| Edited by Minoru Murata. Takashi Kagami: Corresponding author. E-mail: takashi_kagami@ags.pref.aomori.jp |
The control of Na+ influx, efflux, and vacuolar compartmentation are important in the adaptation of plants to highly saline conditions (Blumwald et al. 2000). Na+/H+ antiporters in the plasma membrane and the tonoplast catalyze the exchange of Na+ for H+ across membranes, regulating the internal pH, cell volume, and sodium level in the cytoplasm and vacuole (Apse et al. 1999, Shi et al. 2000). Electrochemical H+ gradients generated by H+ pumps in the plasma membrane and tonoplast provide the energy used by the Na+/H+ antiporters that are also found in these locations.
The yeast NHX1 mediates the sequestration of Na+ within intracellular compartments and localizes to prevacuolar compartments (Nass et al. 1997, Nass and Rao 1998). A gene responsible for a vacuolar Na+/H+ antiporter of Arabidopsis thaliana, AtNHX1, was the first plant homolog of the yeast NHX1 to be isolated (Gaxiola et al. 1999). Five other homologs have since been identified (Yokoi et al. 2002). AtNHX1, 2, and 5 suppress the sensitivities to Na+ and hygromycin of the yeast nhx1 mutant, which is defective in endosomal and vacuolar Na+/H+ antiporter activity. The transcription of these genes increases in the presence of NaCl. The role of the rice vacuolar Na+/H+ antiporter OsNHX1 in Na+ transport has also been well studied (Fukuda et al. 1999, 2004). OsNHX1 plays an important role in the compartmentation of cytoplasmic Na+ into the vacuoles, and its expression is increased in the presence of NaCl. These results suggest that vacuolar Na+/H+ antiporters play a role in salt tolerance in plants. Vacuolar Na+/H+ antiporters also contribute to changes in the color of Ipomoea nil flowers from reddish purple to blue by controlling the vacuolar pH of the petal cells (Fukada-Tanaka et al. 2000, Yamaguchi et al. 2001). Because a Tpn4 transposable element is inserted into the first untranslated exon of the InNHX1 gene in this mutant, its petals are purple. The blue sectors that appear on the petals contain footprints generated by the excision of Tpn4.
Overexpression of a vacuolar Na+/H+ antiporter gene confers salt tolerance to plants (Apse et al. 1999, Zhang and Blumwald 2001, Zhang et al. 2001, Ohta et al. 2002, Fukuda et al. 2004, Wu et al. 2004). As shown in I. nil, a vacuolar Na+/H+ antiporter gene is strongly correlated with blue petal coloration. This result shows the possibility of applying genetic engineering to the development of blue flowers through the use of a vacuolar Na+/H+ antiporter gene. We expected that the study of a vacuolar Na+/H+ antiporter gene of Rosa hybrida might provide a useful tool for the molecular breeding of flower color in roses.
The purpose of this study was to investigate the func-tion of a vacuolar Na+/H+ antiporter gene of Rosa hybrida. We report here the molecular and functional analysis of RhNHX1, a vacuolar Na+/H+ antiporter gene of R. hybrida. The RhNHX1 cDNA was isolated, functional complementation tests were performed, and its transcription levels in the presence of NaCl were determined. The results suggest that RhNHX1 functions as a vacuolar Na+/H+ antiporter in rose plants.
Total RNA was extracted from Rosa hybrida cv. Watarase flower buds using a standard procedure (Sambrook et al. 1989), and mRNA was isolated from the RNA using Oligotex dT-30 (TaKaRa). First-strand cDNA synthesis was performed with oligo(dT) primer and SuperScript II reverse transcriptase (Gibco BRL), and the amplification products were used as template for PCR amplification. To isolate the vacuolar Na+/H+ antiporter gene from rose, degenerate primers designed based on sequences conserved in vacuolar Na+/H+ antiporters were used for PCR. The sequences of the primers were 5'- A/C GIGA A/G GTIGCI T/C TIATGATG - 3' and 5'- CIC G/T CATIA A/G ICCIGCCCACC -3'. A full-length cDNA was isolated with rapid amplification of cDNA ends (RACE) using the Gene Racer kit (Invitrogen), Omniscript RT (Qiagen), and gene-specific primers designed based on the sequence of the isolated cDNA. The nucleotide sequence was deposited in the DDBJ database as accession number AB199912.
A multiple sequence alignment and a phylogenetic tree were created using CLUSTAL W (Thompson et al. 1994) and TreeView (Zhai et al. 2002), respectively, based on complete amino acid sequences. The BOXSHADE program (http://www.ch. embnet.org/software/BOX_form.html) was used for further analysis of conserved amino acid sequences after the initial alignment was performed. A hydropathy plot was generated using the Kyte-Doolittle method (Kyte and Doolittle 1982) with the TMpred program (http://www.ch. embnet.org/software/TMPRED_form.html).
