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First published online March 25, 2005; 10.1104/pp.104.051953 Plant Physiology 137:1435-1444 (2005) © 2005 American Society of Plant Biologists
GmN70 and LjN70. Anion Transporters of the Symbiosome Membrane of Nodules with a Transport Preference for Nitrate1Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville, Tennessee 37996 (E.D.V., D.M.R.); and Agriculture and Agri-Food Canada, Southern Crop Protection and Food Research Centre, London, Ontario N5V 4T3, Canada (K.S.)
A cDNA was isolated from soybean (Glycine max) nodules that encodes a putative transporter (GmN70) of the major facilitator superfamily. GmN70 is expressed predominantly in mature nitrogen-fixing root nodules. By western-blot and immunocytochemical analyses, GmN70 was localized to the symbiosome membrane of infected root nodule cells, suggesting a transport role in symbiosis. To investigate its transport function, cRNA encoding GmN70 was expressed in Xenopus laevis oocytes, and two-electrode voltage clamp analysis was performed. Ooctyes expressing GmN70 showed outward currents that are carried by anions with a selectivity of nitrate > nitrite >> chloride. These currents showed little sensitivity to pH or the nature of the counter cation in the oocyte bath solution. One-half maximal currents were induced by nitrate concentrations between 1 to 3 mM. No apparent transport of organic anions was observed. Voltage clamp records of an ortholog of GmN70 from Lotus japonicus (LjN70; K. Szczyglowski, P. Kapranov, D. Hamburger, F.J. de Bruijn [1998] Plant Mol Biol 37: 651–661) also showed anion currents with a similar selectivity profile. Overall, these findings suggest that GmN70 and LjN70 are inorganic anion transporters of the symbiosome membrane with enhanced preference for nitrate. These transport activities may aid in regulation of ion and membrane potential homeostasis, possibly in response to external nitrate concentrations that are known to regulate the symbiosis.
During the formation of legume-rhizobia symbioses, the development of a specialized organ, the root nodule, is induced via the exchange of signals between the bacteria and plant host (Stougaard, 2000  ; Limpens and Bisseling, 2003). Ultimately, the bacteria infect a specialized plant cell (infected cell) within the core of the nodule and become enclosed in an organelle called the symbiosome, the major organelle of this cell type (Roth et al., 1988). This organelle is delimited by the plant-derived symbiosome membrane, which mediates the symbiotic exchange of nutrients (reduced carbon compounds from the plant cytosol in exchange for reduced nitrogen from the bacteroid), and serves to protect the endosymbiont from plant defense responses (for review, see Udvardi and Day, 1997; Day et al., 2001).
The biogenesis of the symbiosome membrane results in the acquisition of a number of transport activities that aid in the establishment and maintenance of the symbiosis. These include a dicarboxylate transport activity that facilitates the uptake of malate or succinate, which serve as carbon sources to support bacteroid nitrogen fixation (Ou Yang et al., 1990
Nodule development is associated with the spatially and temporally regulated expression of a number of nodule-enhanced transcripts (referred to as nodulins) that aid in the establishment of the symbiosis (for review, see Stougaard, 2000
Isolation of a Full-Length cDNA Encoding GmNod70LP
A set of primers for reverse transcription (RT)-PCR were designed based on conserved sequences from LjN70 (Szczyglowski et al., 1998
Northern-blot analysis of total RNA from various tissues of 28-d-old soybean plants shows high expression of this transcript in nodule tissue, with lower amounts of signal detected in leaf and root tissues (Fig. 2A). Because of its similarity to LjN70 and high expression in nodules, the protein encoded by this cDNA is referred to as Glycine max N70-like protein (GmN70).
Preparation of a Site-Specific Antibody against GmNod70-LP and Immunolocalization A unique hydrophilic region within the putative central loop of GmN70 was chosen to prepare a synthetic peptide antigen (CD-18) for antibody production (Fig. 1). Western-blot analysis of nodule membrane extracts shows that the anti-CD-18 antibody cross reacts with a protein with an apparent molecular mass of 68 kD (Fig. 2B), similar to the proposed molecular mass of GmN70 based on its deduced amino acid sequence.
To determine the localization and expression pattern of the GmN70 gene product in soybean nodule tissue, immunocytochemistry was performed with affinity-purified anti-CD-18 antibodies (Fig. 3). Epifluorescent images of nodule tissue showed high levels of GmN70 immunoreactivity in the central infection zone and not in the inner and outer cortical regions of the nodule (Fig. 3C), similar to soybean nodulin 26 (Fig. 3E), a well-characterized nodule-specific membrane protein that is specifically expressed at a high concentration on the symbiosome membrane in infected cells (Fortin et al., 1987
Since immunocytochemical evidence suggests a specific expression pattern of GmN70 in infected cells, the possibility that this protein is localized to the symbiosome membrane was pursued. Immunoblot of isolated, purified symbiosome membranes showed the presence of an immunoreactive band at 68 kD, suggesting that GmN70 is found on the symbiosome membrane (Fig. 2).
