Development of Vacuolar Membranes during Elongation of Cells in ...

1 downloads 0 Views 1MB Size Report
The development of vacuolar membrane in the elongating hypocotyls of the mung bean was investigated. The hypocotyls from 3-day-old seedlings were ...
Plant Cell Physiol. 31(3): 311-317 (1990) JSPP © 1990

Development of Vacuolar Membranes during Elongation of Cells in Mung Bean Hypocotyls Masayoshi Maeshima Institute of Low Temperature Science, Hokkaido University, Sapporo, 060 Japan

The development of vacuolar membrane in the elongating hypocotyls of the mung bean was investigated. The hypocotyls from 3-day-old seedlings were dissected into the dividing, elongating and mature regions. The diameter of protoplasts prepared from the mature regions was about 3-fold greater than the diameter of those from the dividing region. The activity of inorganic pyrophosphatase, an enzyme associated with the vacuolar membrane, was detectable even in the dividing region. The level of pyrophosphatase was quantified by slot-blot analysis with the pyrophosphatase-specific antibody. The relative amount of pyrophosphatase per cell, calculated on the basis of DNA content, increased about 4-fold during cell maturation. When the densities of vacuolar membranes were compared by sucrose density gradient centrifugation, there was no marked difference among the preparations from three regions. Furthermore, most of the major proteins were common to the three purified preparations of vacuolar membranes. From the results, it appears that most components of vacuolar membrane may be synthesized de novo and added to the existing membrane during cell elongation. Furthermore, it is proposed that the H + -pyrophosphatase may actively hydrolyze its substrate to maintain the internal acidity of expanding vacuoles, because pyrophosphate was present at a concentration of more than 70 /JM in the dividing and elongating regions. Key words: Cell elongation — Inorganic pyrophosphatase — Membrane biogenesis — Mung bean — Vacuole.

In the cells of higher plants, vacuoles function as storage organelles, as hydrolytic compartments, and as controllers of turgor pressure (Boiler and Wiemken 1986, Matile 1978). The space-filling role of the vacuole is also important and is essential to the growth of plant cells. In most cases, the increase in cell volume is accounted for by enlargement of the vacuole rather than by that of the cytoplasm. The expanding vacuole must actively accumulate solutes and protons to maintain its osmolarity and acidity. It is impossible to discuss the development of vacuoles without a full understanding of the biogenesis of the membrane. The vacuolar membrane possesses two distinct proton pumps and several secondary active transport systems. Although the molecular details of the secondary active transport systems are not clear, the proton pumps have been purified and well characterized (Nelson and Taiz 1989, Maeshima and Yoshida 1989). One of the proton pumps, the proton-translocating inorganic pyrophosphatase, has been isolated and shown to be composed of a single polypeptide (Maeshima and Yoshida 1989). In the present work, the enzyme was investigated as a maker of

the vacuolar membrane. The form and function of each vacuole depends on the cell type. Studies on the formation of protein bodies, which are specialized vacuoles, have provided us with much information about the dynamics of vacuolar proteins (Akazawa and Hara-Nishimura 1985, Higgins 1984). Biogenesis of the membrane of plant vacuoles has not yet been investigated in detail, although it has been suggested that the tonoplast grows by incorporation of membrane that originates from the Golgi apparatus or the endoplasmic reticulum, by analogy with the growth of lysosomes (Boiler and Wiemken 1986). The growing stems of seedlings have been used for studies of cell growth (Mason et al. 1988). They may also serve as a good experimental system for studies of vacuolar development. In the present study, hypocotyls of the mung bean were separated into the dividing, elongating and mature regions, and the vacuolar membranes were compared between them. The young dividing cells contain the same type of membrane as the central vacuoles in the mature cells and newly synthesized components appear to 311

