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Plant Physiol. (1998) 116: 589-597
Molecular Cloning of Vacuolar H+-Pyrophosphatase and
Its Developmental Expression in Growing Hypocotyl of Mung
Bean1
Yoichi Nakanishi and
Masayoshi Maeshima*
Laboratory of Biochemistry, Graduate School of Bioagricultural
Sciences, Nagoya University, Nagoya, 464-01 Japan (Y.N.,
M.M.); and Department of Cell Biology, National Institute for Basic
Biology, Okazaki, 444 Japan (M.M.)
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ABSTRACT |
Vacuolar proton-translocating
inorganic pyrophosphatase and H+-ATPase acidify the
vacuoles and power the vacuolar secondary active transport systems in
plants. Developmental changes in the transcription of the
pyrophosphatase in growing hypocotyls of mung bean (Vigna
radiata) were investigated. The cDNA clone for the mung bean
enzyme contains an uninterrupted open reading frame of 2298 bp, coding
for a polypeptide of 766 amino acids. Hypocotyls were divided into
elongating and mature regions. RNA analysis revealed that the
transcript level of the pyrophosphatase was high in the elongating
region of the 3-d-old hypocotyl but was extremely low in the mature
region of the 5-d-old hypocotyl. The level of transcript of the 68-kD
subunit of H+-ATPase also decreased after cell maturation.
In the elongating region, the proton-pumping activity of
pyrophosphatase on the basis of membrane protein was 3 times higher
than that of H+-ATPase. After cell maturation, the
pyrophosphatase activity decreased to 30% of that in the elongating
region. The decline in the pyrophosphatase activity was in parallel
with a decrease in the enzyme protein content. These findings indicate
that the level of the pyrophosphatase, a main vacuolar proton pump in
growing cells, is negatively regulated after cell maturation at the
transcriptional level.
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INTRODUCTION |
The vacuole of higher plants is an enormous, acidic organelle that
occupies a large part of the cell. Typical plant vacuoles are 50 to 100 µm in diameter. The acidic condition in the vacuole is maintained by
the two distinct proton pumps, H+-ATPase and
H+-PPase. The H+ gradient
generated by the proton pumps powers the secondary active transporters
of inorganic ions, sugars, and organic acids (Hedrich and Schroeder,
1989 ; Taiz, 1992).
The vacuolar-type H+-PPase attracts considerable
attention from two viewpoints. The H+-PPase
enzyme molecule is a great proton-pump model with which to study
molecularly how the hydrolysis of phosphate ester is coupled with
proton translocation across the membrane, because the enzyme consists
of a single polypeptide of about 73 kD (Maeshima and Yoshida, 1989; Kim
et al., 1994a), and its substrate is also a simple compound (PPi).
Therefore, the biochemical properties of H+-PPase
and the detailed mechanism of the enzyme reaction have been extensively
analyzed (Baykov et al., 1993; Rea and Poole, 1993). Additionally, the
cDNAs for H+-PPase have been cloned from several
species of higher plants (Sarafian et al., 1992; Tanaka et al., 1993;
Kim et al., 1994b; Lerchl et al., 1995; Sakakibara et al., 1996).
From the physiological aspect, an important question is why
H+-PPase coexists with V-ATPase in the same
vacuolar membrane. In other words, why does the
H+-PPase exist in the vacuolar membranes of
plants but not in yeast vacuoles or animal acidic compartments? The
vacuolar membranes prepared from all of the plant species, including
mosses, ferns, and algae, show H+-PPase activity
in addition to V-ATPase activity (Takeshige et al., 1988; Ikeda et al.,
1991; Maeshima et al., 1994). The existence of
H+-PPase in plant cells may be related to the
huge size of the plant vacuole. Quantitative analysis of the levels of
enzymatic activity, enzyme protein, and the transcript of the
H+-PPase under stress conditions may provide
important information for understanding the physiological function of
H+-PPase. Carystinos et al. (1995) reported that
the relative transcript and enzyme activity of
H+-PPase increased notably under anoxia and
chilling in rice seedlings. They proposed that
H+-PPase may replace V-ATPase under energy
stress. An increase in the H+-PPase activity was
also observed in mung bean (Vigna radiata) hypocotyls under
low-temperature stress (Darley et al., 1995; Davies,
1997).
