Polyamines Improve K⁺/Na⁺ Homeostasis in Barley Seedlings by Regulating Root Ion Channel Activities

Abstract

Polyamines are known to increase in plant cells in response to a variety of stress conditions. However, the physiological roles of elevated polyamines are not understood well. Here we investigated the effects of polyamines on ion channel activities by applying patch-clamp techniques to protoplasts derived from barley (Hordeum vulgare) seedling root cells. Extracellular application of polyamines significantly blocked the inward Na⁺ and K⁺ currents (especially Na⁺ currents) in root epidermal and cortical cells. These blocking effects of polyamines were increased with increasing polycation charge. In root xylem parenchyma, the inward K⁺ currents were blocked by extracellular spermidine, while the outward K⁺ currents were enhanced. At the whole-plant level, the root K⁺ content, as well as the root and shoot Na⁺ levels, was decreased significantly by exogenous spermidine. Together, by restricting Na⁺ influx into roots and by preventing K⁺ loss from shoots, polyamines were shown to improve K⁺/Na⁺ homeostasis in barley seedlings. It is reasonable to propose that, therefore, elevated polyamines under salt stress should be a self-protecting response for plants to combat detrimental consequences resulted from imbalance of Na⁺ and K⁺.

Polyamines are small aliphatic polycations found in all living organisms studied and are required for growth and development of both prokaryotes and eukaryotes, although their specific mechanism of action is not well understood (Tabor and Tabor, 1984). In higher plants, the most prevalent polyamines are spermidine, spermine, and their diamine precursor, putrescine. A variety of roles have been proposed for polyamines in modulation of many physiological processes including chromatin organization, DNA replication, gene transcription, mRNA translation, cell proliferation, and apoptosis (Evans and Malmberg, 1989; Paschalidis and Roubelakis-Angelakis, 2005a, 2005b). Recently, a growing number of evidences have shown that polyamine alteration caused by molecular manipulation has profound effects on plant growth and development (Masgrau et al., 1997; Theiss et al., 2002; Vuosku et al., 2006).

Except for their biological functions in plant growth and development, polyamines have been shown to be involved in stress responses since their levels increased significantly under a number of environmental stress conditions (Flores, 1990; Galston and Kaur-Sawhney, 1995). K⁺ deficiency may be the first reported stress condition that resulted in an increase in putrescine level in barley (Hordeum vulgare; Richard and Coleman, 1952). In addition, osmotic shock (Tiburcio et al., 1986), drought (Erdei et al., 1996), chilling (Shen et al., 2000), and air pollution (Wellburn and Wellburn, 1996) also could increase polyamine levels as well. Regarding salt stress, polyamine levels were increased in several crop species including rice (Oryza sativa), mung bean (Vigna radiata), maize (Zea mays), barley, and sorghum (Sorghum bicolor; Friedman et al., 1989; Krishnamurthy and Bhagwat, 1989; Rodriguez-Kessler et al., 2006). Together, upon stressed environments, polyamine levels could elevate to submillimolar and millimolar range from the basal levels of tens to hundreds of micromole. Although these phenomena were well documented by numerous studies, the physiological importance of elevated polyamines in response to stressed conditions was still largely unknown.

Many studies suggested that the increase in polyamine levels may be an integral self-protecting response to salinity environments. This view was further supported by the findings that exogenous application of polyamines could improve plant performances by decreasing shoot Na⁺ levels under saline conditions (Chattopadhayay et al., 2002; Zhao and Qin, 2004; Zhu et al., 2006). High content of Na⁺ in plant tissues is often considered as the most critical factor responsible for salt toxicity in nonhalophytes (Greenway and Munns, 1980; Niu et al., 1995; Hasegawa et al., 2000). Therefore, the reduction in Na⁺ content in shoots induced by polyamines should be an effective strategy for combating high salinity. In addition, it has been demonstrated that exogenous polyamines could increase shoot K⁺ allocation under saline environments (Lakra et al., 2006; Ndayiragije and Lutts, 2007). Under salinity conditions, K⁺ loss is another detrimental consequence resulted from salt stress, and mitigation of this loss strongly correlates with salt tolerance in plants (Carden et al., 2003; Chen et al., 2005). Together, by maintaining K⁺/Na⁺ homeostasis, polyamines possess beneficial effects to plants in their adaptation to salinity. However, the involved mechanism remains unclear. Importantly, polyamines have been shown to be efficient inhibitors of both slow and fast vacuolar cation channels (Brüggemann et al., 1998; Dobrovinskaya et al., 1999). In addition, polyamines also were characterized as potent blockers of inward K⁺ currents across the plasma membrane of guard cells (Liu et al., 2000). The blocking effects of polyamines on ion channel activities shed new light on elucidating the mechanism responsible for the maintenance of K⁺/Na⁺ homeostasis in plants induced by polyamines under salt stress.