312

M. Maeshima

pyrophosphatase activity. Electrophoresis—Electrophoresis on 12% polyacrylamide gels that contained 0.1% SDS was carried out by the method of Laemmli (1970). The samples were dissociated in 2% SDS, 2% /?-mercaptoethanol, 5% glycerol and Materials and Methods 100 mM Tris-HCl (pH 6.8) at 70°C for 15 min. Plant materials—Seeds of the mung bean (Vigna Immunochemical analysis—Antibody against purified radiata, cv. Wilczek) were imbibed with water and ger- pyrophosphatase was prepared as described previously minated in the dark at 26°C. After 3 days, hypocotyls (Maeshima and Yoshida 1989). Immunoblotting was perwere harvested from uniform straight seedlings with 5-cm formed as described previously (Matsuokaand Asahi 1983) stems and dissected into three regions: the 5-mm portion by a modified version of the method of Towbin et al. just below the cotyledons (dividing region), the next 10-mm (1979). The antibody that reacted with the antigen on a niportion (elongating region), and the remainder (mature trocellulose filter was detected with horseradish peroxidaseregion). Elongation was monitored by marking the entire linked protein A (Amersham) and 4-chloro-l-naphtol (Biohypocotyl at 5-mm intervals with ink. Rad). Preparation of protoplasts—About one gram of each The slot-blot technique was used to determine the region was excised from the hypocotyls of 3-day-old seed- amount of antigen in the vacuolar membrane fraction. lings and cut into slices. The slices of tissue were soaked in The crude membrane fractions were suspended in 1% SDS about 10 ml of maceration medium composed of 1% to a concentration of 2 mg fr wt/ml and incubated at 70°C cellulase Onozuka R-10, 0.2% macerozyme R-10, 0.01% for 5 min. Several dilutions of the crude membrane fracPectolyase Y-23, 50 mM CaCl2, 0.3 M sucrose and 10 mil tions and the purified pyrophosphatase were slot-blotted sodium acetate, and the mixture was allowed to stand for onto nitrocellulose using a slot-blot apparatus (Bio-Rad). 6 h at 25°C. After centrifugation at 400 x g for 5 min, the After the filter had been processed for the development of top layer of protoplasts was collected. The protoplasts color by treatment with pyrophosphatase-specific antibody were examined under a light microscope (HBS-2, Olym- and horseradish peroxidase-linked protein A, as described pus). Diameters of protoplasts were measured from above, levels of antigen on the filter were quantified by densitometric scanning. micrographs. Preparation of tonoplast—Segments of the hypocotyls Measurements—Levels of PPS and ATP were determinwere homogenized with a mortar and pestle in 50 mM ed by the method of Smyth and Black (1984). Segments of MOPS-KOH buffer, pH7.6, that contained 0 . 2 5 M sor- the tissue were quickly frozen in liquid nitrogen and bitol, 5 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, pulverized with a mortar and pestle. PPj and ATP were ex1.5% polyvinylpyrolidone and 1% ascorbic acid. The ho- tracted from the frozen powder by treatment with 0.45 N mogenate was filtered and centrifuged at 3600 xg for HC10 4 . The homogenate was centrifuged at 27,000 x g for 10 min. The supernatant was centrifuged at 120,000 xg 20 min, and then the supernatant was neutralized with for 20 min. The resulting precipitate (crude membrane KOH. Insoluble material was removed by centrifugation fraction) was suspended with 20 mM Tris-acetate buffer, at 10,000xg for 1 min. Amounts of PPS and ATP were pH 7.5, that contained 1 mM EGTA, 1 mM dithiothreitol determined enzymatically using commercial reagents; a and 0.3 M sucrose and assayed for pyrophosphatase, pyrophosphate assay reagent (Sigma) and a ATP bioluminitrate-sensitive ATPase and Cyt c oxidase activities. Fur- nescence CLS (Boehringer Mannheim), respectively. ther purification of tonoplast was carried out as described Levels of P, were determined using a molybdate reagent previously (Maeshima and Yoshida 1989). (Taussky and Shorr 1953). DNA was extracted by the Sucrose density gradient centrifugation—The crude method of Blin and Stafford (1976) and quantified using membrane fractions were subjected to somcation for 20 s diphenylamine (Richards 1974). RNA was extracted by after they had been diluted with 20 mM Tris-acetate buffer, treatment with a mixture of phenol/chloroform/isoamyl pH7.5, that contained 1 mM dithiothreitol, 1 mM MgCl2 alcohol, which was followed by double precipitation by and 1 mM EGTA. After centrifugation at 150,000xg for ethanol and lithium chloride. Levels of RNA were 30 min, the precipitates were suspended in 0.4 ml of the estimated from the absorbance at 260 run. Activities of the same buffer. The suspensions were again subjected to inorganic pyrophosphatase associated with the vacuolar sonication and layered on linear gradients of 12 to 40% membrane (Maeshima and Yoshida 1989) and Cytc ox(w/w) sucrose. All solutions of sucrose contained 1 mM idase (Maeshima et al. 1987) were assayed as described predithiothreitol, 1 mM MgCl2, 1 ihM EGTA and 20 mM Tris- viously. Nitrate-sensitive ATPase activity was measured acetate, pH 7.5. After centrifugation for 18 h at by determining the rate of liberation of P( (Dupont and 26,000 rpm (90,000 x g) in a Hitachi RPS 27 rotor, the gra- Leonard 1980). One unit of enzyme activity was defined as dients were collected as 0.9-ml fractions and assayed for the amount of enzyme which hydrolyzed 1 //mol of the be added to the existing membranes in the elongating cells. The physiological role of vacuolar pyrophosphatase is also discussed.

