全 文 ：Received 28 Jun. 2003 Accepted 28 Aug. 2003
Supported by the National Natural Science Foundation of China (30070069, 30270793), the Hi-Tech Research and Development (863)
Program of China (2002AA629080) and the Shandong Provincial Natural Science Foundation (Z2000D01).
* Author for correspondence. Fax: +86 (0)531 6180107; E-mail:.
http://www.chineseplantscience.com
Cloning and Expression Analysis of the B Subunit of V-H+-ATPase in
Leaves of Halophyte Suaeda salsa Under Salt Stress
LI Ping-Hua, WANG Zeng-Lan, ZHANG Hui, WANG Bao-Shan*
(College of Life Sciences, Shandong Normal University, Jinan 250014, China)
Abstract: For salinity stress tolerance in plants, the vacuolar type H+-ATPase (V-H+-ATPase) is of prime
importance in establishing an electrochemical H+-gradient across tonoplast that energizes sodium
sequestration into the central vacuole. In this paper, the sequence of a cDNA encoding the B subunit of
the vacuolar-type H+-ATPase from Suaeda salsa L., a plant that can survive in seawater was reported. The
B subunit cDNA is 1 974 nucleotides long and include a 18 bp poly(A+) tail together with a complete 1 470
bp coding region for a 489 amino acid with a conservative ATP binding site and a predicted molecular mass
of 54.29 kD. Northern and Western blotting analyses indicated that the expression of B subunit was
significantly up-regulated by NaCl treatment. Moreover, the expressions of B subunit were coordinated
with c subunit of V-H+-ATPase at transcript and translation levels under NaCl stress. The increased V-H+-
ATPase subunit amounts and activity of Suaeda salsa provide the energy for the compartmentation of
sodium in response to salinity.
Key words: B subunit; c subunit; NaCl stress; Suaeda salsa ; V-H+-ATPase
Soil salinity is a major constraint to food production
because it limits crop yield and restricts the use of land
previously uncultivated (Flowers and Yeo, 1995). For plant
adaptation to excess sodium chloride concentration，low
Na+ influx, high Na+ efflux and the vacuolar Na+ accumula-
tion are the main strategies. The vacuolar sodium seques-
tration is mediated by a secondary active Na+/H+ antiport
at the tonoplast (Barkla et al., 1995; Apse et al., 1999) and it
is energized by a proton motive force established by the
vacuolar H+-ATPase (EC 3.6.1.3) and H+-PPase (EC 3.6.1.1)
(Dietz et al., 2001).
The V-H+-ATPase is a large, multimeric enzyme com-
posed of a hydrophilic V1 complex on the cytosolic face of
the tonoplast and a membrane-bound V0 complex (Lüttge
and Ratajczak, 1997; Sze et al., 1999). The V1 domain that
functions in ATP hydrolysis is composed of eight subunits
(A-H). The A subunit that is characterized as a catalytic
subunit of ATP hydrolysis and subunit B that seems as a
non-catalytic regulate subunit of ATP hydrolysis are the
two most important subunits in the V1 domain. The V0 do-
main that is in charge of proton transportation is composed
of five subunits (c, c, c, a and d). The V-H+-ATPase is
important not only as a “house-keeping enzyme” to main-
tain cytosolic ion homeostasis and cellular metabolism, but
also as an “eco-enzyme” which functions as a stress re-
sponse enzyme undergoing moderate changes in expres-
sion of subunits and modulations of enzyme activity under
the environmental stress (Ratajczak, 2000).
In plant, cDNAs encoding the subunit B of V-H+-AT-
Pase were reported for Mesembryanthemum crystallinum,
Arabidopsis thaliana, Oryza sativa, Citrus unshiu and Nic-
otiana tabacum. Although some useful information about
the expression of V-H+-ATPase subunit B under salt stress
was gathered, most of these information was at the protein
level and more information about regulation of V-H+-AT-
Pase subunit B at transcription and translation levels under
salt stress was needed to understand the salt-tolerant mecha-
nism of plants.
Suaeda salsa is a C3 halophyte that could accumulate
Na+ in the vacuole to keep the plant surviving in seawater.
