Plant Physiol. (1998) 116: 1487-1495

Reversibility of H⁺-ATPase and H⁺-Pyrophosphatase in Tonoplast Vesicles from Maize Coleoptiles and Seeds¹

Arnoldo Rocha Façanha and Leopoldo de Meis^*

Instituto de Ciências Biomédicas, Departamento de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Cidade Universitária, RJ-21941-590, Brazil

	ABSTRACT

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Tonoplast-enriched vesicles isolated from maize (Zea mays L.) coleoptiles and seeds synthesize ATP from ADP and inorganic phosphate (Pi) and inorganic pyrophosphate from Pi. The synthesis is consistent with reversal of the catalytic cycle of the H⁺-ATPase and H⁺-pyrophosphatase (PPase) vacuolar membrane-bound enzymes. This was monitored by measuring the exchange reaction that leads to ³²Pi incorporation into ATP or inorganic pyrophosphate. The reversal reactions of these enzymes were dependent on the proton gradient formed across the vesicle membrane and were susceptible to the uncoupler carbonyl cyanide p(trifluoromethoxy)-phenylhydrazone and the detergent Triton X-100. Comparison of the two H⁺ pumps showed that the H⁺-ATPase was more active than H⁺-PPase in coleoptile tonoplast vesicles, whereas in seed vesicles H⁺-PPase activity was clearly dominant. These findings may reflect the physiological significance of these enzymes in different tissues at different stages of development and/or differentiation.

	INTRODUCTION

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Two distinct proton pumps are found in the vacuolar membrane of plants, a V-ATPase (EC 3.6.1.3) and a membrane-bound H⁺-PPase (EC 3.6.1.1). Each of these enzymes can generate a transmembrane electrochemical H⁺ gradient using the energy derived from hydrolysis of its substrate (ATP or PPi, respectively). The H⁺ gradient is then used to energize the secondary transport of the different substances needed for the plant's development (Rea and Sanders, 1987). The physiological role of the membrane-bound PPase is not clear at present. It is currently accepted that the V-ATPase plays a predominant role in the maintenance of a transmembrane electrochemical H⁺ gradient, whereas the H⁺-PPase seems to serve as an ancillary backup system for the pumping of protons (Rea et al., 1992; Taiz, 1992; Baykov et al., 1993). Recently, it was found that H⁺-PPase is overexpressed in response to energetic stresses such as chilling and anoxia (Carystinos et al., 1995; Darley et al., 1995). The cellular PPi content, unlike that of ATP, remains stable during marked changes in respiratory state (Weiner et al., 1987; Dancer and ap Rees, 1989). These observations suggest that the H⁺-PPase may exert a key role in the survival strategies of plants under conditions of limited ATP supply (Macrì et al., 1995, and refs. therein).

The coexistence of two different enzymatic systems playing the same role in the same membrane is apparently paradoxical. Based on thermodynamic considerations, Rea and Sanders (1987) and Schmidt and Briskin (1993a) raised the hypothesis that, instead of both enzymes always operating in parallel to pump protons into the vacuole, the gradient generated by one of the proton pumps might drive the reversal reaction of the other, which therefore behaves as a synthase. The catalytic cycles of both enzymes can be reversed using the energy derived from the H⁺ gradient. Dupaix et al. (1989) and Schmidt and Briskin (1993b) demonstrated the synthesis of ATP from ADP and Pi and showed that synthesis was driven by the H⁺ gradient formed by PPi hydrolysis in tonoplast vesicles. Based on ¹⁸O-exchange measurements at the PPi-binding site, Baykov et al. (1994) concluded that PPi formation had occurred in Vigna radiata tonoplast H⁺-PPase during PPi hydrolysis.

The present study shows that the H⁺ gradient generated across the vacuolar membrane by the hydrolysis of either PPi or ATP may drive both PPi and ATP synthesis (as indicated by isotope exchange rather than net synthesis), which is consistent with the reversal of the tonoplast H⁺-PPase and H⁺-ATPase. Furthermore, there is a significant difference between the activities of tonoplast-enriched vesicles from maize coleoptiles and seeds, suggesting a differential expression or regulation of these enzymes depending on plant cell development and/or differentiation.