We examined the developmental change of H+-PPase
in hypocotyls under normal conditions. The hypocotyl of dicotyledonous
seedlings is widely used as a model for the study of cell elongation
and its control mechanisms (Gen-dreau et al., 1997). Elongation of hypocotyl cells is accompanied by quick expansion of central vacuoles in their cells, and there may be a comparatively modest increase in the
actual amount of cytoplasm during cell elongation. The expanding
vacuoles in elongating cells must incorporate solutes and water.
Therefore, the interior acidic condition of the expanding vacuoles must
be maintained to support the secondary active transport systems. We are
conducting a series of experiments to determine whether the vacuolar
proton pumps are essential for quick expansion of vacuoles.
Previously we reported that, even in the young cells of the mung bean
hypocotyl, the relative H+-PPase level on the
basis of membrane protein content was the same as that of the mature
cell (Maeshima, 1990). In this study we focused our attention on the
developmental expression of the H+-PPase gene
during expansion of vacuoles in elongating hypocotyls of mung bean.
RNA-blot analysis revealed the down-regulation of H+-PPase gene expression in hypocotyl
development, supporting the previous observations. Here we discuss the
primary structure of H+-PPase and the
physiological meanings of the regulation of
H+-PPase level in growing plant cells.
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MATERIALS AND METHODS |
Seeds of mung bean (Vigna radiata cv Wilczek) were
soaked in 1 mm CaSO4 and then
germinated at 26°C in the dark. Hypocotyls from 3- and 5-d-old
seedlings were cut into 1- and 1.5-cm segments, respectively. The
segments were numbered from the hypocotyl apex, as shown in Figure 2A
(3-d-old hypocotyl, segment nos. 31-36; 5-d-old hypocotyl, segment
nos. 51-59). To obtain leaves, mung beans were grown in garden soil
for 3 weeks under long-day conditions (16/8 h, 28/20°C, light/dark
regime). The first, second, and third leaf pairs were harvested from
the 3-week-old plants.
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| Figure 2.
Amounts of RNA and DNA in segments of etiolated
mung bean hypocotyls. A, Mung bean seedlings germinated for 3 and
5 d. Three- and 5-d-old hypocotyls were cut into 1- and 1.5-cm
segments, respectively. B and C, Amounts of total RNA and total DNA,
calculated on the basis of fresh weight (fw) of segment. D, Relative
content of RNA on the basis of DNA content.
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cDNA Construction and Screening
Total RNA was isolated from the hook portion of 3-d-old hypocotyls
for construction of a cDNA library. The hypocotyl segments were
immediately frozen in liquid nitrogen, and then RNA was extracted by
the phenol/SDS extraction method. RNA and DNA were precipitated by cold
ethanol and resuspended in TE buffer (10 mm Tris-HCl, pH
8.0, and 1 mm EDTA). Then RNA was precipitated by 4 m LiCl (total RNA fraction). Poly(A+)
RNA was purified with oligo(dT)-latex. A cDNA library was synthesized from 5 µg of the poly(A+) RNA, ligated into
phage vector (Uni-ZAP XR, Stratagene), and then packaged with a
packaging extract (Gigapack II Gold, Stratagene). Recombinant phages
from the library were blotted onto nylon membranes and then screened
with a 32P-labeled DNA probe according to
standard hybridization protocol (Sambrook et al., 1989). Positive
clones were excised in vivo into the pBluescript SK( ) plasmid
(Stratagene).
DNA Preparation
DNA was isolated from hypocotyl segments by the same method as RNA
extraction. After RNA was removed by LiCl precipitation, the remaining
supernatant was used as the DNA fraction. Samples and standard DNA were
diluted to 0.5 mL with TE buffer and 10 ng/mL
4 ,6-diamidino-2-phenylindole dihydrochloride. Fluorescence of the DNA
solution was measured with a fluorescence spectrophotometer set at 348 nm for excitation and 450 nm for emission, and then DNA content was
calculated.
