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Plant Physiol. (1998) 118: 957-964
The Role of Cytosolic Potassium and pH in the Growth of
Barley Roots1
David J. Walker,
Colin R. Black, and
Anthony J. Miller*
Biochemistry and Physiology Department, IACR-Rothamsted, Harpenden,
Hertfordshire AL5 2JQ, United Kingdom (D.J.W., A.J.M.); and School of
Biological Sciences, University of Nottingham, Sutton Bonington Campus,
Leicestershire LE12 5RD, United Kingdom (C.R.B.)
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ABSTRACT |
In an earlier paper we showed that in
fully developed barley (Hordeum vulgare L.) root
epidermal cells a decrease in cytosolic K+ was associated
with an acidification of the cytosol (D.J. Walker, R.A. Leigh, A.J.
Miller [1996] Proc Natl Acad Sci USA 93: 10510-10514). To show that
these changes in cytosolic ion concentrations contributed to the
decreased growth of K+-starved roots, we first measured
whether similar changes occurred in cells of the growing zone.
Triple-barreled ion-selective microelectrodes were used to measure
cytosolic K+ activity and pH in cells 0.5 to 1.0 mm from
the root tip. In plants growing from 7 to 21 d after germination
under K+-replete conditions, the mean values did not change
significantly, with values ranging from 80 to 84 mM for
K+ and 7.3 to 7.4 for pH. However, in
K+-starved plants (external [K+], 2 µM), the mean cytosolic K+ activity and pH
had declined to 44 mM and 7.0, respectively, after 14 d. For whole roots, sap osmolality was always lower in K+-starved than in K+-replete plants, whereas
elongation rate and dry matter accumulation were significantly
decreased after 14 and 16 d of K+ starvation. The rate
of protein synthesis in root tips did not change for
K+-replete plants but declined significantly with age in
K+-starved plants. Butyrate treatment decreased cytosolic
pH and diminished the rate of protein synthesis in
K+-replete roots. Procaine treatment of
K+-starved roots gave an alkalinization of the cytosol and
increased protein synthesis rate. These results show that changes in
both cytosolic pH and K+ can be significant factors in
inhibiting protein synthesis and root growth during K+
deficiency.
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INTRODUCTION |
The functions of K+ in plant cells can be
divided into those that are biophysical, such as osmoregulation, and
those that are biochemical, such as protein synthesis and enzyme
activation (Leigh and Wyn Jones, 1984 ). Although there has been work
demonstrating the inhibitory effect of K+
deprivation on root growth (Asher and Ozanne, 1967; Siddiqi and Glass,
1983; White, 1993), the underlying biophysical and/or biochemical mechanisms have not been explained. Leigh and Wyn Jones (1984) proposed
that a decline in cytosolic [K+] below the
optimum level for protein synthesis (100-150 mM; Wyn Jones
et al., 1979) was the initial cause of growth reduction under
K+ deprivation. "Critical" root-tissue sap
[K+] has been defined as the tissue
K+ concentration at which growth declines below
90% of the maximum (Ulrich and Hills, 1967). This has been measured to
be between 20 and 25 mM (Spear et al., 1978; White, 1993),
providing support for the hypothesis of Leigh and Wyn Jones (1984) that
growth begins to decrease when vacuolar [K+]
reaches a "minimum" value of 10 to 20 mM. Presumably,
at this concentration vacuolar K+ can no longer
be used to maintain cytosolic K+. Declining
vacuolar K+ could inhibit root growth, since
K+ is an important osmoticum and its accumulation
in newly formed vacuoles drives cell expansion (Cram, 1976). Although
the beneficial effect of increased K+ supply on
wall extensibility has been demonstrated (Métraux and Taiz,
1977), the presence of K+ salts in the nutrient
solution can inhibit root-cell elongation by reducing cell wall
plasticity in the root-expansion zone (Pritchard et al., 1987). The
effect of K+ on root elongation seems to be
species specific (Pritchard, 1994), cultivar specific (Hackett, 1968),
and root-type specific (Hackett, 1968; Triboulot et al., 1997).
