This paper is dedicated to Dr Juan Pedro Donaire on the occasion of his retirement.
Overexpression of the tomato K+/H+ antiporter LeNHX2 confers salt tolerance by improving potassium compartmentalization
Article first published online: 28 JUN 2008
© The Authors (2008). Journal compilation © New Phytologist (2008)
Volume 179, Issue 2, pages 366–377, July 2008
How to Cite
Rodríguez-Rosales, M. P., Jiang, X., Gálvez, F. J., Aranda, M. N., Cubero, B. and Venema, K. (2008), Overexpression of the tomato K+/H+ antiporter LeNHX2 confers salt tolerance by improving potassium compartmentalization. New Phytologist, 179: 366–377. doi: 10.1111/j.1469-8137.2008.02461.x
- Issue published online: 28 JUN 2008
- Article first published online: 28 JUN 2008
- Received: 18 January 2008; Accepted: 5 March 2008
- cation/H+ antiporters;
- overexpression and silencing of NHX genes;
- potassium nutrition;
- salt tolerance;
- Solanum lycopersicon (tomato)
- • Here, the function of the tomato (Solanum lycopersicon) K+/H+ antiporter LeNHX2 was studied using 35S-driven gene overexpression of a histagged LeNHX2 protein in Arabidopsis thaliana and LeNHX2 gene silencing in tomato.
- • Transgenic A. thaliana plants expressed the histagged LeNHX2 both in shoots and in roots, as assayed by western blotting. Transitory expression of a green fluorescent protein (GFP) tagged protein showed that the antiporter is present in small vesicles. Internal membrane vesicles from transgenic plants displayed enhanced K+/H+ exchange activity, confirming the K+/H+ antiporter function of this enzyme. Transgenic A. thaliana plants overexpressing the histagged tomato antiporter LeNHX2 exhibited inhibited growth in the absence of K+ in the growth medium, but were more tolerant to high concentrations of Na+ than untransformed controls. When grown in the presence of NaCl, transgenic plants contained lower concentrations of intracellular Na+, but more K+, as compared with untransformed controls.
- • Silencing of LeNHX2 in S. lycopersicon plants produced significant inhibition of plant growth and fruit and seed production as well as increased sensitivity to NaCl.
- • The data indicate that regulation of K+ homeostasis by LeNHX2 is essential for normal plant growth and development, and plays an important role in the response to salt stress by improving K+ accumulation.
Plant NHX antiporters are members of the sodium proton exchanger (NHE/NHX) subfamily of cation/proton exchangers that are present across all taxonomic groups of eukaryotes, and constitute a subgroup of the cation/proton antiporter 1 (CPA1) family (Saier et al., 1999). The NHE/NHX family can be further subdivided based on subcellular localization into a plasma membrane (PM) group and an intracellular (IC) group (Brett et al., 2005a; Pardo et al., 2006). The plasma membrane group is unique to animals, where these proteins are involved in intracellular pH and volume regulation or Na+ and electrolyte reabsorption through electroneutral Na+/H+ antiport (Counillon & Pouysségur, 2000). The IC group can be further subdivided into a vacuolar clade, which is exclusive to plants, an endosomal clade, which is present in plants, fungi and animals, and a separate intracellular clade for sequences related to the Homo sapiens HsNHE8 protein (Brett et al., 2005a; Pardo et al., 2006).
The first NHE/NHX protein to be described in plants was Arabidopsis thaliana AtNHX1, a member of the vacuolar clade of NHE/NHX proteins (Gaxiola et al., 1999; Quintero et al., 2000; Yokoi et al., 2002). It was shown that overexpression of this protein in various plants improves salt tolerance, indicating a role for the protein in vacuolar Na+ accumulation (Apse et al., 1999; Zhang & Blumwald, 2001; Zhang et al., 2001; Ohta et al., 2002; Xue et al., 2004; He et al., 2005). It was, however, shown that the encoded protein also catalyzes K+/H+ exchange with similar activity (Zhang & Blumwald, 2001; Venema et al., 2002). Furthermore, T-DNA insertional mutants of AtNHX1 show reduced leaf area and cell size, which also indicates that the protein is involved in Na+ or K+ accumulation inside vacuoles for maintenance of cell turgor to drive cell expansion (Apse et al., 2003). Notably, the AtNHX1 gene shows very high expression in stomatal cells, suggesting that the protein is involved in the high K+ accumulation in these cells (Shi & Zhu, 2002).
