JXB Advance Access originally published online on November 6, 2006
Journal of Experimental Botany 2006 57(15):4257-4268; doi:10.1093/jxb/erl199
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RESEARCH PAPER |
Expressions of OsHKT1, OsHKT2, and OsVHA are differentially regulated under NaCl stress in salt-sensitive and salt-tolerant rice (Oryza sativa L.) cultivars
1Department of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences, Box 7080, SE 75007 Uppsala, Sweden
2Department of Biochemistry and Physiology of Plants, University of Bielefeld, D-33501 Bielefeld, Germany
* To whom correspondence should be addressed. E-mail: abdul.kader{at}vbsg.slu.se
Received 15 August 2006; Accepted 13 September 2006
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Abstract |
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Under NaCl-dominated salt stress, the key to plant survival is maintaining a low cytosolic Na+ level or Na+/K+ ratio. The OsHKT1, OsHKT2, and OsVHA transporter genes might play important roles in maintaining cytosolic Na+ homeostasis in rice (Oryza sativa L. indica cvs Pokkali and BRRI Dhan29). Upon NaCl stress, the OsHKT1 transcript was significantly down-regulated in salt-tolerant cv. Pokkali, but not in salt-sensitive cv. BRRI Dhan29. NaCl stress induced the expression of OsHKT2 and OsVHA in both Pokkali and BRRI Dhan29. In cv. Pokkali, OsHKT2 and OsVHA transcripts were induced immediately after NaCl stress. However, in cv. BRRI Dhan29, the induction of OsHKT2 was quite low and of OsVHA was low and delayed, compared with that in cv. Pokkali. OsHKT2 and OsVHA induction mostly occurred in the phloem, in the transition from phloem to mesophyll cells, and in the mesophyll cells of the leaves. The vacuolar area in cv. Pokkali did not change under either short- (5–10 min) or long-term (24 h) salt stress, although it significantly increased 24 h after the stress in cv. BRRI Dhan29. When expressional constructs of VHA-c and VHA-a with YFP and CFP were introduced into isolated protoplasts of cvs Pokkali and BRRI Dhan29, the fluorescence resonance energy transfer (FRET) efficiency between VHA-c and VHA-a upon salt stress decreased slightly in cv. Pokkali, but increased significantly in cv. BRRI Dhan29. The results suggest that the salt-tolerant cv. Pokkali regulates the expression of OsHKT1, OsHKT2, and OsVHA differently from how the salt-sensitive cv. BRRI Dhan29 does. Together, these proteins might confer salt tolerance in Pokkali by maintaining a low cytosolic Na+ level and a correct ratio of cytosolic Na+/K+.
Key words: Cytosolic Na+/K+ homeostasis, FRET efficiency, NaCl stress, OsHKT1, OsHKT2, OsVHA, vacuole volumes
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionReferences |
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Salt stress is one of the most important abiotic stresses affecting natural productivity and causes significant crop loss worldwide. For plants, the sodium ion (Na+) is harmful whereas the potassium ion (K+) is an essential ion. The cytosol of plant cells normally contains 100–200 mM of K+ and 1–10 mM of Na+ (Taiz and Zeiger, 2002); this Na+/K+ ratio is optimal for many metabolic functions in cells. Physico-chemically, Na+ and K+ are similar cations. Therefore, under the typical NaCl-dominated salt environment in nature, accumulation of high Na+ in the cytosol, and thus high Na+/K+ ratios, disrupts enzymatic functions that are normally activated by K+ in cells (Bhandal and Malik, 1988; Tester and Davenport, 2003; Munns et al., 2006). Therefore, it is very important for cells to maintain a low concentration of cytosolic Na+ or to maintain a low Na+/K+ ratio in the cytosol when under NaCl stress (Maathuis and Amtmann, 1999).
The most important way to maintain a low cytosolic Na+ concentration is to minimize the influx of Na+ into the cytosol. Na+ influx can be restricted by means of selective ion uptake. Non-selective cation channels (NSCCs) are proposed to be the dominant pathways of Na+ influx in many plant species (Roberts and Tester, 1997; Tyerman et al., 1997; Buschmann et al., 2000; Davenport and Tester, 2000; Demidchik and Tester, 2002; Demidchik et al., 2002; Kader and Lindberg, 2005). However, the molecular identity of these NSCCs is still unknown. It has also been suggested that high-affinity potassium transporters (HKTs) mediate a substantial Na+ influx in some species, including rice (Uozumi et al., 2000; Horie et al., 2001; Golldack et al., 2002a). In a recent study, it was demonstrated that K+-selective channels play a significant role in Na+ uptake in a salt-sensitive rice, cv. BRRI Dhan29 (Kader and Lindberg, 2005). With some exceptions, plant species have multiple HKT transporters. In rice, eight functional HKT homologues (OsHKT1–4 and 6–9) have been identified (Garciadeblás et al., 2003). All of these functional genes encode proteins with distinct transport activities, which might be expressed in various tissues and/or organs. It has been suggested that OsHKT1 is a Na+ transporter (Horie et al., 2001; Mäser et al., 2002; Garciadeblás et al., 2003) and OsHKT2 a Na+/K+ co-transporter (Horie et al., 2001; Mäser et al., 2002). OsHKT2 is believed to be the sole HKT gene in rice involved in K+ transport (http://www.ausbiotech.org/pdf/2006_Honours_booklet.pdf#search=‘Identifying%20expression%20patterns%20of%20the%20HKT’; accessed 30 June 2006). OsHKT8 was very recently shown to be a Na+ transporter that contributes to increased salt tolerance by maintaining K+ homeostasis in the shoot under salt stress (Ren et al., 2005; Rus et al., 2005). This transporter is thought to be analogous to the function of the AtHKT1 gene in Arabidopsis, which is a Na+-transporter and, interestingly, plays a crucial role in controlling cytosolic Na+ detoxification (Berthomieu et al., 2003; Rus et al., 2004; Sunarpi et al., 2005). Therefore, it is likely that the HKT gene family plays an important role in Na+/K+ homeostasis in rice, even though some of its members are evidently Na+ transporters.
