Plant Science 176 (2009) 232–240

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Plant Science
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Overexpression of the Arabidopsis H+-PPase enhanced resistance to salt and

drought stress in transgenic alfalfa (Medicago sativa L.)
Ai-Ke Bao, Suo-Min Wang *, Guo-Qiang Wu, Jie-Jun Xi, Jin-Lin Zhang, Chun-Mei Wang
The Key Laboratory of Grassland Agro-ecosystem of the Ministry of Agriculture, College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou 730000, PR China

A R T I C L E I N F O A B S T R A C T

Article history: Salinity and drought are two major abiotic factors limiting crop production. To generate the legume
Received 12 July 2008 forage adapting to saline and arid soils, we had transformed alfalfa (Medicago sativa) with AVP1, a
Received in revised form 1 October 2008 vacuolar H+-pyrophosphatase (H+-PPase) gene from Arabidopsis thaliana. In this paper, we report that
Accepted 15 October 2008
overexpression of the AVP1 gene confers enhanced salt and drought tolerance to the transformed alfalfa.
Available online 30 October 2008
Transgenic alfalfa grows well in the presence of 200 mM NaCl and also under a water-deprivation
condition, while wild-type plants exhibit chlorosis and growth inhibition, even death. Compared with
Keywords:
wild-type plants, transgenic plants accumulate more Na+, K+ and Ca2+ in leaves and roots. Moreover, the
H+-PPase
leaves of transgenic plants retain more water during drought stress than those of wild-type plants due to
AVP1 gene
Transgenic alfalfa lower solute potential. Increased solute accumulation and water retention, and steady intracellular ion
Salt and drought tolerance homeostasis might also confer other phenotypes of salt and drought tolerance in the transgenic plants,
which include the higher photosynthesis capacity and the lesser cell membrane damage during salt or
water-deficit stress. Furthermore, the increased potassium uptake and root activity in transgenic alfalfa
may be the consequences of rhizosphere acidification resulting from expression of the AVP1. These results
indicated that the expression of AVP1 confers enhanced salt and drought tolerance on alfalfa, a very
important crop. This study provides a way for improving salt and drought tolerance in important legume
forages.
ß 2008 Elsevier Ireland Ltd. All rights reserved.

1. Introduction enhance vacuolar osmoregulatory capacity, which could confer

Salinity and drought are major abiotic factors reducing plant
productivity and quality [1,2]. To cope with salt stress, plants have
developed multifarious adaptation strategies, one of those is the
compartmentalization of Na+ into the vacuole, which might reduce
the deleterious effects of excess Na+ in the cytosol and to maintain
osmotic balance using Na+ as a cheap osmoregulation substance,
thus to enhance water uptake and salt tolerance of plant [3–5]. The
Na+ compartmentalization process is mediated by the vacuolar
Na+/H+ antiporter that is driven by electrochemical gradient of
protons across tonoplast generated by the vacuolar H+-pumps, H+-
ATPase (EC 3.6.1.3) and H+-pyrophosphatase (H+-PPase, EC 3.6.1.1)
[6,7]. The vacuolar H+-PPase is an electrogenic proton pump that
acidifies vacuoles in plant cells by pumping H+ from the cytoplasm
into vacuoles with PPi dependent H+ transport activity [8,9].
Theoretically, overexpression of H+-PPase should increase the
sequestration of ions in the vacuole by increasing the availability of
protons, thus to alleviate toxicity of Na+ and Cl in the cytosol and
* Corresponding author. Tel.: +86 931 8910983; fax: +86 931 8910979.
E-mail address: smwang@lzu.edu.cn (S.-M. Wang).
plants salt and drought tolerance [5]. Previous studies showed that
the overexpression of H+-PPase genes have resulted in enhanced
tolerance to salinity and/or drought stresses in plants. Enhanced
salt and drought tolerance have been achieved in model plant
Arabidopsis through overexpressing the Arabidopsis H+-PPase AVP1
[5], the halophyte Suaeda salsa H+-PPase SsVP [10] or a H+-PPase
TVP1 from wheat [11]. Moreover, D’yakova et al. [12] reported that
the overexpression of a H+-PPase from the bacterium Rhodospir-
illum rubrum conferred salt tolerance in transgenic tobacco. Similar
salt tolerant phenotype was observed in tobacco plants engineered
with the halophyte Thellungiella halophila H+-PPase TsVP [13]. Zhao
et al. [14] compared the salt tolerance between transgenic rice
plants expressing the S. salsa Na+/H+ antiporter SsNHX1 and co-
expressing SsNHX1 with the Arabidopsis AVP1, and found that the
simultaneous expression of the SsNHX1 and AVP1 conferred
greater performance to the transgenic rice plants than that of the
single SsNHX1. These results suggest that the enhanced vacuolar
H+-pumping in the transgenic plants provided additional driving
force for vacuolar solutes accumulation via the secondary
transporters including the vacuolar Na+/H+ antiporter [15].
Of significance, Li et al. [16] reported that in addition to its role
contributing to Na+ sequestration into the vacuole as an efficient

