OPEN ACCESS ARTICLE

This Article Free via Open Access: OA OA Abstract Full Text (PDF) Supplemental Data OA All Versions of this Article: 149/2/1141    most recent pp.108.129494v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Sun, J. Articles by Xu, Y. Search for Related Content PubMed PubMed Citation Articles by Sun, J. Articles by Xu, Y. Agricola Articles by Sun, J. Articles by Xu, Y.

ENVIRONMENTAL STRESS AND ADAPTATION TO STRESS

NaCl-Induced Alternations of Cellular and Tissue Ion Fluxes in Roots of Salt-Resistant and Salt-Sensitive Poplar Species^{1,[C],[W],[OA]}

College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083, People's Republic of China (J. Sun, S.C., S.D., R.W., N.L., X.S., X.Z., C.L.); Key Laboratory of Biological Resources Protection and Utilization in Hubei Province, Hubei University for Nationalities, Enshi 445000, People's Republic of China (S.C., X.Z.); Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, People's Republic of China (Z.H.); and Xuyue (Beijing) Science and Technology Co., Ltd., Haidian District, Beijing 100080, People's Republic of China (Z.Z., J. Song, Y.X.)

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 
Using the scanning ion-selective electrode technique, fluxes of H⁺, Na⁺, and Cl^– were investigated in roots and derived protoplasts of salt-tolerant Populus euphratica and salt-sensitive Populus popularis 35-44 (P. popularis). Compared to P. popularis, P. euphratica roots exhibited a higher capacity to extrude Na⁺ after a short-term exposure to 50 mM NaCl (24 h) and a long term in a saline environment of 100 mM NaCl (15 d). Root protoplasts, isolated from the long-term-stressed P. euphratica roots, had an enhanced Na⁺ efflux and a correspondingly increased H⁺ influx, especially at an acidic pH of 5.5. However, the NaCl-induced Na⁺/H⁺ exchange in root tissues and cells was inhibited by amiloride (a Na⁺/H⁺ antiporter inhibitor) or sodium orthovanadate (a plasma membrane H⁺-ATPase inhibitor). These results indicate that the Na⁺ extrusion in stressed P. euphratica roots is the result of an active Na⁺/H⁺ antiport across the plasma membrane. In comparison, the Na⁺/H⁺ antiport system in salt-stressed P. popularis roots was insufficient to exclude Na⁺ at both the tissue and cellular levels. Moreover, salt-treated P. euphratica roots retained a higher capacity for Cl^– exclusion than P. popularis, especially during a long term in high salinity. The pattern of NaCl-induced fluxes of H⁺, Na⁺, and Cl^– differs from that caused by isomotic mannitol in P. euphratica roots, suggesting that NaCl-induced alternations of root ion fluxes are mainly the result of ion-specific effects.

Active Na⁺ extrusion to the apoplast or external environment is essential for sustaining Na⁺ homeostasis in the cytosol (Blumwald et al., 2000; Tester and Davenport, 2003; Zhu, 2003; Apse and Blumwald, 2007). PM Na⁺/H⁺ antiporters have been widely considered to play a crucial role in active Na⁺ extrusion under saline conditions (Shi et al., 2000, 2002; Qiu et al., 2002; Martínez-Atienza et al., 2007). NaCl-induced activity of PM Na⁺/H⁺ antiporter has been reported in crop species, tomato (Solanum lycopersicum; Wilson and Shannon, 1995), Arabidopsis (Arabidopsis thaliana; Qiu et al., 2002, 2003), and rice (Oryza sativa; Martínez-Atienza et al., 2007). Furthermore, overexpression of the Na⁺/H⁺ antiporter gene AtSOS1 decreases the accumulation of Na⁺ in transgenic Arabidopsis under NaCl stress (Shi et al., 2003). These PM Na⁺/H⁺ antiporters depend on electrochemical H⁺ gradients, which are generated by PM H⁺-ATPase (Blumwald et al., 2000; Zhu, 2003). Using an ion-selective microelectrode, Shabala and a colleague suggested the involvement of PM H⁺-ATPase in the Na⁺/H⁺ antiport according to H⁺ kinetics on salt shock (Shabala, 2000; Shabala and Newman, 2000). Therefore, the NaCl-induced H⁺ pumping is fundamental to Na⁺/H⁺ exchange and salinity tolerance (Ayala et al., 1996; Vitart et al., 2001; Chen et al., 2007; Gévaudant et al., 2007). However, the active Na⁺/H⁺ antiport across PM and the contribution to salt exclusion have been rarely investigated in tree species.

Munns and Tester (2008) claimed that Cl^– toxicity is more important than Na⁺ toxicity in some woody species, e.g. citrus. Similarly, we have noticed that the inability to restrict Cl^– uptake contributes to the NaCl-induced salt damage in salt-sensitive poplar (Populus spp.) species, in addition to toxicity of excess Na⁺ (Chen et al., 2001, 2002, 2003). The differences in Cl^– tolerance exhibited by plants are usually related to the ability to restrict Cl^– transport to the aerial part (Greenway and Munns, 1980; White and Broadley, 2001). Excluding Cl^– from the xylem seems to be an effective mechanism for lotus to cope with the interactive effect of salt and water logging (Teakle et al., 2007). An influx of Cl^–, immediately after addition of NaCl, was observed in bean (Vicia faba) mesophyll tissue (Shabala, 2000). The Cl^– flux response to salt shock is helpful to reveal the rapid adjustments of plants to salinity. However, Cl^– fluxes in salt-adapted roots, which are necessary to clarify plant adaptations to long durations of salinity, have not been examined.

Populus euphratica has been widely considered as a model plant to elucidate physiological and molecular mechanisms of salt tolerance in woody species (Chen et al., 2001, 2002, 2003; Gu et al., 2004; Ottow et al., 2005a, 2005b; Junghans et al., 2006; Wang et al., 2007, 2008; Wu et al., 2007; Zhang et al., 2007). Comparative studies have shown that salt-stressed P. euphratica seedlings accumulate less Na⁺ and Cl^– in root and shoot tissues than salt-sensitive poplar species (Chen et al., 2001, 2002). It is suggested that the greater capacity to exclude NaCl in P. euphratica is likely the result of salt uptake and transport restriction in roots (Chen et al., 2002, 2003). However, this needs further investigations, e.g. by electrophysiology, to clarify.

In this study, we used a noninvasive ion flux technique to measure the tissue and cellular fluxes of H⁺, Na⁺, and Cl^– in roots of the salt-tolerant P. euphratica and salt-sensitive P. popularis 35-44. The aim was to compare the NaCl-induced alternations of ion fluxes in poplar species differing in salt tolerance. Furthermore, we examined the effects of pH, salt shock, and PM transport inhibitors on Na⁺ and H⁺ fluxes in root-derived protoplasts of the salt-tolerant species, P. euphratica, which exhibited an evident Na⁺ exclusion under saline conditions.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 

Tissue and Cellular Ion Fluxes in Response to Salt and Osmotic Stress in Two Poplar Species

Na⁺ Fluxes
The scanning ion-selective electrode technique (SIET) data show that the pattern of Na⁺ fluxes in roots of P. euphratica differs from that in P. popularis after a short-term (ST) exposure to 50 mM NaCl (24 h) and a long-term (LT) exposure to higher salinity, i.e. 100 mM NaCl (15 d; LT). The ST stress caused a net Na⁺ efflux, ranging from 20 to 800 pmol cm^–2 s^–1 in the measured regions of P. euphratica roots (0–3,000 µm from the apex), but Na⁺ fluxes in P. popularis roots varied within the apical zone and elongation region (Fig. 1, A and B ). NaCl induced a net Na⁺ influx (600–2,300 pmol cm^–2 s^–1) at the region of 0 to 1,000 µm (apical zone, meristem plus associated root cap), but a pronounced shift toward an efflux (300–1,200 pmol cm^–2 s^–1) was seen at the region of 1,100 to 1,800 µm from the apex (root elongation region; Fig. 1B). After being subjected to the LT stress, P. euphratica roots exhibited a stable and constant efflux with a mean value of 400 pmol cm^–2 s^–1 (Fig. 1C). However, a net Na⁺ influx was usually detected along P. popularis roots, although there were several exceptions (Fig. 1D).

