Plant Physiol. (1999) 120: 811-820

Abscisic Acid Induction of Vacuolar H⁺-ATPase Activity in Mesembryanthemum crystallinum Is Developmentally Regulated¹

Bronwyn J. Barkla^*, Rosario Vera-Estrella, Minerva Maldonado-Gama, and Omar Pantoja

Departamento de Biología Molecular de Plantas, Instituto de Biotecnología, Universidad Nacional Autónoma de México, A.P. 510-3, Colonia Miraval, Cuernavaca, Morelos, México 62250

	ABSTRACT

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Abscisic acid (ABA) has been implicated as a key component in water-deficit-induced responses, including those triggered by drought, NaCl, and low- temperature stress. In this study a role for ABA in mediating the NaCl-stress-induced increases in tonoplast H⁺-translocating ATPase (V-ATPase) and Na⁺/H⁺ antiport activity in Mesembryanthemum crystallinum, leading to vacuolar Na⁺ sequestration, were investigated. NaCl or ABA treatment of adult M. crystallinum plants induced V-ATPase H⁺ transport activity, and when applied in combination, an additive effect on V-ATPase stimulation was observed. In contrast, treatment of juvenile plants with ABA did not induce V-ATPase activity, whereas NaCl treatment resulted in a similar response to that observed in adult plants. Na⁺/H⁺ antiport activity was induced in both juvenile and adult plants by NaCl, but ABA had no effect at either developmental stage. Results indicate that ABA-induced changes in V-ATPase activity are dependent on the plant reaching its adult phase, whereas NaCl-induced increases in V-ATPase and Na⁺/H⁺ antiport activity are independent of plant age. This suggests that ABA-induced V-ATPase activity may be linked to the stress-induced, developmentally programmed switch from C₃ metabolism to Crassulacean acid metabolism in adult plants, whereas, vacuolar Na⁺ sequestration, mediated by the V-ATPase and Na⁺/H⁺ antiport, is regulated through ABA-independent pathways.

	INTRODUCTION

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The involvement of the plant growth regulator ABA in the responses of plants to water deficit has long been recognized. Endogenous levels of ABA have been shown to be elevated when plants are stressed with drought and/or NaCl, and application of ABA to unstressed plants results in the induction of numerous water-deficit-related activities. These may range from immediate responses, including the triggering of stomatal closure to reduce transpirational water loss by posttranslational modulation of ion channels in guard cells (Grabov and Blatt, 1998), to alterations in gene expression through the induction of ABA-responsive genes encoding for structural, metabolic, or transport proteins, as well as protein kinases and phosphatases (for reviews, see Ingram and Bartels, 1996; Shinozaki and Yamaguchi-Shinozaki, 1996, 1997; Bray, 1997). A role for ABA as a universal water-deficit-sensitizing signal, however, is clearly not the case. With the characterization of ABA-deficient and -insensitive mutants from Arabidopsis, evidence for water-stress-inducible genes that do not require accumulation of endogenous ABA have been identified (Gosti et al., 1995). Moreover, identification of a cis-acting dehydration-responsive element, distinct from ABA-responsive elements, in water-deficit-induced genes that does not respond to ABA treatment provides further support (Yamaguchi-Shinozaki and Shinozaki, 1994). These findings led to the conclusion that both ABA-dependent and -independent pathways transduce the water-deficit signal. However, more recently, a greater level of complexity to ABA signaling has been added with the findings that these ABA-dependent and -independent pathways actually cross-talk and converge to activate genes involved in the stress response to cold, drought, and salinity (Ishitani et al., 1997).

Mesembryanthemum crystallinum L. is a halophytic plant that responds to NaCl and drought stress by shifting its pathway of carbon assimilation from C₃ metabolism to the more water-conserving CAM. This metabolic transition involves the transcriptional induction of genes encoding key enzymes in the CAM pathway (for review, see Cushman and Bohnert, 1997). Several of these genes have also been shown to be up-regulated by exogenous ABA. These include the CAM-specific isoform of PEPC, 2-phospho-D-glycerate hydrolase (enolase), and phosphoglyceromutase (Chu et al., 1990; McElwain et al., 1992; Forsthoefel et al., 1995a, 1995b). Promotor analysis of the PEPC gene (Ppc1) has identified a putative ABA-responsive element that may be involved in ABA-responsive expression of this gene (Schaeffer et al., 1995). Correlated with these findings are the increased levels of endogenous ABA measured in the drought-stressed plant (Thomas et al., 1992).

