JXB Advance Access originally published online on December 20, 2004
Journal of Experimental Botany 2005 56(412):613-622; doi:10.1093/jxb/eri053
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RESEARCH PAPER |
Physiological evidence for a sodium-dependent high-affinity phosphate and nitrate transport at the plasma membrane of leaf and root cells of Zostera marina L.
Departamento Biología Vegetal, Facultad de Ciencias, Universidad de Málaga, Campus de Teatinos s/n, S-29071, Málaga, Spain
* To whom correspondence should be addressed. Fax: +34 952 131944. E-mail: Lrubio{at}uma.es
Received 25 May 2004; Accepted 19 October 2004
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Abstract |
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Zostera marina L. is an angiosperm that grows in a medium in which inorganic phosphate (Pi) and nitrate
are present in micromolar concentrations and must be absorbed against a steep electrochemical potential gradient. The operation of a Na+-dependent 
transport was previously demonstrated in leaf cells of this plant, suggesting that other Na+-coupled systems could mediate the uptake of anions. To address this question, Pi transport was studied in leaves and roots of Z. marina, as well as uptake in roots. Electrophysiological studies demonstrated that micromolar concentrations of Pi induced depolarizations of the plasma membrane of root cells. However, this effect was not observed in leaf cells. Pi-induced depolarizations showed Michaelis–Menten kinetics (Km=1.5±0.6 µM Pi; Dmax=7.8±0.8 mV), and were not observed in the absence of Na+. However, depolarizations were restored when Na+ was resupplied. additions also evoked depolarizations of the plasma membrane of root cells only in the presence of Na+. Both - and Pi-induced depolarizations were accompanied by an increase in cytoplasmic Na+ activity, detected by Na+-sensitive microelectrodes. Pi net uptake (measured in depletion experiments) was stimulated by Na+. These results strongly suggest that Pi uptake in roots of Z. marina is mediated by a high-affinity Na+-dependent transport system. Both and Pi transport systems exploit the steep inwardly directed electrochemical potential gradient for Na+, considering the low cytoplasmic Na+ activity (10.7±3.3 mM Na+) and the high external Na+ concentration (500 mM Na+). Key words: Nitrate uptake, phosphate uptake, sodium-dependent transport, sodium-selective microelectrodes, Zostera marina
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Introduction |
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Seagrasses are unique angiosperms that grow in the sea, Zostera marina L. being one of the most common seagrasses along coasts worldwide (Larkum and den Hartog, 1989
). Zostera marina lives in a medium with a high Na+ concentration (around 0.5 M). Leaf cells of this species exhibit a plasma membrane potential (Em) of –160 mV (Fernández et al., 1999), maintained by the activity of a H+-ATPase (Fukuhara et al., 1996; Fernández et al., 1999; Muramatsu et al., 2002). With this highly negative Em, the uptake of essential anions such as inorganic phosphate (Pi), which usually occurs in seawater at concentrations below 10 µM (Riley and Chester, 1971), and whose concentration in the cytoplasm of aquatic plant cells is considered to be between 1 and 10 mM, must be energized (Raven, 1984).
Most studies on vascular plants have reported that Pi uptake is powered by the electrochemical potential for H+ present across the plasma membrane of plant cells (Schachtman et al., 1998; Rausch and Bucher, 2002). Several lines of evidence support the presence of an H+/Pi co-transport in the plasma membrane of plant cells: Pi transport transiently depolarizes the plasma membrane, which indicates the inward movement of positive charges (Ullrich-Eberius et al., 1984); Pi uptake is accompanied by an increase in extracellular pH while the cytoplasm acidifies (Sakano, 1990; Ullrich and Novacky, 1990; Mimura et al., 1992; Sakano et al., 1992) and inhibitors that dissipate the electrochemical gradient for H+, also inhibit Pi uptake (Lin, 1979). More recent reports using heterologous expression have shown that protonophores inhibit Pi uptake in cells expressing a distinct Pi transporter gene (Mitsukawa et al., 1997; Liu et al., 1998).
