Enhanced Expression of Vacuolar H+-ATPase Subunit E in
the Roots Is Associated with the Adaptation of
Broussonetia papyrifera to Salt Stress
Min Zhang1, Yanming Fang2*, Zhenhai Liang1, Libin Huang1
1Jiangsu Academy of Forestry, Dongshanqiao, Jiangning, Nanjing, Jiangsu, China, 2College of Forest Resources and Environment, Nanjing Forestry University, Nanjing,
Vacuolar H+-ATPase (V-H+-ATPase) may play a pivotal role in maintenance of ion homeostasis inside plant cells. In the
present study, the expression of V-H+-ATPase genes was analyzed in the roots and leaves of a woody plant, Broussonetia
papyrifera, which was stressed with 50, 100 and 150 mM NaCl. Moreover, the expression and distribution of the subunit E
protein were investigated by Western blot and immunocytochemistry. These showed that treatment of B. papyrifera with
NaCl distinctly changed the hydrolytic activity of V-H+-ATPase in the roots and leaves. Salinity induced a dramatic increase in
V-H+-ATPase hydrolytic activity in the roots. However, only slight changes in V-H+-ATPase hydrolytic activity were observed
in the leaves. In contrast, increased H+pumping activity of V-H+-ATPase was observed in both the roots and leaves. In
addition, NaCl treatment led to an increase in H+-pyrophosphatase (V-H+-PPase) activity in the roots. Moreover, NaCl
treatment triggered the enhancement of mRNA levels for subunits A, E and c of V-H+-ATPase in the roots, whereas only
subunit c mRNA was observed to increase in the leaves. By Western blot and immunocytological analysis, subunit E was
shown to be augmented in response to salinity stress in the roots. These findings provide evidence that under salt stress,
increased V-H+-ATPase activity in the roots was positively correlated with higher transcript and protein levels of V-H+-ATPase
subunit E. Altogether, our results suggest an essential role for V-H+-ATPase subunit E in the response of plants to salinity
Citation: Zhang M, Fang Y, Liang Z, Huang L (2012) Enhanced Expression of Vacuolar H+-ATPase Subunit E in the Roots Is Associated with the Adaptation of
Broussonetia papyrifera to Salt Stress. PLoS ONE 7(10): e48183. doi:10.1371/journal.pone.0048183
Editor: Carl Ng, University College Dublin, Ireland
Received January 7, 2012; Accepted September 21, 2012; Published October 25, 2012
Copyright: ? 2012 Zhang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by Scientific Research Fund in Jiangsu Province (BK2011872), the Priority Academic Program Development of Jiangsu High
Education Institutions (PAPD), National Science & Technology Program in the Twelfth Five-year Plan (No. 2011BAD38B0605), 2010 Enterprise Doctor Assemblage
Program in Jiangsu Province, and Special Research Program for Public-welfare Forestry (200904001). The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Plant cells are characterized by the presence of a large central
vacuole in most differentiated tissues; the vacuole plays a crucial
role in plants’ tolerance to salinity [1,2]. Two plant proton pumps,
vacuolar H+-ATPase (V-H+-ATPase) and H+-pyrophosphatase (V-
H+-PPase), participate in acidifying compartments of the vacuoles,
which establishes an electrochemical H+-gradient to drive
sequestration of Na+into the vacuole lumen, compartmentalizing
this toxic ion from the cytoplasm and maintaining low cytoplasmic
Na+concentrations [2,3,4]. V-H+-ATPase is an ATP-dependent
proton pump that couples the energy released upon hydrolysis of
ATP to the active transport of protons from the cytoplasm to the
lumen of the intracellular compartment . V-H+-ATPase is a
multi-subunit complex organized into two distinct sectors. The
first is the peripherally associated, hydrophilic V1domain, which is
composed of eight different subunits (A–H) and hydrolyzes ATP,
and the second is the hydrophobic, membrane-anchored V0
domain consisting of six different subunits, which functions to
translocate protons across the membrane [6,7]. V-H+-PPase
coexists with V-H+-ATPase in the vacuolar membrane, and
together they are the major components of the vacuolar
membrane in plant cells . Unlike V-H+-ATPase, V-H+-PPase
consists of only a single polypeptide and exists as a dimer of
identical subunits .
Accumulating evidence has implicated the regulation of V-H+-
ATPase activity by salt both in glycophytes and halophytes [9–11].
It was reported that in cell suspensions of Populus euphratica, V-H+-
ATPase hydrolytic and H+pumping activities were stimulated in
response to salt stress . The strategy of Suaeda salsa to adapt to
high salinity seems to be an up-regulation of V-H+-ATPase activity
. The V-H+-ATPase hydrolytic and proton pump activity in
tonoplast vesicles derived from the salt-treated leaves of S. salsa
were significantly elevated compared to that of control leaves. Up-
regulated activity of V-H+-ATPase has also been observed in
cucumber  and Vigna unguiculata . Regulation of V-H+-
ATPase transport activity has been suggested to operate at the
transcriptional level as well as the protein level under salt stress
[16–18]. In the halotolerant sugar beet, an increase in mRNA was
paralleled by an increase in the amount of V-H+-ATPase protein
. Contradictory reports have also claimed that the salt-
mediated increase in V-H+-ATPase activity is not associated with
an increase in protein expression [20,21]. Despite the volume of
studies on changes in V-H+-ATPase and plant salt tolerance to
PLOS ONE | www.plosone.org1October 2012 | Volume 7 | Issue 10 | e48183
date, little is known about the correlation between activation of
this proton pump and salt tolerance in woody plants.