Rose plants were prepared by cutting propagation. Plants with elongated shoots bearing five leaves were used for salt stress experiments. Plants grown in soil in a greenhouse were watered every 2 hours with 300 ml of 0, 100, 200, or 300 mM NaCl. Leaflets of the third leaves of each shoots were collected for Northern blot analysis at the times of watering as well as 4 hours later. After collecting the leaves, plants were grown in a greenhouse and watered everyday.
Genomic DNA (10 μg) from young leaves was digested with XbaI, PstI, NdeI, or PvuII and hybridized with probes consisting of the 5'-UTR or the ORF region of the RhNHX1 cDNA labeled with 32P-dCTP. Total RNA (10 μg) from leaves was subjected to Northern blot analysis with a probe corresponding to the 5'-UTR. The analyses were performed according to standard procedures (Sambrook et al. 1989).
The Saccharomyces cerevisiae strains used in this study, BY4741 (MATa his3delta1 leu2delta0 met15delta0 ura3delta0, ATCC number 201388) and an nhx1 deletion mutant of the same strain (ATCC number 4004290), were obtained from the American Type Culture Collection. Yeast cells were grown in YPD (1% yeast extract, 2% peptone, 2% dextrose), YPGal (1% yeast extract, 2% peptone, 2% galactose), or SD (0.67% yeast nitrogen base, 2% dextrose) medium with appropriate amino acid supplements as indicated.
The full-length coding region of RhNHX1 was amplified with the specific primers 5'- ATG GCT TCT CAT TTG GCC ATG TTG ATG AC -3' and 5'- TCA TTG CCA TTG AGT GTT GTT CCG TTC AGT -3' using first-strand cDNA as a template, and the products were cloned into the pGEM-T Easy vector (Promega). To construct the yeast expression vector, the plasmids were digested with EcoRI and SpeI and inserted into pYES2 (Invitrogen) plasmid that had been digested with EcoRI and XbaI. The resulting plasmid was designated pTKY1.
For functional complementation analysis, pYES2 was introduced into the yeast BY4741 strain, and pYES2 or pTKY1 were introduced into the nhx1 mutant strain, using the lithium acetate method (Gietz and Schiestl 1995). Saturated yeast cultures in liquid SD medium containing supplements of appropriate amino acids were harvested and adjusted to OD600 = 0.8 with double-distilled water. Ten-fold serial dilutions of the strains starting at OD600 = 0.8 were prepared, and 10 μl of each dilution were spotted onto solid YPGal medium (1.5% agar) containing or lacking 0.01 mg/ml hygromycin and grown at 30°C for 2 days.
As shown in Fig. 1, the RhNHX1 cDNA contains 2080 nucleotides with an open reading frame of 1632 nucleotides, and encodes 543 amino acids with a calculated molecular mass of 60,045 daltons. The deduced amino acid sequence of RhNHX1 is 74.1% identical to that of AtNHX1. A hydropathy plot generated with the Kyte-Doolittle method using the TMpred program indicated that RhNHX1 contains 12 putative hydrophobic regions (Fig. 1 and Fig. 2). The NHX genes AtNHX1, OsNHX1, and GhNHX1, which confers salt tolerance to transgenic plants, were predicted to have the same number of hydrophobic regions (Fukuda et al. 1999, Yamaguchi et al. 2003, Wu et al. 2004). A phylogenetic analysis of a number of Na+/H+ antiporters is shown in Fig. 3. RhNHX1 appears in the same cluster as AtNHX1 and OsNHX1, which localize to tonoplasts (Apse et al. 1999, Fukuda et al. 2004), in a group distinct from AtSOS1 (Shi et al. 2000) and SynNhaP (Hamada et al. 2001). These findings suggest that RhNHX1 localizes to the tonoplast.