We have previously identified and characterized the Solanum lycopersicon LeNHX2 protein; this protein and the similar A. thaliana AtNHX5 protein constitute the first members of the endosomal clade of antiporters in plants (Yokoi et al., 2002; Venema et al., 2003). In yeast, the histagged LeNHX2 protein cofractionated with Golgi and prevacuolar membrane markers. Using purified and reconstituted histagged LeNHX2 protein, we have determined that the LeNHX2 protein catalyzes K+/H+ antiport (Venema et al., 2003). Other members of this family are the yeast prevacuolar Saccharomyces cerevisiae ScNHX1 protein, which was shown to be essential for vesicle trafficking and protein targeting between vacuole and prevacuole (Bowers et al., 2000; Brett et al., 2005b), the human NHE7 protein, which localizes to the trans-Golgi network (Numata & Orlowski, 2001), and isoforms HsNHE6 and HsNHE9, which localize to different endosomal compartments but can be transiently present on the plasma membrane (Brett et al., 2002; Hill et al., 2006). Both isoforms NHE7 and NHE9 were shown to catalyze K+/H+ exchange (Numata & Orlowski, 2001; Hill et al., 2006). Based on localization and ion specificity, it was proposed that endosomal NHX isoforms are essential to set the pH of endosomal compartments, which is believed to be fundamental for proper functioning of vesicle sorting in the secretory and endocytic membrane system (Pardo et al., 2006). Acidification of these compartments would depend on K+/H+ exchange to prevent accumulation of toxic Na+ ions (Pardo et al., 2006).
Despite its role in vesicle trafficking, the ScNHX1 antiporter is also involved in salt tolerance and tolerance to hyperosmotic shock in yeast (Nass et al., 1997; Nass & Rao, 1999). Mechanistically this can be explained by assuming that Na+ or K+ accumulation driven by the antiporter regulates the pH and volume of the prevacuolar compartments (PVCs) and late endosomes. The maintenance of turgor pressure in late endosomes and PVCs might be important for proper traffic of essential proteins required for osmotolerance and normal cellular function (Nass & Rao, 1999). Also, overexpression of the plant LeNHX2 protein in yeast, with or without histag, confers salt tolerance to these cells, which is concomitant with the accumulation of K+ in intracellular compartments (Venema et al., 2003). This suggests that the K+/H+ antiporter activity of the NHX genes is responsible for increased salt tolerance, rather than Na+/H+ antiporter activity and vacuolar Na+ accumulation. A drawback of this study was that the heterologous expression of plant proteins in yeast might induce changes in ionic specificity, especially if the activity is highly regulated in plant cells, and that the protein might be mislocalized as a result of the high overexpression. Therefore, to evaluate the function of this gene in plants, we have generated transgenic A. thaliana plants overexpressing the histagged LeNHX2 gene as well as tomato plants in which the expression of the LeNHX2 gene is silenced. Our results show that the histagged LeNHX2 antiporter overexpressed in A. thaliana plants also functions as an endosomal K+/H+ antiporter that increases salt tolerance, but reduces growth at suboptimal K+ concentrations. However, silencing of the gene in tomato has a severe effect on growth and fruit and seed production, indicating that, apart from its role in salt tolerance, regulation of K+ and pH homeostasis in the endosomal compartments by the LeNHX2 antiporter is essential for plant development.
Materials and Methods
For stable expression of the LeNHX2 protein in Arabidopsis thaliana (L.) Heynh., the LeNHX2 coding sequence, to which the sequence for a C-terminal RGS(H)10 tag was added (Venema et al., 2003), was cloned under control of the 35S promoter in the pCAMBIA1303 plant expression vector. To this end, the internal NcoI site at position 1346 in LeNHX2 was first removed by overlap extension PCR, changing the sequence TCC (S) ATG (M) GAT (D) to TCT (S) ATG (M) GAT (D) using primers 5′-GGT CAA TCT GGC TCT ATG GAT GAA ACC TTT G-3′ (forward) and 5′-CAA AGG TTT CAT CCA TAG AGC CAG ATT GAC C-3′ (reverse). Terminal BstEII and NcoI sites were added to the gene by PCR, using primer 5′-TCC ACC ATG GAG GAT CAT CTT-3′ (forward) and 5′-AGC TGG TCA CCT CGA GTC AAT G-3′ (reverse) and the Gus-gfp5 fragment of the pCAMBIA1303 vector was replaced by the LeNHX2-RGS(H)10 fragment.
The construct for LeNHX2 silencing was generated by cloning a 602-bp PCR fragment (bp 704–1306) in sense and antisense orientation into the pKANNIBAL vector (Wesley et al., 2001) using primers 5′-CGC GGA TCC TCG AGT CAG CTG GTG TTG GAG-3′ (BamHI and XhoI sites underlined) and 5′-CGA CAA GCT TGA ATT CCA TGG TTC CTG CCG-3′ (HindIII and EcoRI sites underlined). The plant expression cassette was moved from pKANNIBAL into the binary pART27 plant transformation vector and used for Agrobacterium tumefaciens-mediated transformation of tomato (Solanum lycopersicon L.) cotyledons as described by Ellul et al. (2003).