Both high- and low-affinity Pi uptake systems have been detected in plants by different methods. The progress on molecular techniques has allowed the identification of H+/Pi symporters included in the Pht1 family, using models such as Arabidopsis, tomato, barley, Catharanthus, Medicago truncatula, and Lupinus albus (Grossman and Takahashi, 2001; Rausch and Bucher, 2002; Smith et al., 2003). The Pht1 family comprises both high- and low-affinity Pi transport systems (Km
10 µM Pi and Km400 µM Pi, respectively; Rae et al., 2003). A second family, Pht2, includes H+-coupled Pi transporters with low affinity (Km400 µM) that show similarities with Na+-coupled Pi transporters found in fungal species and animals (Daram et al., 1999).
The existence of Na+-dependent transport systems has been demonstrated in several fungi, cyanobacteria, and algae. Na+-dependent uptake of glucose, amino acids, and nitrate has been shown in marine diatoms (Hellebust, 1978; Rees et al., 1980), and the existence of a Na+-dependent transport has also been described in cyanobacteria (Lara et al., 1993). Na+-coupled Pi uptake has been reported in fungal species (Versaw and Metzenberg, 1995; Martínez and Persson, 1998; Zvyagilskaya et al., 2001). Pi uptake in the green alga Ankistrodesmus was found to be stimulated by Na+ (Ullrich and Glaser, 1982), as well as in some other green algae (Raven, 1984). More recently, Na+ and Pi uptake measurements and voltage-clamp experiments carried out in another green alga, Chara corallina, have reported the activity of a high-affinity Na+/Pi transporter in this plant (Km4 µM; Reid et al., 2000).
However, there are few reports about Na+-coupled transport systems in vascular plants. A Na+-dependent K+ uptake has been described in some freshwater angiosperms such as Egeria, Elodea, and Vallisneria (Walker, 1994; Maathuis et al., 1996). The HKT1 transporter from wheat was initially described as a K+/Na+ symporter using heterologous expression (Rubio et al., 1995), although this activity has not been detected in intact plants (Maathuis et al., 1996; Rubio et al., 1996).
As suggested by Rausch and Bucher (2002), although the dependence on Na+ of a Pi uptake system has so far not been demonstrated in vascular plants, the existence of such a transport system cannot be excluded, for example, in halophytes or in plants living in alkaline media, in which the presence of an inwardly directed electrochemical potential difference for Na+ can potentially be exploited for energization of solute transport. Interestingly, the first physiological evidence of a Na+-coupled transport in an angiosperm has been reported in Z. marina, where a high-affinity transport operates in mesophyll leaf cells (García-Sánchez et al., 2000). This result points to the potential relevance of Na+-coupled transport in this halophytic species and raises the question whether other anions, such as Pi, could be transported in the same way.
Zostera marina, as other submerged aquatic angiosperms, is able to take up Pi from the surrounding water by the leaves, as well as from the interstitial water in the sediment through the roots (Pérez-Lloréns and Niell, 1995). Nevertheless, since nutrient concentrations can vary between the seawater surrounding the leaves and the substrate where the roots are anchored (Touchette and Burkholder, 2000), several differences could be found between the Pi uptake characteristics of leaf and root cells.
The aim of this work was to test the existence of a Na+-dependent Pi transport in both leaves and roots of Z. marina. Pi-transport has been investigated at the plasma membrane of leaf and root cells to determine the possible interactions between Pi and Na+. The results obtained are compared with previous and new contributions on Na+-dependent transport in Z. marina.
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Materials and methods |
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Plant material
Zostera marina L. plants were collected off the coast of Málaga (Spain) at a depth of 5 m. Plants were maintained in the laboratory in natural seawater (NSW) at 15 °C and at a light intensity of 150 µmol m–2 s–1, with a photoperiod of 16/8 h light/dark. The NSW was renewed every 3 d. Under these conditions, leaves were suitable for electrophysiological experiments for at least 2 months, while roots from the collected plants necrotized in 2 weeks and even more quickly if the plants were submitted to nutrient starvation. However, some plants developed new secondary roots in the laboratory, which were used in electrophysiological experiments.
