Proton Transport in Maize Tonoplasts Supported by
Fructose-1,6-Bisphosphate Cleavage. PyrophosphateDependent Phosphofructokinase as a PyrophosphateRegenerating System1
Anelise Costa dos Santos, Wagner Seixas da-Silva, Leopoldo de Meis, and Antonio Galina*
Departamento de Bioquı́mica Médica, Instituto de Ciências Biomédicas, Universidade Federal do Rio de
Janeiro, Rio de Janeiro, 21941–590, RJ, Brazil
The energy derived from pyrophosphate (PPi) hydrolysis is used to pump protons across the tonoplast membrane, thus
forming a proton gradient. In a plant’s cytosol, the concentration of PPi varies between 10 and 800 ␮m, and the PPi
concentration needed for one-half maximal activity of the maize (Zea mays) root tonoplast H⫹-pyrophosphatase is 30 ␮m. In
this report, we show that the H⫹-pyrophosphatase of maize root vacuoles is able to hydrolyze PPi (Reaction 2) formed by
Reaction 1, which is catalyzed by PPi-dependent phosphofructokinase (PFP):
Fructose-1,6-bisphosphate (F1,6BP) ⫹ Pi 7 PPi ⫹Fructose-6-phosphate (F6 P)
PPi
H
⫹
cyt
F1,6BP ⫹ H
⫹
cyt
3 2 Pi
3 H
7 H
⫹
⫹
(reaction 2)
(reaction 3)
vac
vac
(reaction 1)
⫹ F6P ⫹ Pi
(reaction 4)
During the steady state, one-half of the inorganic phosphate released (Reaction 4) is ultimately derived from F1,6BP, whereas
PFP continuously regenerates the pyrophosphate (PPi) hydrolyzed. A proton gradient (⌬pH) can be built up in tonoplast
vesicles using PFP as a PPi-regenerating system. The ⌬ pH formed by the H⫹-pyrophosphatase can be dissipated by addition
of 20 mm F6P, which drives Reaction 1 to the left and decreases the PPi available for the H⫹-pyrophosphatase. The maximal
⌬ pH attained by the pyrophosphatase coupled to the PFP reaction can be maintained by PFP activities far below those found
in higher plants tissues.
Plants and some protozoans of clinical importance
possess a PPi-dependent phosphofructokinase (PFP;
EC 2.7.1.90) that converts Fru-6-phosphate (F6P) to
Fru-1,6-bisphosphate (F1,6BP) and inorganic phosphate (Pi) using PPi as phosphoryl donor. In contrast
to the reaction catalyzed by the isoform that uses
ATP as substrate (ATP-dependent phosphofructokinase, EC 2.7.1.11), the equilibrium constant (Keq) of
the reaction catalyzed by PFP is close to 1 and is,
therefore, readily reversible (O’Brien et al., 1975;
Kombrink et al., 1984). PFP is activated by Fru-2,6bisphosphate (F2,6BP), its allosteric modulator. The
1
This work was supported by Programa de Apoio a Núcleos de
Excelência-PRONEX-Financiadora de Estudos e Projetos (grant),
by Conselho Nacional de Desenvolvimento Cientı́fico e Tecnológico (grant and fellowship to A.C.d.S.), and by Fundação de
Amparo à Pesquisa do Estado do Rio de Janeiro (grant and fellowship to W.S.d.-S.).
* Corresponding author; e-mail [email protected]; fax
55–21–2270 – 8647.
Article, publication date, and citation information can be found
at www.plantphysiol.org/cgi/doi/10.1104/pp.103.026633.
cytosolic concentration of F2,6BP can fluctuate depending on the environmental conditions or the level
of PFP expression in plant tissues (Hajirezaei et al.,
1994; Nielsen and Stitt, 2001). Attempts have been
made to ascertain whether PFP plays a role in glycolysis by converting F6P to F1,6BP; in a number of
cases, this has been shown convincingly (Smyth and
Chevalier, 1984; Duff et al., 1989). However, other
studies demonstrated that PFP is unable to support
glycolysis in vivo because it operates as a gluconeogenic enzyme (ap Rees et al., 1985; Black et al., 1987;
Paul et al., 1995). Therefore, there is no consensus
about the metabolic function of this enzyme.
