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Plant Physiol. (1998) 117: 311-319
Adaptation of Active Proton Pumping and Plasmalemma ATPase
Activity of Corn Roots to Low Root Medium pH1
Feng Yan,
Robert Feuerle,
Stefanie Schäffer,
Helge Fortmeier2, and
Sven Schubert2, *
Institute of Plant Nutrition 330, University of Hohenheim,
Fruwirthstrasse 20, D-70599 Stuttgart, Germany
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ABSTRACT |
Corn
(Zea mays L.) root adaptation to pH 3.5 in comparison
with pH 6.0 (control) was investigated in long-term nutrient solution experiments. When pH was gradually reduced, comparable root growth was
observed irrespective of whether the pH was 3.5 or 6.0. After low-pH
adaptation, H+ release of corn roots in vivo at pH 5.6 was
about 3 times higher than that of control. Plasmalemma of corn roots
was isolated for investigation in vitro. At optimum assay pH, in
comparison with control, the following increases of the various
parameters were caused by low-pH treatment: (a) hydrolytic ATPase
activity, (b) maximum initial velocity and Michaelis constant (c)
activation energy of H+-ATPase, (d) H+-pumping
activity, (e) H+ permeability of plasmalemma, and (f) pH
gradient across the membranes of plasmalemma vesicles. In addition,
vanadate sensitivity remained unchanged. It is concluded that
plasmalemma H+-ATPase contributes significantly to the
adaptation of corn roots to low pH. A restricted net H+
release at low pH in vivo may be attributed to the steeper pH gradient
and enhanced H+ permeability of plasmalemma but not to
deactivation of H+-ATPase. Possible mechanisms responsible
for adaptation of plasmalemma H+-ATPase to low solution pH
during plant cultivation are discussed.
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INTRODUCTION |
Acid soils make up to 40% of the world s arable land (Kochian,
1995 ). Plant growth and development on acid soils may be affected by
high levels of Al and Mn, as well as by limited availability of various
nutrients (Adams, 1981). On the other hand, low pH (high
H+ activity) in root medium
(pHe) may directly inhibit plant growth (Islam et
al., 1980; Schubert et al., 1990). Mechanisms of Al toxicity have been
studied extensively during the last decade (Kochian, 1995), whereas the
understanding of H+ toxicity in plants remains
poor. It has been observed that root growth rate was related to net
H+ release, which may be restricted at low
pHe. Therefore, it has been suggested that
H+ homeostasis of plant root cells may be
influenced by low pHe, resulting in the reduction
of root growth rate (Yan et al., 1992). Net H+
release results from H+ efflux driven by
plasmalemma H+-ATPase activity and from
H+ influx following the plasmalemma
H+ gradient. Reduced net H+
release may be attributed to a decrease in H+
pump activity, an increase in plasmalemma H+
permeability, or both. Because of its overall importance in
physiological processes, the plasmalemma
H+-ATPase has been investigated extensively
during the last two decades. This enzyme has been found to respond to a
number of environmental factors, such as saline stress (Braun et al.,
1986; Ayala et al., 1996), nutrient supply (Kuiper et al., 1991; Santi et al., 1995; Schubert and Yan, 1997), high-O2
treatment (Pinton et al., 1996; Xia and Roberts, 1996), mechanical
stimulation (Bourgeade and Boyer, 1994), and fusicoccin, a fungal toxin
(Marré, 1979). Although there are reports in the literature
describing a response of yeast H+-ATPase to low
pHe (Eraso and Gancedo, 1987), investigations
concerning the response of plasmalemma H+-ATPase
of higher plant cells to low pHe are surprisingly
scarce. Also, it remains unknown whether the plasmalemma
H+ permeability changes when roots are exposed to
low pHe.
In this study we investigated the contribution of plasmalemma
H+-ATPase of corn (Zea mays L.) roots
to low-pHe adaptation. In a long-term experiment
with low-pH treatment, pHe was gradually reduced
to 3.5, whereas pHe in the control was kept
constant at 6.0. H+ release by intact roots was
studied in vivo. Furthermore, plasmalemma of corn roots was isolated
from adapted and nonadapted plants. In vitro, plasmalemma
H+-ATPase activity, activation energy, kinetic
characteristics, H+-pumping activity, and
H+ permeability were investigated. The aim of our
investigation was to reveal the mechanisms by which plant root cells
adapt to low pHe.
