Journal of Experimental Botany, Vol. 62, No. 15, pp. 5561–5570, 2011
doi:10.1093/jxb/err237 Advance Access publication 12 August, 2011
RESEARCH PAPER
Co-expression of Pennisetum glaucum vacuolar Na+/H+
antiporter and Arabidopsis H+-pyrophosphatase enhances
salt tolerance in transgenic tomato
Shimna Bhaskaran and D. L. Savithramma*
Department of Genetics and Plant Breeding, University of Agricultural Sciences (UAS), GKVK, Bangalore 560-065, India
* To whom correspondence should be addressed. E-mail: [email protected]
Received 24 March 2011; Revised 2 July 2011; Accepted 6 July 2011
Abstract
Salinity is one of the major abiotic stresses affecting plant productivity. Tomato (Solanum lycopersicum L.), an
important and widespread crop in the world, is sensitive to moderate levels of salt in the soil. To generate tomato
plants that can adapt to saline soil, AVP1, a vacuolar H+-pyrophosphatase gene from Arabidopsis thaliana, and
PgNHX1, a vacuolar Na+/H+ antiporter gene from Pennisetum glaucum, were co-expressed by Agrobacterium
tumefaciens-mediated transformation. A sample of transformants was self-pollinated, and progeny were evaluated
for salt tolerance in vitro and in vivo. It is reported here that co-expression of AVP1 and PgNHX1 confers enhanced
salt tolerance to the transformed tomato compared with the AVP1 and PgNHX1 single gene transgenic plants and
the wild-type. These transgenic plants grew well in the presence of 200 mM NaCl while wild-type plants exhibited
chlorosis and died within 3 weeks. The transgenic line co-expressing AVP1 and PgNHX1 retained more chlorophyll
and accumulated 1.4 times more proline as a response to stress than single gene transformants. Moreover, these
transgenic plants accumulated a 1.5 times higher Na+ content in their leaf tissue than the single gene transformants.
The toxic effect of Na+ accumulation in the cytosol is reduced by its sequestration into the vacuole. The
physiological analysis of the transgenic lines clearly demonstrates that co-expression of AVP1 and PgNHX1
improved the osmoregulatory capacity of double transgenic lines by enhanced sequestration of ions into the vacuole
by increasing the availability of protons and thus alleviating the toxic effect of Na+.
Key words: Co-expression, H+-pyrophosphatase, Na+/H+ antiporter, salt tolerance, sodium sequestration, transgenic tomato.
Introduction
The progressive salinization of soil is estimated at ;20% of
irrigated land (Ashraf, 1994) and it limits future agriculture,
as most crop species are glycophytes, which are usually salt
sensitive. An estimate from the Food and Agriculture
Organization suggested that ;6% of the world’s total land
area and 20% of irrigated land is affected by high salinity
(http://www.fao.org/ag/agl/agll/spush). To cope with salt
stress, plants have developed multifarious adaptation strategies, one of which is the compartmentalization of Na+ into
the vacuole; this might reduce the deleterious effects of
excess Na+ in the cytosol and maintain the osmotic balance
using Na+ as a cheap osmoregulatory substance, thus
enhancing water uptake and salt tolerance of plants
(Gaxiola et al., 1999). The Na+ compartmentalization
process is mediated by the vacuolar Na+/H+ antiporter that
is driven by the electrochemical gradient of protons across
the tonoplast generated by the vacuolar H+ pump, H+pyrophosphatase (Blumwald and Gelli, 1997). The vacuolar
H+-pyrophosphatase is an electrogenic proton pump that
acidifies vacuoles in plant cells by pumping H+ from the
cytoplasm into vacuoles with pyrophosphatase-dependent
H+ transport activity (Maeshima, 2000). Theoretically,
overexpression of H+-pyrophosphatase should increase the
sequestration of ions in the vacuole by increasing the
availability of protons, thus alleviating the toxicity of Na+
and Cl– in the cytosol and enhancing vacuolar
ª The Author [2011]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
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5562 | Bhaskaran and Savithramma
osmoregulatory capacity, which could confer salt tolerance
to plants (Gaxiola et al., 2001).
