JXB Advance Access originally published online on August 28, 2006
Journal of Experimental Botany 2006 57(12):3259-3270; doi:10.1093/jxb/erl090
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© 2006 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER |
Cloning of an H+-PPase gene from Thellungiella halophila and its heterologous expression to improve tobacco salt tolerance
School of Life Science, Shandong University, Jinan 250100, PR China
*To whom correspondence should be addressed. E-mail: nihaohua{at}263.net
Received 1 April 2006; Accepted 21 June 2006
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Abstract |
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An H+-pyrophosphatase (PPase) gene named TsVP involved in basic biochemical and physiological mechanisms was cloned from Thellungiella halophila. The deduced translation product has similar characteristics to H+-PPases from other species, such as Arabidopsis and rice, in terms of bioinformation. The heterologous expression of TsVP in the yeast mutant ena1 suppressed Na+ hypersensitivity and demonstrated the function of TsVP as an H+-PPase. Transgenic tobacco overexpressing TsVP had 60% greater dry weight than wild-type tobacco at 300 mM NaCl and higher viability of mesophyll protoplasts under salt shock stress conditions. TsVP and AVP1, another H+-PPase from Arabidopsis, were heterologously expressed separately in both the yeast mutant ena1 and tobacco. The salt tolerance of TsVP or AVP1 yeast transformants and transgenic tobacco were improved to almost the same level. The TsVP transgenic tobacco lines TL3 and TL5 with the highest H+-PPase hydrolytic activity were studied further. These transgenic tobacco plants accumulated 25% more solutes than wild-type plants without NaCl stress and 20–32% more Na+ under salt stress conditions. Although transgenic tobacco lines TL3 and TL5 accumulated more Na+ in leaf tissues, the malondialdehyde content and cell membrane damage were less than those of the wild type under salt stress conditions. Presumably, compartmentalization of Na+ in vacuoles reduces its toxic effects on plant cells. This result supports the hypothesis that overexpression of H+-PPase causes the accumulation of Na+ in vacuoles instead of in the cytoplasm and avoids the toxicity of excessive Na+ in plant cells.
Key words: Arabidopsis thaliana, H+-PPase, salt tolerance, Thellungiella halophila
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Introduction |
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Salinity is an important factor limiting plant growth. About 20% of the world's irrigated lands are now affected by salinity (Rhoades and Loveday, 1990). The detrimental effects of salt on plants are a consequence of both water deficit that results from the relatively high solute concentrations in the soil and excessive Na+ concentrations in the cytoplasm. Excessive Na+ in the cytoplasm not only alters ion ratios and affects critical biochemical processes (Maathuis and Amtmann, 1999), but also increases plasma membrane injury and causes malondialdehyde (MDA) accumulation (Dionisio-Sese and Tobita, 1998). To deal with the injury, plants have developed multifarious strategies. The accumulation of compatible solutes and reduction of sodium ions in the cytoplasm are two common mechanisms in plants. Compatible solutes mainly include betaine; polyols and sugars, such as mannitol and sorbitol; and amino acids, such as proline (Chen and Murata, 2002). Mechanisms have been developed in plants to avoid the injury due to excessive sodium ion concentrations in the cytoplasm. There are two important ways among these mechanisms for reducing excessive Na+ in the cytoplasm: one is to exclude Na+ from cells by the Na+/H+ antiporter located in the plasma membrane; and the other is to pump Na+ into vacuoles by the Na+/H+ antiporter located in the tonoplast (Zhu, 2003).
The compartmentalization of Na+ into vacuoles provides an efficient mechanism for averting the toxic effects of Na+ in the cytosol. The transport of Na+ into vacuoles is mediated by vacuolar Na+/H+ antiporters that are driven by the electrochemical gradient of protons. The proton-motive force generated by the vacuolar ATPase (V-ATPase) and vacuolar pyrophosphatase (V-PPase) can drive secondary transporters, such as the Na+/H+ antiporter and Ca2+/H+ antiporter, as well as organic acids, sugars, and other compound transporters to maintain cell turgor (Lincoln, 1992). The vacuolar H+-PPase is a single subunit protein located in the vacuolar membrane (Maeshima, 2000). It pumps H+ from the cytoplasm into vacuoles with PPi-dependent H+ transport activity. Theoretically, overexpression of H+-PPase should enhance the ability to form the pH gradient between the cytoplasm and vacuoles, resulting in a stronger proton-motive force for the Na+/H+ antiporter, Ca2+/H+ antiporter, and other secondary transporters. The accumulation of cations, such as Na+, in vacuoles could increase the osmotic pressure of plants, while reducing the toxic effects of these cations (Gaxiola et al., 2001).