Although environmentally triggered, CAM induction in M. crystallinum is developmentally regulated (Cushman et al., 1990). CAM cannot be elicited in NaCl-, drought-, or ABA-treated juvenile plants, and CAM inducibility by these factors coincides with the transition to mature growth (Adams et al., 1998). In contrast, NaCl-induced synthesis of compatible solutes and vacuolar Na⁺ accumulation, mechanisms of adaptation also considered essential for growth of M. crystallinum under these conditions, are observed independently of the age of the plants (Adams et al., 1998). This would indicate distinct signaling pathways involved in the induction of these three major responses.

Well characterized is the synthesis of compatible solutes in NaCl-stressed plants. In M. crystallinum the sugar alcohol pinitol accumulates in stressed plants to become the major compatible solute (Adams et al., 1998). Like enzymes involved in CAM induction, this response is also controlled at the level of gene expression. Both myo-inositol-1-phosphate synthase and IMT, two enzymes in the pathway leading to pinitol synthesis, are transcriptionally induced by salt stress (Ishitani et al., 1996; Nelson et al., 1998). However, transcripts are induced neither by drought nor by exogenous application of ABA, although transcript accumulation for IMT can be elicited by cold stress (Vernon et al., 1993).

Compared with CAM induction and compatible solute synthesis relatively little is known about the vacuolar accumulation of Na⁺ in M. crystallinum and of the means by which this adaptive response is regulated. Secondary active transport of Na⁺ across the tonoplast would be energized by the proton gradient generated by the activity of the V-ATPase. Several groups have reported increases in V-ATPase activity in tonoplasts isolated from leaves of NaCl-treated M. crystallinum plants (Rockel et al., 1994; Barkla et al., 1995; Tsiantis et al., 1996). Furthermore, the NaCl-induced increase in V-ATPase activity can be correlated with changes in the amount of protein or transcripts for specific V-ATPase subunits. Dietz and Arbinger (1996) showed slight increases in antibody recognition of the V-ATPase E-subunit in leaves from NaCl-stressed plants, and transcript amounts for the 16-kD proteolipid c subunit of the enzyme have been shown to be specifically elevated under NaCl stress (Löw et al., 1996; Tsiantis et al., 1996). Recently, it was suggested that NaCl-induced proteolytic processing of the V-ATPase B-subunit may serve to regulate the activity of the enzyme (Zhigang et al., 1996). This NaCl-stimulated increase in V-ATPase activity has been associated with a parallel increase in Na⁺/H⁺ antiport activity in adult M. crystallinum plants exposed to NaCl stress (Barkla et al., 1995).

In the present study we initiated research aimed at understanding the mechanisms regulating tonoplast Na⁺/H⁺ antiport and V-ATPase activity to begin deciphering components of the signal transduction pathway leading to increases in activity of these transporters under NaCl stress. We have investigated the regulatory role of NaCl and ABA and studied the possibility of a developmental program controlling the response of these transporters.

	MATERIALS AND METHODS

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Plant Materials

Isolation of Tonoplast Vesicles

After the samples were centrifuged at 100,000g (2 h at 4°C) using a swinging bucket rotor (model SW 28, Beckman) in an ultracentrifuge (model L8-M, Beckman), membranes at the 0/22% Suc interface, corresponding to the tonoplast (Struve and Lüttge, 1987; Barkla et al., 1995), were removed with a Pasteur pipette. These membranes were diluted with the suspension solution and sedimented at 80,000g (1 h at 4°C) using a fixed-angle rotor (model 55.2 Ti) in an ultracentrifuge (model L8-M), and the final membrane pellet was resuspended in 200 µL of the same solution. Membranes were frozen directly in liquid N₂ and stored at 80°C in 50-µL aliquots. Those used for quinacrine fluorescence measurements were subject to only a single freeze/thaw cycle, because additional cycles increased the leakiness of the vesicles.