0168-9452/$ – see front matter ß 2008 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.plantsci.2008.10.009
 A.-K. Bao et al. / Plant Science 176 (2009) 232–240
H+ pump, the H+-PPase of Arabidopsis (AVP1) also plays an
important role in root development through the facilitation of
auxin fluxes. The overexpression of AVP1 in model plant
Arabidopsis resulted in increased cell division at the onset of
organ formation, hyperplasia, and increased auxin transport
whereas avp1-1 null mutants displayed severely disrupted root
and shoot development and reduced auxin transport [16,17]. In
keeping with these observations, overexpression of the AVP1 in a
commercial cultivar of tomato (Lycopersicon esculentum) resulted
in a more favorable plant water status and less injury. Further-
more, this phenotype resulted from the development of more
robust root systems [18]. Recently, a further study on AVP1
revealed that phosphorus deficiency environment cue can induce
an increased expression of the AVP1, which caused subsequently
an increased rhizosphere acidification and root proliferation
mediated by P-ATPase [19]. Moreover, the overexpression of
AVP1 in Arabidopsis, tomato and rice results in the enhancement of
potassium uptake and the release of organic acids, thus transgenic
plants outperformed controls when challenged with limited
phosphorus [19].
In short, previous studies suggest that there are potential
benefits in generating engineered plants to overexpress the H+-
PPase, i.e. increased tolerance to salinity, water-deficit and low-Pi
stress conditions.
Alfalfa (Medicago sativa L.) is one of the most important legume
forages with high protein content and more than 10 types of
vitamin and a key component of many crop rotation systems all
over the world [20]. However, most alfalfa cultivars are sensitive to
salt and drought [21]. Therefore, breeding salt and drought
resistant alfalfa cultivars is very necessary for this important
forage crop adapting to saline and arid soils. Salt and drought
tolerance of alfalfa can be improved by conventional breeding
methods, but it takes a long time to select salt and drought tolerant
plants. Recent developments in transgenetic technology have
provided an efficient tool for improving alfalfa. Since the first
report of alfalfa genetic transformation [22], many transgenic
alfalfas have been obtained by gene transfer method [23–27].
However, there are no reports regarding improving the stress
tolerance of alfalfa by expressing H+-PPase.
We have previously performed successful Agrobacterium-
mediated transformation of AVP1, the vacuolar H+-PPase gene
from Arabidopsis thaliana, to the hypocotyl explants of a local
alfalfa cultivar, ‘Xingjiang Daye’ in northwest China and the
regeneration of transgenic plants (Bao A.K. and Wang S.M.,
unpublished data). In present study, we show that transgenic
alfalfa expressing the AVP1 is much more resistant to salt stress
and soil water-deficit than wild-type plants. Comparing to the
wild-type, these transgenic plants accumulate more Na+, K+ and
Ca2+ in their leaf and root, and maintain a higher root activity under
either salt or drought stress.
2. Materials and methods
2.1. Preparation of plant materials
To prepare plant materials, nineteen T0 transgenic alfalfa lines
(Bao A.K. and Wang S.M., unpublished data) and the no-
transformed (wild-type) plants were propagated from stem
cuttings (ramets). Excise stem sections with two to three nodes
using a sharp razor blade and place the base into moist vermiculite
and perlite (1:1) under a photoperiod of 16/8 h (light/dark) at
24  2 8C and 90  5% relative humidity in the growth chamber. After
8–10 days when the adventitious roots formed, the same size plants
were chosen for further culture.
233
2.2. RNA extraction and RT-PCR
Total RNA was extracted from 200 mg of young leaves of
transformed lines and non-transformed wild-type plants with the
guanidine isothiocyanate method [28]. DNase-treated RNA sam-
ples were reverse-transcribed using M-MuLV reverse transcription
kit (Shanghai Sangon, China). First strand cDNAs were synthesized
from 4 mg total RNA using 1 ml M-MuLV reverse transcriptase, 1 ml
Oligo(dT)18 primer and 2 ml dNTP Mix at 37 8C for 1 h, then the
reaction was terminated at 70 8C for 10 min. The total volume of
the reverse-transcription reaction system was 20 ml. Four micro-
litres of the cDNA solution was used as templates for PCR
amplification with a pair of gene-specific primers of the AVP1 gene:
50 -AGAGTGTTGTCGCTAAGTG-30 and 50 -CAGTGAAGTCGTGGTT-
