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proton antiporter and pyrophosphatase proton pump
Plant Physiology and Biochemistry 43 (2005) 347–354 www.elsevier.com/locate/plaphy
Cloning and characterization of a wheat vacuolar cation/proton antiporter and pyrophosphatase proton pump Faïçal Brini a, Roberto A. Gaxiola b, Gerald A. Berkowitz b, Khaled Masmoudi a,* a
b
Plant Molecular Genetics Unit, Center of Biotechnology of Sfax, B.P’K’, 3038 Sfax, Tunisia Department of Plant Science, Agricultural Biotechnology Laboratory, University of Connecticut, 1390 Storrs Road, Storrs, CT 06269-4163, USA Received 12 July 2004; accepted 16 February 2005 Available online 17 March 2005
Abstract Sodium at high millimolar levels in the cytoplasm is toxic to plant and yeast cells. Sequestration of Na+ ions into the vacuole through the action of tonoplast proton pumps (an H+-ATPase in the case of yeast, and either a H+-pyrophosphatase (H+-PPase) or H+-ATPase in the case of plants) and a Na+/H+ antiporter is one mechanism that confers salt tolerance to these organisms. The cloning and characterization of genes encoding these tonoplast transport proteins from crop plants may contribute to our understanding of how to enhance crop plant response to saline stress. We cloned wheat orthologs of the Arabidopsis genes AtNHX1 and AVP1 using the polymerase chain reaction and primers corresponding to conserved regions of the respective coding sequences, and a wheat cDNA library as template. The wheat NHX cDNA cloned by this approach was a variant of the previously reported TNHX1 gene. The vacuolar H+-PPase pump we cloned (TVP1) is the first member of this gene family cloned from wheat; it is deduced translation product is homologous to proteins encoded by genes in barley, rice, and Arabidopsis. Function of TNHX1 as a cation/proton antiporter was demonstrated using the nhx1 yeast mutant. TNHX1 was capable of suppressing the hyg sensitivity of nhx1. Functional characterization of the wheat H+-PPase TVP1 was demonstrated using the yeast ena1 (plasma membrane Na+-efflux transporter) mutant. Expression of TVP1 in ena1 suppressed its Na+ hypersensitivity. Expression analysis of salt-stressed wheat plants showed substantial up-regulation of TNHX1 transcript levels as compared to control plants, while transcript accumulation for TVP1 was not greatly affected by exposure of plants to salt stress. © 2005 Elsevier SAS. All rights reserved. Keywords: Na+/H+ antiporter; H+-pyrophosphatase; Sodium sequestration; Salt tolerance; Transcript accumulation; Wheat
1. Introduction Salinity is a major constraint to crop (including wheat [22]) productivity; reducing yields on saline soils and limiting expansion of agriculture onto previously uncultivated land [8]. 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
Abbreviations: H+-PPase, proton pumping pyrophosphatase; hyg, hygromycin; ORF, open reading frame; UTR, untranslated region; YNB, yeast nitrogen base; YPD, yeast extract/peptone/dextrose; YPGAL, yeast extract/peptone/galactose. * Corresponding author. Tel.: +216 74 440 816x1092; fax: +216 74 440 818. E-mail address: [email protected] (K. Masmoudi). 0981-9428/$ - see front matter © 2005 Elsevier SAS. All rights reserved. doi:10.1016/j.plaphy.2005.02.010
ion accumulation in the cytosol [3,12,15,25]. The capacity for vacuolar compartmentalization of Na+ and Cl– is an adaptation mechanism conserved in halophytes and glycophytes [3,15]; 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 intracellular osmotic status of cells in salt grown plants [27]. Cellular ion exclusion cannot provide complete adaptation of plants to high soil salinity, presumably because of the osmotic stress component of salinity stress [3]. Continued cell growth would be restricted under osmotic stress because of an unfavorable water balance, limiting the water uptake necessary for cell expansion. The integrated processes of cell division and expansion (i.e. growth) require substantial water uptake; cells increase their volume after division by as much as 100-fold
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[20]. Vacuolar expansion is a primary mechanism underlying this massive cell enlargement. Energy-dependant Na+ transport (i.e. against a concentration gradient) across plant cell membranes (plasma lemma and tonoplast) is usually coupled to the proton (H+) electrochemical potential established by H+-translocating pumps [3,13,15,31]. H+ transport across these membranes increases with salt treatment and may be attributed both to pump activation and enhanced transcription [15]. Plasma membrane and tonoplast transporters facilitate Na+ efflux from the cytosol by coupling Na+ transport to the (energetically favorable) transport of H+. Both plasma membrane and tonoplast Na+/H+ antiporter activities increase in response to salt treatment, at least in halophytic species [3]. Overexpression of the vacuolar Na+/H+ antiporter and H+-pyrophosphatase pump (H+PPase) has resulted in enhanced plant tolerance to both salinity [2,12,36], and drought stress [12]. In Saccharomyces cerevisiae the primary pathway for Na+ extrusion is mediated by Ena1 [14,28], the plasma membrane Na+-ATPase. Ena1 yeast mutants are hypersensitive to high growth medium Na+, and expression of the Arabidopsis tonoplast H+-PPase suppresses this mutant phenotype presumably due to increased capacity for Na+ sequestration in the yeast vacuole [11]. Sequestration of cytosolic Na+ into the yeast vacuole occurs through the action of Nhx1 [23]. Recent analyses of the genes involved in cation detoxification in yeast have led to a model in which the Nhx1 Na+/H+ exchanger acts in concert with the vacuolar ATPase and the Gef1 anion channel to sequester cations in a prevacuolar compartment [10,24]. This model posits that sequestration of sodium by Nhx1 depends on the vacuolar H+-ATPase and Gef1, the chloride channel. Gef1-mediated anion influx allows
establishment by the vacuolar H+-ATPase of a proton gradient sufficient in magnitude to drive Na+ accumulation in the vacuole (against a concentration gradient) via Na+/H+ exchange. In this study, we describe the molecular cloning of two wheat cDNAs encoding the tonoplast H+-PPase and Na+/H+ antiporter, their expression patterns in salt-stressed plants, and their functional characterization using heterologous expression in Na+-sensitive yeast mutants.
2. Results 2.1. Molecular characterization of wheat TNHX1 and TVP1 The full-length cDNAs of TNHX1 and TVP1 were cloned and sequenced as described (see Section 4). Sequence analysis of the TNHX1 cDNA revealed an open reading frame (ORF) of 1641 bp with a 3′-untranslated region (UTR) of 207 bp. Alignment of this ORF (GenBank accession no. AY296910) with that of the previously cloned TNHX1 cDNA (GenBank accession no. AY040245) indicated differences at six positions. We attribute this to varietal differences in the genomes used to generate the corresponding libraries and consider our clone to be a variant of TNHX1. The TVP1 cDNA we cloned has an ORF of 2289 bp with a 5′-UTR of 30 bp and a 3′ noncoding region of 352 bp. A clade analysis of plant NHX vacuolar Na+/H+ antiporters, including the wheat cDNA cloned here, is presented in the phylogenetic tree shown in Fig. 1A. Wheat TNHX1 (shown in bold) forms a clade with the most closely related plant NHX homolog, HvNHX2 from
Fig. 1. Phylogenetic relationship between deduced protein sequences of plant vacuolar Na+/H+ antiporters (A) and vacuolar H+-PPase pumps (B). The Arabidopsis (’At’), wheat (’T’), barley (’Hv’), and rice (’Os’) Na+/H+ antiporters are included in the analysis shown in (A). The wheat (’T’), barley (’H’), rice (’O’), Arabidopsis (’A’), and tobacco (’Nt’) H+-PPase pumps are included in the analysis shown in (B). The wheat Na+/H+ antiporters (TNHX1 and TNHX2) can be paired into two phylogenetic subgroups, while the wheat H+-PPase forms a unique group with the barley H+-PPase HVP2. Multiple sequence alignment was performed using the CLUSTALW computer program [16]. The accession numbers for the protein sequences are as follows: AtSOS1 (AAF76139), Arabidopsis thaliana; six A. thaliana (AtNhx) isogenes, AtNhx1 (AF510074); AtNhx2 (AF490586); AtNhx3 (AC011623); AtNhx4 (AB015479); AtNhx5 (AC005287) and AtNhx6 (AC010793); OsNhx1 (AB021878), Oryza sativa (rice); HvNhx1 (AB089197) and HvNhx2 (AY247791), Hordeum vulgare (barley); TNhx1(AY296910) and TNhx2 (AY040246), T. aestivum (wheat); TVP-1 (AY296911), T. aestivum; HVP-1 (AB032839) and HVP-2 (D13472), H. vulgare; NtVP5 (X77915), Nicotiana tabacum (tobacco); OVP-1 (D45383), OVP-2 (D45384), OVP-3 (AB126350) and OVP-5 (AB126351), O. sativa; AVP-1 (M81892), AVP-2 (AF182813) and AVP-3 (AB015138), A. thaliana.
