JXB Advance Access originally published online on November 7, 2005
Journal of Experimental Botany 2005 56(422):3149-3158; doi:10.1093/jxb/eri312
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RESEARCH PAPER |
Uptake of sodium in protoplasts of salt-sensitive and salt-tolerant cultivars of rice, Oryza sativa L. determined by the fluorescent dye SBFI
Department of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences, Box 7080, SE 750 07 Uppsala, Sweden
* To whom correspondence should be addressed. Fax: +46 18 673279. E-mail: Sylvia.Lindberg{at}vbsg.slu.se
Received 10 May 2005; Accepted 15 September 2005
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Abstract |
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In this study, the uptake of Na+ into the cytosol of rice (Oryza sativa L. cvs Pokkali and BRRI Dhan29) protoplasts was measured using the acetoxy methyl ester of the fluorescent sodium-binding benzofuran isopthalate, SBFI-AM, and fluorescence microscopy. By means of inhibitor analyses the mechanisms for uptake and sequestration of Na+ in the salt-sensitive indica rice cv. BRRI Dhan29 and in the salt-tolerant indica rice cv. Pokkali were detected. Less Na+ was taken up into the cytosol of Pokkali than into BRRI Dhan29. The results indicate that K+-selective channels do not contribute to the Na+ uptake in Pokkali, whereas they are the major pathways for Na+ uptake in BRRI Dhan29 along with non-selective cation channels. However, non-selective cation channels seem to be the main pathways for Na+ uptake in Pokkali. Protoplasts from Pokkali leaves took up Na+ only transiently in the presence of extracellular Na+ at 5–100 mM. Therefore, it is likely that the protoplasts have a mechanism for fast extrusion of Na+ out of the cytoplasm. Experiments with protoplasts pretreated with NH4NO3 and NH4VO3 suggest that the salt-tolerant Pokkali extrudes Na+ mainly into the vacuole. After cultivation of both cultivars in the presence of 10 or 50 mM NaCl for 72 h, the isolated protoplasts from Pokkali took up less Na+ than the control protoplasts. The results suggest that the salt-tolerance in Pokkali depends on reduced uptake through K+-selective channels and a fast extrusion of Na+ into the vacuoles.
Key words: Benzofuran isophthalate, compartmentalization, fluorescence ratio microscopy, influx, rice, salt stress, sodium uptake
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSummaryReferences |
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Soil salinity is one of the environmental hazards in agriculture worldwide because it limits crop yield and restricts the use of land previously cultivated. Around 20% of the world's agricultural land and nearly 50% of all irrigated land is adversely affected by soil salinity (Flowers and Yeo, 1995
). Moreover, soil salinization due to irrigation is becoming increasingly detrimental to agriculture (Flowers, 1999).
One of the principal adverse effects of high salinity in non-tolerant plants is growth inhibition by toxicity to Na+. Maintenance of a high cytosolic [K+]:[Na+] ratio is critical for the function of cells (Rubio et al., 1995; Zhu et al., 1998). In saline conditions, Na+ competes with K+ for uptake through common transport systems and this happens often, since in the environment [Na+] is usually higher than [K+]. Thus, elevated levels of cytosolic Na+, or in another way a high [Na+]:[K+] ratio, exerts metabolic toxicity by competition between Na+ and K+ for the binding sites of many enzymes (Bhandal and Malik, 1988; Tester and Davenport, 2003). Protection of this Na+-sensitive metabolic mechanism under saline conditions partly depends on the ability to keep cytosolic Na+ levels low.
