全 文 ：Received 25 Dec. 2003 Accepted 19 Mar. 2004
Supported by the State Key Basic Research and Development Plan of China (G1999011705).
* These authors contributed equally to this work.
** Author for correspondence. E-mail: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (9): 1040-1048
Comparison of Plasma Membrane H+-ATPase Activity in Two Ecotypes of
Reed (Phragmites communis) Leaves from Different Habitats
JING Yan*, GONG Hai-Jun*, ZHAO Zhi-Guang, CHEN Guo-Cang, WANG Suo-Min**, ZHANG Cheng-Lie
(Institute of Botany and Plant Physiology, School of Life Sciences, Lanzhou University, Lanzhou 730000, China)
Abstract: Plasma membrane (PM) vesicles of the leaves of two ecotypes of reed (Phragmites communis
Trin.), swamp reed (SR) and heavy salt meadow reed (HSMR) growing in the desert region of Northwest
China, were purified by two-phase partitioning and the properties of their PM H+-ATPases (EC 3.6.1.35)
were compared. The specific activity of this enzyme was greater in HSMR than in SR and the Km lower
(1.27 mmol/L in SR and 0.30 mmol/L in HSMR), and the Vmax of ATP hydrolysis activity showed no
significant difference between the two ecotypes. The PM H+-ATPase was more sensitive to denaturing
temperatures in HSMR than in SR, and the pH profile also showed a slight difference, suggesting that the
catalytic mechanism of this enzyme was different in HSMR compared with that in SR. The p -nitrophenyl
phosphate (PNPP) hydrolysis activity of H+-ATPase was higher in HSMR than in SR at low concentrations
of PNPP, but showed no difference at high PNPP concentration. The Km for PNPP hydrolysis was 3.61
mmol/L and 1.92 mmol/L in SR and HSMR, respectively. And the Vmax of PNPP hydrolysis showed no
significant difference in the two reed ecotypes. An experiment with the inhibitor vanadate showed that the
catalytic mechanisms of the phosphatase domain of the ATPase were different in the two ecotypes. The
data obtained following trypsin treatment showed a difference in the enzyme activity pattern, suggesting
that there existed a possible change in the C-terminus of the ATPase, either in the structure or in the
property or both of them. In addition, compared with SR, the ATP-dependent H+ pumping activity of ATPase
and the coupling between proton transport and ATP hydrolysis in HSMR were increased. These results
indicated that the properties of PM H+-ATPase were changed in HSMR with compared to those in SR,
which might include enzyme modifications and different isoforms expressed. The alterations of the
properties of this enzyme might be an adaptive response to the habitat.
Key words: plasma membrane (PM); H+-ATPase; reed (Phragmites communis) ecotype; saline habitat
The plasma membrane (PM) H+-ATPase (EC 3.6.1.35) is
the “master enzyme” of plant cells and plays a pivotal role
in the physiology and biochemistry of plants. This enzyme
acts as a primary transporter by pumping protons out of
the cell, therefore creating pH and electrical potential dif-
ferences across the plasma membrane. This electrochemi-
cal gradient is subsequently utilized as the driving force for
the secondary transport of ions and nutrients into and out
of cells (Michelet and Boutry, 1995). Therefore, the PM H+-
ATPase plays an important role in the growth and develop-
ment of plants. The PM H+-ATPase belongs to the P-type
ATPase family characterized by a catalytic phosphorylated
intermediate (Serrano, 1989). The enzyme is composed of
one polypeptide with a molecular weight of about 100 kD. It
has 10 transmembrane helixes, and the first six helixes form
the proton channel. The enzyme has six conserved motifs,
which consist of a phosphatase domain, a transduction
domain and a kinase domain (Briskin and Hanson, 1992;
Michelet and Boutry, 1995). The PM H+-ATPase is encoded
by a multigene family, and the gene expression is tissue-
specifically and tightly regulated at the pre-transcriptional,
transcriptional and post-transcriptional levels (Sussman,
1994). The ATPase is also regulated by environmental fac-
tors such as phospholipids, light, toxins and environmen-
tal stresses (Briskin, 1990; Serrano, 1990; Michelet and
Boutry, 1995; Morsomme and Boutry, 2000).