	MATERIALS AND METHODS

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Seeds of maize (Zea mays L.) were soaked in water for 24 h. Afterward, some of the seeds were used for isolation of tonoplast vesicles, and the remainder were sown on wet filter paper and germinated in the dark at 28°C. Coleoptiles of 5-d-old seedlings were harvested for preparation of vesicles. The maize seeds were provided by Sementes Agroceres S.A. (São Paulo, Brazil).

Tonoplast-Enriched Vesicles

For vesicles from seeds a better yield was obtained using a 10/30/46% (w/w) gradient, in agreement with a previous study (Hoh et al., 1995) showing that during the subcellular fractionation of pea cotyledons at early developmental stages, a peak of V-ATPase activity was found in the fractions between 30 and 32% (w/w) on a Suc gradient. After centrifugation at 100,000g for 3 h in a swinging bucket, the vesicles that sedimented at the interface between 10 and 25 or 30% Suc were collected, diluted with 3 volumes of ice-cold water, and centrifuged at 100,000g for 40 min. Bafilomycin A₁ or NO₃-inhibited H⁺-ATPase and K⁺-dependent H⁺-PPase activities were used as marker enzymes for the tonoplast membrane (Sze, 1985). The pellet was resuspended in a medium containing 10 mm Tris-HCl, pH 7.6, 10% (v/v) glycerol, 1 mm DTT, and 1 mm EDTA. The vesicles were either used immediately or frozen under liquid N₂ and stored at 70°C until use. Protein concentrations were determined by the method of Lowry et al. (1951).

ATPase and PPase Activity

Electrochemical Gradient of Protons

Pi  PPi and Pi  ATP Exchange

These two samples were incubated for 30 min at 35°C and then quenched with TCA. The Ca²⁺-dependent ATPase preparation used was able to catalyze the hydrolysis of the -Pi of ATP at the rate of 3.0 µmol Pi mg¹ min¹. In agreement with previous reports (de Meis, 1984, 1988; de Meis et al., 1985, 1986), we did not detect any measurable cleavage of PPi with the Ca²⁺-dependent ATPase. The yeast PPase used was able to catalyze the hydrolysis of PPi only at the rate of 4.0 µmol mg¹ min¹. The ³²Pi was extracted from the different TCA-quenched samples as phosphomolybdate using isobutyl alcohol-benzene, as previously described (de Meis, 1984; de Meis et al., 1985).

The small amount of radioactivity found in controls (not incubated with the tonoplast vesicles) was subtracted from that found in the experiments with vesicles. The radioactivity remaining in the aqueous phase after extraction of the free ³²Pi with isobutyl alcohol-benzene (representing total ³²Pi esterified) was subtracted from that in the sample treated with Ca²⁺-ATPase or yeast PPase; the difference was attributed to either ATP or PPi synthesized by the tonoplast enzymes. Concentrations of radioactive PPi synthesized were calculated on the basis of two ³²Pi incorporated into each PPi. In earlier reports it was shown that this method makes it possible to quantify the amount of radioactive ATP and PPi formed by solubilized enzyme and by chromatophores of Rhodospirillum rubrum. In these reports, the radioactive ATP and PPi remaining in the aqueous phase after extraction were copurified with nonradioactive ATP and PPi, respectively, and characterized by TLC and autoradiography (de Meis, 1984; Behrens and de Meis, 1985; de Meis et al., 1985).

Materials

	RESULTS

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ATPase and PPase Activity in Tonoplast Vesicles from Coleoptiles and Seeds

Tonoplast vesicles catalyzed the hydrolysis of both ATP and PPi, regardless of whether they were derived from coleoptiles or from seeds. The hydrolysis of both substrates was coupled with proton translocation. However, the activities of the two enzymes varied depending on the origin of the vesicles. For the coleoptile vesicles the velocities of ATP hydrolysis and H⁺ accumulation were faster than the rate of H⁺ transport supported by PPi hydrolysis (Table I). In contrast, for the seed vesicles the initial velocities of ATP hydrolysis and H⁺ transport were several times slower than those of PPi hydrolysis and H⁺ transport (Table I). The H⁺ gradient formed by PPi hydrolysis in seed vesicles was always steeper than that formed in coleoptile vesicles (Table I). These data suggest that there is either a differential expression or a differential activation of H⁺-PPase and H⁺-ATPase in coleoptile and seed vacuolar membranes.