For plant cells, the most important way of keeping the cytosolic Na+ concentration at a low level is to minimize Na+ influx into the cytosol, and to maximize the Na+ efflux from the cytosol, either into the apoplast or into the vacuole (Nie et al., 1995; Blumwald et al., 2000; Zhu, 2001; Qiu et al., 2004). Sodium entry into plant cells may be restricted by selective ion uptake. Several classes of cation channels including outward- and inward-rectifying K+-selective channels (Maathius and Sanders, 1995), and non-selective cation channels, NSCCs (Amtmann and Sanders, 1999; Demidchik and Tester, 2002; Demidchik et al., 2002), seem to be involved in mediating the toxic influx of Na+. High-affinity potassium transporters have also been proposed to mediate substantial Na+ entry into plant roots (Uozumi et al., 2000; Horie et al., 2001; Golldack et al., 2002). However, recent findings suggest that non-selective cation channels are the dominant pathways for Na+ influx into root cells (Roberts and Tester, 1997; Tyerman et al., 1997; Buschmann et al., 2000; Davenport and Tester, 2000; Demidchik and Tester, 2002; Demidchik et al., 2002). The functions of these NSCCs are inhibited by Ca2+ at 0.5 mM or higher concentrations (Amtmann and Sanders, 1999; Schachtman and Liu, 1999; Demidchick and Tester, 2002). These channels are insensitive to most organic blockers, which inhibit different classes of cation channels, such as TEA for K+-selective channels (Demidchik and Tester, 2002).
Once Na+ enters the cytosol at a toxic level, plant cells can deal with the internal Na+ by sequestering it either in the apoplast or in the vacuole. Vacuolar compartmentalization is an efficient strategy for plant cells to cope with salinity stress (Fukuda et al., 1998, 2004; Blumwald et al., 2000; Chauhan et al., 2000; Hamada et al., 2001; Tester and Davenport, 2003). Antiporters for Na+/H+ in the plasma membrane and tonoplasts are expected to fulfil this function (Orlowski and Grinstein, 1997; Blumwald, 2000; Blumwald et al., 2000; Hasegawa et al., 2000; Fukuda et al., 2004; Qiu et al., 2004). Sodium extrusion through these Na+/H+ antiporters is driven by an inwardly directed proton gradient created by H+-ATPases (Blumwald et al., 2000; Hamilton et al., 2002).
Rice is the only major cereal crop that is grown in waterlogged conditions and, being a glycophyte, it is especially sensitive to salinity. Both the production and the planting area of rice are greatly affected by soil salinity (Akbar and Ponnamperuma, 1980; Panaullah, 1993). When grown in saline conditions, rice accumulates toxic Na+ levels in the leaves. Although toxicity from Na+ accumulation in the important crop rice is well studied at the organ and tissue levels, the mechanism by which Na+ enters into the cytosol, and its subsequent removal from the cytoplasm via efflux or compartmentalization or both, are still poorly understood.
The aim of this study was to investigate the mechanisms of Na+ transport into the cytosol of both salt-sensitive and salt-tolerant rice cultivars. A second aim was to study the compartmentalization of Na+. The fluorescent sodium-binding benzofuran isophthalate dye (SBFI-AM), introduced by Minta and Tsien (1989), was used to determine changes in intracellular Na+. This probe has been shown as a tool for the investigation of Na+ uptake into root hairs (Halperin and Lynch, 2003), and into the cytosol of plant protoplasts (D'Onofrio et al., 2005), and also for the determination of apoplastic Na+ in intact leaves (Mühling and Läuchli, 2002).
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Materials and methods |
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Plant material
Seeds of rice (Oryza sativa L. indica cvs Pokkali and BRRI Dhan29) were provided by the Bangladesh Rice Research Institute (BRRI, Gazipur, Bangladesh). They were treated with 10% chlorine solution for 15 min and rinsed with distilled water 5–6 times. Seeds were dipped in 5 mM CaSO4 solution for 3 h. Thereafter, seeds were soaked in water for 48 h in the darkness. They were placed on a Miracloth layer covering a metal net, which was placed on a wide beaker with culture solution. The culture solution used was based on that described by Yoshida et al. (1976)
, with modifications made by Khatun and Flowers (1995) in order to reduce the sodium concentration in the basic solution to almost zero. The beaker was covered with white polythene and placed in a Fison growth chamber (Conviron, Manitoba, R3H OW9, USA). The day/night temperature was 30/25 °C under a 12 h photoperiod with 100 µmol m–2 s–1 PAR (Philips 3629 warm white 36 W, Rosendahl, The Netherlands). Humidity was about 75%. Protoplasts were prepared from both leaf and root tips of 9–10-d-old seedlings. For some of the experiments, seedlings were pretreated for 72 h with 10 or 50 mM NaCl in the same culture solution as stated above before the isolation of protoplasts.