There are some reports of the effects of NaCl on the PM
H+-ATPase, which include an inhibition in the roots of sun-
flower (Ballesteros et al., 1998) and tomato (Gronwalk
et al., 1990), stimulation in mung bean roots (Nakamamura
et al., 1992), but no effect in cotton roots (Hassidim et al.,
1986). At the transcriptional level, PM H+-ATPase mRNA
was increased in tobacco cells (Niu et al., 1993), but no
effect was observed in sunflower roots (Roldán et al., 1991)
subjected to salt. In the roots of tomato, Binzel (1995) ob-
served increase of PM H+-ATPase mRNA content, while
JING Yan et al.: Comparison of Plasma Membrane H+-ATPase Activity in Two Ecotypes of Reed (Phragmites communis)
Leaves from Different Habitats 1041
Wimmers et al. (1992) found no accumulation. However,
relatively little is known about the changes of the PM H+-
ATPase catalytic process under NaCl/saline stress
conditions.
Reed (Phragmites communis) is a hydrophilic plant,
whose typical habitats are fresh and brackish water swamps,
riversides and lakesides. However, it is known to adapt to
adverse terrestrial habitats, and various ecotypes have
evolved (Matoh et al., 1988; Wang et al., 1998; Zheng et
al., 2000). In addition to swamp reed (SR), there are three
other terrestrial reed ecotypes, i.e. heavy salt meadow reed
(HSMR), light salt meadow reed (LSMR), and dune reed
(DR) growing in the desert regions of Northwest China
(Ren and Zhang, 1992; Wang and Zhang, 1993). Long-term
investigations on these reed ecotypes conducted in our
laboratory have shown some stable variations of morpho-
logical and physiological characteristics in response to
drought and salinity (Chen and Zhang, 1991; Wang et al.,
1995; Zhu et al., 2001; Chen et al., 2003). In the present
study, two ecotypes of reed, SR and HSMR, were used to
demonstrate possible changes of the catalytic process of
the PM H+-ATPase.
1 Materials and Methods
1.1 Plants and sampling site
Two ecotypes of reed (Phragmites communis Trin.), re-
ferred to as SR and HSMR (Zheng et al., 2000) growing in
Pingchuan Town, Linze County, Gansu Province, China
were selected for the experiments. This region belongs to
the Cold and Arid Regions Environmental and Engineering
Research Institute of The Chinese Academy of Sciences,
Linze Research Area (39º31-39º58 N, 100º4-100º36 E;
1 300 m above sea level). The mean annual precipitation
is 118 mm, while the annual evaporation is 2 392 mm. Air
temperature is characterized by large daily fluctuation;
the annual average of the daily maximum temperature
being 39 ℃ and minimum -27 ℃. The frost season lasts
197 d. The two ecotypes of reed were sampled from dif-
ferent habitats in the same region. The SR grew in pools
with over 2-m-depth water and the salt content in the
root zone was 0.14% (W/W). The HSMR grew in low-
lying salt flats, and the water and salt contents in the
root zone was 48.5% (W/W) and 0.79% (W/W),
respectively. Since all sampling sites are located within
a narrow area (6.5 km2), the examined reed ecotypes share
similar meteorological conditions. On July 1st, 2002, the
second leaves from the top of the two reed ecotypes
were simultaneously collected at noon and immediately
frozen in liquid N2 until analysis.