Here, we applied patch-clamp techniques to protoplasts isolated from barley seedling root cells and investigated the effects of polyamines on Na⁺- and K⁺-conducted currents. The results showed that extracellular polyamines blocked the inward Na⁺ and K⁺ currents (especially Na⁺ currents) in root epidermal and cortical cells. In root xylem parenchyma, the inward K⁺ current also was blocked by extracellular spermidine, while the outward K⁺ current was enhanced. Our results provided a novel insight for understanding the elevated polyamines in plant cells in response to saline conditions.

RESULTS

Exogenous Spermidine Improved K⁺/Na⁺ Homeostasis in Barley Seedling Shoots

To further understand the protective role of spermidine against salt stress, we determined Na⁺ and K⁺ contents in roots and shoots. According to previous studies, polyamine concentrations in plant tissues reach low millimolar range under stress conditions (Slocum and Flores, 1991; Watson and Malmberg, 1996). Therefore, 0.5 mm spermidine was applied to the barley seedlings to investigate K⁺ and Na⁺ distribution. As shown in Figure 1A, salt treatment resulted in a remarkable increase in Na⁺ contents in the roots and shoots. However, the application of exogenous spermidine significantly decreased Na⁺ levels in the roots (P < 0.05) and shoots (P < 0.01). In addition, the levels of K⁺ in roots and shoots were decreased significantly with exposure to NaCl. This reduction in the roots was further strengthened by exogenous spermidine (P < 0.01), while the shoot K⁺ content was not altered significantly (Fig. 1A). Without NaCl treatment, the root K⁺ level also was decreased by spermidine application (P < 0.05). No significant difference in the shoot K⁺ levels was observed between the control and the spermidine-treated seedlings. The shoot [K⁺]/[Na⁺] was increased significantly by exogenous spermidine with or without NaCl treatment (P < 0.01; Fig. 1B).

Figure 1.

Effect of exogenous spermidine on Na⁺ and K⁺ distribution in barley seedlings. A, Na⁺ and K⁺ contents in the roots and shoots of barley seedlings. B, [K⁺]/[Na⁺] in the roots and shoots of barley seedlings. C, Shoot [K⁺]/[Na⁺] was enhanced by exogenous spermidine in a dose-dependent manner. The applied spermidine concentration was 0.5 mm. Spd in A is an abbreviated form of spermidine. Asterisks indicate the difference at P < 0.05 (*) or P < 0.01 (**) by Student's t test. Data represent mean ± sd of three independent experiments.

To investigate effects of spermidine at different concentrations on shoot [K⁺]/[Na⁺], we treated the barley seedlings with several concentrations of spermidine ranging from 0.1 to 2 mm. As shown in Figure 1C, the shoot [K⁺]/[Na⁺] was enhanced by exogenous spermidine in a dose-dependent manner. When spermidine concentrations ranged from 0.1 to 1 mm, the shoot [K⁺]/[Na⁺] increased largely. The shoot [K⁺]/[Na⁺] increased slightly when spermidine concentrations were higher than 1 mm. Therefore, we chose 1 mm spermidine to investigate its roles on the root ion channel activities in the following experiments.

Effects of Extracellular and Intracellular Spermidine on the Inward K⁺ Currents in Root Epidermal Cells

The exogenous application of spermidine could decrease K⁺ accumulation in barley seedling roots, which prompted us to investigate whether spermidine affects K⁺-permeable channel activity. Thus we applied patch-clamp techniques to the protoplasts isolated from barley root epidermal cells. To investigate the precise functional site of exogenous spermidine, we treated the isolated protoplasts with 1 mm spermidine in the bath solution (extracellular application) or in the pipette solution (intracellular application). During the recordings, the membrane potential was clamped at −52 mV and stepped to values from −190 mV to +30 mV with 20-mV increments to activate inward K⁺ current (Fig. 2A). Under the control condition, a typical time course of inward K⁺ currents was recorded from a root epidermal cell protoplast (Fig. 2, B and E). When 1 mm spermidine was included in the bath solution, the magnitude of inward K⁺ currents was reduced immediately following the establishment of the whole-cell configuration (Fig. 2C). Figure 2D summarized the current-voltage (I-V) relationship under control condition and in the presence of 1 mm spermidine in the bath solution. At −190 mV, 1 mm extracellular spermidine reduced the whole-cell current density from 102 ± 16 pA/pF to 61 ± 12 pA/pF, a 40% decrease of the control level (Fig. 2D). When 1 mm spermidine was perfused to the pipette solution, the currents were not affected with comparison of the control (Fig. 2, E and F). A comparable value in K⁺ current density was observed between the intracellular spermidine-treated protoplasts and the control (Fig. 2G). These results indicated that extracellular spermidine inhibited the inward K⁺ currents in barley root epidermal cells, while intracellular spermidine failed to produce such inhibitory effect.