Preparation of DNA Probes
After treatment of mRNA with DNase, cDNAs were synthesized from
the mRNAs with reverse transcriptase using oligo(dT)-16 primer. cDNA
templates were amplified by PCR using pfu DNA
polymerase and gene-specific primers (forward,
5-ACTGGTTATGGTCTTGGTGGGT-3; reverse,
5-GGCAACATCTTGCACAGGGCTGT-3). The primers correspond to the consensus
nucleotide sequences of H+-PPase cDNA of barley
(Tanaka et al., 1993) and Arabidopsis thaliana (Sarafian et
al., 1992). The amplification protocol was 5 min at 95°C (once), 1 min at 95°C, 1 min at 55°C, 2 min at 72°C (40 cycles), and 7 min
at 72°C (once). Amplified DNA fragments (630 bp) were purified and
ligated into the HincII site of the pUC119 plasmid vector
for transformation of Escherichia coli MV1184. The insert
DNA was confirmed to be a cDNA fragment of
H+-PPase by DNA sequencing.
A DNA probe for subunit A of V-ATPase was prepared by PCR using
specific primers (forward, 5-TCCTGATGCCATGGGAAAGAT-3; reverse, 5-CGCATCATCCAAACAGACTTGT-3). Both primers
correspond to the consensus nucleotide sequences of V-ATPase cDNA of
carrot (Daucus carota, Zimniak et al., 1988) and
cotton (Gossypium hirsutum, Wilkins et al., 1993). The cDNA
fragment obtained showed 83 to 84% homology with the cDNAs for the
V-ATPase subunit A from carrot and cotton. The DNA probes were
radiolabeled with  -[32P]dCTP by the
random-priming method. For northern analysis, a fragment of
H+-PPase cDNA digested by restriction enzyme
NcoI and a PCR product of DNA for the V-ATPase subunit A
were used as DNA probes.
DNA Sequencing and Analysis
The DNA sequence was determined from single-strand plasmid DNAs by
the dideoxy chain-termination method using T7 polymerase. Templates
were prepared as single-strand DNA from the pBluescript SK() plasmid
with helper phage M13K07. The DNA sequence was analyzed using a DNA
sequencer (A.L.F., Pharmacia). Sequences were analyzed using the DNASIS
program of Hitachi Software Engineering (Tokyo).
RNA Analysis
Total RNA was isolated from hypocotyl segments and leaves as
described above. RNAs were electrophoresed in a 1.0% agarose gel,
capillary transferred to a nylon membrane, and fixed to the membrane by
UV irradiation. A nylon membrane was prehybridized in a hybridization
buffer containing 0.02% denatured salmon sperm DNA at 65°C for
1 h and was then hybridized with 106 cpm/mL
32P-labeled denatured probe DNA at 65°C
overnight. The blot membrane was washed twice in 2× SSC (0.3 m NaCl and 30 mm sodium citrate) containing
0.1% SDS at room temperature for 5 min and in 0.1× SSC and 0.1% SDS
at 55°C for 1 h. The membrane was exposed to radiographic film
or an imaging plate. The radioimage of the plate was analyzed (BAS2000,
Fuji, Tokyo).
Vacuolar Membrane Preparation, Immunoblotting, and Enzyme Assay
Vacuolar membranes were prepared from hypocotyl segments as
described previously (Maeshima and Yoshida, 1989). Antibodies against
the putative substrate-binding site of mung bean
H+-PPase (Takasu et al., 1997) and subunit A of
the mung bean V-ATPase (Matsuura-Endo et al., 1992) were prepared as
described previously. For immunoblotting, proteins were separated by
SDS-PAGE (10% gel) and transferred to a PVDF membrane by standard
procedures. Antibodies bound to the antigen were detected with
horseradish peroxidase-coupled protein A and chemiluminescent reagents
(Amersham). Levels of the antigens on immunoblots were quantified with
an image analyzer (Bio-Rad). Activities of substrate hydrolysis and
proton transport of H+-PPase and
H+-ATPase were measured as described previously
(Maeshima and Yoshida, 1989; Matsuura-Endo et al., 1990). Protein
content was determined by the method of Bradford (1976).
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RESULTS |
Molecular Cloning of H+-PPase cDNA of Mung Bean
To analyze the expression of a gene for vacuolar
H+-PPase during elongation of hypocotyl, we
constructed a cDNA library for mRNAs in the hypocotyl. Of 120,000 recombinant phages, DNAs of 190 were hybridized with a radiolabeled
H+-PPase cDNA probe, and the inserted DNAs of 20 positive clones were then sequenced. Ten of the clones encoded the same
sequence of H+-PPase, and the remaining clones
did not. The longest of the H+-PPase clones was
denoted VVP2, and both of its strands were fully sequenced.