1.2 PM purification
PM was prepared by two-phase partitioning. Leaves
were ground into power with liquid N2 and immediately
homogenized in an isolation medium (1:3, W/V) containing
0.25 mol/L sucrose, 3 mmol/L EDTA, 0.6% (W/V) PVP, 0.25
mmol/L PMSF, 15 mmol/L mercaptoethanol and 25 mmol/L
Tris-Mes (pH 7.5). The homogenate was filtered through
four layers of cotton gauze and centrifuged at 13 000g for
15 min. The supernatant was then centrifuged at 80 000g
for 30 min and the microsomal pellet suspended in a buffer
containing 0.25 mol/L sucrose, 1 mmol/L DTT, 5 mmol/L K-
phosphate (pH 7.8). The microsomal fraction was added to
a phase mixture to give a phase system consisting of 6.4%
(W/W) PEG 3350, 6.4% (W/W) dextran T-500, 5 mmol/L K-
phosphate (pH 7.8) and 0.25 mol/L sucrose. Partitioning
was obtained by repeated inversion and the phases sepa-
rated by centrifugation at 2 000g for 5 min. The upper phase
was re-partitioned twice with fresh lower phase. The final
upper phases were collected, diluted ten times with sus-
pension buffer (0.25 mol/L sucrose, 1 mmol/L DTT, 2 mmol/L
Tris-Mes (pH 7.2)) and centrifuged at 100 000g for 30 min,
after which the pellet was collected. All of the above steps
were carried out at 4 ℃.
Membrane protein content was determined by the
Bradford method using BSA as a standard (Bradford, 1976).
The purity of the PM was estimated according to Widell
and Larsson (1990). Vanadate inhibition of the H+-ATPase
activity was 64.0%. The inhibition of this enzyme by nitrate,
azide and molybdate were 5.0%, 9.8% and 6.9%, respectively.
In the following assays of ATPase activity, nitrate, azide
and molybdate were included in the reaction medium.
1.3 ATPase activity assay
The H+-ATPase activity was determined by measuring
the release of Pi from ATP according to the method of Qiu
(1999). The assay medium contained 3 mmol/L ATP, 5 mmol/L
MgSO4, 100 mmol/L KCl, 1 mmol/L NaN3, 50 mmol/L NaNO3,
0.1 mmol/L Na2MoO4, 0.02% (W/V) Triton X-100, 25 mmol/
L Tris-Mes (pH 6.5), and 10 mg PM protein. The reaction
was allowed to continue for 30 min, after which the released
Pi was determined.
1.4 Effect of denaturing temperatures on ATPase activity
Temperature stability of the H+-ATPase was evaluated
according to Sánchez-Nieto et al. (1998). The ATPase as-
say medium without ATP was incubated at 35 ℃, 40 ℃, 45
℃, 50 ℃, 55 ℃ or 60 ℃ in a water bath for 10 min, and then
the tubes were immediately transferred to a water bath at 30
℃ and ATP added to a final concentration of 3 mmol/L. The
reaction proceeded for 60 min, after which the released Pi
was determined.
Acta Botanica Sinica 植物学报 Vol.46 No.9 20041042
1.5 p-nitrophenyl phosphate (PNPP) hydrolysis assay
The H+-ATPase PNPP hydrolysis activity was deter-
mined by measuring the released Pi from PNPP according
to Qiu (1999). The reaction medium contained 20 mmol/L
PNPP, 5 mmol/L MgSO4, 100 mmol/L KCl, 1 mmol/L NaN3,
50 mmol/L NaNO3, 0.1 mmol/L Na2MoO4, 0.02% (W/V) Tri-
ton X-100, 25 mmol/L Tris-Mes (pH 6.5), and 10 mg PM protein.
1.6 Trypsin treatment
Trypsin treatment of PM vesicles was performed as de-
scribed by Qiu and Zhang (2001a). The proteolysis medium
contained 25 mmol/L Tris-Mes, pH 7.5, 2 mmol/L DTT, 5
mmol/L EDTA, 0.25 mol/L sucrose, 0.5 µg/µL PM protein,
2.5 mmol/L ATP and trypsin.
1.7 Proton-pumping activity
The PMs obtained by two-phase partition were frozen
in liquid N2 and thawed at 20 ℃ a total four times to obtain
inside-out membrane vesicles (Palmgren et al., 1990a). Pro-
ton-pumping activity was monitored as the decrease in the
absorbance of acridine orange at 492 nm (Klobus and
Buczek, 1995). The assay medium contained 330 mmol/L
sorbitol, 25 mmol/L BTP-Mes (pH 7.0), 3 mmol/L BTP-ATP
(pH 7.0), 100 mmol/L KCl, 1 mmol/L NaN3, 0.1% (W/V) BSA
(fatty-acid free), 20 µmol/L acridine orange and 50 µg PM
protein in a final volume of 1 mL. After 5 min pre-incubation
at 26℃, the reaction was started by adding MgSO4 to a
final concentration of 5 mmol/L.