Apse et al. (1999) have reported that overexpressing AtNHX1, a vacuolar Na+/H+ antiporter from Arabidopsis, resulted in higher salt tolerance in transgenic Arabidopsis, and the salinity tolerance was correlated with the higher levels of AtNHX1 transcripts and vacuolar Na+/H+ antiporter activity. In addition, evidence from Gaxiola et al. (2001) supports the role of the vacuolar electrochemical proton gradient in salt tolerance by overexpressing a vacuolar H+-PPase in Arabidopsis thaliana. Transgenic plants overexpressing AVP1, coding for a single subunit protein for vacuolar H+-PPase, displayed enhanced salt tolerance that was correlated with the increased ion content of the plants. These results suggest that the enhanced vacuolar H+ pumping in the transgenic plants provided additional energy for vacuolar sodium accumulation via the vacuolar Na+/H+ antiporter.
Thellungiella halophila (salt cress; synonymous with Thellungiella salsuginea) (Al-Shebaz et al., 1999), a classical halophyte living in the seashore saline soils in eastern China, is able to survive for several months and produce viable seeds, even in the presence of 500 mM NaCl (Inan et al., 2004). Thellungiella halophila produces neither salt glands nor other complex morphological alterations either before or after salt adaptation. It appears that the salt tolerance comes from its basic biochemical and physiological mechanisms. In the present report, a vacuolar H+-PPase gene involved in basic biochemical and physiological mechanisms was cloned from T. halophila. Then the H+-PPase genes from A. thaliana and T. halophila were heterologously expressed in the yeast mutant ena1 and in tobacco to study the function of the H+-PPase and compare the different effects of H+-PPase from A. thaliana, a typical glycophyte, and T. halophila, a typical halophyte on salt tolerance. The results indicated that the overexpression of TsVP or AVP1 enhanced the salt tolerance of both the yeast mutant ena1 and tobacco. The TsVP transgenic tobacco lines TL3 and TL5 that had higher H+-PPase hydrolytic activity were further studied by analysing dry weight, solute potential, ion content, MDA content, and electrolyte leakage of leaf cells.
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Materials and methods |
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Cloning the H+-PPase gene from a cDNA library and sequence analyses
A pair of primers P1 (5'-GGCGTTCATTGTTGCATTCAGGT-3') and P2 (5'-CCCAGGCTCCTCCAGTGTTAGAT-3') were designed from the sequence of the AVP1 gene from A. thaliana. A DNA fragment was amplified from T. halophila cDNA by reverse transcription–PCR (RT–PCR) with P1 and P2. This DNA fragment was cloned into pGEM®-T easy vector (Promega, USA) and sequenced by Bioasia, Inc. (Shanghai, China). This DNA fragment, labelled using the DIG-High Prime kit (Roche Inc.), was used to screen a cDNA library from 200 mM NaCl-stressed 8-week-old T. halophila. Several clones positive for the H+-PPase gene named TsVP were isolated and sequenced. Alignment was performed using the deduced amino acid sequence of TsVP and H+-PPases from other species.
Construction of yeast expression vectors and yeast transformation
The complete open reading frames (ORFs) of TsVP and AVP1 (GenBank accession no. NM_101437) were amplified with primers TsVP-P3 (5'-GGAGGAGAGATGGTGGCGTC-3') and TsVP-P4 (5'-CAAAACGAAAATAAGAATGG-3'), and primers AVP-P1 (5'-GAGAAGATGGTGGCGCCTGC-3') and AVP-P2 (5'-TCACTGGGGAGTTGCTGGAG-3'), respectively, using Pyrobest® polymerase (Takara Inc., Japan), These full-length cDNA fragments were cloned into pGEM®-T easy vector and sequenced by Bioasia, Inc (Shanghai, China). The DNA fragments of AVP1 and TsVP were digested with EcoRI and cloned into the EcoRI site in the multiple cloning site (MCS) of the pYES2 vector (Invitrogen Inc., USA) between the GAL1 promoter and the CYC1 terminator sequences. These plasmids were named pYES2-TsVP and pYES2-AVP. Ura– yeast strains were transformed by the LiAc/polyethylene glycol method.
Functional assays using the yeast mutant
The ena1 yeast mutant was transformed with three different plasmids, pYES2-TsVP, pYES2-AVP, and pYES2 as a control. Three ena1 yeast mutant strains and the wild type (W303) were grown at 28 °C for 16 h to reach OD600 1.0–2.0 in YPD medium. About 105 yeast cells in 2 µl and two 5-fold serial dilutions of the yeast cells were spotted onto YPGAL plates with different NaCl concentrations (0.25, 0.5, and 0.75 M). The strains were cultured at 28 °C for 60 h. The growth status of these yeast strains was used to identify the TsVP function and compare the salt tolerance.