Our previous results showed that the increase in V-ATPase
activity of S. salsa under NaCl stress is not obtained by
structural changes of the enzyme, but by an increase in its
protein amount (Wang et al., 2001). As a further step to-
wards understanding the molecular events leading to vacu-
olar salt sequestration and salt tolerance, a cDNA encoding
B subunit of V-H+-ATPase from a cDNA library of S. salsa
was isolated and the transcription and translation regula-
tion of this gene under salt stress was analyzed.
1 Materials and Methods
1.1 Plant material
Seeds of halophyte plant Suaeda salsa L. (collected from
the Yellow River Delta, Shandong Province, China) were
Acta Botanica Sinica
植 物 学 报 2004, 46 (1): 93-99
Acta Botanica Sinica植物学报 Vol.46 No.1 200494
germinated in acid-washed sand. Seedlings were grown in
Hoangland solution (mmol/L): 5 Ca(NO3)2, 1 KH2PO4, 2
MgSO4, 5 KNO3, 0.2 Fe-EDTA, 2.5× 10-4 H3BO3，5×
10-4 H2MoO4， 5× 10-4 CuSO4， 2× 10-3 ZnSO4，2
×10-3 MnCl2 under 16 h light /8 h darkness, a temperature
of 25-35 ℃/20-25 ℃, a relative humidity of 60%/80%, and
a proton flux density of 600 mmol.m-2.s-1.
Six-week-old seedlings were treated with 0 (control), 100
and 400 mmol/L NaCl. The final salinity level was achieved
by raising 50 mmol/L NaCl every 12 h in order to avoid
osmotic shock.
1.2 cDNA library construction
Seedlings of S. salsa were treated with 400 mmol/L NaCl
for 48 h; aerial part tissue was collected and ground under
liquid nitrogen using a mortar and pestle. Total RNA was
extracted using RNAgent (Promega), poly(A+) RNA was
selected with Messagemaker kit (Gibco, BRL). First-strand
cDNA synthesis was carried out by an oligo-dT linker-primer
with an XhoⅠ cloning site. The 5 end of each cDNA was
ligated to an adaptor with an EcoRⅠ-compatible overhang.
cDNA was ligated undirectionally into the EcoRⅠ and
XhoⅠ sites of the λ -ZAP express vector (Stratagene) ,
packaged in vitro, and amplified. The amplified library rep-
resents approximately 106 recombinants.
1.3 Cloning and sequence analysis
The phage library was converted to the plasmid form by
mass excision according to the protocol described by
Stratagene. The obtained phagemid of the library was used
to infect Escherichia coli strain XLOLR. The E. coli was
grown for 45 min and then plated at low density on medium
containing Luria-Bertani broth, tetracycline (10 mg/L), and
kanamycin (25 mg/L). Cultured in 37 ℃ overnight, indi-
vidual colonies were selected randomly for plasmid DNA
purification and sequencing.
Sequencing reactions contained the standard T3 se-
quencing primer (5-ATTAACCCTCACTAA AGGGAA-3),
and thus read into the presumed 5 end of the cDNA, T7
primer (5-TAATACGACT CACTATAGGG-3) situated at
opposite ends of the inserts. Double-strand sequencing of
plasmid was performed on an Automated Sequencer (PE-
Applied Biosystems). Sequences were analyzed using
DNASIS software, and databank searches were conducted
through the BLAST program.
1.4 Membrane vesicle isolation
Tonoplast-enriched membrane vesicles were isolated
according to the method of Wang et al. (2000) and Mandala
and Taiz (1985) with modifications. Leaves were washed
with cold deionized water and homogenized in an extrac-
tion medium (50 mmol/L Tricine-Tris (pH 7.8), 3 mmol/L
EGTA, 3 mmol/L MgSO4, 0.5% (V/V) PVP, 2 mmol/L DTT,
0.2 mmol/L PMSF, 5% (V/V) glycerol and mannitol which
had the same osmotic potential with the leaves). Two mL of
the medium were used for each 2 g of fresh material. The
homogenate was filtered through four layers of cheese-
cloth and centrifuged at 10 000g for 20 min (Backman 45Ti).