Although studies on the activity of Na+/H+ antiporters in
plant cells began as early as in 1976 (Ratner and Jacoby,
1976), it was only a decade ago that plasma membrane and
vacuolar Na+/H+ antiporter genes were identified in plants
(Apse et al., 1999; Gaxiola et al., 1999; Shi et al., 2000; Xia
et al., 2002). Overexpression of Arabidopsis thaliana
AtNHX1 conferred enhanced salt tolerance to Arabidopsis
(Apse et al., 1999) and several other plant species such as
tomato (Solanum lycopersicum) (Zhang and Blumwald,
2001), rapeseed (Brassica napus) (Zhang et al., 2001), bread
wheat (Triticum aestivum) (Xue et al., 2002), maize (Zea
mays) (Yin et al., 2004), and buck wheat (Fagopyrum
esculentum) (Chen et al., 2008). Overexpression of Oryza
sativa OsNHX1 in rice plants (Fukuda et al., 1999, 2004)
and transfer of Gossypium hirsutum GhNHX1 to tobacco
(Nicotiana sp.) (Wu et al., 2004) have been shown to confer
salt tolerance. Overexpression of Hordeum brevisubulatum
HbNHX1 in tobacco rendered transgenic plants tolerant to
both salt and drought stress (Lu et al., 2005). Ectopic
expression of Pennisetum glaucum PgNHX1 in rice (O. sativa)
(Verma et al., 2007) and mustard greens (Brassica juncea)
(Rajgopal et al., 2007) conferred higher levels of salinity
tolerance to transgenic plants. Recently, expression of
Medicago sativa MsNHX1 was shown to enhance salt
tolerance in Arabidopsis (An et al., 2009). Together these
studies have demonstrated the potential for use of specific
vacuolar antiporters as candidate genes in imparting salt
tolerance capabilities. Apse et al. (2003) have also shown
that mutant lines of AtNHX1 had a much lower Na+/H+
and K+/H+ exchange activity, altered leaf development,
with reduction in the frequency of large epidermal cells and
a reduction in overall leaf area compared with wild-type
plants, suggesting that NHX1 genes are also required for
development of the plant.
Previous studies have also shown that the overexpression
of H+-pyrophosphatase genes has also resulted in enhanced
tolerance to salinity and/or drought stresses in plants.
Enhanced salt and drought tolerance have been achieved in
the model plant Arabidopsis through overexpressing the
Arabidopsis H+-pyrophosphatase AVP1 (Gaxiola et al.,
2001), Suaeda salsa H+-pyrophosphatase SsVP (Guo et al.,
2006), and a H+-pyrophosphatase TVP1 from wheat (Brini
et al., 2007). Moreover, Dyakova et al. (2006) reported that
overexpression of a H+-pyrophosphatase from the bacterium
Rhodospirillum rubrum conferred salt tolerance to transgenic
tobacco. A similar salt-tolerant phenotype was observed in
tobacco plants engineered with the Thellungiella halophila
H+-pyrophosphatase TsVP (Gao et al., 2006). Recently,
overexpression of AVP1 was shown to enhance salt and
drought tolerance in transgenic alfalfa (Medicago sativa)
(Bao et al., 2009). Of significance, Li et al. (2005) reported
that in addition to its role contributing to Na+ sequestration
into the vacuole as an efficient H+ pump, AVP1 also plays an
important role in root development through the facilitation
of auxin fluxes. The overexpression of AVP1 in the 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 (Yang et al., 2007).
In brief, previous studies suggest that there are potential
benefits in generating engineered plants to overexpress the
H+-pyrophosphatase and Na+/H+ antiporter to increase
tolerance to salinity. From these results, it was also
predicted that co-expression of the vacuolar Na+/H+ antiporter and H+-pyrophosphatase would confer even higher
salt tolerance to transgenic plants than expression of each
gene alone. The evidence for this hypothesis was provided
by Zhao et al. (2006), who compared the salt tolerance
capacity of transgenic rice plants expressing the S. salsa
Na+/H+ antiporter SsNHX1 and co-expressing SsNHX1
with AVP1. It was found that the simultaneous expression
of SsNHX1 and AVP1 conferred greater performance to the
transgenic rice plants than expression of SsNHX1 alone.
Tomato (S. lycopersicum L.), an important and widespread crop in the world, is sensitive to moderate levels of
salt in the soil. Salinity has a negative effect on tomato
yield, as it decreases fruit weight and marketable yield. The
total fruit number per plant has also been observed to
decrease under salinity at high electrical conductivity values
such as 9 dS m1 (Ieperen, 1996). Although, there are
comparatively salt-tolerant relatives of tomato, it has
proved difficult to enrich elite lines with genes from wild
species that confer tolerance because of the large number of
genes involved, most of them with small effects in comparison with the environment, and the high cost of recovering
the genetic background of the receptor cultivar. Recent
developments in transgenic technology have provided an
efficient tool for improving tomato, allowing economically
important genes to be introduced into a host plant.
However, there are no reports regarding improving the salt
tolerance of tomato by co-expression of the Na+/H+
antiporter and H+-pyrophosphatase.
The aim of this work was to investigate the possibility of coexpression of the Na+/H+ antiporter and H+-pyrophosphatase
to enhance salt tolerance in tomato. Plasmids co-expressing
PgNHX1 and AVP1 under the control of the Cauliflower
mosaic virus (CaMV) 35S promoter, which produces a high
level of gene expression in dicot plants, were initially
subcloned into a common binary vector backbone of pBI121
for better comparison of the transgenic plants. Tomato
plants overexpressing these plasmids were developed. For
the first time it is reported that the salt tolerance of the
transgenic tomato plants co-expressing PgNHX1 and AVP1
had been improved in comparison with the PgNHX1 or
AVP1 single gene transgenics and the wild-type.
