic strength of solution. Electrodes were moved
in a slow (10 s cycle) square-wave by a computer-driven
hydraulic micromanipulator between two positions, close to
(10 mm) and distant from (60 mm) the surface. Net ion ¯uxes
were calculated using the MIFE software as described
previously (Shabala et al., 1997) from the measured di€erence in electrochemical potential for these ions between the
two positions using the cylindrical (mesophyll segments) or
spherical ( protoplasts) di€usion geometry.
Signi®cance of di€erence between means was based on
the use of the Student's t-test.
Protoplast
0
−100
−200
90 mM
NaCI
−300
Tissue
B
−400
0 3 6 9 12 15
−500
−600
0
5
10 15 20
25 30 35 40 45 50 55 60
Time (min)
Net H+ flux
682
90 mM NaCl
500 µM
vanadate
0
5
10 15 20
C
25 30 35
30 nmol m−2 s−1
40 45 50 55 60
Time (min)
F I G . 1. Transient changes in the net H ‡ (A) and Ca2‡ (B) ¯uxes in
response to 90 mM NaCl treatment. (s), Mesophyll tissue; (d),
protoplasts. Each point represents average ¯ux over a 30 s interval.
Error bars are + s.e.m. (n ˆ 6). The insert in B shows initial changes
(at 5 s intervals) in the net Ca2‡ ¯ux from one representative plant. C,
Net H ‡ ¯uxes from a mesophyll cell protoplast after 500 mM vanadate
pretreatment. Vanadate was added as Na2VO4 stock solution at 5 min;
salt treatment was given at 28 min.
it is also likely that the protoplast isolation procedure per se
a€ected quantitative characteristics of the measured H ‡
¯uxes. There is a large body of literature reporting that
plasma membrane characteristics of isolated protoplasts are
very di€erent from those of intact cells (Racusen et al.,
1977; Pantoja and Wilmer, 1986; Henriksen et al., 1996).
The most striking is a di€erence in membrane potential
(MP). While typical MP values for higher plant cells are
found to be in the range ÿ100 to ÿ140 mV, for isolated
protoplasts this value is usually close to zero, or even
positive (Racusen et al., 1977). Based on the fact that
electrical gradients at the plasma membrane are normally
Shabala and NewmanÐSalinity and Cell Wall Exchange
maintained by the activity of the electrogenic H ‡ pump, H ‡
extrusion may be diminished in protoplasts following the
isolation procedure. If this is the case, NaCl-induced activation of H ‡ -extrusion also may be diminished in protoplasts
(Fig. 1A). Another point to remember is a strong dependence of protoplast electrophysiological characteristics on
external osmoticum (Pantoja and Willmer, 1986; Shabala
et al., 1998). As the choice of osmolality of the bath
medium is usually empirically-based, even slight deviation
from the `optimum' could cause mechanical tension on the
plasma membrane and therefore modify activity of membrane transporters (Ding and Pickard, 1993).
As already mentioned in the introduction, there are many
reports suggesting that NaCl could stimulate H ‡ -ATPase
activity at the plasma membrane of plant cells (Nakamura
et al., 1992; Ayala et al., 1996; Vera-Estrella et al., 1999).
To test if such H ‡ -pump activity is responsible for the saltinduced e‚ux of H ‡ observed in our experiments, 500 mM
vanadate (a known inhibitor of plasma membrane
H ‡ -ATPase; Lew and Spanswick, 1984) was added to the
protoplast bathing medium. As expected, such vanadate
treatment resulted in suppression of H ‡ pumping and
caused a signi®cant shift towards net H ‡ in¯ux (Fig. 1C).
What is even more important, vanadate treatment also
prevented NaCl-induced H ‡ extrusion (compare Fig. 1A
and C). These results suggest that the ATP-dependent
H ‡ -pump (inhibited by vanadate) forms a signi®cant part
of the H ‡ extrusion mechanism involved in response to salt
stress. Its level of activity is about ÿ50 nmol m ÿ2 s ÿ1 for
the protoplast membrane, and about ÿ170 nmol m ÿ2 s ÿ1
for mesophyll tissue surface. Initially this mesophyll H ‡
¯ux exchanges in the cell wall for Ca2‡ , as discussed below,
stabilizing only after that exchange is completed.