View larger version (23K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Effects of ST salinity (50 mM NaCl for 24 h; A and B), LT salinity (100 mM NaCl for 15 d; C and D), or hyperosmotic stress (85 mM mannitol, for 24 h; E and F) on net Na+ fluxes in roots of P. euphratica and P. popularis. Control roots were treated without NaCl or mannitol. Na+ fluxes were measured along root axes (0–3,000 µm from the apex) at intervals of 50 to 300 µm. Each point is the mean of five to six individual plants and bars represent the standard error of the mean. An asterisk denotes a significant difference at P < 0.05 between treatments. [See online article for color version of this figure.]

Cellular fluxes of Na⁺ were recorded in protoplasts isolated from control and LT-stressed roots of the two species. Control P. euphratica protoplasts showed an inward rectification with a mean value of 60 pmol cm^–2 s^–1 (Fig. 2, A and C ). LT-stressed P. euphratica cells exhibited an enhanced Na⁺ efflux, although the flux rates oscillated during the period of recording (10–12 min; Fig. 2, A and C). In contrast to P. euphratica, salinity did not significantly change the rectification and flux rate of Na⁺ in root protoplasts of P. popularis during the recording periods (Fig. 2, B and C).

View larger version (26K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Net fluxes of Na+ (A and B), H+ (D and E), and Cl– (G and H) in protoplasts isolated from control and LT-stressed roots of P. euphratica and P. popularis. A continuous flux recording of 10 to 12 min was conducted for each protoplast in corresponding measuring solutions (pH 6.0). Each point represents the mean of five to six individual protoplasts and bars represent the standard error of the mean. The mean fluxes of Na+, H+, and Cl– within the measuring periods are shown (C, F, and I). Columns labeled with different letters (a and b) are significantly different at P < 0.05. [See online article for color version of this figure.]

View larger version (24K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. Effects of ST salinity (A and B), LT salinity (C and D), or hyperosmotic stress (E and F) on net H+ fluxes in roots of P. euphratica and P. popularis. Control roots were treated without NaCl or mannitol. H+ fluxes were measured along root axes (0–3,000 µm from the apex) at intervals of 50 to 300 µm. Each point is the mean of seven to eight individual plants and bars represent the standard error of the mean. An asterisk denotes a significant difference at P < 0.05 between treatments. [See online article for color version of this figure.]

At the cellular level, a NaCl-induced H⁺ influx was found in P. euphratica protoplasts; its mean flux rate increased 7-fold (Fig. 2, D and F). However, H⁺ flux in LT-stressed P. popularis cells did not significantly differ from the control (Fig. 2, E and F).

As shown in Figure 4 , transient H⁺ kinetics response to salt shock was compared in protoplasts, isolated from LT-stressed roots of the two species. After exposure to the 50 mM NaCl, H⁺ fluxes in protoplasts exhibited a two-phase response, with an instantaneous increase of efflux, 7 to 10 pmol cm^–2 s^–1, followed by a continuous drift toward larger influxes (Fig. 4A). However, salt-shocked P. euphratica cells exhibited a typically greater influx than P. popularis over the period of recording (Fig. 4B).

View larger version (11K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Transient H+ kinetics of salt shock (50 mM NaCl) in protoplasts isolated from LT-stressed roots of P. euphratica and P. popularis. A, H+ kinetics recorded after the required amount of 1.0 M NaCl stock solution was added to the chamber. Prior to the salt shock, steady H+ fluxes of cells were examined for approximately 5 min. Each point represents the mean of five to six protoplasts and bars represent the standard error of the mean. B, The mean rate of H+ flux in protoplasts of the two poplar species during the period of salt shock. Columns labeled with different letters (a and b) are significantly different at P < 0.05 between the two species. [See online article for color version of this figure.]

View larger version (27K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. Effects of ST salinity (A and B), LT salinity (C and D), or hyperosmotic stress (E and F) on net Cl– fluxes in roots of P. euphratica and P. popularis. Control roots were treated without NaCl or mannitol. Cl– fluxes were measured along root axes (0–3,000 µm from the apex) at intervals of 50 to 300 µm. Each point is the mean of five to six individual plants and bars represent the standard error of the mean. An asterisk denotes a significant difference at P < 0.05 between treatments. [See online article for color version of this figure.]

Compared to control protoplasts, an accelerated Cl^– influx was observed in LT-stressed P. euphratica cells, where the mean flux rate increased to 290 pmol cm^–2 s^–1 (Fig. 2, G and I). However, Cl^– fluxes in salt-stressed P. popularis cells did not significantly differ from controls (Fig. 2, H and I).

Effects of pH, Salt Shock, and PM Transport Inhibitors on Na⁺ and H⁺ Fluxes in P. euphratica

We used root protoplasts of LT-stressed P. euphratica, which exhibited a clear Na⁺/H⁺ exchange, to characterize the effects of pH, salt shock, and PM transport inhibitors on cellular Na⁺ and H⁺ fluxes.

External pH
Cellular fluxes of Na⁺ and H⁺ depend on pH values in measuring solutions and an acidic environment was found to accelerate Na⁺ efflux (Fig. 6A ) and H⁺ influx (Fig. 6B). More pronounced effects were observed at pH 5.5, compared to pH 6.0 and a neutral pH (Fig. 6).

View larger version (20K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 6. Effects of pH on net Na+ and H+ fluxes in protoplasts isolated from LT-stressed P. euphratica roots. Ion flux measurements were carried out in measuring buffers with pH at 5.5, 6.0, and 7.0. Each point represents the mean of five to six protoplasts and bars represent the standard error of the mean. Inserted sections show the mean flux rates of Na+ and H+ within the measuring period of 8 to 10 min. Columns labeled with different letters (a, b, and c) denote significant difference at P < 0.05. [See online article for color version of this figure.]

View larger version (29K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 7. Effects of sodium orthovanadate (500 µM) and amiloride (50 µM) on net Na+ and H+ fluxes in LT-stressed roots (A–D) and derived protoplasts (E and F) of P. euphratica. Roots and protoplasts were pretreated with 500 µM sodium orthovanadate or 50 µM amiloride for 30 min prior to flux measurements. A to D, Na+ and H+ fluxes were measured along root axes (0–3,000 µm from the apex) at intervals of 50 to 300 µm (pH value in the measuring solutions was 6.0). Each point is the mean of five to six individual roots and bars represent the standard error of the mean. An asterisk denotes a significant difference at P < 0.05 between treatments. E and F, Cellular fluxes of Na+ and H+ were recorded in the measuring solutions with pH values at 5.5 and 6.0 (the continuous recording period was 10 min). Each column is the mean of five to six individual protoplasts and bars represent the standard error of the mean. Columns labeled with different letters (a, b, and c) are significantly different at P < 0.05. [See online article for color version of this figure.]