 View Details | Fig. 1. Nucleotide and deduced amino acid sequences of the RhNHX1 cDNA. Amino acid residues are indicated as single letters. Putative transmembrane regions are underlined and indicated by Roman numerals. The asterisk indicates the termination codon. |
 View Details | Fig. 2. Hydropathy plot of RhNHX1. Hydropathy values were calculated using the method of Kyte and Doolittle (1982). The putative transmembrane domains are indicated by numbers. |
 View Details | Fig. 3. Phylogenetic analysis of Na+/H+ antiporters. A multiple sequence alignment and a phylogenetic tree were generated using CLUSTAL W and TreeView, respectively. The origins of the proteins are as follows: AtNHX1-6 (AAD16946, AAM08403, AAF08577, AAM08405, AAM08406, AAM08407) and AtSOS1 (AAF76139), Arabidopsis thaliana; GhNHX1 (AAM54141), Gossypium hirsutum; HsNHE1 and HsNHE6 (AAC60606, AAC39643), Homo sapiens; InNHX1 (BAB16381), Ipomoea nil; OsNHX1 (BAA83337), Oryza sativa; PhNHX1 (BAB56105), Petunia hybrida; RhNHX1, Rosa hybrida; ScNHX1 (AAB64861), Saccharomyces cerevisiae; SynNhaP (BAA17925), Synechocystis sp. PCC 6803. |
An alignment of the deduced amino acid sequences of Na+/H+ antiporters from several organisms reveals that the third, fifth, and sixth transmembrane regions are highly conserved (Fig. 4A and B). The third transmembrane region of RhNHX1 contains the amiloride binding site 87LFFIYLLPPI96 (Fig. 4A). This region, which is highly conserved in the eukaryotic Na+/H+ antiporter family, serves to inhibit the activity of these proteins in the presence of amiloride (Gaxiola et al. 1999, Darley et al. 2000). Darley et al. (2000) reported that the exchange activities of ScNHX1 and AtNHX1 were inhibited 20 to 40% and 100%, respectively, in the presence of 120 μM amiloride. An asparagine residue and a valine residue in ScNHX1 are replaced in RhNHX1 by isoleucine and tyrosine residues, respectively, as in AtNHX1 and OsNHX1. This result suggests that RhNHX1 is sensitive to amiloride, similar to ScNHX1 and AtNHX1.
 View Details | Fig. 4. Alignment of deduced amino acid sequences of Na+/H+ antiporters from several organisms. (A) A highly conserved region containing the putative transmembrane segment TM3. The conserved amiloride-binding site is indicated by the bold bar under the sequences. The amino acid residues related to amiloride affinity are indicated with asterisks. (B) A highly conserved region containing the putative transmembrane segments TM5 and 6. The conserved Asp is indicated by an asterisk. Predicted membrane spanning regions are marked above the alignment. The sequences were aligned using CLUSTAL W. Homologies among these sequences were highlighted using BOXSHADE. Highly conserved amino acid residues are highlighted in black, and conservative substitutions are shown in gray. The proteins used in this analysis are the same as in Fig. 3. |
The fifth and sixth transmembrane regions of AtNHX1, which are important for transport activity and correspond to the sixth and seventh transmembrane regions of NHE1, a mammalian Na+/H+ exchanger, are also highly conserved in RhNHX1 (Fig. 4B). Although the fifth and sixth transmembrane regions of AtNHX1 are thought to play critical roles in the transport activity, Yamaguchi et al. (2003) reported that these regions do not appear to be transmembrane segments, based on protease protection experiments. They suggested that differences in these regions may determine the direction of ion movement mediated by the protein. NHE1 mediates Na+ influx into the cytosol and H+ efflux from the cytosol, whereas AtNHX1 mediates Na+ influx from the cytosol and H+ efflux into the cytosol. As shown in Fig. 4B, an aspartic acid residue is conserved in all of the analyzed sequences. Hamada et al. (2001) suggested that in the protein from Synechocystis sp. 6803, Asp-138 is involved in the exchange activity, and the geometry in the vicinity of Asp-138 plays an important role.
Southern blot analysis was performed using probes synthesized from the 5'-UTR and the coding region of the RhNHX1 cDNA. In all lanes, a single band hybridized to the 5'-UTR probe, indicating that the genome of the rose plant used in this study possessed a single copy of the RhNHX1 gene (Fig. 5). In contrast, multiple signals were detected with the ORF region probe, indicating that homologs of the RhNHX1 gene exist in the rose genome (Fig. 5), possibly forming a multigene family analogous to AtNHX1 and related genes in A. thaliana.