Subcellular localization of a LeNHX2:GFP translational fusion in onion epidermal cells
An LeNHX2:GFP translational fusion was created using the GFPmut1 variant with enhanced fluorescence which is optimized for translation in eukaryotic cells (Cormack et al., 1996). The C-terminus of LeNHX2 and the N-terminus of the GFP polypeptides were modified by PCR using oligonucleotides LeNHX2-Not: 5′-CCA CCG CGG CCG CCG TGG CCG TCA TAA CCA GC-3′ and GFP-Not: 5′-GCT GGC GGC CGC GGT GGT GTG AGC AAG GGC GAG GAG CTG-3′ (NotI recognition sites underlined). NotI digestion of amplified sequences and subsequent ligation generated an in-frame fusion of GFP to the C-terminus of LeNHX2. Subcellular localization in plant cells was performed by monitoring the transient expression of LeNHX2:GFP in onion epidermal cells after DNA particle bombardment using the vector pGreen-35S (http://www.pgreen.ac.uk). Plasmid DNA was coated onto gold particles (1 µm) and delivered into onion cells using a Biolistic PDS-1000-He apparatus (BioRad, Munich, Germany). The parameters used were: rupture disks bursting pressure, 900 psi; distance to macrocarrier, 8 mm; distance to stopping screen, 6 mm; distance to target tissue, 6 cm. Onion epidermal cells were placed onto Murashige and Skoog basal salt medium (MS) (Murashige & Skoog, 1962) containing 2% sucrose before bombardment and then incubated in the dark for 36 h at 28°C after particle delivery. GFP fluorescence was visualized under a Zeiss Axioskop microscope (Zeiss, Hamburg, Germany) equipped with a GFP filter set (exciter 450–490 nm; dichroic 495 nm; emitter 500–550 nm; Chroma set 41017). Confocal microscopy imaging was performed on a Leica TCS SL DMRE (Leica, Wetzlar, Germany) confocal laser scanning microscope. Serial optical sections of 0.5–2 µm were obtained and green fluorescent signal was acquired using a 488-nm excitation laser line with emission signals being collected at 525 ± 50 nm.
Plant transformation and selection
The plasmid pCAMBIA 1303 carrying the LeNHX2-RGS(H)10 fragment was transferred into the GV3501 A. tumefaciens strain and used for A. thaliana (ecotype Columbia) transformation. Transformation of A. thaliana plants was performed by the floral dip procedure (Clough & Bent, 1998). Transgenic plants were screened in vitro on MS medium supplemented with 50 mg l−1 hygromycin. Twelve lines of the T2 progeny, mono-locus for the transgene, were selected from 70 hygromycin-resistant T1 plants. Among those lines, three independent homozygous lines of the T3 progeny were selected randomly for further studies. The presence of the transgene in the selected lines was verified by PCR analysis using LeNHX2-specific primers (5′-CCT TTG AGG GGA ACA ATG G-3′ and 5′-CAT CTT CAT CTT CGT CTC C-3′) and genomic DNA obtained from 50-mg A. thaliana rosettes as described by Dellaporta et al. (1983).
Transgenic S. lycopersicon plants cv. PE-73 harboring the construct for LeNHX2 silencing were obtained as described by Ellul et al. (2003). Following self-fertilization of T0, T1 plants were selected for antibiotic resistance by germinating the seeds on media supplemented with 200 mg l−1 kanamycin. The presence of the integrated intron-hairpin construction in selected tomato transgenic lines was assessed by PCR analysis using pKANNIBAL-specific primers (5′-GTT CAT TTC ATT TGG AGA-3′ and 5′-CGT CTT ACA CAT CAT CAC TTG-3′) and DNA obtained from tomato leaves following a method for quick genomic DNA preparation for PCR (http://iprotocol.mit.edu/protocol/122.htm).
Analysis of silencing by real-time PCR
Real-time PCR was performed using platinum Taq DNA polymerase (Gibco, Gaithersburg, MD, USA) and SYBR-Green as a fluorescent reporter in a BioRad iCycler. RNA was isolated from leaves using the TRI® RNA Isolation Reagent (Sigma, Madrid, Spain) and first-strand cDNA was synthesized using the Transcriptor first-strand cDNA synthesis kit (Roche, Barcelona, Spain) using the random hexamer primers provided. Gene-specific primers were designed for a 173-bp fragment of LeNHX2 (forward 5′-CCT TTG AGG GGA ACA ATG G-3′, reverse 5′-CAT CTT CAT CTT CGT CTC C-3′). The identity of the amplified fragment was confirmed by gel electrophoresis and sequencing. Serial dilutions of cDNA were used to make a standard curve to optimize amplification efficiency. All reactions were performed in triplicate. Melt curves of the reaction products were generated and fluorescence data were collected at a temperature above the melting temperature of nonspecific products. Relative expression data were calculated from the difference in threshold cycle (ΔCt) between the studied gene (LeNHX2) and DNA amplified by primers specific for 18S ribosomal DNA (5′-AAT ATA CGC TAT TGG AGC TGG-3′ and 5′-ATG GCT CAT TAA ATC AGT TAT-3′). The expression level was calculated from 2EXP[ΔCt (control) – ΔCt (sample)].