In addition, secondary roots from seedlings were also used in electrophysiological experiments. Reproductive shoots containing seeds of Z. marina were collected at low tide from the Eems estuary (The Netherlands) in August 2002, and also from the Z. marina population located on the coast of Málaga in June 2003. The shoots collected were maintained in NSW until mature seeds were released. The seeds were stored before germination in NSW at 4 °C in darkness. Seeds were germinated at 20 °C in distilled water and, after 1 or 2 d, the seed coats opened and cotyledons appeared. Immediately, germinated seeds were sequentially transferred to NSW adjusted to increasing salinities, i.e. 0.1, 1, and 10
, and finally to NSW at 35. After 1 month of culture in NSW, the first pair of secondary roots was suitable for electrophysiological experiments.
The criteria for experimental viability of the cells from both mature plants and seedlings were the membrane potential (Em) and the response to the addition of 1 mM sodium cyanide (NaCN) and 1 mM salicylhydroxamic acid (SHAM), two inhibitors of respiration, which consequently depolarized the membrane to the diffusion potential value, ED (Fernández et al., 1999).
Plants were starved of phosphorus for at least 8 d prior to experiments on Pi transport in artificial seawater (ASW) containing 0.01 mM NaNO3. Also, plants were starved of nitrogen for at least 3 d in ASW containing 0.01 mM NaH2PO4 before assays on transport were carried out. The composition of ASW was: 500 mM NaCl, 55 mM MgCl2, 12 mM CaCl2, 10 mM KCl, 2 mM NaHCO3, and pH was adjusted to pH 8 with NaOH.
Electrophysiological experiments
Membrane potential (Em) was measured using the standard glass microelectrode technique as described by Felle (1981). Leaf pieces (2 cm length), in which the epidermis had been partially removed, or excised roots (2 cm length) were mounted in plexiglass chambers (volume1.1 ml). Continuous perfusion of the assay medium was maintained at a constant flux rate of 10 ml min–1. Mesophyll leaf cells or epidermal root cells (located at 
0.5 cm from the root tip) were impaled with single-barrelled microelectrodes. Microelectrodes were backfilled with 500 mM KCl and fixed to electrode holders, containing an Ag/AgCl pellet, that were connected to a high-impedance differential amplifier (FD-223, World Precision Instruments, Sarasota, FL, USA).
To analyse the effect of Pi additions on Em of both leaf and root cells, experiments were carried out in P-free ASW buffered to pH 8 with 10 mM MOPS/bis tris propane and containing 0.01 mM NaNO3. Increasing concentrations of NaH2PO4 from 0.01 to 25 µM were sequentially added to the assay medium. To study the effect of Na+, experiments were performed with Na+-free ASW (sorbitol-ASW) containing 800 mM sorbitol, 55 mM MgCl2, 12 mM CaCl2, 10 mM KCl, 2 mM KHCO3, and adjusted to pH 8 with 10 mM MOPS/bis tris propane. Pi was added as KH2PO4 and Na+ was supplemented as NaCl. Both ASW and sorbitol-ASW showed similar osmolality (1.09 osmol kg–1) measured with a cryoscopic osmometer (Osmomat, model 030, Gonotec GmbH, Germany).
To analyse the effect of additions on Em of epidermal root cells, experiments were carried out in N-free ASW buffered to pH 8 with 10 mM MOPS/bis tris propane, but containing 0.01 mM NaH2PO4. Increasing concentrations of NaNO3 from 0.01 to 100 µM were sequentially added to the assay medium. To study the effect of Na+, experiments were carried out in sorbitol-ASW, was added as KNO3, and Na+ was supplemented as NaCl.
In order to measure cytosolic Na+ activity 
single- and doubled-barrelled microelectrodes containing Na+-selective ionophore ETH227 were used. Preparation of Na+-selective microelectrodes was similar to the protocol described for pH microelectrodes by Fernández et al. (1999), with slight modifications (Carden et al., 2001). After 30 min heating at 180 °C, the microelectrodes were silanized by the addition of one or two drops of dimethyldichlorosilane/benzene solution (0.1% v/v) to the blunt end of the microelectrodes. The microelectrodes were then heated again for 60 min at 180 °C. Once cold, the microelectrodes were backfilled with the Na+-sensor up to 4–5 mm from the tip. The Na+ sensor was prepared by mixing 1 vol. of Sodium Ionophore I-cocktail A (Fluka no. 71176) in 6 vols of tetrahydrofuran (THF) containing solid polivinylchloride (2% w/v). All chemicals were from Fluka, Sigma-Aldrich (St Louis, MO, USA).