Extraction of Total Protein

Protein Determination

H⁺ Transport Assays

PEPC Activity

Chemicals

	RESULTS

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NaCl Regulation of V-ATPase and Na⁺/H⁺ Antiport Activity in Adult Plants

Tonoplast vesicles were isolated from the leaves of adult M. crystallinum plants (older than 6 weeks). As defined by Adams et al. (1998), this mature growth form is characterized by the emergence of side shoots and secondary leaves, which are morphologically distinct from the primary leaves observed in the juvenile phase of development. Adult plants also show progressive development of epidermal bladder cells and are competent to induce CAM (Adams et al., 1998). Leaves from 6-week-old adult plants treated for 2 weeks with 200 mM NaCl (8 weeks old at the time of tonoplast vesicle isolation) showed an increased V-ATPase H⁺ transport activity of 1.7 times that of the control untreated plants of the same age (Fig. 1A; Table I), which agrees with previously reported results (Barkla et al., 1995). Growth of adult plants in 400 mM NaCl for 2 weeks was able to elicit a further induction in the V-ATPase H⁺ transport activity over that measured in plants exposed to 200 mM NaCl to a value 2.2 times that of the untreated plants (Fig. 1A; Table I).

View larger version (13K): [in this window] [in a new window]   Figure 1. ATP-dependent H+ transport into tonoplast vesicles isolated from leaves of adult M. crystallinum plants. Vesicle acidification was monitored by the quenching of quinacrine fluorescence, as described in ``Materials and Methods''. A, Tonoplast vesicles isolated from leaves of 8-week-old control (con) or NaCl-treated plants exposed to 200 mM NaCl (200) or 400 mM NaCl (400) for 2 weeks. B, Tonoplast vesicles isolated from leaves of control (con 1, 7 weeks old; con 3, 9 weeks old) and 200 mM NaCl-treated plants (NaCl 1, 7 weeks old; NaCl 2, 8 weeks old; NaCl 3, 9 weeks old) during a 3-week treatment period beginning when plants were 6 weeks old. For clarity, the trace for the control 8-week-old plant was not included in the figure. The results are original traces from one experiment representative of a total of three as detailed in Tables I and II. F is relative to that prior to addition of ATP (arrow).

When NaCl induction of V-ATPase H⁺ transport activity was measured weekly over a 3-week treatment period, a time-dependent increase was observed (Fig. 1B; Table II). A small factor of this increase could be attributed to the aging of the plants over the treatment period, as values for V-ATPase H⁺ transport in control untreated plants increased approximately 1.3-fold from a value of 668% F mg¹ protein min¹ measured in tonoplast vesicles from leaves of 7-week-old plants to 844% F mg¹ protein min¹ in 9-week-old plants (Table II). However, following 3 weeks of treatment with 200 mM NaCl, V-ATPase H⁺ transport activity in tonoplast vesicles from leaves of treated plants was 1.9 times greater than the activity measured in control plants of the same age (1614% F mg¹ protein min¹ and 844% F mg¹ protein min¹, respectively; Table II), indicating an increase that is independent of the age of the plant but dependent on the duration of NaCl treatment.

The effect of treatment of adult plants with NaCl on the Na⁺-dependent dissipation of a transmembrane pH gradient was tested in isolated tonoplast vesicles. Following the generation of a preset, inside-acid pH gradient by activation and subsequent inhibition of the V-ATPase, Na⁺ (200 mM) was added to the reaction medium, and the initial rate of quinacrine fluorescence recovery was measured. The initial rate of Na⁺/H⁺ exchange was approximately 1.4-fold higher in tonoplast vesicles isolated from leaves of adult plants treated with 200 mM NaCl for 2 weeks, as compared with vesicles from control plants of the same age (Fig. 2A; Table I). Only a slight further induction in Na⁺/H⁺ exchange was observed in plants treated with 400 mM NaCl for 2 weeks, as compared with those treated with 200 mM NaCl for the same period (Fig. 2A; Table I). When studied over a 3-week period, rates of NaCl-induced Na⁺/H⁺ antiport activity remained relatively unchanged and were not affected by the aging of the plants (Fig. 2B; Table II).