Plant growth conditions and phenotypic evaluation of transgenic plants
Phenotypic analysis of A. thaliana plants overexpressing LeNHX2-RGS(H)10 was performed in T3 plants from lines 213, 243 and 357. Seeds were surface-sterilized with 70% ethanol for 1 min and then a solution of 70% commercial bleach and 0.02% Tween 20 for 5 min and then rinsed three times with sterile distilled water. Seeds were germinated both on Petri dishes and on 200-µl pipette tips (Norén et al., 2004). Seedlings were cultivated on Petri dishes in a medium composed of the MS basal salts, 1% sucrose and 0.7% agarose with pH adjusted to 6.0 with KOH or NaOH. Potassium starvation and NaCl treatments in A. thaliana seedlings on Petri dishes consisted of seed germination and seedling growth for 3 wk in media supplemented with 50 mM NaCl or modified to decrease the K+ content to a final concentration of 100 µM NO3 K, while nitrate was supplied in all MS media as NH4 (20 mM). Germination on pipette tips was carried out for plants that were to be transferred to hydroponic cultivation systems. The media used for germination on pipette tips contained a sterile growth gel composed of the basal mineral solution described by Cellier et al. (2004) and 0.7% agarose, with the pH adjusted to 6.0 with KOH or NaOH. Ten-day-old A. thaliana seedlings growing on pipette tips (four leaves) were transferred to hydroponic culture and grown for an additional 4 wk in the same nutrient solution, which was renewed twice a week during the first 2 wk and every 2 d thereafter. Potassium starvation and NaCl treatments were also performed on 4-wk-old hydroponic A. thaliana cultures under the conditions described by Cellier et al. (2004). Briefly, for K+ starvation, plants were transferred to K+-free medium after rinsing the roots in 0.2 mM CaSO4 and for salt treatment plants were transferred to the basal nutrient solution supplemented with 100 mM NaCl. Plants were maintained for 7 d in the K+-free medium or in the NaCl-supplemented medium. The growth-chamber environmental parameters were as follows: light/dark cycle 12/12 h, light intensity 200 µmol s−1 m−2, temperature 21°C.
Phenotypic evaluation of tomato plants carrying the construct for LeNHX2 silencing was performed on T0 and T1 plants. T0 seedlings rooting on the selective rooting medium (Ellul et al., 2003) were cultivated in seedbeds containing a mixture of peat:vermiculite (1 : 1) for 2 wk and then transferred to 10-l pots containing peat. Plants were cultivated in a glasshouse with supplemental lighting of 120 µmol s−1 m−2, 16 h light per day and temperature controlled to 26°C, and were watered every 2 d with 1/8 Hoagland mineral solution (Hoagland & Arnon, 1950) until fruits were harvested. T1 seedlings from lines 7, 11, 12, 14 and 44 were selected by in vitro germination in media composed of MS basal salts and supplemented with 200 mg l−1 kanamycin. Seedlings with four leaves were transferred from the selective medium to polystyrene boxes containing quartz sand, and watered for 1 wk with 1/10 Hoagland nutrient solution and for another week with a 1/4 dilution of the same solution. Plants were then transferred to pots for hydroponic cultivation for an additional 2 wk. Typically, four plants were cultivated in 3-l pots containing an aerated 1/4 Hoagland nutrient solution. For salt treatment, 130 mM NaCl was added to the nutrient solution for the last 10 d of the experiment. Cultivation medium was renewed every 2 d to avoid contamination.
Phenotypes of transgenic A. thaliana and tomato were evaluated on five randomly selected plants per treatment in terms of fresh weight of roots, shoots, stems and leaves at the end of the experiment.
Preparation of internal membrane fractions and determination of K+/H+ and Na+/H+ exchange in membrane vesicles
Endomembrane-enriched vesicles were prepared from A. thaliana root or shoot tissues according to Cheng et al. (2003). Antiporter activity was assayed by monitoring the relaxation of a pre-established ΔpH gradient created by the action of the vacuolar H+-ATPase in membrane vesicles. One hundred µg of protein was resuspended in 2 ml of assay medium containing 5 mM bis-tris propane (BTP)-4-morphdineethanesulfonic acid (MES), pH 7.5, 50 mM tetramethyl ammonium (TMA)-Cl, 1 mM ATP-BTP, pH 7.5, 300 mM sorbitol and 1 µM 9-amino-6-chloro-2-methoxyacridine (ACMA). Upon addition of 3 mM MgSO4 to the reaction medium, a pH gradient was established by the action of the V-type H+-ATPase, resulting in quenching of the ACMA fluorescence. When a stable level of fluorescence quenching was obtained, the antiporter reaction was started by the addition of 50 mM K2SO4 or Na2SO4 to the medium. At the end of the reaction, the pH gradient was abolished by addition of the K+/H+ ionophore nigericin (0.5 µM) or the Na+/H+ ionophore monensin (0.5 µM). Reactions were performed in disposable cuvettes to avoid contamination with ionophores. The total fluorescence recovery, denoted as the difference between the maximum fluorescence quenching obtained after energization of the ATPase and the final level of fluorescence observed after addition of the ionophores, was normalized to 100%. Fluorescence was monitored in a thermostatted cell at 27°C, using a fluorimeter (model QM2000; Photon Technology International, Sussex, UK) at excitation and emission wavelengths of 415 and 485 nm, respectively.