Figure 2.

Effects of extracellular and intracellular spermidine on the inward K⁺ currents in root epidermal cells. A, During the recordings, the holding potential was −52 mV, the currents were recorded at the membrane potentials from −190 to 30 mV with increment of 20 mV. B, Whole-cell inward K⁺ currents (capacitance = 6.5 pF). C, The same cell as in B was treated with 1 mm spermidine in the bath solution for 15 min. D, The current amplitudes (mean ± sd) from control cells (▪) and cells treated with 1 mm spermidine in the bath solution (▵) are presented as I-IV curves (n = 15). E, Whole-cell inward K⁺ currents (capacitance = 5.4 pF). F, The same cell as in E was treated with 1 mm spermidine in the pipette solution (capacitance = 6.3 pF). G, The current amplitudes (mean ± sd) from 21 control cells (▪) and 25 cells treated with 1 mm spermidine in the pipette solution (▵) are presented as I-V curves.

Characteristics of the Na⁺-Conducted Currents in Root Cell Protoplasts

Both inward and outward Na⁺ currents could be detected in all protoplasts derived from barley root epidermal cells, and these currents were increased with increasing external Na⁺ (Fig. 3A). The inward Na⁺ currents exhibited no visible time dependence in 67% of total protoplasts studied (n = 160). These protoplasts having no time-dependent component were applied for studying Na⁺-conducted current in the following experiments. However, in all protoplasts used, there was a Cl⁻ influx component, which might also contribute the outward current. To exclude this possibility, we calculated the reversal potential (E_rev) of the currents according to the I-V curves (Fig. 3A). The results showed that E_rev for the detected currents was about −30 mV to approximately −10 mV. This value was much closer to the E_rev for Na⁺ in the solution (E_Na = −20 mV) than to the E_rev for Cl⁻ (E_Cl = −51 mV). This finding indicated that the detected currents were conducted by Na⁺ not Cl⁻. In addition, some cations also could induce detectable currents upon hyperpolarization. Thus we employed K⁺ channel blocker tetraethylammonium (TEA) to further confirm our measured currents were conducted by Na⁺. As shown in Figure 3B, the application of 10 mm TEA in the presence of 50 mm NaCl could not decrease the currents, implying the detected currents were not conducted by K⁺. Furthermore, we applied 0.5 mm quinine to the bath solution and in which the magnitude of recorded currents was completely blocked (Fig. 3B). This result suggested that the detected currents were conducted by Na⁺ via nonselective cation channels (NSCCs) since quinine is a well recognized blocker of NSCCs (Demidchik and Tester, 2002; Essah et al., 2003). These inward currents were blocked obviously by 0.5 mm Ca²⁺ (Fig. 3B). Similarly, the currents in root cortical and xylem parenchyma also exhibited the same characteristics (data not shown). Together, the above results strongly suggested that the detected currents in the Na⁺-dominated bath solution in root cells were mainly conducted by Na⁺.

Figure 3.

Characteristics of the Na⁺-conducted currents in root epidermal cells. A, The dependence of instantaneous Na⁺ currents in root epidermal cells on external NaCl concentration. Concentrations are given in mm. Na⁺ was present as the chloride salt. Arrows indicate E_rev. The data represent mean ± sd. B, I-V relations of inward instantaneous currents in root epidermal cells treated with extracellular application of Ca²⁺, TEA⁺, and quinine. The current amplitudes from control cells (♦, n = 15), Ca²⁺-treated cells (○, n = 15), TEA⁺-treated cells (▵, n = 18), and quinine-treated cells (▴, n = 21) are presented as I-V curves. Concentrations are indicated in mm. Na⁺ was present as the Na gluconate. Data were obtained after 10-min exposure to Ca²⁺, TEACl, or quinine. The data represent mean ± sd.

Effects of Extracellular and Intracellular Spermidine on the Inward Na⁺ Currents in Root Epidermal Cells

Since exogenous spermidine could decrease Na⁺ accumulation in the roots of NaCl-treated barley seedlings, we speculate that Na⁺ influx into root cells is blocked by spermidine. To address this speculation, we therefore investigated effect of spermidine on inward Na⁺ currents in root epidermal cells. The holding potential was kept at −52 mV and the currents were recorded at the membrane potential from −170 to +70 mV with increments of 20 mV (Fig. 4A). Instantaneous inwardly Na⁺ currents were detected under control condition (Fig. 4, B and E). As we expected, the inward Na⁺ currents were reduced obviously in the presence of spermidine in the bath solution as compared with control (Fig. 4C). The whole-cell current densities were significantly decreased 61% (from 20.2 ± 2.8 pA/pF to 7.1 ± 0.8 pA/pF, P < 0.01) at −170 mV by extracellular spermidine (Fig. 4D). However, when spermidine was perfused in the pipette solution, the magnitude of inward Na⁺ currents were affected slightly (Fig. 4F). Accordingly, the whole-cell current densities were comparable (Fig. 4G). These data suggested that the inward Na⁺ currents in root epidermal cells were blocked by extracellular spermidine, whereas intracellular spermidine could not produce such blocking effect.