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barley. Fig. 1B shows a phylogenetic analysis of plant vacuolar H+-PPases. TVP1 (shown in bold) forms a clade with the barley vacuolar H+-PPase HVP2. The deduced amino acid sequence of TNHX1, and sequences of representative homologs from other species are shown in Fig. 2. Hydropathy analysis (not shown) of the TNHX1 sequence confirms the presence of the 12 transmembrane domains present in members of this protein family; these domains are identified in Fig. 2. Also shown (as a boxed area within transmembrane domain III in Fig. 2) is the amiloride binding motif that is common to these proteins [5]; this motif is present in TNHX1. The deduced amino acid sequence of the variant of TNHX1 that we cloned (GenBank accession no. AY296910) is 99% similar to the amino acid sequence of the TNHX1 previously reported (GenBank accession no. AY040245). Sequence comparison between TNHX1 and TNHX2 showed 75% identity at the nucleotide level, while no similarity was found between the 3′-UTR of TNHX2 and the 3′-UTR of TNHX1. Regions of the deduced amino acid sequence of TVP1 (and corresponding sequences of some homologs) are shown in Fig. 3. A region of the plant vacuolar H+-PPases that is highly conserved (G240-M287 in TVP1) is shown in Fig. 3A. Within this region, a putative pyrophosphatase catalytic site [20] is present (boxed region in Fig. 3A); this motif can also be found in the TVP1 sequence. Another functional domain present in plant vacuolar H+-PPases is the ’EYYTS’ motif shown in Fig. 3B that is involved in H+ transport [35]. It should be noted that all the plant vacuolar H+-PPases, including TVP1 (see Fig. 3B), contain a conserved glutamate residue (E221 in TVP1; E229 in AVP1) [37]. It has been suggested that this residue plays a role in coupling pyrophosphate hydrolysis to H+ translocation across the vacuolar membrane [7,35]. 2.2. Functional characterization of the wheat antiporter TNHX1 and vacuolar H+-PPase using yeast mutants Heterologous expression in yeast has been used in several studies to characterize the Arabidopsis vacuolar Na+/H+ antiporter AtNHX1 [6,11,34]. However, little functional analysis work has been done with NHX1 homologs from crop plants. Xia et al. [34] have recently expressed the sugar beet (Beta vulgaris) BvNHX1 antiporter in yeast for functional characterization. Here, we used a similar strategy to undertake the first characterization of a Na+/H+ antiporter cDNA cloned from the cereal crop wheat. Gaxiola et al. [11] and Darley et al. [6] demonstrated function of the Arabidopsis AtNHX1 cDNA as a vacuolar Na+/H+ antiporter by partial suppression of hygromycin (hyg) hypersensitivity of the nhx1 yeast mutant. Hygromycin is a cationic antibiotic that is toxic to cells upon accumulation in the cytosol. The basis for hyg hypersensitivity of the yeast nhx1 mutant is likely a reduction in NHX-mediated sequestration of the toxic cation in the yeast vacuole [1,35]. As shown in Fig. 4A, the expression of the wheat cDNA (TNHX1) we have identified as a putative vacuolar Na+/H+ antiporter does in fact partially complement
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the nhx1 mutant phenotype, suppressing hyg hypersensitivity. In the absence of hyg, the nhx1 mutant transformed with any of the plasmids (along with the wild type yeast) grew on YPD medium, but in the presence of hyg, the nhx1 mutant grew only when transformed with the plant TNHX1 cDNA (Fig. 4A). Yokoi et al. [35] has demonstrated that several of the six Arabidopsis NHX genes (AtNHX2, AtNHX5) in addition to AtNHX1 encode vacuolar Na+/H+ antiporters that complement yeast nhx1 sensitivity to hyg, as we report here for the wheat Na+/H+ antiporter TNHX1. In previous studies, Gaxiola et al. [11] used the Na+ hypersensitivity of the ena1 yeast mutant to demonstrate function of the Arabidopsis vacuolar H+-PPase. Results of this work are consistent with a model whereby expression of a plant vacuolar H+-PPase would allow for greater growth of ena1 in the presence of high external Na+ due to increased sequestration into the vacuole of Na+ that enters the yeast cytosol. In this prior work with the Arabidopsis H+-PPase AVP1 [11] a gain-of-function (E427D) mutant construct of AVP1 was used in studies employing the yeast ena1 for functional characterization. Here, we studied the wild type TVP1. We find that the TVP1 cDNA also suppresses the Na+ hypersensitivity of ena1 (Fig. 4B). All ena1 mutants along with the wild type yeast grew on YPGAL medium with no added NaCl, while the ena1 mutant grew in the presence of 0.5 M NaCl only when transformed with the plant TVP1 cDNA (Fig. 4B). Our results with TVP1 are consistent with prior studies of AVP1 expression in yeast [11,17], in suggesting that TVP1 is also an H+-PPase pump localized to the vacuolar and/or prevacuolar tonoplast membrane. In work with AVP1, Kim et al. [17] localized the plant protein to the yeast vacuole membrane as well as demonstrating PPase activity. However, we cannot discount the possibility that TVP1 suppression of the ena1 Na+ hypersensitivity occurs by TVP1 H+-pump action at the yeast plasma membrane. This could possibly suppress ena1 hypersensitivity to Na+ through the action of the yeast cell membrane Na+/H+ antiporter NHA1 [26]. The results shown in Fig. 4 suggest that the two wheat cDNAs TNHX1 and TVP1 can function in a model eukaryote (yeast) cell as a Na+/H+ antiporter and H+-PPase, respectively. 2.3. Southern blot analysis Southern blot analyses of genomic DNA isolated from wheat (Triticum durum) leaves were performed using probes corresponding to 3′-UTR sequences of the TVP1 and TNHX1 cDNAs (Fig. 5). As shown in Fig. 5A, a single band was detected in the case of TVP1 when DNA was subjected to digestion with HindIII, EcoRI, SacI, or XhoI. We conclude from this hybridization pattern that the wheat genome has a single copy of the TVP1 gene. Southern analysis of HindIII, EcoRI and XhoI-digested DNA probed with TNHX1 also identified one hybridizing band (Fig. 5B). This results shown in Fig. 5B indicate that the wheat genome has a single copy of the TNHX1 gene and further, that TNHX1 and TNHX2 are not allelic; i.e. that they are encoded by two separate genes in the
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Fig. 2. Multiple alignment of the deduced amino acid sequence of TNHX1, the Na+/H+ antiporter we cloned from wheat, along with the barley (HvNhx1), rice (OsNhx1), Arabidopsis (AtNhx1), human (HsNHE1), and yeast (ScNhx1) sequences. Identical residues are highlighted in black, residues with conservative substitutions are shaded in gray, and dashes indicate gaps in a sequence. Putative transmembrane domains are indicated by Roman numerals. The amiloride binding motif is framed and the consensus sequence is indicated by the boxed residues above the alignment in the transmembrane domain III region. Numbers flanking the sequences correspond to positions of amino acids directly next to the number.