View larger version (20K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 8. Effects of sodium orthovanadate (500 µM) and amiloride (50 µM) on salt shock-induced transient H+ kinetics in protoplasts isolated from LT-stressed roots of P. euphratica. Protoplasts were pretreated with 500 µM sodium orthovanadate or 50 µM amiloride for 30 min prior to fluxes measurements. A, H+ kinetics recorded after the required amount of 1.0 M NaCl stock solution was added to the chamber. Prior to the salt shock, steady H+ fluxes were measured for approximately 5 min in protoplasts pretreated with or without inhibitors. Each point represents the mean of five to six protoplasts and bars represent the standard error of the mean. B and C show the mean flux rate of H+ at 0 to 10 min and 10 to 35 min after the onset of salt shock. Columns labeled with different letters (a, b, and c) denote significant difference at P < 0.05. [See online article for color version of this figure.]

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

Na⁺/H⁺ Antiport across the PM under NaCl Stress

ST and LT salinity caused a net Na⁺ efflux in P. euphratica roots, although the more pronounced effect was observed in LT-stressed plants (Fig. 1, A and C). Similarly, a net Na⁺ efflux was obtained in bean leaf mesophyll after NaCl addition (Shabala, 2000). Salt-stressed P. euphratica roots exhibited a net H⁺ influx in the apical zone (ST) or along the root axis (LT; Fig. 3, A and C). NaCl-induced H⁺ influx was also seen in the root apex of a wild-type Arabidopsis (Shabala et al., 2005). In accord with root tissues, LT-stressed P. euphratica cells exhibited an evident net Na⁺ efflux (Fig. 2, A and C) and a corresponding influx of H⁺ (Fig. 2, D and F). In this study, the increase in H⁺ influx corresponding to the Na⁺ efflux suggests that the Na⁺ extrusion in salt-stressed P. euphratica roots is mainly the result of an active Na⁺/H⁺ antiport across the PM. The experimental evidence is briefly listed below. (1) Pharmacological evidence at tissue and cellular levels. The PM transport inhibitors, amiloride (inhibitor of the Na⁺/H⁺ antiporter) and sodium orthovanadate (inhibitor of the PM H⁺-ATPase) simultaneously decreased Na⁺ efflux and H⁺ influx along the root axis of LT-stressed plants (Fig. 7, A–D). At cellular levels, the rectification of Na⁺ flux was correspondingly reversed when H⁺ influx was restricted by the inhibitors, regardless of pH values (5.5 or 6.0) in the measuring buffers (Fig. 7, E and F). (2) Experimental results from multiple cycles of salt stress. In this study, LT-stressed cells were shocked with 50 mM NaCl to resalinize these cells to confirm that the enhanced H⁺ influx was the result of NaCl stress. Protoplast isolation and purification were carried out in enzymatic solutions without NaCl addition and thus, the salt stress was alleviated during the period of protoplast preparation (approximately 7 h). Noteworthy is that the enhanced H⁺ influx was refound when these cells were resalinized with 50 mM NaCl (Fig. 8). Moreover, the NaCl-induced H⁺ influx in these restressed cells was inhibited by inhibitors of the Na⁺/H⁺ antiporter and PM H⁺-ATPase, suggesting a contribution of H⁺ influx to Na⁺ extrusion in P. euphratica cells (Fig. 8).

PeNhaD1 and PeSOS1 likely function as Na⁺/H⁺ antiporters in the PM of P. euphratica. The transcriptional level of PeNhaD1 was maintained at the control levels in P. euphratica but declined in the salt-sensitive poplar after 12-h salt stress (Ottow et al., 2005a). Yeast complementation experiments have shown that the mutant strain ANT3 (Saccharomyces cerevisiae, ena1-4::HIS3 nha1::LEU2), which lacks the PM Na⁺/H⁺ antiporter gene ScNHA1, resumes the capacity for salt exclusion after PeNhaD1 transformation (Lü et al., 2007). Furthermore, PeNhaD1 overexpression in salt-sensitive poplar species enhanced salt exclusion in transgenic plants (Chen, 2007). An acidic environment, e.g. pH 5.5 or 6.0, promoted Na⁺/H⁺ exchange in P. euphratica protoplasts, compared to a neutral pH (Fig. 6). This agrees with Ottow et al. (2005a), who have confirmed that PeNhaD1 is strictly pH dependent and only functions under acidic conditions of a pH 5.5 (the tested pH values were 5.5, 7.0, and 8.0). The gene PeSOS1, encoding the putative PM Na⁺/H⁺ antiporter of P. euphratica, has been recently characterized by Wu et al. (2007). PeSOS1 levels in P. euphratica leaves were found to increase after NaCl treatment (Wu et al., 2007). Given these results, we conclude that Na⁺ extrusion in salt-stressed P. euphratica cells is correlated with increasing activity of PM Na⁺/H⁺ antiporters.

PM H⁺-ATPase pumps protons and maintains electrochemical H⁺ gradients, thus promoting the secondary active Na⁺/H⁺ antiport at the PM (Blumwald et al., 2000; Zhu, 2003). Shabala and a colleague suggested the involvement of PM H⁺-ATPase in Na⁺/H⁺ antiport (Shabala, 2000; Shabala and Newman, 2000). In this study, pharmacological experiments at tissue and cellular levels showed that the Na⁺/H⁺ exchange in LT-stressed P. euphratica was associated with the PM H⁺-ATPase (Fig. 7). Evidence by western blotting also proved that the expression of PM H⁺-ATPase was up-regulated by NaCl stress in calluses and leaves of P. euphratica (Yang et al., 2007; Zhang et al., 2007).

In contrast to P. euphratica, P. popularis roots did not exhibit a marked Na⁺ efflux along the root axis in LT-stressed plants, although a net Na⁺ efflux was observed in the elongation region of ST-stressed roots (Fig. 1, B and D). These results are consistent with our previous findings that P. popularis plants have a lower capacity to exclude Na⁺ under LT saline conditions (Chen et al., 2002). Compared with controls, NaCl did not induce a marked H⁺ influx in either ST- or LT-stressed roots (Fig. 3, B and D). The same trends of Na⁺ and H⁺ fluxes were seen in the protoplasts isolated from LT-stressed P. popularis roots (Fig. 2, B, C, E, and F). These results suggest that the PM Na⁺/H⁺ antiport system in this species is insufficient to exclude the entry of Na⁺, which presumably occurs through voltage-independent nonselective cation channels (Maathuis and Sanders, 2001; Demidchik and Tester, 2002; Demidchik et al., 2002; White and Davenport, 2002; Tester and Davenport, 2003; Maathuis, 2006; Shabala et al., 2006). When provided with a salt shock, LT-stressed P. popularis cells exhibited a significant lower H⁺ influx, compared with P. euphratica (Fig. 4). This suggests that the inability to extrude Na⁺ from root tissues and cells likely results from its lower H⁺ pumping activity of H⁺-ATPase. An electron microscopic-cytochemical study showed that PM H⁺-ATPase was down-regulated in P. popularis after exposure to NaCl stress (L. Deng, X. Ma, J. Li, and S. Chen, unpublished data).