View larger version (11K): [in this window] [in a new window]   Figure 2. Na+-dependent H+ efflux from tonoplast vesicles isolated from leaves of adult M. crystallinum plants. A preset steady-state pH gradient (acidic inside) was generated in vesicles by activation of the V-ATPase, as described in ``Materials and Methods''. The recovery of quinacrine fluorescence, indicative of Na+/H+ exchange, was measured upon addition of 200 mM NaCl (arrow). A, Tonoplast vesicles isolated from leaves of 8-week-old control (con) or NaCl-treated plants treated with 200 mM NaCl (200) or 400 mM NaCl (400) for 2 weeks. B, Tonoplast vesicles isolated from leaves of control (con1, 7 weeks old; con3, 9 weeks old) and 200 mM NaCl-treated plants (NaCl1, 7 weeks old; NaCl3, 9 weeks old) during a 3-week treatment period beginning when plants were 6 weeks old. For clarity, the traces for the control and NaCl-treated 8-week-old plants were not included in the figure. The results are original traces from one experiment representative of a total of three, as detailed in Tables I and II. F is relative to that prior to addition of NaCl.

ABA Regulation of V-ATPase and Na⁺/H⁺ Antiport Activity in Adult Plants

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View larger version (14K): [in this window] [in a new window]   Figure 3. ATP-dependent H+ transport into tonoplast vesicles isolated from leaves of ABA-treated adult M. crystallinum plants. Vesicle acidification was monitored by the quenching of quinacrine fluorescence, as described in ``Materials and Methods''. Tonoplast vesicles were isolated from leaves of 8-week-old control (con) M. crystallinum plants or plants treated for 2 weeks with either 200 mM NaCl (NaCl) or foliar applications of 25 µM ABA (ABA) or with both (ABA + NaCl). The results are original traces from one experiment representative of a total of three, as detailed in Table I. F is relative to that prior to addition of ATP (arrow).

In contrast to these results, ABA treatment of adult M. crystallinum plants had no stimulatory effect on Na⁺/H⁺ antiport activity. Treatment of 6-week-old plants with 25 µM ABA for 2 weeks resulted in a slightly lower level of Na⁺-dependent H⁺ transport when compared with control untreated plants of the same age (Fig. 4; Table I). Treatment with ABA in the presence of 200 mM NaCl gave rates of Na⁺/H⁺ exchange equivalent to those obtained from treatment with NaCl alone (Fig. 4; Table I). These results indicate that, unlike NaCl, ABA treatment does not result in parallel increases in V-ATPase and Na⁺/H⁺ antiport activites; rather, ABA appears to regulate only V-ATPase activity.

View larger version (13K): [in this window] [in a new window]   Figure 4. Na+-dependent H+ efflux from tonoplast vesicles isolated from leaves of ABA-treated adult M. crystallinum plants. A preset steady-state pH gradient (acidic inside) was generated in vesicles by activation of the V-ATPase, as described in ``Materials and Methods''. The recovery of quinacrine fluorescence, indicative of Na+/H+ exchange, was measured upon addition of 200 mM NaCl (arrow). Tonoplast vesicles were isolated from leaves of 8-week-old control (con) M. crystallinum plants or plants treated for 2 weeks with either 200 mM NaCl (NaCl) or foliar applications of 25 µM ABA (ABA) or with both (ABA + NaCl). The results are original traces from one experiment representative of a total of three, as detailed in Table I. F is relative to that prior to addition of NaCl.

The ability of ABA to regulate V-ATPase activity with no stimulatory effect on Na⁺/H⁺ antiport activity would suggest that ABA induction of V-ATPase activity is not linked to vacuolar Na⁺ accumulation. However, it is possible that the ABA-dependent increase in V-ATPase activity is necessary to drive the passive uptake of malate into the vacuole, which occurs during CAM induction. Moreover, many of the genes up-regulated during the establishment of CAM are also induced by ABA (Cushman and Bohnert, 1997). To investigate whether this could be an explanation for the increase in V-ATPase activity observed in the adult plants treated with ABA, it was necessary to attempt to separate regulatory processes involved in CAM induction from those involved in vacuolar Na⁺ accumulation in M. crystallinum. One way to do this was to study the regulation by NaCl and ABA of the V-ATPase in juvenile plants in which, in this facultative CAM plant, stress-inducible CAM activity is absent (Adams et al., 1998). Whereas CAM cannot be elicited in juvenile plants, other NaCl-adaptive mechanisms, including vacuolar accumulation of Na⁺, are still present (Adams et al., 1998).