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MATERIALS AND METHODS |
Plant Cultivation
Corn (Zea mays L. cv Blizzard) seeds were soaked in
aerated 0.5 mm CaSO4 for 1 d and
germinated at 25°C in the dark between two layers of filter paper
moistened with 0.5 mm CaSO4. After 4 d seedlings were transferred to a container with 400 L of
one-fifth-concentrated nutrient solution. Plants were grown in a growth
chamber under controlled conditions. Fluorescent lamps (66% L58/31 and
34% L58 W/78, Osram, Frankfurt, Germany) gave a light intensity of
approximately 50 W m 2, with a day/night cycle
of 16 h/8 h at 25°C. RH was 80%. After 2 and 4 d of cultivation
the concentration of the nutrient solution was increased to a one-half
and a full-strength concentration, respectively. The full-strength
nutrient solution had the following composition: 1 mm
NH4NO3, 0.2 mm
NaH2PO4, 1 mm
K2SO4, 2 mm
CaCl2, 3 mm
MgSO4, 0.2 µm
H3BO3, 0.2 µm
CuSO4, 0.01 µm
(NH4)6Mo7O24, 5 µm MnSO4, 0.2 µm
ZnSO4, and 200 µm Fe-EDTA. During
the growth period the concentrations of nitrogen, phosphate, and
potassium were controlled every 3 d and substituted if necessary.
There was no significant depletion of other nutrients in the solution. The pH of the control solution was kept constant at pH 6.0 by continuous titration with 0.1 m NaOH using a pH stat
system. In the low-pH treatment, acidity was gradually enhanced by
increasing H+ activity by increments of 100 µm daily to a final pH of 3.5. Plants were allowed to
grow at the final pH for 4 d and were then used for proton release
studies and plasmalemma isolation at the age of about 3 weeks. To
investigate the effect of low pHe on root growth,
plant cultivation was extended to 33 d.
Measurement of Root Growth and Net Proton Release by Roots
To measure root growth, plants were harvested three times after
cultivation for 19, 27, and 33 d. Root fresh weights and the number of laterals were determined. Main root length was measured with
a ruler. Roots of intact plants used for the study of net H+ release were washed three times for 5 min each
time in a solution composed of 1 mm
CaSO4, 0.5 mm
K2SO4, and 0.5 mm Na2SO4. In
an identical solution net H+ release was
quantified in 2.5 L per four plants. The initial pH of this test
solution was adjusted to 5.6 and 3.4, respectively, by titration with
H2SO4. Net
H+ release was calculated from the pH change of
the solution.
Plasmalemma Isolation
A microsomal membrane fraction was prepared as described by
Faraday and Spanswick (1992) with some modifications. Roots of 3-week-old plants were cut and washed three times with chilled, deionized water. Roots were cut into 1-cm segments and ground in
ice-cold homogenization buffer with a mortar and pestle. The homogenization buffer contained 250 mm Suc, 2 mm EGTA, 10% (v/v) glycerol, 0.5% (w/v) BSA, 2 mm DTT, 1 mm PMSF, 5 mm
2-mercaptoethanol, and 50 mm BTP, adjusted to pH 7.8 with
Mes. The homogenate (adjusted to a grinding medium/tissue ratio of 4 mL/g fresh weight) was filtered through two layers of Miracloth
(Calbiochem) and centrifuged in a swinging bucket rotor at
11,500g (Sorvall AH-629 rotor, 36 mL) for 10 min at 0°C.
The supernatants were centrifuged at 87,000g for 35 min. The
microsomal pellets were resuspended in phase buffer (250 mm
Suc, 3 mm KCl, and 5 mm
KH2PO4, pH 7.8).
The microsomal membrane preparation was fractionated by two-phase
partitioning in aqueous dextran T-500 (Sigma) and PEG-3350 (Sigma)
according to the method of Larsson (1985). Phase separations were
carried out in a series of 32-g phase systems that contained 6.2%
(w/w) of each polymer dissolved in phase buffer (see above). Stock
solutions of polymers were prepared with concentrations of 20 and 40%
(w/w) for dextran and PEG, respectively. The concentration of dextran
stock solution was determined by optical rotation (Larsson, 1985). The
phase stock was weighed and diluted to 6.2% (w/w, each polymer) with
phase buffer to a final weight of 32 g. Polymers in "start
tubes" were, however, diluted to 26 g. Six grams of microsomal
resuspension (in phase buffer) was added to the upper phase of each
start tube. The tubes were sealed with Parafilm (American National Can,
Greenwich, CT) and mixed by inversion (30 times). Phase separation was
achieved at 4°C by centrifugation at 720g (Sorvall AH-629
rotor, 36 mL) for 23 min followed by two washing steps in identical
phases. Centrifugation times for the second through fourth separation
were 15, 10, and 5 min, respectively. The upper phases obtained after
four separations were diluted with phase buffer (see above) and
centrifuged at 151,200g for 40 min. The pellets were washed
with resuspension buffer (250 mm Suc, 3 mm KCl,
and 5 mm BTP/Mes, pH 7.8) and pelleted again. The pellets
were resuspended in resuspension buffer, divided into aliquots, and
immediately stored in liquid nitrogen. Protein was quantified according
to the method of Bradford (1976) using BSA (Sigma) as a standard.