The aim of this work was to relate measurements of
pHc and K+ activity to the
growth of barley (Hordeum vulgare L.) roots. We previously
used triple-barreled microelectrodes to measure aK, pH, and Em
simultaneously in mature cortical and epidermal cells 10 to 20 mm from
the tip of seminal roots of barley (Walker et al., 1995, 1996). The
cytosolic aK and pH of epidermal, but not
cortical, cells declined during K+ deprivation
(Walker et al., 1996). However, it is the meristematic and rapidly
expanding cells within 2 mm of the root tip where the rates of protein
synthesis are highest in roots (Wareing and Phillips, 1981; Curl and
Truelove, 1986). A decrease in cytosolic aK
in expanding cells would be expected to diminish rates of protein synthesis, leading to a decrease in the rates of root elongation and
dry matter production (Leigh and Wyn Jones, 1984; White, 1993). Changes
in pHc have been shown to alter the rate of
protein synthesis in both animal (Grandin and Charbonneau, 1989) and
plant cells (Webster et al., 1991). To test this hypothesis,
pHc and protein synthesis were measured in root
tips treated with butyrate or procaine to alter
pHc in seedlings growing under
K+-starved or K+-replete
conditions. The effects of K+ starvation on
pHc and cytosolic aK
in expanding cells of seminal roots of barley and on the rates of
protein synthesis in root tips were measured; the results indicate that
decreases in pHc under K+
starvation may be the mechanism for growth inhibition.
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MATERIALS AND METHODS |
Plant Growth Conditions, K+ Content, and Measurements
of Root Growth
Barley (Hordeum vulgare L. cv Klaxon) plants were grown
hydroponically in FNS at different K+
concentrations, as described previously (Walker et al., 1996). The dry
weights of entire seminal root systems were recorded following drying
at 80°C for 3 d. Extension growth was measured by marking seminal roots 10 mm from their tips and measuring the distance of the
mark from the root tip again after 5 to 6 h at the same time of
the day (Cohen and Lepper, 1977). The total K+
content of soaked barley seeds and growing seedlings was determined as
described previously (Walker et al., 1996) using 50 seeds or 50 plants
at each growth stage.
Tissue Sap Osmolality
Whole seminal root sap was extracted by the freeze-thaw method
(Tomos et al., 1984) and its osmolality was measured using a vapor
pressure osmometer (model 5500, Wescor, Logan, UT).
Rates of Protein Synthesis
The rate of protein synthesis in the apical 10- to 15-mm lengths
of seminal roots was estimated by measuring the incorporation of
14C-labeled Leu into protein at 20°C. Any
possible bacterial contamination was first removed by a 60-min
incubation in FNS containing 6 mg L 1
tetracycline (Memon and Glass, 1987). This antibiotic pretreatment was
important, because a comparison between roots treated with the protein
synthesis inhibitor cycloheximide and nontreated roots showed that
14C-labeled Leu uptake by microorganisms
accounted for almost all of the apparent
14C-labeled Leu incorporation into roots (data
not shown). Plants were then transferred to 15 mL of FNS labeled with
74 kBq of L-Leu [U-14C] (specific
activity 11.84 GBq mmol1; ICN) and 0.2 mM unlabeled L-Leu. After a 60-min incubation
(20°C), free-space 14C-labeled Leu was desorbed
for 10 min in FNS containing 1.0 mM unlabeled
L-Leu (4°C). The roots were then gently blotted dry, the
root cap (0.5 mm) was excised and discarded, and the remaining apical
10 to 15 mm of the root was excised, weighed, and frozen in liquid
nitrogen. Root tips were defrosted and homogenized in 0.3 mL of
ice-cold extraction buffer (Davies et al., 1996) using a
microhomogenizer (Biomedix Ltd., Pinner, Middlesex, UK). The homogenate
was centrifuged at 3,000g for 10 min (4°C). A sample of
the resulting supernatant ("total 14C