Once filled, the microelectrodes were stored vertically in a desiccator at room temperature to allow the loss of air bubbles and THF to evaporate. The microelectrode barrel containing the Na+ sensor was backfilled with a 500 mM NaCl solution using a 70-mm-long Microfil needle. The tip was also dipped into 500 mM NaCl for 30 min in order to condition the microelectrode prior to the experiments. The voltage-barrel was filled with 500 mM KCl. The signals from the Na+-selective and voltage barrels were measured and simultaneously subtracted by the high impedance differential amplifier. The difference was calibrated before and after the experiments with different NaCl solutions (from 1 to 500 mM NaCl) containing a fixed background KCl concentration (96 mM KCl), as described by Carden et al. (2001). Calibration slopes were 52 mV/pNa, similar to that reported for the Na+-selective ionophore ETH227 (Carden et al., 2001). Moreover, no interference effects were observed on Na+ measurements when Na+-selective microelectrodes were tested for pH and K+ using a NaCl solution (100 mM NaCl) adjusted to different pH values with 10 mM MOPS/bis tris propane (pH 5, 6, 7, 8, 9), or using a 100 mM NaCl solution containing different KCl concentrations (1, 10, 50, and 100 mM KCl).
Depletion experiments
To determine Pi net uptake rates, whole plants were submitted for 8 d to P-free ASW. Experiments were carried out after 1 h of preincubation in the assay media. The composition of the assay media was the same as for impalements, except for the osmoticum used in Na+-free ASW. Since the presence of sorbitol interfered with Pi analytical determination, Na+-free ASW was made with 0.5 M Cl-choline (choline-ASW). Excised leaf and roots (0.3–0.6 g fresh weight) were placed separately in 250 ml flasks. The assay was carried out with gentle and constant agitation at 25 °C. Three replicates were assayed for each treatment (ASW, choline-ASW, and choline-ASW supplemented with 20 mM NaCl). At the beginning of the experiment, 10 µM KH2PO4 was added to the assay medium, and samples were taken at 0, 0.5, 1, 2, 4, 8, 12, and 24 h. Pi was analysed colorimetrically (Fernández et al., 1985) and Pi net uptake rates were estimated as the slope of the Pi depletion curves.
To determine net uptake rates in Z. marina roots, whole plants were incubated for 3 d in N-free ASW. As above, experiments were carried out after 1 h of preincubation in the assay media, whose compositions were the same as used for impalements (ASW, sorbitol-ASW, and sorbitol-ASW supplemented with 20 mM NaCl). Excised roots (0.3–0.6 g fresh weight) were incubated in 250 ml flasks and three replicates were assayed for each treatment. The assay was carried out with gentle constant agitation at 25 °C. At the onset of the experiment 100 µM KNO3 was added to the assay media and samples were taken at 0, 0.5, 1, 2, 4, 8, 12, and 24 h. was analysed colorimetrically using the Shinn method (Strickland and Parsons, 1972) and uptake rates were estimated as the slope of the depletion curves.
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Results |
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Basic electrical membrane characteristics of Z. marina root cells
Epidermal root cells of both plants and seedlings showed basic electrical membrane characteristics similar to those found in mesophyll leaf cells (Fernández et al., 1999
). The resting plasma membrane potential (Em), in both NSW and ASW was –150±11 mV (n=17), and the membrane rapidly depolarized after the addition of 1 mM CN– and 1 mM SHAM, reaching the diffusion potential, ED=–72±8 mV (n=15).