Plants, protozoans, and certain bacteria have a
membrane-bound H⫹-translocating inorganic pyrophosphatase (H⫹-PPase) that works as a proton
pump. This enzyme couples the hydrolysis of PPi
with electrogenic translocation of protons across the
membrane (Baykov et al., 1999). In plants, this enzyme is located in the tonoplast membrane, where it
supports vacuolar functions, such as preventing the
harmful accumulation of Ca2⫹ and Na⫹ in the cytosol
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885
Costa dos Santos et al.
and allowing storage of organic metabolites that can
be recovered by the cytosol when needed by the H⫹energized tonoplast membranes. The cytosolic PPi
concentration in plant cells is approximately 200 ␮m
(Weiner et al., 1987; Maeshima, 2000). This concentration is sufficient to promote the maximal activity of
vacuolar H⫹-PPase in plant cells (Maeshima, 2000).
The PPi concentration is determined by the balance
between the rates of PPi hydrolysis and synthesis. The
hydrolysis of PPi is catalyzed by the H⫹-PPase (Davies
et al., 1993). On the other hand, PPi is synthesized as a
by-product of various biosynthetic reactions such as
the synthesis of nucleic acids, polysaccharides, coenzymes, and proteins; activation of fatty acids; and
isoprenoid synthesis. Under stress conditions, such as
anoxia, the biosynthetic reactions are slowed down,
Pi⫹F1,6BP 7 PPi⫹F6P
thereby limiting the rate of PPi formation. Thus, the
use of the reaction catalyzed by PFP in the direction of
PPi formation may be an auxiliary mechanism to prevent a drop in the cytosolic levels of PPi under the
metabolic conditions of the living cell (Davies et al.,
1993). As far as we know, this proposal has never been
demonstrated experimentally.
The mass to action ratio measured for the substrates and products of PFP in plants is close to its Keq
value (Kubota and Ashihara, 1990). This implies that
in the plant cytosol, the direction of the reaction may
be determined by the [PPi] to [Pi] ratio and that the
PPi formed at the expense of F1,6BP could ultimately
be used to pump H⫹ across the tonoplast membrane
as follows:
(reaction 1)
⌬Go ⫽ ⫹0.7 kcal mol⫺1
(reaction 2)
⌬Go ⫽ ⫺5.1 kcal mol⫺1
H⫹cyt 7 H⫹vac (⌬pH ⫽ 2)
共reaction 3)
⌬G ⫽ ⫹1.5 kcal mol⫺1
F1,6BP ⫹ H⫹cyt 3 F6P ⫹ Pi⫹H⫹vac
(reaction 4)
⌬Go ⫽ ⫺2.9 kcal mol⫺1
PPi 3 2Pi
release of Pi when the two enzymes needed to regenerate PPi or the substrates F1,6BP and Pi were omitted from the assay medium. The rate of cleavage
varied with the concentration of F1,6BP (Fig. 1B), and
the half-maximal rate was observed with 123 ␮m
F1,6BP. PFP and H⫹-PPase both require Mg2⫹ for
their activities (Maeshima and Yoshida, 1989; Givan,
1999; Maeshima, 2000). Magnesium was needed for
the coupled reaction in Figure 1; the maximal rate of
F1,6BP cleavage was obtained in the presence of 0.6
to 1 mm MgCl2. Higher MgCl2 concentrations were
found to be inhibitory (Fig. 1A). The findings of
Figure 1 indicate that the true substrate for the maize
root H⫹-PPase was the very low PPi concentration
derived from Reaction 1 and that the Pi released into
the medium originated from F1,6BP by the action of
PFP, which continuously regenerated the PPi hydrolyzed (Reactions 1 and 2).
The aim of this study was to evaluate whether the
maize (Zea mays) root H⫹-PPase is able to bind and
hydrolyze the PPi formed by Reaction 1 catalyzed by
PFP in vitro under conditions that mimic the concentrations of F1,6BP, Pi, Mg2⫹, F2,6BP, and F6P found in
the plant cell cytosol (Rebeille et al., 1983; Ukaji and
Ashihara, 1987; Weiner et al., 1987; Kubota and Ashihara, 1990; Davies et al., 1993; Stitt, 1998; Table I).
