INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Zostera marina L. is an aquatic angiosperm that grows in a medium with a high salinityseawater itselfwith NaCl concentrations in the region of 0.5 M. Leaf cells of this plant exhibit a plasma membrane potential (E_m) of around 160 mV in natural seawater (Fernández et al., 1999). Molecular and physiological evidence indicates that the E_m in this halophyte is maintained by the activity of a H⁺-pump (Fukuhara et al., 1996; Fernández et al., 1999). With this highly negative E_m, the uptake of essential anions such as NO₃, which usually occurs in seawater at concentrations of 1 to 500 µM (Riley and Chester, 1971) and is probably below 10 µM in the close environment of the plant (Hernández et al., 1993), must be energized.

Most studies on vascular plants and algae have reported that NO₃ transport is powered by the electrochemical potential for protons present across the plasma membrane of plant cells (Ullrich, 1992). Evidence includes simultaneous measurements of NO₃ and H⁺ fluxes (Mistrik and Ullrich, 1996), NO₃-evoked membrane depolarizations (McClure et al., 1990; Ullrich and Novacky, 1990; Glass et al., 1992), and pH dependence of NO₃-elicited inward currents both in intact plants (Meharg and Blatt, 1995) and in oocytes expressing plant NO₃ transporters (Tsay et al., 1993; Zhou et al., 1998; Liu et al., 1999). Four different NO₃ uptake systems have been identified in higher plants: both low- and high-affinity systems are present and can be constitutive or inducible (Wang and Crawford, 1996; Liu et al., 1999). High-affinity systems show saturation kinetics and operate at concentrations lower than 0.5 mM with K_m values in the range of 7 to 100 µM; low-affinity systems show linear kinetics and function at concentrations above 0.5 mM (Wang and Crawford, 1996; Crawford and Glass, 1998).

Energization of solute transport by H⁺ coupling is prevalent among bacteria, fungi, and plants. Nevertheless, for the plasma membrane of most cells the presence of an inwardly directed electrochemical potential difference for Na⁺ can potentially be exploited for energization of solute transport through Na⁺ coupling. Accordingly, a number of Na⁺-dependent transport systems have been identified. Na⁺-dependent uptake of Glc and amino acids has been shown in the marine diatom Cyclotella (Hellebust, 1978), and phosphate transport has been reported to be stimulated by Na⁺ in several green algae (Raven, 1984). In the case of NO₃, Na⁺-dependent uptake has been described only in the marine diatom Phaeodactylum tricornutum (Rees et al., 1980) and in cyanobacteria (Lara et al., 1993).

During the past decade an emerging number of Na⁺-coupled transport systems have also been discovered at the plasma membranes of multicellular plants. In freshwater charophyte algae, the influx of K⁺, urea, and Lys has been shown to be Na⁺ dependent and/or directly coupled to the transport of Na⁺ (Smith and Walker, 1989; Walker and Sanders, 1991), and Na⁺-dependent K⁺ uptake has also been demonstrated in some aquatic angiosperms such as Egeria, Elodea, and Vallisneria (Walker, 1994; Maathuis et al., 1996). A Na⁺,K⁺ symporter, HKT1, has been cloned from wheat (Rubio et al., 1995), but it remains unclear whether Na⁺ coupling comprises the dominant mode of energizing K⁺ uptake in terrestrial angiosperms (Maathuis et al., 1996).

The elevated Na⁺ concentration present in seawater and the highly negative E_m in Z. marina makes feasible the existence of this type of alternative transport system powered by the putative electrochemical potential for Na⁺. To test this hypothesis, we have investigated NO₃ transport at the plasma membrane of leaf cells of Z. marina to determine the possible interactions between Na⁺ and NO₃. These interactions have been studied with respect to the electrophysiological properties of the plasma membrane and to NO₃ depletion in media surrounding whole leaves.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Plant Material

Zostera marina L. plants were collected off the coast of Málaga (Spain) at a 5-m depth. Plants were maintained in the laboratory in natural seawater at 15°C and a light intensity of 150 µmol m² s¹, with a photoperiod of 16 h of light and 8 h of darkness.

Plants were N-starved prior to experiments for at least 3 d in N-free artificial seawater (ASW) adjusted to pH 8.0 with NaOH. The composition of ASW was 0.010 mM KH₂PO₄, 1.2 mM NaHCO₃, 5 mM K₂SO₄, 12 mM CaCl₂, 15 mM MgSO₄, 42.5 mM MgCl₂, and 500 mM NaCl.