Immunological detection of LeNHX2 in transgenic plants
Endomembrane-enriched vesicles were obtained from transgenic and untransformed control A. thaliana roots or shoots according to Cheng et al. (2003). The histagged LeNHX2 protein was partially purified from this membrane fraction by Ni2+-affinity chromatography, essentially according to Venema et al. (2003), with some modifications. Briefly, the membrane fraction (2 ml, 0.5 mg protein ml−1 in 100 mM Tris-HCl, pH 7.5, 20% glycerol, 0.1 mM dithiothreitol and 0.1 mM ethylenediaminetetraacetic acid (EDTA)) was mixed with 20 ml of solubilization buffer (50 mM KH2PO4, pH 7.4, 500 mM NaCl, 10 mM imidazole, 20% glycerol and 0.5% n-dodecyl-β-D-maltoside) supplemented with 40 µl of protease inhibitor cocktail (Sigma) and incubated for 30 min at 4°C under gentle shaking. Unsolubilized material was removed by centrifugation for 30 min at 30 000 g. The supernatant was mixed with 100 µl of Ni-NTA resin (Qiagen, Chatsworth, CA, USA) and incubated overnight at 4°C. The resin was then poured into a Pasteur pipette, sealed with glass wool at the bottom, and prewashed with an imidazole step-gradient of 400-µl fractions containing 20, 50, 75 and 100 mM imidazole (pH 7.4) in 50 mM KH2PO4 (pH 7.4), 500 mM NaCl, 10% glycerol, 0.075% n-dodecyl-β-D-maltoside, 2 µg ml−1 pepstatin and 0.2 mM phenylmethylsulfonyl fluoride (PMSF). Finally, bound protein was eluted slowly in 400 µl of 20 mM BTP-MES, pH 7.4, 500 mM imidazole, pH 7.4, 10% glycerol supplemented with 2 µg ml−1 pepstatin and 0.2 mM PMSF. Two hundred µl of the eluted protein was precipitated with trichloroacetic acid and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) on 10% acrylamide using the system of Laemmli (1970) and electrotransferred to polyvinylidene difluoride (PVDF) membranes as described previously (Kerkeb et al., 2002; Venema et al., 2003). After protein electrotransfer, the blot was incubated with a monoclonal antibody raised against the RGSH4 epitope (Qiagen).
Determination of ion content
For ion content measurements, plant material was dried for 48 h at 80°C. Dried material of separated roots and shoots was milled to powder and digested in a concentrated HNO3:HClO4 (2 : 1, volume/volume (v/v)) solution. K+ and Na+ concentrations were determined in the digested material by inductively coupled plasma spectrometry (Iris Intrepid II; Thermo Electron Corporation, Franklin, MA, USA).
Protein was determined by the method of Schaffner & Weissmann (1973) with bovine serum albumin as standard.
All data in this report were obtained from at least three independent experiments with two or three replicates each. For data analyzed with Student's t-test the differences between treatments were considered as significant when P < 0.05 or 0.01 in a two-tailed analysis.
Characterization of A. thaliana plants overexpressing LeNHX2
The histagged LeNHX2 protein was overexpressed in A. thaliana plants using a 35S promotor. The T3 plants used in this study were homozygous and mono-locus for the transgene as demonstrated by hygromycin-resitance segregation analysis of the transgenic plants (data not shown). In addition to selection on MS media (Murashige & Skoog, 1962) supplemented with 50 mg l−1 hygromycin, the presence of the LeNHX2 construct in the three randomly selected A. thaliana lines used in this study (213, 243 and 357) was confirmed by PCR analysis of genomic DNA extracted from these plants (data not shown).
To demonstrate the presence of the LeNHX2-RGS(H)10 protein in the transgenic A. thaliana plants, western blotting on internal membrane fractions was performed, using a monoclonal antibody against the RGS(H)4 epitope. Especially in shoots, this antibody cross-reacted with many proteins (data not shown). For this reason, a partial purification of the protein by Ni2+-affinity chromatography was performed. After purification the protein was clearly detected in internal membrane fractions of both shoots and roots in transformed but not in untransformed plants (Fig. 1). The apparent size of the protein coincided with the size of the protein obtained from microsomal membrane fractions of yeast expressing LeNHX2-RGS(H)10. (K+,Na+)/H+ antiporter activity was assayed in internal membrane fractions of untransformed control and transgenic plants, by following the relaxation of the pH gradient created by the V type H+-ATPase (Fig. 2). Although transgenic plants also exhibited slightly lower rates of ATP-dependent H+ transport (Fig. 2a–d), calculation of initial rates of fluorescence relaxation after addition of K2SO4 or Na2SO4 clearly showed that plants expressing LeNHX2 exhibited a higher Na+/H+ and an even higher K+/H+ exchange activity (Fig. 2e). As it was previously shown that the LeNHX2 protein expressed in yeast does not localize to the vacuolar membrane, but fractionates together with Golgi and prevacuolar markers (Venema et al., 2003), we studied the localization of LeNHX2 in plants in more detail by transient expression of a GFP fusion protein in onion epidermis cells. GFP fluorescence was observed in discrete regions, and appeared present in small vesicles but not in the vacuolar membrane itself (Fig. 3a,f). The fluorescence pattern was clearly different from the pattern observed for the tonoplast-localized proteins AtNHX1 and AtNHX2 (Fig. 3b,c) or soluble cytoplasmic GFP (Fig. 3d).