Construction of plant expression vectors and tobacco transformation
TsVP and AVP1 full-length cDNAs were cloned into the plant expression vector pROKII under the control of the cauliflower mosaic virus (CaMV) 35S promoter with standard protocols described in Sambrook et al. (2000). Leaf discs from Nicotiana tabacum Wisconsin38 were transformed with Agrobacterium tumefaciens strain LBA4404 (Voelker et al., 1987) carrying the above recombinant plasmids. Regenerated shoots with kanamycin resistance were selected in MS medium (Murashige and Skoog, 1962) supplemented with 170 µM kanamycin, 4.44 µM 6-benzyladenine (6-BA), and 0.571 µM indoleacetic acid (IAA), and rooted in MS medium without growth regulators. These kanamycin-resistant plantlets were confirmed by PCR with primers TsVP-P5 (5'-CAGAACTCGCCGTAAAGACT-3') and TsVP-P6 (5'-GCAGAAACCGAAGATAACG-3') for TsVP transgenic tobacco, and primers AVP-P3 (5'-GCCGTAAAGACTGGCGAACA-3') and AVP-P4 (5'-AGCACCAAGCACGAAAGCAA-3') for AVP1 transgenic tobacco. The PCR-positive plantlets were transplanted into soil and grown in a greenhouse at 27/23 °C (day/night) with a 16 h light (600 µmol m–2 s–1)/8 h dark cycle and 
70% relative humidity. Eight weeks later, these plants were collected for Southern and northern blotting analysis. The transgenic tobacco plants with different levels of TsVP gene expression were used for determination of PPi hydrolysis activity and salt tolerance assays.
Germination and growth of transgenic tobacco
Sterilized seeds from the homozygous T2 transgenic and wild-type plants of tobacco were germinated on MS medium. The seedlings were grown under conditions of 25/20 °C (day/night) temperature, 16 h photoperiod with 200 µmol m–2 s–1 light intensity. Twenty days later, some seedlings were transferred to MS medium supplemented with different concentrations of NaCl (0, 100, 200, or 300 mM, respectively). After 25 d salt treatment, the seedlings were used to measure the parameters such as dry weight, ion content, MDA content, electrolyte leakage of leaf cells, and the protoplast viability. Other seedlings were transplanted into soil and grown in a greenhouse under the same conditions as above. Eight weeks later, these plants were collected for measurement of PPi hydrolysis activity and proton transport assays.
Southern and northern blotting
DNA of tobacco plants was isolated from 1 g of leaves by the cetyltrimethyl ammonium bromide (CTAB) method. Southern blotting was performed with the digoxigenin (DIG)-labelled full-length TsVP cDNA probe as described in the DIG System Manual (Roche, Inc.). RNA was extracted with Trizol reagent from the leaves of transgenic and non-transgenic plants, and treated with RNase-free DNase. Northern blotting was performed with denatured total RNA (20 µg per lane) in the standard protocol with the [
-32P]dCTP-labelled full-length cDNA of TsVP as a probe in Church buffer at 65 °C.
Real-time RT–PCR
The 6-week-old plants of A. thaliana and 8-week-old plants of T. halophila were treated with half-strength MS salt solution containing 200 mM NaCl. The roots and aerial parts of these plants were collected at different times after salt treatment. Total RNAs were extracted by Trizol reagent from these samples and treated with RNase-free DNase. cDNA synthesis was performed with the RT reagent kit (Takara, Dalian, China) according to the manufacturer's protocol. Real-time quantitative RT–PCRs were done on chromsome 4 (MJ Research, USA) with the SYBR® RT–PCR Kit (Takara, Dalian, China), in a 10 µl reaction volume, which contained 5 µl of SYBR® Green I PCR mix, 0.2 µM of each forward and reverse primer, 1 µl of diluted cDNA template, and appropriate amounts of sterile ddH2O. Amplification conditions were: 2 min at 95 °C; 40 cycles of 15 s at 95 °C, 30 s at 58 °C, and 30 s at 72 °C. Fold changes of RNA transcripts were calculated by the 2–
Ct method (Livak and Schmittgen, 2001) with ß-tubulin as an internal control. The entire experiments were repeated at least three times.
Membrane vesicle isolation
Tobacco tonoplast vesicles were prepared by sucrose density gradient ultracentrifugation as described previously by Lüttge et al. (2000) with minor modifications. About 30 g of leaf slices were homogenized in 90 ml of ice-cold buffer containing 100 mM Tricine/Tris, pH 8.0, 300 mM mannitol, 3 mM MgSO4, 3 mM EGTA, and 0.5% (w/v) polyvinylpolypyrrolidone. Prior to use, 5 mM dithiothreitol (DTT) and 1 mM phenylmethylsulphonyl fluoride (PMSF) were added to the buffer. After filtration and pre-centrifugation of the homogenate at 4200 g for 10 min, the pellets were resuspended in a small volume of ice-cold buffer containing 100 mM Tricine/Tris, pH 8.0, 300 mM mannitol, 3 mM MgSO4, 3 mM EGTA, and 5 mM DTT. The suspension containing the vesicles was layered over a 10/25% (w/w) discontinuous sucrose gradient solution with 5 mM HEPES/Tris, pH 7.5, and 2 mM DTT. After centrifugation at 100 000 g for 2 h, the vesicles at the interface between the 10% and 25% sucrose solutions were collected, and diluted with 3 vols of ice-cold buffer containing 10 mM HEPES/NaOH, pH 7.0, 3 mM MgSO4, and 1 mM DTT. Membranes were collected by centrifugation at 100 000 g for 60 min and resuspended in storage buffer with 10 mM HEPES/NaOH, pH 7.0, 40% (v/v) glycerol, 1 mM DTT, and 1 mM PMSF, and then were stored at –70 °C, ready for use.