Vacuolar development in elongating cells

313

substrate per min under the assay conditions. Protein content was determined by the method of Lowry et al. (1951) after precipitation by trichloroacetic acid, with bovine serum albumin as a standard. The tissue segments were extracted with 0.2% SDS to quantify the total protein content. The tissue segments (2 g) were frozen in liquid nitrogen and pulverized with a mortar and pestle. Protein was extracted with 10 ml of 0.2% SDS and 100 nui Tris-HCl (pH 7.5). The homogenate was stirred at 20°C for 30 min and centrifuged at 10,000 xg for 10 min. The supernatants were used for determination of protein. Results Cell growth—During the germination of mung beans the hypocotyls grew extensively, as shown in Figure 1. The rate of stem elongation was highest on day 3 under the growth conditions. Therefore, 3-day-old seedlings were used in this investigation. The hypocotyls of etiolated seedlings were divided into three parts: the dividing, elongating and mature regions. In order to compare the sizes of cells among these three regions of the hypocotyl, protoplasts were prepared from the regions and the diameters of protoplasts were measured from micrographs (Fig. 2). The mean diameters of 60 protoplasts from the dividing, elongating and mature regions were 40, 86 and 126 ftm, respectively. The increase in cell volume during cell maturation was estimated to be greater than 20-fold, as judged from the diameter of protoplasts. As shown in Figure 2, the major increase in cell volume was accounted for by enlargement of the vacuole rather than by any increase in volume of the cytoplasm. Levels of enzymes of the vacuolar membrane and of some metabolites in different regions of the hypocotyl— Table 1 shows the levels of DNA, RNA, protein, ATP, PPj,

0

20 40 60 80 100 Time after imbibition (h)

Fig. 1 Growth of mung bean seedlings. Mung bean seeds were germinated in the dark at 26°C for the indicated periods. Hypocotyl (•), cotyledon (•) and root (A) tissues were carefully taken from uniformly germinating seedlings and weighed. The length of stems is indicated by a broken line. Data represent means of measurements from ten seedlings.

P i( two enzymes associated with the vacuolar membrane and Cytc oxidase in dividing, elongating and mature regions. DNA content, calculated on the basis of the fr wt of the dividing region, was 12 times greater than that of the mature region, indicating that a remarkable increase in cell volume takes place during cell maturation. Although the dividing region accounted for only a small fraction of the total weight of hypocotyl tissue, levels of protein and RNA

Table 1 Levels of metabolites and enzymes in different regions of the mung bean hypocotyl Metabolites and enzymes Fresh weight (mg/seedling) DNA (//g/g fr wt) RNA (mg/g fr wt) Protein (mg/g fr wt) PPi (nmol/g fr wt) ATP (//mol/g fr wt) Pi (j/mol/g fr wt) Pyrophosphatase (units/g fr wt) (mg/g fr wt) (Mg/f*S of DNA)

Nitrate-sensitive ATPase (units/g fr wt) Cyt c oxidase (units/g fr wt)

Dividing 7.70 59.9 1.66 17.6 84 0.93 4.92 0.874 0.20 3.3 0.124 1.60

Region of hypocotyl Elongating

Mature

41.2 16.0 0.462 4.75 70 0.30 4.24 0.463 0.15

193 5.16 0.228 2.65 64 0.18 4.49 0.280 0.070

7.2

0.116 0.483

13

0.0674 0.288

M. Maeshima

B Fig. 2 (A) Three major regions of the mung bean hypocotyl. The hypocotyl from a 3-day-old seedling was separated into dividing (D), elongating (E) and mature (M) regions. (B) Photomicrographs of protoplasts from the three regions of the hypocotyl. Protoplasts were prepared from dividing (a), elongating (b) and mature (c) regions as described in "Materials and Methods". Bar represents 100 fim.