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Fig. 3. Alignment of several conserved regions of the deduced amino acid sequence of the wheat H+-PPase TVP1 with corresponding regions of a barley (HVP1) and an Arabidopsis (AVP1) H+-PPase. Sequences were aligned using the Clustal X program. (A) A conserved region of the proteins flanking the putative H+-PPase catalytic site (shown within the box). (B) The conserved motif found in plant H+-PPases that is critical for H+-pumping by the protein. Numbers flanking the sequences correspond to positions of amino acids directly next to the number.
Fig. 4. Functional characterization of the wheat Na+/H+ antiporter TNHX1 (A) and the wheat H+-PPase TVP1 (B) using yeast mutants. (A) Growth of the nhx1 yeast mutant transformed with either the empty plasmid (Nhx1), or the TNHX1 cDNA. The yeast strains were grown on YPD medium with (+), or without (–) 0.05 mg ml–1 hyg. (B) Growth of the ena1 yeast mutant transformed with either the empty plasmid (ena1), or the TVP1 cDNA. The yeast strains were grown on YPGAL medium with (+) or without (–) 0.5 M NaCl. Growth of the isogenic wild type yeast strain (WT) is shown for both experiments. For both TNHX1 and TVP1, results are shown for two independent yeast clones (TNHX1-2; TNHX1-3; TVP1-1; TVP1-2). In all cases, growth of fourfold serial dilutions of each culture is shown. Experiments shown in this figure were repeated, with similar results (data not shown).
wheat genome. The nucleotide sequences of TNHX1 and TNHX2 have 75% identity. 2.4. Expression pattern of TVP1 and TNHX1 in wheat under salt stress In order to examine the effect of salt stress on the expression of the vacuolar Na+/H+ antiporter and H+-PPase genes, total RNA isolated from roots and shoots of wheat seedlings was subjected to Northern blot analyses. A Northern analysis of RNA isolated from control and salt-stressed plants using the 3′-UTR of TNHX1 as a probe is shown in Fig. 6A. Salt stress significantly increased the transcript levels of TNHX1 in roots and, to a lesser extent, in leaves. As suggested by the results of the Southern analysis shown in Fig. 5B, we can discount the possibility that the 3′-UTR TNHX1 probe hybridizes to TNHX2 message. In prior studies of Arabidopsis, Gaxi-
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Fig. 5. Genomic Southern blot analysis of wheat (T. durum) vacuolar H+-PPase (A) and Na+/H+ antiporter (B). Wheat genomic DNA (10 µg) was digested with the restriction endonucleases HindIII (H), EcoRI (E), XhoI (X) or SacI (S), and hybridized with either the 32P-labelled 3′-UTR specific TVP1 (A) or 3′-UTR specific TNHX1 (B). DNA fragments of a 1 kb ladder were used as molecular markers (indicated on the left of each panel).
Fig. 6. Analysis of TNHX1 (A) and TVP1 (B) expression under salt stress. Expression in roots and leaves of control wheat seedlings (C) and seedlings subjected to irrigation with 200 mM NaCl was monitored. Ribosomal RNA (rRNA) was used as a control. Experiments shown in this figure were repeated a second time with similar results (data not shown).
ola et al. [11] found increases in transcript level of the TNHX1 homolog AtNHX1 in salt-stressed Arabidopsis seedlings. Subsequently, Yokoi et al. [35] found that the transcript levels of several, but not all of the AtNHX genes increase in response to salinity stress. In contrast to the strong up-regulation of TNHX1 in roots of salt-stressed wheat seedlings, we found only a modest (if at all) effect of salinity stress on TVP1 expression in roots (Fig. 6B). Results shown in Fig. 6B also show no effect of salinity stress on the level of TVP1 transcript in leaves of saltstressed wheat seedlings.
3. Discussion Work presented in this report focuses on two vacuolar tonoplast membrane transport proteins of wheat, a Na+/H+ antiporter (TNHX1) and an H+-PPase (TVP1). It has been previously demonstrated that overexpression of the Arabidopsis H+-pyrophosphatase (AVP1) confers salt tolerance to yeast strains only when the yeast contains a functional vacuolar Na+/H+ exchanger (Nhx1) [11]. Thus, the Na+/H+ exchanger
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acts in concert with the vacuolar H+-PPase (and ATPase) to sequester cations in the vacuole (and prevacuolar compartment). Heterologous expression in yeast mutants allowed for an indirect characterization of their functions. The approach we take here to identify the cDNAs as encoding tonoplast ion transport proteins is consistent with the strategy used in prior reports to demonstrate function of the Arabidopsis homologs AVP1 and AtNHX1. Both genes were shown to be present in the T. durum genome. This work represents the first cloning and functional characterization of a vacuolar H+-PPase from the important cereal crop wheat. It has been over a decade since the first report of the cloning of a cereal crop (barley) H+-PPase [32]; since this time no functional characterization of a cereal crop vacuolar H+-PPase has been published. In addition to the characterization of TVP1 from wheat included in our work, we became aware during the preparation of this manuscript of a recently published paper reporting the functional characterization of the barley vacuolar H+-PPase [9]. The H+-PPases are considered to form a multigene family. Two cDNA clones (OVP1 and OVP2) encoding vacuolar H+-PPases isolated from rice were reported [29]. Indeed, there are more than five isoforms in rice, three isoforms in barley [32,21] and at least three isoforms in Arabidopsis. A highly conserved colinearity of genes between rice and wheat is well established [19]. Here, we isolate the first isoform from wheat. Further screening of the wheat cDNA library may lead to the isolation of other vacuolar H+-PPase isoforms. Recently, it has been reported in barley that salt stress increased the transcript level of a tonoplast H+-PPase (HVP1) in roots for a short time (5 h), and subsequently increased that of another isoform (HVP10) after treatment for 24 h [9]. In our case with TVP1, we find only a modest (if at all) increase in the transcript level in roots of salt-stressed plants as compared to TVP1 levels in roots of control plants. As was the case in barley, we found no detectable effect of salinity stress on expression of the vacuolar H+-PPase (TVP1) in leaves. In contrast, we found that the transcript level of the wheat Na+/H+ antiporter gene (TNHX1) increased greatly in roots and to some extent in leaves of wheat seedlings treated with 200 mM NaCl. It seems that the results of transcript accumulation indicate that the expression of TVP1 is not coordinated with that of TNHX1 in roots of wheat seedlings treated with NaCl. The greater increase of TNHX1 expression in roots treated with salt might be due to the accumulation of Na+ in the vacuoles of root cells. Unlike the results with wheat reported here, expression of the vacuolar H+-PPase and Na+/H+ antiporter appear to be coordinated in barley [9] subjected to salinity stress. Members of these families of plant proteins, tonoplast Na+/H+ antiporters and H+-PPases, are known to be important regulators of plant response to salinity stress. Salinity stress limits growth and yields of wheat in many regions of the world; enhanced sequestration of Na+ taken up into wheat leaves could be one strategy to engineer salinity tolerance. The results included in this report, therefore, are relevant to this important agricultural challenge. Further study of these
wheat ion transporter genes could lead to a better understanding of the molecular basis for salinity tolerance in an important cereal crop. In addition, over-expression of the Arabidopsis homologs of these genes has led to salinity (for both the antiporter and pyrophosphatase) and drought (in the case of the pyrophosphatase) tolerance. Our finding that both wheat genes, the Na+/H+ antiporter and H+-PPase, confer salt tolerance in yeast suggests it may be worthwhile to elucidate the contribution of these proteins to salt tolerance in a staple crop plant such as wheat. Engineering plants to increase functional levels of these proteins may be one strategy to develop tolerance to this important abiotic stress.