Cl^– Fluxes under NaCl Stress

Excessive accumulations of Cl^– contribute to salt damage in woody species (Chen et al., 2001, 2002, 2003; Munns and Tester, 2008). P. euphratica exhibits a net Cl^– efflux in LT-stressed roots (Fig. 5C). Similarly, NaCl-induced Cl^– efflux was observed in Arabidopsis (Lorenzen et al., 2004). However, protoplasts isolated from LT-stressed P. euphratica roots did not show a corresponding Cl^– efflux; instead, an influx of Cl^– was usually registered (Fig. 2, G and I). Therefore, Cl^– efflux in stressed roots is presumably, at least in part, the result of ion release from Cl^– sources. We found a marked Cl^– compartmentation in cell walls and vacuoles of salt-stressed P. euphratica roots and Cl^– concentrations were much higher than those of Na⁺ (Chen et al., 2002, 2003). The flux rates of Cl^– in roots may reveal the concentration of Cl^– sources. Salt-induced Cl^– efflux in P. popularis was only seen in ST-stressed plants, but not observed in LT-stressed ones (Fig. 5, B and D), implying that Cl^– was less retained in roots after a longer duration of stress. The Cl^– had presumably been transported to the leaves via the transpiration stream, since P. popularis roots are unable to block efficiently the salt radical translocation through both the symplastic and apoplastic pathways (Chen et al., 2002).

Osmotic and Ionic Effects on Ion Fluxes

Fluxes of H⁺ and Na⁺ upon salt stress (50 mM NaCl, 24 h) differ from the response to hyperosmotic stress (85 mM mannitol, 24 h). In the two tested species, NaCl-induced Na⁺ fluxes were not seen in the isotonic mannitol treatment (Fig. 1, A, B, E, and F). Hyperosmotic stress accelerated the H⁺ efflux along the root axes in the two poplar species (Fig. 3, E and F), but the iso-NaCl induced an H⁺ influx at the root apex of P. euphratica or had no effects on P. popularis (Fig. 3, A and B). Our results indicate that ionic responses of roots to a hyperosmotic treatment were highly stress specific (Shabala, 2000). However, there were marked differences between the two species in Cl^– flux given the mannitol treatment. Hyperosmotic stress caused a drastic Cl^– efflux in roots of P. popularis but not in P. euphratica (Fig. 5, E and F). The Cl^– efflux in osmotically stressed P. popularis was absent when the isotonic mannitol was introduced to the measuring solution (Supplemental Fig. S1), suggesting that the Cl^– efflux enhanced by hyperosmotic stress is likely mediated by stretch-activated anion channels. ST-stressed P. popularis roots showed a trend similar to mannitol treatment (Fig. 5, B and F), indicating that stretch-activated anion channels contributed to salt-induced Cl^– efflux (Falke et al., 1988; Cosgrove and Hedrich, 1991; Teodoro et al., 1998; Qi et al., 2004). Cl^– efflux in P. euphratica roots was not significantly enhanced at a hyperosmotic stress, irrespective of measuring solutions supplemented with or without mannitol (Fig. 5E; Supplemental Fig. S1), suggesting that NaCl-induced Cl^– efflux in P. euphratica roots accounted for the specific salt effects.

	CONCLUSION

TOP ABSTRACT RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 
In conclusion, SIET data show that P. euphratica roots have a higher capacity to exclude Na⁺ in ST and LT treatments. Steady fluxes of H⁺ and Na⁺ in protoplasts of LT-stressed P. euphratica roots indicate that the active Na⁺/H⁺ antiport across a PM contributes to Na⁺ extrusion, especially under acidic conditions. Inhibition of Na⁺/H⁺ antiporter by amiloride (a Na⁺/H⁺ antiporter inhibitor) or sodium orthovanadate (a PM H⁺-ATPase inhibitor) at tissue and cellular levels verified the involvement of Na⁺/H⁺ antiporters and a H⁺ pump (PM H⁺-ATPase) in Na⁺ extrusion. In comparison, the Na⁺/H⁺ antiport system in P. popularis roots is insufficient to exclude Na⁺ at tissue and cellular levels, leading to perturbations of ion homeostasis over long periods of salinity. Moreover, salt-treated P. euphratica roots retain a higher capacity for Cl^– exclusion than P. popularis, especially under LT salinity. The greater capacity to exclude NaCl in P. euphratica roots may restrict the root-to-shoot salt transport and the subsequent accumulation in aerial tissues. Noteworthy, the pattern of root H⁺, Na⁺, and Cl^– flux in NaCl-stressed P. euphratica differs from the response to hyperosmotic stress, suggesting that NaCl-induced alternations of root ion fluxes in this species are mainly the result of ion-specific effects.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 

Plant Materials

In April (2007, 2008), 1-year-old seedlings of Populus euphratica, obtained from the Xinjiang Uygur Autonomous Region of China, and hardwood cuttings of Populus popularis 35-44 (P. popularis), from the nursery of Beijing Forestry University, were planted in individual pots (10 L) containing loam soil and placed in a greenhouse at Beijing Forestry University. Potted plants were well irrigated according to evaporation demand and watered with 1 L full-strength Hoagland nutrient solution every 2 weeks. Plants were raised 3 months prior to the beginning of hydroponic culture. In mid-July, uniform plants were washed free of soil and transferred to individual porcelain pots containing 2 L one-quarter strength Hoagland nutrient solution. Plants were continuously aerated by passing air through the solution. The temperature in the greenhouse was 20°C to 25°C with a 16-h photoperiod (7 AM–11 PM), 150 µmol m^–2 s^–1 of photosynthetically active radiation. Nutrient solutions were renewed every 48 h and plants were raised for 15 to 20 d under hydroponic conditions prior to salt and hyperosmotic treatments.

Treatments

Plants of the two species were subjected to a ST 50 mM NaCl solution for 24 h and a LT salinity of 100 mM NaCl, for 15 d. The required amounts of NaCl were added to the Hoagland nutrient solution. After 15 d of salt treatment (100 mM NaCl), P. popularis exhibited visible necrosis in old leaves and chlorosis generally occurred at the margin of upper leaves. However, no salt injury was seen in P. euphratica during the period of salt stress. The number of newly built roots in P. popularis was decreased by salinity but NaCl did not reduce root growth in P. euphratica. In a 24-h hyperosmotic treatment, plants of the two species were exposed to 85 mM mannitol, instead of iso-NaCl (50 mM). Control plants were not treated with NaCl or mannitol. Young roots with apices of 2 to 3 cm were sampled from the control and from stressed plants of the two species and used for H⁺, Na⁺, and Cl^– flux measurements.

Isolation of Root Protoplasts

Protoplasts were isolated from control and LT-stressed plants of P. euphratica and P. popularis. The procedures for protoplasts isolation were followed, as described in Shabala et al. (1998) and Demidchik and Tester (2002). A brief description is given below. Root tips (2–3 cm) were chopped into 0.5 mm in length and incubated in 4 mL of a basic solution (10 mM CaCl₂, 2 mM MgCl₂, 2 mM MES, 0.1% bovine serum albumin [w/v], and 330 mM mannitol, pH 5.7 was adjusted with Tris) supplemented with the following enzymes (in w/v): 1.5% cellulase Onozuka R-10 (Yakult Honsha, lot no. 216013), 1% Cellylysin (CalBiochem, lot no. B70487), and 0.1% pectolyase Y-23 (Yakult Honsha, lot no. Y-007). Segments of roots were gently shaken at 60 rpm in the enzyme solution at 28°C for 6 h. Then protoplasts were filtered through a nylon mesh with 50-µm-diameter pores. Undigested tissues were placed in a small beaker containing 3 mL of a holding solution (5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM Suc, 10 mM Glc, 2 mM MES, pH 5.7 was adjusted with Tris). Osmotic potentials of basic and holding solutions, 290 to 300 mOsM kg^–1, were adjusted by 330 mM mannitol with a vapor pressure osmometer (Wescor 5520). By gentle agitation with a glass rod, additional protoplasts were released from the undigested tissues and collected by filtering through the nylon mesh. Thereafter, a duplicate dilution method was adopted to minimize mechanical damage caused by the enzyme solution. The collected filtrate was centrifuged at 300g for 5 min to remove the enzyme solution. Then protoplasts were resuspended and washed with 2 to 3 mL of a holding solution (x2). Finally, protoplast suspensions (1 mL) were diluted with a 2-mL holding solution and used for ion flux measurements, including the steady-state measurements (Chen et al., 2005) and transient kinetics (Shabala, 2000).