The experiments were repeated three times with similar
results.
2 Results
2.1 ATPase activity in two ecotypes of reed leaves
As shown in Fig.1A, the ATP hydrolysis activity of the PM
H+-ATPase in HSMR is higher than that in SR at different ATP
concentrations. Kinetic analysis (Fig.1B) showed that the Km
was 1.27 mmol/L in SR and 0.30 mmol/L in HSMR, which
showed significant difference, while the Vmax of ATP hy-
drolysis showed no difference between the two reed
ecotypes.
2.2 Temperature stability
Enzyme stability at denaturing temperatures is a useful
parameter to indicate differences between protein struc-
tures (Sánchez-Nieto et al., 1998). The activity of PM H+-
ATPase in the two ecotypes of reed was measured as a
function of temperature (Fig.2). The enzyme in SR was
stable and active up to 40 ℃ for 10 min, but its activity
progressively lost as the temperature was raised. In contrast,
the activity of the PM H+-ATPase from HSMR suffered
progressive loss in activity as the temperature was raised
above 35 ℃. At higher temperatures, the activity decreased
more rapidly than that in SR.
2.3 pH profile
The pH profile of the PM H+-ATPase activity in the two
ecotypes of reed is shown in Fig.3. The ATPase showed
maximum activity at pH 6.5 in both reed ecotypes. In SR,
the activity was relatively high in the pH range of 6.0 to 6.5,
but at higher and lower pH values, its activity decreased
greatly compared with that at pH 6.5. In contrast, in HSMR,
although the maximum ATPase activity occurred at pH 6.5,
the decrease in activity at higher and lower values was less
than that seen in SR. That is to say, the PM H+-ATPase in
HSMR could maintain relatively high activities across a
broad pH range.
2.4 PNPP hydrolysis by PM H+-ATPase
PNPP is hydrolysed by phosphatases (Lowry, 1957).
Fig.1. ATPase activity of plasma membrane (PM) from two
ecotypes of reed leaves. The ATPase activity of plasma mem-
brane as a function of increasing ATP concentration (A). The
activities were assayed as described in Materials and Methods.
ATP was added as indicated in the figure. The values are the
means of three experiments. The kinetic analysis of this enzyme
is also presented (B). HSMS, heavy salt meadow reed; SR, swamp
reed.
JING Yan et al.: Comparison of Plasma Membrane H+-ATPase Activity in Two Ecotypes of Reed (Phragmites communis)
Leaves from Different Habitats 1043
Qiu (1999) demonstrated that the PM H+-ATPase had PNPP
hydrolysis activity and confirmed that the catalysis pro-
cess was executed by the phosphatase domain of this
enzyme. In our study, we also observed that the PMs of
reed leaves had PNPP hydrolysis activity (Fig. 4A).
Vanadate is a specific inhibitor of PM H+-ATPase and its
site of action is the phosphatase domain of this enzyme
(Serrano, 1989). Our experiments showed that vanadate
could inhibit the PNPP hydrolysis rate (Fig.5), indicat-
ing that the PNPP was hydrolysed by the phosphatase
domain of the ATPase. The H+-ATPase PNPP hydrolysis
activity was higher in the HSMR than that in the SR at
low concentrations of PNPP, but there was no difference
between them at high PNPP concentrations (Fig.4A). Ki-
netic analysis (Fig.4B) showed that the Km for PNPP hy-
drolysis was 3.61 mmol/L in SR and 1.92 mmol/L in
HSMR, while the Vmax of PNPP hydrolysis showed no
significant difference between the two reed ecotypes.