Figure 1 shows the nucleotide sequence
and the deduced amino acid sequence of the clone VVP2. The cDNA
consists of 2531 bp upstream of the polyadenylate tail, which includes
a 13-bp 5 leader sequence, followed by 2298 bp of an open reading
frame encoding 766 amino acids, and, finally, a 220-bp 3-noncoding region. The polyadenylate addition site lies 38 bp downstream from the
AAGAAA polyadenylation signal. The presumed translation start site is
the first ATG encountered downstream from the 5 end. The deduced amino
acid sequence of the N-terminal part showed agreement with the direct
sequence of the purified H+-PPase, although the
purified enzyme lacks the N-terminal Met residue (Maeshima and Yoshida,
1989). There is no cleaved signal peptide in the deduced
H+-PPase sequence. From gel-blot analysis of the
total RNA fraction of hypocotyls, the H+-PPase
mRNA was estimated to be 2.7 kb (Fig. 3), and this size corresponds to
that of the insert DNA. Hung et al. (1995) registered the cDNA sequence
of mung bean H+-PPase without the triplet of GGT
encoding the Gly-688 found in the present study. There is a possibility
that the difference is derived from plant materials. The sequence of a
part including Gly-688 in the present study was identical to that of
the other H+-PPases from many plant species.
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| Figure 1.
Nucleotide sequence and deduced amino acid
sequence of H+-PPase cDNA of mung bean (clone VVP2). The
underlined amino acid residues were identical to those of the direct
sequence obtained from H+-PPase purified from mung bean.
Thirteen putative membrane-spanning domains predicted from the
hydropathy profile are boxed. The termination codon is marked with an
asterisk.
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| Figure 3.
Developmental change in the content of mRNA for
H+-PPase in hypocotyls. Hypocotyls from more than 100 seedlings germinated for 3 or 5 d were cut into segments and RNA
fractions were prepared. Aliquots (4.5 µg) of the total RNA were
electrophoresed and blotted onto a nylon membrane. The membrane was
subjected to northern analysis with a 32P-labeled cDNA
fragment of H+-PPase, and the radioactivity was measured as
described in ``Materials and Methods''. A, Ethidium bromide staining
of rRNAs (control). B, Northern blot with a DNA probe for
H+-PPase. C and D, Relative amounts of H+-PPase
mRNA on the basis of RNA or DNA content, respectively. Values shown are
relative to that of segment no. 31.
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Structural Characteristics of the H+-PPase Polypeptide
The calculated molecular mass of the derived polypeptide was
80,002 D, which was larger than the value of 73 kD for the purified H+-PPase on SDS-PAGE (Maeshima and Yoshida, 1989;
Kim et al., 1994b). Rapid migration of the enzyme on the SDS gel may be
due to the high hydrophobicity of the H+-PPase.
Hydrophobic amino acids (481 residues) account for about 63% of the
total 766 residues. In the sequence of mung bean
H+-PPase, there are 13 hydrophobic domains of
sufficient length and hydrophobicity to span a lipid bilayer (Fig. 1).
The deduced sequence of mung bean H+-PPase has
high identity to those of H+-PPases of other
plant species: A. thaliana (87.5%; Sarafian et al., 1992),
barley (88.6%; Tanaka et al., 1993), red beet (two isoforms, 89.2 and
89.3%; Kim et al., 1994b), tobacco (four isoforms, 88.6-91.5%;
Lerchl et al., 1995), and rice (two isoforms; 88.1 and 86.9%;
Sakakibara et al., 1996). The mung bean enzyme has a putative
substrate-binding site (residues 253-263), as was also described for
Arabidopsis H+-PPase by Rea and Poole (1993).
Decrease in mRNAs for H+-PPase and
H+-ATPase Subunit A during Elongation of Hypocotyl
Mung bean hypocotyls can be separated into a hook part (the
elongating region) and a lower part under the hook (the mature region).