Broussonetia papyrifera, a tree belonging to the Moraceae family, is
important as a source of fiber for cloth and paper. The tree is a
vigorous pioneer species, which can rapidly colonize forest
clearings and is widely favored because of its fast growth . B.
papyrifera is tolerant to drought and resistant to salt stress, which
makes it an ideal tree species to use for controlling salinity .
In the present study, we exploited RT-PCR and Western blot
analysis as well as immunocytochemistry to investigate tissue-
specific expression of V-H+-ATPase in the leaves and roots of the
woody plant B. papyrifera in response to NaCl stress. In addition,
the hydrolytic activities of V-H+-ATPase and V-H+-PPase were
determined by spectrophotometric analysis, and proton pumping
activity of V-H+-ATPase was assayed by monitoring the quench-
ing of ACMA fluorescence. Moreover, vacuolar pH was examined
using the fluorescent pH probe BCECF AM by laser scanning
Materials and Methods
Plant material and growth conditions
In vitro regenerated B. papyrifera rooting plantlets of uniform size
were grown in plastic pots filled with 500 ml of 1/2MS solutions.
All experiments were conducted under controlled conditions
(light/dark cycle of 16/8 h at 2562uC, illumination of 2000 Lx).
Salinity treatments were initiated by adding NaCl to 1/2MS
solution to achieve final concentrations of 50 mM, 100 mM or
150 mM. The nutrient solution was changed every other day. The
roots and leaves were harvested five days after NaCl exposure.
Unstressed plants grown in parallel served as the control and were
harvested at the same time.
Preparation of vacuolar membrane vesicles
Tonoplast-enriched vesicles were isolated according to the
method of Giannini and Briskin  with some modifications.
Fresh leaves or roots were homogenized in homogenization buffer
(70 mM Tris/HCl, pH 8.0, 250 mM sucrose, 2 mM EDTA,
2 mM ATP-Na2, 1% BSA, 0.5% PVP-40, 4 mM DTE, 10%
glycerol, 250 mM KCl) containing protease inhibitor cocktail
(Roche, Indianapolis, IN, USA). The homogenate was centrifuged
at 13,000 g at 4uC for 15 min, and the supernatant was then
centrifuged at 80,000 g for 30 min in a Beckman 70Ti rotor. The
membrane pellet was resuspended in 4 ml suspension buffer
(2 mM BTP/Mes, pH 7.0, 250 mM sucrose, 0.2% BSA, 10%
glycerol, 1 mM DTE) and layered over a 25/38% (w/w)
discontinuous sucrose density gradient. After centrifuging at
100,000 g for 2 h in a Beckman Optima L-80XP ultracentrifuge
with an SW 41Ti rotor, the vacuolar membrane vesicles were
removed from the 8/25% interface and stored at 280uC. The
membrane protein concentration was assayed by the method of
Lowry et al , and bovine serum albumin was used as the
Assay of V-H+-ATPase and V-H+-PPase hydrolytic activities
The hydrolytic activities of V-H+-ATPase and V-H+-PPase
were determined by measuring the amount of inorganic phosphate
released . The reaction was initiated by adding 20 mg of
vacuolar membrane protein into the reaction buffer. For V-H+-
ATPase, the reaction medium contained 25 mM Tris-Mes
(pH 7.0), 4 mM MgSO4?7H2O, 50 mM KCl, 1 mM NaN3,
0.1 mM Na2MoO4, 0.1% Brij 35, 500 mM NaVO4, and 2 mM
ATP-Na2, whereas the reaction medium for V-H+-PPase consisted
of 25 mM Tris-Mes (pH 7.5), 2 mM MgSO4 H2O, 0.1 mM
Na2MoO4, 0.1% Brij 58, and 0.2 mM K4P2O7. The reaction
mixture was incubated at 28uC for 40 min, and then terminated
by the addition of 3% TCA. Inorganic phosphate was assayed
according to Ames . For the determination of V-H+-ATPase
activity, Pi release was measured in the presence and absence of
100 nM concanamycin A (specific inhibitor of V-H+-ATPase) and
the difference between these two activities was attributed to V-H+-
ATPase activity. And K+-dependent H+-PPase activity was
calculated as the difference in activity in the presence and absence
of 50 mM KCl. The enzymatic activities are presented in mmol
Proton pumping assay
The proton pumping activity of the isolated tonoplast vesicles
was measured spectrophotometrically by monitoring the quench-
ing of ACMA (9-amino-6-chloro-2-methoxyacridine) fluorescence
as described previously with minor modification . The assay
buffer contained 10 mM Mes-Tris (pH 7.5), 250 mM sorbitol,
50 mM KCl, 3 mM ATP, 50 mM NaVO4, 1 mM NaN3 and
2 mM ACMA. The reaction was initiated by adding 3.5 mM
MgSO4, and fluorescence quenching (415 nm excitation and
485 nm emission) was measured in a Hitachi 850 fluorescence
spectrometer at 22 uC. Proton pumping activity was expressed as
% quench mg21protein min21.
