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JXB Advance Access published online on January 17, 2007
Journal of Experimental Botany, doi:10.1093/jxb/erl251
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© The Author [2007]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Integrated Approaches to Sustain and Improve Plant Production under Drought Stress Special Issue

Overexpression of wheat Na+/H+ antiporter TNHX1 and H+-pyrophosphatase TVP1 improve salt- and drought-stress tolerance in Arabidopsis thaliana plants

Faïçal Brini1, Moez Hanin1, Imed Mezghani1, Gerald A. Berkowitz2 and Khaled Masmoudi1,*

1Plant Molecular Genetics Unit, Centre of Biotechnology of Sfax (CBS), B.P'K', 3038 Sfax, Tunisia
2Department of Plant Science, Agricultural Biotechnology Laboratory, University of Connecticut, 1390 Storrs Road, Storrs, CT 06269-4163, USA

* To whom correspondence should be addressed. E-mail: khaled.masmoudi{at}cbs.rnrt.tn

Received 30 December 2005; Accepted 18 October 2006


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Transgenic Arabidopsis plants overexpressing the wheat vacuolar Na+/H+ antiporter TNHX1 and H+-PPase TVP1 are much more resistant to high concentrations of NaCl and to water deprivation than the wild-type strains. These transgenic plants grow well in the presence of 200 mM NaCl and also under a water-deprivation regime, while wild-type plants exhibit chlorosis and growth inhibition. Leaf area decreased much more in wild-type than in transgenic plants subjected to salt or drought stress. The leaf water potential was less negative for wild-type than for transgenic plants. This could be due to an enhanced osmotic adjustment in the transgenic plants. Moreover, these transgenic plants accumulate more Na+ and K+ in their leaf tissue than the wild-type plants. The toxic effect of Na+ accumulation in the cytosol is reduced by its sequestration into the vacuole. The rate of water loss under drought or salt stress was higher in wild-type than transgenic plants. Increased vacuolar solute accumulation and water retention could confer the phenotype of salt and drought tolerance of the transgenic plants. Overexpression of the isolated genes from wheat in Arabidopsis thaliana plants is worthwhile to elucidate the contribution of these proteins to the tolerance mechanism to salt and drought. Adopting a similar strategy could be one way of developing transgenic staple crops with improved tolerance to these important abiotic stresses.

Key words: H+-pyrophosphatase, Na+/H+ antiporter, salt and drought tolerance, sodium sequestration, transgenic Arabidopsis plants


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Drought and salinity are major constraints on crop production and food security, and adversely impact the socio-economic fabric of many developing countries. Water scarcity, declining water quality for irrigation, and soil salinity are problems which are becoming more acute (Ghassemi et al., 1995; Flowers, 2004). It is estimated that 20% of all cultivated land and nearly half of irrigated land is affected by salt, greatly reducing the yield of crops to well below their genetic potential (van Schilfgaarde, 1994; Munns, 2002; Flowers, 2004). There is limited evidence at present that new strategies to enhance crop yield stability on saline soils, based on remediation of salinized soils, are feasible (Tester and Davenport, 2003). Increasing salt tolerance of crop plants either by genetic introgression or use of transgenic technology for gene transfer is a realistic strategy (Hasegawa et al., 2000; Koyama et al., 2001; Borsani et al., 2003; Flowers, 2004). Salinity-stress effects on crop growth are manifested by impairment of photosynthetic capacity. High amounts of sodium in the soil solution impair cell metabolism and photosynthesis by imposing an osmotic stress on cell water relations and by increasing the toxicity of sodium in the cytosol. When challenged with salinity stress, one way in which plants can adapt is through a system that allows cellular sodium to be used for osmotic adjustment. Adaptation of plants to salt stress (i.e. resumption of growth after exposure to high soil salinity) requires cellular ion homeostasis involving net intracellular Na+ and Cl– uptake and subsequent vacuolar compartmentalization without toxic ion accumulation in the cytosol (Niu et al., 1995; Blumwald et al., 2000; Hasegawa et al., 2000; Gaxiola et al., 2001). The capacity for vacuolar compartmentalization of Na+ and Cl– is an adaptation mechanism conserved in halophytes and glycophytes (Blumwald et al., 2000; Hasegawa et al., 2000); however, the process is more efficient in halophytes. Vacuolar partitioning of Na+ and Cl– contributes to the maintenance of cellular water status. Together with K+ and organic solute accumulation in the cytosol and organelles, Na+ and Cl– sequestration in the vacuole balances the intracellular osmotic status of cells in plants grown in salt (Rhodes and Hanson, 1993). Cellular ion exclusion cannot provide complete adaptation of plants to high soil salinity, presumably because of the osmotic stress component of salinity stress (Blumwald et al., 2000). Continued cell growth would be restricted under osmotic stress because of an unfavourable water balance, limiting the water uptake necessary for cell expansion.