Here, we show that the affinity of maize root H⫹PPase for PPi under these conditions is sufficient to
shift Reaction 1 toward PPi formation, making it possible to build up a proton gradient in the tonoplast
even in the presence of very low amounts of PFP.
RESULTS
Cleavage of F1,6BP, pH, and Mg2ⴙ Dependence
PFP and maize root H⫹-PPase catalyzed the release
of Pi from F1,6BP (Fig. 1). There was no measurable
Table I. Levels of metabolites in plant cytosol; PFP and H⫹-PPase activities from different plants and experimental conditions used in this study
Plants and Conditions
Metabolites in Plant Cytosola
F1,6BP
Pi
Fru-6P
Enzyme Activity Level
PPi
H⫹-PPasec
milliunits g fresh wt⫺1
mM
Wild-type plants
Antisense transformant plants (lower levels)
Experimental conditions (Figs. 1, 2, and 4)
PFPb
0.6
–
5
–
1
–
0.33
–
293–1,813
20 –58
15–25
–
–
–
–
–
0.5– 60d
18 –25d
a
The concentrations of F1,6BP, Pi, and F6P are those reported in the literature for plant cell cytosol (Rebeille et al., 1983; Ukaji and Ashihara,
b
1987; Weiner et al., 1987; Kubota and Ashihara, 1990; Davies et al., 1993; Stitt, 1998).
The PFP activities for wild-type and antisense
transformant tobacco (Nicotiana tabacum) plants were taken from Nielsen and Stitt (2001) for antisense transformant potato (Solanum tuberosum)
c
plants. Values are from Hajirezaei et al. (1994).
The H⫹-PPase activities for wild-type mung bean, tomato (Lycopersicon esculentum) fruit,
d
and maize leaves were taken from Maeshima and Yoshida (1989), Milner et al. (1995), and Clayton et al. (1993), respectively.
The PFP and
⫹
⫺1
H -PPase activities shown in this report were converted to milliunits per gram fresh wt computing the enzyme activity level (milliunits) present
in 1 mL of reaction medium. This volume was converted to grams to obtain the activities shown.
886
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Plant Physiol. Vol. 133, 2003
Tonoplast Proton Transport Coupled to F1,6BP Cleavage
Figure 1. Cleavage of F1,6BP and Mg2⫹ dependence. The F1,6BP cleavage was dependent on MgCl2 (A) and F1,6BP (B)
concentrations. The MgCl2 dependence was measured in a medium containing: 50 mM MOPS-Tris buffer (pH 7.0), 60
milliunits mL⫺1 mung bean (Vigna radiata) PFP, 0.1 mg mL⫺1 maize vacuolar microsomal protein, 100 mM KCl, 2 ␮M
F2,6BP, and 10 mM F1,6BP (A). When the F1,6BP dependence was measured, the MgCl2 concentration was fixed at 0.6 mM
(B). The reaction was performed at 30°C and started by addition of 0.1 mM Pi. Aliquots of 0.8 mL were removed, and the
reaction was stopped by the addition of 0.2 mL of 50% (w/v) trichloroacetic acid. The figure shows a representative
experiment. Similar results were obtained with three different vesicle preparations.
The concentration of PPi that is attained when the
PPi-regenerating reaction reaches a steady state varied depending on the pH of the medium. The rate of
F1,6BP cleavage was maximal in the pH range of 6.0
to 7.0 and decreased by 50% when the pH was raised
from 7.0 to 8.0 (data not shown).
Transmembrane Proton Gradient
The assay in Figure 2 shows that the energy derived from the steady-state cleavage of F1,6BP was
sufficient to form and maintain a transmembrane
proton gradient in tonoplast vesicles. In the presence
of PFP, the addition of F2,6BP, Pi, and F1,6BP promoted a quenching of ACMA fluorescence, indicating that a proton gradient was formed across the
tonoplast membrane (Fig. 2A). The gradient was
abolished when 1 ␮m FCCP, a proton ionophore, was
added to the medium (Fig. 2, A and B).
The proton gradient formed depended on the concentration of Pi present in the reaction medium (Fig.