Total RNA was extracted from fresh roots and leaves using
TRIzol reagent (Invitrogen, Carlsbad, California, USA) according
to the manufacturer’s instructions. cDNA was synthesized with the
GoScriptTMReverse Transcription System (Promega, Madison,
Wisconsin) following the manufacturer’s protocol. The resulting
cDNA was then used as template for the PCR reaction. PCR
cycling was performed with an ABI 2720 thermocycler (Applied
Biosystems). The PCR program was as follows : predenatura-
tion at 94uC for 1.5 min and then a total of 25 cycles of
denaturation at 94uC for 1 min, annealing at 55uC for 1 min and
extension at 72uC for 2 min, followed by one cycle of final
extension at 72uC for 10 min. PCR products were run on 1%
agarose gels and stained with ethidium bromide. Primers used for
the PCR reactions are shown in Table 1.
Western blot analysis
Membrane proteins were separated by sodium dodecylsulfate
polyacrylamide gel electrophoresis (SDS-PAGE) according to
previously described procedures  and the separated proteins
Table 1. Nucleotide sequence of the primers used in this
V-H+-ATPase in B. papyrifera under Salt Stress
PLOS ONE | www.plosone.org2 October 2012 | Volume 7 | Issue 10 | e48183
were electrophoretically transferred onto polyvinylidene difluoride
(PVDF) membranes (Bio-Rad, Hercules, CA, USA). Subsequently,
the membranes were blocked with 1% bovine serum (BSA) in Tris-
buffered saline (TBS) for 20 min. The membranes were then
incubated with a rabbit anti-VHA-E antibody from Arabidopsis
thaliana (a kind gift from Prof. Karl-Josef Dietz, Lehrstuhl fu ¨r
Biochemie und Physiologie der Pflanzen, Universita ¨t Bielefeld,
Bielefeld, Germany) overnight at 4uC. The antibody was used at a
1:1000 dilution. After washing with TBS (containing 0.1% Tween-
20), membranes were incubated with horseradish peroxidase-
conjugated goat anti-rabbit secondary antibody (Jackson Immu-
noResearch Laboratories, Inc., West Grove, PA, USA) for 1 h at
room temperature. Membrane-bound V-H+-ATPase was detected
with SuperSignal West Pico Chemiluminescent Substrate (Thermo
Fisher Scientific Inc., Rockford, IL, USA). The images were
acquired with a ChemiDOC XRS instrument (Bio-Rad), and
band intensity was analyzed with Quantity One-1D analysis
Immunolocalization of subunit E of V-H+-ATPase (VHA-E)
was performed according to Golldack and Dietz with minor
modifications . In brief, leaf and root (within the root hair
zone) tissues were fixed in 4% paraformaldehyde and embedded in
OCT compound (Sakura Finetek, CA, USA). Then, 7 mm sections
were cut using a Leica CM1950 cryostat (Leica Biosystems
Nussloch GmbH, Heidelberger, Germany) and mounted on poly-
L-Lys-coated microscopic slides. The slides were blocked with 1%
BSA in phosphate-buffered saline (PBS; 150 mM NaCl, 5 mM
KCl, 0.8 mM KH2PO4, 3.2 mM Na2HPO4, pH 7.2) for 15 min.
The sections were incubated with rabbit anti-VHA-E antibody
(1:500 dilution) overnight at 4uC. After being washed twice with
PBS, the sections were incubated with Alexa fluo-635 conjugated
anti-rabbit secondary antibody (Molecular Probes, Eugene, OR) at
1:500 dilution for 30 min. Parallel sets processed without primary
antibody were used as the negative controls. Nuclei were stained
with 49,69-diamidino-2-phenylindole (DAPI) (Molecular Probes,
Eugene, OR). Microscopic images were obtained with a Leica
TCS SP5 confocal scanning microscope (Leica Microsystems,
Heidelberg GmbH, Mannheim, Germany).
Vacuolar pH was determined using the pH-sensitive fluorescent
dye BCECF AM (Molecular Probes, Eugene, OR) in accordance
with Krebs et al . Loading of the dye was performed by
incubating the plantlets in 1/10 MS medium containing 0.5%
sucrose, 10 mM Mes-KOH (pH 5.8) and 0.02% Pluronic F-127
(Molecular Probes, Eugene, OR), for 1 h at 22uC in the dark. The
final concentration of BCECF AM was 10 mM. Dye loading was
terminated by washing the plant material in 1/10 MS medium.
Subsequently, root segments (within the root hair zone) were
examined longitudinally with a Leica TCS SP5 confocal scanning
microscope. The specimens were excited at 488 and 458 nm, and
the emission was detected between 530 and 550 nm. Ratio images
(488/458 nm) were generated using the Leica LAS AF Lite
software. An average ratio was calculated and used to determine
the pH value using a pH-ratio calibration.