The supernatant was loaded on a 0:25% (W/W) sucrose
gradient solution (5 mmol/L Hepes adjusted to pH 7.5 with
Tris, 1 mmol/L DTT) and centrifuged at 100 000 g for 2 h
(Backman SW 40Ti). The vesicles located at the 0:25%
(W/W) sucrose interface (tonoplast-enriched vesicles) were
carefully collected and diluted 3-4 folds with a dilution
buffer (3 mmol/L MgSO4, 10 mmol/L Hepes-Tris pH 7.5, 1
mmol/L DTT) and centrifuged at 100 000g for 30 min (Backman
45Ti). The pellets were suspended in a storage buffer (40%
(V/V) glycerol, 2 mmol/L DTT, 10 mmol/L Hepes, adjusted to
pH 7.5 with Tris). The tonoplast-enriched vesicles were fro-
zen in liquid nitrogen and stored at –75 ℃ for further use. All
steps of the procedure were performed at 2-4 ℃.
1.5 Measurement of protein content
Protein content was determined as Bradford (1976) us-
ing bovine serum album as a protein standard.
1.6 V-H+-ATPase activity assay
V-H+-ATPase hydrolysis activity of tonoplast-enriched
membrane vesicles were calculated from the amount of in-
organic phosphate released in the absence and presence
of 50 mmol/L KNO3. Enzyme reactions were run at 37 ℃ for
30 min with a protein concentration of 5 µg in a assay me-
dium containing 30 mmol/L Tris/Mes, pH 7.5, 0.1 mmol/L
(NH4) 4 MoO4, 1 mmol/L NaN3, 1 mmol/L MgSO4, 0.03%
(V/V) TritionX-100, 50 mmol/L KCl, 3 mmol/L ATPNa2. Inor-
ganic phosphate was assayed using the method of Lin and
Morales (1977).
1.7 RNA extraction and Northern blotting analysis
Total RNA of leaves of S. salas was isolated by
guanidinium thioisocyanate extraction (Chomczynski and
Sacci, 1987). RNA was quantified by optical density at 260
nm, and the concentration was confirmed by electrophore-
sis on an RNA formaldehyde gel (Sambrook et al., 1989).
Total RNA was loaded on 1.2% (W/V) agarose denaturing
formaldenhyde gel. It was then transferred to Hybond-N+
membrane. In order to affirm uniformity in loading for RNA
blots, RNA was stained by ethidium bromide. RNA was
cross-linked to the membrane by 254 nm UV irradiation.
RNA Northern blot hybridization was performed as de-
scribed by Sambrook et al. (1989). For high stringency in
the presence of 50% (W/V) formamide, A 32P-labeled DNA
probe (3 non-coding region) was prepared using a random
primer labeled kit (Random Primers Systems; TaKaRa,
95 LI Ping-Hua et al.: Analysis of V-H+-ATPase B Subunit
Japan). Hybridization and washes were carried out at 65 ℃.
After drying the blots, autoradiography of the filters was
obtained on Kodak Xar-5 X-ray films using an intensifying
screen at –70 ℃.
1.8 SDS-PAGE and Western blotting analysis
Tonoplast protein was extracted by 30% (V/V) ethanol/
70% (V/V) acetone. Sodium-dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE) was performed as described
in Ratajczak (1994) on slab gels containing 12% (W/V)
acrylamide and by using the Lammli buffer system (Laemmli,
1970). For Western blotting analysis, proteins were trans-
ferred electronically from acrylamide gels to Immoblion
Hybond-P membranes under conditions previously de-
scribed (Fischer-schliebs et al., 1997). After blocking free
protein binding sites for 1 h in 1% (W/V) fat free milk power
dissolved in Tris buffered saline (TBS), the membranes were
incubated with the antisera ATP95 against the V-H+-AT-
Pase holoenzyme of Kalanchoe daigremontiana (Fischer-
schliebs et al., 2000). An alkaline phosphatase-coupled
goat-anti rabbit IgG was used as a secondary antibody.