2B). In the presence of 30 ␮m Pi, a small proton
Figure 2. Membrane proton gradient supported by PFP, F1,6BP, and Pi. The assay conditions were identical to those
described in Figure 1B, except that the 9-amino-6-chloro-2-methoxyacridine (ACMA; 3 ␮M) was present and F1,6BP,
F2,6BP, and Pi concentrations are listed below for each panel. At the end of gradient formation, 1 ␮M carbonyl cyanide
p-(trifluoromethoxy)phenylhydrazone (FCCP) was added to dissipate the H⫹ gradient in A and B. In C, the addition of PFP
activator, F2,6BP, enhanced the proton gradient formation, and the addition of 20 mM F6P dissipated it. The arrows indicate
the sequential addition of: A, 25 milliunits of PFP, 2 ␮M F2,6BP, 2 mM Pi, and 0.6 mM F1,6BP; B, 30 ␮M Pi was included
in the reaction medium before the sequential addition of: 25 milliunits of PFP; 4 ␮M F2,6BP; 0.6 mM F1,6BP, and 2 mM Pi;
C, 10 milliunits of PFP, 2 mM Pi, 0.1 mM F1,6BP, 4 ␮M F2,6BP, and 20 mM F6P. The figure shows a representative experiment.
Similar results were obtained in three different vesicle preparations.
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887
Costa dos Santos et al.
gradient was observed after the addition of 0.1 mm
F1,6BP (Fig. 2B). Further addition of Pi (2 mm), promoted both an increase in the initial rate of proton
pumping and an enhancement of the proton gradient. This result suggests that the addition of Pi to the
medium displaces Reaction 1 toward PPi formation,
increasing its steady-state concentration.
The capacity of the system to form the proton
gradient was highly dependent on the presence of
the PFP activator F2,6BP (Fig. 2C). A slow rate of
proton pumping was measured in presence of 2 mm
Pi, 0.1 mm F1,6BP, and 10 milliunits of PFP. However,
when 2 ␮m F2,6BP was added to the medium, the
rate of proton pumping was accelerated by more
than 2-fold. This result confirms that the rate of the
PPi formation by PFP is stimulated by F2,6BP and
that the proton gradient is also dependent on the rate
of PPi formation (Fig. 2C). Finally, the transmembrane proton gradient was abolished when 20 mm
F6P was added to the medium, indicating that when
the equilibrium is driven toward F1,6BP formation,
the steady-state concentration of PPi available to the
maize root H⫹-PPase is greatly reduced, a condition
that led to a decrease of the H⫹ gradient.
Equilibrium Concentrations of PPi Formed from F1,6BP
and Pi and PPi Dependence of Maize Root Hⴙ-PPase
The equilibrium concentration of PPi formed in the
reaction catalyzed by PFP varied depending on the
initial concentrations of F1,6BP, Pi, and F6P. To evaluate if the range of PPi concentrations formed in our
experiments was close to that needed for one-half
maximal activation of the maize root H⫹-PPase, the
PPi concentration was calculated in two ways: by
using the concentrations of substrates and products
found in plant cytosol (Kubota and Ashihara, 1990;
Davies et al., 1993) or by using the results obtained
under the conditions of the experiments of Figure 2.
In addition, we measured the apparent affinity of
maize root H⫹-PPase for PPi (Fig. 3). Table II shows
the PPi concentrations that would be formed under
these different conditions. These values were calculated using the Keq of 0.31 (Kubota and Ashihara,
1990) for the reaction catalyzed by PFP.
The PPi concentration needed for one-half maximal
rate activity of maize root H⫹-PPase was 3.2 ⫻ 10⫺5
m (Fig. 3). This value is similar to that previously
reported by Maeshima (2000), is 10-fold lower than
the PPi equilibrium concentrations calculated in Table II (conditions A and B), and is close to the equilibrium values calculated for condition C. This means
that the rate of proton pumping (Fig. 2) responds
readily to the equilibrium of the reaction catalyzed
by PFP. The addition of 20 mm F6P (Fig. 2C) reduces
the PPi concentration to such a low level (Table II,
condition D) that can no longer be used by the maize
root H⫹-PPase to accumulate H⫹ inside the tonoplast
vesicles.