Energy-dependent Na+ transport (i.e. against a concentration gradient) across plant cell membranes (plasmalemma and tonoplast) is usually coupled to the proton (H+) electrochemical potential established by H+-translocating pumps (Blumwald et al., 2000; Hasegawa et al., 2000; Shi et al., 2000; Gaxiola et al., 2002). H+ transport across these membranes increases with salt treatment and may be attributed both to pump activation and enhanced transcription (Hasegawa et al., 2000). Plasma membrane and tonoplast transporters facilitate Na+ efflux from the cytosol by coupling Na+ transport to the (energetically favourable) transport of H+. Both plasma membrane and tonoplast Na+/H+ antiporter activities increase in response to salt treatment, at least in halophytic species (Blumwald et al., 2000). Overexpression of the vacuolar Na+/H+ antiporter and H+-pyrophosphatase pump (H+-PPase) has resulted in enhanced plant tolerance to both salinity (Apse et al., 1999; Zhang and Blumwald, 2001) and drought stress (Gaxiola et al., 2001; Park et al., 2005). These results suggest that the enhanced vacuolar H+-pumping in the transgenic plants provided additional driving force for vacuolar sodium accumulation via the vacuolar Na+/H+ antiporter. Recently, Li et al. (2005) showed that overexpression of the Arabidopsis H+-PPase results in increased cell division at the onset of organ formation, hyperplasia, and increased auxin transport.

As salinity stress negatively affects survival, growth, and development of crop plants, owing to irrigation practices and increasing demands on fresh water supply, engineering of salt-tolerant crop plants could provide an acceptable solution to the reclamation of farmlands lost to agriculture because of salinity and lack of rainfall. In this study, it is shown that transgenic Arabidopsis thaliana plants overexpressing one of the two wheat cDNAs encoding the tonoplast H+-PPase (TVP1) or the Na+/H+ antiporter (TNHX1) are much more resistant to high concentrations of NaCl and to water deprivation than the isogenic wild-type strains. These transgenic plants accumulate more Na+ and K+ in their leaf tissue than the wild type.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Plant material, NaCl- and drought-stress treatment, and Na+ and K+ content determination
Seeds of wild-type and transgenic Arabidopsis thaliana (ecotype Columbia) were allowed to germinate in sterile culture on Murashige and Skoog (MS) agar medium (Murashige and Skoog, 1962) under light/dark cycle conditions of 16/8 h at 22 °C. After 10 d, young seedlings were transplanted to a new medium supplemented with 50, 100, 150, and 200 mM NaCl. In the greenhouse, plants were grown in pots containing compost soil (leaf-mould:stable-litter:sand; 1:1:1) for 4–5 weeks, with a light/dark cycle of 16/8 h at 25 °C and 60–70% relative humidity. For salt-stress treatments, the water solution was supplemented with NaCl to a final concentration of 200 mM and plants were placed in a container with capillarity uptake for 3 d. Leaves from the rosette were excised carefully to determine their Na+ and K+ content. After 24 h at 105 °C, the dry weight was measured. The Na+ and K+ contents were extracted with 0.5 N HNO3. After centrifugation to remove debris, the supernatants were analysed by atomic absorption. For drought-stress treatment, 3-week-old plants were transferred to a growth chamber at 25 °C with no further addition of water for 10 d.