Immunostaining was performed using nitroblue tetrazolium
by formation of an indigo dye-precipitate with antigen-
coupled alkaline phosphate (Chow et al., 1990).
2 Results
2.1 Isolation and sequencing of the subunit B of V-H+-
ATPase from S. salsa
The salt-stressed cDNA library of halophyte S. salsa
was sequenced as expressed sequence tags (EST). From
the ESTs, we isolated a cDNA clone (BE859205) that had
high homology with V-H+-ATPase B subunit of Arabidopsis.
To gain insight into the possible biochemical and physi-
ological functions of the V-H+-ATPase subunit B in S. salsa,
sequence of the subunit B was determined. By sequencing
using T3 and T7 primers, we obtained full sequence of the
cDNA (GenBank accession number AY231438). The insert
fragment was 1 974 bp with 1 470 bp open reading frame, a 32
bp 5 un-translated region and a 472 bp non-coding 3 region.
The deduced amino acid was 489 amino acids with a pre-
dicted molecular weight of 54.29 kD. The start codon ATG
was found in a likely initiation sequence GATAATGGG simi-
lar to the consensus initiation sequence AACAATGGC in
plants (Lütcke et al., 1987) and two putative polyadenylation
signal consensus sequences (AATAAA) (Rothnie et al.,
1994) were present in the 3 un-translated end (from 1 583 to
1 588 and from 1 869 to 1 874) of the cDNA.
2.2 Homology analysis of amino acid sequence of V-H+-
ATPase B subunit in S. salsa
Amino acid sequence alignment analysis suggested that
the V-H+-ATPase B subunit of S. salsa had high homology
with other reported V-H+-ATPase subunit B in higher plants,
lower plants and insects. It shared 93%, 92%, 92%, 83%,
78%, 77%, 77% amino acid identity with those of Mesem-
bryanthemum crystallinum, Arabidopsis thaliana, Oryza
sativa, Acetabularia acetabulum, Cyanidium caldarium,
Drosophila melanogaster, Aedes aegypti, respectively and
there was a conservative ATP binding site of “324-SGSIT-
328”.
The cluster results in Fig.1 suggested that the V-H+-
ATPase subunit B coming from higher plants was clus-
tered into a group; the V-H+-ATPase subunit B coming from
insects D. melanogaster and A. aegypti was clusted into a
group; A. acetabulum and C. caldarium were between the
higher plants and insects as lower plants. The data indicted
that the evolution of V-H+-ATPase subunit B was accom-
panied with the biological evolution. In higher plants, the
V-H+-ATPase subunit B from salt-tolerant plants S. salsa
and M. crystallinum was in one branch and V-H+-ATPase
subunit B from glycophytes A. thaliana and O. sativa was
in another branch, which indicated an adaptation of V-AT-
Pase B subunit to environment stress during evolution.
Fig.1. Amino acid sequence cluster analysis of subunit B of V-
H+-ATPase in Suaeda salsa with related sequences from Mesem-
bryanthemum crystallinum, Arabidopsis thaliana, Oryza sativa,
Acetabularia acetabulum, Cyanidium caldarium, Drosophila
melanogaster and Aedes aegypti. Sequence identification num-
bers from the National Center for Biotechnology Information are
gi: 26986106， 17065080, 14626084, 1303677, 7436110, 8810
and 4680480, respectively.
2.3 Effect of NaCl on the activity of tonoplast V-H+-AT-
Pase in the leaves of S. salsa seedlings
The activity of the leaf V-H+-ATPase was significantly
induced by NaCl stress (Fig.2). The activities were 2.16-
and 2.58-fold higher than that of the control under 100 and
400 mmol/L NaCl treatment for 2 d, respectively.
Acta Botanica Sinica植物学报 Vol.46 No.1 200496
2.4 Changes in mRNA levels for the subunit B from
leaves of S. salsa in response to salt stress
In order to investigate transcript inducibility of V-H+-
ATPase B subunit in leaves of S. salsa during NaCl stress,
Northern blot hybridization was carried out with total RNA
isolated from plant leaves stressed by the addition of 400
mmol/L NaCl for 12, 24, 48,72, 96 and 120 h.