Measurements of Net Na⁺, H⁺, and Cl^– Fluxes with SIET

Net fluxes of Na⁺, H⁺, and Cl^– were measured noninvasively using SIET (the SIET system BIO-001A; Younger USA Sci. & Tech. Corp.; Applicable Electronics Inc.; and ScienceWares Inc.; Kühtreiber and Jaffe, 1990; Kochian et al., 1992; Zonia et al., 2002; Vincent et al., 2005; Xu et al., 2006). The concentration gradients of the target ions were measured by moving the ion-selective microelectrode between two positions close to the plant material in a preset excursion (30 µm for excised roots and 10 µm for protoplasts in our experiment) at a programmable frequency in the range of 0.3 to 0.5 Hz. The SIET can measure ionic fluxes down to picomolar levels but must be measured slowly at approximately 1 to 2 s per point. This is mainly due to the mechanical disturbance of the gradient by the electrode movement, although the time constant of the liquid ion exchanger (LIX) electrodes is also a factor. The ionic gradient is accurately measured due to the following reasons: (1) while conducting the experiment, the electrode stays in one position for a certain time (from 0.8 to a few seconds) to let the gradient recover before it takes a reading of that position; (2) the time the electrode spends in one position is also needed for the electrode to get stabilized to give a reliable measurement. In fact, the time spent on electrode stabilization is longer than the time for gradient recovery, which is one of the key factors determining the efficiency of the electrode (Kunkel et al., 2006). The electrode is stepped from one position to another in a predefined sampling routine while also being scanned with the three-dimensional microstepper motor manipulator (CMC-4).

Prepulled and silanized glass micropipettes (2–4 µm aperture, XYPG120-2; Xuyue Sci. and Tech. Co., Ltd.) were first filled with a backfilling solution (Na: 100 mM NaCl; Cl: 100 mM KCl; H: 40 mM KH₂PO₄ and 15 mM NaCl, pH 7.0) to a length of approximately 1 cm from the tip. Then the micropipettes were front filled with approximately 15-µm columns of selective liquid ion-exchange cocktails (LIXs; Na: Fluka 71178; H: Fluka 95293; Cl: Fluka 24902). An Ag/AgCl wire electrode holder (XYEH01-1; Xuyue Sci. and Tech. Co., Ltd.) was inserted in the back of the electrode to make electrical contact with the electrolyte solution. DRIREF-2 (World Precision Instruments) was used as the reference electrode. Ion-selective electrodes of the following target ions were calibrated prior to flux measurements: (1) Na⁺: 0.9 mM, 2.0 mM, 5.0 mM (Na⁺ concentration was usually 0.9 mM in the measuring buffer for root and cell samples); (2) H⁺: pH 5.5, 6.0, 6.5 (pH of the measuring buffer was usually adjusted to 6.0 for root and cell samples; pH tests of protoplasts were carried out at pH 5.5, 6.0, and 7.0); (3) Cl^–: 0.25 mM, 0.5 mM, 2.0 mM (Cl^– concentration was usually 0.5 mM in the measuring buffer for root and cell samples).

Only electrodes with Nernstian slopes >50 mV/decade (–50 mV/decade for Cl^– electrodes) were used in our study. Ion flux was calculated by Fick's law of diffusion:

where J represents the ion flux in the x direction, dc/dx is the ion concentration gradient, and D is the ion diffusion constant in a particular medium. Data and image acquisition, preliminary processing, control of the three-dimensional electrode positioner, and stepper-motor-controlled fine focus of the microscope stage were performed with ASET software, part of the SIET system.

Experimental Protocols

Root Tissues
After exposure to the saline (ST and LT) and hyperosmotic treatments, root segments with 2 to 3 cm apices were sampled for ion flux measurements. To decrease the effect of salt release on flux recording (preloaded Na⁺ and Cl^– would diffuse from the surface of NaCl-stressed roots in a buffer with lower Na⁺ and Cl^– concentrations), roots were rinsed with redistilled water and immediately incubated in the measuring solution to equilibrate for 30 min (as shown in Fig. 9 , a rapid efflux of Na⁺ and Cl^– took place after the roots were immediately sampled); then the flux rate decreased gradually and reached a steady level within 10 min, although P. euphratica exhibited a typical higher efflux rate than P. popularis. The measuring site was 500 µm from the root apex, in which a vigorous flux of Na⁺ or Cl^– was usually observed. Afterward, roots were transferred to the measuring chamber containing 10 to 15 mL of a fresh measuring solution. After the roots were immobilized on the bottom, ion flux measurements were started from the apex and went along the root axis until 3,000 µm (ion flux rates in the region of 0–3,000 µm are significantly larger than mature zones; data not shown). The measured positions of roots could be visualized and defined under the SIET microscope because young roots were semitransparent under light. Generally, control roots have an approximately 0.5- to 1.0-mm apex length and an 8- to 10-mm-long elongation zone. For LT-stressed P. popularis roots, the position and length of the root apex were similar to those of the controls but the full length of the elongation zone was reduced to 4–7 mm. Nevertheless, this did not affect the flux measurement in the elongation region since the SIET recordings were performed at a root length of 3 mm from the apex. For P. euphratica, there was no morphological difference between control and stressed roots. Na⁺, H⁺, and Cl^– were monitored in the following solutions, respectively: (1) Na⁺: 0.1 mM KCl, 0.1 mM CaCl₂, 0.1 mM MgCl₂, 0.5 mM NaCl, 0.3 mM MES, 0.2 mM Na₂SO₄, pH 6.0, adjusted with choline and HCl; (2) H⁺: 0.1 mM KCl, 0.1 mM CaCl₂, 0.1 mM MgCl₂, 0.5 mM NaCl, 0.3 mM MES, 0.2 mM Na₂SO₄, pH 6.0 adjusted with NaOH and HCl; (3) Cl^–: 0.05 mM KCl, 0.05 mM CaCl₂, 0.05 mM MgCl₂, 0.25 mM NaCl, 0.2 mM Na₂SO₄, pH 6.0, adjusted with choline and H₃PO₃ (MES was absent in the Cl^– measuring solution because it had negative effects on the selectivity of Cl^– LIX; Messerli et al., 2004).

View larger version (26K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 9. Fluxes of Na+ (A) and Cl– (B) in excised roots of LT-stressed P. euphratica and P. popularis. Na+ and Cl– fluxes were measured immediately after roots were sampled and the measuring site was 500 µm from the root apex, in which a vigorous flux of Na+ or Cl– was usually observed. Each point is the mean of five to six individual plants and bars represent the standard error of the mean. [See online article for color version of this figure.]