Generation of transgenic Arabidopsis plants
The full-length TNHX1 and TVP1 open reading frames (ORFs) were amplified with PfuTurbo DNA polymerase (Stratagene, La Jolla, CA, USA) using wheat cDNA library as template and primers corresponding to the 5' and 3' ends of the wheat genes TNHX1 and TVP1 with added BamHI and EcoRI restriction sites. These oligonucleotide primers were: 5'-CGGGATCCGGCATGGGGCTCGATTT-3' and 5'-GAACGAGATTAATT-TACAGA-3' for the TNHX1 ORF; 5'-CGGGATCCGGCATGGCGATCCTCGGG-3' and 5'-GAATTCCTTCTAGATGTACTTGAACAG-3' for the TVP1 ORFs. The TNHX1 and TVP1 ORF were cloned separately into the BamHI and EcoRI sites of the pCB302.2 plasmid (Xiang et al., 1999). This binary vector contains a tandem repeat of the cauliflower mosaic virus (CaMV) 35S promoter, the 35S terminator, and the BAR gene for resistance to the herbicide Basta as a selectable marker, between the NOS promoter and terminator. Agrobacterium-mediated transformation was performed via the floral dipping technique of Arabidopsis thaliana (ecotype Columbia) (Clough and Bent, 1998). Transgenic plants for TNHX1 and TVP1 were selected by spraying the herbicide Basta (40 mg l–1) on the seedlings three times at 3 or 4 d intervals. Plants were subsequently selected for two further generations to identify transgenic plants homozygous for the transgene.

RNA extraction and RT-PCR
Total RNA was extracted from 200 mg of young leaves of Arabidopsis transgenic lines using the RNeasy total RNA isolation kit (Qiagen). To remove contaminating DNA, RNAs (10 µg) were treated with RNase-free DNase (Promega). DNase-treated RNA samples (0.5 µg) were reverse-transcribed using M-MLV reverse transcriptase (Invitogen). The reverse transcription (RT) reactions were performed at 37 °C for 1 h using 2 µM oligo-dT18. Two microlitres of first strand cDNAs were used as templates for PCR amplification with a pair of gene-specific primers: 5'-AACGAGATTAATTTACAG-3' and 5'-CACAATCATTGTTCTGTTCT-3' for TNHX1; 5'-AAGAGTTGGTAGAACTCTCG-3' and 5'-GTTCTTTACATCACCATC-3' for TVP1. An Arabidopsis Actin gene fragment, used as an internal control, was amplified with the primers: 5'-GGCGATGAAGCTCAATCCAAACG-3' and 5'-GGTCACGACCAGCAAGATCAAGACG-3'. Samples were denatured for 5 min at 94 °C and then run for 35 cycles of 1 min each at 94 °C and 55 °C, 1 min 30 s at 72 °C with a final extension of 5 min at 72 °C. The PCR products were separated by agarose gel electrophoresis.

Water potential measurement
Leaf thermocouple psychrometers were used to determine water potential of leaves. Leaf discs (5 mm) from fully watered and salt- and drought-stressed wild-type or transgenic Arabidopsis thaliana plants were used with a Wescor PsyPro vapour pressure psychrometer and C-52 sample chambers.

Relative water content (RWC) measurement
Leaves from the rosette stage of plants were excised and their fresh weight was taken immediately. After floating leaves in deionized water at 4 °C overnight, their rehydrated weight was determined. Finally, they were dried in an oven at 70 °C overnight and weighed. The RWC was calculated as follows: RWC=(fresh weight–dry weight)/(rehydrated weight–dry weight).