The difference in Km for PNPP hydrolysis of the PM H+-
ATPases suggested that the catalytic mechanism of the
phosphatase domain of the ATPase was different in the
Fig.2. Comparison of sensitivity of PM H+-ATPase to high
temperature in two ecotypes of reed leaves. The PM vesicles
were preincubated in the assay mixture containing 25 mmol/L
Tris-Mes, pH 6.5, 5 mmol/L MgSO4, 100 mmol/L KCl, 1 mmol/
L NaN3, 50 mmol/L NaNO3, 0.1 mmol/L Na2MoO4, 0.02% (W/
V) Triton X-100 for 10 min at the indicated temperature. Then
the mixture was transferred to an incubation temperature of 30℃
to measure ATPase activity by adding 3 mmol/L ATP-Tris (pH
6.5). The data are the means of three experiments and expressed
as percentage of the maximal activity (100%). Abbreviations are
the same as in Fig.1.
Fig.3. Effect of pH on the ATP hydrolysis activity of PM in
two ecotypes of reed leaves. The ATPase activity was deter-
mined as described in Materials and Methods, except that the
pH was as indicated in the figure. The data are the means of three
experiments and expressed as percent of the activity assayed at
pH 6.5 (100%). Abbreviations are the same as in Fig.1.
Fig.4. P-nitrophenyl phosphate (PNPP) hydrolysis activity of
PM from two ecotypes of reed leaves. The PM PNPP hydroly-
sis activity as a function of increasing PNPP concentration is
shown (A). The activities were assayed as described in Materi-
als and Methods. PNPP was added as indicated in the figure.
The values are the means of three experiments. The kinetic analy-
sis of the PNPP hydrolysis is also presented (B). Abbreviations
are the same as in Fig.1.
Acta Botanica Sinica 植物学报 Vol.46 No.9 20041044
two reed ecotypes.
The inhibitory effect of vanadate on the PNPP hy-
drolysis activity of the PM H+-ATPases in the two
ecotypes of reed was also different. As shown in Fig.5,
the sensitivity of the ATPase to vanadate in HSMR was
higher than that in SR. In HSMR, 0.3 mmol/L vanadate
inhibited about 50% of the PNPP hydrolysis activity,
while in SR an inhibitor concentration of about 1 mmol/L
was required to inhibit 50% of the activity. This result
also suggested that the catalytic mechanism of the
ATPase phosphatase domain was altered in the HSMR
compared with that in SR.
2.5 Effect of trypsin treatment on PM H+-ATPase
There is now convincing evidence that the C-terminus
of PM H+-ATPase acts as an auto-inhibitory domain, which
can be moved by trypsin proteolysis, resulting in increased
activity (Palmgren et al., 1990b). In the present study, we
also observed stimulation following treatment with trypsin
(Fig.6). However, the effect on activity in SR was greater
than that in HSMR. Moreover, the trypsin concentration
for maximum stimulation was also different, being 0.5
mg/mL in SR and 1 mg/mL in HSMR.
2.6 Proton-pumping activity
As shown in Fig.7, the ATP-dependent H+ pumping ac-
tivity of PM in HSMR was higher than that in SR.
Moreover, the coupling between proton transport and ATP
hydrolysis was also different, being more tightly coupled
in HSMR than in SR (Fig.8).
3 Discussion
PM H+-ATPase plays an important role in the growth
and development of plants. Much work has shown that
this enzyme is involved in the response to NaCl/saline stress
(Hassidim et al., 1986; Nakamura et al., 1992; Ballesteros et
al., 1998). It was suggested that PM H+-ATPase may func-
tion simultaneously, both as a detector and as an effector
in response to NaCl (Reinhold et al., 1984). However,
Fig.5. Comparison of sensitivity of P-nitrophenyl phosphate
(PNPP) hydrolysis to vanadate in two ecotypes of reed leaves.
The activities were assayed as described in Materials and
Methods. Vanadate was added as indicated in the figure. The
values are the means of three experiments and expressed as per-
cent of the activity without vanadate (100%). Abbreviations are
the same as in Fig.1.
Fig.6. Effect of trypsin treatment on the PM H+-ATPase in two
ecotypes of reed leaves. Trypsin treatment and the activity assay
are as described in Materials and Methods. Trypsin was added
as indicated in the figure. The values are the means of three experi-
ments and expressed as percent of the activity without trypsin
treatment (100%). Abbreviations are the same as in Fig.1.