GAT-30 , which amplify a 765 bp fragment. A 286 bp fragment of
alfalfa GPDH (glyceraldehyde-3-phosphate dehydrogenase) gene,
used as an internal control, was amplified with the specific
primers: 50 -GTGGTGCCAAGAAGGTTGTTAT-30 and 50 -CTGGGAAT-
GATGTTGAAGGAAG-30 . Amplification conditions for the AVP1
fragment were an initial denaturation at 94 8C for 2 min, followed
by 30 cycles of 94 8C for 30 s, 54 8C for 60 s, 72 8C for 60 s, then a
final extension at 72 8C for 10 min. Amplification conditions for the
GPDH fragment were an initial denaturation at 94 8C for 2 min,
followed by 25 cycles of 94 8C for 30 s, 54 8C for 30 s, 72 8C for 45 s,
then a final extension at 72 8C for 8 min. The PCR products were
electrophoresed on a 1.2% agarose gel containing ethidium
bromide.
2.3. Salt and drought stress treatments
For salt stress treatments, plants from alfalfa transgenic lines
and the wild-type were transplanted to plastic culture pots
(cubical-shaped pot with 4 cm in width, 5 cm in length and 5 cm in
height, 1 plant/pot) containing soil under a photoperiod of 16/8 h
(light/dark, the light flux density during the light period was
approximately 600 mmol m2 s1) at 24  2 8C and 60  5% relative
humidity, watered every 2 days with 1/8 Hoagland nutrient solution
for 4 weeks, and then the nutrient solution was supplemented with
NaCl. NaCl concentrations were incrementally increased with 50 mM
2 day1 increments until final concentrations (0, 100, 200 mM) were
achieved. After salt treatment for 7 days, the plants were used for
further physiological analysis.
For drought stress treatments, plants from alfalfa transgenic
lines and the wild-type were transplanted to plastic culture pots
(cylindrical pot with 8 cm in diameter and 10 cm in height, 1 plant/
pot) containing soil under a photoperiod of 16/8 h (light/dark, the
light flux density during the light period was approximately
600 mmol m2 s1) at 24  2 8C and 60  5% relative humidity,
watered every 2 days with 1/8 Hoagland nutrient solution to field
capacity for 4 weeks, then the water was withheld until all plants
showed severe drought stress symptoms (i.e., visible wilting, see
Fig. 2b). During drought-stress treatments, the physiological indexes
of transgenic and wild-type plants were measured at 1-day or 2 days
interval (this varied depending on the experiment conditions at
the time).
2.4. Measurement of biomass and Na+, K+ and Ca2+
contents in root and leaf
Shoot dry weight of the transgenic and wild-type plants of each
treatment was determined after drying samples at 80 8C for 48 h.
Na+, K+ and Ca2+ were measured according to the method
described by Flowers and Hajibagheri [29] with slight modifica-
tions. Roots were rinsed with deionized water for 10 s, then
washed with cold LiNO3 solution isotonic. The leaves and roots
234 A.-K. Bao et al. / Plant Science 176 (2009) 232–240
from the transgenic and wild-type plants of each treatment were
dried at 80 8C for 48 h, the dry weight was measured. Na+, K+ and
Ca2+ were extracted from dried plant tissue with 100 mM acetic
acid at 90 8C for at least 2 h, then the cation contents were
determined using a flame spectrophotometer (2655-00, Cole-
Parmer Instrument Co., USA).
2.5. Quantification of solutes in leaf tissue
Quantification of solutes in leaf tissue was detected as
described by Gaxiola et al. [5]. Extended leaves excised from the
transgenic and wild-type alfalfa plants of each treatment were
rinsed with deionized water and blotted on sterile filter paper
immediately. The leaves were then frozen in liquid nitrogen and
thawed to extrude sap by a syringe. The resulting sap was
determined with a cryoscopic osmometer (OSMOMAT-030,
GONOTEC GmbH, Germany). The readings (mmol kg1) were used
to calculate the solute potential (Cs) in MPa (mega Pascales) with
the formula Cs = moles of solute  RK, where R = 0.008314 and
K = 298 8C.
2.6. Measurement of the relative water content
During water-deficit stress, the relative water content (RWC)
of transgenic and wild-type alfalfa plants were measured
according to the method described by Gaxiola et al. [5]. Leaves
from alfalfa plants were excised, and their fresh weight was
measured immediately. After floating them in deionized water at
4 8C overnight, their rehydrated weight was determined. Finally,
they were dried in an oven at 80 8C for 48 h and weighed. The RWC
was calculated as indicated: RWC = (fresh weight  dry weight)/
(rehydrated weight  dry weight).
2.7. Measurement of the net photosynthetic rate (Pn)
The Pn was measured using an automatic photosynthetic