For the pH-ratio calibration in situ, the plantlets were incubated
in pH equilibration buffers to equilibrate the vacuolar pH to that
of the externa1 solution . The equilibration solution contained
50 mM Mes-BTP (pH 5.2–6.4) or 50 mM Hepes-BTP (pH 6.8–
7.6), 50 mM ammonium acetate and 450 mM sorbitol. The mean
ratio values were obtained by measuring three plantlets at each pH
and were used to generate the calibration curve.
Data are presented as the means 6 SEM of three replicates. All
data were subjected to one-way ANOVA analysis using Sigma-
Stat3.5 software, and significant differences among treatments
were calculated with Duncan’s multiple range tests (P=0.05).
Phenotypes of salt tolerance
After 5 days of exposure to NaCl, the phenotypes of B. papyrifera
plantlets were recorded (Fig. S1). Plants treated with 50 mM and
100 mM NaCl grew well without obvious symptoms of salt injury.
However, plants treated with 150 mM NaCl exhibited symptoms
of salt injury, including chlorosis of leaves and leaf tip necrosis.
These findings indicated that B. papyrifera could tolerate up to
100 mM NaCl.
Effects of NaCl stress on V-H+-ATPase and V-H+-PPase
hydrolytic activity in the leaves and roots of B. papyrifera
Because previous studies have shown that salt stress induced
enhancement of V-H+-ATPase and H+-PPase activities in some
plant species [4,11,12,14], we first determined changes in
tonoplast H+-ATPase and H+-PPase activity in the woody plant
B. papyrifera under NaCl stress. Our preliminary experiment
showed that 100 nM concanamycin A resulted in 83% inhibition
of V-H+-ATPase hydrolytic activity, indicating that the isolated
membrane vesicles were enriched in tonoplasts without significant
contamination by other cellular membranes. A distinct activity
profile for V-H+-ATPase was observed in the leaves and roots in
response to NaCl. In the leaves, NaCl only induced a slight
increase in V-H+-ATPase activity, whereas it markedly stimulated
V-H+-ATPase activity in the roots. ATP hydrolysis activity was
increased by 6.8%, 8.2% and 4.5% at the 50 mM, 100 mM and
150 mM NaCl treatments, respectively, in the leaves, relative to
those of untreated plants (Fig. 1A). In contrast, in the roots, salt-
induced stimulation of hydrolytic activity reached 19.1% and
26.1% at 50 mM and 100 mM NaCl, respectively, while 150 mM
NaCl treatment did not induce a significant increase in V-H+-
ATPase activity (5.8%) (Fig. 1A).
The alterations in H+-PPase activity induced by salt stress were
very similar to that of H+-ATPase. Tonoplast H+-PPase activity
remained relatively constant under different concentrations of
NaCl stress in the leaves (Fig. 1B). In comparison to the leaves, salt
stress led to a sharp increase in H+-PPase activity in the roots. The
increase in H+-PPase activity was more evident at 100 and
150 mM NaCl (21.6% and 22.1%, respectively) (Fig. 1B).
NaCl treatment affected proton transport activity of
tonoplast H+-ATPase and vacuolar pH
The proton transport activity of V-H+-ATPase was measured as
the fluorescence quenching of ACMA. As shown in Fig. 2A and C,
fluorescence quenching in the roots of control plantlets was 11.2%,
while it reached 23.4% at 50 mM NaCl, an increase of 108.9%.
Proton transport activity exhibited 122.3% stimulation when the
NaCl concentration rose to 100 mM. However, proton transport
activity showed only a slight change in comparison to the control
when NaCl reached 150 mM. The results also showed that proton
transport activity in the leaves was activated by NaCl treatment in
a similar manner to that in the roots (Fig. 2 B and C). Proton
transport activity was increased by 76.2% and 80.4% at 50 mM
and 100 mM NaCl, respectively. In contrast, 150 mM NaCl only
induced a minimal change in proton transport activity.
V-H+-ATPase in B. papyrifera under Salt Stress
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To determine the influence of NaCl on vacuolar pH, the pH-
sensitive fluorescent dye BCECF was loaded in the plant roots for
in situ pH measurements. As indicated in Fig. 2D–F, significant
pH changes were observed after exposure to NaCl. In control
roots the vacuolar pH was 5.9; in contrast, 50 mM and 100 mM
NaCl treatment resulted in vacuolar acidification by 0.4 and
0.5 pH units, respectively. In addition, 150 mM NaCl caused only
a slight decrease in vacuolar pH, from 5.9 to 5.8, compared with
Expression of V-H+-ATPase subunit genes in the leaves
and roots is differentially regulated under NaCl stress
To examine whether the expression of the genes encoding V-
H+-ATPase could be modulated by salinity, the transcript levels of
subunits A, B, E and c were determined using RT-PCR. Our
results indicated that the relative mRNA levels of subunits A, B, E
and c were differentially regulated by salinity in the leaves and
roots (Fig. 3). As shown in Fig. 3A and C, transcript levels of
subunits A and E in the roots were significantly enhanced by salt
stress. There was 1.4-, 2.1- and 1.9-fold more mRNA for subunit
A under the conditions of 50, 100 and 150 mM NaCl,
respectively, in comparison to control roots. Transcript levels of
subunit E were increased by 1.5-, 1.6- and 1.4-fold, respectively,
when exposed to the same concentrations of NaCl. However, the
transcript levels of subunits A and E did not change significantly in
salt-stressed leaves (Fig. 3B and D). Moreover, the mRNA levels
for subunit B showed only slight changes in both leaves and roots
during salt treatment. Consistent with previous studies [31,32], we
observed an enhancement in transcript levels of subunit c with
NaCl treatment, especially at 100 and 150 mM NaCl, which
induced 5.6- and 4.9-fold increases in the mRNA level,
respectively, in the roots, while mRNA expression was elevated
by 1.4- and 1.6-fold, respectively, in the leaves.