Measurement of ATPase and H+-PPase activity
ATPase and PPase activity were measured as the release of Pi from ATP and PPi, respectively, during an incubation period of 30 min at 37 °C according to the method of Wang et al. (2001). Inorganic phosphate was determined using the method of Lin and Morale (1977). V-ATPase hydrolytic activity is presented as the difference of the measured values in the absence or in the presence of 50 mM 
V-PPase hydrolytic activity was calculated as the difference in activity measured in the presence or absence of 50 mM KCl (K+-stimulated PPase activity).
Proton transport assays
An inside-acid pH gradient (pH) in vacuolar membrane vesicles produced by the vacuolar H+-PPase was measured at 25 °C by quenching of quinacrine fluorescence using a spectrofluorometer at 495 nm after excitation at 420 nm (Churchill and Sze, 1983). The complete 300 µl reaction mixture contained 10 mM HEPES/Tris (pH 7.5), 0.33 mM EGTA, 2.0 µM quinacrine, 250 mM mannitol, 50 mM KCl, 1 mM PPi, and 50 µg of membrane protein. Proton pumping was initiated by the addition of 3 mM MgSO4. The rate of fluorescence quenching was used as a relative estimate of the rate of H+ pumping.
Tobacco leaf disc salt tolerance assay
The fourth fully expanded leaves acrofugally of wild-type and transgenic plants were briefly washed twice in distilled water. Leaf discs of 1 cm in diameter were cut and floated on 400 mM NaCl and 600 mM NaCl solution for 60 h (Veena et al., 1999) under continuous white light at 25 °C. The chlorophyll was extracted with 80% acetone and the content of chlorophyll was measured by a spectrometer. The experiment was performed with three TsVP transgenic lines TL2, TL3, and TL5, two AVP1 transgenic lines, AL3 and AL7, and one line of wild-type tobacco, and repeated three times on every plant.
Tobacco protoplast isolation and protoplast viability under salt tolerance
The healthy mature leaves of sterilized tobacco seedlings without salt stress were used to isolate mesophyll protoplasts; these leaves were sliced into 2 mm wide strips and added to 5 ml volumes of CPW9M (Patat-Ochatt et al., 1988) solution containing 9% mannitol, 1.5% Onozuka R-10 cellulase (Yakult Honsha Co., Japan), and 0.5% macerozyme. The pH of the CPW9M solution was adjusted to 5.8. The incubation was carried out in darkness for 1.5–2.0 h at 27 °C. After digestion, the enzyme mixtures were filtered through nylon mesh, and the protoplasts were purified as described by Pan et al. (2003). Finally, the mesophyll protoplasts were collected and resuspended in CPW9M solution with 9.0% mannitol. These purified mesophyll protoplasts were treated with CPW9M solution containing different concentrations of NaCl (100, 200, 300, and 400 mM) for 5 min. The viability of mesophyll protoplasts was assessed with the fluorescein diacetate (FDA) staining method (Widholm, 1972). The viability of mesophyll protoplasts was calculated by the following formula: viability of mesophyll protoplasts=the number of protoplasts with green fluorescence after FDA staining/the number of total mesophyll protoplasts x 100%.
Quantification of solutes in leaf tissue
Extended leaves from non-NaCl-stressed tobacco seedlings were frozen in liquid nitrogen and thawed to extrude sap by a glass syringe. The osmotic potential of the leaf sap was determined with a cryoscopic osmometer. The readings (mmol kg–1) were used to calculate the solute potential (
S) in MPa (mega Pascales) using the formula S=moles of solutexRK, where R = 0.008314 and K = 298.
Na+ absorption by the leaf discs of tobacco
Na+ absorption was performed as described by Smith and Epstein (1964a, b) with minor modifications. Healthy and fully expanded leaves from wild-type and transgenic plants were washed briefly in distilled water twice. The leaf discs of 1 cm in diameter were cut and floated on distilled water for 2 h, then they were floated on 300 mM NaCl for various times in continuous white light at 25 °C; five discs as a group were picked out every 2 h. These groups of discs were washed twice with distilled water and transferred into the solution containing 100 mM KCl and 200 mM mannitol for 20 min to remove Na+ in the outer space of mesophyll cells, then these discs were again rinsed twice with distilled water. These leaf discs were used for Na+ content assays. The Na+ content of leaf discs was determined by atomic absorption, and the speed of uptake of the Na+ was calculated by the following formula: Na+ uptake speed (µmol h–1 per disc)=(TN+2 Na+ content–TN Na+ content)/2, where TN Na+ content presents the Na+ content per disc at different time points.