Enzyme Assays
Hydrolytic ATPase activity was determined in 0.5 mL of 30 mm BTP/Mes buffer containing 5 mm
MgSO4, 50 mm KCl, 0.02% (w/v) Brij
58 (Sigma), and 5 mm disodium-ATP. Reactions were initiated by the addition of 1 µg of membrane protein, proceeded for 30 min at
30°C, and stopped with 1 mL of stopping reagent (2% [v/v] concentrated H2SO4, 5%
[w/v] SDS, 0.7% [w/v]
(NH4)2MoO4) followed immediately by
50 µL of 10% (w/v) ascorbic acid. After 10 min 1.45 mL of
arsenite-citrate reagent (2% [w/v] sodium citrate, 2% [w/v]
sodium m-arsenite, and 2% [w/v] glacial acetic acid) was
added to prevent the measurement of phosphate liberated because of
ATPase activity from ATP hydrolysis under acidic conditions (Baginski
et al., 1967). Color development was complete after 30 min and
A820 was measured by means of a
spectrophotometer (U3200, Hitachi, Tokyo, Japan). ATPase activity was
calculated as phosphate liberated in excess of boiled-membrane control.
Plasmalemma-bound ATPase activity was determined as the difference in
activity between assays with and without addition of 0.1 mm
Na3VO4. The kinetic characteristics of plasmalemma ATPase were studied in the presence of
an ATP-generating system that included 5 units of pyruvate kinase
(Sigma) and 5 mm PEP (Boehringer Mannheim; Sekler and Pick, 1993). Vmax and
Km were determined by means of a
regression analysis. Activation energy of ATPase was calculated
using the Arrhenius equation from Vmax
values determined at 20 and 30°C, respectively.
pH Gradient
The formation of a pH gradient across the plasmalemma of
inside-out vesicles was measured as the quenching of
A492 by AO. The assay mixture contained 5 mm BTP/Mes (pH 6.5), 7.5 µm AO, 100 mm KCl, 1 mm NaN3, 1 mm Na2MoO4,
0.05% (w/v) Brij 58, 50 µg of membrane protein in a final volume of
1.5 mL. Brij 58 was used to create inside-out vesicles (Johansson et
al., 1995). After equilibration of the membrane vesicles with the
reaction medium (about 10 min), the reaction was initiated by the
addition of Mg-ATP (mixture of MgSO4 and
disodium-ATP, adjusted to pH 6.5 with BTP) to give a final
concentration of 5 mm. The reaction temperature was 25°C.
Determination of Membrane Lipids
Total plasmalemma lipids were determined according to the work of
Bligh and Dyer (1959). Lipids were extracted by adding 1.6 mL of
membrane to a solution consisting of 4 mL of chloroform, 4 mL of
isopropanol, and 2 mL of water. After chloroform was evaporated, lipids
were determined gravimetrically. Then, lipids were digested with
perchloric acid and the released phosphate was quantified with the
method of Fiske and SubbaRow (Dittmer and Wells, 1969). The amount of
phospholipids was calculated by assuming an average Mr of 800.
Statistical Treatment
Variation is indicated by ± se (if bars exceed
symbols in figures). Significant differences between treatments were
calculated by using the Student's t test.
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RESULTS |
Adaptation of Corn Roots to Low pHe
We reported previously that an abrupt decrease in solution pH
markedly reduced the root growth rate of corn seedlings (Yan et al.,
1992). In contrast, in the present study we found that corn roots were
capable of growing normally even at pHe 3.5 when nutrient solution pH was decreased gradually. During an experimental period from 19 to 33 d no difference between
pHe 3.5 and 6.0 was found for root fresh weight,
main root length, or the number of laterals (Table
I). In addition, roots grown at
pHe 3.5 were harder and looked brighter than
those grown at pHe 6.0. In a simple test solution
(pH 5.6) roots grown at pHe 3.5 showed about 3 times higher net H+ release than control roots
(Fig. 1A). This could not be attributed to passive release of protons from the apoplast because roots were
thoroughly washed with the test solution three times before being
transferred into test solution. Also, the fact that substantial H+ release lasted for more than 8 h
indicates enhanced active H+ pumping of corn root
cells after adaptation to low pHe. This holds
true even when the test solution pH was initially adjusted to 3.4 (Fig.