4. Methods 4.1. Yeast strains and plasmids All strains used are isogenic to W303 (ura3-1 can1100 leu2-3, 112trp1-1 his3-11, 15). Plasmids pRS8 and pYES2 (Invitrogen) were used to construct PMA1/TNHX1 and pGAL1/TVP1, respectively. The pRS8 plasmid was constructed (Mascorro-Gallardo and Gaxiola, unpublished data) using the pRS424 plasmid [4] as a backbone with the high expression level PMA1 (S. cerevisiae plasma membrane H+-ATPase) promotor [30] driving expression. The wild type W303, and the yeast mutant strains nhx1::HIS3 and ena1::HIS3 (see [11] for construction and/or source) were used for the work reported here. Cells were grown in YPD (1% (w/v) yeast, 2% (w/v) peptone, and 2% (w/v) dextrose; Difco; Franklin Lakes, NJ), or YPGAL (1% (w/v) yeast, 2% (w/v) peptone, and 2% (w/v) galactose; Difco). Transformation was performed using the EZ-Yeast transformation kit (BIO 101; Carlsbad, CA). 4.2. Plasmid constructs PCR products were generated from a wheat (Triticum aestivum) root tissue cDNA library. The full-length TNHX1 ORF was amplified with PfuTurbo DNA polymerase (Stratagene, La Jolla, CA) using wheat cDNA library as template and primers corresponding to the 5′ and 3′ ends of the wheat gene TNHX1 (GenBank accession no. AY040245) with BamHI and SphI, restriction sites added, respectively. These oligonucleotide primers were 5′-ATAAGAATGCGGCCGCGGCATGGGGCTCGATTT-3′, and 5′-ACATGCATGCCTGGGCTTCGACTTAACTAC-3′, respectively. The PCR product was cloned into the pCR2.1-Topo vector (Invitrogen, Carlsbad, CA), generating plasmid pTNHX1. After sequencing, the TNHX1 cDNA was digested with BamHI and SphI and cloned into pRS8 digested with the same restriction enzymes. The wheat vacuolar H+-PPase cDNA ’TVP1’ was cloned as follows. The 5′ end of the cDNA was amplified from the wheat library using a degenerate primer ’HvP2’ corresponding to the 5′ end of the barley H+-PPase HVP1 (accession no. AB032839) sequence (5′-TGGCAAGGYTGRCTCTTTG-
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TGCTGAAYCC-3′) and the T7 primer (5′-TAATACGACTCACTATAGGG-3′). The missing 3′ end of the TVP1 cDNA was amplified from the same wheat cDNA library using a 5′ primer corresponding to a conserved region of plant (barley, rice, Arabidopsis) vacuolar H+-PPases, and a 3′ primer corresponding to the polylinker region of the pYES2 plasmid. Forward and reverse primer sequences were 5′-ACCCGTACAGCATTGTTC-3′ and 5′-TGAATGTAAGCGTGACATAA-3′, respectively. The two PCR products generated from the above reactions (corresponding to the 5′ and 3′ regions of the target TVP1 cDNA) were cloned into the pCR2.1-Topo vector, generating plasmids pTVP5′.1 and pTVP3′.2. Sequencing of pTVP5′.1 and pTVP3′.2 allowed for the generation of a new set of oligonucleotide primers corresponding to the 5′ end and 3′ end of the TVP1 ORF. The forward primer (with an added BamHI restriction site) and reverse primer sequence, respectively, were 5′-CGGGATCCGGCATGGCGATCCTCGGG-3′ and 5′-CTTCTAGATGTACTTGAACAG-3′. The PCR product was first cloned into the pCR2.1-Topo vector, generating plasmid pTVP1. The insert, corresponding to the full-length TVP1 ORF was isolated with BamHI and EcoRI digestion of pTVP1, and then cloned into pYES2 plasmid digested with the same restriction enzymes. The reconstituted TVP1 ORF was sequenced (GenBank accession no. AY296911). 4.3. Functional assays using yeast mutants The nhx1 yeast mutant was transformed with either the empty pRS8 plasmid, or the pRS8 plasmid containing TNHX1. The two nhx1 strains, along with the wild type (W303) strain were grown at 28 °C for 2 days in selective yeast nitrogen base (YNB) medium lacking tryptophan (Qbiogene/Bio 101, Carlsbad, CA) (pH 6.5). Aliquots (5 µl) of the saturated cultures, and fourfold serial dilutions of the cultures were spotted onto YPD plates supplemented with (+) or without (–) 0.05 mg ml–1 hyg. The strains were cultured at 28 °C for 2 days on YPD medium before growth evaluation. The ena1 yeast mutant was transformed with either the empty pYES2 plasmid, or the pYES2 plasmid containing TVP1. The two ena1 strains, along with the wild type (W303) strain were grown at 28 °C for 2 days in selective YNB-ura medium. Aliquots (5 µl) of the saturated cultures, and fourfold serial dilutions of the cultures were spotted onto YPGAL plates supplemented with (+) or without (–) 0.5 M NaCl. The strains were cultured at 28 °C for 2 days before growth evaluation. The assays used here to functionally characterize both TNHX1 and TVP1 are based on work [11] done with the same yeast mutants to functionally characterize the Arabidopsis homologs of TNHX1 and TVP1 (i.e. AtNHX1 and AVP1, respectively). 4.4. Plant growth conditions Seeds of T. durum cv. ’Oum Rabiaa’ were germinated on wet filter paper, transferred to hydroponic culture with one
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quarter-strength Murashige and Skoog (MS) medium, and grown in a growth chamber (16 h photoperiod, 25 °C) for 1 week prior to initiation of salinity stress. A group of plants were maintained on the same medium (control treatment), and for a second group of plants, the growth medium was replaced with one quarter-strength MS medium with 200 mM NaCl added. Plants were grown for 3 days further under these conditions. 4.5. Genomic blot hybridization Genomic DNA was isolated from leaves of wheat as described previously [18]. Samples (10 µg) of the DNA were digested separately with HindIII, EcoRI, XhoI and SacI restriction endonucleases. Digested DNA was electrophoretically fractionated on a 1% (w/v) agarose gel and transferred to Hybond N+ nylon membranes (Amersham) by capillary transfer. After prehybridization with 200 µg ml–1 of salmon sperm DNA, the immobilized DNAs were hybridized overnight with probes that were 32P-labeled using the Rediprime random primer labeling kit (Amersham). A PCR amplification product corresponding to the 3′-UTR was used as a probe for TNHX1 and TVP1. The primers used to generate the TNHX1 probe are 1481:5′-GCTCGGCTCATCGTGTA-3′ and 1840:5′-CTGAACGAGATTAATTTACAG-3′. The primers used to generate the TVP1 probe are 2302:5′-CTGTTCAAGTACATCTAGAAG-3′ and 2620:5′-CAAGGGTTCAAACATTATCG-3′. After hybridization and washing, membranes were exposed to an imaging plate, and the radioimage of the plate was analyzed using the Molecular Imager FX-Plus (Bio-Rad, France). 4.6. Northern blot hybridization Total RNA (10 µg) of each sample (roots and leaves from control and salt-stressed wheat seedlings) was extracted by phenol/LiCl precipitation [33]. The RNA was electrophoresed on 1% (w/v) formaldehyde-agarose gels, and transferred to Hybond N+ membranes. Northern blot hybridizations (using the 3′-UTR TNHX1 and TVP1 DNA probes mentioned above) were performed at 42 °C in 50% (v/v) formamide, 0.12 M Na2HPO4, pH 7.2, 0.25 M NaCl, 7% (w/v) SDS and 1 mM EDTA. After hybridization with the DNA probes described above, membranes were exposed to an imaging plate and the radioimage of the plate was analyzed using the Molecular Imager FX-Plus.
