Journal of Experimental Botany, Vol. 54, No. 383, pp.

657661, February 2003

DOI: 10.1093/jxb/erg072

RESEARCH PAPER

Potassium activities in cell compartments of salt-grown

barley leaves

Tracey Ann Cuin1, Anthony J. Miller2, Sophie A. Laurie3 and Roger A. Leigh1,4
1
Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, UK
2
Agriculture and Environment Division, Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK
3
Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Long Ashton, Bristol
BS18 9AF, UK

Received 6 June 2002; Accepted 3 October 2002

Downloaded from http://jxb.oxfordjournals.org/ by guest on September 24, 2013

Abstract limitation to crop productivity because the majority of crop
+ plants are not particularly salt tolerant (Flowers and Yeo,
Triple-barrelled microelectrodes measuring K activity
1995). Therefore, much effort is being directed towards
(aK), pH and membrane potential were used to make
identifying the physiological and biochemical processes
quantitative measurements of vacuolar and cytosolic
that are affected by NaCl with the aim of modifying these
aK in epidermal and mesophyll cells of barley plants
to increase salt tolerance (Apse and Blumwald, 2002).
grown in nutrient solution with 0 or 200 mM added
Compartmentation of Na+ and Cl in the vacuole is
NaCl. Measurements of aK were assigned to the
considered to be important in ameliorating the effects of
cytosol or vacuole based on the pH measured. In
salt on plants, and increasing Na+ transport into the vacuole
epidermal cells, the salt treatment decreased aK in
has been shown to increase salt tolerance (Apse et al.,
the vacuole from 224 to 47 mM and in the cytosol
1999). In addition, the maintenance of a high cytosolic
from 68 to 15 mM. In contrast, the equivalent changes
K+:Na+ ratio is also considered important (Maathuis and
in the mesophyll were from 235 to 150 mM (vacuole)
Amtmann, 1999). Thus, the capacity of plants to counter-
and 79 to 64 mM (cytosol). Thus mechanisms exist to
act salinity stress may strongly depend on their ability to
ameliorate the effects of salt on aK in compartments
compartment ions in the vacuole and to minimize ionic
of mesophyll cells, presumably to minimize any
changes in the cytosol.
deleterious consequences for photosynthesis.
Although many studies have conrmed the sequestra-
Thermodynamic calculations showed that K+ is
tion of Na+ and Cl in the vacuole (Storey et al., 1983;
actively transported into the vacuole of both epider-
Fricke et al., 1996), the behaviour of ions in the cytosol has
mal and mesophyll cells of salinized and non-
been more difcult to determine because the small volume
salinized plants. Comparison of the values of aK in
of this compartment makes quantitative measurements
K+-replete, non-salinized leaf cells with those previ-
technically difcult. Those approaches that have been
ously measured in root cells of plants grown under
used, such as X-ray microanalysis (Storey et al., 1983;
comparable conditions indicates that cytosolic aK is
Flowers and Hajibagheri, 2001) and compartmental tracer
similar in cells of both organs, but vacuolar aK in leaf
efux analysis (Yeo, 1981), generally support the hypoth-
cells is approximately twice that in roots. This sug-
gests differences in the regulation of vacuolar aK, but
esis that maintenance of the cytosolic K+:Na+ ratio is
not cytosolic aK, in leaf and root cells.
important in salt tolerance. However, none of these
techniques is fully quantitative and so accurate estimates
Key words: Barley, cytosol, potassium, salinity, vacuole. of cytosolic ion concentrations in salinized plant cells are
still lacking. In particular, measurements are needed in leaf
cells because adverse effects of salinity in these are likely
to have signicant consequences for plant productivity.
Introduction
The lack of such measurements represents a signicant
Soil salinity is estimated to affect between 340 and 950 impediment to testing hypotheses about responses of plants
million hectares of land worldwide and is a serious to salt and hence to identifying targets for manipulation.
4
To whom correspondence should be addressed. Fax: +44 (0)1223 333953. E-mail: RL225@cam.ac.uk