Effect of Pi additions on the membrane potential in the presence and absence of Na+
The addition of micromolar Pi concentrations (0.1–25 µM NaH2PO4) evoked rapid depolarizations in the plasma membrane of epidermal root cells from P-starved seedlings (Fig. 1). This effect was also observed in epidermal root cells from P-starved mature plants, on the few occasions in which healthy secondary roots were available (data not shown). However, the Em of mesophyll leaf cells from both seedlings and mature plants did not show any shift following Pi additions (data not shown). Membrane depolarizations, which are an estimate of the transport activity, showed saturation kinetics and were fitted to the Michaelis–Menten model (Fig. 1). Curve fitting of the seedling data rendered a Km value of 1.5±0.6 µM Pi and a maximum depolarization (Dmax) of 7.8±0.8 mV.
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The effect of Pi additions on the Em of epidermal root cells was also analysed in Na+-free ASW, in which NaCl was substituted by sorbitol (sorbitol-ASW). Replacement of NaCl for isosmotic sorbitol produced a slight depolarization of the membrane (data not shown). In sorbitol-ASW, Pi-induced depolarizations were not observed. However, once the medium was supplemented with 20 mM NaCl, Pi-induced depolarizations were restored (Fig. 2).
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Effect of NO3– additions on the membrane potential of epidermal root cells
Since the maximum depolarizations induced by saturating Pi concentrations in epidermal root cells were not higher than 10 mV, and no response could be detected in leaf cells, the effect of
additions on the Em of epidermal root cells from mature plants was analysed to check if this nutrient also showed an electrophysiological response in these cells different from that observed in mesophyll cells from these plants (García-Sánchez et al., 2000). The addition of micromolar concentrations of from 0.1 to 100 µM NaNO3 induced plasma membrane depolarizations in root cells of N-starved plants. These membrane depolarizations showed saturation kinetics (Fig. 3) as detected previously in leaf cells (García-Sánchez et al., 2000). Curve fitting of the data to the Michaelis–Menten model rendered a Km value of 8.9±3.9 µM 
higher than that reported for leaf cells (Km=2.3±0.78 µM 
García-Sánchez et al., 2000). Furthermore, -induced Dmax in epidermal root cells was 7.0±0.8 mV, only 45% of the Dmax value observed in leaf cells (15.6±0.9 mV; García-Sánchez et al., 2000). This response was also detected in epidermal root cells from seedlings which showed similar Km and Dmax values (data not shown).
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As in the case of leaf cells,
-induced depolarizations were abolished in Na+-free medium (sorbitol-ASW). Nevertheless, if the Na+-free medium was supplemented with 20 mM NaCl, the NO3-induced depolarizations were restored (Fig. 4).
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Effect of Pi or NO3– additions on the cytoplasmic Na+ activity of epidermal root cells
Figure 5A and B illustrates two examples of simultaneous measurements of Em and cytoplasmic Na+ activity
in epidermal root cells of Z. marina using double-barrelled microelectrodes. The impalements were stable for at least 30 min, the measured membrane potentials being similar to those recorded with single microelectrodes (–153±14 mV, n=5). The mean value of 
calculated from the calibration curves (Fig. 5C), was 10.7±3.3 mM (n=5).
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It should be noted that the addition of saturating concentrations of Pi (Fig. 5A) or
(Fig. 5B) produced the depolarization of the plasma membrane (around 8 mV in both cases) and simultaneously an increase of the 
Calibrations curves showed that 
increased a maximum of 0.6 mM after the addition of 10 µM KH2PO4, and the initial value was restored after Pi was washed from the medium (Fig. 5A). On the other hand, the addition of 50 µM KNO3 evoked an increase of 0.4 mM in the followed by the restoration of the initial value, once was washed from the medium (Fig. 5B).
Pi and NO3– net uptake rates in the presence and absence of Na+
Pi depletion experiments revealed that both leaves and roots from mature plants take up Pi from the medium (Table 1), although Pi net uptake rates were 3-fold higher in roots than in leaves in ASW containing 0.5 M NaCl (ANOVA, 
=0.05). In both cases, Pi net uptake rates were higher in the presence than in the absence of Na+. In Na+-free ASW (choline-ASW), Pi net uptake rates decreased both in leaves and roots by at least 90% compared with the rates obtained in ASW. When choline-ASW was supplemented with 20 mM NaCl, Pi was depleted from the medium at higher rates, almost to 50% of the rate observed in ASW (ANOVA, =0.05).