1B). Under this condition control roots showed net H+ uptake.
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Table I.
Effect of adaptation to low pHe on corn
root growth
Main root length and the number of laterals were determined after
27 d of cultivation at different nutrient solution pH. In the
low-pHe treatment nutrient solution pH was gradually
reduced to 3.5 within the first 14 d of cultivation. The values
represent means (± se) of six plants.
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| Figure 1.
Effect of low nutrient-solution pH during
cultivation on the time course of net H+ release by roots
of intact corn plants. Plants were adapted for 3 weeks in nutrient
solution at pHe 6.0 ( ) and 4 d at pHe
3.5 after gradually decreasing pHe ( ). Test medium
consisted of 1 mm CaSO4, 0.5 mm
K2SO4, and 0.5 mm
Na2SO4 with pH 5.6 (A) and 3.4 (B). Negative
values denote H+ uptake. Values represent means ± se of six replications. FW, Fresh weight.
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Effect of Low pHe on the Isolation of Plasmalemma from
Corn Roots
A summary of relative ATPase-specific activities associated with
phase-partitioned corn root plasmalemma is presented in Table II. At assay pH 6.5 both nitrate and
azide showed no inhibition effect on the ATPase activities. Similar
results were obtained at assay pH 8.0 (not shown). Molybdate inhibited
specific ATPase activity by about 5%, which indicates the presence of
soluble phosphatase. On the other hand, specific ATPase activity of the isolated membrane fraction was highly sensitive to vanadate. Fifty percent inhibition was measured at 3.8 (± 0.2) and 3.3 (±0.2) µm vanadate for adapted and nonadapted membrane,
respectively, and more than 95% inhibition was achieved at 500 µm vanadate (not shown). These data indicate that the
isolated membrane fraction from corn roots is highly enriched with
plasmalemma. This is in agreement with results of Faraday and Spanswick
(1992). Furthermore, it is also evident from Table II that
pHe during plant cultivation did not affect the
isolation of plasmalemma from corn roots by aqueous polymer two-phase
partitioning.
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Table II.
Effect of corn root adaptation to low
pHe on relative ATPase activities after treatment
with various inhibitors
Assays were conducted at pH 6.5. Membranes were isolated from corn
roots cultivated at pHe 6.0 and 3.5, respectively. The values represent means (± se) of four isolations.
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Effect of Low pHe during Plant Cultivation on
Plasmalemma ATPase Activity
To investigate the effect of low pHe on
plasmalemma ATPase activity, we measured the difference in specific
ATPase activities with and without 100 µm vanadate and
defined this difference as plasmalemma ATPase activity. In an assay pH
range from 5.6 to 6.2 the plasmalemma derived from corn roots grown at
pHe 3.5 showed about 20% higher ATPase activity
than that grown at pHe 6.0 (Fig. 2). This increase in plasmalemma ATPase
activity by low pHe during plant cultivation was
less pronounced at higher assay pH and vanished at assay pH 7.0. Figure
2 also indicates a small variation of pH optimum for ATPase activity
after adaptation to low pH. Maximum ATPase activity for roots grown at
pHe 3.5 was reached at assay pH 6.2, whereas
ATPase activity for control roots showed an optimum between 6.2 and
6.4. This is very close to the optimum pH 6.5 reported earlier for
plasmalemma H+-ATPase activity of corn roots (De
Michelis and Spanswick, 1986; Cowan et al., 1993). In addition,
plasmalemma ATPase of corn roots grown at
pHe 3.5 showed higher sensitivity to a change in
assay pH from 7.0 to 6.4. In this range the pH dependence of ATPase activity was 0.40 and 0.58 µmol Pi mg1
protein min1 for control and adapted roots,
respectively.
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| Figure 2.
Effect of low nutrient-solution pH during plant
cultivation on pH-dependent plasmalemma ATPase activity. Plasmalemma
was isolated from 3-week-old corn roots grown at pHe 6.0 () and 3.5 (). Plasmalemma ATPase activity is expressed as the
difference of activity assayed with and without 0.1 mm
Na3VO4. Values represent means ± se of four replications. *, Significant difference at 5%
level.