To compare the mRNA level of vacuolar proton pumps among hypocotyl
segments, 1- or 1.5-cm segments were prepared from seedlings germinated
for 3 and 5 d (Fig. 2A). The top
segment, no. 31, consists of small, young cells; the lower segments,
nos. 35 and 36, consist of large, mature cells (Maeshima, 1990). Rapid
elongation took place at segment no. 31, in which the elongation rate
was about 400%/d in 3-d-old seedlings. The lower part did not elongate
so rapidly; the rate at segment no. 32 was about 30%. The amount of
total RNA per gram fresh weight was highest in the elongating region
(segment no. 31). Figure 2C shows the total amount of DNA in each
segment on the basis of fresh weight. The level of DNA was highest in
segment no. 31, which has a small cell volume. To compare the relative
amounts of RNA in cells, the ratio of RNA to DNA was calculated. The
RNA to DNA ratio decreased during hypocotyl elongation, as shown in
Figure 2D.
The levels of H+-PPase mRNA during hypocotyl
elongation were determined by northern analysis (Fig.
3). Figure 3, C and D, shows the
H+-PPase mRNA levels on the basis of the amount
of RNA and DNA, respectively. In both cases, the
H+-PPase mRNA content was the highest in the
elongating region (segment nos. 31 and 32) and lowest in the mature
region (segment nos. 34 and 35). The level of
H+-PPase mRNA per DNA content in segment no. 57 of 5-d-old hypocotyl was 5% of that in the elongating region.
To determine the levels of mRNA for the V-ATPase subunit A, which is a
nucleotide-binding subunit, its cDNA was prepared and used as a probe.
In contrast to the H+-PPase mRNA, the level of
subunit-A mRNA was highest in the middle part (segment nos. 32 and 33)
of 3-d-old hypocotyls (Fig. 4). These
findings suggest that the expression of genes for
H+-PPase and the V-ATPase subunit A are regulated
independently. Expression in the mature regions (segment nos. 34-36
and no. 57) was very low, as was the H+-PPase
mRNA.
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| Figure 4.
Level of mRNA for the subunit A of V-ATPase in
hypocotyl segments. Aliquots (20 µg) of the total RNA fractions from
the hypocotyl segments were subjected to northern analysis with a
32P-labeled cDNA fragment of the V-ATPase subunit A. A,
Ethidium bromide staining of rRNAs. B, Northern analysis. C and D,
Relative amount of mRNA for the V-ATPase subunit A on the basis of RNA or DNA content, respectively. Values shown are relative to that of
segment no. 31.
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Change in Levels of mRNAs for H+-PPase and V-ATPase
Subunit A in Leaves
RNA was extracted from leaves of different ages to determine the
levels of mRNA for the two vacuolar proton pumps. As shown in Figure
5, the levels of mRNAs for
H+-PPase and V-ATPase subunit A were highest in
the immature third leaf and decreased during leaf growth. The relative
levels of the mRNAs for both proteins in the first, old leaf were less
than 5% of that of the third, young leaf.
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| Figure 5.
Levels of mRNAs for H+-PPase and
subunit A of V-ATPase in mung bean leaves. Total RNA fractions were
prepared from eight pairs of the first, second, and third leaves of
3-week-old mung bean plants. RNAs (20 µg) were subjected to northern
analysis with a radiolabeled DNA probe for H+-PPase (A) or
the subunit A of V-ATPase (B).
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Levels of H+-PPase and H+-ATPase in
Vacuolar Membranes of Hypocotyl
We determined the levels of H+-PPase and
H+-ATPase in the vacuolar membranes of the
hypocotyl segments. Immunoblot analysis with the antibody against mung
bean H+-PPase revealed that the amount of
H+-PPase molecules on the basis of membrane
protein was highest in segment no. 31 and decreased during cell
elongation (Fig. 6, A and B). As presumed
from quantification of mRNA, the amount of
H+-PPase protein in the mature part of the
hypocotyl (segment no. 58) was less than one-third of that in the
elongating region (segment no. 31). The activities of PPi hydrolysis
and PPi-dependent H+ transport in the membrane
vesicles on the basis of membrane protein decreased during cell
elongation (Fig. 6, C and D). This also indicates that there was no
inactivation or suppression of H+-PPase activity
in the membranes during cell elongation. The data are the relative
levels of the enzyme on the basis of the membrane protein and reflect
the relative density of the enzyme in a unit area of vacuolar membrane.
Since the amount of vacuolar membrane in each cell increases as cells
grow, the total amount of H+-PPase in the cell
increased during cell growth, although the relative content of the
enzyme on the basis of membrane protein decreased markedly.
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| Figure 6.