Ca2‡ responses
Even more striking was the di€erence between tissue and
protoplast Ca2‡ ¯ux responses (Fig. 1B); moreover, the
observed di€erence was qualitative, not only quantitative
(as in case of the H ‡ ¯ux). While mesophyll tissue
responded to the NaCl treatment by an immediate large
net Ca2‡ e‚ux, with the peak Ca2‡ e‚ux at 2±4 min after
NaCl treatment started (see insert to Fig. 1B), there were
virtually no changes in the net Ca2‡ ¯ux measured from the
protoplast surface. These data suggest that the presence of
the cell wall was crucial for the measured NaCl-induced
Ca2‡ ¯uxes at the tissue level. This is a strong indication
that observed Ca2‡ e‚ux originated from the cation/
calcium exchange in the cell wall, but not from the activity
of plasma membrane Ca2‡ transporters per se.
To verify this hypothesis, mesophyll tissue was treated
with 100 mM La3‡ , a known inhibitor of plasma membrane
Ca2‡ channels. It is known that Ca2‡ in¯ux into the cell is
mediated by Ca2‡ -permeable ion channels which facilitate
the rapid movement of Ca2‡ down its electrochemical
gradient (White, 1998), while Ca2‡ movement out of the
cell requires active transport mechanisms such as a Ca2‡
pump. Therefore, the net Ca2‡ ¯ux measured in our experiments represents a balance between these two oppositely
directed processes.
100 µM
La3+
90 mM
NaCl
A
Net Ca2+ flux
0
683
10
20
200 nmol m−2 s−1
30
40
50
60
70
80
90
Time (min)
B
90 mN
KCl
Net Ca2+ flux
200 nmol m−2 s−1
0
5
10
15
20
25
30
35
40
45
50
55
60
Time (min)
F I G . 2. A, E€ect of 100 mM La3‡ on net Ca2‡ ¯ux from bean
mesophyll tissue. La3‡ was added as LaCl3 stock solution at 5 min;
90 mM NaCl treatment was given at 30 min. B, KCl-induced kinetics of
net Ca2‡ ¯ux from bean mesophyll tissue. One representative example
out of four is shown.
When Ca2‡ channels were blocked by La3‡ in our
experiments (Fig. 2A), a signi®cant shift towards net Ca2‡
e‚ux (up to ÿ30 nmol m ÿ2 s ÿ1 30 min after La3‡ treatment was given) was observed, in full agreement with the
above statement. However, blocking the Ca2‡ channels
with La3‡ had little e€ect on the NaCl-induced Ca2‡ ¯ux
transient (Fig. 2A). Neither the qualitative course of Ca2‡
¯ux responses, nor the magnitude of the peak Ca2‡ e‚ux
were signi®cantly di€erent from the control (at the P ˆ 0.05
level). Therefore, we conclude that most of the net Ca2‡
e‚ux measured from NaCl-treated bean mesophyll tissue
originated from the cell wall and not from the activity of
plasma membrane Ca2‡ transporters. Hence, we suggest
that NaCl-induced changes in cytosolic free Ca2‡ reported
elsewhere (Okazaki et al., 1996; Cramer and Jones, 1996)
are likely to have originated from Ca2‡ released from
internal stores. This conclusion is in good agreement with
other reports where indirect methods were employed to
evaluate the origin of [Ca2‡ ]cyt changes (Lynch et al., 1987).