Another way for plant cells to deal with high cytosolic Na+ is to transport it out of the cytosol, either into the vacuole or into the apoplast. Apoplastic sequestration of Na+, however, plays no role in salt tolerance in rice, since most Na+ in the rice shoot arrives there through apoplastic streaming (Yeo et al., 1999). Conversely, vacuolar compartmentalization is an efficient strategy for plant cells to cope with salt stress (Fukuda et al., 1998, 2004; Apse et al., 1999; Blumwald, 2000; Chauhan et al., 2000; Hamada et al., 2001; Tester and Davenport, 2003). When compartmentalized into the vacuole, Na+ is no longer toxic to cells (Flowers and Läuchli, 1983; Subbarao et al., 2003), but is even advantageous for growth and osmotic adjustment (Zhu, 2003; Rodriguez-Navarro and Rubio, 2006), particularly since the vacuole may occupy over 95% of the volume of mature cells. In a recent study, it was also demonstrated that vacuolar compartmentalization is evident under salt stress in the salt-tolerant rice, cv. Pokkali, whereas apoplastic sequestration of cytosolic Na+ is dominant in the salt-sensitive cv. BRRI Dhan29 (Kader and Lindberg, 2005). The candidate protein for compartmentalizing Na+ in the vacuole is the tonoplast Na+/H+-antiporter, which is energized to do so by the vacuolar H+-ATPase (VATPase or VHA). Both VHA and pyrophosphatase are generally required for the maintenance of intracellular pH and ion homeostasis (Padmanaban et al., 2004). Through the hydrolysis of ATP, VHA generates a proton motive force that energizes secondary transport across the vacuolar membrane (like that of Na+) under high salt conditions. VHA was shown to be important for salt tolerance in Saccharomyces cerevisiae (Hamilton et al., 2002) and in many plant species as well (Golldack and Dietz, 2001; Kluge et al., 2003a; Senthilkumar et al., 2005; Vera-Estrella et al., 2005).
Like other salt-tolerant species, the salt-tolerant rice cv. Pokkali contains less Na+ both in its roots and shoots under salt stress than do salt-sensitive rice cultivars (Golldack et al., 2003; Kader and Lindberg, 2005). Several Na+/K+ -transporters could be involved in conferring the ability to maintain a low cytosolic Na+ level in Pokkali. To clarify the regulatory mechanisms involved in maintaining cytosolic Na+ homeostasis in rice, the expression of OsHKT1, OsHKT2, and OsVHA were compared in both a salt-sensitive (cv. BRRI Dhan29) and a salt-tolerant (cv. Pokkali) rice at different time points under NaCl stress. The transcripts of these transporter genes were quantified using real-time reverse transcription polymerase chain reaction (RT-PCR). Cell-specific expressions of the transporter genes were examined using in situ PCR to investigate their specific roles in cytosolic Na+ accumulation/detoxification.
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Materials and methods |
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Plant materials
Seeds of rice (Oryza sativa L. indica cvs Pokkali and BRRI Dhan29) were provided by the Bangladesh Rice Research Institute (BRRI, Gazipur, Bangladesh). The seeds were soaked in water for 48 h in darkness. Afterwards, they were germinated on vermiculite soaked in half-strength Hoagland's solution. After a week, the seedlings were transferred to hydroponic tanks containing half-strength Hoagland's solution. Seedlings were grown for another 2 weeks in a controlled environment chamber with day/night temperatures of 25/21 °C under 14 h of light (300 µE m–2 s–1); humidity was approximately 50%. Afterwards, the plants were stressed by adding NaCl at a final concentration of 150 mM to the nutrient solution for between 1 h and 72 h. Non-stressed control plants were grown concurrently and harvested at the same time.
RNA extraction and cDNA synthesis for qRT-PCR
RNA was isolated from the shoot and root tissues of rice cvs Pokkali and BRRI Dhan29. For qRT-PCR, total RNA was isolated using the guanidinium isothiocyanate method. Synthesis of cDNA was performed as described by Golldack et al. (2002b). For PCR amplification, the following sequence-specific forward and reverse oligonucleotide primers were used: 5'-GAGTCGTCTCAGAAATGA-3' and 5'-TGAACTTTCAGGCAGAAC-3' (OsHKT1), 5'-GAGTCGTCTCAGAAATGA-3' and 5'-TTCTACGATTCAAAAGGC-3' (OsHKT2), and 5'-CTTCTGGCAATCTTGGAG-3' and 5'-CAGTGTAGACGAAGTGCA-3' (OsVHA). The following conditions were used for the PCR reactions: 1 cycle consisted of 1 min 30 s at 94 °C, 1 min at 94 °C, 1 min at 55 °C, 2 min at 72 °C, and a final extension of 10 min at 72 °C. OsHKT1 was amplified for 30 cycles, OsHKT2 for 45 cycles, and OsVHA for 28 cycles, for samples from both the roots and shoots of rice. The PCR products from RT-PCR amplifications were separated on 1.7% (w/v) agarose gels and stained with ethidium bromide. Photographic documentation was performed using a gel documentation system (INTAS, Göttingen, Germany).
RNA extraction and cDNA synthesis for real-time RT-PCR
For real-time RT-PCR, total RNA was extracted using the RNeasy plant mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. A total of 5 µg of RNA was reverse transcribed to cDNA with oligo (dT) 20 mer with a T7 promoter in the 5' position and two randomized nucleotides in the 3' position, using SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA). The cDNA was diluted to 2 ng µl–1 and 5 µl of the diluted cDNA (10 ng cDNA) was used as a template in each well for quantitative real-time PCR analysis. The cDNA was amplified using SYBR Green PCR Master Mix (Finnzymes, Espoo, Finland) on the ABI 7000 thermocycler (Applied Biosystems, Foster City, CA, USA). Primers for the actin gene were used as an internal control to normalize the expression data for each gene. The following primer sequences were used: 5'-ACACCCAATATTATTCCTCTTAA-3' and 5'-CGGGAATACGCTAAAGG-3' (OsHKT1), 5'-CAAAGGCAGGTGAATCAAG-3' and 5'-CGATTCAAAAGGCCCTAA-3' (OsHKT2), and 5'-TTTCTTTTGCTACTGCCTTTATATTGC-3' and 5'-AGTGTAGACGAAGTGCATGAAGGA-3' (OsVHA).
The PCR conditions were as follows: 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. A dissociation kinetic analysis was performed at the end of the experiment to check the specificity of annealing. Three replications were performed for each sample in each experiment; each experiment was independently performed twice. Standard determination curves were generated using serial dilutions of 50, 10, 2, and 0.4 ng cDNA in each well for every experiment. Results were analysed according to Muller et al. (2002).
In situ PCR
Sections of leaves and roots were fixed with FAA, dehydrated, and embedded in Paraplast Plus (Fisher Scientific, Pittsburgh, PA, USA) as described by Golldack et al. (2002b). Microtome sections of 12 µm were mounted on microscopic slides coated with aminoalkylsilane (S4651 silane-prep slides; Sigma-Aldrich, Germany). The tissue sections were deparaffinized and rehydrated, and then treated with proteinase K and RNase-free DNase I. Synthesis of cDNA was performed with oligo-dT primers and SuperScript II Reverse Transcriptase (Invitrogen, Breda, the Netherlands). PCR reactions were performed using the same gene-specific oligonucleotide primers used for the qRT-PCR reactions described earlier. The PCR conditions were as follows: 94 °C for 1 min 30 s in the first cycle followed by 1 min at 94 °C, 1 min at 55 °C, 2 min at 72 °C (with the same number of cycles for each gene as in qRT-PCR), and a final extension at 72 °C for 10 min. For signal detection Alexa Fluor 488–5-dUTP (Molecular Probes, Leiden, the Netherlands) was used as a label. Negative control reactions were performed without adding the gene-specific oligonucleotide primers to the PCR reactions. After PCR amplifications, the tissue sections were stained with 10 µM propidium iodide for 10 min. Microscopic images were taken with a cooled CCD-Camera coupled to an Axioskop fluorescence microscope (Zeiss, Germany). In the microscopic images the specific signals were indicated by green to yellow fluorescence and the negative background signals by red.