Protoplast isolation
The protoplasts from rice cvs Pokkali and BRRI Dhan29 were prepared as described by Shishova and Lindberg (1999) with some modifications. Leaves were sliced into 0.5 mm pieces and treated with 1% (w/v) cellulase (lyophilized powder; 10 units mg–1 solid) from Trichoderma resei (Sigma, EC 3.2.1.4
[EC]
) and 0.6% (w/v) macerase (lyophilized powder; 0.6 units mg–1 solid), Macerozyme R-10 (Serva, EC 3.2.1.4
[EC]
) for 2 h. Protoplasts were also isolated from root tips. The top 1 cm of root tips was sliced in 0.5 mm pieces and treated with 4% (w/v) cellulysin® cellulase (lyophilized powder; 1.0 units mg–1 solid) from Trichoderma viride (Calbiochem, LabKemi, Sweden, EC 3.2.1.4
[EC]
) and 0.2% (w/v) pectolyase Y23 (lyophilized powder; 3.6 units mg–1 solid) from Aspergillus japonicus (Kemila, Sollentuna, Sweden, EC 3.2.1.15
[EC]
) for 3 h as described by Lindberg and Strid (1997).
Dye loading
The protoplasts were washed twice in the loading medium containing 0.5 M sorbitol (Sigma, St Louis, MO, USA), 0.1 mM CaCl2, 0.2% (w/v) polyvinylpolypyrrolidone (PVP, Sigma, St Louis, MO, USA) and a buffer (pH 5.5: medium A) containing 5 mM TRIS (Labassco, Germany) and 5 mM MES (Sigma, St Louis, MO, USA). The SBFI-AM (Molecular Probes, Eugene, OR, USA) was dissolved in dimethylsulphoxide (DMSO, Merck, Eurolab AB, Stockholm, Sweden (<0.1% water) to give a 5 mM stock solution. Two microlitres of the stock solution was diluted with 6.75 µl ethanol (Kemetyl, Stockholm, Sweden) and 1.25 µl pluronic F-127 (Molecular Probes) as described by Poenie et al. (1986), and added to 1 ml of protoplast suspension to get a final concentration 10 µM. Dye loading was performed in medium A for 4 h at room temperature in darkness. After loading, the samples were centrifuged and pellets were resuspended into 1 ml of a solution similar to medium A, but with TRIS-MES buffer at pH 7 (medium B). Before measurements, samples were kept in darkness at room temperature for 25 min.
Fluorescence measurements
An epi-fluorescence microscope (Axiovert 10; Zeiss, Oberkochen, Germany), supplied with an electromagnetic filter-exchanger (Zeiss), Xenon lamp (Zeiss XBO 75), photometer (Zeiss 01), microprocessor (MSP 21, Zeiss), and a personal computer was used to determine fluorescence intensity after excitation at 340/380 nm. These wavelengths represent the maximum and minimum points of the excitation spectrum of the dye SBFI (Minta and Tsien, 1989). Emission wavelengths were 530–550 nm. All measurements were performed with a Planneofluar x40/0.75 objective (Zeiss) for phase contrast. Adjustment of signals and noise were made automatically. By means of ratio microscopy (Tsien and Poenie, 1986; Bright et al., 1987), the effect of different dye concentration can be eliminated. Microslides were covered with poly-L-lysine (MW 150 000–300 000, Sigma) 0.15%, to attach protoplasts to their surface.
Standard determinations of [Na+] using SBFI-AM were made in situ with the different types of protoplasts in TRIS-MES buffer (pH 7) containing five different concentrations of Na+ (added as NaCl): 0, 5, 10, 50, and 100 mM. KCl was added to the solutions to give a final concentration of 100 mM [Na++K+] to approximate physiological ionic strengths (Haugland, 2002). Gramicidin (Sigma) was added to the buffer with protoplasts at a final concentration of 10 µM (Negulescu and Machen, 1990). As gramicidin is a monovalent cation ionophore, forming transmembrane pores through which cations can pass, it can equilibrate extracellular and intracellular concentrations of Na+ as well as of K+ (Harootunian et al., 1989). As salt stress also induces cytosolic acidification, nigericin (Sigma) was added at a final concentration of 5 µM to avoid pH effect (Negulescu and Machen, 1990). Protoplasts of similar size that showed fluorescence only from the cytosol and without fluorescence from the chloroplasts (in the case of leaf protoplasts) were chosen for the fluorescence measurements. Measurements were undertaken 5–10 min after the addition of gramicidin and nigericin. The fluorescence ratio 340/380 nm increased in a linear way with an extracellular concentration of Na+ up to 100 mM (Fig. 1). It was considered that the initial cytosolic Na+-concentration was zero, since the rice seedlings were cultivated in a solution containing no sodium.