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Effect of Low pHe during Plant Cultivation
on the Kinetic Characteristics and the Activation Energy of
Plasmalemma ATPase
To gain a deeper insight into the effect of low
pHe on the enzymatic properties of corn root
ATPase, we analyzed the kinetic characteristics of plasmalemma ATPase
at ATP concentrations from 20 to 4000 µm. In this
concentration range plasmalemma ATPase revealed typical
Michaelis-Menten kinetics (Fig. 3), as
found before by other authors (Cowan et al., 1993). At an assay
temperature of 30°C, plasmalemma ATPase from
low-pHe (adapted) roots showed higher
Vmax and Km
than that from high-pHe (control) roots (Table III). This was found both at assay pH 6.5 and 7.0. However, at an assay temperature of 20°C, the difference
between treatments (pHe 3.5 and 6.0) became
insignificant. Because quantification of ATPase activity was based on
total membrane protein but not pure ATPase enzyme protein, it was not
evident whether the increase in Vmax was
caused by a relative increase in the amounts of ATPase molecules or in
the hydrolytic efficiency of the enzyme per se.
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| Figure 3.
Effect of low nutrient-solution pH during plant
cultivation on the kinetic characteristics of corn root plasmalemma
ATPase. Plasmalemma was isolated from 3-week-old corn roots grown at
pHe 6.0 () and 3.5 (). Activity of plasmalemma ATPase
was determined in an assay solution of pH 6.5, consisting of 100 mm KNO3, 1 mm NaN3, and
1 mm Na2MoO4. The concentration of
Mg-ATP in the assay solution was kept constant in the range of 20 to
4000 µm. The inset represents a double-reciprocal plot of
the effect of ATP concentration on ATPase activity. A representative
example of four replications is presented.
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Table III.
Effect of corn root adaptation to low
pHe on the kinetic characteristics of plasmalemma
ATPase
Membranes were isolated from 3-week-old corn plants grown at
pHe 6.0 and 3.5. Vmax and
Km were determined using ATP concentrations from
20 to 4000 µm. The values represent means (± se) of four isolations.
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Therefore, we calculated the activation energy of plasmalemma ATPase
according to the Arrhenius equation using
Vmax determined at both 20 and 30°C. At
assay pH 6.5 the activation energy of the plasmalemma ATPase derived
from corn roots grown at pHe 3.5 (107.0 ± 5.4 kJ mol1) was significantly higher than that
from control roots (93.5 ± 3.1 kJ mol1).
Similar to ATPase activity (Fig. 2), the difference in activation energy between plasmalemma ATPase from corn roots grown at
pHe 3.5 and those grown at
pHe 6.0 almost disappeared at assay pH 7.0 (not
shown). The fact that, despite increased activation energy, at assay pH
6.5 hydrolytic ATPase activity from pHe 3.5 roots under saturating substrate concentrations was higher than ATPase activity from pHe 6.0 roots implies that
adaptation of roots to low pHe increased the
number of ATPase molecules in the plasmalemma.
Phospholipids have been reported to stimulate plasmalemma ATPase
activity (Palmgren and Sommarin, 1989; Kasamo and Yamanishi, 1991).
Therefore, we determined the concentration of phospholipids and the
lipid-to-protein ratio of the corn root plasmalemma after differential
pHe treatment. The results presented in
Table IV reveal no difference in the
lipid-to-protein ratio or in the phospholipid-to-protein ratio. Also,
there was no significant effect on the phospholipid-to-lipid ratio
(Table IV).
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Table IV.
Effect of corn root adaptation to low
pHe on the lipid-to-protein, the phospholipid-to-protein,
and the phospholipid-to-lipid ratios in the plasmalemma
Ratios were calculated from concentrations determined in the
plasmalemma of 3-week-old corn roots grown at pHe 6.0 and
3.5, respectively. The values represent means (± se) of
four isolations.
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Effect of Low pHe during Plant Cultivation on
H+-Pumping Activity and Plasmalemma H+
Permeability
Plasmalemma H+-pumping activity was
monitored by the  A492 of AO. After
initiation of H+ pumping by addition of Mg-ATP
there was rapid quenching and it eventually reached a constant level
and completely collapsed by 5 µm gramicidin
(Fig. 4). In addition, this
H+-pumping activity was almost completely
inhibited by 500 µm vanadate (Fig. 4A). Compared with
control (Fig. 4B), absorbance quenching of AO caused by plasmalemma
vesicles from low pHe roots was more rapid at the
beginning and reached a higher level after 100 min (Fig. 4C). We used
initial rate and maximum quenching (pH gradient) to characterize the
plasmalemma H+ pump. The initial rate of
H+ pumping was determined according to the
quenching rate within the 1st min, which may reflect active
H+ influx into plasmalemma vesicles. Maximum
quenching was measured 100 min after initiation of the
H+ pump. At this time net
H+ transport across the plasmalemma was 0 and
H+ influx due to active pumping and
H+ efflux because of leakage reached equilibrium.