uptake") was mixed with an equal volume of 10% (w/v) SDS. An aliquot
of this mixture was transferred to a scintillation vial, to which 4 mL
of liquid-scintillation cocktail (Ultima Gold; Packard, Pangbourne, UK)
was added, and 14C dpm were counted with a
liquid-scintillation analyzer (model 2500 TR, Packard). A
170-µL sample of the remaining supernatant was diluted to 1.0 mL with
extraction buffer and centrifuged at 150,000g for 30 min
(4°C). The resulting supernatant ("soluble fraction") was
removed, mixed with 200 µL of 47% (w/v) TCA, and left for 60 min
(4°C). This mixture was then centrifuged at 12,000g for 10 min (4°C), to pellet the precipitated protein, which (after removal
of the supernatant) was solubilized in 100 µL of solubilization buffer (5% SDS in 50 mM Tris-HCl, pH 6.8; 20°C). A
sample of this preparation was added to a scintillation vial for
counting. The 150,000g pellet ("particulate fraction")
was resuspended in 150 µL of solubilization buffer (20°C) and a
sample was added to a scintillation vial for counting. The dpm values
for these two fractions were added together to give the total protein
value.
To study the effect of cytosolic acidification on protein synthesis,
7-d plants growing in FNS containing 0.5 mM
K+ were incubated in FNS containing 10 mM sodium butyrate for 90 min before transfer to FNS
containing 14C-labeled Leu (plus butyrate) for a
further 60 min (Sanders et al., 1981). The effect of procaine on
protein synthesis in the root tips was studied by incubating the plants
for 10 min in 2 µM K+ FNS
containing 0.2 mM procaine (pH 8.95) prior to a 60-min
incubation in the same solution to which
14C-labeled Leu was added. The percentage
incorporation of 14C-labeled Leu into the protein
was calculated as:
Measuring the rate of protein synthesis as the rate of
incorporation of 14C-labeled Leu into proteins
removes any effect of different treatments on the uptake of Leu into
cells.
Electrophysiology
Simultaneous measurements of intracellular
aK, pH, and Em
in expanding cells 0.5 to 1.0 mm from the tips of barley seminal roots
(Jeschke and Stelter, 1976; Huang and van Steveninck, 1989) were
performed using triple-barreled microelectrodes (Walker et al., 1995,
1996). The impaled cells were those destined to become mature cortical
cells, being the second, third, and fourth layers of cells encountered
by the microelectrode tips as they penetrated into the root (Huang and
van Steveninck, 1989). These electrode impalements may have produced
tissue damage, but stable membrane potential recordings were obtained
from the cells, and the value of these was similar in magnitude to
those obtained from mature cells growing under the same conditions
(Walker et al., 1996). Measurements were also made in fully vacuolate
epidermal and cortical cells 10 to 20 mm from the tips of adventitious
roots of 21-d plants. Butyrate-treated plants (grown for 7 d in
FNS containing 0.5 mM KCl) were transferred for 2 h to
the same solution (but containing 10 mM sodium butyrate, pH
5.8) prior to microelectrode measurements. Procaine-treated plants (21 d, K+ starved) were incubated for 20 to 50 min,
before the electrode impalements, in a nutrient solution containing (in
mM) 0.5 NaH2PO4, 0.5 NaNO3, 1.0 MgSO4, 0.5 Ca(NO3)2, 0.025 NaFeEDTA,
and 0.2 procaine (Felle and Bertl, 1986), pH 8.95, and with added
micronutrients (Hoagland and Arnon, 1950). The output from the
pH-selective barrel changed slightly after the procaine treatment (data
not shown). Therefore, 0.2 mM procaine was included in the
pH-calibration solutions used to calibrate microelectrodes before and
after measurements in procaine-treated roots.
Results are shown as mean values ± SE
(n), except for aK and
[H+], which are shown as means with confidence
limits obtained following conversion from the corresponding
 log(aK) and pH values (Fry et al., 1990).