Measurement of ion concentrations
The amounts of Na+, K+, Ca+, and Cl– in tobacco leaves and roots were measured. All leaves without midribs of the tobacco seedlings were used to measure the Na+ concentration. The roots were rinsed in deionized water for 10 s and then washed with cold LiNO3 solution isotonic with MS medium containing different concentrations of NaCl according to the method described by Flowers et al. (1986). Tobacco leaves and roots were dried for 48 h at 70 °C, and the dry weight was measured. The dried leaves and roots were extracted with 1 N HNO3 as described by Storey (1995). The supernatants were analysed by atomic absorption for Na+, K+, and Ca2+. The amount of Cl– was determined by silver titration according to the method described previously by Chen et al. (2001).
Assay of malondialdehyde concentration
The MDA content was determined using the protocol described by Peever and Higgins (1989). Tobacco leaves (100–300 mg) were used to measure MDA content. The absorbance at 450, 532, and 600 nm was determined with a spectrometer. The concentration of MDA was calculated by the following formula:
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Determination of leaf cell membrane damage
Leaf cell membrane damage was measured as the leakage of electrolytes from leaf cells using a conductivity meter, according to the method described by Gibon et al. (1997). The cell membrane damage represented as electrolytes leakage (%) was calculated using the following equation described by Premachandra et al. (1991): electrolytes leakage (%)=[1–(1–S1/S2)/(1–C1/C2)]x100, where the conductivity measurements correspond to NaCl-treated leaves (S1), boiled NaCl-treated leaves (S2), non-NaCl-treated leaves (C1), and boiled non-NaCl-treated leaves (C2).
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Results |
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Cloning of the H+-PPase gene from the T. halophila cDNA library
A pair of primers, P1 and P2, was designed to amplify the T. halophila double-stranded cDNA. A DNA fragment was obtained and sequenced. The result indicated that the fragment was a part of the H+-PPase gene in T. halophila. Then the fragment was used to screen the T. halophila cDNA library. A positive clone containing a full-length cDNA for the putative H+-PPase gene named TsVP was isolated. The full-length cDNA of TsVP was registered in GenBank (accession no. AY436553).
TsVP gene sequence analyses
Sequence analyses by the NCBI ORF finder revealed an ORF of 2316 bp with a 5'-untranslated region of 100 bp and a 3'-untranslated region of 344 bp. The deduced amino acid sequence is 771 amino acids and contains 14 transmembrane domains predicted by the online analyses tool, TopPred software from http://us.expasy.org. The topological model was very similar to that of a previous report on H+-PPase by Maeshima (2000). Multiple alignments of the deduced amino acid sequence with vacuolar H+-PPases from other species were performed with the Clustal W program. The results indicated that the TsVP protein shared 96% identity with the AVP1 protein of A. thaliana at the amino acid level, and the five conserved domains reported by Drozdowicz and Rea (2001) were also present in the deduced amino acid sequence of TsVP (Fig. 1).
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Analyses of expression patterns under salt stress
Northern blotting analyses indicated that TsVP mRNA was present in both the leaves and roots of 8-week-old T. halophila seedlings. As shown in Fig. 2A and B, the level of mRNA accumulation in the leaves was relatively higher than in the roots. Total RNA was isolated at various times (0, 2, 4, 8, 16, and 24 h) under salt stress. The accumulation of TsVP mRNA increased over time during the period 0–16 h, and reached a maximum at 16 h. TsVP mRNA decreased from 16 h to 24 h.
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The expression patterns of AVP1 and TsVP under salt stress were compared by real-time RT–PCR with ß-tubulin as an internal control. The fold change in gene expression is shown in Fig. 2C for AVP1 and Fig. 2D for TsVP. The increase in AVP1 expression was <2.5-fold, while TsVP expression increased 3-fold and 5-fold during the first 16 h in the aerial parts and roots, respectively.
Functional assays of TsVP and AVP1 using the yeast mutant ena1
In Saccharomyces cerevisiae, the primary pathway for Na+ extrusion is mediated by ENA1 (Haro et al., 1991; Rios et al., 1997), a plasma membrane Na+-ATPase. The yeast mutant strain ena1 lacks the plasma membrane sodium efflux pump; therefore, it must rely on an internal detoxification system to overcome sodium toxicity. Growth of the ena1 strain is sensitive to a low concentration of sodium (200 mM), whereas the growth of the wild-type strain is not inhibited by such low NaCl concentrations. Gaxiola et al. (1999) have reported that overexpression of the E229D (AVP1-D) gain-of-function mutant of the AVP1 gene (Zhen et al., 1997) with enhanced H+ pumping capability in the yeast Na+ hypersensitive strain ena1 can restore the salt tolerance to salt-sensitive ena1 mutants. The work of Gaxiola et al. (1999) supports a model whereby expression of a plant vacuolar H+-PPase could contribute to better growth of ena1 in the presence of a high external Na+ concentration by pumping more Na+ from the cytosol into vacuoles. This work demonstrates that the yeast ena1 mutant strain can be used to identify the function of the plant vacuolar H+-PPase (Brini et al., 2005). In the present work, overexpression of TsVP can restore the salt tolerance of the salt-sensitive ena1 mutant in a similar way to AVP1. As shown in Fig. 3, all yeast strains grew well on YPGAL medium without NaCl. When these yeast strains grew on YPGAL medium that had different concentrations of NaCl (0.25, 0.50, and 0.75 M), the ena1 mutants carrying the pYES2-TsVP or pYES2-AVP1 plasmids showed a similar growth status, which was much better than the ena1 mutants carrying empty pYES2 plasmids but still weaker than the wild-type yeast. These findings were consistent with previous work on AVP1-D heterologous expression in the yeast ena1 mutant, suggesting that TsVP is an H+-PPase similar to AVP1.