888
Figure 3. PPi saturation kinetics of maize root H⫹-PPase. The assay
medium composition was 50 mM MOPS-Tris (pH 7.0), 100 mM KCl,
0.6 mM MgCl2, 3 ␮M FCCP, and 0.030 mg mL⫺1 maize vesicle
protein. The PPi concentrations are shown on the abscissa. The
reaction was started by addition of vesicles. The amount of PPi
hydrolyzed never exceeded 5% of the total PPi added to the medium.
The kinetic parameters shown in the figure were calculated from a
nonlinear regression analysis applied to the Hill equation using the
program ENZFITTER. The specific activities shown are means ⫾ SE of
at least four independent preparations.
PFP Dependence for Formation of the Transmembrane
Proton Gradient
The rate of vacuolar proton pumping varied depending on the amount of PFP added to the reaction
medium (Fig. 4). The proton pumping was blocked
by 12 mm fluoride, an anion that inhibits the vacuolar
maize root H⫹-PPase (Fig. 4, black circles). The assay
condition of 2 mm Pi, 0.5 mm F1,6BP, 2 ␮m F2,6BP,
and 18 milliunits of PFP was sufficient to stimulate
the proton pumping to a level similar to that observed using 0.3 mm PPi, a saturating concentration
(Fig. 4, white circles and dotted line). The amount of
PFP used in Figure 4 is very small and is close to the
levels observed in antisense transformant plants in
which more than 95% of the total PFP activity has
been abolished (Hajirezaei et al., 1994; Nielsen and
Stitt, 2001).
DISCUSSION
In this report, the capacity of maize root vacuolar
H⫹-PPase to use the PPi formed at equilibrium by
PFP was evaluated. It was found that in the presence
of Mg2⫹, Pi, F1,6BP and F2,6BP concentrations similar
to those available in plant cytosol (Rebeille et al.,
1983; Ukaji and Ashihara, 1987; Weiner et al., 1987;
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Plant Physiol. Vol. 133, 2003
Tonoplast Proton Transport Coupled to F1,6BP Cleavage
Table II. PPi formation in presence of F1,6BP, Pi, and F6P
After PPi formation catalyzed by PFP, PPi is hydrolysed by H⫹ PPase, forming a pH gradient. The respective free energy change (⌬G1 and ⌬G2)
are calculated. In the table, the concentrations of PPi found after the reaction F1,6BP ⫹ Pi 3 PPi ⫹ F-6P (reaction 1, ⌬G1) reaches equilibrium
were calculated using the value of 0.31 for the apparent equilibrium constant (Keq) as previously described (Kubota and Ashihara, 1990). The
equilibrium PPi concentrations were calculated using the following equation: 关PPi兴 ⫽ 关F1,6BP兴 ⫻ 关Pi兴 ⫻ Keq/关F6P兴. The PPi formed in the presence
of 0.6 mM MgCl2 is further hydrolyzed by the H⫹-PPase in the following reaction: H⫹cyt ⫹ PPi 3 2Pi ⫹ H⫹vac (reaction 2, ⌬G2). The ⌬G° ⫽ ⫺5.1
kcal mol⫺1 was taken from the values measured by de Meis (1984) in the presence of 0.6 mM MgCl2 to calculate the ⌬G2.
Conditions
⌬G
Concentrations
F1,6BP
PI
F6P
PPi formed
⌬G1
a
kcal mol
M
Ab
Bc
Cc
Dc
6⫻10⫺4
6⫻10⫺4
6⫻10⫺4
1⫻10⫺4
5⫻10⫺3
2⫻10⫺3
3⫻10⫺5
2⫻10⫺3
⌬G2a
1⫻10⫺3
3⫻10⫺5
3⫻10⫺5
2⫻10⫺2
3.3⫻10⫺4
3.5⫻10⫺4
2.3⫻10⫺5
3.0⫻10⫺6
⫺0.6
⫺2.1
⫺1.3
⫺0.02
⫺3.9
⫺5.0
⫺8.4
⫺2.2
⌬G1a⫹ 2
⫺1
⫺4.5
⫺7.2
⫺9.7
⫺2.2
a
The ⌬G1 values (kilocalories per mole) for different conditions were calculated from the equation ⌬G1 ⫽ ⌬G° ⫹ R⌻ ln 关PPi兴 ⫻ 关F6P兴/关F1,6BP兴
⫻ 关Pi兴 using the concentrations shown in the table, and ⌬G2 values were calculated from the equation ⌬G2 ⫽ ⌬G° ⫹ R⌻ ln 关Pi兴2 ⫻ 关H⫹vac兴/关PPi兴
⫻ 关H⫹cyt兴, assuming a ⌬pH of 2 units. The final concentration of F6P derived from contamination of F1,6BP and its hydrolysis was estimated to
be 5% (3 ⫻ 10⫺5 M). This concentration of F6P was used to calculate the concentration of PPi formed in experiments in which extra amounts
b
of F6P were not added to the assay medium (conditions B and C).