In the presence of 5 mm MgCl₂, the concentration of PPi needed for maximal rates of substrate hydrolysis (Fig. 1) was found to vary between 0.1 and 0.2 mm. In agreement with previous reports (White et al., 1990; Leigh et al., 1992), a decrease of the PPase was observed in the presence of an excess of substrate (Fig. 1). The half-maximal ATPase activity was reached at about 0.2 to 0.4 mm ATP (data not shown), a K_m similar to that found for tonoplast H⁺-ATPase from oat roots.

View larger version (18K): [in this window] [in a new window]   Figure 1. H+-PPase of seed vesicles: substrate dependence for PPi hydrolysis and 32Pi incorporation. The assay medium composition was 50 mm Mops-Tris buffer, pH 7.0, 0.1 mm ADP, 100 mm KCl, 5 mm 32Pi, 5 mm MgCl2, and 0.05 mg/mL tonoplast vesicles, at 35°C. , PPi hydrolysis; , 32Pi incorporation.

PPi and ATP Synthesis in Tonoplasts from Coleoptiles and Seeds

View larger version (17K): [in this window] [in a new window]   Figure 2. Time course of 32Pi incorporation by tonoplast vesicles from coleoptiles (a) and seeds (b). The assay medium composition was 50 mm Mops-Tris buffer, pH 7.0, 0.1 mm ADP, 100 mm KCl, 5 mm 32Pi, 5 mm MgCl2, and 0.05 mg/mL tonoplast vesicles at 35°C. Triangles show when 0.02% Triton X-100 was present in the assay medium. The reaction was started by the addition of either 1 mm ATP (, ) or 1 mm PPi (, ) and was arrested by the addition of TCA. Essentially the same results were obtained in five experiments using different vesicle preparations.

View larger version (18K): [in this window] [in a new window]   Figure 3. PPi dependence of 32Pi incorporation by tonoplast vesicles from seeds. The assay medium composition was 50 mm Mops-Tris buffer, pH 7.0, 0.1 mm ADP, 100 mm KCl, 5 mm MgCl2, and 0.05 mg/mL tonoplast vesicles at 35°C, using as the substrate either 1 mm ATP () or 1 mm PPi (). The reaction was carried out in the presence of increasing Pi concentrations, maintaining the radioactive 32Pi: nonradioactive Pi proportions constant at 20,000 cpm/nmol of nonradioactive Pi. Essentially the same results were obtained in four independent experiments.

The [³²P]phosphate-exchange assay adopted in this study does not allow us to identify directly which of the two compounds (ATP or PPi) has been synthesized. To this end, the reaction medium containing the Pi esterified during the exchange assay was subjected to enzymatic hydrolysis by PPase purified from yeast or by sarcoplasmic reticulum Ca²⁺-ATPase isolated from rabbit skeletal muscle. When the substrate was ATP, most of the ³²Pi incorporated into products during the exchange reaction disappeared after treatment with PPase from yeast, indicating that a large fraction of PPi had been formed (Fig. 4). On the other hand, when the substrate was PPi, the ³²Pi incorporation was significantly affected by the Ca²⁺-ATPase as well as by the PPase (Fig. 4). Since two molecules of Pi were incorporated for each PPi molecule synthesized, similar amounts of PPi and ATP were synthesized under these conditions (Table II). The same proportions were observed with tonoplast vesicles derived from coleoptiles as with vesicles from seeds.