measuring apparatus (LI-6400, LI-COR Biosciences, USA) as
described by Qiu et al. [30].
2.8. Assay of malondialdehyde (MDA) content and leaf
cell membrane damage
Extended leaves excised from the transgenic and wild-type
alfalfa plants of each treatment were used to measure MDA
content. The MDA content was determined using thiobarbituric
acid (TBA) protocol [31]. The absorbance at 450, 532, and 600 nm
was determined using an ultraviolet spectrophotometer (UV-
2102C, Unico (Shanghai) Instrument Co., Ltd., China). The content
of MDA in nmol g1 was calculated by the following formula: MDA
content (nmol g1) = C (mmol l1)  V (l)/fresh weight (g)  1000,
where C (mmol l1) = 6.45  (A532  A600)  0.56 A450, and V refer
to the volume (l) of extracting solution.
Leaf cell membrane damage was determined as the relative
membrane permeability of leaf cells using a conductivity meter
(EC215, HANNA, Italy), according to the method described by
Gibon et al. [32] with slight modifications. The cell membrane
damage represented as relative membrane permeability (%) was
calculated using the following equation: relative membrane
permeability (%) = S1/S2  100, where S1 and S2 refer to con-
ductivity of alfalfa live leaves and boiled leaves, respectively.
2.9. Measurement of root activity (a-NA oxidation rate)
Roots were carefully taken from the soil and washed. Root fresh
weight (FW) was recorded and a-naphthylamine (a-NA) oxidation
rate (mg 100 g1 FW h1) was measured as described by Zhong
and Huang [33].
2.10. Data analysis
Data for this study were analyzed by One-way Analysis of
Variance (ANOVA) using SPSS 13.0 (SPSS Inc., USA) and Duncan’s
Multiple Range test was used to detect significant differences
between means. Least significant differences were evaluated at the
5% level of probability.
3. Results
3.1. AVP1 expression in transgenic alfalfa plants
To detect the expression of AVP1 in transgenic alfalfa lines,
RT-PCR was performed on young leaves of the transgenic alfalfa
together with wild-type. As expected, AVP1 expression was
observed in all transgenic alfalfa lines, but its levels are different
(data not shown). Compared with other lines, line 1 and line 8
showed the highest and lowest expression level of AVP1,
respectively (Fig. 1). So, line 1 (now called L1) and line 8 (now
called L8) were then chosen for further physiological assays.
3.2. Overexpression of AVP1 increases salt and drought
tolerance of alfalfa
Phenotypically, the transgenic plants did not differ from wild-
type plants under normal growth conditions (Fig. 2a). When plants
were exposed to 200 mM NaCl for 10 days, however, the transgenic
plants overexpressing AVP1 are more salt tolerant than wild-type
plants. After 10 days in the presence of up to 200 mM NaCl, wild-
type plants exhibit chlorosis, even die, whereas both L1 and L8
transgenic lines continue to grow well (Fig. 2b). To further study
the difference between transgenic and wild-type alfalfa under salt
treatment, shoot biomass (dry weight) was measured under
different NaCl concentrations. Shoot biomass of all plants decreased
gradually with increased salt concentrations. But the shoot biomass
of wild-type plants was significantly lower than transgenic plants
under the same NaCl concentration (100 and 200 mM). For example,
in presence of 200 mM NaCl, the shoot dry weight of L1 and L8
were 2.5-fold (P < 0.01) and 2.2-fold (P < 0.01) in comparison that of
wild-type, respectively. Moreover, the difference in shoot biomass
between L1 and L8 became more distinct at higher salt concentra-
tions (200 mM NaCl) (Fig. 2c).
Drought stress phenotype was assayed by imposing a 8-day
water-deficit stress period. After 6 days of drought stress, wild-
type plants showed growth reduction and exhibited chlorosis,
whereas the transgenic lines continued normal growth (Fig. 3a)
until day 8 after subjected to water-deficit stress (Fig. 3b). After 8
days of water deprivation, plants from each group were rewatered.
Fig. 1. Analysis by RT-PCR of AVP1 expression in transgenic alfalfa. Specific PCR
products of 765 bp were detected transgenic alfalfa using a 1.2% agarose gel
containing ethidium bromide. The photograph show representative in transgenic
lines (line 1 and line 8) which have the highest and lowest expression levels of AVP1,
respectively. (-RT): without reverse transcriptase; WT: non-transformed plants; L1,
L8: transgenic plants overexpressing AVP1. A 286 bp GPDH gene fragment was
amplified by RT-PCR as an internal control.
 A.-K. Bao et al. / Plant Science 176 (2009) 232–240 235