Journal of Experimental Botany, Vol. 54, No. 383, Society for Experimental Biology 2003; all rights reserved
658 Cuin et al.
Another confounding aspect is that different cell types in
leaves can be quite distinct in the ionic composition of
their vacuoles (Leigh and Tomos, 1993; Karley et al.,
2000), raising the possibility that cytosolic ion concentra-
tions may also vary between cells, or may respond
differently to salinity in different cell types. Differential
responses of cytosolic K+ in epidermal and cortical cells of
K+-decient barley roots show that this is a real possibility
(Walker et al., 1996).
Triple-barrelled microelectrodes measuring K+ activity
(aK), pH, and membrane potential (Em) (Walker et al.,
1995, 1996) can be used to make quantitative intracellular
measurements of aK in the vacuole and cytosol of cells
in situ. The inclusion of the pH-sensing barrel allows
assignment of aK values to either the vacuole or the cytosol
(Walker et al., 1995). In the study described here, such
electrodes were used to measure quantitatively aK in the
vacuole and cytosol of leaf cells of barley plants grown in
nutrient solutions with or without added NaCl. The results
show that there is regulation of cytosolic aK in response to
salinity, but this is restricted to mesophyll cells; cytosolic
aK declines to very low levels in epidermal cells, although
these remain viable. Comparison of the results with
published data (Walker et al., 1996) indicates important
similarities and differences in the regulation of cytosolic
and vacuolar aK in K+-replete leaf and root cells.
Materials and methods
Plant material and whole-tissue measurements
Barley (Hordeum vulgare L. cv. Gerbel) seeds were germinated and
grown in a modied Hoagland's nutrient solution containing 5 mM
K+ (Walker et al., 1996). Plants were grown at a constant
temperature of 20 C with a photoperiod of 16 h, a photon ux
density of approximately 300 mmol m2 s1 at plant height, and a
relative humidity of 75%. Plants were grown in unamended nutrient
solution for the rst 7 d after germination and then the NaCl
concentration in the nutrient solution of some of the plants was
increased by 50 mM d1 to a nal concentration of 200 mM. When
treated in this way, the cultivar used, Gerbel, is relatively salt
resistant (Flowers and Hajibagheri, 2001). All measurements were
made on the third leaf from 21-d-old plants. This leaf had emerged
and expanded after the salt treatment was imposed, and its responses
to salt are similar to those of other leaves in barley (Greenway,
1962). The fresh weight and dry weight of the whole shoot and the
third leaf were recorded for both control and salt-treated plants.
Tissue was dried at 80 C for 48 h. Measurements of tissue K+ and
Na+ concentrations were made by atomic absorption spectro-
photometry on sap extracts prepared according to Fricke et al.
(1994) from 1 cm leaf sections taken from the region where
measurements were made with microelectrodes. Tissue ion concen-
trations are expressed as mM in tissue water.
Electrophysiology
Triple-barrelled microelectrodes measuring aK, pH and Em were
made and calibrated as described previously (Walker et al., 1995)
except that the K+ sensor contained dibutyl sebacate as the
plasticizer (Cuin et al., 1999). Potassium concentrations in calibra-
tion solutions were converted to aK using activity coefcients of
between 0.75 and 0.78 (Robinson and Stokes, 1959; Walker et al.,
1995). Electrodes were inserted into cells half-way along the length
of the third leaf of intact plants. Leaves were mounted on a Perspex
holder with their lower edge touching a reservoir of dilute nutrient
solution that also contained the reference bath electrode (Miller et al.,
2001). This arrangement ensured that an electrical circuit was
completed when the triple-barrelled microelectrode was inserted into
the leaf. Leaves were placed on the holder at least 30 min before the
measurements were made, and stomates remained open throughout
the experiment. Impalements were made in cells of the `trough'
region of the upper epidermis (Fricke et al., 1995; Fricke, 1997) and
on the rst layer of mesophyll cells immediately beneath these.
Mesophyll cells were accessed by pushing the electrode through an
epidermal cell. Successful penetration was monitored by following
the Em, which became more negative when the electrode was within
a cell. Values of aK are expressed as mean and 95% condence limits
(Fry et al., 1990).
Results
Treatment of the barley plants with NaCl adversely
affected growth and signicantly changed leaf ion con-
centrations. Compared with control (non-salinized) plants,
the fresh weights of the whole shoot and third leaf
decreased by 47% and 35%, respectively, in plants grown
in 200 mM NaCl (Fig. 1A). This change was accompanied
by a decrease in leaf K+ concentration and an increase in
leaf Na+ concentration (Fig. 1B). These results are
comparable with those reported by Flowers and
Hajibagheri (2001) for the same barley variety grown
under similar conditions.
Measurements of aK were assigned to the vacuole or the
cytosol on the basis of the accompanying pH measure-
ments. In both epidermal and mesophyll cells, impale-
ments yielded two populations of pH measurements with
mean values of 7.1 and 5.1 (Table 1), which were taken to
indicate insertions in the cytosol and vacuole, respectively
(Walker et al., 1995, 1996). Compartmental pH values
were not statistically different between epidermal and
mesophyll cells, and were also unaffected by salinity. The
mean values of Em ranged from 83 to 91 mV in cells of
non-salinized plants, but were 16 to 28 mV less negative in
salinized plants, with the greatest effect on epidermal cell
Em (Table 1). There were no statistically-signicant
differences between Em for impalements into the cytosol
and the vacuole, suggesting that the Em across the
tonoplast was close to zero. This contrasts with
Arabidopsis where Miller et al. (2001) measured a trans-
tonoplast Em of about 30 mV (cytosol with respect to the
vacuole; Bertl et al., 1992).
In non-salinized plants, mean vacuolar aK was 224 and
235 mM in epidermal and mesophyll cells, respectively,
while the corresponding values in the cytosol were 68 and
79 mM (Table 2). Following growth in 200 mM NaCl, the
mean vacuolar aK in epidermal cells declined to 47 mM
and aK in the cytosol of these cells to 15 mM. In contrast,
vacuolar aK in mesophyll cells decreased only to 150 mM,
while, in the cytosol, aK declined to 64 mM.