Phenotypic analysis of transgenic plants in response to salt stress and K starvation
In normal growth conditions, no difference between untransformed control and transgenic plants was observed (Figs 4, 5). As the functional analysis had shown that the histagged LeNHX2 protein has a high K+/H+ exchange activity, the effect of varying K+ concentrations in the growth medium was studied. At a low K+ concentration (100 µM) and in the absence of K+, plants expressing LeNHX2-RGS(H10) remained smaller than untransformed controls. This effect was seen particularly in seedlings grown in Petri dishes, while in hydroponically grown plants root growth seemed to be especially affected (Figs 4, 5). The effect of LeNHX2-RGS(H)10 expression on A. thaliana salt tolerance was also studied. Control plants were greatly affected by 50 and 100 mM NaCl. In all tested conditions, transgenic plants were more tolerant to salt stress than untransformed controls, which was reflected in better growth and higher fresh weight (Figs 4, 5).
Analysis of ion content in A. thaliana
The Na+ and K+ content of control and transgenic A. thaliana plants was analysed in plants grown hydroponically (Fig. 6). In roots, only minor differences in K+ and Na+ content between transgenic and control plants were observed. An NaCl-induced increase in root Na+ concentration was accompanied by a decrease in K+ concentrations to similar extents in control and transgenic plants. In shoots, however, control plants accumulated more Na+ than transgenic plants, while K+ concentrations were higher in transgenic plants, leading to much more favorable K+:Na+ ratios in shoots of transgenic plants cultivated with NaCl. When plants were grown in low K+ concentrations, the K+ content of shoots especially was affected. The difference in total K+ content between control and transgenic plants was, however, not significant, although shoots of transgenic plants contained marginally more K+ than control plants.
Study of LeNHX2 silencing in tomato
The degree of silencing of LeNHX2 in the primary transgenic lines (T0) was studied by real-time PCR (Table 1). Phenotypic analysis showed that the T0 plants that exhibited a high degree of silencing grew less than untransformed controls in normal growth conditions (Table 1, Fig. 7). Plants that showed a high degree of silencing (> 70%) did produce much fewer or no fruits and no seeds (Table 1). Only from line 20, which exhibited a high degree of silencing (93%), were some seeds obtained. The correlation between LeNHX2 silencing and plant growth was confirmed in T1 plants derived from this line. As seen in Fig. 8, significant growth inhibition was observed for the strongly silenced line 20 T1 plants but not for azygous T1 plants of the same line. The effect of LeNHX2 silencing on tomato salt sensitivity was studied in hydroponic cultures of T1 plants. As strongly silenced T0 plants did not produce seeds, this experiment could be performed only in lines 7, 11, 12, 14 and 44 with a moderate degree of silencing. Plants harboring the silencing construct were selected by germination on 200 mg l−1 kanamycin-supplemented medium. In both silenced and untransformed tomato plants, treatment with 130 mM NaCl decreased the fresh and dry weight of roots, leaves and stems (Table 2). However, the NaCl-induced decrease in fresh and dry weight was higher in leaves and stems of the silenced lines than in leaves and stems of the untransformed control.
|Plant line||Expression (%)||Fruit number|
|Line||0 mM NaCl||130 mM NaCl|
|Control||43.4 ± 4.2||55.3 ± 6.5||33.8 ± 2.6||23.8 ± 2.9||45||12.6 ± 1.0||77||12.2 ± 1.2||64|
|L7||34.8 ± 3.0||41.0 ± 4.3||19.5 ± 1.7||17.6 ± 2.2||49||7.2 ± 0.8||83||7.6 ± 0.6||61|
|L11||26.7 ± 3.1||30.0 ± 2.6||17.3 ± 1.4||10.2 ± 1.0||62||4.1 ± 0.5||86||5.8 ± 0.7||66|
|L12||38.2 ± 2.7||45.4 ± 6.0||23.6 ± 2.4||18.0 ± 1.6||53||7.7 ± 1.0||83||9.6 ± 1.2||59|
|L14||33.3 ± 2.3||38.2 ± 2.8||23.5 ± 1.6||12.7 ± 0.9||62||6.6 ± 0.8||83||8.4 ± 0.3||64|
|L44||23.1 ± 2.2||26.4 ± 2.5||15.6 ± 1.1||8.0 ± 0.8||65||4.4 ± 0.4||83||6.0 ± 0.7||62|
We have recently identified the first intracellular K+/H+ antiporter in plants, LeNHX2. Although the LeNHX2 gene, when expressed in yeast, improves salt tolerance, we showed that the histagged protein catalyzes K+/H+ exchange in vitro, and that yeast cells accumulate K+ in intracellular compartments upon expression of the gene (Venema et al., 2003). In this study the function of this gene was analyzed in more detail by overexpression and silencing in plants. Arabidopsis thaliana has a very efficient, fast, and high-throughput transformation system when compared with that available for tomato (Clough & Bent, 1998). This advantage has prompted many researchers to express tomato genes in A. thaliana as a rapid method of analyzing their potential function (Mysore et al., 2001), although caution is still necessary when interpreting the results.