Membrane Potential Measurements

Leaf pieces were excised to remove partially the epidermis, and were mounted in a plexiglass chamber (volume 1.1 mL). Continuous perfusion of the assay medium was maintained at a flux of approximately 10 mL/min. Both epidermal and mesophyll cells were impaled with single-barrel electrodes. Membrane potentials were measured using the standard glass microelectrode technique as described by Felle (1981). Micropipettes were backfilled with 0.5 M KCl. Microelectrodes were fixed to electrode holders containing an Ag/AgCl pellet, and connected to a high-impedance differential amplifier (FD-223, World Precision Instruments, Sarasota, FL). Tip potentials never exceeded 10 mV.

Experiments were carried out in N-free ASW (as above) buffered with 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-Tris(hydroxymethyl)-amino-methane (Tris) to pH 8.0. NO₃ was added as KNO₃. To check the effect of pH, the same buffer was adjusted to pH 6.0 or 7.0. To test the effect of Na⁺, N-free ASW with a slightly different composition was used (NaCl-ASW: 2.5 mM KHCO₃, 2.5 mM K₂SO₄, 5 mM CaSO₄, 5 mM MgSO₄, 500 mM NaCl, and 10 mM HEPES-Tris). In Na⁺-free ASW, NaCl was substituted by 0.8 M sorbitol (sorbitol-ASW). Na⁺ was added to the medium at increasing concentrations as Na₂SO₄ or NaCl.

NO₃ Depletion Experiments

Whole plants were maintained for 3 d in N-free ASW and were preincubated in the assay medium for 1 h before starting the experiment. The composition of assay medium was the same as for impalements. Whole leaves (0.6-0.9 g fresh weight) were incubated in 100-mL flasks. Three replicates were assayed for each treatment (NaCl-ASW, sorbitol-ASW, sorbitol-ASW + 5 mM Na₂SO₄, and sorbitol-ASW + 10 mM NaCl). The assay was carried out with slow and constant agitation at 25°C and at a light intensity of 50 µmol m² s¹. The first treatment (NaCl-ASW) was also assayed in darkness. KNO₃ was added at an initial concentration of 100 µM. Samples were taken at 0, 1, 2, 4, 8, 12, and 24 h. NO₃ was analyzed colorimetrically as described in García-Sánchez et al. (1993).

Chemicals

pH buffers and sorbitol were from Sigma-Aldrich (St. Louis). Monensin (Sigma) was dissolved in ethanol at a concentration of 50 mM.

Data Analysis

Data are given as means ± SE. Membrane depolarization data were fitted with the Michaelis-Menten equation using a non-linear regression computer program (KaleidaGraph, Synergy Software, Reading, PA).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Effect of NO₃ Additions on the Membrane Potential

Figure 1A demonstrates that NO₃ evoked a rapid membrane depolarization in N-starved plants in the light. Measurable changes in E_m were evident even at 1 µM NO₃ (not shown). When NO₃ was washed from the medium, the membrane first hyperpolarized so that E_m was transiently more negative than the original resting potential, and then depolarized with another overshoot to a value close to that before NO₃ addition (Fig. 1A). Depolarizations showed saturation at concentrations around 50 µM NO₃ (Fig. 2). Because depolarization is an integral function of the net charge carried by the NO₃ transport system, the concentration dependence of the depolarization can be taken as a rough guide to the affinity of this system for its substrate. Fitting the data with the Michaelis-Menten equation yielded a K_m value of 2.31 ± 0.78 µM NO₃ and a maximum depolarization of 15.6 ± 0.9 mV (Fig. 2).

View larger version (15K): [in this window] [in a new window]   Figure 1.   Effect of the addition of NO3 on Em. A, Tissue in the light, incubated in ASW, showing the response of a single mesophyll leaf cell to the addition of 5 and 100 µM KNO3. B, Tissue in the dark with KNO3 added at the concentrations indicated. Small downward arrowheads indicate the onset of the NO3 wash.

View larger version (17K): [in this window] [in a new window]   Figure 2.   Membrane depolarizations induced by increasing the NO3 concentration. Mesophyll leaf cells were impaled in ASW in the light () or in the dark (). Data are the means ± SE of three independent replicates. Values in the light were fitted with the Michaelis-Menten relationship, as shown by the curve.

NO₃-induced depolarizations were lower in the dark than in the light, with maximum values around 10 mV, and they did not exhibit saturation kinetics (Figs. 1B and 2). The response of E_m to NO₃ additions was also different (Fig. 1B): the depolarization was very rapid and when NO₃ was washed from the medium the E_m did not exhibit the overshoot in the depolarizing direction before returning to the resting value.

The depolarizations elicited by NO₃ suggest that uptake of this anion involves flow of positive charge into the cell.