FRET analysis
Protoplast isolation:
Protoplasts from the leaves of both Pokkali and BRRI Dhan29 were prepared as described by Shishova and Lindberg (1999), but with some modifications. Leaves from approximately 10-d-old seedlings were sliced into 0.5 mm pieces and treated with 1% (w/v) cellulase (lyophilized powder; 10.0 units mg–1 solid) from Trichoderma resei (Sigma, EC 3.2.1.4
[EC]
; Sigma-Aldrich, St Louis, MO, USA) and 0.6% (w/v) macerase (lyophilized powder; 0.6 units mg–1 solid) Maceroenzyme R-10 (EC 3.2.1.4
[EC]
; Serva Electrophoresis, Heidelberg, Germany) for 2 h. The protoplasts were washed twice in the loading medium containing 0.5 M sorbitol (Sigma-Aldrich, St Louis, MO, USA), 0.1 mM CaCl2, 0.2% (w/v) polyvinylpolypyrrolidone (PVP; Sigma-Aldrich, St Louis, MO, USA), and a buffer (pH 5.5) containing 5 mM TRIS (Labassco, Germany) and 5 mM MES (Sigma-Aldrich, St Louis, MO, USA).
Protoplast transfection:
Constructs of the V-ATPase subunits VHA-a, VHA-c, VHA-E, and VHA-B of Mesembryanthemum crystallinum fused to fluorescent proteins (Seidel et al., 2004, 2005) were used for the transient transfection of rice protoplasts. The transfection was performed as previously described in the case of Arabidopsis protoplasts (Seidel et al., 2004).
FRET measurement:
Fluorescence resonance energy transfer (FRET) was measured using a confocal microscop (Leica SP2; Leica Microsystems, Heidelberg, Germany). Cyan fluorescence protein (CFP) was excited by the 458 nm line of an argon-ion-laser and detected in the range of 470–510 nm as a control for the CFP crosstalk into the FRET channel (530–600 nm detection and 458 nm excitation bandwidth). Yellow fluorescence protein (YFP) was detected in the range of 530–600 nm using the 514 nm line of the argon-ion-laser for excitation. The YFP -signal was required for estimating YFP by direct excitation by the 458 nm laser line (Seidel et al., 2005).
FRET-efficiency was calculated by relating the corrected emission intensity in the FRET channel to the sum of the CFP emission and the FRET emission (Seidel et al., 2005):
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For each FRET-pair, the FRET-effect of 40 protoplasts was measured in two independent experiments.
Measurement of vacuolar area
Protoplasts from shoots of cvs Pokkali and BRRI Dhan29 were isolated as described above. Protoplasts were stained with 3 µl of 5 mM 6-carboxyfluorescein diacetate (6-CFDA; Sigma-Aldrich, St Louis, MO, USA) in DMSO through PEG-mediated osmotic shock. Non-specific esterase activity in the vacuolar lumen cleaves the non-fluorescent 6-CFDA to the fluorescent, pH-dependent dye, 6-carboxyfluorescein (6-CF; Preston et al., 1989). The 6-CF fluorescence in the vacuoles was imaged using a laser scanning confocal microscop (Leica SP2; Leica Microsystems, Heidelberg, Germany). The dye was excited sequentially by the 458 nm and 488 nm lines of an argon-ion-laser. Images of the vacuoles were taken, showing a signal in the 500–530 nm range after passing through a Leica RSP500 short -pass filter (Leica). The areas of the vacuolar images were measured using the Leica Confocal Software LCS Lite (ftp://ftp.llt.de/pub/softlib/LCSLite/).
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Results |
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The transcript levels of OsHKT1 (a Na+-transporter), OsHKT2 (a K+-Na+ co-transporter), and OsVHA (an energizer for tonoplast Na+/H+ antiporter under salt stress) were quantified using real-time RT-PCR in the salt-sensitive rice cv. BRRI Dhan29 and the salt-tolerant rice cv. Pokkali after 0, 1, 6, 24, 48, and 72 h of salt stress with 150 mM NaCl. The cell-specificity of these transcripts was also studied using in situ PCR. The conformational change and energy transfer efficiency of OsVHA upon NaCl stress was studied by measuring FRET efficiencies between the peripheral stalk subunit VHA-E and the catalytic head subunit VHA-B and between the proteolipid VHA-c and the C-terminal transmembrane domain of VHA-a, respectively. The changes in vacuolar area in leaf cells (protoplasts) upon salt stress were also investigated.
Expression of OsHKT1
OsHKT1 transcripts displayed variable expression with time up to 72 h after 150 mM NaCl stress, in both root and shoot tissues of rice cvs Pokkali and BRRI Dhan29 (Fig. 1). There was clear up-regulation of the transcript in BRRI Dhan29 in the root, 1 h after the stress, and in the shoot 6 h after the stress. In the root tissue, the up-regulation increased substantially 6 h after the stress. However, 6 h after the stress, when the transcript displayed the highest induction in both the root and shoot tissues, induction was approximately four times higher in the root and approximately two times higher in the shoot than their respective control levels. In both the root and shoot tissues, the up-regulation of the transcript compared with the control expression continued up to 72 h after the stress, but with some variation in terms of degree of up-regulation. On the other hand, there was significant up-regulation of the transcript in cv. Pokkali only 24 h after the stress in both the shoot and root tissues, and the expression was less than 1-fold higher in both cases than the respective control levels. Although the transcript displayed an up-regulation compared with the control expression up to 72 h after the stress in the salt-sensitive cv. BRRI Dhan29, there was significant down-regulation of this transcript in the salt-tolerant cv. Pokkali starting 48 h after the stress in both the shoot and root tissues. Seventy-two hours after the stress, the down-regulation of this transcript was approximately 62% in the Pokkali shoot and approximately 72% in the Pokkali root compared with the control expression. In situ expression of the OsHKT1 transcript confirmed its expression in both the root and shoot tissues of BRRI Dhan29 and Pokkali (Figs 4, 5). Under NaCl stress conditions, in root tissue of BRRI Dhan29 (Fig. 4), a strong signal was detected from the epidermis and vascular cylinder, but only a weak signal from the cortex. In root tissue of Pokkali, the signal was weaker upon stress than the control signal. Under control conditions, a strong signal in BRRI Dhan29 leaf tissue came from the mesophyll cells and the phloem, a weaker signal from the epidermal cells, and no signal from the xylem; in Pokkali leaves, a strong signal came from the mesophyll cells, phloem and xylem. After 48 h of NaCl stress, the OsHKT1 transcript in BRRI Dhan29 disappeared from the epidermis and also, notably, from the phloem, while a somewhat higher signal came from the mesophyll cells. In Pokkali, however, down-regulation of the transcript was noted in all types of cells, most notably from the phloem, xylem, and mesophyll cells.