PEPC Activity in Adult and Juvenile M. crystallinum Plants

NaCl and ABA Regulation of V-ATPase Activity in Juvenile Plants

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View larger version (13K): [in this window] [in a new window]   Figure 5. ATP-dependent H+ transport into tonoplast vesicles isolated from leaves of juvenile M. crystallinum plants. Vesicle acidification was monitored by the quenching of quinacrine fluorescence, as described in ``Materials and Methods''. A, Tonoplast vesicles isolated from leaves of 5-week-old control plants (cont) or plants treated with either 200 mM NaCl (NaCl) or 25 µM ABA (ABA) for 2 weeks. The results are original traces from one experiment representative of a total of three. F is relative to that prior to addition of ATP (arrow).

	DISCUSSION

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The mechanisms by which M. crystallinum senses changes in the environment and transduces these signals into physiological responses resulting in drought and salinity tolerance, including CAM induction, compatible solute synthesis, and vacuolar Na⁺ accumulation, have only recently begun to be investigated. It is unlikely that there exists a single signaling pathway that coordinately regulates these key adaptive responses; rather, it is becoming increasingly clear that the response to environmental factors is complex, requiring several distinct pathways to explain the intricate patterns of gene expression and enzyme responses elicited by a range of environmental stimuli (Bohnert et al., 1995). To better understand the signaling pathways involved in M. crystallinum stress tolerance we have investigated the role played by both NaCl and ABA in initiating changes in the V-ATPase activity in both adult and juvenile plants.

Independently of the age of the plant, treatment with NaCl resulted in substantial increases in V-ATPase H⁺ transport activity to a maximum induction of 1.7- and 1.9-fold in juvenile and adult plants, respectively (Figs. 1 and 5; Table I), in agreement with previously reported results (Rockel et al., 1994; Barkla et al., 1995; Tsiantis et al., 1996). Similar treatment periods of adult and juvenile plants resulted in a comparable increase in V-ATPase activity (1.7-fold following 2 weeks of treatment), indicating, despite developmental differences, that plant age does not affect the response of the V-ATPase to salinity. In adult plants the maximal level of induction was reached after a treatment period of 3 weeks, suggesting a sustained effect of NaCl treatment on enzyme activity. This NaCl-induced increase in V-ATPase activity in adult plants was paralleled by increases in activity for the Na⁺/H⁺ antiport (Fig. 2A; Table I), providing a link between increased tonoplast energization and increased vacuolar Na⁺ accumulation via Na⁺/H⁺ exchange. However, maximal levels of Na⁺/H⁺ antiport activity were reached after 1 week of treatment with 200 mM NaCl, suggesting that increases in NaCl concentration or age of plant had no effect on antiport activity (Fig. 2B; Table II).

In adult plants application of exogenous ABA to the leaves of well-watered plants also induced V-ATPase H⁺ transport activity by 1.8-fold, a level similar to that observed for plants treated with 200 mM NaCl (Fig. 3; Table I). This increase was not due to direct effects of ABA on enzyme activity, because V-ATPase activity in tonoplast vesicles isolated from leaves of control M. crystallinum plants preincubated in the presence of ABA showed no such induction (data not shown). ABA-induced V-ATPase H⁺ transport activity has also been reported for barley roots treated with ABA (Kasai et al., 1993), and transcripts for the V-ATPase A-subunit have been shown to be up-regulated by ABA application to tobacco cell-suspension cultures and leaves of Brassica napus (Narasimhan et al., 1991; Orr et al., 1995). ABA application to leaves of M. crystallinum increased transcript levels of the V-ATPase c-subunit (Tsiantis et al., 1996).

In contrast to the stimulation of V-ATPase activity, ABA application to leaves of M. crystallinum failed to induce Na⁺/H⁺ antiport activity in either adult or juvenile plants (Fig. 4; Table I; also data not shown). These results suggest that ABA is not involved as a signal mediating NaCl-induced antiport activity.