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Generation of TsVP and AVP1 transgenic tobacco plants
TsVP and AVP1 were each inserted into the plant expression vector pROKII under the control of the CaMV 35S promoter and with the plant selective marker gene nptII. The resulting plasmids were named pROKII-TsVP and pROKII-AVP1. Nicotiana tabacum Wisconsin38 was transformed with A. tumefaciens (LBA4404) carrying pROKII-TsVP or pROKII-AVP1, and 15 independent TsVP transformants were identified by PCR. Eleven TsVP transgenic tobacco lines were subjected to Southern and northern blotting to test DNA integration and RNA expression further. The hybridization results from six transgenic tobacco plantlets are shown in Fig. 4A and B. Three TsVP transgenic tobacco lines TL2, TL3, and TL5 are shown in lanes 2, 3, and 5 in Fig. 4A and B. Two AVP1 transgenic tobacco lines AL3 and AL7 with the highest levels of expression were used in further assays.
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DNA/HindIII molecular weight marker; lanes 1–6 show genomic Southern blotting analysis of different transgenic tobacco plants. Genomic DNA from the leaves of transgenic tobacco was digested with HindIII, which has one cut site in the T-DNA region and no cut site in the TsVP cDNA region, and hybridized with labelled full-length TsVP cDNA as probe. (B) CK, an untransformed tobacco plant; lanes 1–6, different transgenic tobacco plants corresponding to transgenic tobacco lines 1–6. Northern blotting analysis was performed using total RNA from the extended leaves of the plants. Hybridization was performed with the 32P-labelled full-length TsVP cDNA as probe.ATPase and PPase activity
The TsVP gene was transferred into tobacco and expressed at different levels. To confirm the function of TsVP, the hydrolytic activity of H+-PPase was measured using tobacco tonoplast vesicles. The purity of the tonoplast vesicles was between 52% and 63% as determined by the hydrolytic activity of V-ATPase in the presence or absence of the specific V-ATPase inhibitor 50 mM
The hydrolytic activity of ATPase and PPase, which was determined by measuring the release of inorganic phosphate, is shown in Fig. 5A and B. The V-ATPase activity of wild-type tobacoo and the three TsVP transgenic tobacco lines was comparable, whereas the H+-PPase hydrolytic activity in transgenic tobacco lines was higher than that of wild-type tobacco. The level of H+-PPase hydrolytic activity in the transgenic tobacco lines was correlated with the level of TsVP gene expression. The H+-PPase hydrolytic activity was significantly higher in transgenic tobacco lines TL3 and TL5, which had higher levels of TsVP gene expression than TL2, and was significantly different from that in wild-type tobacco (P <0.05) by t-test, while the hydrolytic activity of H+-PPase in TL2 was only modestly higher than that in the wild-type tobacco. PPi-dependent proton transport of homozygous T2 transgenic tobacco lines TL3 and TL5 was measured using a quinacrine fluorescence assay in which the decrease (quenching) of fluorescence intensity accompanied the formation of inside-acid pH. The rate and magnitude of pH formation were always higher in reactions with vesicles isolated from TsVP transgenic tobacco (Fig. 5C).
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Leaf discs from transgenic tobacco with overexpression of TsVP or AVP1 are salt tolerant
Leaf discs from transgenic tobacco TL2, TL3, TL5, AL3, and AL7, and wild-type tobacco were floated on 400 and 600 mM NaCl solution, respectively, for 60 h. The amount of chlorophyll was compared between the transgenic and wild-type tobacco plants. The results indicated that chlorophyll was lost from the TsVP and AVP1 transgenic tobacco leaves at a slower rate than from leaves of wild-type tobacco under two different NaCl concentrations (Fig. 6A), and no distinct difference between TsVP and AVP1 transgenic tobacco plants was observed. The result also reflected that the transgenic plants with higher expression of TsVP and higher hydrolytic activity of vacuolar H+-PPase possessed a better salt tolerance, and the transgenic tobacco TL2 that had lower expression of TsVP was less salt tolerant than TL3 and TL5. The transgenic tobacco lines TL3 and TL5 were used for further assays.