Acknowledgements We thank Dr. Daniel Schachtman (Donald Danforth Plant Science Center, St Louis, MO) for the gift of the wheat cDNA library. This work was supported jointly by grants from the Secrétariat d’Etat à la Recherche Scientifique et à la Technologie (SERST), Tunisia, and the USDA-RSED awards FG-TN102 and 5831483022. R.A.G. was supported by the
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National Research Initiative of the USDA Cooperative State Research, Education, and Extension Service under grant no. 2001-35100-10772. G.A.B. was supported by NSF award no. MCB-0344141. References [1]
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Cloning and characterization of a wheat vacuolar cation/proton antiporter and pyrophosphatase proton pump Faïçal Brini a, Roberto A. Gaxiola b, Gerald A. Berkowitz b, Khaled Masmoudi a,* a
b
Plant Molecular Genetics Unit, Center of Biotechnology of Sfax, B.P’K’, 3038 Sfax, Tunisia Department of Plant Science, Agricultural Biotechnology Laboratory, University of Connecticut, 1390 Storrs Road, Storrs, CT 06269-4163, USA Received 12 July 2004; accepted 16 February 2005 Available online 17 March 2005
Abstract Sodium at high millimolar levels in the cytoplasm is toxic to plant and yeast cells. Sequestration of Na+ ions into the vacuole through the action of tonoplast proton pumps (an H+-ATPase in the case of yeast, and either a H+-pyrophosphatase (H+-PPase) or H+-ATPase in the case of plants) and a Na+/H+ antiporter is one mechanism that confers salt tolerance to these organisms. The cloning and characterization of genes encoding these tonoplast transport proteins from crop plants may contribute to our understanding of how to enhance crop plant response to saline stress. We cloned wheat orthologs of the Arabidopsis genes AtNHX1 and AVP1 using the polymerase chain reaction and primers corresponding to conserved regions of the respective coding sequences, and a wheat cDNA library as template. The wheat NHX cDNA cloned by this approach was a variant of the previously reported TNHX1 gene. The vacuolar H+-PPase pump we cloned (TVP1) is the first member of this gene family cloned from wheat; it is deduced translation product is homologous to proteins encoded by genes in barley, rice, and Arabidopsis. Function of TNHX1 as a cation/proton antiporter was demonstrated using the nhx1 yeast mutant. TNHX1 was capable of suppressing the hyg sensitivity of nhx1. Functional characterization of the wheat H+-PPase TVP1 was demonstrated using the yeast ena1 (plasma membrane Na+-efflux transporter) mutant. Expression of TVP1 in ena1 suppressed its Na+ hypersensitivity. Expression analysis of salt-stressed wheat plants showed substantial up-regulation of TNHX1 transcript levels as compared to control plants, while transcript accumulation for TVP1 was not greatly affected by exposure of plants to salt stress. © 2005 Elsevier SAS. All rights reserved. Keywords: Na+/H+ antiporter; H+-pyrophosphatase; Sodium sequestration; Salt tolerance; Transcript accumulation; Wheat
1. Introduction Salinity is a major constraint to crop (including wheat [22]) productivity; reducing yields on saline soils and limiting expansion of agriculture onto previously uncultivated land [8]. 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
Abbreviations: H+-PPase, proton pumping pyrophosphatase; hyg, hygromycin; ORF, open reading frame; UTR, untranslated region; YNB, yeast nitrogen base; YPD, yeast extract/peptone/dextrose; YPGAL, yeast extract/peptone/galactose. * Corresponding author. Tel.: +216 74 440 816x1092; fax: +216 74 440 818. E-mail address: [email protected] (K. Masmoudi). 0981-9428/$ - see front matter © 2005 Elsevier SAS. All rights reserved. doi:10.1016/j.plaphy.2005.02.010
ion accumulation in the cytosol [3,12,15,25]. The capacity for vacuolar compartmentalization of Na+ and Cl– is an adaptation mechanism conserved in halophytes and glycophytes [3,15]; 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 intracellular osmotic status of cells in salt grown plants [27]. Cellular ion exclusion cannot provide complete adaptation of plants to high soil salinity, presumably because of the osmotic stress component of salinity stress [3]. Continued cell growth would be restricted under osmotic stress because of an unfavorable water balance, limiting the water uptake necessary for cell expansion. The integrated processes of cell division and expansion (i.e. growth) require substantial water uptake; cells increase their volume after division by as much as 100-fold
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[20]. Vacuolar expansion is a primary mechanism underlying this massive cell enlargement. Energy-dependant Na+ transport (i.e. against a concentration gradient) across plant cell membranes (plasma lemma and tonoplast) is usually coupled to the proton (H+) electrochemical potential established by H+-translocating pumps [3,13,15,31]. H+ transport across these membranes increases with salt treatment and may be attributed both to pump activation and enhanced transcription [15]. Plasma membrane and tonoplast transporters facilitate Na+ efflux from the cytosol by coupling Na+ transport to the (energetically favorable) transport of H+. Both plasma membrane and tonoplast Na+/H+ antiporter activities increase in response to salt treatment, at least in halophytic species [3]. Overexpression of the vacuolar Na+/H+ antiporter and H+-pyrophosphatase pump (H+PPase) has resulted in enhanced plant tolerance to both salinity [2,12,36], and drought stress [12]. In Saccharomyces cerevisiae the primary pathway for Na+ extrusion is mediated by Ena1 [14,28], the plasma membrane Na+-ATPase. Ena1 yeast mutants are hypersensitive to high growth medium Na+, and expression of the Arabidopsis tonoplast H+-PPase suppresses this mutant phenotype presumably due to increased capacity for Na+ sequestration in the yeast vacuole [11]. Sequestration of cytosolic Na+ into the yeast vacuole occurs through the action of Nhx1 [23]. Recent analyses of the genes involved in cation detoxification in yeast have led to a model in which the Nhx1 Na+/H+ exchanger acts in concert with the vacuolar ATPase and the Gef1 anion channel to sequester cations in a prevacuolar compartment [10,24]. This model posits that sequestration of sodium by Nhx1 depends on the vacuolar H+-ATPase and Gef1, the chloride channel. Gef1-mediated anion influx allows
establishment by the vacuolar H+-ATPase of a proton gradient sufficient in magnitude to drive Na+ accumulation in the vacuole (against a concentration gradient) via Na+/H+ exchange. In this study, we describe the molecular cloning of two wheat cDNAs encoding the tonoplast H+-PPase and Na+/H+ antiporter, their expression patterns in salt-stressed plants, and their functional characterization using heterologous expression in Na+-sensitive yeast mutants.
2. Results 2.1. Molecular characterization of wheat TNHX1 and TVP1 The full-length cDNAs of TNHX1 and TVP1 were cloned and sequenced as described (see Section 4). Sequence analysis of the TNHX1 cDNA revealed an open reading frame (ORF) of 1641 bp with a 3′-untranslated region (UTR) of 207 bp. Alignment of this ORF (GenBank accession no. AY296910) with that of the previously cloned TNHX1 cDNA (GenBank accession no. AY040245) indicated differences at six positions. We attribute this to varietal differences in the genomes used to generate the corresponding libraries and consider our clone to be a variant of TNHX1. The TVP1 cDNA we cloned has an ORF of 2289 bp with a 5′-UTR of 30 bp and a 3′ noncoding region of 352 bp. A clade analysis of plant NHX vacuolar Na+/H+ antiporters, including the wheat cDNA cloned here, is presented in the phylogenetic tree shown in Fig. 1A. Wheat TNHX1 (shown in bold) forms a clade with the most closely related plant NHX homolog, HvNHX2 from
Fig. 1. Phylogenetic relationship between deduced protein sequences of plant vacuolar Na+/H+ antiporters (A) and vacuolar H+-PPase pumps (B). The Arabidopsis (’At’), wheat (’T’), barley (’Hv’), and rice (’Os’) Na+/H+ antiporters are included in the analysis shown in (A). The wheat (’T’), barley (’H’), rice (’O’), Arabidopsis (’A’), and tobacco (’Nt’) H+-PPase pumps are included in the analysis shown in (B). The wheat Na+/H+ antiporters (TNHX1 and TNHX2) can be paired into two phylogenetic subgroups, while the wheat H+-PPase forms a unique group with the barley H+-PPase HVP2. Multiple sequence alignment was performed using the CLUSTALW computer program [16]. The accession numbers for the protein sequences are as follows: AtSOS1 (AAF76139), Arabidopsis thaliana; six A. thaliana (AtNhx) isogenes, AtNhx1 (AF510074); AtNhx2 (AF490586); AtNhx3 (AC011623); AtNhx4 (AB015479); AtNhx5 (AC005287) and AtNhx6 (AC010793); OsNhx1 (AB021878), Oryza sativa (rice); HvNhx1 (AB089197) and HvNhx2 (AY247791), Hordeum vulgare (barley); TNhx1(AY296910) and TNhx2 (AY040246), T. aestivum (wheat); TVP-1 (AY296911), T. aestivum; HVP-1 (AB032839) and HVP-2 (D13472), H. vulgare; NtVP5 (X77915), Nicotiana tabacum (tobacco); OVP-1 (D45383), OVP-2 (D45384), OVP-3 (AB126350) and OVP-5 (AB126351), O. sativa; AVP-1 (M81892), AVP-2 (AF182813) and AVP-3 (AB015138), A. thaliana.