The inhibitory effects of PM transport inhibitors on ion fluxes were examined in P. euphratica. LT-stressed P. euphratica roots were subjected to 500 µM sodium orthovanadate (a PM H⁺-ATPase inhibitor) or 50 µM amiloride (a Na⁺/H⁺ antiporter inhibitor) for 30 min. Then measuring solutions containing sodium orthovanadate were removed slowly with a pipette and a 10-mL fresh solution was slowly added to the measuring chamber. Measuring solutions containing amiloride were not replaced because amiloride had no obvious effect on the Nernstian slopes of Na⁺ and H⁺ electrodes. Fluxes of Na⁺ and H⁺ were scanned along the root axes (0–3,000 µm) at intervals of 50 to 300 µm.

Protoplasts
Prior to cellular ion flux recording, protoplasts were first fixed on the bottom of the measuring chamber to reduce the mobility caused by moving the electrode during measurements. Glass slips treated with a poly-L-Lys (Sigma) solution (Mazea et al., 1975) were used to affix individual protoplasts. Briefly, 20- x 20-mm coverslips were placed in a potassium bichromate washer for several hours and rinsed with redistilled water, then dried at 160°C for 2 to 3 h in an oven. After that, the coverslips were treated with poly-L-Lys solutions at concentrations of 0.002% (w/v). Slips were finally stored in a sealed container for air drying at room temperature. A 100 to 200 µL of protoplast suspensions was put in the middle of the poly-L-Lys-pretreated coverslips in the measuring chamber. When protoplasts had been settled on the surface (requiring several minutes), a 10-mL measuring solution of Na⁺, H⁺, or Cl^– was slowly added to the measuring chamber. The ionic compositions and pH of the measuring solutions were the same as those used for tissue flux measurements but for two changes: (1) The concentration of MES was decreased from 0.3 to 0.05 mM (Na⁺, H⁺), and (2) 330 mM mannitol was introduced to the measuring solutions (Na⁺, H⁺, and Cl^–). The steady-state flux measurements were started and continued for 10 to 12 min. The effects of salt shock, pH, and PM transporter inhibitors on ion fluxes were examined, which are briefly described as follows. (1) Salt shock: Transient H⁺ flux kinetics in response to NaCl (50 mM) were measured in protoplasts isolated from LT-stressed root tips of the two species. A steady-state H⁺ flux was recorded (5–6 min) prior to the salt shock. Then NaCl stock (1 M) was slowly added to the measuring solution until the final NaCl concentration in the buffer reached 50 mM. After that, the H⁺ flux recording was restarted and continued for a further 40 to 50 min. The data measured during the first 2 to 3 min was discarded due to the diffusion effects of stock addition (Shabala, 2000; in our study, blank measurements, i.e. without protoplast addition, were carried out to exclude the disturbance of NaCl addition on H⁺ flux measurements). (2) pH: Protoplasts of LT-stressed P. euphratica roots were used in our experiment and pH values of measuring solutions were adjusted to 5.5, 6.0, and 7.0, respectively. Steady-state Na⁺ and H⁺ fluxes were recorded and the continuous recording period was 8 to 10 min. (3) Inhibitor treatments: LT-stressed P. euphratica protoplasts were subjected to sodium orthovanadate (500 µM) or amiloride (50 µM) for 30 min in the measuring solutions. Prior to the flux recording, the measuring solutions containing sodium orthovanadate were replaced with a 10-mL fresh solution, but measuring solutions with amiloride were not renewed (amiloride had no clear effect on the Nernstian slopes of the Na⁺ and H⁺ electrodes). Then the steady-state Na⁺ and H⁺ fluxes were measured in protoplasts pretreated with or without inhibitors at pH 5.5 and 6.0. Moreover, the H⁺ kinetics of the salt shock (50 mM NaCl) were recorded in these cells as described above.

Data Analysis

Three-dimensional ionic fluxes were calculated using MageFlux developed by Yue Xu (http://www.youngerusa.com/mageflux or http://xuyue.net/mageflux). Positive values in figures represent cation efflux or anion influx and vice versa.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Effects of hyperosmotic stress (85 mM mannitol, 24 h) on net Cl^– fluxes in roots of P. euphratica and P. popularis.

	ACKNOWLEDGMENTS

 
We thank Dr. Joseph G. Kunkel from the University of Massachusetts at Amherst, Dr. Marshall Porterfield from Purdue University, and Mr. Alan Shipley from Applicable Electronics Inc. for their professional advice and technical support. Prof. Dr. Gerrit Hazenberg (Faculty of Forestry, Lakehead University) and Dr. Thomas Teichmann (Department of Cell Biology, Faculty of Biology, Georg-August-University) are sincerely acknowledged for English correction.

Received September 7, 2008; accepted November 18, 2008; published November 21, 2008.

	FOOTNOTES

 
¹ This work was supported by projects of the National Natural Science Foundation of China (grant nos. 30430430 and 30872005), the HI-TECH Research and Development Program of China (863 Program, grant no. 2006AA10Z131), the Foundation for the Authors of National Excellent Doctoral Dissertations of China (grant no. 200152), the Foundation for the Supervisors of Excellent Doctoral Dissertations of Beijing (grant no. YB20081002201), the Teaching and Research Award Program for Outstanding Young Teachers in the Institution of Higher Education of the Ministry of Education, China (grant no. 2002–323), the Fok Ying Tung Education Foundation (grant no. 91031), and the Natural Science Foundation of Hubei Province (grant no. 2007ABB003).

² Present address: Tianjin Landscape-gardening Institute, Tianjin 300181, China.

³ Present address: The Centre for Research Ecotoxicology and Environmental Remediation, Institute of Agricultural Environmental Protection, Ministry of Agriculture, Tianjin 300191, China.

The author responsible for the distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Shaoliang Chen (lschen{at}bjfu.edu.cn).

^[C] Some figures in this article are displayed in color online but in black and white in the print edition.

^[W] The online version of this article contains Web-only data.

^[OA] Open access articles can be viewed online without a subscription.

www.plantphysiol.org/cgi/doi/10.1104/pp.108.129494

^* Corresponding author; e-mail lschen{at}bjfu.edu.cn.

	LITERATURE CITED

TOP ABSTRACT RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 
Apse MP, Blumwald E (2007) Na⁺ transport in plants. FEBS Lett 581: 2247–2254[CrossRef][Web of Science][Medline]

Arif I, Newman IA, Keenlyside N (1995) Proton flux measurements from tissues in buffered solution. Plant Cell Environ 18: 1319–1324[CrossRef]

Ayala F, O'Leary JW, Schumaker KS (1996) Increased vacuolar and plasma membrane H⁺-ATPase activities in Salicornia bigelovii Torr. in response to NaCl. J Exp Bot 47: 25–32[Abstract/Free Full Text]

Blumwald E, Aharon GS, Apse MP (2000) Sodium transport in plant cells. Biochim Biophys Acta 1465: 140–151[Medline]

Chen CX (2007) PeNhaD1 gene transformation and salt resistance of transgenic Populus tomentosa. PhD dissertation. Beijing Forestry University, Beijing, China (in Chinese)

Chen S, Li J, Fritz E, Wang S, Hüttermann A (2002) Sodium and chloride distribution in roots and transport in three poplar genotypes under increasing NaCl stress. For Ecol Manage 168: 217–230[CrossRef]

Chen S, Li J, Wang S, Fritz E, Hüttermann A, Altman A (2003) Effects of NaCl on shoot growth, transpiration, ion compartmentation, and transport in regenerated plants of Populus euphratica and Populus tomentosa. Can J For Res 33: 967–975[CrossRef]