on the basis of fr wt in this region were higher than in the elongating and mature regions. The levels of the substrates for inorganic pyrophosphatase and ATPase were also quantified. The levels of PPj and ATP on the basis of fr wt in the dividing and elongating regions were higher than in the mature region, while the level of Pj was constant among the regions. The levels of enzymes associated with the vacuolar membrane in the three regions were determined. The

dividing region had higher levels of activity of the vacuolar ATPase and pyrophosphatase than did the elongating and mature regions. The level of pyrophosphatase protein on the basis of fr wt was determined by slot-blot analysis using a pyrophosphatase-specific antibody (Fig. 3A). The level of pyrophosphatase protein in each region was consistent with the enzymatic activity, as shown in Table 1. Thus, activation or inactivation of pyrophosphatase probably does not take place during cell elongation. As shown in Figure

315

Vacuolar development in elongating cells

Vs

V16 32 '64

B Fig. 3 (A) Quantification of the antigen in three regions of the hypocotyl with pyrophosphatase-specific antibody. The crude membrane fractions prepared from dividing (lane 1), elongating (lane 2) and mature (lane 3) regions of 3-day-old seedlings were treated with \% SDS and subjected to slot-blot analysis, as described in "Materials and Methods". Samples were spotted to correspond the same amount of fresh tissue from the three regions. Figures on the ordinate indicate the dilutions of samples. (B) Immunoblot analysis of the pyrophosphatase subunit. Crude membrane fractions, equivalent to 6 mg fr wt, were subjected to SDS-polyacrylamide gel electrophoresis, and then the proteins were transferred to a nitrocellulose filter. Antigen on the filter was detected with pyrophosphatase-specific antibody and horseradish peroxidase-linked protein A. Lanes: 1, dividing region; 2, elongating region; 3, mature region.

3B, there was no difference, during electrophoresis, in the mobility of the pyrophosphatase from the various regions, i.e. there was no change in its apparent molecular mass in the three regions. However, the amount of pyrophosphatase protein on the basis of DNA content increased from 3.3 to 13/ig//ig of DNA during cell maturation. The values may correspond to the relative amount of the enzyme per cell. This increase in the amount of enzyme suggests that active biogenesis of vacuolar membranes occurs in the elongating cells. Apparent density and protein composition of vacuolar membrane—The crude membrane fractions were sonicated and subjected to sucrose density gradient centrifugation to compare the densities of vacuolar membranes. As shown in Figure 4 there was no significant difference in the density of the peak fraction of the pyrophosphatase activity among the three regions. The apparent densities of the three membrane fractions were about 1.13 g/ml. In order to compare the protein components of vacuolar membranes, the vacuolar membranes from the three regions were purified and subjected to SDS-polyacryl-

s to Fraction number

Fig. 4 Distribution of pyrophosphatase activity after sucrose density gradient centrifugation. The crude membrane fractions were subjected to sonication and centrifuged at 150,000 x g for 30min. The precipitates were suspended with l n u i dithiothreitol, 1 mM EGTA, 1 mu MgCl2 and 20 mM Tris-acetate, pH 7.5 and again subjected to sonication. Then the suspensions were layered on linear sucrose gradients (15 ml, 12-40% w/w), as described in "Materials and Methods". After centrifugation at 90,000 x g for 18 h, the gradients were collected as 0.9-ml fractions. Inorganic pyrophosphatase activity was assayed in the presence of 1 mM sodium molybdate. a, Dividing region (0.3 g fr wt); b, elongating region (0.6 g fr wt); c, mature region (1.2 g fr wt).

amide gel electrophoresis (Fig. 5). The bands of pyrophosphatase (73 kDa) and the two major subunits (68 and 57 kDa) of the vacuolar ATPase were distinguishable on the gel. There were no significant differences in the protein composition of the membrane among the three regions. However, the relative levels of some proteins, such as the 24-kDa polypeptide (band d in Fig. 5), increased during cell maturation. Discussion Stems of germinating seedlings grow rapidly and this growth is accompanied by elongation of cells. A large increase in cell volume cannot be obtained without vacuolar development. In this study, the growing stems of mung bean seedlings were divided into three regions to investigate the mechanism of development of the vacuolar memb-

316

M. Maeshima

94 _a ~b -c

67

45

30

Mfe mm 20

14Fig. 5 SDS-polyacrylamide gel electrophoresis of the purified vacuolar membranes. The vacuolar membranes were prepared from dividing, elongating and mature regions of 3-day-old seedlings. The protein component of each membrane fraction (60 /ig of protein) was analyzed by SDS-polyacrylamide gel electrophoresis. The gel was stained with Coomassie brilliant blue R. Lanes: 1, dividing region; 2, elongating region; 3, mature region. Letters a, b, c and d indicate the positions of the pyrophosphatase, subunit A of the vacuolar ATPase, subunit B of the vacuolar ATPase and the 24-kDa polypeptide, respectively.