View larger version (20K): [in this window] [in a new window]   Figure 4. Identification of the products synthesized by coleoptiles (a) and seeds (b) using the sarcoplasmic reticulum Ca2+-ATPase and the PPase from yeast. The assay medium composition was 50 mm Mops-Tris buffer, pH 7.0, 100 mm KCl, 0.1 mm ADP, 5 mm MgCl2, 5 mm 32Pi, and 0.05 mg/mL tonoplast protein. The reactions were started by adding either 1 mm ATP or 1 mm PPi. After 30 min the reactions were stopped by filtration to remove vesicles, and the filtrates were divided into four aliquots. Three of them were incubated with 0.05 mg/mL Ca2+-ATPase from skeletal muscle sarcoplasmic reticulum (hatched bars), 0.01 mg/mL PPase from yeast (gray bars), or both (white bars); one aliquot (control; black bars) was not incubated with either enzyme. The Ca2+-ATPase reaction medium was supplemented with 0.15 mm CaCl2, 2 µm A23187, and 0.1 mm ATP when ATP was not already present. The PPase reaction medium was supplemented with 0.1 mm PPi when PPi was not already present. The reaction was stopped after 30 min by the addition of TCA (10%, w/v). The medium was subjected to phosphomolybdate extraction, and the radioactivity remaining in the aqueous phase was counted. Values represent the means + se of six independent experiments.

Additional evidence for the identification of the products synthesized in these reactions was obtained using inhibitors of vacuolar ATPase and PPase. Bafilomycin A₁ was able to inhibit the ATP synthesis promoted by PPi hydrolysis, as reported previously by Schmidt and Briskin (1993b). Preincubation of vesicles with fluoride blocked the PPi synthesis dependent on ATP hydrolysis. Regardless of the origin of vesicles (coleoptiles or seeds), the amount of ³²Pi incorporation recovered after treatment with fluoride was essentially identical to the ³²Pi incorporation recovered after treatment with PPase from yeast (Table II). Likewise, when Bafilomycin A₁ was used, seed vesicles showed the same ³²Pi incorporation as that recovered after treatment with sarcoplasmic reticulum Ca²⁺-ATPase. However, in coleoptile vesicles the amount of ³²Pi incorporation found in the presence of Bafilomycin A₁ was higher than that recovered after treatment with Ca²⁺-ATPase (Table II). It may be that eliminating the synthesis of ATP driven by PPi hydrolysis increases the H⁺ gradient available for reversal of the reaction catalyzed by the H⁺-PPase, thus increasing PPi synthesis.

ADP and KCl Dependence of the Reversal Reaction of Tonoplast ATPase and PPase from Coleoptiles and Seeds

View larger version (16K): [in this window] [in a new window]   Figure 5. ADP dependence for 32Pi incorporation in tonoplast vesicles from coleoptiles (a) and seeds (b). The assay medium composition was 50 mm Mops-Tris buffer, pH 7.0, 100 mm KCl, 5 mm MgCl2, 5 mm 32Pi, 0.05 mg/mL tonoplast protein, and the concentration of ADP is shown on the abscissa. The reactions were started by either 1 mm ATP () or 1 mm PPi (). Essentially the same results were obtained in three experiments using different vesicle preparations.

Figures 6 and 7 show the K⁺ dependence of the forward and reverse reactions in tonoplast vesicles derived from coleoptiles and from seeds. With PPi as the substrate (Fig. 6) both reactions were strongly stimulated by K⁺. This indicates that if the PPase is not activated by K⁺; there is no H⁺ gradient formation and the medium ³²Pi cannot be used to synthesize either PPi or ATP. By contrast, with ATP as the substrate (Fig. 7), the forward reaction (Fig. 7, insets) exhibited only a small stimulation by KCl, whereas most of the ³²Pi incorporation showed a K⁺ dependence. In this case, the gradient is formed by the ATPase, but the energy derived from the gradient can be used only to synthesize PPi if the PPase is activated by K⁺.

View larger version (20K): [in this window] [in a new window]   Figure 6. KCl dependence for PPi hydrolysis and Pi-exchange reactions catalyzed by tonoplast vesicles from coleoptiles and seeds. The assay medium composition was 50 mm Mops-Tris buffer, pH 7.0, 5 mm MgCl2, 5 mm 32Pi, 0.1 mm ADP, and 0.05 mg/mL vacuolar membrane protein. , 32Pi incorporation when 1 mm PPi was used as the substrate. In the case of the Pi-exchange assay the medium also contained 0.1 mm ADP. , Hydrolysis activities. Essentially the same results were obtained in four experiments using different vesicle preparations.