Discussion
As far as is known, this paper reports the rst quantitative
measurements of cytosolic aK in leaf cell compartments of
plants subjected to salt stress. Overall, the results indicate
that both vacuolar and cytosolic aK in the mesophyll are
Fig. 1. Fresh weights of the whole shoot and third leaf (A) and ion
concentrations in the third leaf (B) of 21-d-old barley plants grown in
nutrient solution containing 0 mM or 200 mM added NaCl. Results
are mean 6SD (n=9).
Potassium in cell compartments of barley 659
maintained at the expense of the supply to the epidermis.
The results are consistent with the observations by Fricke
et al. (1996) who found, using single-cell sampling, that
decreases in K+ concentrations and increases in Na+
concentrations were less in vacuoles of mesophyll cells
than in those of epidermal cells when plants were grown at
NaCl concentrations of 100 mM or more. This indicates
that mechanisms exist to ameliorate ionic changes in leaf
mesophyll cells, but not in epidermal cells. This may be a
device to protect and maintain the photosynthetic activity
of the mesophyll cells. However, since the fresh weight of
the third leaf declined by 35% in the salt-treated plants,
other changes must have affected growth detrimentally.
For instance, it is possible that the decreases in aK in
epidermal cell compartments may ultimately be affecting
some fundamental growth process. Testing the importance
of changes in subcellular ion activities in response to
salinity will require investigation to relate the ion activities
to the rates of key physiological processes, as done for
cytosolic aK and protein synthesis in K+-decient barley
roots (Walker et al., 1998).
The differential behaviour of mesophyll and epidermal
cells is similar to that seen in K+-decient barley roots
where cytosolic aK declined to a much greater extent in
epidermal cells than in cortical cells (Walker et al., 1996).
These collective observations indicate that ion supplies to
key cell types in both leaves and roots are protected in
order to maintain their function in response to deciencies
or excesses of ions. How this is achieved in leaves is
unclear, but the observation is consistent with work
showing that epidermal and mesophyll cells have quite
distinct ionic compositions (Leigh and Tomos, 1993;
Karley et al., 2000). It seems clear that supply and/or
retention of ions in different cell types is important in leaf
function and in responses to ionic stresses.
Leigh and Wyn Jones (1984) proposed that cytosolic K+
concentrations are buffered by the mobilization of
vacuolar K+. Measurements of cytosolic and vacuolar aK
in barley roots grown under a range of external K+
concentrations supported this contention, with cytosolic
aK declining only when the vacuolar aK fell below about
25 mM (Walker et al., 1996). This does not seem to occur