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Viability of tobacco mesophyll protoplasts under salt tolerance
Tobacco mesophyll protoplasts were treated with CPW9M containing different concentrations of NaCl (100, 200, 300, and 400 mM). Under NaCl shock stress, the protoplasts showed changes in appearance and some of them lost the ability to fluoresce after staining with FDA. The viability of mesophyll protoplasts decreased with higher NaCl concentrations (Fig. 6B). Both the TsVP and the AVP1 transgenic tobacco had higher viability (P <0.01) of mesophyll protoplasts at different NaCl concentrations, which indicated a higher salt tolerance of the cells. The assay for viability of mesophyll protoplasts also reflected that TsVP and AVP1 transgenic tobacco plants have the same level of salt tolerance.
More biomass of transgenic plants under salt treatment
To study further the difference between transgenic and wild-type tobacco under salt treatment, biomass (dry weight) was measured under three different NaCl concentrations. Biomass decreased gradually with increased salt concentrations. The differences in biomass between wild type and TL3, TL5, AL3, and AL7 at the same NaCl concentration became more distinct at higher salt concentrations (Fig. 6C). For example, at 300 mM NaCl, the biomass of transgenic tobacco discs was 60% greater than that of wild type, with P <0.01 (n=6) by t-test.
Faster Na+ uptake in transgenic tobacco than in the wild type
Leaf discs from transgenic lines TL3 and TL5 showed faster uptake of Na+, and the speed of Na+ uptake by the leaf discs was significantly different compared with wild-type tobacco from 2 to 8 h, with P <0.05, as shown in Fig. 7.
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Transgenic plants retain more solutes under non-salt stress conditions
The amount of solutes in the leaves was measured under non-NaCl stress conditions. The leaves of transgenic tobacco lines TL3 and TL5 retained 25% more solutes than those of wild-type tobacco. The osmotic pressure or solute potential (
S, refers to the concentration of osmotically active particles dissolved in water) of the wild-type plants was less negative than that of the transgenic lines TL3 and TL5 under non-NaCl stress conditions. The values of the wild-type plants, and transgenic lines TL3 and TL5 were –0.807 MPa (SE=0.026, n=6), –0.986 MPa (SE=0.068, n=6), and –1.012 MPa (SE=0.067, n=6), respectively. The data showed that a significant difference existed in solutes between the wild-type and transgenic plants, with 0.01 <P <0.05 by t-test under non-NaCl stress conditions.
Ion concentrations in plant tissues
The amounts of Na+, K+, Ca2+, and Cl– were measured in the leaves and roots of plants under different NaCl concentrations (0, 100, 200, and 300 mM). In the leaves, the Na+ and Cl– concentration increased, and the K+ or Ca2+ concentration decreased with increasing NaCl concentrations from 0 to 300 mM. The Na+ content of leaves was from 20% to 30% higher in transgenic lines TL3 and TL5 than that in wild-type plants under different NaCl treatments, and the difference between the transgenic and wild-type plants was significant with the 300 mM NaCl treatment (P <0.05) (Table 1). Furthermore, the K+ or Ca2+ content was also slightly higher than that in the wild type with different salt treatments, but the difference was not significant. In the roots, the Na+ or Cl– content increased with the increase of salinity. However, the K+ or Ca2+ content increased with the increase of salinity, which is different from the situation in leaves.
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To study further the effects of excessive Na+ on plants, leaf cell electrolyte leakage (%), that represents the degree of cell membrane damage under stress, and MDA content were determined. MDA content and leaf cell membrane damage increased with the increase in salt concentration. In transgenic tobacco lines TL3 and TL5, the amount of MDA was 43% and 76%, respectively, less than that of wild-type plants (Fig. 8A). Leaf cell electrolytes leakage was 29% and 34% respectively, less than that of wild-type plants at 300 mM NaCl (Fig. 8B).
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Discussion |
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Thellungiella halophila is closely related to Arabidopsis and its genome size is approximately twice that of Arabidopsis. In recent years, T. halophila has become an important model for abiotic stress research (Amtmann et al., 2005). The present work focuses on an H+-PPase, which is located in tonoplasts and plays an important role in the plant's basic biochemical and physiological systems. The gene TsVP from T. halophila is the first gene encoding an H+-PPase isolated from a halophyte. The deduced amino acid sequence contains 771 amino acids and encodes 14 transmembrane domains. The transmembrane model is identical to H+-PPases of other species (Maeshima, 2000), and five conserved domains used to identify H+-PPase homologues by Drozdowicz and Rea (2001) were also found in the deduced amino acid sequence. TsVP protein shares 96% identity with the AVP1 protein at the amino acid level; the different amino acids were mainly clustered near the N-terminus from residue 4 to 150. The function of TsVP was characterized by heterologous expression in the yeast salt-sensitive mutant ena1, which was also used to demonstrate the function of Arabidopsis AVP1 by Gaxiola et al. (1999).