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net uptake rates in Z. marina depletion experiments were also carried out in Z. marina roots and net uptake rates were estimated in media with different Na+ concentrations (Table 1). net uptake rates were always higher in the presence than in the absence of Na+ (ANOVA, =0.05). The highest net uptake rate was measured in ASW containing 0.5 M NaCl. In Na+-free ASW (sorbitol-ASW), uptake rates decreased by 87%. However, when sorbitol-ASW was supplemented with 20 mM NaCl, was depleted at higher rates, around 30% of the values measured in ASW (ANOVA, =0.05). |
Discussion |
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Electrophysiological experiments performed in Z. marina have shown that the addition of Pi micromolar concentrations induces rapid membrane depolarizations in epidermal root cells from both P-starved seedlings and mature plants (data not shown). It seems then that there is no developmental difference in the response of the cells to Pi. The induced depolarizations indicate that Pi uptake is coupled with the inward movement of positive charge and several lines of evidence suggest that Pi transport is coupled with the entrance of Na+. Thus, Pi-induced depolarizations were only recorded in media containing Na+ and there was an increase of
concurrently to the Pi-induced depolarizations. In addition, depletion experiments have shown that, in Na+-free media, Pi net uptake rates were only 4% of the rates measured in ASW containing normal seawater Na+ concentrations (0.5 M NaCl).
A Na+-coupled transport system has been described previously in this plant, where a Na+-dependent high-affinity transport operates in mesophyll leaf cells (García-Sánchez et al., 2000). This high-affinity system also seems to be present in epidermal root cells of Z. marina. In fact, micromolar concentrations induced membrane depolarizations in epidermal root cells of N-starved plants and these depolarizations disappeared in the absence of Na+ in the medium. In addition, there was an increase of accompanying the -induced depolarizations. On the other hand, net uptake rates were higher in the presence than in the absence of Na+.
Maximum Pi-induced depolarizations were not higher than 10 mV and the calculated Dmax value was 7.8±0.8 mV. These Pi-induced depolarizations are low compared with the values obtained in frond cells of the aquatic higher plant Lemna gibba, where the depolarizations were in the range 18–66 mV depending on the Em value (Ullrich-Eberius et al., 1981), or in root hairs of Limnobium stoloniferum, where depolarizations of about 60 mV were recorded (Ullrich and Novacky, 1990). These values were obtained with 1–1.8 mM Pi concentrations; however, Pi-depolarizations were saturated at 50–100 µM Pi in Lemna gibba (Ullrich-Eberius et al., 1981). These saturating values are higher than those found in epidermal root cells of Z. marina, which are around 10 µM Pi. In this species, the addition of 1 mM Pi did not produce higher membrane depolarizations (data not shown) and the low Pi saturation value could be considered an adaptation to the diluted Pi concentrations in seawater.
Pi-induced depolarizations in Z. marina could only be observed after 8 d of P starvation. This result agrees with the observation of maximum P influx in Chara corallina after 10 d of P starvation (Mimura et al., 1998) and also with the P-induced depolarizations observed in Lemna gibba submitted to P starvation for 10 d (Ullrich-Eberius et al., 1981). In the same way, N-starvation was necessary for to induce membrane depolarizations, but only for 3 d. This contrasts with the results observed in other plants where some transporters increased their activity after exposure to (Kronzucker et al., 1995; Glass et al., 2002).
Although maximum Pi-induced depolarizations values are low, they are quite similar to those produced by in epidermal root cells (Dmax=7.01±0.8 mV). However, this Dmax value was half of the value of the Dmax induced by in leaf cells (Dmax=15.6±0.9 mV, García-Sánchez et al., 2000), although the range of assayed concentrations and the Em of the cells were similar in both tissues. On the contrary, no Pi induced depolarizations were detected in mesophyll leaf cells following Pi additions. In this way, the difference in Dmax values induced by suggests that the number of active transporters could be lower in root than in leaf cells. On the other hand, there are also differences in the affinity of the transporter for that is lower in root (Km=8.9±3.9 µM ) than in leaf cells (Km=2.3±0.78 µM ; García-Sánchez et al., 2000). These results indicate that leaves and roots could have a different role in Pi and transport.