rane. The cell volume increased at least 10-fold during cell elongation, as judged from the size of protoplasts and the DNA content. During cell maturation the protein components and the apparent density of the vacuolar membranes did not change significantly (Figs. 4 and 5). The evidence presented here indicates that the young cells in the dividing region contain vacuolar membranes that are similar to the membranes of the central vacuoles in the mature region. Inorganic pyrophosphatase is a major component of the vacuolar membrane (Maeshima and Yoshida 1989). The preparations of vacuolar membrane from the three regions of the hypocotyl contained pyrophosphatase at high levels (Table 1), and the relative level of the enzyme with respect to the total membrane protein did not change during maturation (Fig. 5). The major subunits of the vacuolar ATPase were also detected in every preparation of vacuolar membranes. The presence of these components in the dividing region indicates that biogenesis of vacuolar membrane takes place just after cell division. The increase in the amount of the vacuolar pyrophosphatase on the basis of DNA content indicates that net syn-

thesis of the enzyme occurs during cell maturation. The level of enzyme on the basis of DNA content in the mature region was 4 times that in the dividing region. Therefore, more than half of the pyrophosphatase in the mature cells may be synthesized in the elongating cells. In recent preliminary experiments, when the mRNA isolated from the dividing and elongating regions of mung bean seedlings was translated in a cell-free translation system, pyrophosphatase was immunoprecipitated by the pyrophosphatasespecific antibody from the translation products. This result suggests that the increase in levels of pyrophosphatase protein may be due to active translation of the mRNA in the elongating cells. Furthermore, the absence of any change in the apparent density of the membrane suggests that the membrane phospholipid is supplied at the same rate as the membrane protein. In conclusion, it is proposed that the vacuolar membrane in the mature cells may be constructed from both the existing proteins in the dividing cell and the proteins that are synthesized de novo during cell elongation. However, no data are available about the pre-existing vacuoles in the young cells since it is difficult to collect only "newborn" cells from the elongating stems. Data for the dividing region obtained in this study represent the mean of data for newborn cells and cells that are several hours old. It is generally assumed that vacuoles arise initially in young dividing cells by progressive fusion of vesicles driven from the Golgi apparatus or the endoplasmic reticulum (Boiler and Wiemken 1986, Klausner 1989). The following two mechanisms of vacuolar development are possible, (a) Most proteins of the vacuolar membrane are synthesized just after cell division, and the membrane components of the central vacuole in the mature cell are supplied by fusion of the small, primary vacuoles. In this case, the total surface area of vacuolar membrane in a cell may not change during cell elongation, (b) The membrane proteins are actively synthesized, even in elongating cells, and are supplied to the existing vesicles. The results of the present study revealed an increase of the amount of membrane proteins, and this observation supports the second suggested mechanism of vacuolar development. Levels of PPj and ATP in the hypocotyl tissue of mung bean seedlings were similar to those in soybean stems (Smyth and Black 1984). Takeshige and Tazawa (1989) reported that, in Chara cells, PPj was present predominantly in the cytosol at a concentration of about 0.2 mu. In spinach, the level of PPj in the cytosol was found to be in the range of 0.2 to 0.3 mM (Weiner et al. 1987). By contrast, over 90% of the cellular PPj is located in the mitochondria of rat hepatocytes (Davidson and Halestrap 1988). Compartmentation of PPj in the mung bean hypocotyl must be determined. If PPj is compartmentalized in the cytosol, the local concentration of PPj may be greater than 0.2 mM. Taiz (1986) estimated that the level of PPj in

Vacuolar development in elongating cells the cytosol may reach 0.39 mM. At 0.2 mil PP;, the vacuolar pyrophosphatase can manifest its hydrolytic activity at about 40% of the maximum rate (Maeshima and Yoshida 1989). In order to maintain the turgor pressure required for the continuing elongation of the cell, solutes must be actively accumulated in the growing vacuole to maintain its osmolarity, and the growing vacuole must actively take up protons at a very much higher rate than does the small vacuole. Therefore, it is likely that the H + -pyrophosphatase is essential for maintaining the proton gradient across the vacuolar membrane in the elongating cells. The protein components of the H + -ATPase and H + pyrophosphatase have been characterized. The vacuolar ATPase is composed of several subunits (Nelson and Taiz 1989, Matsuura-Endo et al. 1990), while the pyrophosphatase consists of a single polypeptide tightly bound to the membrane (Maeshima and Yoshida 1989). Thus, pyrophosphatase is a good marker of the vacuolar membrane. Further studies, using biochemical and immunocytochemical techniques, may provide us more information, not only about the formation and development of vacuoles but also about the physiological significance of the pyrophosphatase. This work was supported in part by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan.