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Expression of OsHKT2
Real-time analysis indicated a substantial increase in the OsHKT2 transcript immediately after stress with 150 mM NaCl in Pokkali, but not in BRRI Dhan29 (Fig. 2). There was an approximately 15-fold higher expression of OsHKT2 in Pokkali shoot- tissue 1 h after the stress than in the control. Although the up-regulation of OsHKT2 in Pokkali leaf tissue gradually decreased by the ensuing time points, at 72 h after stress it still represented a 158% higher expression than that of the control. In the shoot tissue of BRRI Dhan29, a significant up-regulation of the transcript was found at 6 h after stress, representing nearly a 2-fold higher expression than that of the control. The up-regulation decreased slightly afterwards up to 72 h after the stress. There was also an up-regulation of OsHKT2 in root tissue of Pokkali, which continued up to 72 h after the stress. However, the up-regulation in Pokkali roots was not as high as in the shoot tissue; it was less than 50% higher than the control level up to 48 h after the stress, and approximately 177% higher 72 h after the stress. On the other hand, OsHKT2 was down-regulated in the root tissue of BRRI Dhan29 up to 24 h after the stress (to nearly 50% of the control expression), after which a slight up-regulation was evident 48 h and 72 h after the stress.
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As demonstrated by in situ analysis (Fig. 4), under control conditions OsHKT2 was mainly expressed in the epidermis and vascular cylinder of the root with some low expression in the cortex. After stress with 150 mM NaCl, a weaker signal was detected from all these types of root cells from BRRI Dhan29, whereas a stronger signal was obtained from Pokkali, in line with the real-time quantification of this transcript. Under control conditions, the expression of OsHKT2 in leaf tissue was confined mainly to the mesophyll cells (Fig. 5). After NaCl stress for 24 h, however, a very strong signal appeared from the phloem and the connecting area between the phloem and mesophyll cells. The signal in mesophyll cells was also somewhat stronger in stressed cells than in control cells.
Expression of OsVHA
As shown in Fig. 3, there was an induction of OsVHA in both the roots and shoots of Pokkali 1 h after the stress, which increased further 6 h after the stress. The induction was also noted in both the root and shoot tissues of BRRI Dhan29, but not until 6 h after the stress, and to a lesser extent than in Pokkali. Induction of the transcript 6 h after the stress was approximately four times higher in Pokkali shoot tissue and nearly twice as high in BRRI Dhan29 shoot tissue than their respective control expressions. Afterwards, the induction in both the cultivars started to decrease with time. In the shoot tissue of both cultivars, the lowest induction occurred 48 h after the stress, which then gave some induction again at 72 h after the stress. On the other hand, in comparison with the shoot tissue, the initial induction of OsVHA in root tissue was lower in both Pokkali and BRRI Dhan29, though 72 h after the stress the induction was close to that of the shoot.
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Under control conditions, OsVHA in root tissue produced a strong signal from the epidermis and vascular cylinder, but a very weak signal from the cortex region in both cultivars (Fig. 4). Moreover, after 24 h of stress with 150 mM NaCl, the signal from the root vascular cylinder in BRRI Dhan29 became weaker, whereas the signal from the root epidermis in Pokkali was stronger. In leaf tissue of BRRI Dhan29, OsVHA was expressed under control conditions in phloem cells, at the transition from phloem to mesophyll cells, and in the mesophyll cells (Fig. 5). On the other hand, in leaf tissue of Pokkali, no signal was detected from the phloem cells or from the transition from the phloem to mesophyll cells; the expression was instead confined to the mesophyll cells alone. After 1 h of stress with 150 mM NaCl, however, a very strong signal was detected in the Pokkali leaf tissue from the phloem cells and from the transition from the phloem to mesophyll cells, along with comparatively stronger expression in the mesophyll cells. A stronger signal from the aforesaid cells was also found in BRRI Dhan29 leaf tissue, but not to the same extent as in the Pokkali leaf tissue.
Effect of salt stress on vacuolar area
Vacuoles, comprising up to 95% of cell volume in mature cells (Epimashko et al., 2004), are thought to be a good sink for the compartmentalization of cytosolic Na+. So changes in vacuole area could be evident under salt stress. However, no significant change were found in the vacuolar area in the salt-tolerant cv. Pokkali upon salt stress, after either short-term (5–10 min) or long-term (24 h) treatment (Fig. 6). On the other hand, compared to the control (91.1±16.06), the vacuolar area in the salt-sensitive cv. BRRI Dhan29 decreased (62.62±10.5) after 5–10 min of NaCl stress, but then increased (115.16±0.81) after 24 h of stress. The vacuolar area in unstressed tissue was the same in both cultivars: 102.4±6.26 µm2 in Pokkali and 91.1±16.06 µm2 in BRRI Dhan29.