Leaf surface determination
UTHSCSA image tool is a free image processing and analysis program. It can acquire, display, edit, and analyse images (http://ddsdx.uthscsa.edu/dig/itdesc.html). Total leaf area of Arabidopsis seedlings in plates was calculated in square millimetres using the image tool program. Photographs of plants were taken after 10 d of salt-stress treatment.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Molecular characterization of Arabidopsis transgenic lines overexpressing the wheat vacuolar THNX1 and TVP1 genes
The wheat vacuolar TNHX1 and TVP1 transporters (GenBank accession nos AY296910 and AY296911 for TNHX1 and TVP1, respectively) were previously characterized in yeast through complementation experiments in nhx1 and ena1 mutants (Brini et al., 2005). The aim was to investigate the overexpression effect of these genes in Arabidopsis. For this purpose, full ORFs were cloned in the binary vector pCB302.2 under the control of the double promoter (P2x35S) and terminator (T35S) (Fig. 1A). After Agrobacterium-mediated transformation of Arabidopsis plants and selection with the herbicide Basta, several transformants were produced. Four transgenic lines (lines 1–4 for TNHX1 and lines 5–8 for TVP1) for each construct were grown up to the T3 generation from which homozygous plants were isolated for further analysis. The expression level of both transgenes has been monitored by RT-PCR performed on young leaves of the eight transgenic lines together with those of control plants (wild-type and plants transformed with the empty vector pCB302.2). As expected, the lines 1–4 and 5–8 express TNHX1 and TVP1, respectively. Line 1 shows a slightly higher level of expression of TNHX1 than the three other lines (Fig. 1B), whereas, lines 5–8 seem to have similar steady levels of TVP1 transcript (Fig. 1C). Line 1 (now called TNHX1) and line 6 (now called TVP1) were then chosen for physiological studies. Genetic analysis of the two lines shows that the BAR marker gene segregates at the 3:1 ratio suggesting that they carry single inserts as confirmed by Southern blot analysis (data not shown).


Figure 1
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  		Fig. 1 Analysis of the transgenic TNHX1 and TVP1 lines. (A) Physical map of pTNHX1 and pTVP1 constructs. Both transgenes were inserted between the CaMV double promoter (P2x35S) and terminator (T35S). The BAR marker is flanked by the nopaline synthase promoter (PNOS) and terminator (TNOS). Analysis by RT-PCR of TNHX1 (B) and TVP1 (C) expression in Arabidopsis transgenic lines. Specific PCR products of 0.5 kb and of 1.2 kb were detected in four TNHX1 (lanes 1–4) and four TVP1 (lanes 5–8) transgenic lines, respectively. (–), H2O; Wt, wild type; (–RT), without reverse transcriptase. A 380 bp Actin fragment was amplified by RT-PCR as an internal control.