View larger version (19K): [in this window] [in a new window]   Figure 7. KCl dependence for ATP hydrolysis and Pi-exchange reactions catalyzed by tonoplast vesicles from coleoptiles and seeds. The assay medium composition was 50 mm Mops-Tris buffer, pH 7.0, 5 mm MgCl2, 5 mm 32Pi, 0.1 mm ADP, and 0.05 mg/mL vacuolar membrane protein. , 32Pi incorporation when 1 mm ATP was used as the substrate. In the case of the Pi-exchange assay the medium also contained 0.1 mm ADP. , Hydrolysis activities. Essentially the same results were obtained in four experiments using different vesicle preparations.

	DISCUSSION

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In the present work we examined the activities of tonoplast H⁺ pumps from coleoptiles and seeds that represent different stages of plant development. Mature coleoptiles manifest a premature senescence, since their function is linked to the early stages of growth of the seedlings. The coleoptile protects the leaf primordia and has been shown to serve as a gravi-guiding system for shoots of germinating seedlings (Edelmann, 1996). The balance of activity between the two proton pumps in coleoptile tonoplast vesicles is consistent with the pattern established for most vegetative tissue preparations, in which the tonoplast H⁺-ATPase usually can generate a pH gradient across the vacuolar membrane of similar or greater magnitude than the H⁺-PPase (Chanson et al., 1985; Giannini and Briskin, 1987; Rea and Sanders, 1987). The remarkable preponderance of H⁺-PPase activity in preparations from whole seeds contrasts with what has been found in coleoptiles and in most vegetative tissues.

In the early stages of seed hydration, the mitochondria are functionally deficient and the adenylate energy of the cell is low (Ivanov and Khavkin, 1976; Bewley and Black, 1985, and refs. therein). The prevalence of H⁺-PPase in developing seeds supports the idea that the physiological significance of this enzyme would be to maintain the proton gradient under conditions of limited ATP supply (Carystinos et al., 1995; Darley et al., 1995; Macrì et al., 1995). Suzuki and Kasamo (1993) showed that H⁺-PPase activities are about 3-fold higher than the V-ATPase activities in 7-d-old pumpkin cotyledons. However, this situation changes over the next few days, as the V-ATPase assumes the major role in proton pumping.

Maeshima et al. (1994) studied the induction of H⁺-PPase and H⁺-ATPase in germinating pumpkin seeds. Their data showed an abundance of the H⁺-PPase in 2-d-old pumpkin cotyledons as well as the remarkable activation of the H⁺-PPase compared with the H⁺-ATPase activity in tonoplasts from 2- to 6-d-old pumpkin cotyledons. These findings, as well as ours with maize seeds, support the hypothesis that PPi may serve as the key energy source during seed germination and/or early developmental stages in plant cells.

In hydrating seeds another source of energy might be derived from the presence of another pathway for the production of ATP. Here we have extended previous studies of the reversal reaction of both tonoplast H⁺ pumps (Dupaix et al., 1989; Schmidt and Briskin 1993b; Baykov et al., 1994). We show that the proton gradient generated by hydrolysis of either PPi or ATP can be used to promote reversal of the catalytic cycle of both H⁺-PPase and H⁺-ATPase. In accordance with predictions based on thermodynamic calculations (Roberts, 1990, and refs. therein; Schmidt and Briskin, 1993a), our assessment of the products synthesized shows that reversal of H⁺-PPase is favored over reversal of H⁺-ATPase (Table II). Although the analysis of mass-action ratios and equilibrium constants for tonoplast H⁺-PPase activities by these authors indicates that in vivo H⁺-PPase may be operating near equilibrium, this is the first time to our knowledge that PPi synthesis has been verified experimentally in tonoplasts.

ATP synthesis coupled to an electrochemical gradient generated by H⁺-PPase has been described in red beet tonoplasts (Schmidt and Briskin, 1993b). However, these authors concluded that under most physiological conditions reversal of the V-ATPase would be unlikely because of the difference in the free energy values for ATP synthesis and PPi hydrolysis. They suggested that high PPi and low Pi concentrations would be required to form a gradient of the necessary magnitude. On the contrary, under our conditions ATP synthesis was driven by PPi hydrolysis in the presence of a Pi concentration 5 times higher than that of PPi. These results may reflect a difference in the conditions required for isotope exchange compared with those required for net synthesis.