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Effect of salt stress on VHA structure
VHA is a protein complex of 13 different subunits with a bipartite structure similar to that of F-ATP -synthases (Seidel et al., 2005). The membrane integral sector, V0, is composed of subunits VHA-a, c, d, and e and the cytoplasmically exposed V1 sector contains the subunits VHA-A to VHA-H. Sector V0 catalyses the proton transport, whereas sector V1 is involved in ATP binding and hydrolysis. To test whether salt has an effect on the stator structure of the VHA, the FRET-efficiency between the peripheral stalk subunit VHA-E and the catalytic head subunit VHA-B was measured. No changes were identified as due to NaCl stress in either cv. Pokkali (48.34±1.73 and 46.76±1.32) or cv. BRRI Dhan29 (38.57±1.86 and 40.81±2.26). The VHA of cv. Pokkali displayed a significantly (t test; P=0.000128) higher FRET efficiency between VHA-E and VHA-B than BRRI Dhan29, indicating structural differences between these two enzymes. To investigate the structural flexibility of the transmembrane sector, V0, of the V-ATPase, the FRET efficiency between the proteolipid VHA-c and the C-terminal transmembrane domain of VHA-a was measured. As shown in Fig. 7, under control conditions the FRET efficiency was similar in Pokkali (46.16±1.84) and BRRI Dhan29 (44.6±1.82); however, the efficiency of energy transfer increased significantly (t test, P=0.00154) due to NaCl stress in cv. BRRI Dhan29 (51.54±1.45) and decreased a little in cv. Pokkali (43.43±1.76).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionReferences |
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The present study detected the induction of OsHKT1 in both the root and shoot tissue of BRRI Dhan29 under high NaCl conditions, although the degree of induction compared with the control expression varied with time. On the other hand, in Pokkali the transcript of OsHKT1 remained unaffected up to 6 h after NaCl stress in both the root and shoot tissues and displayed a down-regulation 48 h and 72 h after the stress. There was, however, some induction of OsHKT1 in both the root and shoot tissues of Pokkali 24 h after the stress. As shown before, OsHKT1 mediates Na+ influx, but not K+ influx (Horie et al., 2001). It has also been shown that OsHKT1 specifically mediates Na+ uptake in rice roots when the plants are K+ deficient (Garciadeblás et al., 2003), and is induced by low-K+ conditions (Horie et al., 2001). In the present study, the induction of OsHKT1 in root epidermis and vascular cylinder cells, as well as in shoot mesophyll cells of salt-sensitive BRRI Dhan29, might indicate its involvement in Na+ uptake by the root and in the subsequent circulation of Na+ in the leaf mesophyll cells, where Na+ causes damage. Since the experimental plants were grown with an optimal K+ concentration in the growth medium, there should be no K+ deficiency in cells under control conditions. However, under high NaCl conditions, Na+ competition at K+ binding sites may result in K+ deficiency (Maathuis and Amtmann, 1999), and thus might cause the induction of OsHKT1 in both the cultivars observed here. Another possibility is that under high NaCl conditions, excess Na+ entering the cytosol increases the optimal cytosolic Na+/ K+ ratio, which cells might recognize as a K+ deficiency, thus inducing OsHKT1 as suggested by Horie et al. (2001) in cases of K+ deficiency. The higher uptake of Na+ into the cytosol of cv. BRRI Dhan29 than into that cv. Pokkali (Kader and Lindberg, 2005) is probably caused by a faster induction of OsHKT1 in BRRI Dhan29 (1 h and 6 h after the stress) than in Pokkali (24 h after the stress).
With a longer stress period, the salt-tolerant cv. Pokkali starts to down-regulate OsHKT1 expression in both roots and shoots, as found here. The down-regulation of OsHKT1 in leaf mesophyll cells in Pokkali explains the salt-tolerance of this cultivar, possibly by hindering Na+-influx into these metabolically very important cells. The down-regulation of OsHKT1 in response to salt stress was also demonstrated by Horie et al. (2001) and Golldack et al. (2002a). However, the initial induction of OsHKT1 in the salt-tolerant cv. Pokkali, for whatever reason, counters its salt-tolerance ability. Possibly, some post-transcriptional changes, or any conformational changes of the protein, hinder Na+ transport into Pokkali by means of this Na+ transporter. In a recent study, it was shown that the uptake of Na+ by K+-selective channels/transporters was not found in this cultivar (Kader and Lindberg, 2005).
This study also suggests the involvement of OsHKT2 in the salt-stress response, especially in the salt-tolerant cv. Pokkali. There was a substantial induction (some 15-fold higher than in the control) of OsHKT2 in shoot tissue of Pokkali and to a lesser extent in root tissue of the same cultivar, but not in the salt-sensitive cv. BRRI Dhan29. Although OsHKT2 (a K+-Na+ coupled transporter) does not mediate K+ influx from a high K+ solution in the absence of Na+, it does confer tolerance of salt stress under high Na+ conditions, probably by an increased K+ uptake ability, as demonstrated in Saccharomyces cerevisiae (Horie et al., 2001). In the present study, the induction of OsHKT2 in the epidermis, exodermis, and xylem tissue of roots might indicate its involvement in K+ uptake and transport through xylem. The induction in the mesophyll cells and in the transition from the phloem to mesophyll cells may indicate its involvement in the recirculation of K+ within the mesophyll cells through the phloem. In addition to metabolites such as sugars, minerals and salts also can use the phloem pathway to be redistributed from old source leaves towards young and expanding sink leaves (Sondergaard et al., 2004). Thus, the induction of OsHKT2 in the salt-tolerant cv. Pokkali might confer salt tolerance by increasing its expression in leaf tissue, through contributing to a low cytosolic Na+/K+ ratio, as suggested by Horie et al. (2001). Maathuis (2006) recently reported a greater than 3-fold change of Na+-K+ symport in response to salt stress. Several other K+-transporter genes have also been shown to be up-regulated under high NaCl conditions, which possibly reflects a plant's ability to maintain specific cytosolic K+ levels at various capacities. Salt stress increases the transcript of the K+-transporter genes AtKC1 (Pilot et al., 2003), KMT1 and various HAK/KUP (Su et al., 2001, 2002). The ability to maintain ionic homeostasis was shown to be an important salt-tolerance determinant in barley when it was compared with a moderate salt-sensitive rice cultivar IR64 (Ueda et al., 2006). This study also revealed that the regulatory mechanism for controlling K+/Na+ homeostasis in cells of the salt-tolerant cv. Pokkali seemed to work by increasing K+ uptake (by inducing the expression of OsHKT2), and then also by reducing Na+ influx (by decreasing the expression of OsHKT1). These mechanisms were not as efficient in BRRI Dhan29 as in Pokkali. Since K+, at a high concentration, also is inhibitory for enzymatic functions (Greenway and Osmond, 1972), the induction of OsHKT2 in Pokkali leaves decreased over the course of the stress period.
In addition to the increased uptake of K+ and decreased Na+-influx, as proposed, Pokkali might maintain K+/Na+ homeostasis in the cytosol by compartmentalizing cytosolic Na+ into the vacuole. Upon NaCl stress in Pokkali, relatively quick (1 h after the stress) induction of OsVHA was found in this cultivar. Although the expression of OsVHA was induced in the salt-sensitive cv. BRRI Dhan29, the induction was much delayed (until 6 h after the stress) and was less than that of the salt-tolerant cv. Pokkali. This result is consistent with an earlier study, where we demonstrated the compartmentalization of Na+ into the vacuole after few minutes of NaCl stress in Pokkali, but not in BRRI Dhan29 (Kader and Lindberg, 2005). The induced expression of OsVHA upon salt stress was found mainly in the epidermis of the roots. This is quite rational, since these cells first encounter the excess of cytosolic Na+, before it enters into the xylem through the symplastic pathway to be transported into the shoot. The induction in the phloem and the transition from the phloem to mesophyll cells in leaves might also indicate the effort of these cells to keep the harmful Na+ away from the mesophyll cells.