Effect of pH on NO₃-Induced Depolarizations

Many H⁺-coupled transport systems in plants are activated by a rise in external H⁺ concentration (Bush, 1993), and this enhancement of electrophoretic transport can be reflected in a larger transport-related depolarization as the pH is lowered. We therefore examined the pH dependence of the NO₃-induced depolarization. In contrast to many H⁺-coupled transport systems, the depolarizations induced by saturating NO₃ concentrations (100 µM) were lower at acidic than at alkaline pH (Fig. 3). A shift of external pH from 8.0 to 6.0 decreased the magnitude of the depolarization by a factor of about 3. 

View larger version (10K): [in this window] [in a new window]   Figure 3.   Effect of pH on NO3-induced depolarizations. Mesophyll leaf cells were impaled in ASW buffered at different pHs and challenged with saturating NO3 concentrations (100 µM KNO3). Values are the means ± SE of five independent replicates.

Effect of Na⁺ on NO₃-Evoked Depolarizations

To determine whether Na⁺ was involved as the coupling ion, we examined the capacity of NO₃ to depolarize the membrane in the absence of Na⁺. Replacement of NaCl in the medium with isosmotic sorbitol produced a slight hyperpolarization of the membrane (data not shown). In Na⁺-free conditions, NO₃-induced membrane depolarizations were abolished (Fig. 4). The addition of Na⁺ (as Na₂SO₄) at concentrations as low as 250 µM to NaCl-free ASW (sorbitol-ASW) restored the NO₃-induced depolarizations observed in NaCl-ASW (Figs. 4 and 5). In contrast, NO₃ additions in the presence of KCl did not produce any effect (Fig. 4) and depolarizations were of the same order when Na⁺ was added as NaCl or Na₂SO₄ (data not shown).

View larger version (19K): [in this window] [in a new window]   Figure 4.   NO3-induced depolarizations in the presence and absence of Na+. Lines show the response of Em of a single mesophyll leaf cell to 100 µM KNO3 addition (). The tissue was incubated sequentially in different media: ASW containing NaCl (NaCl-ASW), NaCl-free ASW (sorbitol-ASW), and sorbitol-ASW to which KCl or Na2SO4 were added as indicated. Downward arrows indicate onset of NO3 wash.

View larger version (16K): [in this window] [in a new window]   Figure 5.   Effect of increasing Na+ concentrations on NO3-induced depolarization. Leaf cells were incubated in NaCl-free ASW (sorbitol-ASW) and challenged with saturating NO3 concentrations (100 µM KNO3). Na+ was added as Na2SO4. The data are the means ± SE of three independent replicates. The solid line represents a non-linear least squares fit of the data to the Michaelis-Menten equation.

The kinetics with respect to Na⁺ ions was further investigated, with depolarization in response to saturating NO₃ concentrations (100 µM) monitored over a range of Na⁺ concentrations (Fig. 5). The results demonstrate that the response is effectively saturated at around 5 mM Na⁺. Fitting of the data with the Michaelis-Menten equation yielded a K_m of 0.72 ± 0.18 mM Na⁺ and a maximum depolarization of 13.1 ± 0.7 mV.

Effect of Monensin on NO₃-Induced Depolarizations

The ionophore monensin, which dissipates the electrochemical potential for Na⁺ (Pressman, 1976), was added to the assay medium at a concentration of 50 µM to determine its effects on the NO₃-induced depolarization. The presence of monensin in absence of NO₃ failed to evoke significant changes in E_m (data not shown). However, monensin partially inhibited the depolarization induced by NO₃ (Fig. 6), showing a mean inhibition value in tests with 100 µM NO₃ of 85.0% ± 20.7% (n = 8).

View larger version (9K): [in this window] [in a new window]   Figure 6.   Response of Em of a single mesophyll leaf cell to the addition of 100 µM KNO3 (indicated by the long upward arrows) incubated in ASW before (control) and after the addition of 50 µM monensin. , Onset of NO3 wash.

NO₃ Uptake Rates in the Absence and Presence of Na⁺

If NO₃ transport in Z. marina is Na⁺ coupled, then net uptake from the medium should be enhanced in the presence of Na⁺. Table I shows that NO₃ was depleted from the medium by leaves at higher rates when Na⁺ was present. NO₃ uptake rates were measured in NaCl-free ASW (sorbitol-ASW) and in the same medium but with the addition of 10 mM Na⁺ as Na₂SO₄ or NaCl. The differences between the mean NO₃ uptake rates in the presence and absence of Na⁺ were significant (t test, P < 0.05), and the addition of Na⁺ more than doubled the net NO₃ uptake rate. Furthermore, there were no significant differences between the means obtained in the presence of Na₂SO₄ or NaCl.