Expression of V-H+-ATPase subunit E protein in the roots
is enhanced by NaCl stress
We next performed immunodetection of V-H+-ATPase subunit
E by Western blot analysis. As shown in Fig. 4A, in parallel with
the increase in the level of subunit E transcripts, a dramatic
elevation in the protein level of subunit E in the roots was observed
following NaCl treatments. Densitometric analysis revealed
95.9%, 166.8% and 89.9% induction in the protein level with
50, 100 and 150 mM NaCl, respectively, in the roots (Fig. 4C). In
contrast, NaCl treatments failed to stimulate the expression of V-
H+-ATPase protein in the leaves, consistent with the RT-PCR
findings (Fig. 4B, D).
Immunolocalization of V-H+-ATPase subunit E in leaves
Cytolocalization of subunit E in leaf and root cross-sections was
conducted using immunofluorescency. In control and NaCl-
stressed leaves, subunit E was found in leaf mesophyll cells of
both palisade tissue and spongy parenchyma (Fig. 5). The signal
intensity of subunit E after each treatment was similar, indicating
that NaCl had little effect on the distribution of subunit E in leaf
tissues. Signals for subunit E were detected in all cell types of the
epidermis, cortex and vascular cylinder in control roots, with the
strongest expression present in the vascular cylinder (Fig. 6A).
When exposed to 50 mM NaCl, the signal intensity of subunit E
was clearly enhanced in all tissues (Fig. 6B). The expression of
subunit E was further enhanced with elevated NaCl concentration,
especially in the vascular cylinder, where a large amount of
subunit E protein accumulated (Fig. 6C). Moreover, at 150 mM
NaCl the signal intensity of subunit E was lower in the epidermis
and cortex compared with the 100 mM NaCl treatment; however,
it was still stronger than that in the control roots (Fig. 6D), and the
accumulation of subunit E protein in the vascular cylinder was not
Sequestration of Na+into the vacuole has been considered one
of the most effective ways to maintain intracellular ion homeostasis
. The exclusion of Na+from the cytosol by the vacuole is driven
by an electrochemical gradient in the membranes generated by V-
H+-ATPase and V-H+-PPase. Thus, regulation of V-H+-ATPase
might play an essential role in plant salt tolerance. In the present
study, we observed enhancements in V-H+-ATPase hydrolytic and
H+pumping activities in the roots of B. papyrifera in response to
NaCl stress. Moreover, transcript analysis of subunits A, B, E and
c of V-H+-ATPase showed an increase in the expression of
subunits A, E and c gene. And Western blot analysis using the
antibody to V-H+-ATPase subunit E revealed an elevation in the
protein level of subunit E. This NaCl-induced stimulation of V-
Figure 1. Effect of NaCl stress on the hydrolytic activity of V-H+-
ATPase (A) and V-H+-PPase (B). Tonoplasts isolated from the leaves
and roots of control and salt stressed plants were used. V-H+-ATPase
activity was determined by measuring the amount of inorganic
phosphate released in the presence and absence of concanamycin A,
and V-H+-PPase activity was measured in the presence and absence of
KCl. The results are presented as the means 6 SEM of three replicates,
and different letters indicate significant differences among treatments
V-H+-ATPase in B. papyrifera under Salt Stress
PLOS ONE | www.plosone.org4October 2012 | Volume 7 | Issue 10 | e48183
H+-ATPase may be associated with transcriptional activation of
subunits A, E and c and up-regulation of the protein level of
An array of evidence has demonstrated that the activity of V-
H+-ATPase is increased in most plants in response to salt stress
[12,13,15]. However, contrary findings have also been document-
Figure 2. NaCl stress-induced changes in H+-Pump activity of V-H+-ATPase (A–C) and vacuolar pH (D–F). Fluorescence quenching of
ACMA in the root (A) and leaf (B) and changes in H+-Pump activity of V-H+-ATPase (C) were presented. (D) Confocal images of root cell vacuoles
loaded with BCECF AM. Pseudocolor in the ratio image enhances visualization of dye distribution and fluorescence intensity of the dye. (Scale bar
=100 mm) (E) In situ pH-ratio calibration curve. The calibration curve was obtained by plotting the fluorescence ratios (488/458 nm) against the pH of
the equilibration buffers. (F) Changes in vacuolar pH with different concentrations of NaCl treatment.
V-H+-ATPase in B. papyrifera under Salt Stress
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ed, such as data from wheat roots under severe NaCl stress, where
V-H+-ATPase activity was significantly depressed . Moreover,
a recent study showed that V-H+-ATPase activity increased a short
time (24 h) after treatment with NaCl, whereas the activity
decreased with prolonged exposure time (4 d and 8 d) . In our
study, V-H+-ATPase activity was stimulated in the roots, but only
slight changes were observed in the leaves of B. papyrifera. Together
this suggests that the activity of V-H+-ATPase responds differently
to salinity in different plant species, and changes in V-H+-ATPase
activity are concentration and time dependent.