The molar concentrations of F1,6BP, Pi, and F6P are those reported in
the literature for plant cell cytosol (Rebeille et al., 1983; Ukaji and Ashihara, 1987; Weiner et al., 1987; Kubota and Ashihara, 1990; Davies et
c
al., 1993; Stitt, 1998).
The molar concentrations of F1,6BP, Pi, and F6P are those used in Figure 2.
Kubota and Ashihara, 1990; Stitt, 1998), the PPi
formed by even a small amount of PFP was sufficient
to activate the vacuolar H⫹-PPase and to form a
proton gradient across the tonoplast membrane. This
reaction is coupled to a decrease in the F1,6BP concentration, a compound that has a lower energy of
hydrolysis than PPi (de Meis, 1989, 1993). Table II
shows that the free energy change (associated with
Reaction 4) is markedly negative (Table II, ⌺⌬G1 ⫹ 2
varying from ⫺2.19 to ⫺9.68 kcal mol⫺1), despite
different levels of PPi formed under the various conditions used to calculate the equilibrium PPi concentration. Even when the PPi concentration was as low
as 3 ␮m (Table II, condition D), the calculated ⌺⌬G1 ⫹
⫺1
2 was ⫺2.19 kcal mol
, a value that indicates a
favorable shift to PPi hydrolysis. In maize roots tips,
the contribution to the steady-state level to PPi in the
cytosol of only one of the reactions that produce PPi,
the formation of UDP-Glc from Glc-1 P and UTP, was
estimated to be 10 ␮m (Roberts, 1990) a value that is
more than 3 times higher than that used to obtain a
value of ⌺⌬G1 ⫹ 2 of ⫺2.19 kcal mol⫺1 (Table II,
condition D). This means that there are no thermodynamic constraints for the pumping of protons into
the vacuole lumen using F1,6BP, Pi and 20 mm F6P to
achieve the equilibrium concentration of PPi of 3 ␮m.
The observation that the protons leak out of the
vesicles when 20 mm F6P is added to the medium
(Fig. 2C) is related to the decrease of the steady-state
PPi concentration to a level far below that needed by
the tonoplast H⫹-PPase (Fig. 3).
Based on reported concentrations of glycolytic intermediates of plant cells (Kubota and Ashihara,
1990), the ⌬G for the formation of PPi (Reaction 1;
Kubota and Ashihara, 1990) and the ⌬G of PPi hydrolysis (Reactions 2 and 3; de Meis, 1984; Davies et
al., 1993), it was possible to calculate the overall ⌬G
Plant Physiol. Vol. 133, 2003
Figure 4. PFP dependence for the formation of the membrane proton
gradient. The reaction was carried out in medium containing 10 mM
MOPS-Tris (pH 7.0), 3 ␮M ACMA, 1 mM MgCl2, 100 mM KCl, 2 ␮M
F2,6BP, 2 mM Pi, 0.1 mg mL⫺1 maize vacuolar microsomal protein,
and 0.6 mM F1,6BP. The initial rate of H⫹ pumping was taken in the
linear phase of fluorescence quenching of ACMA and was expressed
as percentage ⌬F per minute after the addition of 1, 3, 8, and 18
milliunits of PFP to the cuvette (2 mL) in the absence (E) or in the
presence (F) of 12 mM NaF or 2 ␮M FCCP (⌬). The figure shows a
representative experiment. Similar results were obtained in three
different vesicle preparations. The dotted line represents the maximal
rate of H⫹ pumping measured when 0.3 mM PPi was added to the
reaction mixture in the absence of the PPi-PFP regenerating system.