Materials and methods
Vector construction
Plasmid pBI121/PgNHX1 construction: The Na+/H+ antiporter
gene from P. glaucum (PgNHX1) was digested with XhoI and
BamHI restriction enzymes from PCAMBIA1301/PgNHX1 and
Co-expression of PgNHX1 and AVP1 enhances salt tolerance in transgenic tomato | 5563
the resulting fragment was inserted into pRT100 between the
CaMV 35S promoter and the poly(A) site. The resulting plasmid
was named pRT100/PgNHX1. The positive clone was digested
with HindIII to release the PgNHX1 gene flanked on either side by
the CaMV 35S promoter and the terminator cassette, respectively.
This HindIII fragment was cloned at the HindIII site of pBI121
(Fig. 1A). The clone was named pBI121/PgNHX1 (Fig. 1B).
Plasmid pBI121/AVP1 construction: The H+-pyrophosphatase gene
from Arabidopsis (AVP1) was PCR amplified from the mini binary
vector pCB302/AVP1 using specific primers such that a XhoI site is
created at the 5’ end and an XbaI site is created at the 3’ end of the
amplified product. The eluted and purified fragment was then
digested with XhoI and XbaI and inserted into pRT100 between the
CaMV 35S promoter and the poly(A) site, and the resultant clone
was named pRT100/AVP1. The positive clone was digested with
HindIII to release the AVP1 gene flanked on either side by the
CaMV 35S promoter and the terminator cassette, respectively. This
HindIII fragment was cloned at the HindIII site of pBI121. The
clone was named pBI121/AVP1 (Fig. 1C).
Plasmid pBI121/AVP1+PgNHX1 construction: The AVP1 gene
flanked on either side by the CaMV 35S promoter and the
terminator cassette from pBI121/AVP1 was amplified using
specific primers such that a HindIII site is created at the 5’ end
and a NotI site is created at the 3’ end of the amplified product.
Similarly, the PgNHX1 gene flanked on either side by the CaMV
35S promoter and the terminator cassette from pBI121/PgNHX1
was amplified using specific primers such that a NotI site is created
at the 5’ end and a HindIII site is created at the 3’ end of the
amplified product. The PCR-amplified products thus obtained
were purified and digested with NotI and HindIII, and ligated to
HindIII-digested pBI121, and the resulting plasmid was named
pBI121/AVP1+ PgNHX1 (Fig. 1D).
The three plasmids constructed above were mobilized to
Agrobacterium tumefaciens strain LBA4404 and used for plant
transformation.
Tomato transformation
Transformation of tomato (S. lycopersicum L. cv. Pusa Ruby,
a salt-sensitive cultivar) cotyledon was carried out according to
McCormick et al. (1986) with minor modifications. The transformed explants and plantlets were screened on MS medium
(Murashige and Skoog, 1962) supplemented with 100 mg l1
kanamycin. The resistant shoots were separated and transferred to
MS medium supplemented with 0.5 mg l1 indole acetic acid
(IAA) for initiation of rooting. This was followed by hardening of
pot cultures.
PCR and Southern blot analysis
Putative transformed plants were screened by PCR analysis using
tomato genomic DNA from the wild-type and various transgenic
lines as template and PgNHX1 forward (5#-ATGGCTGTGTTCAGCAGGAC-3#) and reverse (5#-TCACCAAAAACATGTCTTCA-3#)
primers or AVP1 forward (5#-ATGGTGGCGCCTGCTTTGTTACCGG-3#) and reverse (5#-TTAGAAGTACTTGAAAAGGATACCA-3#) primers. For Southern blot hybridization, 10 lg of
genomic DNA from PCR-positive tomato lines was digested with
EcoRI and separated by electrophoresis on a 0.8% agarose gel, then
transferred to a nylon membrane and hybridized (Sambrook et al.,
1989) with the Alkphos-labelled PCR product of PgNHX1 or AVP1.
Fig. 1. Binary vector constructs used for tomato transformation. (A) pBI121 binary vector. This vector harbours neomycin
phosphotransferase II (NPTII) as the selectable marker and b-glucuronidase (GUS) as the reporter gene. (B) pBI121/PgNHX1 [PgNHX1
inserted between the CaMV 35S promoter and poly(A) at the HindIII site]. (C) pBI121/AVP1 [AVP1 inserted between the CaMV 35S
promoter and poly(A) at the HindIII site]. (D) pBI121/AVP1+PgNHX1 [both transgenes flanked by the CaMV 35S promoter and poly(A)
inserted at the HindIII site].