Level of H+-PPase in vacuolar
membranes of hypocotyl segments. Vacuolar membranes were prepared from
more than 200 pieces of segment (segment nos. 31, 35, and 58). Aliquots
(1 µg) of vacuolar membranes were subjected to SDS-PAGE and
immunobloted with antibody to mung bean H+-PPase (73 kD). A, Immunoblot. B, Amounts of H+-PPase protein relative
to segment no. 31. C and D, PPi-hydrolysis activity (C) and
H+-transport activity (D) of H+-PPase in the
vacuolar membranes. The data shown are means ± sd for
two experiments, each with triplicate assays.
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In contrast to H+-PPase, the relative content of
V-ATPase subunit-A protein did not decrease in the mature part of
hypocotyls (Figs. 7, A and B); neither
did that of subunit B (Fig. 7, D and E). The activities of ATP
hydrolysis and ATP-dependent H+ transport of the
membranes were constant during growth (Fig. 7, C and F). Consequently,
the V-ATPase activity in 5-d-old hypocotyls was slightly higher than
the H+-PPase activity, although the
H+-PPase activity in 3-d-old hypocotyls was several times
greater than the ATPase activity. The present data suggest that the
distribution density of V-ATPase on vacuolar membranes did not change
during development of the vacuole.
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| Figure 7.
Levels of V-ATPase in vacuolar membranes of
hypocotyl segments. Aliquots (1 µg) of vacuolar membranes of each
segment were subjected to SDS-PAGE and immunoblotting with antibodies
to subunits A (68 kD, A) and B (57 kD, D) of V-ATPase. B and E, Amounts
of subunit A (B) and B (E) of V-ATPase relative to that in segment no.
31. C and F, ATP-hydrolysis activity (C) and H+-transport
activity (F) of V-ATPase in the vacuolar membranes. The data shown are
means ± sd for two experiments, each with triplicate assays.
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DISCUSSION |
We cloned the cDNA of the vacuolar membrane
H+-PPase of mung bean and examined its expression
in elongating hypocotyls. Many positive clones for
H+-PPase obtained from a cDNA library of
hypocotyl have the same sequence. Genomic Southern analysis suggested
the presence of two copies of H+-PPase genes, and
we sequenced about 1500 bp of the two genes. The nucleotide sequences
of their protein-coding regions were identical to each other (data not
shown). Furthermore, immunoblot analysis of the vacuolar membranes
separated by two-dimensional PAGE showed only a single spot with the
antibody to H+-PPase (Takasu et al., 1997).
Therefore, we concluded that there may be only one species of mRNA for
H+-PPase and no isoform of the enzyme in mung
bean hypocotyl.
The cDNA VVP2 encodes a polypeptide of 80,002 with 766 amino acids. We
compared it with several cDNA clones of H+-PPase
among plant species. The deduced polypeptides consisted of 761 to 771 amino acids with a pI of approximately 5.0. Their molecular masses
ranged from 79,841 to 80,800 D. A detailed amino acid comparison
between the mung bean H+-PPase polypeptide and
the published sequences from A. thaliana (Sarafian et al.,
1992), Hordeum vulgare (Tanaka et al., 1993), Beta
vulgaris (Kim et al., 1994b), Nicotiana tabacum (Lerchl
et al., 1995), and Oryza sativa (Sakakibara et al., 1996)
revealed that the polypeptides are well conserved, with 86 to 91%
identity. The least conserved region is the amino-terminal part (the
first 56 residues), with 29% homology among 11 H+-PPases. The carboxyl-terminal half from the
405th residue showed a high homology of 90%. The vacuolar
H+-PPase is one of the most highly conserved
polypeptides among higher plants.
Mung bean H+-PPase shows a higher similarity to
H+-PPases of other plant species. The vacuolar
H+-PPase is a common enzyme among green plants,
including nonvascular and vascular plants, Chara corallina,
and Acetabularia acetabulum (Ikeda et al., 1991; Maeshima et
al., 1994). In addition to green plants, the
H+-PPase was purified from a photosynthetic
bacterium Rhodospirillum rubrum (Nyrén et al., 1991),
and it has been shown to react with antibodies against mung bean
H+-PPase (Nore et al., 1991). Molecular cloning
of the H+-PPases of other organisms, such as
mosses, ferns, C. corallina, A. acetabulum, and
R. rubrum, may provide information to help gain an
understanding of the molecular evolution of
H+-PPase.