This parameter indicates the steepest pH gradient that can be created
by H+-pumping activity.
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| Figure 4.
Effect of low nutrient-solution pH during plant
cultivation on H+ transport of plasmalemma vesicles of
3-week-old corn roots. Plasmalemma vesicles were isolated from corn
roots grown at pHe 6.0 (B) and 3.5 (C). The pH gradient
formation was monitored by A492 of AO. At
assay solution pH 6.5, intravesicular acidification was initiated by
addition of 5 mm Mg-ATP. The pH gradient formation was
almost completely inhibited by 500 µm
Na3VO4 (A). The established pH gradient was
completely collapsed by 5 µm gramicidin (Gram).
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At assay pH 6.5 the initial rate of H+ pumping by
plasmalemma ATPase from low-pHe-adapted roots was
increased by 42.4% in comparison with control. This increase became
less pronounced when assay pH was increased to 7.0 (Table
V). Furthermore, at assay pH 6.5 the
plasmalemma from low-pHe roots created a 35.2%
steeper H+ gradient than control roots (Table V).
A less pronounced increase in pH gradient of
low-pHe root plasmalemma in comparison with control was measured when assayed at pH 7.0, a similar trend as found
for initial rate of H+ pumping.
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Table V.
Effect of corn root adaptation to low
pHe on H+ transport across plasma membrane
Membranes were isolated from 3-week-old corn roots grown at solution
pHe 6.0 and 3.5. The assay was conducted at 25°C and at
pH 6.5 and pH 7.0. The values represent means (± se) of
four independent isolations.
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For inside-out vesicles the establishment of a pH gradient is
determined by both active H+ influx (pumping) and
passive H+ efflux (leakage). To determine
H+ efflux from plasmalemma vesicles, we measured
the degradation of the pH gradient after stopping
H+ pumping by addition of 500 µm
vanadate. Because the degradation rate of pH gradient depends on the
gradient itself, a comparison between treatments should be made at the
same pH gradient. Therefore, we stopped H+
pumping when the pH gradient of plasmalemma vesicles reached 0.0900 A units. After addition of vanadate the established pH gradient was degraded quickly and then reached a relative constant level. This resting pH gradient was completely collapsed by gramicidin (Fig. 5). It is evident from Figure 5
that the gradient degradation was faster for
low-pHe roots (Fig. 5A) than for control roots (Fig. 5B). Two parameters were used to characterize the pH gradient degradation. The degradation rate within the 1st min after addition of
vanadate was measured as the initial rate to describe how rapidly the
pH gradient degradation starts, and t1/2, the
time in which half of the established pH gradient was degraded, was
determined to characterize the time course of degradation. Compared
with control, the initial degradation rate for plasmalemma vesicles from low-pHe roots was increased by 42%.
Furthermore, a 35% decrease in t1/2 was caused
by low-pHe treatment (Table V). Both parameters indicate a higher H+ permeability of plasmalemma
derived from low-pHe roots.
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| Figure 5.
Effect of low nutrient-solution pH during plant
cultivation on passive H+ transport across the plasmalemma
of corn roots. Plasmalemma was isolated from 3-week-old corn roots
grown at pH 3.5 (A) and 6.0 (B). The change of pH gradient across the
plasmalemma was monitored by A492 of AO.
Plasmalemma ATPase hydrolytic activity was initiated by addition of
Mg-ATP (5 mm) to create a pH gradient across plasmalemma vesicles. For a reliable comparison, ATPase activity was stopped by
addition of Na3VO4 (500 µm) after
quenching had reached 0.0900 A units for both membranes.
The resting pH gradient was collapsed by gramicidin (Gram, 5 µm).
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To investigate the coupling of ATPase hydrolytic activity with pumping
activity, we determined hydrolytic ATPase activity for 100 min under
the same conditions as for H+-pumping
measurement. Within 100 min the activity of plasmalemma H+-ATPase was very constant (Fig.
6), although there must have been a large
increase in pH gradient during this time (Fig. 4). This result
indicates that the hydrolytic activity of plasmalemma
H+-ATPase is independent of the pH gradient
across the membrane of plasmalemma vesicles. Furthermore, under this
condition only 13% stimulation of hydrolytic ATPase activity (0.53 and
0.47 µmol Pi mg1 min1
for pHe 3.5 and 6.0 vesicles, respectively) was
estimated, a result similar to that found for the kinetic parameters of
the enzyme assayed at 20°C (Table III). The fact that
low-pHe treatment caused differential stimulation
in H+-pumping activity (42% increase) and
hydrolytic activity (13% increase) suggests that
low-pHe treatment of corn root cells improved transport-coupling efficiency of plasmalemma
H+-ATPase.