Further evidence supporting a cell wall origin of the
measured Ca2‡ e‚ux came from experiments when
di€erent NaCl concentrations were used to induce ¯ux
responses from the mesophyll tissue (Fig. 3). No signi®cant
di€erence was found in the magnitude of the Ca2‡ ¯ux
responses to a wide range of NaCl concentrations ( from
50 to 120 mM; Fig. 3). A possible explanation for such
`saturation' kinetics could be that at concentrations above
50 mM most of the exchangeable cell wall Ca2‡ has been
replaced by Na ‡ and H ‡ ions, and further increase in
684
Shabala and NewmanÐSalinity and Cell Wall Exchange
100
100
0
0
Net Ca2+ flux (nmol m−2 s−1)
−100
−100
20 mM
−200
−200
−300
−300
−400
−400
−500
−500
−600
−600
−700
50 mM
−700
3
5
7
9
11
13
15
3
100
100
0
0
−100
−100
90 mM
−200
5
−300
−400
−400
−500
−500
−600
−600
−700
9
11
13
15
150 mM
−200
−300
7
−700
3
5
7
9
11
13
15
3
5
7
9
11
13
15
Time (min)
F I G . 3. Transient changes in the net Ca2‡ ¯uxes in response to di€erent NaCl treatment in bean mesophyll segments. Average data from ®ve to
seven individual segments are shown for each variant. Each point represents average ¯ux over a 5 s interval. Error bars are + s.e.m.
external Na ‡ concentration had little e€ect on this process.
If plasma membrane transporters were involved, a rising
dose-dependent response would be expected.
Also small was the e€ect of variation in the external Ca2‡
concentrations on the NaCl-induced Ca2‡ e‚ux. Figure 4
shows typical records obtained for three di€erent Ca2‡
concentrations in the bath (0.1 mM, 1 mM and 10 mM,
respectively). No signi®cant di€erence was found at the
P ˆ 0.05 level, indicating that activity of plasma membrane
(PM) Ca2‡ transporters contributed insigni®cantly to the
observed kinetics of the net Ca2‡ ¯uxes in response to salt
treatment.
The peak value of the Ca2‡ e‚ux was roughly
ÿ570 nmol m ÿ2 s ÿ1 when 90 mM NaCl was applied
(Fig. 1B). The steady level of H ‡ e‚ux at the end of the
transient (when H ‡ exchange with wall Ca2‡ was completed) was only ÿ170 nmol m ÿ2 s ÿ1. As two protons
exchange for one Ca2‡ in the cell wall, only 15% of the
observed peak Ca2‡ e‚ux can be explained by extruded H ‡
exchanging for wall Ca2‡ . The rest of the Ca2‡ ¯ux due to
NaCl treatment must be attributed to displacement of wall
Ca2‡ by sodium ions. This Ca2‡ displacement from the cell
wall is not speci®c to the Na ‡ ion. We have found that
90 mM NaCl
0.1 mM
1.0 mM
10 mM
200 nmol m−2 s−1
0
10
20
30
40
50
60
Time (min)
F I G . 4. Transient Ca2‡ ¯ux responses to 90 mM NaCl treatment for
di€erent Ca2‡ concentrations in the bath. One representative example
(out of ®ve) is given for each variant. Each point represents average
¯ux over a 30 s interval. (s), 0.1 mM Ca2‡ in the bath; (d), 1.0 mM
Ca2‡ ; (h), 10 mM Ca2‡ .
90 mM KCl induces Ca2‡ e‚ux very similar to that caused
by 90 mM NaCl (Fig. 2B).
Shabala and NewmanÐSalinity and Cell Wall Exchange
685
T A B L E 1. Comparison between WADM modelled ion concentrations in the cell wall, amount of the wall Ca2‡ exchanged due
to solution change, and the observed net Ca2‡ e‚ux measured in experiments with di€erent concentrations of NaCl applied
to the leaf mesophyll (Fig. 3)
External Na ‡ concentration, mM
Parameter
0
Donnan potential of the wall, VW (mV)
Wall pH
Wall Na‡ concentration (mM)
Wall Ca2‡ concentration (mM)
Amount of Ca2‡ exchanged (mM)
Ca2‡ ¯ux changes* 2 min after salt stress
(nmol m ÿ2 s ÿ1)
ÿ95
3.87
0
329
N/A
N/A
20
50
90
120
ÿ76
4.19
435
148
182
ÿ500
ÿ59
4.48
534
117
212
ÿ550
ÿ47
4.69
563
114
215
ÿ570
ÿ37
4.86
588
114
215
ÿ580
Parameters for the model and external ion concentrations are given in the text.
*Measured experimentally as shown in Fig. 3.