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barley. Fig. 1B shows a phylogenetic analysis of plant vacuolar H+-PPases. TVP1 (shown in bold) forms a clade with the barley vacuolar H+-PPase HVP2. The deduced amino acid sequence of TNHX1, and sequences of representative homologs from other species are shown in Fig. 2. Hydropathy analysis (not shown) of the TNHX1 sequence confirms the presence of the 12 transmembrane domains present in members of this protein family; these domains are identified in Fig. 2. Also shown (as a boxed area within transmembrane domain III in Fig. 2) is the amiloride binding motif that is common to these proteins [5]; this motif is present in TNHX1. The deduced amino acid sequence of the variant of TNHX1 that we cloned (GenBank accession no. AY296910) is 99% similar to the amino acid sequence of the TNHX1 previously reported (GenBank accession no. AY040245). Sequence comparison between TNHX1 and TNHX2 showed 75% identity at the nucleotide level, while no similarity was found between the 3′-UTR of TNHX2 and the 3′-UTR of TNHX1. Regions of the deduced amino acid sequence of TVP1 (and corresponding sequences of some homologs) are shown in Fig. 3. A region of the plant vacuolar H+-PPases that is highly conserved (G240-M287 in TVP1) is shown in Fig. 3A. Within this region, a putative pyrophosphatase catalytic site [20] is present (boxed region in Fig. 3A); this motif can also be found in the TVP1 sequence. Another functional domain present in plant vacuolar H+-PPases is the ’EYYTS’ motif shown in Fig. 3B that is involved in H+ transport [35]. It should be noted that all the plant vacuolar H+-PPases, including TVP1 (see Fig. 3B), contain a conserved glutamate residue (E221 in TVP1; E229 in AVP1) [37]. It has been suggested that this residue plays a role in coupling pyrophosphate hydrolysis to H+ translocation across the vacuolar membrane [7,35]. 2.2. Functional characterization of the wheat antiporter TNHX1 and vacuolar H+-PPase using yeast mutants Heterologous expression in yeast has been used in several studies to characterize the Arabidopsis vacuolar Na+/H+ antiporter AtNHX1 [6,11,34]. However, little functional analysis work has been done with NHX1 homologs from crop plants. Xia et al. [34] have recently expressed the sugar beet (Beta vulgaris) BvNHX1 antiporter in yeast for functional characterization. Here, we used a similar strategy to undertake the first characterization of a Na+/H+ antiporter cDNA cloned from the cereal crop wheat. Gaxiola et al. [11] and Darley et al. [6] demonstrated function of the Arabidopsis AtNHX1 cDNA as a vacuolar Na+/H+ antiporter by partial suppression of hygromycin (hyg) hypersensitivity of the nhx1 yeast mutant. Hygromycin is a cationic antibiotic that is toxic to cells upon accumulation in the cytosol. The basis for hyg hypersensitivity of the yeast nhx1 mutant is likely a reduction in NHX-mediated sequestration of the toxic cation in the yeast vacuole [1,35]. As shown in Fig. 4A, the expression of the wheat cDNA (TNHX1) we have identified as a putative vacuolar Na+/H+ antiporter does in fact partially complement
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the nhx1 mutant phenotype, suppressing hyg hypersensitivity. In the absence of hyg, the nhx1 mutant transformed with any of the plasmids (along with the wild type yeast) grew on YPD medium, but in the presence of hyg, the nhx1 mutant grew only when transformed with the plant TNHX1 cDNA (Fig. 4A). Yokoi et al. [35] has demonstrated that several of the six Arabidopsis NHX genes (AtNHX2, AtNHX5) in addition to AtNHX1 encode vacuolar Na+/H+ antiporters that complement yeast nhx1 sensitivity to hyg, as we report here for the wheat Na+/H+ antiporter TNHX1. In previous studies, Gaxiola et al. [11] used the Na+ hypersensitivity of the ena1 yeast mutant to demonstrate function of the Arabidopsis vacuolar H+-PPase. Results of this work are consistent with a model whereby expression of a plant vacuolar H+-PPase would allow for greater growth of ena1 in the presence of high external Na+ due to increased sequestration into the vacuole of Na+ that enters the yeast cytosol. In this prior work with the Arabidopsis H+-PPase AVP1 [11] a gain-of-function (E427D) mutant construct of AVP1 was used in studies employing the yeast ena1 for functional characterization. Here, we studied the wild type TVP1. We find that the TVP1 cDNA also suppresses the Na+ hypersensitivity of ena1 (Fig. 4B). All ena1 mutants along with the wild type yeast grew on YPGAL medium with no added NaCl, while the ena1 mutant grew in the presence of 0.5 M NaCl only when transformed with the plant TVP1 cDNA (Fig. 4B). Our results with TVP1 are consistent with prior studies of AVP1 expression in yeast [11,17], in suggesting that TVP1 is also an H+-PPase pump localized to the vacuolar and/or prevacuolar tonoplast membrane. In work with AVP1, Kim et al. [17] localized the plant protein to the yeast vacuole membrane as well as demonstrating PPase activity. However, we cannot discount the possibility that TVP1 suppression of the ena1 Na+ hypersensitivity occurs by TVP1 H+-pump action at the yeast plasma membrane. This could possibly suppress ena1 hypersensitivity to Na+ through the action of the yeast cell membrane Na+/H+ antiporter NHA1 [26]. The results shown in Fig. 4 suggest that the two wheat cDNAs TNHX1 and TVP1 can function in a model eukaryote (yeast) cell as a Na+/H+ antiporter and H+-PPase, respectively. 2.3. Southern blot analysis Southern blot analyses of genomic DNA isolated from wheat (Triticum durum) leaves were performed using probes corresponding to 3′-UTR sequences of the TVP1 and TNHX1 cDNAs (Fig. 5). As shown in Fig. 5A, a single band was detected in the case of TVP1 when DNA was subjected to digestion with HindIII, EcoRI, SacI, or XhoI. We conclude from this hybridization pattern that the wheat genome has a single copy of the TVP1 gene. Southern analysis of HindIII, EcoRI and XhoI-digested DNA probed with TNHX1 also identified one hybridizing band (Fig. 5B). This results shown in Fig. 5B indicate that the wheat genome has a single copy of the TNHX1 gene and further, that TNHX1 and TNHX2 are not allelic; i.e. that they are encoded by two separate genes in the
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Fig. 2. Multiple alignment of the deduced amino acid sequence of TNHX1, the Na+/H+ antiporter we cloned from wheat, along with the barley (HvNhx1), rice (OsNhx1), Arabidopsis (AtNhx1), human (HsNHE1), and yeast (ScNhx1) sequences. Identical residues are highlighted in black, residues with conservative substitutions are shaded in gray, and dashes indicate gaps in a sequence. Putative transmembrane domains are indicated by Roman numerals. The amiloride binding motif is framed and the consensus sequence is indicated by the boxed residues above the alignment in the transmembrane domain III region. Numbers flanking the sequences correspond to positions of amino acids directly next to the number.