 
Increased resistance of TNHX1- and TVP1- expressing Arabidopsis seedlings under salt- and drought-stress conditions in vitro and in vivo
The homozygous transgenic lines TNHX1 and TVP1 were tested for salt- and drought-stress tolerance. Ten days after germination, Arabidopsis seedlings were transferred to MS medium containing 50–200 mM NaCl, and plant survival was monitored. The transgenic plants overexpressing TNHX1 and TVP1 are much more salt tolerant than wild-type or transgenic plants transformed with empty plasmid (Fig. 2A). Plants from TNHX1 and TVP1 transgenic lines continue to grow well in the presence of 100–200 mM NaCl, whereas wild-type plants and the empty plasmid transgenic line (pCB) exhibit chlorosis and die after 10 d of salt-stress treatment. The toxic effects of 150 mM NaCl, with inhibition of growth and development of chlorotic leaves after 10 d in control plants, were delayed and attenuated in the transgenic plants. In plants grown on soil under salt stress (200 mM NaCl) or drought (10 d of water deprivation), controls showed growth reduction and exhibited chlorosis, whereas the transgenic lines survived and continued normal growth (Fig. 2B). After 2 weeks of stress treatment, control plants died, whereas the other transgenic plants continued to grow normally. The total leaf area (TLA) decreased much more in wild-type plants than in transgenic plants subjected to salt stress (Fig. 3). In the absence of salt, similar TLA values were scored in transgenic and wild-type plants ({approx}6.8–6.9 mm2). These values decrease proportionally with increasing NaCl concentrations. At 50 mM NaCl, a reduction of {approx}34% in the TLA value was registered in wild-type plants, whereas in TNHX1 and TVP1 transgenic lines the TLA reduction was 15% and 23%, respectively. At 200 mM NaCl, the decrease in the TLA values was {approx}70%, 40%, and 48% in wild-type, TNHX1, and TVP1 transgenic plants, respectively. The ability of the transgenic plants (TNHX1 and TVP1) to survive high salt-stress conditions and continue to grow is consistent with the possibility that expression of these vacuolar transport proteins in the transgenic plants resulted in increased sodium sequestration into the vacuole (and pre-vacuolar compartments).


Figure 2
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  		Fig. 2 Effect of stress conditions on TNHX1 and TVP1 transgenic Arabidopsis plants cultured in vitro and in vivo. (A) Arabidopsis plants grown in standard culture medium for 1 week (the first two true leaves had already emerged) were transferred to culture plates containing 50 mM, 100 mM, 150 mM, or 200 mM NaCl. The photograph was taken 10 d after transplantation, when the transgenic salt-stressed seedlings showed a higher survival rate compared with the controls (PCB and Wt). (B) Wild-type plants and transgenic lines were grown on soil in standard culture conditions (16 h light/8 h dark cycle at 24 °C). Plants were watered continuously by capillarity for 3 weeks. The photograph corresponds to plants at day 10 of water-deficit stress and to plants after 3 d of continuous salt treatment (200 mM NaCl).

 

Figure 3
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  		Fig. 3 Total leaf area of individual leaves from wild-type and transgenic Arabidopsis plants. Values are means of five replicates of one expanded leaf per plant. White columns depict wild-type plants, grey columns depict TVP1 plants, and black columns represent TNHX1 plants. At 50 mM NaCl, P=4.5E-07 for wild type, P=2.4E-05 for TVP1, and P=1.02E-06 for TNHX1 plants. At 200 mM NaCl, P=0.12 for TVP1, and P=0.07 for TNHX1 plants.

 
Water potential, RWC, and water loss rate of TNHX1 and TVP1 transgenic plants
The leaf water potential ({Psi}w) of wild-type and transgenic plants overexpressing TNHX1 and TVP1 was determined. When the plants were continuously irrigated, values of the {Psi}w were less negative for wild-type plants (–0.865 MPa, SD=0.06) and transgenic plants transformed with empty plasmid (–0.89 MPa, SD=0.05) than the values for transgenic lines TNHX1 (–1.08 MPa, SD=0.05) and TVP1 (–1.245 MPa, SD=0.075) (Fig. 4A). When challenged with salt stress (200 mM NaCl), the leaf {Psi}w was lowered in control and transgenic plants. However, the {Psi}w was much more negative in transgenic lines than in control plants (Fig. 4B). Under drought stress, the {Psi}w decline was greater in transgenic plants than in control plants (Fig. 4C). This could be explained by an enhanced osmotic adjustment in the transgenic plants. Osmotic adjustment lowers leaf {Psi}w, leading to an increase in the leaf:soil {Psi}w gradient which is the driving force for water uptake into the plant. This leads to greater water uptake at low soil solution {Psi}w (i.e. salinization of the soil solution lowers soil solution {Psi}w) which can occur due to salinization.