The role of the gradient in both the ATP  Pi exchange and the PPi  Pi exchange is still not clear. In earlier reports it was assumed that a transport ATPase could catalyze only an exchange reaction when an ionic gradient was formed across the vesicle membrane. The notion was that the energy derived from the gradient represented an absolute energetic requirement for the exchange reaction. Later experimentation revealed that during the exchange reaction the catalytic cycle of some, but not all, transport enzymes can be reversed in the absence of a gradient. An example is the membrane-bound Ca²⁺-ATPase found in different animal tissues. This enzyme can catalyze an ATP  Pi exchange in both the presence and absence of a transmembrane Ca²⁺ gradient (de Meis and Carvalho, 1974; de Meis and Vianna, l979; Plank et al., 1979; de Meis et al., 1986).

For other enzymes, such as the ATP synthase of mitochondria, an ATP  Pi exchange can be measured only in the presence of an H⁺ gradient. For most transport enzymes so far studied, the gradient seems to be needed to increase the affinity of the enzyme for Pi and to improve the ratio between the rates of hydrolysis and the synthesis of ATP (for reviews, see de Meis, 1993; Boyer, 1997; Weber and Senior, 1997). During the exchange reaction some of the energy derived from the cleavage is retained by the enzyme for the synthesis of a new ATP or PPi molecule. This is observed in enzymes that undergo a conformational change during the catalytic cycle. In these enzymes the products of the hydrolysis are released from the enzyme surface before returning to the conformation that allows the beginning of a new catalytic cycle. At this stage of the cycle the enzymes re-bind the products, reverse the catalytic cycle, and synthesize a new ATP or PPi molecule (de Meis, 1981, 1989; Sakamoto and Tonomura, 1983; de Meis et al., 1986).

In aqueous solutions under conditions close to those found in vivo, the hydrolysis of both ATP and PPi is accompanied by a large change in free energy (George et al., 1970; Hayes et al., 1978). During the catalytic cycle of several enzymes involved in energy transduction, there are steps in which the hydrolysis of these compounds is accompanied by only a small energy change (George et al., 1970; Hayes et al., 1978; de Meis, 1984). It has been proposed that the large change in energy of hydrolysis of the Pi compounds is promoted by a small change in water structure in the microenvironment on the enzyme (de Meis, 1984; de Meis et al., 1985, and refs. therein). These studies have important implications for the equilibrium of enzyme reaction, especially for seeds in which water concentration is low. The present data provide further evidence for the reversibility of the reaction mechanisms of the tonoplast ATP- and PPi-dependent proton pumps. Given the appropriate thermodynamic conditions in vivo, these pumps may operate as systems of energy conservation, with a role in maintaining the cytosolic ATP and PPi levels.

This notion is consistent with structural studies showing that the vacuolar H⁺-PPase and the R. rubrum H⁺-PPase (Nore et al., 1991) have evolved from a common ancestral gene, just as V- and F-type ATPases seem to have evolved from a single enzyme present in a common ancestor (Kibak et al., 1992). The coupling observed between the proton gradient and the Pi exchange suggests that the tonoplast H⁺-PPase and H⁺-ATPase share functional as well as structural similarities with the H⁺-PPase of R. rubrum and the F-type ATP synthases, both of which can couple the synthesis of their own products to the electrochemical H⁺ gradient generated in the membranes in which they are embedded.

	FOOTNOTES

   Received October 9, 1997; accepted December 4, 1997.

	ABBREVIATIONS

Abbreviations: ACMA, 9-amino-6-chloro-2-methoxyacridine. FCCP, carbonyl cyanide p(trifluoromethoxy)-phenylhydrazone. PPase, pyrophosphatase. V-ATPase, vacuolar ATPase.

	ACKNOWLEDGMENT

The authors are grateful to Dr. Martha Sorenson for the helpful discussion of the manuscript.

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