Chen S, Li J, Wang S, Hüttermann A, Altman A (2001) Salt, nutrient uptake and transport, and ABA of Populus euphratica; a hybrid in response to increasing soil NaCl. Trees (Berl) 15: 186–194[CrossRef]

Chen Z, Newman I, Zhou M, Mendham N, Zhang G, Shabala S (2005) Screening plants for salt tolerance by measuring K⁺ flux: a case study for barley. Plant Cell Environ 28: 1230–1246[CrossRef]

Chen Z, Pottosin II, Cuin TA, Fuglsang AT, Tester M, Jha D, Zepeda-Jazo I, Zhou M, Palmgren MG, Newman IA, et al (2007) Root plasma membrane transporters controlling K⁺/Na⁺ homeostasis in salt stressed barley. Plant Physiol 145: 1714–1725[Abstract/Free Full Text]

Cosgrove DJ, Hedrich R (1991) Stretch-activated chloride, potassium, and calcium channels coexisting in plasma membranes of guard cells of Vicia faba. Planta 186: 143–153[Web of Science][Medline]

Demidchik V, Davenport RJ, Tester M (2002) Nonselective cation channels in plants. Annu Rev Plant Biol 53: 67–107[CrossRef][Medline]

Demidchik V, Maathuis FJM (2007) Physiological roles of nonselective cation channels in plants: from salt stress to signalling and development. New Phytol 175: 387–404[CrossRef][Web of Science][Medline]

Demidchik V, Tester M (2002) Sodium fluxes through nonselective cation channels in the plasma membrane of protoplasts from Arabidopsis roots. Plant Physiol 128: 379–387[Abstract/Free Full Text]

Falke LC, Edwards KL, Pickard BG, Misler S (1988) A stretch-activated anion channel in tobacco protoplasts. FEBS Lett 237: 141–144[CrossRef][Web of Science][Medline]

Gévaudant F, Duby G, Stedingk EV, Zhao R, Morsomme P, Boutry M (2007) Expression of a constitutively activated plasma membrane H⁺-ATPase alters plant development and increases salt tolerance. Plant Physiol 144: 1763–1776[Abstract/Free Full Text]

Greenway H, Munns R (1980) Mechanisms of salt tolerance in nonhalophytes. Annu Rev Plant Physiol 31: 149–190[Web of Science]

Gu R, Fonseca S, Puskás LG, Hackler L Jr, Zvara Á, Dudits D, Pais MS (2004) Transcript identification and profiling during salt stress and recovery of Populus euphratica. Tree Physiol 24: 265–276[Abstract]

Hasegawa PM, Bressan RA, Zhu JK, Bohnert HJ (2000) Plant cellular and molecular responses to high salinity. Annu Rev Plant Biol 51: 463–499[CrossRef][Web of Science]

Junghans U, Polle A, Düchting P, Weller E, Kuhlman B, Gruber F, Teichmann T (2006) Adaptation to high salinity in poplar involves changes in xylem anatomy and auxin physiology. Plant Cell Environ 29: 1519–1531[CrossRef][Medline]

Kochian LV, Shaff JE, Kühtreiber WM, Jaffe LF, Lucas WJ (1992) Use of an extracellular, ion-selective, vibrating microelectrode system for the quantification of K⁺, H⁺, and Ca²⁺ fluxes in maize roots and maize suspension cells. Planta 188: 601–610[Web of Science]

Kühtreiber WM, Jaffe LF (1990) Detection of extracellular calcium gradients with a calcium-specific vibrating electrode. J Cell Biol 110: 1565–1573[Abstract/Free Full Text]

Kunkel JG, Cordeiro S, Xu Y, Shipley AM, Feijó JA (2006) The use of non-invasive ion-selective microelectrode techniques for the study of plant development. In AG Volkov, ed, Plant Electrophysiology—Theory and Methods. Springer-Verlag, Berlin/Heidelberg, pp 109–137

Lorenzen I, Aberle T, Plieth C (2004) Salt stress-induced chloride flux: a study using transgenic Arabidopsis expressing a fluorescent anion probe. Plant J 38: 539–544[CrossRef][Web of Science][Medline]

Lü PP, Hu J, Chen SL, Shen X, Yin WL, Chen YH, Sun YR, Hu ZM (2007) Function of the putative Na⁺/H⁺ antiporter gene PeNhaD1 from salt-resistant Populus euphratica Oliv. J Plant Physiol Mol Biol 33: 173–178 (in Chinese with English abstract)

Maathuis FJM (2006) The role of monovalent cation transporters in plant responses to salinity. J Exp Bot 57: 1137–1147[Abstract/Free Full Text]

Maathuis FJM, Sanders D (2001) Sodium uptake in Arabidopsis thaliana roots is regulated by cyclic nucleotides. Plant Physiol 127: 1617–1625[Abstract/Free Full Text]

Martínez-Atienza J, Jiang X, Garciadeblas B, Mendoza I, Zhu JK, Pardo JM, Quintero FJ (2007) Conservation of the salt overly sensitive pathway in rice. Plant Physiol 143: 1001–1012[Abstract/Free Full Text]

Mazea D, Schatten G, Sale W (1975) Adhension of cells to surfaces coated with polylysine. J Cell Biol 66: 198–200[Abstract/Free Full Text]

Messerli MA, Smith PJS, Lewis RC, Robinson KR (2004) Chloride fluxes in lily pollen tubes: a critical reevaluation. Plant J 40: 799–812[CrossRef][Web of Science][Medline]

Munns R, Tester M (2008) Mechanisms of salinity tolerance. Annu Rev Plant Biol 59: 651–681[CrossRef][Medline]

Ottow EA, Brinker M, Teichmann T, Fritz E, Kaiser W, Brosché M, Kangasjärvi J, Jiang X, Polle A (2005b) Populus euphratica displays apoplastic sodium accumulation, osmotic adjustment by decreases in calcium and soluble carbohydrates, and develops leaf succulence under salt stress. Plant Physiol 139: 1762–1772[Abstract/Free Full Text]

Ottow EA, Polle A, Brosché M, Kangasjärvi J, Dibrov P, Zörb C, Teichmann T (2005a) Molecular characterization of PeNhaD1: the first member of the NhaD Na⁺/H⁺ antiporter family of plant origin. Plant Mol Biol 58: 73–86

Qi Z, Kishigami A, Nakagawa Y, Iida H, Sokabe M (2004) A mechanosensitive anion channel in Arabidopsis thaliana mesophyll cells. Plant Cell Physiol 45: 1704–1708[Abstract/Free Full Text]

Qiu QS, Barkla BJ, Vera-Estrella R, Zhu JK, Schumaker KS (2003) Na⁺/H⁺ exchange activity in the plasma membrane of Arabidopsis. Plant Physiol 132: 1041–1052[Abstract/Free Full Text]

Qiu QS, Guo Y, Dietrich MA, Schumaker KS, Zhu JK (2002) Regulation of SOS1, a plasma membrane Na⁺/H⁺ exchanger in Arabidopsis thaliana, by SOS2 and SOS3. Proc Natl Acad Sci USA 99: 8436–8441[Abstract/Free Full Text]

Roberts SK (2006) Plasma membrane anion channels in higher plants and their putative functions in roots. New Phytol 169: 647–666[CrossRef][Web of Science][Medline]

Shabala L, Cuin TA, Newman IA, Shabala S (2005) Salinity-induced ion flux patterns from the excised roots of Arabidopsis sos mutants. Planta 222: 1041–1050[CrossRef][Web of Science][Medline]