Table 1. Vacuolar and cytosolic pH and membrane potentials (Em) in `trough' cells of the upper epidermis and in mesophyll cells
of the third leaf of 21-d-old barley plants grown in nutrient solution containing 0 or 200 mM added NaCl
Values of Em were all measured with respect to the bath solution in which the lower edge of the leaf was immersed (see Materials and
methods). Results are given as mean 6SD. The number of separate measurements is given in brackets.
NaCl (mM) Epidermis Mesophyll

Vacuole Cytosol Vacuole Cytosol

pH Em (mV) pH Em (mV) pH Em (mV) pH Em (mV)

0 5.160.4(19) 86610(21) 7.260.2(10) 8369(10) 5.060.3(9) 87615(10) 7.060.1(11) 91615(12)
200 5.160.2(12) 58610(13) 7.060.2(9) 5768(12) 5.060.4(7) 71619(7) 7.260.1(8) 64616(10)
660 Cuin et al.
Table 2. Compartmental aK in `trough' cells of the upper epidermis and in mesophyll cells of the third leaf of 21-d-old barley
plants grown in nutrient solution containing 0 or 200 mM added NaCl
Results are given as mean and 95% condence limits (Fry et al., 1990). The number of separate measurements is indicated in square brackets.
NaCl (mM) Compartmental aK (mM)
Epidermis
Vacuole Cytosol
0 224 (259, 193) [8] 68 (77, 61) [6]
200 47 (55, 25) [7] 15 (20, 11) [9]
in leaf epidermal cells of salt-grown barley where
cytosolic aK declined to 15 mM despite the vacuole still
containing about 50 mM K+ (Table 2). Why the vacuolar
pool is not fully mobilized to maintain cytosolic aK is
unclear. It is unlikely to be due to the existence of a
minimum K+ concentration below which the vacuolar pool
cannot decline (as proposed by Leigh and Wyn Jones,
1984) because very low K+ concentrations can be meas-
ured in sap extracted from epidermal vacuoles of leaves of
barley plants grown in 150 mM NaCl (Fricke et al., 1996).
Therefore, it seems more likely that the maintenance of a
high cytosolic aK is not important in these cells, and so it
declines, despite the availability of vacuolar K+. Fricke
et al. (1996) observed large differences in K+ concentra-
tions in vacuolar sap extracts from different epidermal
cells of salt-grown leaves, so it is important that other cells
are investigated in order to determine whether the
behaviour of vacuolar and cytosolic aK observed in
upper epidermal `trough' cells is typical of all epidermal
cells.
At 15 mM, the cytosolic aK in the `trough' epidermal
cells from the salt-grown plants was at levels considered
inadequate to support key K+-dependent functions such as
protein synthesis (Lubin and Ennis, 1964; Leigh and Wyn
Jones, 1984; Walker et al., 1998). It therefore seems
unlikely that these cells are particularly metabolically
active. Nonetheless, they appear to retain their viability, as
evident by (1) a relatively negative Em, indicating that their
plasma membrane is intact and ion transporters function-
ing; (2) a near-neutral cytosolic pH, which, at the measured
values of Em and a typical leaf apoplastic pH of about 5.5
(Muhling and Lauchli, 2000), could only be maintained by
active H+ extrusion; and (3) a turgor of approximately 0.9
MPa with correspondingly high sap osmotic pressure and
ion concentrations (Fricke, 1997; Fricke et al., 1996).
Thus, cytosolic aK values below those normally considered
optimal for protein synthesis do not necessarily lead to a
loss of viability of epidermal cells.
The compartment-specic values of Em, pH and aK
obtained with the triple-barrelled microelectrodes provide