Fig.7. ATP-dependent H+ pumping activities of PM in two
ecotypes of reed leaves. The activities were assayed as described
in Materials and Methods. Data are the means of three experi-
ments and expressed as △A492.mg-1 protein.min-1. △A492 is
the absorbance change of acridine orange at 492 nm. Abbrevia-
tions are the same as in Fig.1.
JING Yan et al.: Comparison of Plasma Membrane H+-ATPase Activity in Two Ecotypes of Reed (Phragmites communis)
Leaves from Different Habitats 1045
relative little is known about the changes of the catalytic
mechanism for the PM H+-ATPase under NaCl/saline stress.
Moreover, in studies on responses of PM H+-ATPase to
NaCl/saline stress, artificially stressed plants have usually
been used. We suggest that it is very important to use
plants possessing natural and stable tolerance/resistance
to soil salinity to study the responses of PM H+-ATPase, in
order to clarify the adaptation and resistance/tolerance
mechanisms of plants to long-term saline stress. Therefore,
in some sense, the two ecotypes of reed, swamp reed and
heavy salt meadow reed growing in the northwest of China,
are such ideal materials.
In this work, some properties of ATP and PNPP hydroly-
sis of the PM H+-ATPase in the two ecotypes of reed leaves
were investigated. Compared with SR, the Km for ATP hy-
drolysis of this enzyme in HSMR was lower, but there was
no significant difference in the Vmax. These results are simi-
lar to those of Kerkeb et al. (2001), who observed that NaCl-
sensitive and 50 mmol/L NaCl -tolerant tomato calli had
similar Vmax of PM H+-ATPase, but the Km value was lower
in the tolerant calli than in the resistant ones. Therefore,
the change of Km in HSMR might be involved in salinity
tolerance. As is known, in the catalytic process of PM H+-
ATPase, ATP-binding is conducted by the kinase domain
(Serrano, 1989). The decrease of Km for ATP hydrolysis in
HSMR indicated that the affinity of the kinase domain to
ATP was increased compared with that in SR, suggesting
that the phosphorylation step and the property of the ki-
nase domain of this enzyme were different between the two
reed ecotypes. Higher affinity of the kinase domain to ATP
in HSMR contributed to its higher ATPase activity. An in-
crease of PM H+-ATPase can enhance the Na+ transport
from the cytosol across the PM (Kalampanayil and
Wimmersk, 2001). Therefore, the increase of the ATPase
activity and decrease of Km in HSMR could be viewed as
an adaptive response to habitat.
Compared with the SR, the temperature stability of the
PM H+-ATPase in HSMR was altered (Fig.2), implying that
the enzyme structure was different in the two reed ecotypes.
The pH profile also showed a slight difference between the
two reed ecotypes and in HSMR the PM H+-ATPase could
maintain relatively higher activities in a broad pH range
(Fig.3) than in SR. This alteration in the catalytic property
of the enzyme might be essential for HSMR to survive ad-
verse saline stress.
It has been demonstrated that plant PM H+-ATPase has
PNPP hydrolysis activity and that the hydrolysis of this
substrate is conducted by the phosphatase domain of this
enzyme (Qiu, 1999). In our work, the higher PNPP hydroly-
sis activity and lower Km for PNPP hydrolysis by PM H+-
ATPase in HSMR (Fig.4) as compared to SR suggests a
difference in the phosphatase domain between the two
enzymes. The sensitivity of the ATPase to vanadate, a spe-
cific inhibitor of the PM H+-ATPase acting on the phos-
phatase domain (Serrano, 1989), was greater in HSMR than
in SR (Fig.5), also suggesting differences in the phosphatase
domain between the two ecotypes. Increased catalytic ac-
tivity of the phosphatase domain of PM H+-ATPase in
HSMR contributed to its increased ATPase activity, which
might be beneficial for this ecotype in adaptation to its
saline habitat.