Shabala S (2000) Ionic and osmotic components of salt stress specifically modulate net ion fluxes from bean leaf mesophyll. Plant Cell Environ 23: 825–837[CrossRef]

Shabala S, Demidchik V, Shabala L, Cuin TA, Smith SJ, Miller AJ, Davies JM, Newman IA (2006) Extracellular Ca²⁺ ameliorates NaCl-induced K⁺ loss from Arabidopsis root and leaf cells by controlling plasma membrane K⁺-permeable channels. Plant Physiol 141: 1653–1665[Abstract/Free Full Text]

Shabala S, Newman I, Whittington J, Juswono U (1998) Protoplast ion fluxes: their measurement and variation with time, position and osmoticum. Planta 204: 146–152[CrossRef][Web of Science]

Shabala S, Newman IA (2000) Salinity effects on the activity of plasma membrane H⁺ and Ca²⁺ transporters in bean leaf mesophyll: masking role of the cell wall. Ann Bot (Lond) 85: 681–686[Abstract/Free Full Text]

Shi H, Ishitani M, Kim C, Zhu JK (2000) The Arabidopsis thaliana salt tolerance gene SOS1 encodes a putative Na⁺/H⁺ antiporter. Proc Natl Acad Sci USA 97: 6896–6901[Abstract/Free Full Text]

Shi H, Lee BH, Wu SJ, Zhu JK (2003) Overexpression of a plasma membrane Na⁺/H⁺ antiporter gene improves salt tolerance in Arabidopsis thaliana. Nat Biotechnol 21: 81–85[CrossRef][Web of Science][Medline]

Shi H, Quintero FJ, Pardo JM, Zhu JK (2002) The putative plasma membrane Na⁺/H⁺ antiporter SOS1 controls long distance Na⁺ transport in plants. Plant Cell 14: 465–477[Abstract/Free Full Text]

Teakle NL, Flowers TJ, Real D, Colmer TD (2007) Lotus tenuis tolerates the interactive effects of salinity and waterlogging by ‘excluding’ Na⁺ and Cl^– from the xylem. J Exp Bot 58: 2169–2180[Abstract/Free Full Text]

Teodoro AE, Zingarelli L, Lado P (1998) Early changes of Cl^– efflux and H⁺ extrusion induced by osmotic stress in Arabidopsis thaliana cells. Physiol Plant 102: 29–37[CrossRef]

Tester M, Davenport R (2003) Na⁺ tolerance and Na⁺ transport in higher plants. Ann Bot (Lond) 91: 503–527[Abstract/Free Full Text]

Vincent P, Chua M, Nogue F, Fairbrother A, Mekeel H, Xu Y, Allen N, Bibikova TN, Gilroy S, Bankaitis VA (2005) A sec14p-nodulin domain phosphatidylinositol transfer protein polarizes membrane growth of Arabidopsis thaliana root hairs. J Cell Biol 168: 801–812[Abstract/Free Full Text]

Vitart V, Baxter I, Doerner P, Harper JF (2001) Evidence for a role in growth and salt resistance of a plasma membrane H⁺-ATPase in the root endodermis. Plant J 27: 191–201[CrossRef][Web of Science][Medline]

Wang R, Chen S, Deng L, Fritz E, Hüttermann A, Polle A (2007) Leaf photosynthesis, fluorescence response to salinity and the relevance to chloroplast salt compartmentation and anti-oxidative stress in two poplars. Trees (Berl) 21: 581–591[CrossRef]

Wang R, Chen S, Zhou X, Shen X, Deng L, Zhu H, Shao J, Shi Y, Dai S, Fritz E, et al (2008) Ionic homeostasis and reactive oxygen species control in leaves and xylem sap of two poplars subjected to NaCl stress. Tree Physiol 28: 947–957[Abstract/Free Full Text]

White PJ, Broadley MR (2001) Chloride in soils and its uptake and movement within the plant: a review. Ann Bot (Lond) 88: 967–988[Abstract/Free Full Text]

White PJ, Davenport RJ (2002) The voltage-independent cation channel in the plasma membrane of wheat roots is permeable to divalent cations and may be involved in cytosolic Ca²⁺ homeostasis. Plant Physiol 130: 1386–1395[Abstract/Free Full Text]

Wilson C, Shannon MC (1995) Salt-induced Na⁺/H⁺ antiport in root plasma membrane of a glycophytic and halophytic species of tomato. Plant Sci 107: 147–157[CrossRef][Web of Science]

Wu Y, Ding N, Zhao X, Zhao M, Chang Z, Liu J, Zhang L (2007) Molecular characterization of PeSOS1: the putative Na⁺/H⁺ antiporter of Populus euphratica. Plant Mol Biol 65: 1–11[Medline]

Xu Y, Sun T, Yin LP (2006) Application of non-invasive microsensing system to simultaneously measure both H⁺ and O₂ fluxes around the pollen tube. J Integr Plant Biol 48: 823–831[CrossRef][Web of Science]

Yang Y, Zhang F, Zhao M, An L, Zhang L, Chen N (2007) Properties of plasma membrane H⁺-ATPase in salt-treated Populus euphratica callus. Plant Cell Rep 26: 229–235[CrossRef][Web of Science][Medline]

Zhang F, Wang Y, Yang YL, Wu H, Wang D, Liu JQ (2007) Involvement of hydrogen peroxide and nitric oxide in salt resistance in the calluses from Populus euphratica. Plant Cell Environ 30: 775–785[CrossRef][Medline]

Zhu JK (2001) Plant salt tolerance. Trends Plant Sci 6: 66–71[CrossRef][Web of Science][Medline]

Zhu JK (2003) Regulation of ion homeostasis under salt stress. Curr Opin Plant Biol 6: 1–5[Medline]

Zonia L, Cordeiro S, Tup J, Feijó JA (2002) Oscillatory chloride efflux at the pollen tube apex has a role in growth and cell volume regulation and is targeted by inositol 3,4,5,6-tetrakisphosphate. Plant Cell 14: 2233–2249[Abstract/Free Full Text]

This article has been cited by other articles:

				J.-L. Horng, P.-P. Hwang, T.-H. Shih, Z.-H. Wen, C.-S. Lin, and L.-Y. Lin Chloride transport in mitochondrion-rich cells of euryhaline tilapia (Oreochromis mossambicus) larvae Am J Physiol Cell Physiol, October 1, 2009; 297(4): C845 - C854. [Abstract] [Full Text] [PDF]

				F. Zeng, H. Yan, and S. K. Arndt Leaf and whole tree adaptations to mild salinity in field grown Populus euphratica Tree Physiol, October 1, 2009; 29(10): 1237 - 1246. [Abstract] [Full Text] [PDF]

				J. Sun, S. Dai, R. Wang, S. Chen, N. Li, X. Zhou, C. Lu, X. Shen, X. Zheng, Z. Hu, et al. Calcium mediates root K+/Na+ homeostasis in poplar species differing in salt tolerance Tree Physiol, September 1, 2009; 29(9): 1175 - 1186. [Abstract] [Full Text] [PDF]

This Article Free via Open Access: OA OA Abstract Full Text (PDF) Supplemental Data OA All Versions of this Article: 149/2/1141    most recent pp.108.129494v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Sun, J. Articles by Xu, Y. Search for Related Content PubMed PubMed Citation Articles by Sun, J. Articles by Xu, Y. Agricola Articles by Sun, J. Articles by Xu, Y.