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The fluorescence intensity ratio of the cytosol at 340/380 nm was determined in single protoplasts before, and after, the addition of NaCl (5, 50, and 100 mM final concentrations) to the protoplast suspension. Measurements were undertaken up to 4 h after loading of the dye, because the SBFI fluorescence intensity in the cytosol may not change for at least 4 h (Halperin and Lynch, 2003
). As a control for the addition of sodium, mannitol of the same osmolarity was added to determine whether the change in fluorescence intensity ratio is an artefact caused by protoplast shrinking. To investigate the interference of K+ binding to the Na+-sensitive fluorescent dye SBFI-AM, measurements of the fluorescence intensity ratio were made after the addition of 5, 50, and 100 mM KCl. By means of regression analysis of the in situ calibration curve (Fig. 1), fluorescence intensity ratios were converted to the cytosolic Na+-concentration. The measurements were performed with protoplasts of similar size (approximately 10 µm).
Statistics
Each plot is a copy of printer plots and shows representative traces of a specific experiment repeated 
7 times with protoplasts from independent cultivations. Each value is the average of around 100 fluorescent-ratio determinations. The tables show data from experiments repeated 5–6 times with protoplasts from independent cultivations. Data are presented as means ±standard error (SE).
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Loading of leaf and root protoplasts with the SBFI-AM dye took 3 h and 4 h, respectively, at room temperature before any fluorescence was detectable. No visible difference in the efficiency of taking up the dye was found between the two cultivars. For measurements, only root protoplasts with a dense cytoplasm and no, or little, vacuolation were selected and all protoplasts from leaves were chosen from the mesophyll cells (distinguished by the presence of chloroplasts; Fig. 2a). In both the cases, protoplasts of similar size showing fluorescence only from the cytosol (Fig. 2b, c) were chosen for the measurements. Upon the addition of extracellular NaCl, the cytosolic fluorescence intensity ratio increased. On the other hand, addition of 5, 50, and 100 mM KCl or 10, 100, and 200 mM mannitol did not increase the fluorescence intensity ratio (Fig. 3a, b).
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Upon the addition of 5, 50, and 100 mM NaCl to the external solution, less Na+ was taken up into the cytosol of Pokkali, compared with that of BRRI Dhan29 (Table 1; Fig. 4). The total cytosolic concentration of Na+ in Pokkali was approximately half of that in BRRI Dhan29 after each addition. Moreover, the increase in Na+ concentration in the cytosol of Pokkali leaf protoplasts was transient upon the addition of 5, 50, and 100 mM NaCl, whereas the increase in cytosolic Na+ concentration in BRRI Dhan29 was stable after the addition of 50 and 100 mM NaCl. Compared to shoot protoplasts, root protoplasts of Pokkali showed even less uptake of Na+ upon addition of external NaCl, although the increase of the concentration was almost stable after each addition of NaCl. Both root and shoot protoplasts of BRRI Dhan29 took up Na+ into the cytosol almost at the same concentration.
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The uptake of Na+ in the cytosol of leaf protoplasts of Pokkali and BRRI Dhan29 was different in the two cultivars after pretreatment of protoplasts with some of the known inhibitors for K+-selective channels and NSCCs (Fig. 5a, b). When pretreated with 1.0 mM TEA, an inhibitor for K+-selective channels, the protoplasts from Pokkali leaf took up Na+ almost in the same way as protoplasts from untreated controls. TEA caused, however, a significant inhibition of the uptake of Na+ into the cytosol of BRRI Dhan29. A Hanes plot (Fig. 6a, b) shows that TEA inhibited the Na+ uptake in BRRI Dhan29 in a non-competitive way, whereas the uptake in Pokkali was not affected by TEA. No significant inhibition of cytosolic Na+ uptake was found in protoplasts from Pokkali leaf when they were pretreated with 10 mM Cs+, another inhibitor of K+-selective channels. However, treatment with 1 mM Ba2+ inhibited the uptake of Na+ by about one-third of that of control protoplast. Both Cs+ and Ba2+ also exerted partial inhibition in the uptake of Na+ into the protoplasts from leaves of BRRI Dhan29 when the protoplasts were pretreated with them.