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889
Costa dos Santos et al.
for the glycolytic conversion of F1,6BP to either pyruvate or PPi and the subsequent hydrolysis of PPi by
the H⫹-PPase needed to maintain a ⌬pH of 2 units
across the tonoplast membrane. From these estimates, it becomes apparent that thermodynamic pull
for the utilization of F1,6BP toward H⫹ pumping
(⌬G1 ⫹ ⌬G2 in Fig. 5) is slightly more favorable than
the formation of pyruvate (⌬G3 ⫹ ⌬G4 in Fig. 5). The
readily reversible PFP reaction may provide an adaptive pathway for glycolysis and gluconeogenesis in
higher plant cells (Black et al., 1987). The result
shown in Figure 2 suggests a new role of PFP, the
formation of PPi to be used for the formation of a
proton gradient in tonoplast vesicles.
One of the possible limiting factors for the utilization of F1,6BP and Pi to form PPi would be the
amount of PFP and/or H⫹-PPase present in the cells.
A simultaneous up-regulation in the levels of H⫹PPase and PFP have been detected during anoxia in
rice (Oryza sativa) seedlings (Mertens et al., 1990;
Carystinos et al., 1995; Drew, 1997). Table I shows
that the levels of PFP and H⫹-PPase activities detected in different plants (Hajirezaei et al., 1994;
Nielsen and Stitt, 2001) were sufficient in our assays
to promote the formation of a H⫹ gradient. We found
that it was possible to detect a proton gradient in
tonoplast vesicles even in the presence of a PFP activity similar to that observed in antisense transformant plants (Hajirezaei et al., 1994; Nielsen and Stitt,
2001). The H⫹-PPase levels used in our experiments
are similar to those detected in maize roots and other
plants (Table I). These data suggest that, given the
low ⌬G values of Reactions 1 and 2 (Fig. 5), the low
ratio in the activity levels of PFP to H⫹-PPase would
limit the vacuolar proton pumping when the concentration of PFP decreases to levels below 4 milliunits g
fresh weight⫺1. In recent reports (Hajirezaei et al.,
1994; Nielsen and Stitt, 2001) using tobacco and potato plant transformants that lead up to a 95% reduction in the PFP activity (see also Table I), there were
no major changes in carbon fluxes or leaf or plant
growth. Nevertheless, there was a compensatory increment in the F2,6BP levels both in tobacco leaves
and potato tubers (Hajirezaei et al., 1994; Nielsen and
Stitt, 2001). In our study, the levels of PFP activity are
similar to those detected in tobacco and potato plant
transformants (Fig. 4; Table I). This raises the possibility that under steady-state conditions in the plant
cytosol, different enzymes involved in energy transduction, such as maize root vacuolar H⫹-PPase, may
use phosphate compounds with a low energy of hydrolysis (de Meis, 1989, 1993; de Meis et al., 1992a,
1992b; Montero-Lomeli and de Meis, 1992; Galina et
al., 1995) to perform the work that is usually associated with the consumption of higher energy phosphate compounds such as PPi (Romero and de Meis,
1989).
MATERIALS AND METHODS
Materials and Chemicals
All chemicals were obtained from Sigma (St. Louis). PPi:Fru-6-phosphate
1-phosphotransferase was from mung bean (Vigna radiata; Sigma F-8757).
FCCP was dissolved in ethanol. The amounts used were such that the
Figure 5. Free-energy changes (⌬G) associated with the conversion of F1,6BP to build up a H⫹ gradient in tonoplast vesicles
or with conversion to pyruvate by the glycolytic pathway. After Glc or Fru phosphorylation, the F1,6BP formed can be
diverted to PPi synthesis by PPi-PFP (⌬G1), and the PPi is hydrolyzed by H⫹-PPase to build up a proton gradient of 2 units
of pH in the tonoplast vesicles (⌬G2). Alternatively, the F1,6BP is converted into triose-P by aldolase (⌬G3). The triose-P is
converted by glycolytic enzymes to pyruvate (⌬G4). ⌬G1 and ⌬G2 were calculated as shown in Table II (condition A) using
concentrations in the plant cell cytosol. The ⌬G3 and ⌬G4 were calculated from glycolytic reactions described by Kubota
and Ashihara (1990).