B. papyrifera grew well in 50 mM and 100 mM NaCl, whereas
when NaCl reached 150 mM, chlorosis of the leaves and leaf tip
necrosis were observed. Consistent with these phenotypes,
increases in the H+pumping activity of V-H+-ATPase in leaves
Figure 3. RT-PCR analysis of the expression of genes for V-H+-ATPase subunits A, B, E and c. RNA was isolated from roots (A) and leaves
(B), and the transcript levels of V-H+-ATPase subunits were analyzed by RT-PCR. PCR products of the roots (C) and leaves (D) were quantified using
Quantity One-1D software. RT-PCR results were normalized to their respective actin bands and expressed as fold changes.
V-H+-ATPase in B. papyrifera under Salt Stress
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Figure 4. Western blot analysis of V-H+-ATPase subunit E protein expression in response to NaCl stress. Immunostaining was
performed using rabbit anti-VHA-E antibody. Western blots of tonoplast vesicles from roots (A) and leaves (B) are presented. Subunit E protein levels
in the roots (C) and leaves (D) were quantified. The protein level of subunit E in the control was set to 100, and the protein levels of NaCl stressed
plants were compared with control.
Figure 5. Immunolocalization of V-H+-ATPase subunit E in the leaves of B. papyrifera. V-H+-ATPase subunit E was stained red with rabbit
anti-VHA-E antibody and the nuclei were stained blue with DAPI. The merged images of VHA-E, nuclei and the DIC image are also presented. DIC,
differential interference contrast; ad, adaxial epidermis; ab, abaxial epidermis; p, palisade tissue; s, spongy parenchyma. Scale bar =50 mm.
V-H+-ATPase in B. papyrifera under Salt Stress
PLOS ONE | www.plosone.org7 October 2012 | Volume 7 | Issue 10 | e48183
and roots from 50 mM and 100 mM NaCl treated plants were
observed. In contrast, no obvious changes in the H+pumping
activity of V-H+-ATPase were detected at 150 mM NaCl.
Meanwhile, acidification of vacuoles occurred, paralleling the
increase in H+pumping activity. Vacuolar pH was decreased by
0.4–0.5 pH units compared to control plants.
It has been suggested that changes in plant V-H+-ATPase
activity occur in parallel to alterations in transcript levels and/or
the amounts of different protein subunits of V-H+-ATPase after
exposure to salinity stress. In this report, we analyzed the effects of
NaCl exposure on the gene expression of subunits A, B, E and c
and the protein levels of subunit E by RT-PCR and Western blot
analysis. These revealed that salinity triggered a tissue-specific
expressional response in B. papyrifera plantlets. A coordinated up-
regulation of the mRNA levels for subunits A, E and c was noticed
in the roots but not in the leaves of plants exposed to NaCl stress.
This increase in mRNA levels was in parallel with the augmented
V-H+-ATPase activity, suggesting the increased transcript levels
may be partially responsible for the stimulation of V-H+-ATPase
activity. Coordinated up-regulation of V-H+-ATPase subunits has
also been shown in other plant species, including halotolerant
sugar beet  and the common ice plant [34,35]. Consistent with
the enhancement of subunit E mRNA expression, an increase in
its protein level occurred in the roots of salt-exposed B. papyrifera,
indicating that the increase in protein expression may also be
involved in the regulation of V-H+-ATPase activity.
In addition to translational regulation of V-H+-ATPase activity,
some other mechanisms by which V-H+-ATPase activity may be
regulated have been proposed. A recent study provided evidence
that a WNK kinase, AtWNK8, could phosphorylate subunit C of
V-H+-ATPase, indicating post-translational modifications were
also involved in the regulation of V-H+-ATPase activity .
Moreover, the Ser/Thr kinase SOS2 was reported to promote salt
tolerance by interacting with V-H+-ATPase and up-regulating its
transport activity . More recent research has found that the
Cdc42 effector Ste20 stimulates V-H+-ATPase activity by forming
a complex with Vma13, a regulatory subunit of V-H+-ATPase
. Several reports have suggested that V-H+-ATPase activity
may also be modulated by assembly-disassembly of the V1and V0
sectors [38,39]. Additionally, changes in the lipid microenviron-
ment of the vacuolar membrane may account for the regulation of
V-H+-ATPase activity because it was reported that alterations in
the membrane lipid composition and structure were associated
with modulation of tonoplast transport proteins [40,41]. Whether
these mechanisms are involved in the regulation of V-H+-ATPase
activity in B. papyrifera needs further investigation.
Altogether, we have shown the differential and tissue-specific
expression of V-H+-ATPase subunits in response to salt stress. This
indicates that the enhanced expression of V-H+-ATPase subunit E
in the roots may confer salt tolerance to the woody plant B.
papyrifera. These findings may provide insights into understanding
the salt resistance of plants.