Gaxiola et al. (2001) have reported that the overexpression of the vacuolar H+-PPase gene AVP1 in transgenic Arabidopsis plants resulted in drought and salt tolerance, and ascribed these properties to the increased accumulation of solutes. In the study of Gaxiola et al., the transgenic plants accumulated more solutes without salt stress and more Na+ and K+ in their leaf tissues under salt stress conditions. In the present experiments, H+-PPase genes from A. thaliana or T. halophila were heterologously expressed in the yeast mutant ena1 and tobacco. Salt tolerance in transgenic tobacco was correlated with TsVP gene expression and H+-PPase activity. The results also indicated that the salt tolerance produced by heterologous expression of TsVP or AVP1 was at a similar level. With no NaCl stress, the transgenic tobacco leaves accumulated more solutes than the wild-type leaves, which was consistent with Gaxiola's report. The higher viability of tobacco mesophyll protoplasts under NaCl stress in the transgenic tobacco plants may be due to more solutes in leaf tissues. Under salt stress conditions, more Na+ accumulated in the leaves of the transgenic plants than in the leaves of the wild type. The difference was significant when treatment was with 300 mM NaCl. K+ and Ca2+ were only modestly increased in transgenic tobacco leaf tissues. With increasing salinity, the K+ and Ca2+ content decreased in the leaves and increased in the roots, especially for K+. This may be due to increased uptake of K+ and Ca2+ by the roots under salt stress, and the excess Na+ may have disrupted the transport of K+ and Ca2+ from the roots to the leaves. In the case of Cl–, there was no significant difference between the wild-type and transgenic tobacco.
To study further the effects of excessive Na+ on tobacco, MDA content and cell membrane damage were measured in leaf tissues. MDA content and cell membrane damage were less in transgenic tobacco than in wild-type tobacco under salt stress conditions. These results demonstrated that transgenic tobacco was healthier or subjected to less damage under salt stress. The correlation in our results between higher Na+ and less damage in transgenic tobacco leaf tissues supports the hypothesis that the overexpression of H+-PPase can enhance the accumulation of Na+ in vacuoles. Accumulation of Na+ in vacuoles instead of in the cytoplasm can avoid the toxicity of excessive Na+ to plant cells. In view of the higher viability of tobacco mesophyll protoplasts in the transgenic tobacco plants under NaCl shock stress, it is concluded that higher salt tolerance in the transgenic tobacco was the result of better adaptability to salt stress for shorter times and more Na+ accumulation in vacuoles instead of in the cytoplasm. The successful heterologous expression of TsVP in tobacco and enhanced tobacco salt tolerance demonstrate that this gene from a halophyte can be beneficial for plant genetic engineering to enhance plant salt tolerance.
A comparative study of the genetic effects of salt tolerance between T. halophila and A. thaliana could reveal differences in the mechanisms of plant salt tolerance between the glycophyte and halophyte. In T. halophila, the expression pattern of TsVP was analysed under salt stress at different time points. TsVP mRNA levels in both roots and leaves increased during the first 16 h and decreased after 16 h. The expression of AVP1 in Arabidopsis showed no distinct change under salt stress. This suggests that the regulation of expression of the H+-PPase gene is different between T. halophila and A. thaliana under salt stress. This difference might result from two possibilities: one is that some transcription factors are different in T. halophila and A. thaliana, the other is that the promoter sequence or enhancer sequence of the H+-PPase gene is different in T. halophila and A. thaliana.
In the present report, the salt tolerance from heterologous expression of TsVP or AVP1 was at similar levels; this might suggest that TsVP and AVP1 have almost the same biochemical function. However, the expression patterns were different between T. halophila and A. thaliana under salt stress. Although it is not confirmed that different gene expression patterns contribute to the difference in salt sensitivity between T. halophila and A. thaliana, it is concluded that the differences in salt sensitivity between A. thaliana and T. halophila cannot be attributed to the function of H+-PPase. It could be the result of some subtle regulatory networks correlated with basic biochemical and physiological mechanisms in plants under salt stress. Of course, it cannot be absolutely excluded that there are some specialized genes for salt tolerance in T. halophila.
It was reported that the hydrolytic activity of H+-PPase firstly increased and then decreased in the leaves of halophytic Suaeda salsa L. under 400 mM NaCl stress (Wang et al., 2001). Recently, mRNA from a tonoplast H+-PPase gene was found to increase in the roots for a short time and there was no detectable increase in the level of transcript in the leaves under salt stress in barley (Fukuda et al., 2004). Combining TsVP expression patterns in T. halophila in the present report, it is concluded that the changes of H+-PPase activity were different in different tissues of different species under salt stress conditions.
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Acknowledgements |
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We are grateful to Gerald R Fink of the Whitehead Institute for Biomedical Research, who generously offered the yeast mutant ena1. We thank Dr Roberta Greenwood for her help in editing this manuscript. This research was supported by Hi-Tech Research and Development (863) Program of China (2002AA212071) and the National Project for Transgenic Plant Research and Industrialization of China (JY2002-B-006)
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Abbreviations |
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CaMV, cauliflower mosaic virus; DIG, digoxigenin; DTT, dithiothreitol; FDA, fluorescein diacetate; MDA, malondialdehyde; ORF, open reading frame; PMSF, phenylmethylsulphonyl fluoride; PPase, pyrophosphatase.
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