Despite the fact that sediment pore water is generally considered the primary nutrient source for seagrasses (McRoy and McMillan, 1977; Marschner, 1995; Pérez-Lloréns and Niell, 1995), recent evidence suggests that uptake of both and Pi by below-ground tissues can be limited by diffusion, and that roots may lack the capacity to support the total nutrient requirement (Touchette and Burkholder, 2000). Thus, in Z. marina, as in most seagrasses, supplies would be mainly provided by leaf absorption from the water column (Terrados and Williams, 1997; Lee and Dunton, 1999) and Pi supplies would rely upon Pi uptake from the sediment only when Pi is negligible in the water column (Touchette and Burckholder, 2000). Depletion experiments in Z. marina have shown that roots have higher Pi and net uptake rates than leaves; however, as the depletion experiments were carried out in artificial seawater and not in the real substrate where the roots are anchored, the uptake capacity of the roots could have been overestimated. In fact, the very low Km for in leaves points to the importance of uptake from the surrounding seawater. On the contrary, the small Km value for Pi observed in epidermal root cells (1.5±0.6 µM Pi), and the lack of any Pi-induced depolarization in leaf cells, suggests the relevance of Pi uptake through the roots.
While the Km value calculated for Pi transport in epidermal root cells is in the range of high-affinity values described for Pi transporters in higher plants (Km10 µM Pi; Mimura, 2001; Rae et al., 2003), it is lower than the reported value for system I of Lemna gibba (KT=7.3 µM Pi; Ullrich-Eberius et al., 1989) and even lower than the Km (3.1 µM Pi) measured in cultured tobacco cells expressing the gene encoding for the Pth1 transporter of Arabidopsis (Mitsukawa et al., 1997). The Km for Pi in Z. marina is closer to the values observed in some fungal species found in mycorrhizal associations such as Gigaspora margarita (Km=1.8–3.1 µM Pi for the high-affinity transporter; Thomson et al., 1990) or in the unicellular alga Chlamydomonas reinhardtii (0.1–0.5 µM Pi; Shimogawara et al., 1999). Zostera marina is an angiosperm that evolved from a terrestrial to a marine habitat. High-affinity transport systems of terrestrial plants, such as barley or maize, exhibit Km values ranging from 10 to 100 µM (Guo et al., 2002), which are slightly higher than the Km showed by epidermal root cells of Z. marina and much higher than the value observed in leaf cells (García-Sánchez et al., 2000). A compilation of data on the nutrient environment of seagrass meadows worldwide (Hemminga, 1998) shows that the average Pi concentration is only 1 µM in the water column and 12 µM in pore water, while in the case of average concentrations are not much different between water column and pore water (2.7 µM and 3.4 µM respectively). Thus, the high affinity of both Pi and transporters in Z. marina could be related to the development of mechanisms to improve survival in a P- and N-diluted medium like seawater.
On the other hand, the Na+-dependence of both Pi and transporters could have been evolved as an adaptation to a medium with a high salinity and alkaline pH, considering the high electrochemical potential for Na+ that can be developed in cells of Z. marina. The free energy relationship (
G'/F) for a plasma membrane cation-coupled transport system operating with a stoichiometry of n cations per transported anion is given (in millivolts) as:
 | (1) |
), and subscripts ‘o’ and ‘c’ refer to external medium and cytoplasm, respectively.
Pi concentration in the cytoplasm of aquatic plants is in the range of 1–10 mM (Raven, 1984), similar to the values reported for terrestrial plants (5–10 mM Pi; Mimura, 2001). Nevertheless, Pi exists in different forms (H3PO4, 
and 
) and the concentration of each species varies as a function of pH. The cytosolic pH value measured both in leaf (Fernández et al., 1999) and in root cells (data not shown) of Z. marina is around 7.3. Since the pK value for the dissociation of 
into 
is 7.2, the most abundant species of Pi in the cytoplasm are (44% Pi) and (56% Pi). On the other hand, under the alkaline conditions of seawater (pH 8) the most abundant form is (86% Pi).