Analysis of variance was performed using Microsoft Excel and linear
regression analysis was performed using Sigmaplot (Jandel Scientific,
Erkrath, Germany).
Thermodynamic Calculations
Uptake of K+ at the plasma membrane involves
two different mechanisms (for review, see Maathuis and Sanders, 1996).
For K+-starved plants growing in 2 µM K+ solution, only the
high-affinity plasma membrane uptake system in root cells can retrieve
K+. The mechanism for this is cotransport with
either Na+ or H+ (Rubio et
al., 1995). The free-energy relationship for high-affinity K+ uptake in millivolts across the plasma
membrane via H+ or Na+
symport was calculated as:
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(1)
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where n is the stoichiometry of
H+ or Na+ to
K+ transported by a high-affinity
K+ transporter, M is the activity of
H+ or Na+, and the
subscripts o and c refer to the external medium and cytosol,
respectively. The mean values of Em,
aK, and pHc were measured with ion-selective microelectrodes for seedlings growing in
FNS at 2 µM
[K+]o.
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RESULTS |
Root Dry Matter Production, Elongation Rate, and Osmolality
Figure 1A shows the changes in the
root dry weight of barley seedlings growing in either 2 µM or 5 mM K+ FNS for 6 to 21 d after germination. Whole-root dry weight for K+-starved plants was not significantly lower
than that for K+-replete plants until d 16 (Fig.
1A). Shoot dry weight for the same plants showed a similar
relationship (data not shown). The dry weights of barley seedlings
growing in 0.5 mM K+ FNS were not
significantly different from those of roots growing in 5 mM
K+ (data not shown), although root-elongation
rates were significantly lower in K+-starved
plants from 14 d on (Fig. 1B). This change in growth and
elongation rates at 14 to 16 d may occur at the developmental stage when seed K+ reserves have been consumed
and K+-starved and
K+-replete plants both begin to develop
adventitious roots (Russell, 1970). Figure 1C shows the osmolality
values of extracts from both K+-starved and
K+-replete plants. In contrast to the effects on
growth shown in Figure 1, A and B, the osmolality of
K+-replete plants was always significantly higher
than that of K+-starved plants, but in both cases
there was a general decline with time (Fig. 1C). Because the
differences in sap osmolality occurred from 6 d on, before
significant differences in growth were detected, this result suggests
that there is no relationship between this parameter and the growth
inhibition that occurs in the K+-starved roots.
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| Figure 1.
The effect of K+ supply on the growth
and sap osmolality of barley roots growing from d 5 in FNS containing
either 2 µM ( ) or 5 mM ( )
K+. A, Seminal root dry weights; B, root-elongation rate;
and C, osmolality of whole seminal root sap.
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To prove that K+ uptake from the external
solution continued throughout the 21-d experimental period for plants
growing in 2 µM K+, the total
K+ of the seed and whole plants was measured
throughout the experiment. These measurements showed that the mean
total quantity of K+ in the seed allowed to soak
was 4.1 µmol per seed but had increased to 6.8 µmol per plant at
6 d and to 12.2 µmol per plant by 21 d.
The Effect of K+ Supply on Cytosolic
aK and pHc
Continued growth of barley seedlings at a
[K+]o of 2 µM resulted in a decline in the cytosolic
aK of expanding cells of seminal roots from
78 mM at 7 d to 44 mM at 21 d (Fig.
2A). For K+-replete
plants growing at a [K+]o
of 0.5 or 5 mM, the mean cytosolic
aK values of expanding cells did not change
significantly between 6 to 21 d. The mean
Em values for plants growing in 2 µM K+ decreased from 141 ± 4 mV at 7 d to 124 ± 8 mV at 21 d, whereas for plants
growing in 5 mM K+, the mean
Em values were in the range 80 to 89
mV. Figure 2B shows the effect of changing the K+
supply on the pHc of expanding cells. The mean
pHc was 7.4 ± 0.1 in the expanding cells of
seminal roots of K+-replete plants until d 16 and
was still 7.3 ± 0.1 at 21 d (Fig. 2B). The decrease in mean
pHc, from 7.4 ± 0.1 on 7 d to 7.0 ± 0.1 by 16 d, for K+-starved plants
occurred in parallel with a decrease in cytosolic aK (Fig. 2A); the latter value is similar
to that obtained for fully vacuolate K+-starved
epidermal cells (Walker et al., 1996).