5564 | Bhaskaran and Savithramma
RT-PCR analysis
Total RNA was extracted from 200 mg of young leaves of tomato
transgenic lines using the RNeasy total RNA isolation kit (Qiagen,
Germany). Reverse transcription-PCR (RT-PCR) was performed using
total RNA pre-treated with RNase-free DNase I. A 5 lg aliquot of
total RNA from each sample was used in a 10 ml reverse transcription
reaction system with oligo(dT) as primer (Takara, Japan). A 1 ll
aliquot of the reverse transcription reaction mixture after the completion of cDNA synthesis was used as the template for PCR amplification
with a pair of gene-specific primers: 5#-AGAGTGTTGTCGCTAAGTG-3# and 5#-CAGTGAAGTCGTGGTTGAT-3# for AVP1
and 5#-ATCGAACTTGCGCCAGTAGT-3# and 5#-CAGCACAATCACTGTCGTCC-3# for PgNHX1. The actin gene fragment, used as
an internal control, was amplified with the primers: 5#-TGGGTCG
TCCCAGGCACACA-3# and 5#-ACCAGTGGTACGACCGCTAGCAT-3#.
Salt stress treatments
Seedling growth under salt stress was studied by subjecting the
uniformly grown tomato seedlings (36 h old) to NaCl (200 mM
and 300 mM) stress after induction for 16 h on 100 mM NaCl.
The seedlings were subjected to stress for 3 d followed by recovery
on half-strength MS medium without NaCl for 5 d. The measurements of seedling weight were recorded and expressed as a percentage reduction in comparison with the respective unstressed control.
To study the salt tolerance in vivo, different transgenic lines and
the wild-type were planted into plastic pots containing soil under
a photoperiod of 16/8 h, watered every 2 d with 1/8 Hoagland
nutrient solution for 4 weeks, and then the nutrient solution was
supplemented with NaCl. NaCl concentrations were incrementally
increased by 50 mM every 2 d until a final concentration of
200 mM was achieved. After salt treatment for 7 d, the plants
were used for physiological analysis. The stress treatment was
continued for 6 weeks.
Leaf senescence assay
The healthy and fully expanded youngest leaves from wild-type
and transgenic plants (45 d old) were briefly washed in deionized
water and 1 cm diameter leaf discs were finely cut and floated in
a 5 ml solution of NaCl (150, 300, and 600 mM, 96 h) or sterile
distilled water (which served as the experimental control) for the
leaf senescence assay (Fan et al., 1997). The effects of salt stress
treatment on leaf discs were assessed by observing phenotypic
changes and quantified by estimating their chlorophyll content
(Arnon, 1949).
Proline
Proline concentration was determined as described by Bates et al.
(1973). Fully expanded matured leaf segments were homogenized
with 3% sulphosalicylic acid and the homogenates were centrifuged
at 3000 g for 20 min. The supernatant was treated with acetic acid
and acid ninhydrin; after boiling for 1 h, the absorbance at 520 nm
was determined. Contents of proline were expressed as lM g1
fresh weight (FW).
Determination of ion content
Fully expanded matured leaves of transgenic and wild-type plants
were rinsed briefly in deionized water and tissues were dried in an
oven at 80 C for 24 h. The samples were digested in a concentrated solution of HNO3 and the reaction was stopped by the
addition of 2 N HCl. Samples were suitably diluted prior to
estimating the concentration using simultaneous inductively coupled argon-plasma emission spectrometry (ICP trace analyser,
Labtam, Australia).
Seed germination assay
The seeds from transgenic and wild-type plants were surface
sterilized. The sterilized seeds were cultured in half-strength MS
medium with and without addition of NaCl. The percentage
germination was recorded after 10 d.
Statistical analysis
The significance of salt treatment effects and the effects of the
transgenes were determined using analysis of variance (ANOVA).
Variation among treatment means was analysed using the LSD
(least significant difference) procedure at 5% probability.
Results
Production of transgenic tomato overexpressing
PgNHX1 and AVP1, and co- expressing PgNHX1 and
AVP1
The aim of the work was to investigate the effect of coexpression of the vacuolar Na+/H+ antiporter gene
PgNHX1 and the H+-pyrophosphatase AVP1 gene in
tomato. For this purpose, the full open reading frames of
these genes were cloned into the binary vector pBI121 under
the control of the CaMV 35S promoter and poly(A). Single
gene constructs of PgNHX1 and AVP1 under the control of
the CaMV 35S promoter were also made for better
comparison of transgenic plants.
Ten-days-old cotyledons from in vitro grown seedlings
were co-cultivated with Agrobacterium LBA4404 carrying
the recombinant plasmids pBI121/PgNHX1, pBI121/AVP1,
or pBI121/AVP1+PgNHX1. A total of 20, 26, and 15
independent PgNHX1, AVP1, and AVP1+PgNHX1 transgenic tomato lines, respectively, were obtained which were
transferred to pots and raised to maturity to obtain T1
seeds. Morphologically, no noticeable difference was observed in the transgenic plants compared with the wild-type
plants.
Molecular characterization of transgenic plants
overexpressing PgNHX1 and AVP1, and co-expressing
AVP1 and PgNHX1
PCR analysis confirmed the presence of transgenes in all 61
independent lines when total genomic DNAs from various
independent transformed lines were used as the template
with PgNHX1- or AVP1-specific primers (data not shown).
Five transgenic lines (lines N1–N5 for PgNHX1, lines A1–
A5 for AVP1, and lines AN1–AN5 for AVP1+PgNHX1)
for each construct were grown to the T2 generation and
used for further analysis.