Figure 6. Distribution of V-H+-ATPase subunit E protein in root tissues of B. papyrifera grown under NaCl stress. (A) Control, (B) 50 mM
NaCl treated plants, (C) 100 mM NaCl treated plants and (D) 150 mM NaCl treated plants. Localization of VHA-E was examined by
immunofluorescency using rabbit anti-VHA-E antibody. ep, epidermis; ct, cortex; vc, vascular cylinder. Scale bar =50 mm.
V-H+-ATPase in B. papyrifera under Salt Stress
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under different concentrations of NaCl. (A) Whole plants of
control and NaCl treated B. papyrifera. (B) Leaves from the
corresponding plants. Note the leaf tip chlorosis and necrosis in
150 mM NaCl treated plants.
Phenotype of Broussonetia papyrifera grown
The authors would like to thank Dr. Yin Ding (State Key Laboratory of
Analytical Chemistry for Life Science, Department of Chemistry, Nanjing
University, P.R.China) for her assistance in the confocal microscopic
Conceived and designed the experiments: YMF. Performed the experi-
ments: MZ ZHL. Analyzed the data: LBH. Wrote the paper: YMF MZ.
1. Kluge C, Lahr J, Hanitzsch M, Bolte S, Golldack D, et al (2003) New insight into
the structure and regulation of the plant vacuolar H+-ATPase. J Bioenerg
Biomembr 35: 377–388.
2. Barkla BJ, Vera-Estrella R, Herna ´ndez-Coronado M, Pantoja O (2009)
Quantitative proteomics of the tonoplast reveals a role for glycolytic enzymes
in salt tolerance. Plant Cell 21: 4044–4058.
3. Schnitzer D, Seidel T, Sander T, Golldack D, Dietz KJ (2011) The cellular
energization state affects peripheral stalk stability of plant vacuolar H+-ATPase
and impairs vacuolar acidification. Plant Cell Physiol 52: 946–956.
4. Silva P, Gero ´s H (2009) Regulation by salt of vacuolar H+-ATPase and H+-
pyrophosphatase activities and Na+/H+exchange. Plant Signal Behav 4: 718–
5. Jefferies KC, Cipriano DJ, Forgac M (2008) Function, structure and regulation
of the vacuolar (H+)-ATPases. Arch Biochem Biophys 476: 33–42.
6. Cipriano DJ, Wang YR, Bond S, Hinton A, Jefferies KC (2008) Structure and
regulation of the vacuolar ATPases. Biochim Biophys Acta 1777: 599–604.
7. Toei M, Saum R, Forgac M (2010) Regulation and isoform function of the V-
ATPases. Biochemistry 49: 4715–4723.
8. Maeshima M (2000) Vacuolar H+-pyrophosphatase. Biochimica et Biophysica
Acta 1465: 37–51.
9. Batelli G, Verslues PE, Agius F, Qiu Q, Fujii H (2007) SOS2 promotes salt
tolerance in part by interacting with the vacuolar H+-ATPase and upregulating
its transport activity. Mol Cell Biol 27: 7781–7790.
10. Popova OV, Golldack D (2007) In the halotolerant Lobularia maritime
(Brassicaceae) salt adaptation correlates with activation of the vacuolar H(+)-
ATPase and the vacuolar Na+/H+antiporter. J Plant Physiol 164: 1278–1288.
11. Queiro ´s F, Fontes N, Silva P, Almeida D, Maeshima M (2009) Activity of
tonoplast proton pumps and Na+/H+exchange in potato cell cultures is
modulated by salt. J Exp Bot 60: 1363–1374.
12. Silva P, Fac ¸anha AR, Tavares RM, Gero ´s H (2010) Role of tonoplast proton
pumps and Na+/H+antiport system in salt tolerance of Populus euphratica Oliv.
J Plant Growth Regul 29: 23–34.
13. Qiu N, Chen M, Guo J, Bao H, Ma X (2007) Coordinate up-regulation of V-
H+-ATPase and vacuolar Na+/H+antiporter as a response to NaCl treatment in
a C3 halophyte Sueda salsa. Plant Sci 172: 1218–1225.
14. Kabala K, Klobus G (2008) Modification of vacuolar proton pumps in
cucumber roots under salt stress. J Plant Physiol 165: 1830–1837.
15. Otoch MLO, Sobreira ACM, Araga ˜o MEF, Orellano EG, Lima MGS (2001)
Salt modulation of vacuolar H+-ATPase and H+-Pyrophosphatase activities in
Vigna unguiculata. J Plant Physiol 158: 545–551.
16. Hanitzsch M, Schnitzer D, Seidel T, Golldack D, Dietz KJ (2007) Transcript
level regulation of the vacuolar H(+)-ATPase subunit isoforms VHA-a, VHA-E
and VHA-G in Arabidopsis thaliana. Mol Membr Biol 24: 507–518.
17. Golldack D, Dietz KJ (2001) Salt-induced expression of the vacuolar H+-ATPase
in the common ice plant is developmentally controlled and tissue specific. Plant
Physiol 125: 1643–1654.
18. Senthilkumar P, Jithesh MN, Parani M, Rajalakshmi S, Praseetha K (2005) Salt
stress effects on the accumulation of vacuolar H+-ATPase subunit c transcripts in
wild rice, Porteresia coarctata (Roxb.) Tateoka. Curr Sci 89: 1386–1394.