Following NaCl addition, amounts of subunit B tran-
scripts in leaves increased for up to 48 h and then decreased
(Fig.3). From 72 to 120 h, the mRNA levels of B subunit in
salt-stressed leaves were still higher than those of controls.
Fig.2. Effect of NaCl on the activities of leaf tonoplast V-H+-
ATPase of Suaeda salsa under NaCl treatment. Data are means
(n=15) of three independent experiments ± SE.
Fig.3. Northern blotting analysis of transcript levels for V-H+-
ATPase B subunit under 400 mmol/L NaCl stress from 0 to 120
h. Thirty mg of total RNA per lane was loaded on a 1.2% (W/V)
agarose denaturing formaldenhyde gel. In a lower panel, equal
loading of RNA was verified on the gel by ethidium bromide
staining of the agarose gel.
Fig.5. Western blotting analysis of tonoplast vesicle prepara-
tions isolated from Suaeda salsa leaves exposed to 0, 100 and 400
mmol/L NaCl treatment for 2 d. Immunstaining was performed
using antisreum ATP95. Letters on the left margin indicated the
position of subunits B and c, and letters on the right margin
showed the molecular weight. Lane 1, control; lane 2, 100
mmol/L NaCl treatment; lane 3, 400 mmol/L NaCl treatment.
Fifteen mg protein was added per pocket.
Fig.4. Northern blotting analysis of transcript levels for sub-
units B and c of V-H+-ATPase from Suaeda salsa under 100 and
400 mmol/L NaCl stress for 2 d. Twenty mg of total RNA per lane
were loaded on a 1.2% (W/V) agarose denaturing formaldenhyde
gel. In a lower panel, equal loading of RNA was verified on the gel
by ethidium bromide staining of the agarose gel.
2.6 Effect of NaCl treatment on the subunit amounts of V-
H+-ATPase in leaves of S. salsa
Western blotting analysis with the antiserum ATP95 was
directed against the V-H+-ATPase holoenzyme of K.
daigremintiana cross-reacted with four polypeptides
(Fig.5). It revealed a significant increase of protein of 54 kD
subunit B and 16 kD subunit c under NaCl stress compared
to that of the control and the protein was increased with
the increase of NaCl concentration.
2.5 Coordinate of subunits B and c of V-H+-ATPase from
S. salsa
cDNA encoding c subunit (BE240898) of V-H+-ATPase
was also isolated from cDNA library of S. salsa. In order to
investigate the possible relationship of subunits of V-H+-
ATPase, Northern blotting analysis was performed using
subunit c cDNA as probe. The markedly induced mRNA
increases of both subunit B and c were detected after addi-
tion of 100 and 400 mmol/L NaCl (Fig. 4).
97 LI Ping-Hua et al.: Analysis of V-H+-ATPase B Subunit
3 Discussion
V-H+-ATPase B subunit belongs to a conserved AT-
Pase family and Gogarten et al. (1989) concluded that V-H+-
ATPase subunits could be useful molecular markers for
ear ly evolut ion. Our results a lso indicated that
V-H+-ATPase B subunit is a conservative subunit and its
evolution was accompanied with the biological evolution.
For plants, the great divergence in amino acid sequences
of V-H+-ATPase subunit B apparents in the amino and car-
boxyl termini indicates that these regions are more subject
to evolutionary changes and methods such as point muta-
tion should be used to discern the importance of these
amino acids divergence. While, the amino acid sequence of
V-H+-ATPase subunit B from halophyte S.salsa had the
highest identity with that of M. crystallinum, which is also
a halophilic species as its growth rate is maximal at moder-
ate salt concentrations (Tsiantis et al., 1996), indicating
that V-H+-ATPase subunit B plays an important role in plants
adapted to salinity soil and its evolution is also accompa-
nied by environmental adaptation.