Genomic Southern blot analysis using an Alkphoslabelled PgNHX1 or AVP1 probe also indicated the transgenic nature of these lines. However, no cross-hybridizing
band was seen in the wild-type plants (data not shown).
The expression level of transgenes was monitored by RTPCR performed on young leaves of the 15 transgenic lines
together with those of wild-type plants. As expected, the
lines N1–N5 and A1–A5 expressed PgNHX1 and AVP1,
respectively. Line N5 and N4 showed a slightly higher level
Co-expression of PgNHX1 and AVP1 enhances salt tolerance in transgenic tomato | 5565
of expression of PgNHX1 than the three other lines (Fig. 2A),
whereas lines A1–A5 seem to have similar steady levels of
AVP1 transcript (Fig. 2B). The RT-PCR for the lines AN1–
AN5 were performed in two separate experiments with
PgNHX1- and AVP1-specific primers. The lines AN4 and
AN1 showed a slightly higher expression level of PgNHX1,
whereas the expression level of AVP1 was similar (Fig. 2C)
in all the lines. Thus, the lines N5, N4, A1, A2, AN4, and
AN1 were chosen for further studies.
Co-expression of PgNHX1 and AVP1 confers greater
salt tolerance to transgenic tomato than expression of
only PgNHX1 or AVP1
The transgenic lines and wild-type plants were tested for salt
tolerance capacity. Three-day-old seedlings were tested for
seedling growth performance by subjecting the uniformly
grown seedlings to NaCl (200 mM and 300 mM) stress for
3 d. After 5 d of recovery, reduction in seedling weight was
calculated as the percentage reduction compared with the
respective unstressed control. Significant difference was
noticed among different transgenic lines and the wild-type.
The transgenic plants co-expressing PgNHX1 and AVP1
exhibit significantly lower reduction in seedling weight than
the plants transformed separately with PgNHX1 and AVP1.
At 200 mM NaCl, a reduction of 7.67% and 8.06% in
Fig. 2. Analysis by RT-PCR of (A) PgNHX1, (B) AVP1, and (C)
AVP1 and PgNHX1 expression in tomato transgenic lines. Specific
PCR products of 250 bp and of 765 bp were detected in five
PgNHX1 (lanes N1–N5) and five AVP1 (lanes A1–A5) transgenic
lines, respectively. Five transgenic lines co-expressing AVP1 and
PgNHX1 (lines AN1–AN5) showed the PCR product of both
PgNHX1 and AVP1. WT, wild-type. A 380 bp actin fragment was
amplified by RT-PCR as an internal control.
seedling weight was registered in AN4 and AN1, respectively,
whereas in single PgNHX1 and AVP1 transgenic lines the
seedling weight reduction was 20.19% and 22.93% (N5 and
N4, respectively) and 31.33% and 30.91% (A1 and A2,
respectively). At 300 mM NaCl, the decreases in the seedling
weight were 21.62% and 23.61% (AN4 and AN1, respectively), 34.57% and 38.07% (N5 and N4, respectively), and
45.86% and 48.37% (A1 and A2, respectively; Fig. 3).
All the transgenic lines grew well, flowered normally, and
set fruits and viable seeds even when watered with 200 mM
NaCl (Table 1). Although no significant difference was
noticed among different transgenic lines for plant height, fruits
per plant, and average fruit weight in non-stress conditions,
the values were reduced significantly in plants grown at
200 mM NaCl. The wild-type plants displayed severe chlorosis,
withered, and died after 3 weeks (Fig. 4). The percentage
germination of the seeds collected from stressed transgenic
plants was recorded. No significant difference in germination
was noticed between transgenic lines and the wild-type under
non-stress conditions, indicating that the viability of the
seeds was not affected during stress (Fig. 5).
Leaf senescence assay and proline content of
transgenic lines and the wild-type
The leaf disc senescence assays of wild-type versus transgenic plants was performed as a bioassay for estimation of
salt tolerance potential. Leaf discs of 1 cm diameter made
from wild-type and transgenic lines were floated on saline
solutions of different concentrations for 96 h to investigate
the effect of overexpression of PgNHX1 or AVP1 or coexpression of AVP1 and PgNHX1 in ameliorating the toxic
effect of NaCl. In these tests, generally a higher
Fig. 3. Response of transgenic tomato seedlings to salt stress.
Uniformly grown tomato seedlings (36 h old) were subjected to
NaCl stress for 3 d after an induction for 16 h on 100 mM NaCl.
The seedling weight during the recovery period was recorded and
expressed as the percentage reduction compared with the
respective unstressed control. Values are means 6SD (n¼5), and
vertical bars indicate the SD. In each plant, mean values followed
by different letters are significantly different according to the LSD
test (P <0.05). AN4, AN1, transgenic plants co-expressing
PgNHX1 and AVP1; N5, N4, transgenic plants overexpressing
PgNHX1; A1, A2, transgenic plants overexpressing AVP1; WT,
non-transgenic controls.