Fig. 2. Transgenic alfalfa plants expressing AVP1 showed enhanced salt tolerance. Plants were watered every 2 days with 1/8 Hongland nutrient solution for 4 weeks, and then
the nutrient solution was supplemented with NaCl (the solution was changed every day). NaCl concentrations were incrementally increased with 50 mM 1 day1 increments
until final concentrations (0, 100, 200 mM) were achieved. The photographs show representative plants of wild-type alfalfa and two AVP1-overexpressing transgenic lines at
the 10th day without (a) and with 200 mM NaCl treatment (b). (c) Shoot biomass of transgenic and wild-type alfalfa under different NaCl concentrations. The shoot biomass
were measured after 7 days of salt treatment. Values are means  S.D. (n = 18) and bars indicate S.D. Columns with different letters indicate significant difference at P < 0.05
(Duncan test). WT: non-transgenic controls; L1, L8: transgenic plants overexpressing AVP1.

Wild-type plants died, but plants from both transgenic lines
survived and resumed normal growth (Fig. 3c and d). These results
showed that the overexpression of AVP1 confers transformed
alfalfa an increased drought tolerance. However, the performance
of plants from L1 was better than L8 during water-deficit, for
example, the shoot dry weight of L1 was 1.7-fold than that of L8
(P < 0.05) after rewatered for 4 days (data not shown), which is
similar to the phenotype under 200 mM NaCl, suggesting that the
expression levels of AVP1 are correlated with the salt and drought
tolerant phenotype.
3.3. AVP1 transgenic alfalfa accumulate more cations
than wild-type plants
The contents of Na+, K+, and Ca2+ were determined in the leaves
and roots of plants from transgenic lines and wild-type grown
under different NaCl concentrations (0, 100, and 200 mM) and
different water-deficit treatments (0, 3, and 6 days). The results
showed that both L1 and L8 accumulated more Na+, K+, and Ca2+ in
their leaves and roots than wild-type under either salt or water-
deficit treatments. Comparatively, the contents of cations in L1
plants were highest under salt or water-deficit stress, which is also
correlated with the expression levels of AVP1.
Under salt treatments, Na+ contents significantly increased in
leaves and roots of both transgenic and wild-type plants as the
media NaCl concentration increased. At 100 and 200 mM NaCl,
however, there were significantly more Na+ in both L1 and L8 than
wild-type (P < 0.05) (Fig. 4a and b). Though the amounts of K+
decreased with increasing NaCl concentrations in the leaves and
roots of both transgenic and wild-type plants, the K+ content in
transgenic lines were significantly higher than that in wild-type,
regardless NaCl level (Fig. 4c and d). With increasing NaCl
concentrations, it was observed that Ca2+ contents in leaves and
roots of wild-type did not exhibited obviously change, but
increased in both transgenic lines, which resulted in the significant
difference of Ca2+ contents between both transgenic lines and
wild-type at 100 and 200 mM NaCl (P < 0.05) (Fig. 4e and f). Under
water-deficit treatments, there are significantly more Na+, K+, and
Ca2+ in both L1 and L8 than that in wild-type, which are similar to
the situation of NaCl treatments (Fig. 5). After 6 days of drought
stress, contents of all three cations in leaves and roots of both L1
and L8 significantly increased, but only Na+ content exhibited an
remarkably increase in wild-type (Fig. 5a and b). These results
suggest that the enhanced salt and drought tolerance in transgenic
alfalfa plants might be related to an increased cation accumulation
capacity, since other transgenic plants overexpressing AVP1 have
been shown to have an enhanced H+ electrochemical gradient
across their tonoplast, leading to increased vacuolar solute
accumulation by H+ co- and counter-transporters [5].
Furthermore, it was observed that both transgenic lines
absorbed more K+ than wild-type under normal conditions
(P < 0.05) (Fig. 4c and d; Fig. 5c and d). During water-deficit,
236 A.-K. Bao et al. / Plant Science 176 (2009) 232–240