In addition to maintaining cytosolic ionic homeostasis and pH, VHA was also shown to be important for salt tolerance in Saccharomyces cerevisiae (Hamilton et al., 2002) and in many plant species (Golldack and Dietz, 2001; Kluge et al., 2003a; Senthilkumar et al., 2005; Vera-Estrella et al., 2005). Some variable expression of OsVHA with time was observed under salt stress in both cultivars studied (Fig. 3). This increase–decrease–increase induction of OsVHA in both the cultivars might indicate certain limitations of the vacuole as a sink for cytosolic Na+. Immediately after the stress, the salt-tolerant cv. Pokkali increased the expression of VHA; the induction continued to increase until 6 h after the stress, and thus possibly compartmentalized cytosolic Na+ into the vacuole. Thereafter, a high amount of Na+ in the vacuole might have reduced OsVHA induction, which decreased nearly to the control level 48 h after the stress. At 72 h after the stress OsVHA is again induced, which might be correlated with cell growth.
The FRET-measurements between two different subunits of a particular protein fused with fluorophores, such as CFP and YFP, facilitate quantitative estimates of distance in terms of the efficiency of resonance energy transfer (Seidel et al., 2005). Thus, the changes in FRET efficiency between two different subunits of VHA upon salt stress might be a good indication of any structural change of the protein due to salt stress. In general, salinity affects the expression of VHA-genes in plants. One important mechanism of salt-tolerance in plants seems to be the adaptation of transport capacities via changes in the amount of VHA and therefore of VHA activity (Ratajczak, 2000; Wang et al., 2001; Kluge et al., 2003b). However, so far no report has demonstrated any structural change of VHA due to salt stress, though some ATP-hydrolysis and proton transport data do reveal changes in the coupling ratio of plant VHA upon exposure to salt stress (Ratajczak, 2000).
The FRET efficiency between the proteolipid VHA-c and the C-terminal transmembrane domain of VHA-a decreased slightly in cv. Pokkali, but increased significantly in cv. BRRI Dhan29 upon NaCl stress. Signalling for the regulation of VHA is transduced from the V1 to V0 domain via conformational changes in VHA-a (Landolt-Marticorena et al., 1999). On the other hand, flexibility in the stoichiometry of the proteolipid VHA-c within the complex might also explain the observed structural changes. These changes could be due either to an increase in the diameter of the proteolipid-ring (Klink et al., 1990; Ratajczak, 2000) or to the replacement of VHA-c by isoforms, as indicated by differential immunological cross reactions (FischerSchliebs et al., 1997). Changes in the coupling ratio of the V-ATPase from Mesembryanthemum crystallinum (Rockel et al., 1994) support this hypothesis. Although the analysed VHA subunits are derived from M. crystallinum, artificial effects can be excluded, because the increase of FRET efficiency was specific to cv. BRRI Dhan29 upon salinity exposure. Nevertheless, the differential changes of FRET efficiency between VHA-a and VHA-c in Pokkali and BRRI Dhan29 might be correlated with their differential levels of Na+-compartmentalization into the vacuole.
Under salt stress, both the increased vacuolar compartmentalization ability of Na+ (by inducing the expression of VHA) and decreased uptake of Na+ into the cytosol (by decreasing the expression of OsHKT1) seem to work more efficiently in the salt-tolerant cv. Pokkali than in the salt-sensitive cv. BRRI Dhan29. Maathuis (2006) suggested that both the down-regulation of HKT1 and the up-regulation of an NHX isoform (tonoplast Na+/H+ antiporter) could contribute greatly to limiting Na+ loading in plant tissue, particularly when cytosolic Na+ contents are concerned.
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionReferences |
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With respect to OsHKT1, OsHKT2, and OsVHA expression data, the regulatory mechanism of cytosolic Na+/K+ homeostasis seems to be an important salt-tolerance determinant in the salt-tolerant rice cv. Pokkali. This mechanism is less efficient in the salt-sensitive cv. BRRI Dhan29. At the onset of NaCl stress, Pokkali increases the expression of OsHKT2 in both the root and shoot tissues. Since the induction is very strong in the shoot, in particular in the mesophyll cells, the OsHKT2 possibly enhances the recirculation of K+ in metabolically active leaf mesophyll cells under high NaCl conditions, to maintain an adequate cytosolic Na+/K+ ratio. Although the cells also take up Na+ along with K+ by means of this transporter, cells might transport Na+ back from the mesophyll cells by means of OsHKT8, as suggested by Rus et al. (2005) and as delineated in Fig. 8. As with OsHKT2, Pokkali also induces the expression of OsVHA at the onset of high NaCl conditions, most likely to compartmentalize cytosolic Na+ into the vacuole. The induction of OsVHA decreases nearly to the control expression levels in root tissue 24 h after the stress and in shoot tissue 48 h after the stress, although OsVHA expression increases 72 h after the stress. Moreover, Pokkali induces the expression of the Na+ transporter OsHKT1 24 h after NaCl stress. This might occur either because of K+ deficiency in cells (caused by Na+ competition at transport sites), or by interruption of the cytosolic Na+/K+ ratio, which cells might sense as a K+-deficiency. However, at a certain stage later on, Pokkali down-regulates the expression of OsHKT1. It is concluded that, at the onset of high NaCl conditions, Pokkali maintains cytosolic Na+/K+ homeostasis by increasing the Na+-K+ coupled uptake through the induction of OsHKT2, as well as by increasing the compartmentalization of cytosolic Na+ into the vacuole. The latter is facilitated by the induced expression of OsVHA. There is, however, no structural change of OsVHA, as estimated in terms of the FRET efficiency between VHA-a and VHA-c, in cv. Pokkali. Pokkali might also maintain a low influx of cytosolic Na+, either by means of a conformational change of the OsHKT1 protein and/or any post-transcriptional changes of the OsHKT1 gene. On the other hand, 48 h after the stress, Pokkali maintains cytosolic K+/Na+ homeostasis by down-regulating OsHKT1. However, to understand the mechanism of K+/Na+ homeostasis in rice fully, the cell- and tissue-specific expression patterns of other members of the HKT family need to be investigated under conditions of NaCl stress.
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Acknowledgements |
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We gratefully acknowledge financial support from the Islamic Development Bank (IDB) and from the Lennart Hjelm's foundation, SLU. We thank Dr Vladislav V Yemelianov for his help to convert the figures as TIFF file.
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionReferences |
|---|
Apse MP, Aharon GS, Snedden WA, Blumwald E. (1999) Salt tolerance conferred by overexpression of a vacuolar Na+/H+ antiport in Arabidopsis. Science 285 1256–1258.
Berthomieu P, Conejero G, Nublat A, et al. (2003) Functional analysis of AtHKT1 in Arabidopsis shows that Na+ recirculation by the phloem is crucial for salt tolerance. EMBO Journal 22 2004–2014.[CrossRef][Web of Science][Medline]
Bhandal IS and Malik CP. (1988) Potassium estimation, uptake, and its role in the physiology and metabolism of flowering plants. International Review of Cytology 110 205–254.