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| Figure 2.
The effect of K+ supply on cytosolic
aK and pHc of expanding barley
root cells. Triple-barreled microelectrodes were used to measure
cytosolic aK (A) and pHc of seminal root cells
(B) of plants growing in either 2 µM () or 5 mM () K+.
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In contrast, the mean values for cytosolic
aK (70 mM) and pHc
(7.0) in the epidermal cells of adventitious roots (Table
I) were higher than those of analogous
cells in the seminal roots of 21-d K+-starved
plants (39 mM and 6.7, respectively; Walker et al., 1996). The growth of adventitious roots was decreased in seedlings growing in
2 µM K+ when compared with plants
grown in 5 mM K+ (data not shown).
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Table I.
pHc and cytosolic aK and
Em of epidermal cells 10 to 20 mm from the tips of
adventitious roots of 21-d barley plants growing in FNS containing 2 µM K+
Em and pH values are means ± SE, and aK values are means with
confidence limits in parentheses and the number of measurements in the
final parentheses.
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For 7-d plants growing in 0.5 mM K+,
a 2-h treatment with butyrate had no effect on cytosolic
aK or Em but
decreased the mean pHc from 7.3 ± 0.1 to
6.8 ± 0.1 (Table II). For 21-d
K+-starved plants, procaine treatment increased
the mean pHc of expanding cells from 7.0 ± 0.1 to 7.3 ± 0.1 and altered the mean Em by 16 mV but had no significant effect
on cytosolic aK (Table II). In Figure
3A, growth, shown as the percentage of
maximum dry weight, is plotted against cytosolic
aK; the results show that for expanding
cells there was a correlation between the two parameters
(r = 0.997). Furthermore, the "critical" cytosolic aK for growth was 73 mM,
equivalent to a [K+] of almost 100 mM (Robinson and Stokes, 1970), which is similar to the
predicted value (Leigh and Wyn Jones, 1984). Figure 3B shows the
relationship between growth and pHc in expanding
root-tip cells and the acidification of the cytosol; like the decline
in cytosolic aK, the change in
pHc may also cause decreased growth.
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Table II.
Effect of treatment with either 10 mM
sodium butyrate or 0.2 mM procaine on pHc and
the cytosolic aK and Em of expanding cells in
barley root tips
Em and pHc values are means ± SE, and aK values are means with
confidence limits in parentheses and the number of measurements in the
final parentheses.
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| Figure 3.
Relationship between seminal root growth
(expressed as a percentage of the dry weight of the seminal roots of
plants growing in FNS containing 5 mM K+) and
cytosolic aK (A) and pHc (B) of
expanding barley root cells. In A, the fitted linear regression is
y = 1.359x 10.976 (r2 = 0.997, n = 11).
Critical aK is defined as the concentration
at which growth was 90% of the maximum (Ulrich and Hills, 1967). In B,
the fitted linear regression is y = 102.46x 658.08 (r2 = 0.976, n = 11). The root dry weight of plants
growing in 5 mM K+ was assumed to represent
maximum growth and was not significantly different from that of plants
growing in 0.5 mM K+.