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Although Pokkali protoplasts did not respond to TEA and Cs+ regarding Na+ uptake, the Na+ uptake was inhibited in the presence of 1 mM Ca2+, an inhibitor of NSCCs (Fig. 5b). In Pokkali, other inhibitors of NSCCs like 1 mM Zn2+ or 1 mM La3+ also showed the same type of inhibition of uptake of Na+ as by 1 mM Ca2+. On the other hand, in BRRI Dhan29 all of these inhibitors (1 mM Ca2+ or Zn2+ or La3+) exerted a partial inhibition of the Na+ uptake.
By the use of NH4VO3, an inhibitor of plasma membrane H+-ATPase, or NH4NO3, an inhibitor of tonoplast H+-ATPase, or both NH4VO3 and NH4NO3, it was investigated whether the cytosolic Na+ taken up was extruded into the vacuole or into the apoplast. When the protoplasts were pretreated with either 0.1 mM NH4VO3 or 1.0 mM NH4NO3, or both, the cytosolic Na+ concentration varied between these cultivars upon the addition of external NaCl (Table 2; Fig. 7a, b). Ammonium vanadate did not affect the cytosolic concentration of Na+ in the protoplasts of Pokkali upon the addition of NaCl. However, the increase in cytosolic Na+ concentration in the protoplasts of the same cultivar was much higher when the protoplasts were pretreated with NH4NO3, compared with the untreated protoplasts. Accordingly, when the protoplasts were pretreated with both NH4VO3 and NH4NO3, the increase in cytosolic Na+ concentration was the same as for pretreatment with NH4NO3 alone. The change in cytosolic Na+-concentration in BRRI Dhan29 was different from that of Pokkali. Sodium accumulation in the cytosol was much higher when protoplasts were pretreated with NH4VO3 compared with the untreated protoplasts. Cytosolic Na+ concentration also became somewhat higher in the protoplasts pretreated with NH4NO3 compared with the control protoplasts. Moreover, when the protoplasts from BRRI Dhan29 were pretreated with both NH4NO3 and NH4VO3, Na+ absorption into the cytosol was slightly higher after the addition of 5 and 50 mM NaCl, but was similar after the addition of 100 mM NaCl compared with the protoplasts treated with NH4VO3 alone.
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When the seedlings of both the cultivars were grown in saline conditions of 10 and 50 mM NaCl for 72 h, protoplasts from Pokkali showed less Na+ uptake at both concentrations, whereas BRRI Dhan29 showed a similar uptake of Na+ to that of untreated protoplasts (Fig. 8a, b). This observation was further evaluated by a growth experiment in which 10-d-old seedlings of both these cultivars were grown for 3 weeks at the same culture solution as stated in the Materials and methods, but containing 0, 5, 10, and 50 mM NaCl. Pokkali continued to increase its fresh weight up to 50 mM NaCl solution, whereas BRRI Dhan29 could survive after treatment with 10 mM NaCl (data not shown). The seedlings of BRRI Dhan29 died after treatment with 50 mM NaCl solution during the cultivation.
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Uptake of Na+ into the cytosol
These results show that less Na+ was taken up into the cytosol of leaf protoplast of the salt-tolerant cultivar Pokkali than in that of the salt-sensitive cultivar BRRI Dhan29 (Table 1; Fig. 4). By use of ion selective microelectrodes, Carden et al. (2003)
also showed that the tolerant cultivar of barley after short-term Na+-treatment contained lower concentrations of Na+ in the cytosol than the sensitive cultivar. The addition of membrane-permeable cyclic nucleotides to Arabidopsis during growth assays improved plant salinity tolerance, which corresponded to lower levels of Na+ accumulation in plants (Maathuis and Sanders, 2001). Golldack et al. (2003) showed a similar difference in Na+ uptake between Pokkali and a salt-sensitive cultivar of rice, IR29.