 View Details | Fig. 5. Southern blot analysis of the RhNHX1 gene. Ten µg of genomic DNA from young leaves were digested with XbaI (1), PstI (2), NdeI (3), or PvuII (4), separated by gel electrophoresis, transferred to a membrane, and hybridized with probes corresponding to the 5'-UTR or ORF regions of the RhNHX1 cDNA. Size standards are indicated in kilobases to the left of the panel. |
AtNHX1 expression has been shown to increase in the presence of 250 mM NaCl (Gaxiola et al. 1999). To examine the effect of salinity on RhNHX1 expression, total RNAs from rose leaves collected at 0 and 4 hours after watering with 0, 100, 200, or 300 mM NaCl were subjected to Northern blot analysis with the 5'-UTR region probe used in the Southern blot analysis. The results showed that RhNHX1 expression is increased by salt treatment (Fig. 6). The RhNHX1 transcripts were more abundant in leaves treated with 200 and 300 mM NaCl than in those treated with 100 mM NaCl. rRNA stained with ethidium bromide was monitored as a loading control. Following the salt-treatment experiments, the plants were allowed to continue to grow in the greenhouse. At 2 weeks after the NaCl treatment, the plants exhibited delayed growth and reduced leaf size compared to the control plants (data not shown). The plants treated with 200 or 300 mM NaCl appeared particularly heavily damaged, shedding leaves that had not yet turned brown, and the plants treated with 300 mM NaCl died within 2 months. Although it was expected that the RhNHX1 expression in plants treated with 300 mM NaCl would be greater than or similar to that in plants treated with 200 mM NaCl, the RhNHX1 expression at 4 hours after watering with 300 mM NaCl was slightly lower than in plants watered with 200 mM NaCl (Fig. 6). A potential explanation for the plants not responding more strongly to NaCl stress is that the roots of the plants treated with 300 mM NaCl might have been damaged by osmotic stress, leading to impaired functioning. The NaCl tolerance limit for plants is around 200 mM. In A. thaliana, the growth of wild-type plants is generally inhibited by treatment with 50 to 200 mM NaCl (Apse et al. 1999). The increased expression of RhNHX1 during salt stress indicates that RhNHX1 expression is regulated by NaCl.
 View Details | Fig. 6. Northern blot analysis of RhNHX1. Ten-μg aliquots of total RNA from leaves were subjected to Northern blot analysis using a probe corresponding to the 5'-UTR region of the RhNHX1 cDNA. The plants were watered with solutions of 0 mM (1), 100 mM (2), 200 mM (3), or 300 mM (4) NaCl, and leaves were collected immediately and after 4 hours. rRNA stained with ethidium bromide is shown as a loading control. |
A functional complementation test was performed by monitoring the hygromycin-sensitive phe-notype of the yeast nhx1 mutant. Yeast cells are hypersensitive to the toxic cation hygromycin, which accumulates intracellularly in response to an electrochemical proton gradient (Darley et al. 2000). The hygromycin sensitivity of the yeast nhx1 mutant is correlated with the Na+ sensitivity of this mutant (Gaxiola et al. 1999, Darley et al. 2000, Yokoi et al. 2002, Venema et al. 2002, Yamaguchi et al. 2003, Fukuda et al. 2004). Yeast NHX1 plays an important role in the compartmentation of hygromycin into vacuoles (Gaxiola et al. 1999). Therefore, we employed the hygromycin-resistance test to determine the vacuolar Na+/H+ antiport activity of the product of the RhNHX1 gene in nhx1 cells. The yeast nhx1 mutant was found to be more sensitive to hygromycin than the BY4741 strain, whereas a strain harboring the full-length RhNHX1 cDNA in pTKY1 grew normally. These results indicate that the RhNHX1 protein functions in endosomal compartmentation and as an Na+/H+ antiport, as does the yeast NHX1 (Fig. 7).
 View Details | Fig. 7. Expression of RhNHX1 in the yeast nhx1 mutant. The pYES2 vector was introduced into the wild type and the nhx1 mutant strain, and the vector pTKY1, which carries the RhNHX1 gene, was introduced into nhx1. Ten-fold serial dilutions were spotted onto YPGal or YPGal supplemented with 0.01 mg/ml hygromycin, and the plates were incubated at 30°C for 2 days. |
The above results demonstrate that the RhNHX1 gene isolated in this study functions as a vacuolar Na+/H+ antiporter in rose. Vacuolar Na+/H+ antiporters are involved in salt tolerance and blue flower coloration in plants. Transgenic salt-tolerant crops have been developed by genetic transformation with vacuolar Na+/H+ antiporter genes (Zhang and Blumwald 2001, Zhang et al. 2001, Ohta et al. 2002, Fukuda et al. 2004, Wu et al. 2004). No studies have reported the genetic engineering of flower color using vacuolar Na+/H+ antiporter genes. In our preliminary data, the transformation of Lobelia erinus, which is a potential model system useful for rapid in planta studies of various functions (Tsugawa et al. 2004), with the RhHNX1 gene resulted in a slight change in flower color, from red to light reddish-purple, but no blue flowers were obtained (data not shown). This result suggests that in L. erinus, it may not be possible to achieve a blue flower color solely by introduction of the RhNHX1 gene. Further analysis of RhNHX1 and other related vacuolar transporters should add to the understanding of ion homeostasis, not only in salt tolerance but also in the regulation of pH resulting in petal color modification. In the future, the rose vacuolar Na+/H+ antiporter may be used in molecular breeding for blue flower color.
We thank Yoko Tamura and Syouko Tanaka for technical assistance and Hajime Hasegawa and Syouichi Ozaki for their encouragement to pursue this study.
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