Figure 4.

Effects of extracellular and intracellular spermidine on the inward Na⁺ currents in root epidermal cells. A, Plasma membrane currents were recorded with voltage pulses ranging from −170 to 70 mV at 20 mV increments. Holding potential was kept at −52 mV. B, Whole-cell inward Na⁺ currents (capacitance = 4.5 pF). C, The same cell as in B was treated with 1 mm spermidine in the bath solution for 15 min. D, The current amplitudes (mean ± sd) from control cells (▪) and cells treated with 1 mm spermidine in the bath solution (▵) are presented as I-V curves (n = 16). E, Whole-cell inward Na⁺ currents (capacitance = 6.5 pF). F, The same cell as in E was treated with 1 mm spermidine in the pipette solution (capacitance = 5.7 pF). G, The current amplitudes (mean ± sd) from 14 control cells (▪) and 23 cells treated with 1 mm spermidine in the pipette solution (▵) are presented as I-V curves.

Effects of Three Natural Polyamines on the Inward Na⁺ and K⁺ Currents in Root Epidermal Cells

To test whether other natural polyamines inhibit inward Na⁺ current, we included 1 mm putrescine, spermidine, or spermine, in the bath solution. The results showed that the inward Na⁺ currents were blocked by all three natural polyamines (Fig. 5A). Moreover, these blocking effects were closely related to charge values, since spermine was found to be the most potent blocker, orderly followed by spermidine and putrescine. In addition, we also investigated effects of these polyamines on inward K⁺ current. As shown in Figure 5B, all three polyamines blocked inward K⁺ currents, and these blocking effects were increased with increasing the polycation charge of each polyamine. These data showed that both inward Na⁺ and K⁺ currents were blocked by three natural polyamines applied extracellularly. And such blocking potency was positively correlated with their charges. Moreover, we previously found that spermidine was increased significantly in NaCl-treated barley roots, while spermine was changed slightly (Zhao et al., 2003). In the following experiments, therefore, we just chose extracellular application of spermidine to investigate effects of polyamines on K⁺- and Na⁺-conducted currents in other root cell protoplasts.

Figure 5.

Effects of natural polyamines on the inward Na⁺ and K⁺ currents in root epidermal cells. A, The responses of inward Na⁺ current to different polyamines in the bath solution are shown as I-V curves (control, n = 13; putrescine, n = 15; spermidine, n = 17; spermine, n = 19). Each polyamine concentration was 1 mm. The data were presented as mean ± sd. B, The responses of inward K⁺ current to different polyamines in the bath solution are shown as I-V curves (control, n = 16; putrescine, n = 14; spermidine, n = 18; spermine, n = 17).

Extracellular Spermidine Blocked the Inward K⁺ and Na⁺ Currents in Root Cortical Cell Protoplasts

As mentioned above, extracellular spermidine could block inward K⁺ and Na⁺ currents in root epidermal cells. We next investigated whether spermidine produces similar effect in root cortical cells. At control condition, a typical inward K⁺ current was recorded as shown in Figure 6B. When spermidine was applied in the bath solution, a decrease in inward K⁺ currents was observed (Fig. 6C). According to I-V curve, the whole-cell K⁺ current density was reduced from 68.3 ± 9.6 pA/pF to 42.2 ± 8.7 pA/pF (P < 0.05) at −190 mV by application of spermidine in the bath solution (Fig. 6D). With respect to inward Na⁺ currents, extracellular spermidine also caused a remarkable reduction with comparison of the current recorded at control condition (Fig. 7, B and C). Correspondingly, the whole-cell Na⁺ current density was decreased 71% by spermidine treatment (28.4 ± 5.1 pA/pF) with comparison of control (8.3 ± 3.7 pA/pF) at the membrane potential of −170 mV (P < 0.01; Fig. 7D). These results clearly indicate that inward K⁺ and Na⁺ currents, especially Na⁺ currents, were significantly blocked by extracellular spermidine.

Figure 6.

Extracellular spermidine blocked the inward K⁺ currents in root cortical cells. A, The holding potential was −52 mV and the currents were recorded at the membrane potentials from −190 to 50 mV with increments of 20 mV. B, Whole-cell inward K⁺ currents (capacitance = 15.5 pF). C, The same cell as in B was treated with 1 mm spermidine in the bath solution for 15 min. D, The current amplitudes (mean ± sd) from control cells (▪) and cells treated with 1 mm spermidine in the bath solution (▵) are presented as I-V curves (n = 17).