Blumwald E. (2000) Sodium transport and salt tolerance in plants. Current Opinion in Cell Biology 12 431–434.[CrossRef][Web of Science][Medline]
Buschmann PH, Vaidynathan R, Gassmann W, Schroeder JI. (2000) Enhancement of Na+ uptake currents, time-dependent inward-rectifying K+ channels currents, and K+ channel transcripts by K+ starvation in wheat root cells. Plant Physiology 122 1387–1397.
Chauhan S, Forsthoefel N, Ran Y, Quigley F, Nelson DE, Bohnert HJ. (2000) Na+/myo-inositol symporters and Na+/H+-antiport in Mesembryanthemum crystallinum. The Plant Journal 24 511–522.[CrossRef][Web of Science][Medline]
Davenport RJ and Tester M. (2000) A weakly voltage-dependent, nonselective cation channel mediates toxic sodium influx in wheat. Plant Physiology 122 823–834.
Demidchik V, Davenport RJ, Tester M. (2002) Nonselective cation channels in plants. Annual Review of Plant Physiology and Plant Molecular Biology 53 67–107.[CrossRef][Medline]
Demidchik V and Tester M. (2002) Sodium fluxes through nonselective cation channels in the plasma membrane of protoplasts from Arabidopsis roots. Plant Physiology 128 379–387.
Epimashko S, Meckel T, Fischer-Schliebs E, Lüttge U, Thiel G. (2004) Two functionally different vacuoles for static and dynamic purposes in one plant mesophyll leaf cell. The Plant Journal 37 294–300.[Web of Science][Medline]
FischerSchliebs E, Ball E, Berndt E, BesemfelderButz E, Binzel ML, Drobny M, Mühlenhoff D, Müller ML, Rakowski K, Ratajczak R. (1997) Differential immunological cross-reactions with antisera against the V-ATPase of Kalanchöe daigremontiana reveal structural differences of V-ATPase subunits of different plant species. Biological Chemistry 378 1131–1139.[Web of Science][Medline]
Flowers TJ and Läuchli A. (1983) Sodium versus potassium: substitution and compartmentation. In Läuchli A and Bieleski RL (Eds.). Inorganic plant nutritionBerlin Springer-Verlag Vol. 15B pp. 651–681.
Fukuda A, Nakamura A, Tagiri A, Tanaka H, Miyao A, Hirochika H, Tanaka Y. (2004) Function, intracellular localization and the importance in salt tolerance of a vacuolar Na+/H+ antiporter from rice. Plant and Cell Physiology 45 146–159.
Fukuda A, Yazaki Y, Ishikawa T, Koike S, Tanaka Y. (1998) Na+/H+ antiporter in tonoplast vesicles from rice roots. Plant and Cell Physiology 39 196–201.
Garciadeblás B, Senn ME, Banuelos MA, Rodriguez-Navarro A. (2003) Sodium transport and HKT transporters: the rice model. The Plant Journal 34 788–801.[CrossRef][Web of Science][Medline]
Golldack D and Dietz KJ. (2001) Salt-induced expression of the vacuolar H+-ATPase in the common ice plant is developmentally controlled and tissue specific. Plant Physiology 125 1643–1654.
Golldack D, Quigley F, Michalowski CB, Kamasani UR, Bohnert HJ. (2003) Salinity stress-tolerant and -sensitive rice (Oryza sativa L.) regulate AKT-type potassium channel transcripts differently. Plant Molecular Biology 51 71–81.[CrossRef][Web of Science][Medline]
Golldack D, Popova OV, Dietz KJ. (2002b) Mutation of the matrix metalloproteinase At2-MMP inhibits growth and causes late flowering and early senescence in Arabidopsis. Journal of Biological Chemistry 277 5541–5547.
Golldack D, Su H, Quigley F, Kamasani UR, Munoz-Garay C, Balderas E, Popova OV, Bennett J, Bohnert HJ, Pantoja O. (2002a) Characterization of a HKT-type transporter in rice as a general alkali cation transporter. The Plant Journal 31 529–542.[CrossRef][Web of Science][Medline]
Greenway H and Osmond CB. (1972) Salt responses of enzymes from species differing in salt tolerance. Plant Physiology 49 256–259.
Hamada A, Shono M, Xia T, Ohta M, Hayashi Y, Tanaka A, Hayakawa T. (2001) Isolation and characterization of a Na+/H+ antiporter gene from the halophyte Atriplex gmelini. Plant Molecular Biology 46 35–42.[CrossRef][Web of Science][Medline]
Hamilton CA, Taylor GJ, Good AG. (2002) Vacuolar H+-ATPase, but not mitochondrial F1F0-ATPase, is required for NaCl tolerance in Saccharomyces cerevisiae. FEMS Microbiology Letters 208 227–232.[CrossRef][Web of Science][Medline]
Horie T, Yoshida K, Nakayama H, Yamada K, Oiki S, Shinmyo A. (2001) Two types of HKT transporters with different properties of Na+ and K+ transport in Oryza sativa. The Plant Journal 27 129–138.[CrossRef][Web of Science][Medline]
Kader MA and Lindberg S. (2005) Uptake of sodium in protoplasts of salt-sensitive and salt-tolerant cultivars of rice, Oryza sativa L. determined by the fluorescent dye SBFI. Journal of Experimental Botany 56 3149–3158.
Klink R, Haschke HP, Kramer D, Lüttge U. (1990) Membrane-particles, proteins and ATPase activity of tonoplast vesicles of Mesembryanthemum crystallinum in the C3 and CAM state. Botanica Acta 103 24–31.
Kluge C, Lahr J, Hanitzsch M, Bolte S, Golldack D, Dietz KJ. (2003a) New insight into the structure and regulation of the plant V-ATPase. Journal of Bioenergetics and Biomembranes 35 377–388.[CrossRef][Web of Science][Medline]
Kluge C, Lamkemeyer P, Tavakoli N, Golldack D, Kandlbinder A, Dietz KJ. (2003b) cDNA cloning of 12 subunits of the V-type ATPase from Mesembryanthemum crystallinum and their expression under stress. Molecular Membrane Biology 20 171–183.[CrossRef][Web of Science][Medline]
Landolt-Marticorena C, Kahr HW, Zawarinski P, Correa J, Manolson MF. (1999) Substrate- and inhibitor-induced conformational changes in the yeast V-ATPase provide evidence for communication between the catalytic and proton-translocating sectors. Journal of Biological Chemistry 275 15449–15457.
Maathuis FJM. (2006) The role of monovalent cation transporters in plant responses to salinity. Journal of Experimental Botany 57 1137–1147.
Maathuis FJM and Amtmann A. (1999) K+ nutrition and Na+ toxicity: the basis of cellular K+/Na+ ratios. Annals of Botany 84 123–133.
Mäser P, Hosoo Y, Goshima S, et al. (2002) Glycine residues in potassium channel-like selectivity filters determine potassium selectivity in four-loop-per-subunit HKT transporters from plants. Proceedings of the National Academy of Sciences, USA 99 6428–6433.