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Fig. 3. Alignment of several conserved regions of the deduced amino acid sequence of the wheat H+-PPase TVP1 with corresponding regions of a barley (HVP1) and an Arabidopsis (AVP1) H+-PPase. Sequences were aligned using the Clustal X program. (A) A conserved region of the proteins flanking the putative H+-PPase catalytic site (shown within the box). (B) The conserved motif found in plant H+-PPases that is critical for H+-pumping by the protein. Numbers flanking the sequences correspond to positions of amino acids directly next to the number.
Fig. 4. Functional characterization of the wheat Na+/H+ antiporter TNHX1 (A) and the wheat H+-PPase TVP1 (B) using yeast mutants. (A) Growth of the nhx1 yeast mutant transformed with either the empty plasmid (Nhx1), or the TNHX1 cDNA. The yeast strains were grown on YPD medium with (+), or without (–) 0.05 mg ml–1 hyg. (B) Growth of the ena1 yeast mutant transformed with either the empty plasmid (ena1), or the TVP1 cDNA. The yeast strains were grown on YPGAL medium with (+) or without (–) 0.5 M NaCl. Growth of the isogenic wild type yeast strain (WT) is shown for both experiments. For both TNHX1 and TVP1, results are shown for two independent yeast clones (TNHX1-2; TNHX1-3; TVP1-1; TVP1-2). In all cases, growth of fourfold serial dilutions of each culture is shown. Experiments shown in this figure were repeated, with similar results (data not shown).
wheat genome. The nucleotide sequences of TNHX1 and TNHX2 have 75% identity. 2.4. Expression pattern of TVP1 and TNHX1 in wheat under salt stress In order to examine the effect of salt stress on the expression of the vacuolar Na+/H+ antiporter and H+-PPase genes, total RNA isolated from roots and shoots of wheat seedlings was subjected to Northern blot analyses. A Northern analysis of RNA isolated from control and salt-stressed plants using the 3′-UTR of TNHX1 as a probe is shown in Fig. 6A. Salt stress significantly increased the transcript levels of TNHX1 in roots and, to a lesser extent, in leaves. As suggested by the results of the Southern analysis shown in Fig. 5B, we can discount the possibility that the 3′-UTR TNHX1 probe hybridizes to TNHX2 message. In prior studies of Arabidopsis, Gaxi-
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Fig. 5. Genomic Southern blot analysis of wheat (T. durum) vacuolar H+-PPase (A) and Na+/H+ antiporter (B). Wheat genomic DNA (10 µg) was digested with the restriction endonucleases HindIII (H), EcoRI (E), XhoI (X) or SacI (S), and hybridized with either the 32P-labelled 3′-UTR specific TVP1 (A) or 3′-UTR specific TNHX1 (B). DNA fragments of a 1 kb ladder were used as molecular markers (indicated on the left of each panel).
Fig. 6. Analysis of TNHX1 (A) and TVP1 (B) expression under salt stress. Expression in roots and leaves of control wheat seedlings (C) and seedlings subjected to irrigation with 200 mM NaCl was monitored. Ribosomal RNA (rRNA) was used as a control. Experiments shown in this figure were repeated a second time with similar results (data not shown).
ola et al. [11] found increases in transcript level of the TNHX1 homolog AtNHX1 in salt-stressed Arabidopsis seedlings. Subsequently, Yokoi et al. [35] found that the transcript levels of several, but not all of the AtNHX genes increase in response to salinity stress. In contrast to the strong up-regulation of TNHX1 in roots of salt-stressed wheat seedlings, we found only a modest (if at all) effect of salinity stress on TVP1 expression in roots (Fig. 6B). Results shown in Fig. 6B also show no effect of salinity stress on the level of TVP1 transcript in leaves of saltstressed wheat seedlings.
3. Discussion Work presented in this report focuses on two vacuolar tonoplast membrane transport proteins of wheat, a Na+/H+ antiporter (TNHX1) and an H+-PPase (TVP1). It has been previously demonstrated that overexpression of the Arabidopsis H+-pyrophosphatase (AVP1) confers salt tolerance to yeast strains only when the yeast contains a functional vacuolar Na+/H+ exchanger (Nhx1) [11]. Thus, the Na+/H+ exchanger
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acts in concert with the vacuolar H+-PPase (and ATPase) to sequester cations in the vacuole (and prevacuolar compartment). Heterologous expression in yeast mutants allowed for an indirect characterization of their functions. The approach we take here to identify the cDNAs as encoding tonoplast ion transport proteins is consistent with the strategy used in prior reports to demonstrate function of the Arabidopsis homologs AVP1 and AtNHX1. Both genes were shown to be present in the T. durum genome. This work represents the first cloning and functional characterization of a vacuolar H+-PPase from the important cereal crop wheat. It has been over a decade since the first report of the cloning of a cereal crop (barley) H+-PPase [32]; since this time no functional characterization of a cereal crop vacuolar H+-PPase has been published. In addition to the characterization of TVP1 from wheat included in our work, we became aware during the preparation of this manuscript of a recently published paper reporting the functional characterization of the barley vacuolar H+-PPase [9]. The H+-PPases are considered to form a multigene family. Two cDNA clones (OVP1 and OVP2) encoding vacuolar H+-PPases isolated from rice were reported [29]. Indeed, there are more than five isoforms in rice, three isoforms in barley [32,21] and at least three isoforms in Arabidopsis. A highly conserved colinearity of genes between rice and wheat is well established [19]. Here, we isolate the first isoform from wheat. Further screening of the wheat cDNA library may lead to the isolation of other vacuolar H+-PPase isoforms. Recently, it has been reported in barley that salt stress increased the transcript level of a tonoplast H+-PPase (HVP1) in roots for a short time (5 h), and subsequently increased that of another isoform (HVP10) after treatment for 24 h [9]. In our case with TVP1, we find only a modest (if at all) increase in the transcript level in roots of salt-stressed plants as compared to TVP1 levels in roots of control plants. As was the case in barley, we found no detectable effect of salinity stress on expression of the vacuolar H+-PPase (TVP1) in leaves. In contrast, we found that the transcript level of the wheat Na+/H+ antiporter gene (TNHX1) increased greatly in roots and to some extent in leaves of wheat seedlings treated with 200 mM NaCl. It seems that the results of transcript accumulation indicate that the expression of TVP1 is not coordinated with that of TNHX1 in roots of wheat seedlings treated with NaCl. The greater increase of TNHX1 expression in roots treated with salt might be due to the accumulation of Na+ in the vacuoles of root cells. Unlike the results with wheat reported here, expression of the vacuolar H+-PPase and Na+/H+ antiporter appear to be coordinated in barley [9] subjected to salinity stress. Members of these families of plant proteins, tonoplast Na+/H+ antiporters and H+-PPases, are known to be important regulators of plant response to salinity stress. Salinity stress limits growth and yields of wheat in many regions of the world; enhanced sequestration of Na+ taken up into wheat leaves could be one strategy to engineer salinity tolerance. The results included in this report, therefore, are relevant to this important agricultural challenge. Further study of these
wheat ion transporter genes could lead to a better understanding of the molecular basis for salinity tolerance in an important cereal crop. In addition, over-expression of the Arabidopsis homologs of these genes has led to salinity (for both the antiporter and pyrophosphatase) and drought (in the case of the pyrophosphatase) tolerance. Our finding that both wheat genes, the Na+/H+ antiporter and H+-PPase, confer salt tolerance in yeast suggests it may be worthwhile to elucidate the contribution of these proteins to salt tolerance in a staple crop plant such as wheat. Engineering plants to increase functional levels of these proteins may be one strategy to develop tolerance to this important abiotic stress.