Figure 7.

Extracellular spermidine blocked the inward Na⁺ currents in root cortical cells. A, The currents were recorded at the membrane potentials from −170 to 70 mV (in 20 mV steps) with the holding potential of −52 mV. B, Whole-cell inward Na⁺ currents (capacitance = 18 pF). C, The same cell as in B was treated with 1 mm spermidine in the bath solution for 15 min. D, The current amplitudes (mean ± sd) from control cells (▪) and cells treated with 1 mm spermidine in the bath solution (▵) are presented as I-V curves (n = 18).

Effects of Extracellular Spermidine on the Inward and Outward K⁺ Currents in Root Xylem Parenchyma

We observed the blocking effects of spermidine on the inward K⁺ and Na⁺ currents in epidermal and cortical cells. It is also necessary to clarify whether extracellular spermidine affects K⁺ currents in root xylem parenchyma. It is worthy to note that both inward and outward K⁺ currents could be detected in root xylem parenchyma. Among all the tested protoplasts (n = 130), 70% protoplasts exhibited outwardly rectifying K⁺ channel activity, and 30% protoplasts produced inwardly rectifying K⁺ channel activity. During the recordings, the membrane potential was clamped at −52 mV and stepped to values from −90 mV to +130 mV (for activating outward K⁺ current) or −190 mV to +90 mV (for activating inward K⁺ current) with 20-mV increments (Figs. 8A and 9A). An increase in the outward K⁺ currents was occurred by application of 1 mm extracellular spermidine (Fig. 8, B and C). The whole-cell K⁺ current density was 28.4 ± 4.6 pA/pF under control condition at membrane potential of +130 mV. It was significantly increased to 44.7 ± 7.7 pA/pF by spermidine (P < 0.05; Fig. 8D). Unlike the outward K⁺ currents, the inward K⁺ currents were decreased by applying 1 mm spermidine at the bath solution (Fig. 9, B and C). Accordingly, the whole-cell current density was reduced from 26.1 ± 4.8 pA/pF to 15.4 ± 3.4 pA/pF (P < 0.05) at −190 mV (Fig. 9D). These data showed that extracellular spermidine affected the inward and outward K⁺ currents in xylem parenchyma in a contrary manner, since the inward K⁺ currents were blocked by spermidine while the outward K⁺ currents were enhanced.

Figure 8.

Effect of extracellular spermidine on the outward K⁺ currents in root xylem parenchyma. A, The membrane potential was clamped at −52 mV and stepped to values from −90 to 130 mV with 20 mV increments. B, Whole-cell inward K⁺ currents (capacitance = 7.2 pF). C, The same cell as in B was treated with 1 mm spermidine in the bath solution for 15 min. D, The current amplitudes (mean ± sd) from control cells (▪) and cells treated with 1 mm spermidine in the bath solution (▵) are presented as I-V curves (n = 14).

Figure 9.

Effect of extracellular spermidine on the inward K⁺ currents in root xylem parenchyma. A, During the recordings, the holding potential was −52 mV, the currents were recorded at the membrane potentials from −190 to 90 mV with increments of 20 mV. B, Whole-cell inward K⁺ currents (capacitance = 9 pF). C, The same cell as in B was treated with 1 mm spermidine in the bath solution for 15 min. D, The current amplitudes (mean ± sd) from control cells (▪) and cells treated with 1 mm spermidine in the bath solution (▵) are presented as I-V curves (n = 18).

Effects of Extracellular Spermidine on the Outward Na⁺ Currents in Barley Root Xylem Parenchyma

To test whether outward Na⁺ currents in xylem parenchyma are affected by spermidine, we recorded outward Na⁺ currents in the bath solution containing 1 mm spermidine. The holding potential was kept at −52 mV and the currents were recorded at the membrane potentials from −130 to +110 mV with increment of 20 mV (Fig. 10A). A typical weakly rectifying outward Na⁺-dependent current in the xylem parenchyma was recorded under control condition (Fig. 10B). The outward Na⁺ currents were decreased slightly by spermidine (Fig. 10C). According to I-V curve, no significant difference in the whole-cell Na⁺ current density was observed between the spermidine-treated protoplasts and the control (Fig. 10D). These findings showed that the outward Na⁺ currents in xylem parenchyma were not affected significantly by extracellular spermidine.

Figure 10.

Effect of extracellular spermidine on the outward Na⁺ currents in root xylem parenchyma cells. A, During the recordings, the holding potential was −52 mV, the currents were recorded at the membrane potentials from −130 to 110 mV with increments of 20 mV. B, Whole-cell outward Na⁺ currents (capacitance = 8.6 pF). C, The same cell as in B was treated with 1 mm spermidine in the bath solution for 15 min. D, The current amplitudes (mean ± sd) from control cells (▪) and cells treated with 1 mm spermidine in the bath solution (▵) are presented as I-V curves (n = 15).