Fig. 3. Transgenic alfalfa plants expressing AVP1 showed enhanced drought tolerance. Representative plants of wild-type alfalfa and two AVP1-overexpressing transgenic lines
were watered with 1/8 Hoagland nutrient solution every 2 days to field capacity for 4 weeks. After that, the soil was allowed to dry by withholding water for 6 days (a), and for
8 days (b) when all plants were wilting. Then soil was rewatered to field capacity for 1 day (c) and 4 days (d). WT: non-transgenic controls; L1, L8: transgenic plants
overexpressing AVP1.

Fig. 4. Cation contents in leaf and root of transgenic and wild-type alfalfa under different NaCl concentrations. The Na+ (a, b), K+ (c, d), and Ca2+ (e, f) contents were measured
after 7 days of salt treatment. Values are means  S.D. (n = 6) and bars indicate S.D. Columns with different letters indicate significant difference at P < 0.05 (Duncan test). WT:
non-transgenic controls; L1, L8: transgenic plants overexpressing AVP1.
 A.-K. Bao et al. / Plant Science 176 (2009) 232–240 237

Fig. 5. Cation contents in leaf and root of transgenic and wild-type alfalfa under water-deficit stress. The Na+ (a, b), K+ (c, d), and Ca2+ (e, f) contents were measured 0, 3, and 6
days of water-deficit treatments. Values are means  S.D. (n = 6) and bars indicate S.D. Columns with different letters indicate significant difference at P < 0.05 (Duncan test). WT:
non-transgenic controls; L1, L8: transgenic plants overexpressing AVP1.

interestingly, the amounts of K+ have dramatically enhanced in
both transgenic plants (P < 0.05), but no significant increasing
could be seen in the wild-type (P > 0.05) (Fig. 5c and d). These
results suggest that expression of the AVP1 might confer transgenic
alfalfa an enhanced K+ uptake capacity.
3.4. AVP1 transgenic alfalfa retain more solutes and water
than wild-type plants
The amount of solutes in the leaves was assayed by measuring
leaf solute potential (Cs, refers to the concentration of osmotically
active particles dissolved in water, conventionally defined as
osmotic pressure). Under normal growth conditions, the osmotic
pressure or solute potential of the wild-type plants was less
negative than that of the transgenic lines L1 and L8 (P < 0.05)
(Fig. 6a). The Cs values of the wild-type plants, and both transgenic
lines L1 and L8 were 1.13 MPa (S.D. = 0.05), 1.83 MPa
(S.D. = 0.09), and 1.75 MPa (S.D. = 0.12). When challenged with
salt or drought stress, similarly, the leaf Cs droped in wild-type and
transgenic plants, but reached significant lower values in transgenic
lines than in wild-type (data not shown). These results are consistent
with a higher osmoregulatory capacity in transgenics than in wild-
type alfalfa.
To study further the osmoregulatory capacity in alfalfa, leaf
relative water content (RWC) was evaluated during water
deprivation. RWC were reduced in both transgenic and wild-
type plants, but RWC in wild-type plants shown a faster decline
than in transgenic alfalfa with time lapse. After 6 days of water-
deficit stress, a higher water loss rate was observed in wild-type
plants (RWC dropped 45% to a value of 0.51), whereas the RWC of
the transgenic plants decreased only slightly to a value of 0.82 (for
L1, decreased 13%) and 0.75 (for L8, decreased 20%). At day 8, the
RWC of wild-type plants continued to sharply decrease to a value
of 0.26, whereas the RWC of the transgenic plants still maintained

Fig. 6. Leaf solute potential and water retention in wild-type and transgenic plants. (a) Leaf solute potential of transgenic and wild-type alfalfa under fully watered conditions.
(b) Relative water content in leaf of wild-type and transgenic alfalfa under water-deficit stress. The RWC were determined at days 0, 3, 6, and 8 of water-deficit stress. Values
are means  S.D. (n = 6) and bars indicate S.D. WT: non-transgenic controls; L1, L8: transgenic plants overexpressing AVP1.
238 A.-K. Bao et al. / Plant Science 176 (2009) 232–240

Fig. 7. Net photosynthetic rate of transgenic and wild-type alfalfa under different NaCl concentrations and water-deficit stress. The net photosynthetic rate was measured
after 7 days of salt treatments (a) or 0, 3, and 6 days of water-deficit treatments (b). Values are means  S.D. (n = 6) and bars indicate S.D. Columns with different letters indicate
significant difference at P < 0.05 (Duncan test). WT: non-transgenic controls; L1, L8: transgenic plants overexpressing AVP1.

at 0.62 (for L1) and 0.52 (for L8) (Fig. 6b). These data further
suggest that overexpression of AVP1 triggers an enhanced
capacity for osmotic adjustment of transgenic alfalfa resulting
in greater water uptake at low soil water content during water-
deficit stress.
3.5. Overexpression of AVP1 protects photosynthetic machinery
in transgenic alfalfa
To further evaluate the increased salt and drought tolerance of
transgenic alfalfa overexpressing AVP1, net photosynthetic rate
(Pn) of wild-type and both transgenic lines was determined.
Though the net photosynthetic rate declined in all experimental
lines with the salt concentration and drought time increase, but the
Pn of wild-type exhibited an greater decrease compared with the
both transformed alfalfa lines (Fig. 7). For example, at 200 mM
NaCl, there was 74.1% reduction of the Pn in wild-type, whereas
only 42.8% and 50.9% in transgenic L1 and L8 (P < 0.05),
respectively (Fig. 7a); after 6 days of water-deficit stress, similarly,
the decline of the Pn in control plants was 68.2%, but only 30.8%
and 44% in transgenic L1 and L8 (P < 0.05), respectively (Fig. 7b).
These results are consistent with a role for AVP1 in vacuolar Na+
sequestration and the accumulation of solutes, thus protecting
photosynthetic machinery from Na+ toxicity and dehydration
during salt and drought stress.
3.6. AVP1 overexpression maintains the stability of cell
membrane in transgenic alfalfa
To study the stability of membrane in transgenic and wild-type
plants under salt and drought stress, relative membrane perme-
ability and MDA content that represent the degree of cell
membrane damage under stress were determined. The results
indicated that MDA content and relative membrane permeability
increased with the increase in salt concentration or water-deficit
time, but they showed a faster rise in wild-type than that in both
transgenic lines (Fig. 8). In transgenic lines L1 and L8, the amount
of MDA was 34.3–44.7% and 18.4–23.1%, respectively, lower than
that in wild-type alfalfa (P < 0.05); relative membrane perme-
ability was 50.8–52.8% and 31.4–39.8%, respectively, less than that
of wild-type plants (P < 0.05) at 200 mM NaCl and day 6 of water-
deficit. These results suggest that transgenic alfalfa was healthier
or subjected to less damage under salt or drought stress.
3.7. AVP1 transgenic alfalfa has higher root activity than wild-type
To study further the effects of salt and water-deficit on alfalfa,
the a-naphthylamine (a-NA) oxidation rate in roots, as an
indicator of root activity, was determined. With increased salt
concentration and longer drought times the a-NA oxidation rate of
wild-type plants showed a sharp decrease, whereas only slightly