Possible channels for the uptake of Na+
By use of inhibitors for K+-selective channels such as TEA, Cs+, and Ba2+, and for non-selective cation channels (NSCCs) such as Ca2+, Zn2+, and La3+, it was found that Na+ influx into the cytosol was mediated by different channels or transporters in these cultivars (Fig. 5a, b). The inhibition of Na+ uptake in BRRI Dhan29 by all of these inhibitors indicates that both K+-selective channels and NSCCs are involved in mediating Na+ uptake in this cultivar. The non-competitive inhibition of Na+ uptake in protoplasts of BRRI Dhan29 (Fig. 6a) suggests that TEA binds to the K+-channel protein. This result is consistent with other studies, suggesting that K+-selective channels contribute to Na+ influx in rice. Horie et al. (2001) isolated two isoforms of HKT transporters from rice and suggested that they are a Na+ transporter (OsHKT1) and a Na+- and K+-coupled transporter (OsHKT2). Rice homologues to the wheat HKT1 are also known to contribute to substantial Na+ influx (Uozumi et al., 2000). In addition to the HKT type transporters, other proteins for non-specific Na+ uptake include HAK/KT/KUP-type transporters, inward-rectifying potassium channels, and low-affinity cation transporters (Schachtman et al., 1997). Although inward rectifying potassium channels are highly selective for K+, they could also mediate comparable uptake of Na+ at high salinity (Amtmann and Sanders, 1999). Garciadeblas et al. (2003) suggested that a member of the HKT family of transporters could mediate Na+ uptake in rice. Recent studies of Arabidopsis mutants with extreme NaCl sensitivity have shown that suppression of this phenotype by a defect in AtHKT1 leads to lowered Na+ influx. Therefore, AtHKT1 could be the major protein for Na+ uptake, at least in some species (Rus et al., 2001).
Except for Ba2+, the inhibitors for K+-selective channels of Na+ uptake did not significantly affect the uptake of Na+ in cv. Pokkali (Fig. 5b). It is, however, shown that Ba2+ may also block NSCCs (Demidchik and Tester, 2002). Therefore, the inhibition of Na+ uptake by Ba2+ in this cultivar might be the result of blocking of NSCCs, since other K+-selective channels did not inhibit the uptake. The Hanes plot (Fig. 6b) showing a lack of inhibition also indicates that TEA does not bind to K+-selective channels in cv. Pokkali. Thus, K+-selective channels are probably not involved in Na+ uptake in this cultivar. Instead the inhibitor analyses indicate that NSCCs are the main pathways for Na+ influx in cv. Pokkali, since inhibitors for NSCCs almost totally blocked the uptake of Na+. The NSCCs have been shown to be the major pathways for Na+ influx for many species (Roberts and Tester, 1997; Tyerman et al., 1997; Buschmann et al., 2000; Davenport and Tester, 2000; Demidchik and Tester, 2002).
Uptake in root and shoot protoplasts
Upon the addition of NaCl to protoplasts, the concentration of cytosolic Na+ in root protoplasts of Pokkali was less than that of leaf protoplasts (Table 1; Fig. 4). On the other hand, the uptake of Na+ was the same in both root and shoot protoplasts of BRRI Dhan29. Golldack et al. (2003) found a clear correlation of OsAKT expression in differentiated roots with whole-plant Na+-selectivity in the salt-sensitive and salt-tolerant rice cultivars IR29 and Pokkali, respectively. In response to salinity, a down-regulation occurred in cells of Pokkali. Therefore, the differences in cytosolic Na+ concentration in leaf and root protoplasts of Pokkali obtained in this study might depend on a different expression of NSCCs in root and leaf protoplasts.
In the present study, it was found that the sequestration process in the salt-tolerant cultivar Pokkali is less efficient in root protoplasts, compared with that in shoot protoplasts (Fig. 4). The result corroborates the study of Fukuda et al. (2004), which indicated that the transcript levels of OsNHX1 expression were higher in shoots, than those in roots. Thus, it is likely that rice OsNHX1 may play an important role in the salt tolerance of shoots, rather than in roots.