It has been shown that cleavage of the C-terminal end
of the PM H+-ATPase with trypsin increased the enzyme
activity (Palmgren et al., 1990b). Our data with trypsin treat-
ment showed a difference in the pattern of activity of this
enzyme, both in terms of maximum activation, and in terms
of dependence of trypsin concentration (Fig.6). These re-
sults suggested that there existed a possible change in the
C-terminus of the PM H+-ATPase, either in the structure or
in the property or both of them in HSMR compared with
those in SR. It was suggested that the C-terminal end of
PM H+-ATPase may regulate its phosphatase and kinase
domains (Qiu and Zhang, 2001a; 2001b). Thus, the differ-
ence in properties in the phosphatase domain and kinase
domain of PM H+-ATPase in HSMR and SR might be par-
tially attributed to an altered structure or property of the C-
terminus. The changes might be essential in adaptation of
the HSMR to its saline habitat.
The differences observed in the enzymatic properties in
the two reed ecotypes could also be due to the switching
on and off of different isoforms of the genes. Palmgren and
Fig.8. The ratio of the H+ pumping activity to the ATPase
hydrolysis. The ATPase activity was assayed as described in
Materials and Methods with 3 mmol/L ATP in the reaction
medium. The proton-pumping activity was as shown here. Ab-
breviations are the same as in Fig.1.
Acta Botanica Sinica 植物学报 Vol.46 No.9 20041046
Christensen (1994) observed three AHA isoforms have dif-
ferent pH profile, vanadate sensitivity and LPC activation.
Therefore, the differences, at least regarding values of vana-
date sensitivity in the two reed ecotypes may be due to
differences in the isoenzyme expressed. The isoforms of
the PM H+-ATPase are differentially expressed and regu-
lated both developmentally and by environmental condi-
tions (Kasamo and Sakibara, 1995; Michelet and Boutry,
1995; Ballesteros et al.., 1998). Therefore, we speculate that
different isoform(s) of this enzyme expressed in HSMR com-
pared with those in SR might be involved in adaptation to
the saline habitat.
The proton-pumping activity of the PM ATPase, which
is tightly associated with the ATP hydrolysis activity is
also involved in responses of plants to environmental
stresses. In sunflower roots, it was observed that salt stress
decreased the PM H+-ATPase hydrolysis activity, whereas
the proton-pumping activity was not changed, resulting in
increased H+/ATP coupling efficiency (Ballesteros et al.,
1998). In tomato, similar phenomena were also observed
(Rodriguez-Rosales et al., 1999). Kerkeb et al. (2001) found
that the NaCl-sensitive and 50 mmol/L NaCl-tolerant to-
mato calli had similar PM H+-ATPase hydrolysis activity,
while the proton-pumping activity was higher in the toler-
ant calli. In addition, the H+/ATP coupling was higher in
the tolerant calli than that in the sensitive ones. Therefore,
higher proton-pumping activity and H+/ATP coupling were
related to salt tolerance. In the present work, both the AT-
Pase hydrolysis and proton-pumping activity in HSMR were
increased compared with those in SR, as was the H+/ATP
coupling ratio. The increase of proton-pumping activity
can enhance the transport of Na+ out of the cells
(Kalampanayil and Wimmersk, 2001). Therefore, the increase
of proton-pumping activity and a tighter H+/ATP coupling
in HSMR might contribute to adaptation to the long-term
saline habitat.
What contributes to the observed changes, genetic or
environmental (saline habitat) factors or both? It is known
that reed plants can adapt to adverse habitats and evolve
various ecotypes, which show genetic differences (Matoh
et al., 1988; Wang et al., 1998). In general, genetic variation
within a species is brought about by ecological adaptation
to various habitats. Thus, genetic variation strongly de-
pends on the habitats. Therefore, compared with SR, the
variations in the structural and catalytic properties of the
PM H+-ATPase in HSMR might be due to the changes of
gene expression during long-term adaptation to extreme
habitats, and these changes contributed to higher saline
tolerance.
Overall, in adaptation of the HSMR to the saline habitat,
the properties of the PM H+-ATPase were changed, which
might include enzyme modifications of the functional do-
mains and different isoforms expressed. Regulations of the
properties of this enzyme might be an adaptive response to
salinity.
Acknowledgements: We thank Prof. T J Flowers, School
of Life Sciences, Sussex University, UK, for his critical cor-
recting the English version of the manuscript.
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