5566 | Bhaskaran and Savithramma
Table 1. Plant and fruit yield of wild-type and transgenic plants
grown in the presence or absence of 200 mM NaCl
Line
NaCl (mM)
Plant height
(cm)
Fruits per
plant
Fruit weight
(g)
AN4
0
200
0
200
0
200
0
200
0
200
0
200
0
200
46.062.1 a
41.862.9 a,b,c
45.265.1 a,b
40.863.7 b,c,d
44.862.3 a,b
35.263.4 e
42.265.0 a,b
33.863.7 e
46.462.1 a
36.264.5 d,e
43.265.9 a,b
37.463.5 c,d,e
44.664.3 a,b
–
44.861.9 a
43.862.6 a
43.264.4 a
42.064.6 a,b
44.662.4 a
37.663.2 b,c,d
40.667.1 a,b,c
36.664.4 c,d
45.461.5a
36.863.7 c,d
43.664.2 a
35.464.3 d
45.264.1 a
–
45.661.7 a
40.56 1.5 b,c
43.162.4 a,b,c
39.561.4 c
42.863.2 a,b,c
30.761.9 d
43.362.7 a,b
31.563.4 d
41.063.9 b,c
29.763.6 d
43.163.1 a,b,c
29.064.6 d
43.364.1 a,b
–
AN1
N5
N4
A1
A2
WT
Values are means 6S.D (n¼5). In each plant, mean values followed by
different letters are significantly different according to the LSD test
(P <0.05). AN4, AN1, transgenic plants co-expressing PgNHX1 and
AVP1; N5, N4, transgenic plants overexpressing PgNHX1; A1, A2,
transgenic plants overexpressing AVP1; WT, non-transgenic controls.
Fig. 4. Determination of salt tolerance at the whole-plant level.
The different transgenic lines and the wild-type were watered with
200 mM NaCl. Wild-type plants show severe chlorosis caused by
sodium toxicity, whereas the transgenic plants display normal
growth.
concentration of NaCl is used to see the results in a shorter
period of time and these concentrations do not reflect the
tolerance limits of plants in soil conditions. The transgenic
line co-expressing AVP1 and PgNHX1 showed a clear
advantage in overcoming the deleterious effect elicited by
NaCl toxicity in a concentration-dependent manner.
Biochemical investigations for the estimation of chlorophyll
indicated that under high salinity conditions, significant
differences in chlorophyll content between the different
transgenic lines and the wild-type were also noticed at
different concentrations of NaCl. On average, the transgenic lines co-expressing PgNHX1 and AVP1 could retain
as much as 66.80% chlorophyll as compared with the non-stress
Fig. 5. Effect of salt stress on seed germination. Values are
means 6SD (n¼5), and vertical bars indicate the SD. In each
plant, mean values followed by different letters are significantly
different according to the LSD test (P <0.05). AN4, AN1, transgenic plants co-expressing PgNHX1 and AVP1; N5, N4, transgenic plants overexpressing PgNHX1; A1, A2, transgenic plants
overexpressing AVP1; WT, non-transgenic controls.
conditions. This is in contrast to only 59.20%, 53.07%, and
28.07% chlorophyll retention by the PgNHX1- and AVP1overexpressing plants, and the wild-type, respectively, under
similar conditions (Fig. 6). This documented the usefulness
of co-expression of PgNHX1 and AVP1 over single gene
transgenics and the wild-type to survive under toxic NaCl
levels.
Furthermore, it was observed that transgenic lines produced more proline than the wild-type under normal
conditions. During salt stress, the amounts of proline were
dramatically enhanced in all transgenic plants and in the
wild-type (Fig. 7). On average, the transgenic plants coexpressing AVP1 and PgNHX1 when stressed with 200 mM
NaCl displayed a 2.33-fold increase in proline content
which was significantly higher than the single PgNHX1 and
AVP1 transgenics which showed 1.91- and 1.67-fold
increases in their proline content.
The transgenic lines co-expressing PgNHX1 and AVP1
accumulate more Na+ and less K+ than PgNHX1 and
AVP1 single gene transgenics
At 200 mM NaCl, there was significantly more Na+ in all
transgenic lines than in the wild-type. However, no significant differences were seen among transgenics and
wild-type plants in the content of Na+ under non-stress
conditions (Fig. 8A). At 200 mM NaCl, the Na+ concentration in leaves of the transgenic line co-expressing AVP1 and
PgNHX1 was 1.19 mg 100 mg1 DW, while it was only
0.782 mg 100 mg1 DW, 0.765 mg 100 mg1 DW, and
0.353 mg 100 mg1 DW, respectively in PgNHX1 and
AVP1 transgenic lines and wild-type plants. This indicated
that the transgenic lines absorbed more Na+ than the wildtype in the same conditions. Despite showing a high Na+
content in leaves when the transgenic plants were stressed
with 200 mM NaCl, these plants were able to grow, flower,
and set seeds. These results clearly demonstrated that
overexpression of the vacuolar Na+/H+ antiporter or
H+-pyrophosphatase and co-expression of the Na+/H +
Co-expression of PgNHX1 and AVP1 enhances salt tolerance in transgenic tomato | 5567
Fig. 6. Retardation of salt stress-promoted senescence in detached leaves of transgenic tomato co-expressing AVP1 and
PgNHX1 compared with single gene transgenics of PgNHX1 or
AVP1 and the wild-type indicating the tolerance at the cellular level
towards toxic levels of salt. Chlorophyll content (lg g1 FW) from
NaCl-treated leaf segments of wild-type and transgenic plants
after incubation in 150, 300, and 600 mM NaCl for 96 h is shown.