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| Figure 6.
Time course of plasmalemma H+-ATPase
activity of 3-week-old corn roots grown at pH 6.0 () and 3.5 ().
The H+-ATPase activity of plasmalemma was assayed at 25°C
in a solution with 5 mm BTP/Mes (pH 6.5), 5 mm
Mg-ATP, 100 mm KCl, 7.5 µm AO, and 0.05%
(w/v) Brij 58. A representative example of four replications is
presented.
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DISCUSSION |
Adaptation of Corn Roots to Low Solution pH
In a previous study we reported that the growth rate of corn roots
was markedly inhibited by an abrupt decrease of
pHe (Yan et al., 1992). When, however, corn roots
were adapted to low pHe by gradually increasing
H+ activity in the root medium, corn roots
attained a growth rate at pHe 3.5, which was
comparable to that at pHe 6.0 (Table I). After
this adaptation period no apparent damage to corn root physiology or
morphology was observed. This is in contrast to faba beans, which
showed a marked decrease in root growth after identical adaptation
conditions (not shown). Therefore, it may be concluded that species
differences between corn and faba beans with respect to
low-pHe tolerance are not only constitutive but
also adaptive. As shown previously for constitutive
low-pHe tolerance (Yan et al., 1992), adaptive
low-pHe tolerance was associated with the ability
of corn roots to release higher amounts of protons into the external
medium (Fig. 1). Although it cannot be excluded that there was
additional release of protons that were stored in vacuoles during the
cultivation at low pHe (Fig. 1A), the fact that
higher net H+ extrusion was also observed at
pHe 3.4 (Fig. 1B) indicates that the major
contribution to low-pHe tolerance may be an
improvement of H+ pumping at low
pHe. This is in agreement with a tight
relationship between net H+ release and root
growth rate (Schubert et al., 1990; Yan et al., 1992).
Involvement of Plasmalemma H+-ATPase during
Adaptation of Corn Roots to Low Solution pH
In the present study we demonstrated the involvement of
plasmalemma H+-ATPase in adaptation of corn root
cells to low solution pH. This conclusion is supported by the following
results obtained at optimum assay pH in vitro: compared with control,
(a) the hydrolytic activity of plasmalemma
H+-ATPase of adapted root cells was increased by
about 20% (Fig. 2); (b) the initial rate of
H+-pumping activity of adapted plasmalemma was
enhanced by 42% (Table V; Fig. 4); (c) adapted plasmalemma
H+-ATPase created and maintained a 35% higher pH
gradient (Fig. 4; Table V); and (d) this higher pH gradient was not
explained in terms of a reduced H+ permeability
of this membrane; rather, low-pHe plasmalemma
showed 42% higher H+ permeability (Fig. 5; Table
V).
The possible mechanisms involved in the adaptation of plasmalemma
H+-ATPase to low-pHe
conditions may include (a) an increase in the number of
H+-ATPase enzymes per unit membrane, (b) a
modulation of the turnover rate of hydrolysis of this enzyme by lipid
environment change (Cooke and Burden, 1990), (c) a modification of the
autoinhibitory domain in the C terminus of
H+-ATPase (Palmgren et al., 1991), and (d)
differential expression of isoforms of this enzyme (Palmgren and
Christensen, 1994).
Although an increase in plasmalemma H+-ATPase
activity has been reported under a number of environmental conditions,
in most cases it is not clear whether the observed changes in
H+-ATPase activity reflect modulation of either
the amount or the turnover rate of hydrolysis of the enzyme (Serrano,
1989). Identifying these two components is a prerequisite for
understanding plant responses to environmental conditions on a
molecular basis. Therefore, in the present study we attempted to
separate these two components by additional measurement of activation
energy of this enzyme. Higher activation energy of
H+-ATPase in low-pHe
plasmalemma indicates a reduced substrate turnover rate of this enzyme.
The increase in hydrolytic activity of plasmalemma ATPase under
acid-stress conditions (Fig. 2) may thus be attributed to higher
abundance of H+-ATPase molecules in adapted
plasmalemma, not to higher substrate turnover rate per mole of enzyme.
This conclusion can be supported by a recent finding that the amount of
the H+-ATPase protein in plasmalemma and the
level of its mRNA transcript in Dunaliella acidophila (an
acid-tolerant alga) are far higher than in Dunaliella salina
(a salt-tolerant alga) and that the level of mRNA transcript of
H+-ATPase in both algae displays
pHe dependence (Weiss and Pick, 1996).