References Akazawa, T. and Hara-Nishimura, I. (1985) Topographic aspects, biosynthesis, extracellular secretion, and intracellular storage of proteins in plant cells. Annu. Rev. Plant Physiol. 36: 441-472. Blin, N. and Stafford, D. W. (1976) A general method for isolation of higher molecular weight DNA from eukaryotes. Nucl. Acids Res. 3: 2303-2308. Boiler, T. and Wiemken, A. (1986) Dynamics of vacuolar compartmentation. Annu. Rev. Plant Physiol. 37: 137-164. Davidson, A. M. and Halestrap, A. P. (1988) Inorganic pyrophosphate is located primarily in the mitochondria of the hepatocyte and increases in parallel with the decrease in lightscattering induced by gluconeogenic hormones, butyrate and ionophore A23187. Biochem. J. ISA: 379-384. Dupont, F. M. and Leonard, R. T. (1980) Solubilization and partial purification of the adenosine triphosphatase from a corn root plasma membrane fraction. Plant Physiol. 65: 931-938. Higgins, T. J. V. (1984) Synthesis and regulation of major proteins in seeds. Annu. Rev. Plant Physiol. 35: 191-221. Klausner, R. D. (1989) Sorting and traffic in the central vacuolar

317

system. Cell 57: 703-706. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T 4 . Nature 277: 680685. Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275. Maeshima, M., Hattori, T. and Asahi, T. (1987) Purification of complex II and IV from plant mitochondria. Meth. Enzymol. 148:491-501. Maeshima, M. and Yoshida, S. (1989) Purification and properties of vacuolar membrane proton-translocating inorganic pyrophosphatase from mung bean. J. Biol. Chem. 264: 2006820073. Mason, H. S., Mullet, J. E. and Boyer, J. S. (1988) Polysomes, messenger RNA, and growth in soybean stems during development and water deficit. Plant Physiol. 86: 725-733. Matile, P. (1978) Biochemistry and function of vacuoles. Annu. Rev. Plant Physiol. 29: 193-213. Matsuura-Endo, C , Maeshima, M. and Yoshida, S. (1990) Subunit composition of vacuolar membrane H + -ATPase from mung bean. Eur. J. Biochem. 187: 745-751. Matsuoka, M. and Asahi, T. (1983) Mechanism of the increase in cytochrome c oxidase activity in pea cotyledons during seed hydration: the presence of free cytochrome-c-oxidase subunits in dry seeds and their probable assembly into the holoenzyme during seed hydration. Eur. J. Biochem. 134: 223-229. Nelson, N. and Taiz, L. (1989) The evolution of H + -ATPases. Trends Biochem. Sci. 14: 113-116. Richards, G. M. (1974) Modification of the diphenylamine reaction giving increased sensitivity and simplicity in the estimation of DNA. Anal. Biochem. 57: 369-376. Smyth, D. A. and Black, C. C , Jr. (1984) Measurement of the pyrophosphate content of plant tissues. Plant Physiol. 75: 862-864. Taiz, L. (1986) Are biosynthetic reactions in plant cells thermodynamically coupled to glycolysis and the tonoplast protonmotive force. J. Theor. Biol. 123: 231-238. Takeshige, K. and Tazawa, M. (1989) Determination of the inorganic pyrophosphate level and its subcellular localization in Chara corallina. J. Biol. Chem. 264: 3262-3266. Taussky, H. H. and Shorr, E. (1953) A microcolorimetric method for the determination of inorganic phosphorus. J. Biol. Chem. 202: 675-685. Towbin, H., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gel to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76: 435(M354. Weiner, H., Stitt, M. and Heldt, H. W. (1987) Subcellular compartmentation of pyrophosphate and alkaline pyrophosphatase in leaves. Biochim. Biophys. Ada 893: 13-21. (Received August 7, 1989; Accepted January 11, 1990)