Leaf segments floated in water served as the experimental control.
Values are means 6SD (n¼5), and vertical bars indicate the SD.
In each plant, mean values followed by different letters are
significantly different according to the LSD test (P <0.05). AN4,
AN1, transgenic plants co-expressing PgNHX1 and AVP1; N5, N4,
transgenic plants overexpressing PgNHX1; A1, A2, transgenic
plants overexpressing AVP1; WT, non-transgenic controls
Fig. 8. Na+ (A) and K+ (B) concentration in leaves of transgenic
and wild-type tomato in the presence or absence of 200 mM
NaCl. The transgenic lines were tested for ion content by atomic
ICP trace analyser, and values are indicated as mg 100 mg1 DW.
Values are means 6SD (n¼5), and vertical bars indicate the SD. In
each plant, mean values followed by different letters are significantly different according to the LSD test (P <0.05). AN4, AN1,
transgenic plants co-expressing PgNHX1 and AVP1; N5, N4,
transgenic plants overexpressing PgNHX1; A1, A2, transgenic
plants overexpressing AVP1; WT, non-transgenic controls.
the growth of the transgenic plants was not significantly
affected by high salinity, suggesting that K+ nutrition was
not compromised in the transgenic plants.
Fig. 7. Proline content in leaves of transgenic and wild-type plants
in the presence or absence of 200 mM NaCl. Three independent
transgenic lines were tested for proline content by a colorimetric
method and the values are indicated as lM g1 FW. Values are
means 6SD (n¼5), and vertical bars indicate the SD. In each
plant, mean values followed by different letters are significantly
different according to the LSD test (P <0.05). AN4, AN1, transgenic plants co-expressing PgNHX1 and AVP1; N5, N4, transgenic plants overexpressing PgNHX1; A1, A2, transgenic plants
overexpressing AVP1; WT, non-transgenic controls
antiporter and H+-pyrophosphatase could enhance the
accumulation of Na in vacuoles. The K+ content in leaves
of transgenic lines and wild-type plants decreased as the
NaCl concentration increased (Fig. 8B). At the same NaCl
concentration, the leaves of the wild-type plants contained
significantly more K+ than those of transgenic lines. Under
high salinity conditions, Na+ may displace K+ from its carrier
binding sites and this competition results in impaired K+
uptake and lower K+ cytosolic concentrations. Nevertheless,
Discussion
In the present work it was shown that co-expression of
P. glaucum vacuolar Na+/H+ antiporter (PgNHX1) and
Arabidopsis H+-pyrophosphatase (AVP1) in transgenic
tomato resulted in enhanced salt tolerance compared with
the PgNHX1 or AVP1 single gene transgenics. wild-type
tomato displayed growth inhibition, chlorosis, and even
death when treated with high salt concentrations, whereas
the transgenic plants exhibit normal development and
survival. This can be attributed to ion homeostasis due to
overexpression of PgNHX1 and AVP1. This conclusion is
supported by the measurements of ion accumulation; the
transgenic plants accumulated more Na+ and less K+ in
their leaf tissue. The increased accumulation of Na+ is likely
to be a consequence of the activity of the vacuolar Na+/H+
antiporter in PgNHX1 transgenics. The enhanced tolerance
to salinity in AVP1 transgenic plants might be ascribed to
5568 | Bhaskaran and Savithramma
the increased sequestration of Na+ into the vacuole and
enhanced vacuolar osmoregulatory capacity, because of the
overexpression of H+-pyrophosphatase. Presumably, the
greater AVP1 activity provides increased H+ to drive
the secondary active uptake of cations into the lumen of the
vacuole and any toxic effects intrinsic to Na+ are mitigated
by this sequestration into the vacuole. The transgenic line
co-expressing AVP1 and PgNHX1 accumulated more Na+
compared with AVP1 or PgNHX1 single gene transgenics.
The increased accumulation of sodium is likely to be
a consequence of the enhanced transport efficiency of
the vacuolar Na+/H+ antiporter in the presence of increased proton supply resulting from the overexpression of
H+-pyrophosphatase (Gaxiola et al., 2001). These results
corroborate earlier findings of salinity tolerance by coexpressing SsNHX1 and AVP1 in rice (Zhao et al., 2006).