890
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Plant Physiol. Vol. 133, 2003
Tonoplast Proton Transport Coupled to F1,6BP Cleavage
ethanol concentration in the reaction mixture was never higher than 0.5%
(v/v). At this concentration, ethanol had no effect on either the H⫹ gradient
or PPi hydrolysis.
Preparation of Plant Material and Isolation of
Microsomes from Maize Root Cells
Maize (Zea mays) seeds were surface sterilized with sodium hypochlorite
(approximately 10 min in a 3% [v/v] solution), then washed with sterile
water and soaked in water for 24 h. Radicles were harvested for preparation
of vesicles from seeds allowed to germinate for 5 d on wet filter paper in the
dark at 28°C.
Vacuolar membrane vesicles were isolated from whole roots using differential centrifugation. Roots (approximately 100 g wet weight) were homogenized in an IKA-Euroturrax T25 basic (speed 3 for 15 s using an
S25N-18G probe, Wilmington, NC) with 200 mL of an ice-cold extraction
buffer containing 10% (v/v) glycerol, 0.5% (v/v) polyvinylpyrrolidone-40,
0.13% (w/v) bovine serum albumin, 5 mm EDTA, and 0.1 m Tris-HCl (pH
8.0). Just before use, 3.3 mm dithiothreitol, 150 mm KCl, and 1 mm phenylmethylsulfonyl fluoride (final concentrations) were added to the buffer. The
homogenate was strained through four layers of cheesecloth and centrifuged at 8,000g for 20 min. The supernatant was centrifuged at 100,000g for
40 min. The pellet was resuspended in 80 mL of the extraction buffer and
centrifuged once more at 100,000g for 40 min. The pellet was resuspended in
a small volume of ice-cold buffer containing 10 mm Tris-HCl (pH 7.6), 10%
(v/v) glycerol, 1 mm EDTA, and 1 mm dithiothreitol. The vesicles were
frozen under liquid N2. Protein concentrations were determined by the
method of Lowry et al. (1951).
Measurement of F1,6BP Cleavage
The cleavage of F1,6BP was measured colorimetrically at 30°C by determining the rate of liberation of Pi (Fiske and Subbarow, 1925) in reaction
media containing: 50 mm MOPS-Tris buffer (pH 7.0), 60 milliunits mL⫺1
mung bean PFP, 0.1 mg mL⫺1 maize vacuolar microsomal protein, 100 mm
KCl, 2 ␮m F2,6BP, 0.6 mm MgCl2, and different F1,6BP concentrations. When
the MgCl2 dependence was measured, the F1,6BP was fixed at 10 mm. When
the pH dependence was measured, 50 mm MOPS-Tris and 50 mm glycyl-Gly
buffer were added (pH from 6.0–8.5), and the concentrations of MgCl2 and
F1,6BP were 1 and 5 mm, respectively. The reaction was started by the
addition of 0.1 mm Pi. Aliquots of 0.8 mL were removed and the reaction
was stopped by addition of 0.2 mL of 50% (w/v) trichloroacetic acid.
Reaction times were adjusted so as to limit the cleavage of F 1,6 BP to ⬍10%.
Cleavage was linear with time under these conditions.
Measurement of PPi-Dependent Proton Transport into
Maize Tonoplast Vesicles
The accumulation of H⫹ by the vesicles was determined by measuring
the fluorescence quenching of ACMA (Molecular Probes, Eugene, OR) using
a spectrofluorimeter (model F-3010, Hitachi, Tokyo). The excitation wavelength was set at 415 nm, and the emission wavelength was set at 485 nm.
The reaction was carried out in 2 mL of medium containing 10 mm MOPSTris (pH 7.0), 3 ␮m ACMA, 1 mm MgCl2, and 100 mm KCl.
ACKNOWLEDGMENT
We are grateful to Dr. Martha Sorenson for critical reading of the manuscript and valuable suggestions.
Received May 8, 2003; returned for revision June 20, 2003; accepted June 27,
2003.
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