Fig. 8. MDA contents and relative membrane permeability in leaf of transgenic and wild-type alfalfa under different NaCl concentrations and water-deficit stress. The MDA
contents and relative membrane permeability were measured after 7 days of salt treatments (a, b) or 0, 3, and 6 days of water-deficit treatments (c, d). Values are means  S.D.
(n = 6) and bars indicate S.D. Columns with different letters indicate significant difference at P < 0.05 (Duncan test). WT: non-transgenic controls; L1, L8: transgenic plants
overexpressing AVP1.
 A.-K. Bao et al. / Plant Science 176 (2009) 232–240
Fig. 9. Root activity of transgenic and wild-type alfalfa under different NaCl concentrations and water-deficit stress. The root activity was measured after 7 days of salt
treatments (a) or 0, 3, and 6 days of water-deficit treatments (b). Values are means  S.D. (n = 6) and bars indicate S.D. Columns with different letters indicate significant
difference at P < 0.05 (Duncan test). WT: non-transgenic controls; L1, L8: transgenic plants overexpressing AVP1.
drop in both transgenic lines under salt stress (Fig. 9a), and
significantly increased at day 3 of water-deficit (P < 0.05) (Fig. 9b).
Under either salt or drought stress, the root activity of both
transgenic lines were higher than wild-type plants. At 200 mM
NaCl, the a-NA oxidation rate of L1 and L8 were 2.2 folds (P < 0.01)
and 1.6 folds (P < 0.01) in comparison that of wild-type,
respectively (Fig. 9a). After 6 days of water-deficit stress, similarly,
the a-NA oxidation rate of L1 and L8 were 2.1 folds (P < 0.01) and
1.9 folds (P < 0.01) than that of wild-type, respectively (Fig. 9b).
These results imply that the expression of AVP1 resulted in an
enhanced root activity in transgenic alfalfa when challenge with
abiotic stress conditions.
4. Discussion
Previous studies demonstrated that overexpressing the H+-
PPase of A. thaliana [5], S. salsa [10], or wheat [11] in A. thaliana, and
the H+-PPase of R. rubrum [12] and T. halophila [13] in tobacco led to
enhanced salt and/or drought tolerance in transgenic plants. In this
study, overexpression of H+-PPase from Arabidopsis in transgenic
alfalfa plants also resulted in increased salt and drought tolerance.
Wild-type alfalfa displayed growth inhibition, reduced biomass,
chlorosis, and even death when treated with high salt concentra-
tions or water deprivation, whereas the transgenic plants
exhibited survived and normal development (Figs. 2 and 3);
interestingly, the AVP1 expression levels are consistent with
tolerant phenotypes. Line 1 (highest expression level of the AVP1)
show higher tolerance than Line 8 (lowest expression level of the
AVP1). All of these properties of transgenic alfalfa might be
ascribed to the increased sequestration of Na+ into vacuole,
maintained ion homeostasis especially intracellular K+ and Na+
homeostasis, and enhanced vacuolar osmoregulatory capacity,
because of the overexpression of H+-PPase. This conclusion was
supported by our measurements on cations accumulation levels in
leaves and roots of the transgenic alfalfa; there are more Na+, K+
and Ca2+ in leaves and roots of transgenic alfalfa compared to wild-
type line (Figs. 4 and 5). The increased accumulation of sodium
(Fig. 4a and b; Fig. 5a and b) is likely to be a consequence of the
enhanced transport efficiency of the vacuolar Na+/H+ antiporter
[3,5,34], in the presence of increased proton supply resulting from
the overexpression of AVP1.
In addition to reduce the toxicity of excess Na+ in the cytosol [3–
5], compartmentation of Na+ can also facilitate cellular K+ [5,35]
and Ca2+ uptake [14]. On one hand, therefore, this compartmenta-
tion maintained intracellular ion homeostasis. Regulating ion
homeostasis especially intracellular K+ and Na+ homeostasis are of
critical importance for plant adaptation to salt and drought stress
[36]. Potassium is required for plant growth, tropisms, cell
expansion, enzyme activity, ion homeostasis and stomatal move-
ments [14]. In the present study, the relatively more K+ in
transgenic alfalfa than in wild-type under salt and water-deficit
239
stress conditions (Fig. 4c and d; Fig. 5c and d) might contribute to
stress resistances, similar results were also observed in almost all