DISCUSSION

It is well established that polyamine levels increased upon salt stress in a variety of plants (Flores, 1990; Galston and Kaur-Sawhney, 1995). However, the physiological role of stress-induced polyamine elevation remains unknown. Here, we showed that the inward Na⁺ and K⁺ currents (especially Na⁺ currents) in barley root epidermal and cortical cells were inhibited by extracellular spermidine. In root xylem parenchyma, the outward K⁺ currents were enhanced by spermidine, while the inward K⁺ currents were blocked. Thus, by repressing Na⁺ influx into roots and by preventing K⁺ loss from shoots, polyamines improved K⁺/Na⁺ homeostasis in barley seedlings to cope with high saline conditions. Therefore, we proposed that elevated polyamines in plant cells under salinity should be a self-protecting response.

Root epidermal and cortical cells are responsible for net ion uptake into the root symplasm (Enstone et al., 2003). Most ion transport in these root cells is mediated by plasma membrane ion channels. In the present study, a time- and voltage-dependent current was detected in the K⁺-dominated bath solution in the protoplasts derived from barley seedling root epidermal and cortical cells. In addition, an instantaneous current was recorded when K⁺ was replaced by Na⁺ in bath solution. These voltage-independent currents also exhibited no visible time-dependent manner. Moreover, these currents were unaffected by TEA but were sensitive to Ca²⁺ and quinine. These features of Na⁺-conducted currents were quite similar to the characteristics of NSCCs. Up to now, a growing number of evidence suggests that NSCCs are the major pathway for Na⁺ influx into root cells in a variety of plants (Roberts and Tester, 1997; Tyerman et al., 1997; Buschmann et al., 2000; Davenport and Tester, 2000; Demidchik and Tester, 2002; Demidchik et al., 2002). Importantly, the inward Na⁺ currents in root epidermal and cortical cells were significantly blocked by spermidine in the bath solution. Consistently, exogenous application of spermidine could decrease Na⁺ accumulation in the barley seedling roots especially under salt stress (Fig. 1A). These results suggested the process of Na⁺ influx into roots was blocked by spermidine. The blockage of Na⁺ influx into root epidermal and cortical cells by spermidine, therefore, is very important for barley plants to cope with external high saline environments.

In contrast to epidermal and cortical cells, root xylem parenchyma mediates the net loss of ions from roots symplasm into the xylem vessels for transport to shoots (Enstone et al., 2003). In this study, the outward K⁺ current in root xylem parenchyma was enhanced by spermidine (Fig. 8, B and C), indicating the release of K⁺ from xylem parenchyma to xylem vessels was increased. In contrast, the inward K⁺ current was blocked by extracellular spermidine (Fig. 9, B and C), suggesting the recirculation of K⁺ from shoots to roots was decreased. Similar to our results, polyamines were well characterized as potent blockers of inward K⁺ channels in both plant cells (Brüggemann et al., 1998; Dobrovinskaya et al., 1999; Liu et al., 2000) and mammalian cells (Shin and Lu, 2005). Thus, by regulating inward and outward K⁺ currents at different mode, spermidine prevented K⁺ loss from shoots. Previous studies showed that mitigation of K⁺ loss strongly correlates with the level of salt tolerance in barley cultivars (Carden et al., 2003; Chen et al., 2005). Therefore, the decrease in K⁺ loss from shoots induced by spermidine should be of great importance for improving plant salt tolerance. However, at the whole-plant level, no significant difference in the shoot K⁺ contents was observed when compared to the spermidine-treated plants with the control (Fig. 1A). This discrepancy is probably due to the remarkable decrease in root K⁺ content caused by spermidine application (Fig. 1A). Additionally, the involvement of other K⁺-permeable channels in barley plants also might be considered (Rodriguez-Navarro, 2000).

A large number of reports indicated that polyamine levels are increased in response to salt-stressed conditions. Furthermore, the increases in polyamine titers by chemical or genetic approaches can alleviate salt injuries in a variety of plants (Zhao and Qin, 2004; Liu et al., 2006; Wi et al., 2006; Yamaguchi et al., 2006). These findings implied that polyamines play an important role against salinity and increase of polyamines under saline conditions seems to be a self-protecting response of plants. However, the precise mechanism for alleviating effects of polyamines against salinity still remains elusive. Many explanations responsible for such beneficial roles against salinity were proposed, including stabilization of membrane integrity (Mansour and Al-Mutawa, 1999), maintenance of enzyme structures and activities (Zhao and Qin, 2004), adjustment of osmotic pressure (Jouve et al., 2004), and clearance of free radical species (Verma and Mishra, 2005). In the present study, we found that exogenous application of polyamines could decrease Na⁺ accumulation in roots and maintain a high level of K⁺ in shoots by affecting Na⁺ and K⁺ currents in specific root cells. Therefore, these data raised another explanation for increased levels of polyamines in response to salt stress.