A comparison between vacuolar H+-PPase and
soluble PPases suggested that the configuration
(E/D)(X)7KXE is a putative catalytic site of the PPases
(Rea et al., 1992; Kim et al., 1994b). Indeed, this motif, which is at
position 253 of mung bean H+-PPase, is conserved
among 11 H+-PPases from six plant species. The
polyclonal antibodies specific to a peptide of DVGADLVGKVE, which
corresponds to the motif, markedly inhibited the activities of PPi
hydrolysis and the PPi-dependent H+ transport
(Takasu et al., 1997). These findings support the hypothesis that the
motif (E/D)(X)7KXE is involved in substrate binding and/or substrate hydrolysis at the cytosolic surface of
H+-PPases.
The vacuolar H+-PPase is sensitive to the
sulfhydryl reagent NEM. Free Mg2+ or substrate
(Mg2+ plus PPi) protects the reversible
inhibition by NEM (Zhen et al., 1994; Kim et al., 1995; Gordon-Weeks et
al., 1996). Kim et al. (1995) demonstrated that substrate-protectable
NEM inhibition is due to NEM binding to Cys-634, which is located on a
cytoplasmic loop between membrane-spanning domains X and XI of
Arabidopsis H+-PPase. Recently, Gordon-Weeks et
al. (1996) investigated the Mg2+-protectable
sensitivity of mung bean H+-PPase to NEM and
proposed that Glu and Asp residues in the loop containing Cys-634 are
involved in Mg2+ binding to the enzyme. The
sequence of mung bean enzyme shows that Glu and Asp residues are
conserved in the loop containing Cys-630 of mung bean enzyme. The
hypothesis must be tested by site-directed mutagenesis and direct
binding experiments.
The aim of this study was to examine whether the vacuolar
H+-PPase gene is expressed constantly in the
process of vacuole development. The hypocotyl of mung bean seedlings is
a good system for the study of vacuole development and cell elongation.
The amount of the vacuolar membranes, which reflects the surface area
of vacuoles, increased at least four times during cell elongation in
mung bean hypocotyls (Maeshima, 1996). High expression of the
H+-PPase gene and its active biosynthesis are
believed to be essential to quick expansion of the vacuole in growing
cells (Maeshima et al., 1996). Measurements of enzymatic activities and
the immunochemical quantification of the two proton pumps clearly
showed that H+-PPase is the main proton pump of
vacuoles in elongating hypocotyls. In 3-d-old hypocotyls,
proton-pumping activity of H+-PPase per membrane
protein content was 2 to 3 times greater than that of V-ATPase (Figs. 6
and 7). This is in agreement with a previous study (Maeshima, 1990). In
5-d-old hypocotyls, however, the proton-pumping activity of V-ATPase
was slightly higher than that of H+-PPase. The
change in ratio of the activity between the two proton pumps is due to
the decrease in H+-PPase activity. A marked
decrease in the amount of H+-PPase was also
observed in radish roots (Maeshima et al., 1996).
In this study we determined the levels of transcripts of genes for
H+-PPase and the V-ATPase subunit A in segments
of hypocotyls. The decrease of the gene expression resulted in the low
activity of H+-PPase. The genes for
H+-PPase and the V-ATPase subunit A are highly
transcribed in the elongating parts of the hypocotyl, and the levels of
transcripts are lower in mature parts. In the same hypocotyls, the
levels of mRNAs for H+-PPase and the V-ATPase
subunit A in the mature part were, on the basis of DNA, less than 50%
of that in the elongating part. A similar phenomenon was observed in
the developing leaf. Immature leaves contained a high level of mRNAs
for H+-PPase and the V-ATPase subunit A, but the
levels decreased in mature leaves. Our findings are in agreement with
those reported by Lerchl et al. (1995). In tobacco the expression of
H+-PPase was active in young sink leaves, and the
transcripts decreased in the mature source leaves. The decrease
in transcription of the H+-PPase gene
resulted in low levels of H+-PPase protein
and activity. The H+-PPase may be gradually
degraded after cell maturation.