Figure 4
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  		Fig. 4 Water potential for wild-type and transgenic plants. (A) Water potential of fully hydrated leaves from fully watered plants (control). (B) Water potential of leaves from salt-stressed plants (200 mM NaCl, 3 d). (C) Water potential of leaves from drought-stressed plants (10 d of water deprivation). White columns depict wild-type water potential values, dotted columns represent pCB lines, black columns represent TNHX1 lines, and grey columns represent TVP1 lines. Values are means ±standard deviation (n=5).

 
Transgenic and wild-type plants also show a significant reduction in their RWC during water stress. After 7 d of water-deficit stress, a higher water loss rate was observed in wild-type plants (RWC dropped to a value of 0.52), whereas the RWC of the transgenic plants decreased slightly to a value of 0.84 (for TNHX1) and 0.8 (for TVP1) (Fig. 5). At day 9, the RWC of wild-type plants continued to decrease to a value of 0.45, whereas the RWC of the transgenic plants stabilized at 0.7. The maintenance of greater RWC in the transgenic plants is also consistent with the possibility that expression of TNHX1 and TVP1 led to enhanced capacity for osmotic adjustment, altering the relationship between water content and {Psi}w.


Figure 5
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  		Fig. 5 Relative water content of wild-type and transgenic plants under a water-deficit stress. Ten wild-type plants and 10 of each transgenic line were grown and stressed as described in the Materials and methods. The RWC of wild-type (open circles) and transgenic plants TVP1 (closed triangles) and TNHX1 (open squares) is shown. One leaf per plant (three plants of each group) was removed from the seedlings for analysis. Values are means ±standard deviation (n=4 for each).

 
Transgenic TNHX1 and TVP1 plants retain more cations than wild-type plants
The Na+ and K+ contents from leaves of wild-type and transgenic plants grown in the presence of 200 mM NaCl were analysed. The Na+ content was much higher in the transgenic lines than in wild-type plants (Fig. 6A). The K+ content of transgenic plants was also higher than in the wild-type plants (Fig. 6B). TNHX1 plants exhibit the highest Na+ content. This could be explained by sodium sequestration in the vacuole. The resulting elevated vacuole solute content would confer greater water retention ({Psi}w=–1.85 MPa), permitting plants to survive under conditions of low soil water potential. Overexpresssion of TNHX1 and TVP1 improved the salt tolerance of transgenic Arabidopsis cells. Plants from the transgenic lines grow well in the presence of up to 200 mM NaCl, whereas wild-type plants grow poorly and exhibit chlorosis. This improvement in salt tolerance was correlated with an increase in the Na+ content of Arabidopsis cells grown in a medium with a high NaCl concentration. These results indicate that TNHX1 acts in concert with the vacuolar H+-PPase TVP1 (and ATPase) to sequester cations in the vacuole (and pre-vacuolar compartments) and resulted in the increased tolerance of the transgenic plants to a high NaCl concentration.


Figure 6
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  		Fig. 6 Na+ (A) and K+ (B) content from leaves of wild-type and transgenic plants grown in the presence of 200 mM NaCl. White columns correspond to wild-type plants, grey columns correspond to TVP1 plants, and black columns correspond to TNHX1 plants. Values are means ±standard deviation (n=5).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
In the present work it was shown that overexpression of the wheat vacuolar Na+/H+ antiporter TNHX1 and H+-pyrophosphatase TVP1 in transgenic Arabidopsis plants results in salt and drought tolerance. Wild-type plants displayed progressive chlorosis, reduced leaf area, and a general growth inhibition when treated with high salt concentrations or when deprived of water. The transgenic plants accumulate more Na+ and K+ in their leaf tissue. The increased accumulation of Na+ and K+ is likely to be a consequence of the activity of the vacuolar Na+/H+ antiporter. It is speculated that the antiporter facilitates K+ as well as Na+ uptake into vacuoles in exchange for H+ into the cytoplasm. This compartmentalization may prevent Na+ toxicity and facilitate cellular K+ uptake (Wu et al., 1996). The Na+/H+ antiporter of Arabidopsis thaliana (AtNHX1) was shown to mediate the transport of K+ as well as Na+ in tomato tonoplast vesicles (Zhang and Blumwald, 2001). The capacity of AtNHX1 to mediate both K+/H+ and Na+/H+ transport was also demonstrated on reconstituted liposomes with purified AtNHX1 (Venema et al., 2002). Moreover, vacuoles isolated from leaves of the Arabidopsis nhx1 mutant plants had much lower Na+/H+ and K+/H+ exchange activity (Apse et al., 2003). Alternatively, the increase in K+ content of leaves in the transgenic lines (TNHX1 and TVP1) may also result from increased Na+ compartmentalization in leaf cells leading to increased transpiration rates and therefore increased delivery of K+ to the leaves.