In this study, extracellular application of spermidine could inhibit the inward Na⁺ and K⁺ currents in root epidermal and cortical cells. However, intracellular application of spermidine in the pipette solution failed to produce such inhibitory effects on the Na⁺ and K⁺ currents. This finding led to a suggestion that the functional site(s) for polyamines might be located in extracellular spot(s). In contrast, both intracellular and extracellular application of polyamines could inhibit inward K⁺ currents in guard cells (Liu et al., 2000). And the authors suggested that polyamine target(s) located in intracellular site(s) because extracellular polyamines could transport into cytoplasm rapidly (Pistocchi et al., 1987). The process involved in polyamine transport out of plant cells is rarely reported. Some evidence came from the animal literature showed that polyamine export is a selective process regulated by the growth rate of the cell (Wallace and Keir, 1981; Wallace and Mackarel, 1998). In this study, we detected Na⁺ and K⁺ currents immediately by using spermidine-contained pipette solution after the capsule was sealed. This short time course might limit spermidine export. Therefore, the intracellular application of polyamines could not transport out of cells to regulate ion channel activities. At the whole-plant level, the increase in cytoplasmic polyamine levels induced by environmental stresses (e.g. salt stress) could transport out of cells with long time durations. In addition, it has been demonstrated in cultured mammalian cells that polyamine export could be increased by contact inhibition of growth (Wallace and Keir, 1981) or treatment with antiproliferative drugs (Melvin et al., 1978). Therefore, the external factor(s) (e.g. NaCl) also might activate polyamine export. Thus, the increased polyamines in stressed plant cells could transport to extracellular site to regulate ion channel activities.

Although numerous studies observed the blockage effects of polyamines on ion channels, the responsible molecular mechanisms are still largely unknown. At physiological pH, polyamines are positively charged and thus can interact electrostatically with negatively charged nucleic acids and proteins, including ion channels. Here we observed that extracellular application of polyamines inhibited Na⁺-conducted currents in root cell protoplasts in a charge-dependent manner (spermine, 4+ > spermidine 3+ ≫ putrescine 2+). Similar to our results, previous studies also showed that polyamines blocked the slow- and fast-activating vacuolar cation channel in a charge-dependent manner (Brüggemann et al., 1998; Dobrovinskaya et al., 1999). These results suggested that polyamines may modulate ion channel activities through direct binding to the channel proteins. This view is consistent with previous findings carried out in both animal and bacterial cells (Delavega and Delcour, 1995; Johnson, 1996). It has been reported that several specific polyamine-binding proteins in plasma membranes from plant cells were well characterized (Tassoni et al., 1998, 2002). These proteins may be the potential targets for polyamines to produce their biological functions on ion channel activities. Previous studies showed that phosphorylation or dephosphorylation of ion channel proteins is closely related with ion channel activities (Bethke and Jones, 1997; Michard et al., 2005). Thus, external polyamines also may affect some membrane-bound protein kinase and/or phosphatase activities to regulate ion channel activities. This speculation awaits further validation, and some experiments toward such issue are going to proceed. Identification of ion channel structural elements and/or receptor molecules regulated by polyamines seems to be of great importance for elucidating the involved molecular mechanisms.

MATERIALS AND METHODS

Plant Materials

Seeds of barley (Hordeum vulgare) ‘J4’ were geminated as described previously (Zhao and Qin, 2005). The 5-d-old seedlings were transplanted into 8 L plastic pots containing four mesh-washed perlite. The plants were grown in one-half-strength Hoagland solution at a photon flux density of 800 to approximately 900 μmol m⁻² s⁻¹ with 12/12 h day/night cycle at 28°C in the phytotron. Plants at the three leaf stage were treated with 200 mm NaCl and various concentrations of spermidine as indicated. Control plants were grown in one-half-strength Hoagland solution with or without spermidine. After 7-d treatment, plant roots and shoots were harvested for experiments.

Na⁺ and K⁺ Determination

The roots and shoots of the plants were rinsed with deionized water three times and then dried at 80°C to a constant weight after filtration with Whatman paper. A total of 0.1 g dry powder samples were then extracted with 5 mL 4 m HCl at 37°C overnight to release the free cations and centrifuged at 10,000g for 10 min. The resulting supernatants of the extracts were diluted and Na⁺ and K⁺ were determined with a Shimadzu AA-680 atomic absorption/flame spectrophotometer.