Plant plasmalemma H+-ATPase has an absolute
requirement for a lipid environment (Serrano et al., 1988). Although
the mechanism by which ATPase activity is activated is still uncertain,
it has been suggested that a direct lipid-protein binding is involved (Cooke and Burden, 1990). It has been demonstrated that a stimulation of plasmalemma H+-ATPase may be achieved by
phospholipids (Palmgren et al., 1988; Serrano et al., 1988; Kasamo and
Yamanishi, 1991). However, in the present study the stimulation of
H+-ATPase and H+-pumping
activities induced by low pHe was not mediated by
changes in lipid composition. Neither the ratio of phospholipid to
protein nor the ratio of phospholipid to total lipid of corn root
plasmalemma was changed by low-pHe treatment
(Table IV). This, of course, does not rule out the possibility that the
composition of individual phospholipids or fatty acids may be changed
by low-pHe treatment, which may be involved in
the modulation of H+-ATPase activity (Palmgren et
al., 1988).
It is well established that a part of the C-terminal region of
H+-ATPase constitutes an autoinhibitory domain. Removal of
this domain by fusicoccin, lysophospholipids, and trypsin can activate the enzyme (Palmgren et al., 1991; Johansson et al., 1993). The activation effect of lysophospholipids on the plasmalemma
H+-ATPase is of physiological relevance because a
phospholipase that generates lysophosphatidylcholine from
phosphatidylcholine is found in the plant plasmalemma (Palmgren et al.,
1988; Palmgren and Sommarin, 1989). Therefore, an attractive hypothesis
for adaptation of corn root cells to low pHe may
be a stimulation of phospholipase in plasmalemma by low pH, resulting
in the release of lysophospholipids that may interact with
H+-ATPase to remove the autoinhibitory domain.
Lysophospholipids may induce several effects on plasmalemma
H+-ATPase (Palmgren et al.,1988; Palmgren and
Sommarin, 1989): (a) a higher degree of stimulation in
H+ pumping than in hydrolytic ATPase activity,
(b) an increase in Vmax and a decrease in
Km, (c) a shift of pH optimum toward more alkaline values, or (d) sensitivity of the enzyme to vanadate remains
unchanged. After adaptation to low-pHe, plasmalemma
H+-ATPase showed a 20% higher hydrolytic
activity and 42% higher H+-pumping activity
(Figs. 2 and 4; Table V). This may indicate a changed degree of
coupling between ATP hydrolysis and H+ pumping
(Baunsgaard et al., 1996). In addition, adapted
H+-ATPase displayed the same sensitivity to
vanadate as the control (Table II). These results are compatible with
effects (a) and (d) induced by lysophospholipids. However, in
comparison with control, adapted plasmalemma
H+-ATPase showed increased
Km (Table III) and a slight shift of pH optimum toward more acid values (Fig. 2). These changes in
H+-ATPase in adapted plasmalemma are not
consistent with effects (b) and (c), which are caused by
lysophospholipids.
Alternatively, differential expression of
H+-ATPase isoforms may be responsible for the
adaptation of plasmalemma H+-ATPase to low
pHe. Palmgren and Christensen (1994) compared
functions between plant plasmalemma H+-ATPase
isoforms expressed in yeast and found a significant difference in
Km, turnover rate for ATP hydrolysis, and
vanadate sensitivity between investigated isoforms. Existence of
biochemical heterogeneity was reported for native corn root plasmalemma
H+-ATPase (Gallagher and Leonard, 1987; Grouzis
et al., 1990). The increase in Km of
low-pHe plasmalemma
H+-ATPase may imply differential expression of
H+-ATPase isoforms in the plasmalemma as a
response of the root cells to low pHe. With
higher ATP supply in vivo (Yan et al., 1992) more efficient isoforms
may significantly contribute to H+ extrusion
against a steeper H+ gradient across the
plasmalemma of corn root cells exposed to low
pHe.
| |
FOOTNOTES |
1
This work was supported by German Science
Foundation grant Schu 589/5-1.
2
Present address: Institute of Plant Nutrition,
Justus Liebig University, Suedanlage 6, D-35390 Giessen, Germany.
*
Corresponding author; e-mail
sven.schubert{at}ernaehrung.uni-giessen.de; fax 49-641-99-39-169.
Received November 17, 1997;
accepted February 18, 1998.
| |
ABBREVIATIONS |
Abbreviations:
AO, acridine orange.
BTP, 1,3-bis[tris(hydroxylmethyl)-methylamino]propane.
pHe, external pH.
| |
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Top
Abstract
Introduction