Compartmentalization of Na+
A distinct difference was found in the increase in cytosolic Na+ concentration for both the cultivars when the protoplasts were pretreated with inhibitors for either plasma membrane H+-ATPase or tonoplast H+-ATPase. The tonoplast H+-ATPase energizes the Na+/H+ antiporter at the same membrane for compartmentalization of cytosolic Na+ into the vacuole, and plasma membrane H+-ATPase does the same for the sequestration of cytosolic Na+ into the apoplast (Blumwald et al., 2000; Hamilton et al., 2002). In Pokkali, an increase in cytosolic Na+ concentration was obtained in the presence of the tonoplast H+-ATPase inhibitor (Table 2; Fig. 7a) compared with the control. This indicates an involvement of tonoplast Na+/H+ antiporter in the compartmentalization of cytosolic Na+ into the vacuole. On the other hand, apoplastic Na+ sequestration is probably absent in this cultivar, since inhibition of plasma membrane H+-ATPase did not increase the cytosolic Na+ concentration compared with the untreated control protoplasts. Experiments with inhibitors for both tonoplast and plasma membrane H+-ATPase also suggest a vacuolar compartmentalization of cytosolic Na+ in this cultivar, rather than apoplastic Na+ sequestration.
In the sensitive cultivar BRRI Dhan29, the mechanisms for keeping cytosolic Na+ concentration low was quite opposite to that of Pokkali. Inhibition of plasma membrane H+-ATPase in this cultivar resulted in a higher cytosolic Na+ concentration. This indicates the involvement of the plasma membrane Na+/H+ antiporter for apoplastic Na+ sequestration in this cultivar. However, the inhibitor analyses of the tonoplast H+-ATPase shows that the vacuolar compartmentalization is also involved to some extent, especially at a high external Na+ concentration. From these studies it can be suggested that there is a fast efflux (vacuolar compartmentalization) of cytosolic Na+ from leaf protoplasts of the salt-tolerant Pokkali, and some sequestration of Na+ into the apoplast along with some vacuolar compartmentalization from the sensitive cultivar BRRI Dhan29. The salt-tolerant cultivar Pokkali does not use plasma membrane Na+/H+ antiporters for Na+ extrusion into the apoplast, whereas the sensitive cultivar does. This might make the latter cultivar sensitive to salt stress, since sodium transported from cells by plasma membrane Na+/H+ antiporters would cause a problem for the neighbouring cells. Yeo et al. (1999) showed that Na+ leakage into the transpiration stream through the apoplast may account for a major part of Na+ entry into rice plants. On the other hand, Fukuda et al. (2004) found that an OsNHX1 (Na+/H+ antiporter) is localized in the tonoplast of rice and, at high salt concentrations, plays an important role in the compartmentalization of Na+ into the vacuoles. Therefore, a large amount of the tonoplast antiporter is one of the most important salt tolerance factors in rice.
Salt stress also induces a Na+/H+ antiport mechanism at the tonoplast of the salt-tolerant Plantago species, but not in the salt-sensitive species (Prins, 1995). For the salt-tolerant species quince, it was suggested that the main Na+ extrusion operates at internal membranes and not at the plasmalemma (D'Onofrio et al., 2005).
In some of the protoplasts, a subcellular compartmentalization of the dye, as in chloroplasts, can be found. The AM ester loading may cause intracellular compartmentalization of SBFI, especially if the loading is done at a higher temperature than room temperature (Haugland, 2002). It is reported that dye movement from the cytosol to other cell compartments does not necessarily affect the validity of measurements (Grynkiewicz et al., 1985; Brauer et al., 1995). Moreover, as only protoplasts loaded in the cytosol were considered for measurements, and not those in the chloroplasts or vacuoles, there can be confidence that these results are free from any retranslocation of the dye from the cytosol.