Root Cortical and Xylem Parenchyma Protoplast Preparation

Seeds of barley ‘J4’ were germinated as described previously (Zhao and Qin, 2005). Plants were harvested after 5 to 7 d when roots were typically 4- to 5-cm long. Protoplasts were isolated using a protocol modified from two previously described procedures (Wegner and Raschke, 1994; Roberts and Tester, 1995). For root cortical protoplast preparation, barley seedling roots were selected from the first node (without lateral root formation and up to a length of about 3 cm). After removing the tips, the cortex was stripped from the stele by hand. The tissue was finely chopped in a solution of 10 mm KCl, 2 mm MES (2-[N-morpholino]ethanesulfonic acid)/KOH, 1 mm CaCl₂, 0.2% (w/v) bovine serum albumin, 1% (w/v) cellulase (Onozuka RS, Yakult Honsha Co.), and 0.02% (w/v) pectolyase Y23 (Yakult Honsha). The pH was adjusted to 5.7 with Tris and d-sorbitol was added to a total osmolality of 570 mOsm. Stelar sections were agitated at 30°C in a shaking water bath (100 rpm) for 30 min (60 to 80 min for cortex). The digest was filtered using 100 μm nylon mesh and centrifuged at 80g for 8 min. The pellet was resuspended in 8 mL of ice-cold bath solutions and centrifuged again. Large protoplasts (about 30 μm in diameter for stele and 50 μm for cortex) with visible cytoplasmic streaming were selected for patch clamping.

Epidermal Protoplasts Isolation

Seeds of barley ‘J4’ were surface sterilized in an aqueous solution of 1% NaClO for 5 min. The seeds were then washed several times with filter-sterilized water and placed on wet filter paper in petri dishes that had been exposed to UV light. The petri dishes were sealed with Parafilm and stored in the dark for 2 d at 25°C, by which time the germinated seeds had roots approximately 3- to 4-cm long with visible root hair growth. Protoplasts were isolated using a protocol modified from previously described procedure (Gassmann and Schroeder, 1994): Whole intact seedlings with roots were placed in 5 mL of enzyme solution in a water bath shaker (100 rpm) at 30°C for 10 min. The enzyme solution contained (in mm): 10 KCl, 2 MES/KOH, 1 CaCl₂, pH 5.7, and 0.8% (w/v) cellulase (Sigma), 0.02% (w/v) pectolyase Y23 (Yakult Honsha), and 0.1% bovine serum albumin. The osmolality of the enzyme solution was adjusted to 650 mOsm with d-sorbitol. After enzyme treatment, the seedlings were pulled out and the digest solution was filtered using 50 μm nylon mesh and centrifuged at 100g for 10 min. The pellet was resuspended in 8 mL of ice-cold bath solutions and centrifuged again. Large protoplasts (about 15 to 20 μm in diameter) with visible cytoplasmic streaming were selected for patch clamping.

Patch-Clamp Electrophysiology

Patch-clamp pipettes were pulled from glass capillaries on a puller (P-87, Sutter Instrument) and fire polished to a tip resistance of 3 to approximately 8 mΩ. Whole-cell currents across barley root epidermal, cortical, and xylem parenchyma protoplasts were measured using the patch-clamp technique with an amplifier (EPC-9, HEKA Elektronik). Whole-cell data were low-pass filtered with a cutoff frequency of 2.9 kHz and analyzed with the software PLUSE and PLUSEFIT (version 8.3).

Low Cl⁻ intracellular and extracellular solutions were used in experiments to exclude possible Cl⁻ currents. The patch-clamp pipette solution consisted of (in mm): for K⁺ currents, 100 K-gluconate, 1 MgCl₂, 1 CaCl₂, 1.4 EGTA, 1.5 Mg-ATP, 2 HEPES (4-[2-hydroxyethyl]-1-piperazineethanesulfonic acid) adjusted to pH 7.2 with Tris and 550 mOsm using d-sorbitol; for Na⁺ currents, 100 Na-gluconate, 10 EGTA, 5 HEPES, 2 MgCl₂ (adjusted to pH 7.2 with NaOH and 580 mOsm using d-sorbitol). Bath solution consisted of (in mm): for K⁺ currents, 30 K-gluconate, 2 KCl, 10 HEPES, 2 MgCl₂, 2 CaCl₂ (adjusted to pH 5.8 with MES and 500 mOsm using d-sorbitol); for Na⁺ currents, 50 Na-gluconate, 2 NaCl, 0.05 CaCl₂, 2 MES (adjusted to pH 5.8 with Tris and 500 mOsm using d-sorbitol). The final whole-cell currents were expressed as currents per unit capacitance (pA/pF) to account for variations in the cell surface area. Polyamines and some specific ion channel blockers were applied to bath solution or pipette solution as mentioned in the figure legends. Data are presented as means ± sd and the statistical significance of differences between currents was determined by the Student's t test.