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The decline in the mean pHc of expanding cells,
from 7.3 to 6.8, caused by the 2-h butyrate treatment resembles that
observed previously in the epidermal and cortical cells of the seminal roots of barley (Walker et al., 1996) and other butyrate-treated tissues (Sanders et al., 1981; Reid et al., 1985b). Although
deleterious effects of prolonged butyrate treatment (>6 h) on the cell
cycle have been reported (Lanzagorta et al., 1988; Tramontano et al., 1991), Reid et al. (1985a) observed no effect of a 2-h exposure to 10 mM butyrate on ATP concentrations in barley roots. In the current study the root-elongation rates at 9 d for plants growing in 5 mM K+ and exposed for 2 h
to 10 mM butyrate on d 7 (0.46 ± 0.03 mm h1, n = 9) were no different
from those of control plants (0.40 ± 0.03 mm
h1, n = 44).
The thermodynamic calculations for two different high-affinity
K+ uptake mechanisms at the plasma membrane are
shown in Table III. Both are symport
mechanisms with transport coupled to either H+ or
Na+, and in the calculations of
Na+ cotransport two different cytosolic
Na+ concentrations were used. It has been
suggested that the mechanism of high-affinity K+
uptake in wheat is a Na+:K+
symport with a 1:2 stoichiometry (Rubio et al., 1995), but such a
mechanism could not have maintained the measured
aK gradient across the plasma membrane of
expanding barley root cells (Table III). However, assuming a cytosolic
Na+ concentration of 1 mM and a 1:1
stoichiometry, this mechanism becomes feasible (Table III). A
H+:K+ symport mechanism
with a 1:1 stoichiometry (Schachtman and Schroeder, 1994) might also
have maintained the measured trans-plasma membrane aK gradient in the expanding barley root
cells (Table III).
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Table III.
Free-energy values (mV) for the uptake of
K+ by H+ or Na+ symport mechanisms
into expanding cells of seminal roots of barley plants growing in FNS
containing 2 µM K+ for various times
The values were calculated using Equation 1 and assumed two different
cytosolic Na+ concentrations of 1 and 20 mM.
Negative values indicate that the mechanism could maintain the observed
trans-plasma membrane K+ gradient.
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Protein Synthesis
The rate of protein synthesis, measured as the percentage
incorporation of 14C-labeled Leu into protein in
the apical regions of roots within a 1-h period, was independent of
seedling age for K+-replete plants (data not
shown). However, for K+-starved plants, the
incorporation of radiolabeled Leu decreased as the starvation
increased. Figure 4 shows the
relationships between the percentage of
14C-labeled Leu incorporation into protein and
cytosolic aK and pH; different symbols are
used to identify the butyrate or procaine treatments. In both data
sets, the rate of protein synthesis decreased with cytosolic
aK and pHc, but Figure 4A shows
that the butyrate treatment gave a value that does not comply with the
relationship provided by the other points. Figure
5 shows the effect of butyrate or
procaine treatment on radiolabeled Leu incorporation into protein in
barley root tips relative to control tips. Cytosolic acidification following treatment with butyrate (Table II) decreased
14C-labeled Leu incorporation, from 16.9 ± 1.4% to 4.6 ± 0.5% (Fig. 5), whereas alkalinization of the
cytosol of expanding cells of 21-d K+-starved
plants using procaine (Table II) almost doubled
14C-labeled Leu incorporation (Fig. 5). The
decrease in the mean pHc of expanding cells
treated with butyrate (to 6.8) was more pronounced than the effect of
21 d of K+ starvation (decrease to 7.0;
Table II compared with Fig. 5). Furthermore,
14C-labeled Leu incorporation was decreased to a
greater extent in butyrate-treated roots than in
K+-starved roots (Table II). Protein synthesis
was also partially restored by procaine treatment in
K+-starved root tips, whereas butyrate treatment
of K+-replete cells inhibited protein synthesis
(Table II). These results show that cytosolic acidification inhibits
protein synthesis to a greater extent than depletion of cytosolic
aK.
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| Figure 4.
Relationship between the rate of protein synthesis
and cytosolic aK (A) and pHc (B)
of expanding cells of barley root tips. The fitted linear regressions
are: A, y = 0.135x + 3.90 (r2 = 0.183, n = 9); B,
y = 19.59x 128.1 (r2 = 0.790, n = 9).