transgenic plants overexpressing the H+-PPase [5,10,11,13]. More-
over, Our data also showed significantly higher levels of Ca2+
contents in transgenic lines compared with wild-type plants under
salt or drought stress (Fig. 4e and f; Fig. 5e and f), which may be also
correlated to the increased salt and drought tolerance, since Ca2+
plays a number of roles in plant life process such as stabilizing cell
walls and membranes, stimulating net uptake of K+, regulating
H2O2 homeostasis and as a second messenger [37,38]. On the other
hand, compartmentation of Na+ maintained turgor of transgenic
plants, since the net increase in the content of solutes in the cell
must lead to an increase in the uptake of water [5]. Our finding of a
decreased solute potential in leaves of transgenic plants (Fig. 6a)
are consistent with this idea. Gaxiola et al. [5] concluded that the
increased solute accumulation as a consequence of AVP1 over-
expression results in a concomitant increase in retention of water.
Similar result was observed in transgenic alfalfa; under water-
deficit stress, more water was retained in the leaves of transgenic
alfalfa than wild-type plants (Fig. 6b).
There have been the evidences showed that salt and water-
deficit stress caused the reduction on photosynthetic activities
[39]. This was due to that decreased the amount of water and
increased content of Na+ in the cytosol can irreversibly inactivated
PSI and PSII [40]. Our results indicated that the photosynthesis
capacity was higher in transgenic alfalfa than that in wild-type
under salt or drought stress (Fig. 7). This is consistent with the
phenotype observed from transgenic rice co-expressing the
SsNHX1 and AVP1 [14]. Zhao et al. [14] concluded that the higher
photosynthesis capacity of the transgenic plants might be related
to the increased Na+ sequestration and the less generation of H2O2
due to co-expression of the SsNHX1 and AVP1. Furthermore, abiotic
stresses including salt and drought stress can cause oxidative
damage to cell membrane, the prevention capacity to membrane
damage is correlated with stress tolerance [41]. Gao et al. [13]
found that cell membrane damage were lower in transgenic
tobacco expressing the TsVP than in wild-type under salt stress,
because the overexpression of H+-PPase can enhance the
accumulation of Na+ in vacuoles, which can avoid the toxicity of
excessive Na+ to plant cells. In this study, overexpression of AVP1
also contribution to maintain the stability of cell membrane in
transgenic alfalfa under stress conditions (Fig. 8). Theses results
further support that the overexpression of H+-PPase can enhance
the salt and/or drought tolerance of transgenic plants.
Interestingly, a recent study showed that overexpression of the
AVP1 results in the enhanced K+ uptake and the release of organic
acids, which contribute to an increased rhizosphere acidification,
and thus enhanced phosphorus uptake in transgenic plants [19].
We also found that an increased amount of K+ in transgenic alfalfa
than that in wild-type plants (Fig. 4c and d; Fig. 5c and d).
Furthermore, compared with wild-type, transgenic alfalfa has
240 A.-K. Bao et al. / Plant Science 176 (2009) 232–240
higher root activity under stress conditions (Fig. 9), this may be
also contributed to the salt and drought tolerance in transgenic
alfalfa.
Integrating data in this report, we conclude that the over-
expression of Arabidopsis H+-PPase confer salt and drought
tolerance in alfalfa. To our knowledge it was the first report to
overexpress a H+-PPase in alfalfa, which provides a feasible way for
improving agronomically important legume forages to adapt
abiotic stress conditions, such as, saline and arid soils.
Acknowledgements
We thank Dr. R.A. Gaxiola (Arizona State University, U.S.A.) for
his kindly granting bacteria and plasmids which carry AVP1 gene
and his constructive comments on this manuscript. This work was
supported by grants from the National Basic Research Program of
China (973 Program No. 2007CB108901), the National High Tech
Project of China (863 Project No. 2006AA10Z126), the National
Science Foundation of China (Programs No. 30770347 and
30671488) and the program for New Century Excellent Talents,
China (NCET-05-0882).
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