Under the salinity and dehydration conditions used in this study, the differences in the pattern of {Psi}w and RWC decline in wild-type and transgenic plants suggest the possibility of a higher transpiration rate, increases in the uptake of water, and maintainance of turgor for the transgenic Arabidopsis plants. Increases in water uptake when plants are subjected to low soil solution {Psi}w can lead to maintenance of greater turgor (Radin, 1983). Increased turgor can allow for maintenance of greater stomatal aperture (hence CO2 supply to the leaf) as well as increased cell division and expansion (hence leaf growth and development) at low leaf {Psi}w after osmotic adjustment occurs. Osmotic adjustment lowers leaf {Psi}w, leading to an increase in the leaf:soil {Psi}w gradient which is the driving force for water uptake into the plant. This leads to greater water uptake at low soil solution {Psi}w, a condition that occurs due to salinization. Osmotic adjustment also alters the relationship between cell protoplast volume (approximated by RWC) and {Psi}w, as leaf {Psi}w declines due to restricted water uptake from saline soils (and/or soils with low {Psi}w due to drought), relatively greater RWC is maintained. Maintenance of greater leaf turgor at low {Psi}w and shifting the RWC:{Psi}w relationship has been demonstrated to lead to the maintenance of greater photosynthetic capacity and growth in plants exposed to low soil {Psi}w (Morgan, 1984; Gupta and Berkowitz, 1987; Johnson et al., 1987; Gunasekera and Berkowitz, 1992). When challenged with salinity stress, one way in which plants can adapt is through a system that allows for use of cellular sodium for osmotic adjustment. The compartmentalization of Na+ into vacuoles provides an efficient mechanism to avoid the toxic effects of Na+ in the cell cytosol. Prior research work (Apse et al., 1999; Gaxiola et al., 2001; Zhang and Blumwald, 2001; Park et al., 2005) indicated that overexpression of the Arabidopsis vacuolar Na+/H+ antiporter (AtNHX1) and pyrophosphatase H+-pump (AVP1) in Arabidopsis and tomato plants provides enhanced cellular-level tolerance to salinity (and water deficit in the case of AVP1) by increasing the capacity to accumulate cations (Na+ and/or K+) in the vacuole.

The present finding that the wheat Na+/H+ antiporter and H+-PPase genes confer salt and drought tolerance in Arabidopsis suggests that it may be worthwhile to elucidate the contribution of these proteins to salt tolerance in a staple crop plant. The overexpression of these aforementioned genes in crop plants, including wheat, may be a strategy for engineering agriculturally important plants to withstand these important abiotic stresses.


   Acknowledgements
 
We thank Dr Chantal Ebel for helpful assistance with the UTHSCSA image tool processing and analysis program and Jalel Azeza for technical assistance. This work was supported jointly by grants from the Ministry of Research, Technology and Development of Competencies (MRTDC), Tunisia, the USDA-RSED awards FG-TN102 and the International Atomic Energy Agency contract #12999R.


   References
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 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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