sufcient information to make thermodynamic calcula-
tions of the direction of active K+ transport at the tonoplast,
and the feasibility of different active transport mechanisms
Mesophyll
Vacuole Cytosol
235 (259, 213) [7] 79 (91, 68) [7]
150 (175, 128) [8] 64 (75, 55) [8]
(see Walker et al., 1996, for equations). These calculations
(results not presented) show that for both epidermal and
mesophyll cells, under both sets of growth conditions, K+
is always actively transported into the vacuole. Two
possible mechanisms for active transport into the vacuole
have been proposed, a 1:1 K+-H+ antiport (Hassidim et al.,
1990) and the vacuolar K+-dependent, H+-transporting
inorganic pyrophosphatase (Davies et al., 1992).
Calculations of the thermodynamic feasibility of each of
these indicates that both are possible mechanisms in barley
leaf cells (results not shown). Therefore, it seems that
salinization of barley leaf cells does not have any obvious
effect on the direction or mechanism of active K+ transport
at the tonoplast. This contrasts with K+-deprivation in
barley roots which changes the direction of active K+
transport from into the vacuole in K+-replete conditions to
into the cytosol in K+-deciency (Walker et al., 1996). It
was not possible to make similar calculations for active K+
transport at the plasma membrane of leaf cells because aK
in the apoplast of control and salinized leaves was not
measured.
Walker et al. (1996) showed that cytosolic and vacuolar
aK in K+-replete barley root cells are about 80 mM and
120 mM, respectively, with little difference between
epidermal and cortical cells. Comparison of these values
with those for leaf cells from K+-replete, non-salinized
plants (Table 2) shows that the aK in the vacuoles of leaf
cells is approximately twice that in the root cells, but the
cytosolic values are similar. This indicates that cytosolic
aK is regulated to similar values in diferent cell types of
barley while vacuolar aK has different set points in root and
leaf cells (see also Leigh, 2001). It would now be
interesting to determine what regulatory processes operate
on K+ transport at the plasma membrane and tonoplast to
bring about this uniformity of cytosolic aK, but differences
in the vacuolar values. In particular, tonoplast transport
must be under quite different control in root cells
compared to leaf cells.
Finally, the work reported here will assist in attempts to
use Na+-selective microelectrodes to measure the effect of
salinity on cytosolic and vacuolar Na+ activities (Carden
et al., 2001). Currently-available Na+ sensors show some
sensitivity to K+, especially at Na+ activities between 1 and

10 mM (Carden et al., 2001). Therefore, knowledge of the
values and behaviour of subcellular aK in salinized plant
cells is an important prerequisite for such studies. An
approach combining the use of Na+- and K+-selective
microelectrodes would allow the effects of salinity on
cytosolic activities of these ions to be quantied and so
allow hypotheses about the importance of cytosolic
K+:Na+ ratio in salinity tolerance to be tested (Maathuis
and Amtmann, 1999).
Acknowledgements
The research was supported by grant B104CT960775 from the EU
Biotechnology Programme 19941998. Rothamsted Research and
Long Ashton Research Station are grant aided by the Biotechnology
and Biological Sciences Research Council of the UK.
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