S. salsa can accumulate more than 1 mol/L Na+ in leaves
and the vacuolar compartmentation of Na+ is the main strat-
egy for it to cope with salinity (Wang et al., 2001). Vacuolar
Na+ sequestration is known to depend on the activity
of tonoplast Na+/H+ antiport that is mainly energized
by V-H+-ATPase on the tonoplast. Indeed, the tonoplast
Na+/H+ antiport activity of S. salsa is significantly increased
under NaCl stress (Wang, unpublished data) and at the
same time, the activity of V-H+-ATPase is also significantly
increased (Fig.2).
The activity of V-H+-ATPase is often regulated by the
modulating of enzyme structure or the changing in expres-
sion of subunits (Ratajczak, 2000). In order to discern the
possible modulation mechanism of V-H+-ATPase activity
of S. salsa under NaCl stress, the expression of B subunit
of V-H+-ATPase was analyzed. The results in Fig.3 indi-
cated that the transcript of V-H+-ATPase subunit B was
obviously up-regulated by NaCl stress. Under 400 mmol/L
NaCl stress, the mRNA of B subunit reached the maximal
level during 48 h NaCl treatment and kept higher than that
of the control during the next 2 d, which indicated a dy-
namic change of B subunit under salt stress. The mRNA
level of B subunit was also increased with the increase of
NaCl concentration (Fig.4).
The V-H+-ATPase subunit B is an important non-cata-
lytic ATP binding subunit located in the V1 domain. While,
proton-translocation of V-H+-ATPase is mainly charged by
the V0 domain subunit c. Is the NaCl-induced transcript
activation of subunit B in V1 domain accompanied by the
change of subunit c in V0 domain? The expression of V-H+-
ATPase subunit c of S. salsa was also analyzed to resolve
this question. Figure 4 suggests that the expression of sub-
unit c was also increased with the increase of NaCl
concentration. It also indicated a cooperative relationship
between subunits B and c of V-H+-ATPase from S. salsa.
Western blotting analysis with the antiserum ATP95 re-
vealed a significant increase of protein amounts of the V-
H+-ATPase subunits B and c of S. slasa under 100 and 400
mmol/L NaCl treatment, which gave some other evidence
for a salt-induced coordinate up-regulation of V-H+-ATPase
subunits at translation level. The coordinated salt-induced
increase of subunits B and c of V-H+-ATPase from S. salsa
at transcription and translation levels indicated an increase
of V-H+-ATPase holoenzyme amounts, which maybe the
reason for the increase of V-H+-ATPase activity of S. salsa
under salt stress. The salt-induced increase of V-H+-AT-
Pase activity energies the Na+/H+ antiport that finally caused
the Na+ compartmentation of S. salsa.
The salt-induced expression of V-H+-ATPase subunit B
was reported in halotolerant plants such as common ice
plants (Dietz et al., 2001) and sugar beet (Kirsch et al.,
1996). While for glycophytes, salt-reduced protein amount
of V-H+-ATPase subunit B was reported in wheat (Wang et
al., 2000) and the protein amount of subunit B in pea was
not changed by sodium chloride exposure (Yu et al., 2001).
The former data and our results suggested that salt stress
affected V-H+-ATPase subunit B expression differently in
glycophytes and halophytes.
Our results showed that the expression of V-ATPase B
subunit from leaves of S. salsa was significantly up-regu-
lated and was coordinated with subunit c at transcription
and translation level under NaCl stress. The increased V-
H+-ATPase amounts had a close relationship with the ac-
tivity of V-H+-ATPase, which provided the energy for the
compartmentalization of sodium in response to salinity.
More information will be needed to study the structure,
function and regulation of V-H+-ATPase B subunit, which
could be important to discern the real role of subunit B
contributed to V-H+-ATPase and the composition and re-
action mode of V-H+-ATPase in response to environmental
stimuli, and further display the salt-tolerant mechanism of
halophytes such as S. salsa.
Acknowledgements: The authors thank Professor Dr.
Ulrich LÜTTGE (Darmstadt University, Germany) for pro-
viding an antiserum ATP95 directed against the V-H+-AT-
Pase holoenzyme of Kalanchoe daigremontiana.
Acta Botanica Sinica植物学报 Vol.46 No.1 200498
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(Managing editor: ZHAO Li-Hui)