Protein synthesis was measured as the percentage incorporation of
14C-labeled Leu. The roots were treated with butyrate ( )
or procaine ( ). The remaining symbols represent seedlings supplied
with different K+ concentrations (Walker et al., 1996).
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| Figure 5.
Histogram showing the effects on protein synthesis
of treating K+-replete seedlings with butyrate or
K+-starved seedlings with procaine. Protein synthesis was
measured as the incorporation of 14C-labeled Leu into
protein. Seedlings were growing from d 5 in FNS containing 0.5 mM K+ and treated with butyrate (10 mM) and 21-d barley plants were growing from d 5 in FNS
containing 2 µM K+ treated with procaine (0.2 mM).
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DISCUSSION |
For many plants the
[K+]o required to give
optimal growth is small; for example, the concentration needed for
maximal growth in 14 different species was only 30 µM
K+ (Asher and Ozanne, 1967). In barley seedlings
supplied with an external concentration of only 2 µM, the
growth of seminal roots continued but at a rate that at 21 d was
50% of that measured for K+-replete plants (Fig.
1A). Growing plants with a suboptimal K+ supply
has allowed the relationship among cytosolic
aK, pHc, and growth
to be determined.
Root Growth and K+ Supply
The growing root tip is generally described as a sink for
K+ (Silk et al., 1986; Moritsugu et al.,
1993). There are three possible sources of K+ to
support the growth of the root tips in K+-starved
barley seedlings: seed reserve, remobilization of
K+ from other tissues, and uptake from the
external solution.
During the time course of these measurements, the seed reserve of
K+ was being depleted and the growth differences
between K+-starved and -replete roots started to
become significant at 14 to 16 d. This observation suggests that
the seed reserve of K+ may have been consumed,
although adventitious roots also began to appear at this time, which
would have provided a new sink for K+. The
appearance of adventitious roots coincided with a significant slowing
of seminal root growth in K+-starved barley
plants (Fig. 1). To sustain continued growth of adventitious roots,
withdrawal of K+ from the shoot or the seminal
roots may have occurred. The different responses to
K+ starvation shown by fully developed epidermal
cells of seminal roots (Walker et al., 1996) and analogous cells of
adventitious roots (Table I) may be an example of this redistribution
process.
Total K+ content increased with age in the
K+-starved seedlings, showing that
K+ uptake occurred throughout the 21-d
measurement period. This K+ uptake may have been
limited to the cortical cells, because in wheat roots the high-affinity
plasma membrane K+ transporter (HKT1) was mainly
expressed in this tissue (Schachtman and Schroeder, 1994).
Furthermore, the expanding cells of seminal roots were unable to
maintain optimal growth rates by uptake of K+
from an external concentration of 2 µM. The high-affinity
plasma membrane K+-uptake system may be located
only in the growing tip cells of adventitious roots and not in seminal
roots; although uptake of the radiolabeled K+ analog,
86Rb+, by the apical 10 mm
of excised barley seminal roots could be measured when the combined
[Rb+]o and
[K+]o was 23 µM (Moritsugu et al., 1993).
It has been proposed that K+ uptake by HKT1 is
coupled to either H+ or Na+
gradients across the plasma membrane (Rubio et al., 1995), and a
related gene has been identified in barley (Santa-María et al.,
1997), which also mediates low-affinity Na+
uptake. However, no direct evidence for the coupling of
K+ and Na+ uptake by barley
roots has been found (Maathuis et al., 1996). The microelectrode
measurements in the barley cells allow the energetics of these various
high-affinity K+ uptake mechanisms to be
determined. These calculations suggest a 1:1 H+
symport as the most likely mechanism (Table III), but this calculation depends on knowledge of the cytosolic Na+
concentration, so intracellular measurements of this are needed.
The Cellular Mechanism of Growth Inhibition during
K+ Starvation
The cytosolic acidification associated with cytosolic
K+ depletion in expanding cells was similar to
that reported for cells of barley root epidermis (Walker et al., 1996)
and the fungus Neurospora (Blatt and Slayman, 1987). For one
variety of barley, Compana, the rate of protein synthesis
([3H]Leu incorporation) increased 0.5- to
2.7-fold when [K+]o was
increased from 10 to 100 µM (Memon and Glass, 1987).