Modelling of wall ion exchange
Further support for the cell wall origin of the measured
Ca2‡ ¯ux induced by NaCl treatment comes from modelling the Na ‡ -induced exchange of ions in the cell wall using
the Weak Acid Donnan Manning (WADM) model
described by Ryan et al. (1992). This model incorporates
three main components.
(1) The pH-dependent dissociation of weak acids (of
assumed pK) ®xed in the wall, described by the
Henderson-Hasselbalch equation:
Adiss
10… pHW ÿpK†
ˆ
;
Asites 1 ‡ 10… pHW ÿpK†
where Asites ˆ HA ‡ Adiss is the total concentration of
ionizable sites in the wall at pH pHW . We assumed
Asites ˆ 800 mM, and pK ˆ 3 for this study (Arif and
Newman, 1993).
(2) The classical Donnan equilibrium between the wall
(with concentration of available ®xed charges A) and the
external solution, described by the Donnan potential
VW of the wall, which relates the wall and external
concentrations of each ion. If the concentration (strictly
activity) ratio, wall/external, of an ion of valency z is x,
then
VW ˆ …RT=zF†1n…x†
(3) The special character of divalent Ca2‡ in binding to
available weak acid anions is called `condensation'
in the WADM model. The Ca2‡ which binds thereby
reduces the anions available for the Donnan equilibrium from Adiss to A, according to the equation
A ˆ Adiss (0.5/x), where x is a linear charge density
parameter (see Richter and Dainty, 1990, who found a
value of 0.7 for x). Under our conditions the overall
e€ect of this binding is 520% of the calculated results
and does not a€ect their qualitative validity.
The above equations, with overall charge balance and use
of activity coecients with ion concentrations, were solved
iteratively to determine the equilibrium concentrations of
all ions in the cell wall. This was done for the pre-treatment
solutions (0.1 mM CaCl2 ; 1 mM KCl; pH 5.5) and for each
solution having increased Na ‡ concentration ( from 20 to
120 mM). Some results of this modelling are shown in
Table 1.
Application of non zero external Na ‡ concentrations
caused a signi®cant increase in the cell wall pH (up to
1.0 pH for 120 mM NaCl; Table 1) and a dramatic threefold decrease in the wall Ca2‡ concentrations. However, the
amount of exchanged Ca2‡ plateaued for external Na ‡
concentrations larger than 50 mM. The model predicts,
therefore, that the amount of Ca2‡ exchanged in the cell
wall for two di€erent NaCl concentrations, 50 and 120 mM,
will not be signi®cantly di€erent. Being displaced from the
cell wall, this exchangeable Ca2‡ will be measured as net
Ca2‡ ¯ux by the microelectrode technique. Therefore, if no
PM transporters contribute to the Ca2‡ ¯ux, no di€erence
between net Ca2‡ ¯uxes for 50 mM and 120 mM NaCl
treatments is expected. This is exactly what was found in
our experiments (Fig. 3; Table 1).
Physiological implications
An important conclusion from these observations is that
NaCl-induced changes in cytosolic free Ca2‡ reported
elsewhere (Cramer and Jones, 1996; Okazaki et al., 1996)
are likely to have originated from Ca2‡ released from
internal stores, but not from the activity of the PM Ca2‡
transporters. Further study should be aimed at elucidating
the signalling pathways involved in this process. Also,
displacement of Ca2‡ from the cell wall not only confused
assessment of the activity of plasma membrane Ca2‡
transporters in the mesophyll cells, but also masked the
actual magnitude and timing of Na ‡ -stimulated H ‡ ¯ux
from the tissue. Only when Ca2‡ transients are complete
(40±50 min after the start of NaCl treatment), does net H ‡
e‚ux represent the actual H ‡ ¯ux through the plasma
membrane. The cell wall ionic exchange, which can be
quantitatively modelled, must be kept in mind when
mechanisms of salt stress perception are studied.
686
Shabala and NewmanÐSalinity and Cell Wall Exchange
AC K N OW L E D G E M E N T S
This work was supported by the Australian Research
Council grants to Dr Sergey Shabala and Dr Ian Newman.
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