K+-Na+ and pH interferences with SBFI
At a low Na+ concentration and a high K+ concentration there might be some competition between Na+ and K+ for binding to the dye (Minta and Tsien, 1989; Haugland, 2002; Halperin and Lynch, 2003). Similar to Halperin and Lynch (2003), it was found here that the SBFI fluorescence is very weak compared with Fura. In the present study, addition of KCl up to 100 mM did not change the SBFI fluorescence ratio (Fig. 3a), although extracellular KCl addition might change cytosolic or vacuolar K+ concentration. Therefore, the interference of cytosolic or vacuolar K+ on SBFI-Na+ fluorescence is unlikely. Mühling and Läuchli (2002) also reported that SBFI fluorescence was not significantly affected by 40 mM K+, but significantly affected by pH changes from 5.0 to 6.5. Another report, however, shows that SBFI fluorescence is unaffected by pH changes between 6.5 and 7.5 (Haugland, 2002). NaCl stress produces pH changes in cells involved in cell signalling (Gao et al., 2004), and the changes in pH in the cytosol under NaCl stress are between 6.5 and 7.5 (Gao et al., 2004; MA Kader and S Lindberg, unpublished results). Therefore, it is unlikely that the fluorescence ratio in the present study was affected by changes in the cytosolic pH.
Effects by viscosity and osmolarity
The fluorescence of SBFI is affected by changes in viscosity (Minta and Tsien, 1989). In response to osmotic stress caused by salinity, cells accumulate compatible osmolytes like glycinebetaine, proline, and sugars, which could change the viscosity if they are present at high concentration. Several reports showed that the fluorescence of SBFI is significantly affected only at a very high concentration of these osmolytes (Harootunian et al., 1989; Mühling and Läuchli, 2002). In the present study, however, no change in viscosity can be expected since measurements were taken only for 10 min after the addition of NaCl. Moreover, the SBFI ratio may also be affected by ionic strength (Negulescu and Machen, 1990; Haugland, 2002). Using KCl during the in situ calibration approximated the effect of ionic strength in the present study.
There was no increase in the fluorescence intensity ratio upon the addition of mannitol up to 200 mM (Fig. 3b), indicating that the increase in fluorescence intensity ratio upon the addition of extracellular NaCl was not an artefact of protoplast shrinking.
Effects of pretreatment with NaCl during cultivation
Pretreatment of seedlings with NaCl (10 and 50 mM) for 72 h before the uptake experiments resulted in less Na+ uptake into the protoplasts of the tolerant cultivar, but no significant difference in the sensitive cultivar (Fig. 8a, b). The growth experiments indicated that 10 mM NaCl caused some toxicity in the sensitive cultivar BRRI Dhan29, but not in Pokkali (data not shown). Also at 50 mM NaCl concentration, all seedlings of Pokkali survived with a positive growth rate (but with a significant reduction compared with the control). Therefore, the lower uptake of Na+ in Pokkali, after pretreatment with NaCl, might depend on induction of some tolerance mechanisms to Na+. On the other hand, the somewhat higher uptake of Na+ in BRRI Dhan29 might be due to some toxic effect of an endogenous high concentration of Na+. The present results are in agreement with the results of Golldack et al. (2002, 2003), which showed that OsHKT1 transcription was down-regulated in roots and leaves of Pokkali as a response to salt stress. These authors also showed that rice OsAKT1-type potassium channels are regulated in different ways under salt stress in the salt-tolerant and salt-sensitive cultivars. In Pokkali the OsAKT1-type transcripts disappeared in plants treated with 150 mM NaCl for 48 h, but this transcript in cv. IR29 was not repressed.
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From this investigation it can be concluded that Na+ uptake in the cytosol is much higher (approximately double) in the salt-sensitive cultivar BRRI Dhan29, than in the salt-tolerant cultivar Pokkali. The NSCCs seem to be the main pathway for Na+ uptake in Pokkali, whereas K+-selective channels and NSCCs both contribute to the Na+ influx in BRRI Dhan29. Under salt stress, root protoplasts of Pokkali prevent Na+ uptake more efficiently than shoot protoplasts, whereas both root and shoot protoplasts of BRRI Dhan29 take up Na+ at almost the same rate. Pokkali compartmentalizes the internal Na+ into the vacuole more efficiently in shoots than in roots. Therefore, it is likely that the tolerance to salt in Pokkali depends on preventing Na+-influx into the roots, and sequestering of Na+ from cytosol into the vacuole of the shoots.
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We acknowledge the financial support for this project from the Islamic Development Bank, Jeddah, Saudi Arabia. We would also like to thank Göran Wingstrand for his kind help in taking the pictures of the protoplasts.
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