Muller PY, Janovjak H, Miserez AR, Dobbie Z. (2002) Processing of gene expression data generated by quantitative real-time RT-PCR. BioTechniques 32 1372–1379.[Web of Science][Medline]
Munns R, James AJ, Läuchli A. (2006) Approaches to increasing the salt tolerance of wheat and other cereals. Journal of Experimental Botany 57 1025–1043.
Padmanaban S, Lin XY, Perera I, Kawamura Y, Sze H. (2004) Differential expression of vacuolar H+-ATPase subunit c genes in tissues active in membrane trafficking and their roles in plant growth as revealed by RNAi. Plant Physiology 134 1514–1526.
Pilot G, Gaymard F, Mouline K, Cherel I, Sentenac H. (2003) Regulated expression of Arabidopsis shaker K+ channel genes involved in K+ uptake and distribution in the plant. Plant Molecular Biology 51 773–787.[CrossRef][Web of Science][Medline]
Preston RA, Murphy RF, Jones EW. (1989) Assay of vacuolar pH in yeast and identification of acidification-defective mutants. Proceedings of the National Academy of Sciences, USA 86 7027–7031.
Ratajczak R. (2000) Structure, function and regulation of the plant vacuolar H+-translocating ATPase. Biochimica et Biophysica Acta 1465 17–36.[Medline]
Ren ZH, Gao JP, Li LG, Cai XL, Huang W, Chao DY, Zhu MZ, Wang ZY, Luan S, Lin HX. (2005) A rice quantitative trait locus for salt tolerance encodes a sodium transporter. Nature Genetics 37 1141–1146.[CrossRef][Web of Science][Medline]
Roberts SK and Tester M. (1997) Patch clamp study of Na+ transport in maize roots. Journal of Experimental Botany 48 431–440.[Web of Science]
Rockel B, Ratajczak R, Becker A, Lüttge U. (1994) Changes densities and diameters of intermembrane tonoplast particles Mesembryanthemum crystallinum in correlation with NaCl- induced CAM. Journal of Plant Physiology 143 318–324.
Rodriguez-Navarro A and Rubio F. (2006) High-affinity potassium and sodium transport systems in plants. Journal of Experimental Botany 57 1149–1160.
Rus AM, Bressan RA, Hasegawa PM. (2005) Unraveling salt tolerance in crops. Nature Genetics 37 1029–1030.[CrossRef][Web of Science][Medline]
Rus A, Lee BH, Munoz-Mayor A, Sharkhuu A, Miura K, Zhu JK, Bressan RA, Hasegawa PM. (2004) AtHKT1 facilitates Na+ homeostasis and K+ nutrition in planta. Plant Physiology 136 2500–2511.
Seidel T, Golldack D, Dietz KJ. (2005) Mapping of C-termini of V-ATPase subunits by in vivo-FRET measurements. FEBS Letters 579 4374–4382.[CrossRef][Web of Science][Medline]
Seidel T, Kluge C, Hanitzsch M, Ross J, Sauer M, Dietz KJ, Golldack D. (2004) Colocalization and FRET-analysis of subunits c and a of the vacuolar H+-ATPase in living plant cells. Journal of Biotechnology 112 165–175.[CrossRef][Web of Science][Medline]
Senthilkumar P, Jithesh MN, Parani M, Rajalakshmi S, Praseetha K, Parida A. (2005) Salt stress effects on the accumulation of vacuolar H+-ATPase subunit c transcripts in wild rice, Porteresia coarctata (Roxb.) Tateoka. Current Science 89 1386–1394.
Shishova M and Lindberg S. (1999) Auxin-induced cytosol acidification in wheat leaf protoplasts depends on external concentration of Ca2+. Journal of Plant Physiology 155 190–196.
Sondergaard TE, Schulz A, Palmgren MG. (2004) Energization of transport processes in plants. Roles of the plasma membrane H+-ATPase. Plant Physiology 136 2475–2482.
Su H, Golldack D, Katsuhara M, Zhao C, Bohnert HJ. (2001) Expression and stress-dependent induction of potassium channel transcripts in the common ice plant. Plant Physiology 125 604–614.
Su H, Golldack D, Zhao C, Bohnert HJ. (2002) The expression of HAK-type K+ transporters is regulated in response to salinity stress in common ice plant. Plant Physiology 129 1482–1493.
Subbarao GV, Ito O, Berry WL, Wheeler RM. (2003) Sodium: a functional plant nutrient. Critical Reviews in Plant Sciences 22 391–416.
Sunarpi Horie T and Motoda J, et al. (2005) Enhanced salt tolerance mediated by AtHKT1 transporter-induced Na+ unloading from xylem vessels to xylem parenchyma cells. The Plant Journal 44 928–938.[CrossRef][Web of Science][Medline]
Taiz L and Zeiger E. (2002) Plant physiologySunderland, Massachusetts Sinauer Associates, Inc., Publishers.
Tester M and Davenport R. (2003) Na+ tolerance and Na+ transport in higher plants. Annals of Botany 91 503–527.
Tyerman SD, Skerrett M, Garri IIA, Findlay GP, Leigh RA. (1997) Pathways for the permeation of Na+ and Cl– into protoplasts derived from the cortex of wheat roots. Journal of Experimental Botany 48 459–480.[Web of Science]
Ueda A, Kathiresan A, Bennet J, Takabe T. (2006) Comparative transcriptome analyses of barley and rice under salt stress. Theoretical and Applied Genetics 112 1286–1294.[CrossRef][Web of Science][Medline]
Uozumi N, Kim EJ, Rubio F, Yamaguchi T, Muto S, Tsuboi A, Bakker EP, Nakamura T, Schroeder JI. (2000) The Arabidopsis HKT1 gene homolog mediates inward Na+ currents in Xenopus laevis oocytes and Na+ uptake in Saccharomyces cerevisiae. Plant Physiology 122 1249–1259.
Vera-Estrella R, Barkla BJ, Garcia-Ramirez L, Pantoja O. (2005) Salt stress in Thellungiella halophila activates Na+ transport mechanisms required for salinity tolerance. Plant Physiology 139 1507–1517.
Wang B, Lüttge U, Ratajczak R. (2001) Effect of salt treatment and osmotic stress on V-ATPase and V-Ppase in leaves of the halophyte Suaeda salsa. Journal of Experimental Botany 52 2355–2365.
Yeo AR, Flowers SA, Rao G, Welfare K, Senanayake N, Flowers TJ. (1999) Silicon reduces sodium uptake in rice (Oryza sativa L.) in saline conditions and this is accounted for by a reduction in the transpirational bypass flow. Plant, Cell and Environment 22 559–565.
Zhu JK. (2003) Regulation of ion homeostasis under salt stress. Current Opinion in Plant Biology 6 441–445.[CrossRef][Web of Science][Medline]
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