Growth of hypocotyls is impossible without vacuole expansion, since the
central vacuole occupies more than 80% of the volume of the mature
cell. To maintain the high osmotic pressure of the expanding vacuole,
the vacuole must actively incorporate solutes such as sugars and
inorganic ions. Vacuolar H+-PPase and
H+-ATPase provide the power for the secondary
active transporters. Therefore, the vacuolar proton pumps are essential
for vacuole enlargement. The physiological change in the
H+-PPase level is efficient from the perspective
of the cell's energetics. The substrate for
H+-PPase, PPi, is produced as a by-product of
several metabolic processes, such as polymerization of DNA and RNA and
syntheses of aminoacyl-tRNA (protein synthesis), ADP-Glc (starch
synthesis), UDP-Glc (cellulose synthesis), and fatty acyl-CoA
( -oxidation of fatty acid).
In growing cells, RNAs, proteins, and cellulose are actively
synthesized for the construction of cells and as a result, a large
amount of PPi is produced (Fig. 8). PPi
accumulated in the cytosol at high concentrations inhibits the
biosynthesis of macromolecules. In animal and bacteria cells, PPi is
removed by soluble inorganic PPases. In plant cells, the vacuolar
H+-PPase scavenges PPi in the cytosol and
utilizes it as a source of energy for the active transport of protons
into the vacuole. The vacuolar H+-PPase in
growing cells helps to conserve ATP, which is a cell's energy
currency. In mature cells metabolic activity decreases and PPi may not
be available in such large amounts. Thus, the suppression of gene
expression of H+-PPase after cell maturation is
economical for the plant cell.
View larger version (49K):
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| Figure 8.
PPi production and vacuolar membrane
H+-ATPase and H+-PPase. PPi is supplied as a
by-product of biosyntheses of macromolecules such as RNA, proteins, and
cellulose in elongating cells.
|
|
The change in level of the V-ATPase subunit A did not parallel the
change in the mRNA level. The relative content of the subunit A protein
was the same after hypocotyl growth, although the relative mRNA content
in the 5-d-old hypocotyl was about 30% of that in the elongating part
of the 3-d-old hypocotyl (Fig. 4). The enzymatic activity of V-ATPase
was also constant during hypocotyl growth (Fig. 7), suggesting that
neither activation nor inactivation of V-ATPase occurred in hypocotyls.
Probably, molecules of V-ATPase are more stable than those of
H+-PPase. V-ATPase can operate constantly because
ATP is maintained at a high concentration (a few millimolar). In
conclusion, the H+-PPase is a major proton pump
of vacuoles in growing hypocotyls, and its abundance is regulated at
the transcriptional level in response to the supply of PPi. On the
other hand, the V-ATPase activity remains relatively constant as a
fundamental proton pump in the growing hypocotyl.
The level of H+-PPase is regulated under stress
conditions. Carystinos et al. (1995) reported that the relative
transcript and enzyme activity of H+-PPase
increased notably under anoxia and chilling in rice seedlings. They
proposed that H+-PPase may replace V-ATPase under
energy stress. The H+-PPase activity was also
increased in mung bean hypocotyls under low-temperature stress (Darley
et al., 1995). It is obvious that gene expression of
H+-PPase is regulated mainly in response to the
demand of vacuolar acidification and is independent of V-ATPase.
Transgenic plants that lack the H+-PPase gene(s)
or the enzyme activity of H+-PPase should be
analyzed to determine why plant vacuoles have H+-PPase in addition to V-ATPase. Also, the
molecular mechanism involved in the regulation of the
H+-PPase gene expression by the supply of ATP and
PPi requires further study.
| |
FOOTNOTES |
1
Part of the research for this work was supported
by Grants-in-Aid for Scientific Research from the Ministry of
Education, Science, and Culture of Japan to M.M. (nos. 09257221 and
09660114).
*
Corresponding author; e-mail maeshima{at}agr.nagoya-u.ac.jp; fax
81-52-789-4094.
Received June 24, 1997;
accepted October 27, 1997.
The DDBJ accession number for the DNA sequence reported in this article
is DAB009077.
| |
ABBREVIATIONS |
Abbreviations:
H+-PPase, H+-transporting inorganic pyrophosphatase.
NEM, N-ethylmaleimide.
V-ATPase, vacuolar
H+-ATPase.
| |
ACKNOWLEDGMENTS |
We thank Dr. Yoshiyuki Tanaka of the National Institute of
Agrobiological Resources and Dr. Kenzo Nakamura of Nagoya University for their kind advice and stimulating discussions.
| |
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Top
Abstract
Introduction