The transgenic lines co-expressing PgNHX1 and AVP1
accumulated a markedly lower level of K+ in their leaves
under severe saline conditions (200 mM NaCl) as compared
with the PgNHX1 and AVP1 single gene transgenics and
the wild-type. Significant reduction in K+ during salt stress
has been reported on overexpression of AtNHX1 in mustard
greens, tomato, and buck wheat (Zhang and Blumwald,
2001; Zhang et al., 2001; Chen et al., 2008) and of PgNHX1
in mustard greens (Rajgopal et al., 2007). This is likely to be
the result of increased Na+ import, because the rate of K+
transport can be affected by Na+ levels through its
competition for K+-binding sites of transport proteins
which function in acquisition of K+ (Blumwald, 2000).
Nevertheless, the growth of the transgenic plants was not
significantly affected by high salinity, suggesting that K+
nutrition was not compromised in the transgenic plants.
A significant increase in proline was observed in the
transgenic lines co-expressing PgNHX1 and AVP1 in
comparison with single gene PgNHX1 and AVP1 transgenics, and the wild-type upon imposition of salt stress. The
accumulation of proline in response to high salinity is well
documented. Many prokaryotic and eukaryotic organisms
accumulate proline during osmotic and salt stress (Schobert,
1977; Czonka and Hanson, 1991). Proline contributes to
osmotic adjustment and the protection of macromolecules
during dehydration (Yancey et al., 1982) and as a hydroxyl
radical scavenger (Smirnoff and Cumbes, 1989). Evidence
supporting the role of proline during salt stress was
obtained based on salt tolerance in transgenic tobacco
plants with enhanced levels of proline biosynthesis (Kishor
et al., 1995) and salt tolerance of Arabidopsis with suppressed levels of proline degradation (Nanjo et al., 1999).
An increase in proline content during salt stress has also
been reported in transgenic tomato and buck wheat overexpressing AtNHX1 (Zhang and Blumwald, 2001; Chen
et al., 2008). In the present study, the transgenic lines were
found to accumulate more proline than the wild-type even
in non-stress conditions. The cellular content of free proline
increases in most plants in response to osmotic stresses.
However, the regulatory modes for such proline accumulation differ amongst various plants due to variations in
transcript and protein levels of the proline cycle enzymes
and diversity in feedback inhibition of P5CS, the key
enzyme of the route of proline synthesis. The ectopically
expressed genes may have a role in signalling, especially in
the up-regulation of the proline biosynthetic pathway.
However, this has to be investigated further. It may also be
thought that overexpression of these genes under non-stress
conditions protects the plants from subsequent stress. These
results may suggest that transgenic tomato plants possess an
inherent resistance to salinity conditions, which is much
higher than that of the wild-type.
Leaf disc senescence assays of wild-type and transgenic
plants were performed as a bioassay for estimation of salt
tolerance potential. The lines co-expressing PgNHX1 and
AVP1 showed a clear advantage in overcoming the deleterious effect of NaCl toxicity in a concentration-dependent
manner compared with the single gene PgNHX1 and AVP1
transgenics. The wild-type plants showed extensive bleaching, reflecting symptoms of injury due to stress, while the
transgenic lines did not appear to be affected under similar
conditions. Biochemical investigations for the estimation of
chlorophyll, which was taken as an index of the damage
done to the photosynthetic apparatus under stress, indicated that under high salinity conditions the lines coexpressing PgNHX1 and AVP1 exhibited a higher salt
tolerance potential. The leaf disc senescence assay provides
a useful index for estimating salt tolerance potential;
however, the interpretations are limited due to the isolated
nature of the system. Also the response could result from
continuous transport of a high concentration of salt over
a long time inside the leaf tissue, resulting in the death of
the tissue (Munns, 2005). In order to check this and to
complement the results obtained by leaf disc assay, the
efficacy of PgNHX1 or AVP1 overexpression and coexpression of AVP1 and PgNHX1 in transgenic tomato
was evaluated by studies at the whole-plant level. It was
found that all three transgenic plants were able to grow
well, flowered normally, and set viable seeds even when
watered with 200 mM NaCl. Owing to the economic
importance of the tomato crop, the effect of constitutive
overexpression of the transgenes on the growth of transgenic tomato was tested. The tested plant characteristics
were not significantly different between transgenic and wildtype plants. During salt stress, a slight reduction in fruits
per plant and average fruit weight was noted in transgenic
plants, and wild-type plants died before setting fruits.
Taken together, the data clearly demonstrate that coexpression of AVP1 and PgNHX1 improved the osmoregulatory capacity of double transgenic lines by enhanced
sequestration of ions into the vacuole by increasing the availability of protons and thus alleviating the toxic effect of Na+.
Adopting a similar strategy could be one way of developing
transgenic staple crops with improved tolerance to salt stress.
Acknowledgements
We thank Dr R. A. Gaxiola (Arizona State University, USA)
for kindly donating plasmids, which carry the AVP1 gene,
Co-expression of PgNHX1 and AVP1 enhances salt tolerance in transgenic tomato | 5569
and Dr M. K. Reddy (ICGEB, New Delhi, India) for kindly
providing the plasmids which carry the PgNHX1 gene.
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