The histagged LeNHX2 gene under control of a 35S promotor could be expressed in A. thaliana plants. Western blotting experiments indicated that the protein expressed in plants was of the same size as previously obtained by expression in yeast (Fig. 1; Venema et al., 2003). The study of subcellular localization by transitory expression of a GFP fusion protein in onion epidermis cells showed that the LeNHX2 protein is not targeted to the tonoplast (Fig. 3). Fluorescence was observed in small vesicles, clearly different from the pattern observed for AtNHX1 or AtNHX2, which are present in the vacuolar membrane (Fig. 3; Yokoi et al., 2002). The localization pattern observed for the LeNHX2 protein was identical to that observed for the A. thaliana homolog AtNHX5 (B. Cubero & J.-M. Pardo, unpublished results). These data confirm the results obtained when the LeNHX2 protein was expressed in yeast, showing a cofractionation of the functional histagged LeNHX2 protein with Golgi and prevacuolar markers (Venema et al., 2003), and show that the plant NHX proteins of this clade have an endosomal/prevacuolar localization similar to the localization observed for other nonplant NHX isoforms (Pardo et al., 2006). As the LeNHX2 protein does not localize to the tonoplast, we isolated membrane fractions enriched in total endosomal membranes, in order to study antiporter activity. It was shown before that histagged LeNHX2 purified from yeast cells and reconstituted in liposomes mainly catalyzes K+/H+ antiport (Venema et al., 2003). These findings are now confirmed for the histagged LeNHX2 protein expressed in plants, using vesicles from intracellular membranes (Fig. 2). A high background K+/H+ and especially Na+/H+ exchange activity is observed in control plants, attributable to the normal set of cation/proton antiporters present in A. thaliana endomembranes, including the vacuole. In transgenic plants, K+/H+ exchange activity is enhanced approx. 100%, while the increase in Na+/H+ exchange activity is only c. 30% (Fig. 2). In absolute terms, K+/H+ exchange activity increases two times more than Na+/H+ exchange activity in vesicles derived from overexpressing plants as compared with control plants. It can thus be deduced that the LeNHX2-RGS(H)10 protein functions mainly as a K+/H+ antiporter protein in plants also (Fig. 2), although some caution should still be observed, as heterologous expression of proteins could modify the ion selectivity of NHX antiporters, particularly if ion selectivity by individual NHX proteins is under physiological control. The fact that LeNHX2 increases antiport activity substantially in membrane fractions from transgenic plants, as compared with control plants, indicates that the LeNHX2 protein is present in a significant fraction of the isolated subcellular membrane fraction. In conclusion, there appear to be no differences in the localization and molecular characteristics of the LeNHX2 protein expressed in plants and yeast. These findings strengthen the view that the LeNHX2 protein is involved in the regulation of the intracellular or endosomal K+ concentration and pH, having an indirect effect on salt tolerance through the modulation of these parameters. Both silencing and overexpression experiments in plants confirm this hypothesis.
Arabidopsis thaliana plants that overexpressed the histagged LeNHX2 antiporter grew equally well in normal growth conditions compared with control plants, but in limiting K+ concentrations growth was somewhat inhibited (Figs 4, 5). It is well known that, in conditions of limiting K+ supply, vacuolar K+ concentrations tend to decrease in favor of cytoplasmic K+ concentration (Walker et al., 1996). It is thus likely that the increased K+ accumulation in internal compartments in LeNHX2-RGS(H)10 overexpressing plants causes growth inhibition in such conditions, as it would negatively affect cytoplasmic K+ concentrations, causing the observed growth inhibition. At the same time, transgenic plants were more salt tolerant than control plants. Enhanced K+ accumulation would be beneficial in salt stress conditions as it would permit plants to maintain osmotic balance, without the need for uptake and vacuolar accumulation of Na+, with concomitant cytoplasmic intoxication. At the same time, increased K+ reserves could be used in order to maintain cytoplasmic K+ concentrations. Accordingly, we found that transgenic plants were more salt tolerant, and accumulated more K+. As LeNHX2-overexpressing plants also exhibited increased Na+/H+ exchange activity (Fig. 2), transgenic plants could also display enhanced salt tolerance as a result of sequestration of Na+ in vesicles. Strikingly, however, transgenic plants also contained less Na+ in aerial parts in Na+ stress conditions, indicating that reduced Na+ influx or increased Na+ efflux in these plants outweighed increased Na+ accumulation by LeNHX2. Possibly, the altered cytoplasmic K+ concentration or pH in transgenic plants modulates the selectivity and activity of K+ uptake mechanisms, thereby reducing net Na+ uptake. In this respect, it is well known that K+ deficiency induces several high-affinity K+ uptake mechanisms, which are more specific for K+ (Ashley et al., 2006; Rodríguez-Navarro & Rubio, 2006). Another possibility is that LeNHX2 overexpression affects the activity or abundance of other transporters by interfering with protein synthesis or targeting. In this sense, it has been reported that, in yeast, pH regulation in late endosomes or the prevacuolar compartment by the ScNHX1 protein is important for vesicle trafficking and protein targeting (Brett et al., 2005b). This would explain why there are such big changes in ion content, considering that the LeNHX2-RGS(H)10 protein is present only in small vesicles, and not in the big central vacuole. Indeed, the fact that transgenic plants exhibit slightly lower V-ATPase activities in endosomal membrane fractions (Fig. 2a–d) indicates that LeNHX2 expression has some effect on the abundance or activity of other ion transporters, as has been reported before for other ion transporters (Cheng et al., 2003).