View this table: [in this window] [in a new window]   Table I.   Effect of Na+ on NO3 uptake rates Whole leaves were incubated in different assay media: ASW containing 0.5 M NaCl (NaCl-ASW) or ASW in which NaCl was substituted with 0.8 M sorbitol (sorbitol-ASW). Additions of Na+ were made to sorbitol-ASW as shown. NO3 uptake rates were measured in the light except where indicated. The values shown are the means ± SE of three replicates of a representative experiment.

The uptake of NO₃ was also measured in ASW containing 0.5 M NaCl in the light and in the dark. The difference between the mean values was significant (t test, P < 0.1), being 2-fold higher in the light than in the dark (Table I).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The depolarizations induced by NO₃ in Z. marina indicate that the uptake of this anion is coupled with the inward movement of positive charge. However, since the magnitude of the depolarization is decreased at acid pH, it appeared possible that in this plant NO₃ uptake is not coupled with the entrance of H⁺ as is the rule in other angiosperms (Ullrich, 1992). Nevertheless, the decrease in depolarization observed at low pH might be explained by a direct pH effect on transporter kinetics or by an increase in membrane conductance.

In contrast, the lack of any NO₃-induced depolarization in Na⁺-free ASW and the restoring of the depolarization when Na⁺ is added to the medium suggest that NO₃ uptake into leaf cells is coupled with the entrance of Na⁺. The stimulation of NO₃ uptake rates by the leaves in the presence of Na⁺ points to the same conclusion, as does the inhibition of NO₃-evoked depolarizations in the presence of monensin, an ionophore that dissipates the Na⁺ electrochemical gradient. However, because monensin, like other cation-H⁺ exchangers, acts as an uncoupler of photosynthesis and respiration (Rottenberg, 1977), the ionophore might also affect NO₃ transport indirectly via an effect on nitrogen metabolism.

Na⁺-coupled NO₃ transport has been demonstrated previously only in the marine diatom Phaeodactylum tricornutum (Rees et al., 1980) and in cyanobacteria, where it has been well defined (Lara et al., 1993). Calculated K_m values for NO₃ (2.31 ± 0.78 µM) and Na⁺ (0.72 ± 0.18 mM) in Z. marina are quite similar to the values reported in the cyanobacterium Anacystis nidulans, which exhibits a K_m for NO₃ of 1.6 ± 0.2 µM and for Na⁺ of 0.36 ± 0.04 mM (Rodríguez et al., 1994). The K_m for Na⁺ in Z. marina is also close to the value reported for the marine diatom (2.58 ± 0.56 mM, Rees et al., 1980). These low-millimolar K_m values for Na⁺ imply that the NO₃ transporter of Z. marina, as well as those of the other organisms, will be functioning at saturating Na⁺ concentrations in a marine environment. It should be noted, though, that the K_m for Na⁺ reported in the present study was derived at a saturating concentration of NO₃, and that a rise in K_m as the concentration of the substrate ion is lowered cannot be precluded (Sanders et al., 1984).

Other features of NO₃ transport are similar in Z. marina and cyanobacteria. NO₃ uptake in cyanobacteria is inhibited by 60% in the presence of 25 µM monensin (Rodríguez et al., 1992); in Z. marina, 50 µM monensin produces an inhibition of the NO₃-induced depolarizations of about 85%. Moreover, a cyanobacterium mutant lacking NO₃ reductase activity shows an inhibition of NO₃ uptake at concentrations around 25 µM (Rodríguez et al., 1994). In Z. marina NO₃-evoked depolarizations, as well as NO₃ uptake, are smaller in the dark and depolarizations reach saturation at lower concentration values than in the light. The lower NO₃ uptake rate in the cyanobacterium mutant is explained by apparent substrate inhibition of the transporter (Rodríguez et al., 1994), as NO₃ cannot be reduced. This effect might also be invoked here to explain the decrease in NO₃ transport in the dark observed in Z. marina, as it is known that NO₃ reductase activity declines in the absence of light mainly due to a lack of reducing power (Beevers and Hageman, 1980).

Maximum NO₃-induced depolarizations measured in Z. marina are of the same order as the values reported in Arabidopsis root hairs (Meharg and Blatt, 1995; Wang and Crawford, 1996). The response of E_m when NO₃ is washed is more complex in Z. marina than in Arabidopsis root hairs, and it is different in the light and in the dark. This suggests a role of N metabolism in determining the effect of NO₃ additions on E_m (Mistrik and Ullrich, 1996) that could be more important in the light and in photosynthetically active tissue.