19. Kirsch M, An Z, Viereck R, Lo ¨w R, Rausch T (1996) Salt stress induces an
increased expression of V-type H+-ATPase in mature sugar beet leaves. Plant
Mol Biol 32: 543–547.
20. Parks GE, Dietrich MA, Schumaker KS (2002) Increased vacuolar Na+/H+
exchange activity in Salicornia bigelovii Torr. in response to salt. J Exp Bot 53:
21. Vera-Estrella R, Barkla BJ, Bohnert HJ, Pantoja O (1999) Salt stress in
Mesembryanthemum crystallinum L. cell suspensions activates adaptive mechanisms
similar to those observed in the whole plant. Planta 207: 426–435.
22. Li MR, Li Y, Li HQ, Wu GJ (2011) Overexpression of AtNHX5 improves
tolerance to both salt and drought stress in Broussonetia papyrifera (L.) Vent. Tree
Physiol 31: 349–357.
23. Yang F, Ding F, Du TZ (2009) Absorption and allocation characteristics of K+,
Ca2+, Na+and Cl2in different organs of Broussonetia papyrifera seedlings under
NaCl stress. Chin J Appl Ecol 20: 767–772. (In Chinese).
24. Giannini JL, Briskin DP (1987) Proton transport in plasma membrane and
tonoplast vesicles from red beet (Beta vulgaris L.) storage tissue. Plant Physiol 84:
25. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement
with the Folin phenol reagent. J Biol Chem 193: 265–275.
26. Krebs M, Beyhl D, Go ¨rlich E, Al-Rasheid KAS, Marten I, et al. (2010)
Arabidopsis V-ATPase activity at the tonoplast is required for efficient nutrient
storage but not for sodium accumulation. Proc Natl Acad Sci USA 107: 3251–
27. Ames BN (1966) Assay of inorganic phosphate, total phosphate and
phosphatases. Methods Enzymo l8: 115–118.
28. Mu ¨ller ML, Irkens-Kiesecker U, Rubinstein B, Taiz L (1996) On the mechanism
of hyperacidification in lemon. J Biol Chem 271: 1916–1924.
29. Betz M, Dietz KJ (1991) Immunological characterization of two dominant
tonoplast polypeptides. Plant Physiol 97: 1294–1301.
30. Yoshida S (1994) Low temperature-induced cytoplasmic acidosis in cultured
mung bean (Vigna radiata [L.] Wilczek) cells. Plant Physiol 104: 1131–1138.
31. Dietz KJ, Tavakoli N, Kluge C, Mimura T, Sharma SS (2001) Significance of
the V-type ATPase for the adaptation to stressful growth conditions and its
regulation on the molecular and biochemical level. J Exp Bot 52: 1969–1980.
32. Tsiantis MS, Bartholomew DM, Smith JAC (1996) Salt regulation of transcript
levels for the c subunit of a leaf vacuolar H+-ATPase in the halophyte
Mesembryanthemum crystallinum. Plant J 9:729–736.
33. Wang BS, Ratajczak R, Zhang JH (2000) Activity, amount and subunit
composition of vacuolar-type H+-ATPase and H+-PPase in wheat roots under
severe NaCl stress. J Plant Physiol 157:109–116.
34. Dietz KJ, Arbinger B (1996) cDNA sequence and expression of subunit E of the
vacuolar H+-ATPase in the inducible crassulacean acid metabolism plant
Mesembryanthemum crystallinum. Biochim Biophys Acta 1281: 134–138.
35. Lo ¨w R, Rockel B, Kirsch M, Ratajczak R, Hortensteiner S (1996) Early salt
stress effects on the differential expression of vacuolar H+-ATPase genes in roots
and leaves of Mesembryanthemum crystallinum. Plant Physiol 110: 259–265.
36. Hong-Hermesdorf A, Bru ¨x A, Gru ¨ber A, Gru ¨ber G, Schumacher K (2006) A
WNK kinase binds and phosphorylates V-ATPase subunit C. FEBS Lett 580:
37. Lin M, Li SC, Kane PM, Ho ¨fken T (2012) Regulation of vacuolar H+-ATPase
activity by the Cdc42 effector Ste20 in Saccharomyces cerevisiae. Eukaryot Cell
38. Sumner JP, Dow JAT, Earley FGP, Klein U, Jager D, et al. (1995) Regulation of
plasma membrane V-ATPase activity by dissociation of peripheral subunits.
J Biol Chem 270: 5649–5653.
39. Kane PM, Parra KJ (2000) Assembly and regulation of the yeast vacuolar H+-
ATPase. J Exp Biol 203: 81–87.
40. Zhao FG, Qin P (2005) Protective effects of exogenous fatty acids on root
tonoplast function against salt stress in barley seedlings. Environ Exp Bot 53:
41. Liang YC, Zhang WH, Chen Q, Ding RX (2005) Effects of silicon on H+-
ATPase and H+-PPase activity, fatty acid composition and fluidity of tonoplast
vesicles from roots of salt-stressed barley (Hordeum vulgare L.). Environ Exp Bot
V-H+-ATPase in B. papyrifera under Salt Stress
PLOS ONE | www.plosone.org9 October 2012 | Volume 7 | Issue 10 | e48183