4. Methods 4.1. Yeast strains and plasmids All strains used are isogenic to W303 (ura3-1 can1100 leu2-3, 112trp1-1 his3-11, 15). Plasmids pRS8 and pYES2 (Invitrogen) were used to construct PMA1/TNHX1 and pGAL1/TVP1, respectively. The pRS8 plasmid was constructed (Mascorro-Gallardo and Gaxiola, unpublished data) using the pRS424 plasmid [4] as a backbone with the high expression level PMA1 (S. cerevisiae plasma membrane H+-ATPase) promotor [30] driving expression. The wild type W303, and the yeast mutant strains nhx1::HIS3 and ena1::HIS3 (see [11] for construction and/or source) were used for the work reported here. Cells were grown in YPD (1% (w/v) yeast, 2% (w/v) peptone, and 2% (w/v) dextrose; Difco; Franklin Lakes, NJ), or YPGAL (1% (w/v) yeast, 2% (w/v) peptone, and 2% (w/v) galactose; Difco). Transformation was performed using the EZ-Yeast transformation kit (BIO 101; Carlsbad, CA). 4.2. Plasmid constructs PCR products were generated from a wheat (Triticum aestivum) root tissue cDNA library. The full-length TNHX1 ORF was amplified with PfuTurbo DNA polymerase (Stratagene, La Jolla, CA) using wheat cDNA library as template and primers corresponding to the 5′ and 3′ ends of the wheat gene TNHX1 (GenBank accession no. AY040245) with BamHI and SphI, restriction sites added, respectively. These oligonucleotide primers were 5′-ATAAGAATGCGGCCGCGGCATGGGGCTCGATTT-3′, and 5′-ACATGCATGCCTGGGCTTCGACTTAACTAC-3′, respectively. The PCR product was cloned into the pCR2.1-Topo vector (Invitrogen, Carlsbad, CA), generating plasmid pTNHX1. After sequencing, the TNHX1 cDNA was digested with BamHI and SphI and cloned into pRS8 digested with the same restriction enzymes. The wheat vacuolar H+-PPase cDNA ’TVP1’ was cloned as follows. The 5′ end of the cDNA was amplified from the wheat library using a degenerate primer ’HvP2’ corresponding to the 5′ end of the barley H+-PPase HVP1 (accession no. AB032839) sequence (5′-TGGCAAGGYTGRCTCTTTG-
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TGCTGAAYCC-3′) and the T7 primer (5′-TAATACGACTCACTATAGGG-3′). The missing 3′ end of the TVP1 cDNA was amplified from the same wheat cDNA library using a 5′ primer corresponding to a conserved region of plant (barley, rice, Arabidopsis) vacuolar H+-PPases, and a 3′ primer corresponding to the polylinker region of the pYES2 plasmid. Forward and reverse primer sequences were 5′-ACCCGTACAGCATTGTTC-3′ and 5′-TGAATGTAAGCGTGACATAA-3′, respectively. The two PCR products generated from the above reactions (corresponding to the 5′ and 3′ regions of the target TVP1 cDNA) were cloned into the pCR2.1-Topo vector, generating plasmids pTVP5′.1 and pTVP3′.2. Sequencing of pTVP5′.1 and pTVP3′.2 allowed for the generation of a new set of oligonucleotide primers corresponding to the 5′ end and 3′ end of the TVP1 ORF. The forward primer (with an added BamHI restriction site) and reverse primer sequence, respectively, were 5′-CGGGATCCGGCATGGCGATCCTCGGG-3′ and 5′-CTTCTAGATGTACTTGAACAG-3′. The PCR product was first cloned into the pCR2.1-Topo vector, generating plasmid pTVP1. The insert, corresponding to the full-length TVP1 ORF was isolated with BamHI and EcoRI digestion of pTVP1, and then cloned into pYES2 plasmid digested with the same restriction enzymes. The reconstituted TVP1 ORF was sequenced (GenBank accession no. AY296911). 4.3. Functional assays using yeast mutants The nhx1 yeast mutant was transformed with either the empty pRS8 plasmid, or the pRS8 plasmid containing TNHX1. The two nhx1 strains, along with the wild type (W303) strain were grown at 28 °C for 2 days in selective yeast nitrogen base (YNB) medium lacking tryptophan (Qbiogene/Bio 101, Carlsbad, CA) (pH 6.5). Aliquots (5 µl) of the saturated cultures, and fourfold serial dilutions of the cultures were spotted onto YPD plates supplemented with (+) or without (–) 0.05 mg ml–1 hyg. The strains were cultured at 28 °C for 2 days on YPD medium before growth evaluation. The ena1 yeast mutant was transformed with either the empty pYES2 plasmid, or the pYES2 plasmid containing TVP1. The two ena1 strains, along with the wild type (W303) strain were grown at 28 °C for 2 days in selective YNB-ura medium. Aliquots (5 µl) of the saturated cultures, and fourfold serial dilutions of the cultures were spotted onto YPGAL plates supplemented with (+) or without (–) 0.5 M NaCl. The strains were cultured at 28 °C for 2 days before growth evaluation. The assays used here to functionally characterize both TNHX1 and TVP1 are based on work [11] done with the same yeast mutants to functionally characterize the Arabidopsis homologs of TNHX1 and TVP1 (i.e. AtNHX1 and AVP1, respectively). 4.4. Plant growth conditions Seeds of T. durum cv. ’Oum Rabiaa’ were germinated on wet filter paper, transferred to hydroponic culture with one
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quarter-strength Murashige and Skoog (MS) medium, and grown in a growth chamber (16 h photoperiod, 25 °C) for 1 week prior to initiation of salinity stress. A group of plants were maintained on the same medium (control treatment), and for a second group of plants, the growth medium was replaced with one quarter-strength MS medium with 200 mM NaCl added. Plants were grown for 3 days further under these conditions. 4.5. Genomic blot hybridization Genomic DNA was isolated from leaves of wheat as described previously [18]. Samples (10 µg) of the DNA were digested separately with HindIII, EcoRI, XhoI and SacI restriction endonucleases. Digested DNA was electrophoretically fractionated on a 1% (w/v) agarose gel and transferred to Hybond N+ nylon membranes (Amersham) by capillary transfer. After prehybridization with 200 µg ml–1 of salmon sperm DNA, the immobilized DNAs were hybridized overnight with probes that were 32P-labeled using the Rediprime random primer labeling kit (Amersham). A PCR amplification product corresponding to the 3′-UTR was used as a probe for TNHX1 and TVP1. The primers used to generate the TNHX1 probe are 1481:5′-GCTCGGCTCATCGTGTA-3′ and 1840:5′-CTGAACGAGATTAATTTACAG-3′. The primers used to generate the TVP1 probe are 2302:5′-CTGTTCAAGTACATCTAGAAG-3′ and 2620:5′-CAAGGGTTCAAACATTATCG-3′. After hybridization and washing, membranes were exposed to an imaging plate, and the radioimage of the plate was analyzed using the Molecular Imager FX-Plus (Bio-Rad, France). 4.6. Northern blot hybridization Total RNA (10 µg) of each sample (roots and leaves from control and salt-stressed wheat seedlings) was extracted by phenol/LiCl precipitation [33]. The RNA was electrophoresed on 1% (w/v) formaldehyde-agarose gels, and transferred to Hybond N+ membranes. Northern blot hybridizations (using the 3′-UTR TNHX1 and TVP1 DNA probes mentioned above) were performed at 42 °C in 50% (v/v) formamide, 0.12 M Na2HPO4, pH 7.2, 0.25 M NaCl, 7% (w/v) SDS and 1 mM EDTA. After hybridization with the DNA probes described above, membranes were exposed to an imaging plate and the radioimage of the plate was analyzed using the Molecular Imager FX-Plus.
Acknowledgements We thank Dr. Daniel Schachtman (Donald Danforth Plant Science Center, St Louis, MO) for the gift of the wheat cDNA library. This work was supported jointly by grants from the Secrétariat d’Etat à la Recherche Scientifique et à la Technologie (SERST), Tunisia, and the USDA-RSED awards FG-TN102 and 5831483022. R.A.G. was supported by the
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National Research Initiative of the USDA Cooperative State Research, Education, and Extension Service under grant no. 2001-35100-10772. G.A.B. was supported by NSF award no. MCB-0344141. References [1]
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