To silence the LeNHX2 gene, a 613-bp fragment from the C-terminal end of this gene was used. On a nucleotide basis, this fragment shows only 47–50% homology to the other known tomato NHX antiporters of the vacuolar clade, LeNHX1, LeNHX3 and LeNHX4. The observed phenotypes are therefore not likely to be attributable to unspecific inhibition of these LeNHX isoforms (Kumar et al., 2006). LeNHX2 is most similar to the A. thaliana putative endosomal antiporters AtNHX5 and AtNHX6, with 72 and 70% identity on a nucleotide basis, respectively. Therefore, we cannot rule out the possibility that the used fragment results in silencing of more endosomal LeNHX isoforms in tomato.
We have obtained a total of 14 tomato independent primary transformants (T0). We could observe a clear correlation between the degree of silencing of the LeNHX2 gene and the general performance of the plants (Table 1, Fig. 7). Compared with controls, plants that displayed a high degree of silencing were extremely sensitive to watering with nutrient solution, which had to be diluted to 1/8 in order to water silenced plants. Use of nondiluted nutrient solutions gave rise to wilting of the silenced plants, indicating perturbation of osmoregulation in those plants. Most plants in which LeNHX2 expression was strongly silenced produced no or very few fruits, and no seeds (Table 1). In only one case were T1 plants from a strongly silenced line obtained, in which the effects of LeNHX2 silencing on general plant growth were confirmed (Fig. 8). These data indicate that the LeNHX2 gene plays a fundamental role in plant development, affecting both turgor and growth of plants, as well as fruit and seed formation. Physiological studies have shown that K+ nutrition plays an important role in phloem loading and unloading, stimulating transport of assimilates and nutrients to sink tissues, and that, especially in tomato, proper K+ input is essential for fruit development and yield (Usherwood, 1985; Marschner, 1995). Perturbation of K+ homeostasis could thus explain the strong effect of LeNHX2 disruption on fruit formation. Similarly, it has been suggested, based on microarray experiments and T-DNA insertion experiments, that the AtNHX1 gene plays an important role in plant development in A. thaliana (Apse et al., 2003; Sottosanto et al., 2004). Because of this strong effect, it was difficult to assess the role in salt tolerance using this approach, as only moderately silenced plants could be studied in the T1 generation. It was, however, clear that LeNHX2 silencing also renders the plants more sensitive to salt stress (Table 2). The fresh weight of leaves and stems, especially, was affected more by salt treatment in the LeNHX2-silenced plants than in the control plants. We could not, however, detect any significant differences in K+ or Na+ ion content between control and silenced plants.
In summary, our data show that LeNHX2 plays a fundamental role in plant development, possibly by regulating intracellular K+ content. Additionally, it is shown that the LeNHX2 gene plays a role in salt tolerance. While silencing of the gene causes a severe phenotype, overexpression does not have many side effects, except that plants become more sensitive to low K+ concentrations. Plants that overexpress a histagged LeNHX2 protein are, however, markedly more salt tolerant, a phenotype that is related to increased K+, and decreased Na+ accumulation in aerial parts of the plant. This suggests that the LeNHX2 gene could be a suitable candidate for engineering more salt-tolerant crops.
The authors wish to thank Drs Michael Palmgren and José Manuel Pardo for critically reading the manuscript and Miss Ana Vílchez for excellent technical assistance. This work was supported by grants BIO2005-00878 from the Spanish Plan Nacional de Biotecnología (to KV) and AGR-436 from the Consejería de Innovación, Ciencia y Empresa, Junta de Andalucía (to MPR-R). XJ was supported by a grant from the Spanish Ministerio de Educación y Ciencia and BC by a grant of ‘Programa Averroes’ from the Consejería de Innovación, Ciencia y Empresa, Junta de Andalucía. FJG and MNA are fellows of the CSIC I3P Program.
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