As discussed in García-Sánchez et al. (2000), cytosolic concentrations of in Z. marina could be considered to be around 3 mM 
The mean value of reported in this work is 10.7 mM, which is in agreement with the reported range for some marine plants (1–50 mM Na+; Raven, 1984) and is close to the values measured in a salt-tolerant variety of barley (2–28 mM Na+) using Na+-selective microelectrodes (Carden et al., 2003).
The Na+ concentration of natural seawater and seawater in the sediments can be taken as 500 mM Na+ (Siever et al., 1965; Riley and Chester, 1971) and the pH is typically 8. However, it should be considered that the external Na+ concentration and pH could be different surrounding the cells. On the other hand, external Pi and concentrations could be considered to be around 10 µM (Riley and Chester, 1971). Figure 6 shows the free energy relationship (G') for Pi and transport across the plasma membrane of epidermal root cells calculated from equation 1. The cation:Pi or cation: stoichiometry values (n) which render a negative free energy (G'<0) are the only stoichiometries that make the transport thermodynamically feasible. In the case of Pi the free energy relationship was calculated for both species, and 
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It should be noted that Na+-coupled transport is strongly favoured, in both
or and transport systems. A stoichiometry of 
renders sufficient free energy for transport (G'=–13 kJ mol–1) in Z. marina, while the same stoichiometry for 
is not sufficient to drive net uptake of Pi (G'>0, Fig. 6A). Furthermore, if Pi is transported as 
a stoichiometry of 
is sufficient to allow the transport, while a stoichiometry of 
yields only a modest inward driving force of –8 kJ mol–1 (Fig. 6B). This stoichiometry could be different in leaf cells, where Pi net uptake has been detected by depletion experiments, as Pi transport seems to be electrically silent in mesophyll cells. On the other hand, as estimated for the Na+-coupled transport system in leaf cells (García-Sánchez et al., 2000), a stoichiometry of 
renders sufficient energy for transport (G'=–19 kJ mol–1), while a stoichiometry higher than 
is required to generate a similar inward driving force (Fig. 6C).
In vascular plants there have not been reports of functional analysis of Na+-coupled Pi transporters, although several genes of putative Na+-coupled Pi transporters have been identified (Smith et al., 2003). The first quantitative physiological demonstration of such a transport system in plant cells is that described in Chara corallina. This freshwater charophyte plant showed a Km value for Pi around 10 µM and a stoichiometry as high as 6Na+ for each Pi, estimated by the influx of positive charge in voltage-clamp experiments (Reid et al., 2000). The estimated stoichiometry for the Na+-coupled Pi transport in Z marina is much lower, 2Na+ or 3Na+. Na+/Pi transport in C. corallina seems to be induced by low Pi concentrations (0.5–1 µM Pi) and is inactivated by a 6 d treatment with 1 µM Pi (Mimura et al., 2002). However, in Z. marina, Pi-induced depolarizations could be observed only in plants submitted to P starvation for at least 8 d and no depolarizations were ever detected in plants maintained in natural seawater containing 0.5–1 µM Pi.
In conclusion, several lines of evidence indicate that a Na+-coupled high-affinity Pi transport system operates in epidermal root cells of Z. marina. This is the first report of a Na+-dependent Pi transport in an angiosperm and, together with the presence a Na+-coupled transport in this plant, it highlights the importance of Na+-dependent transport in halophytic species. The increment in the accompanying both and Pi transport, as well as the low measured in Z. marina epidermal root cells, suggest that very efficient Na+ homeostatic mechanisms would have been developed in this plant, and studies on this topic are now in progress.
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Acknowledgements |
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We thank Agustín Barrajón for sampling assistance in Málaga and Marieke M. van Katwijk (Department of Environmental Studies, University of Nijmegen, The Netherlands) for technical advice on seed germination. This work was supported by the Spanish Ministry of Science and Technology, grant no. BOS 2001-1855.
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