When adult plants were treated with ABA in the presence of NaCl, a further stimulation in V-ATPase H⁺ transport activity was observed, demonstrating a partially additive effect of treatments on enzyme activity (Fig. 3; Table I). These data, and the lack of effect of ABA on Na⁺/H⁺ antiport activity, suggest that NaCl and ABA do not act through the same signal transduction pathway. However, it is possible that two distinct pathways exist: (a) the first mediated by NaCl and independent of ABA, responsible for up-regulation of V-ATPase and Na⁺/H⁺ antiport activity to energize Na⁺ accumulation, and (b) the second dependent on water-deficit-induced increases in ABA, which stimulates V-ATPase enzyme activity to drive malate accumulation into the vacuole during the establishment of CAM. Additional support for this view comes from results of studies of juvenile M. crystallinum plants. In contrast to adult plants, ABA treatment of juvenile plants failed to induce V-ATPase H⁺ transport activity, whereas NaCl elicited a response similar to that observed in the adult plants (Figs. 3 and 5; Table I). This age-dependent ABA response is reminiscent of the developmentally programmed, stress-induced switch from C₃ metabolism to CAM in M. crystallinum, in which only adult plants are competent in CAM induction (Table III; Cushman et al., 1990). Therefore, in juvenile plants, in which NaCl-stress-induced Na⁺ accumulation is also observed, increases in V-ATPase activity would be required. However, because CAM is not triggered in young plants, V-ATPase energization of vacuolar malate accumulation is not necessary. To justify this hypothesis, it must be possible to elicit two distinct pathways initiating from the recognition by the plant of the salinity signal. For salinity stress, with which a water deficit is also imposed, drought-related responses would be expected (including CAM induction in older plants to increase water-use efficiency). However, there is also the added ionic effects related to the presence of high concentrations of Na⁺ and Cl, which could require survival responses by the plant different from drought stress alone. Evidence for specific responses to the ionic component of NaCl stress, with the ability to differentiate the osmotic component, has recently been shown for an Arabidopsis calcineurin B homolog (Liu and Zhu, 1998) and in M. crystallinum for the induction of IMT and myo-inositosol-1-phosphate synthase, enzymes in the pathway leading to synthesis of the compatible solute pinitol (Nelson et al., 1998). To further support this hypothesis it will be necessary to identify specific components of each signaling pathway.

Recent advances in this field have been made in the glycophyte Arabidopsis, for which NaCl-stress adaptive responses were found to trigger changes in cytoplasmic Ca²⁺ (Knight et al., 1997), with the possible involvement of calcineurin, a Ca²⁺/calmodulin-dependent protein phosphatase (Liu and Zhu, 1998; Pardo et al., 1998). However, this pathway is similar to that characterized in yeast, in which the pathway was shown to primarily regulate ion homeostasis through the coordination of Na⁺ influx and efflux systems (Mendoza et al., 1994) and may not be relevant when studying adaptive mechanisms of halophytes such as M. crystallinum, which selectively accumulate Na⁺ rather than exclude this ion. The different responses of halophytes and glycophytes to NaCl stress strengthens the need to study signaling pathways in NaCl-tolerant plants and to focus research on understanding those mechanisms that are unique to each group. Furthermore, the results presented in this study highlight the importance of interpreting the adaptive responses to salinity of M. crystallinum specifically and plants in general within the context of existing developmental programs.

Whether the NaCl- or ABA-induced increase in V-ATPase activity observed is due to increased pump density or control of existing pump activity is unknown. We do know that there is no coordinate transcriptional up-regulation of genes for V-ATPase subunits in NaCl-stressed M. crystallinum (Löw et al., 1996) and so far, only the c-subunit of the membrane sector has been shown to be transcriptionally regulated by NaCl in both juvenile and adult plants (Tsiantis et al., 1996). At the protein level, antibodies against V-ATPase subunits A, B, E, and Ac39 (yeast vma6 gene product) do not identify NaCl-induced increases in protein for these subunits in tonoplast isolated from M. crystallinum leaves and cell suspensions (Maldonado-Gama, 1997; Vera-Estrella et al., 1999). However, an increase in pump density may not require up-regulation of all subunits; rather, key subunits may direct assembly of the holoenzyme (Stevens and Forgac, 1997). Alternatively, increases in a particular subunit may not increase pump density but, instead, alter the coupling efficiency of H⁺ transport and ATP hydrolysis. Although posttranslational modifications of V-ATPases have not been described in plants, in yeast V-ATPases have been shown to be regulated by Glc through rapid disassembly and reassembly in vivo (Kane, 1995), and evidence has been provided for regulation of V-ATPases by heterotrimeric G-proteins and protein kinase C (for review, see Merzendorfer et al., 1997).

	FOOTNOTES

   Received January 21, 1999; accepted April 2, 1999.

	ABBREVIATIONS

Abbreviations: F, fluorescence intensity. IMT, myo-inositol O-methyltransferase. PEPC, PEP carboxylase. V-ATPase, tonoplast H⁺-translocating ATPase.

	ACKNOWLEDGMENT

We thank Henk Miedema for critical review of the manuscript.

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