However, whole-root compartmental efflux analysis showed that cytosolic [K+] increased only from 133 to 140 mM. In another barley variety, Betzes, the root protein
synthesis increased by 7-fold, whereas cytosolic
[K+] increased from 127 to 187 mM
as [K+]o was increased
from 10 to 100 µM (Memon and Glass, 1987). However, this
averaging technique for measuring cytosolic
aK may have hidden tissue differences and
the observed effect on protein synthesis may have resulted partly from
an increase in pHc. It was found using cytosolic
extracts of Xenopus oocytes that the rates of in vitro
translation at pH 7.0 and 6.8 were reduced by approximately 15% and
65%, respectively, relative to pH 7.3 (Grandin and Charbonneau, 1989).
Furthermore, a strong pH dependence of protein synthesis has been shown
in polyribosome preparations from maize root tips by Webster et al.
(1991), who suggested that the pH dependence of root-tip protein
synthesis could provide a mechanism that enables the cell's
translational machinery to sense changes in pHc, leading directly to selective gene expression. In mammalian cells, a role for
cytosolic acidification in signaling programmed cell death is unlikely
(Schrode et al., 1997), but changes in cytosolic
aK have been implicated (Hughes et al.,
1997). Under these K+-starved conditions, the
seminal root cells may be showing programmed cell death so that
specific sink root meristems are removed and K+
is made available to supply the emerging adventitious roots. Proof of
apoptosis would require the identification of the morphological and
biochemical markers (for review, see Harvel and Durzan, 1996) that are associated with this process in the root cells.
An additional role for K+ is in balancing the net
anionic charge of proteins within the cytosol (Maathuis and Sanders,
1996). The cytosolic acidification that is associated with depletion of
cytosolic K+ could result from
H+ substituting in this function. However, the
depletion of cytosolic K+ from 80 to 45 mM (Fig. 2A) was associated with a relatively small increase in H+ concentration from 40 to 100 nM (pH 7.4-7.0, Fig. 2B). Changes in
pHc will occur only after the
H+-buffering capacity of the cytosol has been
exceeded, and estimates of this parameter range from 18 to 40 mM H+ per pH unit (Roos and Boron,
1981; Reid et al., 1989); thus, H+ can only
partially substitute for K+ in balancing the
charge within the cytosol.
For K+-starved barley seedlings, simultaneous
measurements of growth, cytosolic aK, and
pHc in cells of the root-growth zone have allowed
the relationship among these parameters to be determined. There was a
direct dependence of growth on both pHc and
cytosolic aK in expanding cells (Fig. 3).
Protein synthesis may be altered by changes in cytosolic ion
concentrations, and when this process was assayed in vivo, like growth,
it was found to be dependent on both pHc and
cytosolic ak (Fig. 4). However, artificial
manipulation of pHc by treatment with butyrate or
procaine showed that it is this parameter, rather than cytosolic
aK, that is critical for protein synthesis
and therefore growth.
| |
FOOTNOTES |
1
IACR-Rothamsted receives grant-aided support
from the Biotechnology and Biological Sciences Research Council of the
United Kingdom.
*
Corresponding author; e-mail tony.miller{at}bbsrc.ac.uk; fax
44-1582-760981.
Received May 13, 1998;
accepted August 7, 1998.
| |
ABBREVIATIONS |
Abbreviations:
aK, K+
ion activity.
Em, trans-plasma membrane electrical potential.
FNS, full
nutrient solution.
[K+]o, external
K+ concentration.
pHc, cytosolic pH.
| |
ACKNOWLEDGMENT |
We wish to thank Dr. Emyr Davies (IACR-Rothamsted) for advice
regarding assaying rates of protein synthesis.
| |
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Top
Abstract
Introduction