A kinetic study of Na⁺-dependent NO₃ transport in A. nidulans indicates that Na⁺ and NO₃ ions are the real substrates of the transporter and a minimum stoichiometry of one Na⁺ per NO₃ and a maximum of two has been proposed (Rodríguez et al., 1994). In Z. marina, the stoichiometry must be >1, since we have recorded membrane depolarization in response to NO₃.

Z. marina, as an angiosperm, evolved from terrestrial species that presumably, like extant species, transported NO₃ from the soil using H⁺-coupled transport systems. Therefore the question arises as to why Na⁺-coupled NO₃ transport evolved in Z. marina. This question might best be addressed by considering the thermodynamic aspects of cation-coupled NO₃ transport in a marine environment, and that requires knowledge of the respective NO₃, Na⁺, and H⁺ electrochemical potentials across the plasma membrane. Cytosolic concentrations of NO₃ ([NO₃]_c) and Na⁺ ([Na⁺]_c) have been estimated in some marine plants, showing a range for Na⁺ of 1 to 50 mM and for NO₃ of 0 to 1 mM (Raven, 1984). More accurate cytosolic measurements in non-marine plants give values of 3 to 5 mM for NO₃ in barley root epidermal cells (Walker et al., 1995; van der Leij et al., 1998) and 50 mM for Na⁺ in a salt-tolerant Chara species (Kiegle and Bisson, 1996). (Although total internal Na⁺ concentrations have been estimated in two species of seagrass, being around 100 mM in epidermal leaf cells and showing higher values in other tissues [Beer et al., 1980], such estimates will reflect primarily the composition of the large central vacuole rather than that of the cytosol.) We might therefore take as reasonable estimates of [NO₃]_c and [Na⁺]_c in Z. marina respective values of 3 and 50 mM. The cytosolic pH has been measured as 7.3 in leaf cells of this species (Fernández et al., 1999) and the membrane potential is typically 160 mV. Normal seawater concentrations of NO₃ and Na⁺ can be taken as 10 µM and 550 mM, respectively, and the pH is typically 8.0. The generalized free energy relationship for a plasma membrane cation-NO₃ symporter operating with a stoichiometry of n cations per NO₃ is given (in millivolts) as

(1)

where C⁺ is the coupling cation (either Na⁺ or H⁺) and subscripts o and c denote the external solution and cytoplasm, respectively. Figure 7 shows the resulting free energy relationships for hypothetical cation-NO₃ symporters operating in conditions appropriate to Z. marina. Clearly, in the case of H⁺ coupling, a stoichiometry of two is not sufficient to drive net uptake (free energy > 0), and even a stoichiometry of three yields only a modest inward driving force of 5 kJ mol¹. It is likely that a still higher value of n would be required to guarantee NO₃ influx in varying external conditions. By contrast, Na⁺-coupled transport would be strongly inwardly directed (13 kJ mol¹), with a stoichiometry of just 2Na⁺:NO₃ (Fig. 7).

View larger version (25K): [in this window] [in a new window]   Figure 7.   Thermodynamic relationship of hypothetical NO3 transporters employing either H+ or Na+ as the coupling ion. Calculations were performed with Equation 1, with parameter values assigned as discussed in the text.

NO₃ transport kinetics in Z. marina indicates the existence of a high-affinity transport system, with a much lower K_m (2.31 ± 0.78 µM) than has to date been reported for other angiosperms (where values reside in the range 6-20 µM, Crawford and Glass, 1998), although the value in Z. marina is in the same range (2-13 µM) as that described for marine algae (DeBoer, 1985), the fungus Aspergillus nidulans (1 µM, A.J. Miller, personal communication), and cyanobacteria (Rodríguez et al., 1994). The low K_m observed in Z. marina is very likely to be an adaptation to an environment in which NO₃ concentrations are typically <10 µM.

In higher plants, two high-affinity systems for NO₃ have been defined: one inducible and another one constitutive (Wang and Crawford, 1996; Crawford and Glass, 1998). We have found that the E_m response to NO₃ addition in N-starved plants does not show any lag, which might indicate the existence of a constitutive high-affinity system. However, we have also detected an increase in the magnitude of depolarizations after repeated additions of NO₃ (data not shown), which could be the manifestation of an inducible transport system.

In conclusion, several lines of evidence indicate that a Na⁺-dependent high-affinity NO₃ transport system operates in Z. marina. This is the first report of Na⁺-dependent NO₃ transport in an angiosperm and points to the potential relevance of Na⁺-coupled transport in halophytic species. The Na⁺ dependence of NO₃ transport in Z. marina also suggests that other anions could enter the cell using a similar mechanism, and studies of other anion transporters are now in progress.