To investigate the transport properties of GmN70, cRNA was prepared by in vitro transcription and was microinjected into Xenopus ooctyes. After 3 d of culture, western-blot analysis indicated the expression of GmN70 in oocytes (data not shown). Transfer of these oocytes from Frog Ringer's solution into an identical bath solution in which NaCl was replaced by sodium-gluconate resulted in a depolarization of the resting membrane potential (from –16 mV to –3.7 mV; Table I). Subsequent perfusion with an identical buffer in which gluconate was replaced with nitrate resulted in a hyperpolarization of the oocyte membrane (Vm = –27 mV). Analysis of the membrane currents of the GmN70 oocytes by two-electrode voltage clamp recording showed that nitrate also induced elevated outward steady-state currents (Fig. 4A) and shifted the reversal potential of the oocyte to a more negative value (Table I). In contrast, uninjected control oocytes showed little change in resting Vm (Table I) or in membrane currents (Fig. 4C) in response to these various anions in the recording bath. The data suggest that GmN70 mediates the uptake of nitrate by the oocyte.
Analysis of the currents induced by various concentrations of nitrate in the recording bath showed that increasing the concentration of NO3– resulted in an increase in outward current and a corresponding shift in oocyte reversal potential to more negative values consistent with the increase in the equilibrium potential of nitrate (Fig. 5). Plots of the intensity of this outward current versus nitrate concentration show an Imax with a linear dependence on Vm and a K0.5 value ([NO3–] that induces one-half maximal current) ranging from 1 mM (+75 mV) to 3 mM (+15 mV; Fig. 5C).
Since many members of the MFS exhibit cotransport of protons or other cations as part of their mechanism of transport (Pao et al., 1998 ), the dependence of nitrate transport on pH and the nature of the counter cations was investigated (Fig. 6). The magnitude of the nitrate currents generated at pH 5 was indistinguishable from those at pH 7.6 and showed no difference in response to Na+, K+, or the impermeable N-methyl-D-glucamine, suggesting that the nature of the counter cation has little effect on GmN70-mediated transport.
To investigate the selectivity of transport, currents induced by a series of anions in the bath solution were analyzed (Fig. 7). Oocytes bathed in nitrate and nitrite yielded outward currents of similar intensity. Substitution of chloride generated currents of diminished intensity, whereas currents generated with organic anions (acetate, malate, and succinate) showed little difference from the negative control anion gluconate. In addition to differences in the current magnitude, chloride currents showed more positive reversal potentials than those induced by an equivalent concentration of nitrate (Table I). Based on the difference of these reversal potentials, a permeability ratio (Pnitrate/PCl–) of 2.23 (SEM = 0.21; n = 7) was determined for oocytes expressing GmN70.
Voltage clamp recording of oocytes expressing the orthologous protein LjN70 (Szczyglowski et al., 1998 ) also show outward currents in the presence of nitrate and nitrite but exhibited little apparent permeability to chloride or organic anions. Overall, the data suggest that GmN70 and LjN70 are nodule anion transport proteins with a selectivity preference for nitrate.
GmN70 represents an ortholog of the nodulin LjN70, a protein expressed during the late developmental stages of L. japonicus nodule organogenesis (Szczyglowski et al., 1998 ). Similar to LjN70, GmN70 mRNA shows enhanced expression in mature nodules. By immunolocalization and western-blot analyses, GmN70 shows preferential expression on the symbiosome membrane within infected cells of soybean nodules. Both LjN70 and GmN70 appear to encode a polytopic membrane protein with 12 putative transmembrane regions organized in 2 groups of 6 with a large hydrophilic-loop region between, a topology similar to MFS transporters (Pao et al., 1998). The MFS is a diverse family found in all prokaryotes and eukaryotes, and represents a large collection of membrane transporters of a wide range of substrates including sugars, drugs, organic and inorganic ions, Krebs-cycle intermediates, amino acids, and peptides (Pao et al., 1998).
Xenopus oocytes expressing GmN70 displayed distinct electrophysiological properties. Perfusion of these oocytes with a solution in which chloride was replaced by the impermeant anion gluconate resulted in depolarization of the plasma membrane. Subsequent perfusion with solutions containing chloride or nitrate resulted in restoration of negative membrane potentials and the induction of outward currents measured by two-electrode voltage clamp recording. It has been noted that Xenopus oocytes have an endogenous calcium-induced voltage-activated chloride channel (Dascal, 1987
Functional analysis of plant nitrate transporters of the MFS family shows that they are members of one of two families, the nitrate-nitrite transport family, which encode high-affinity nitrate transporters (NRT2 family) and a lower affinity nitrate transporter family (NRT1 family) with sequence similarity to the peptide transporters of yeast and plants (for review, see Forde, 2000 However, examination of the genomes and EST libraries reveals that a subfamily of proteins with a higher degree of similarity to GmN70 and LjN70 is present in a variety of plant species (Fig. 8). An inspection of the annotated Arabidopsis genome revealed 2 genes (At2g39210 and At2g28120) that encode proteins with 65% and 57% sequence identity to GmN70, respectively. Preliminary transport analyses of the Arabidopsis gene products by expression in Xenopus oocytes suggest that they exhibit transport properties similar to LjN70 and GmN70 (data not shown). This finding suggests that plants contain a subfamily of closely related anion/nitrate transporters related to GmN70 that may have a broader role beyond symbiosis. This is supported by examination of the soybean EST libraries, which shows that GmN70 is among several closely related cDNAs (ranging from 57%–87% identity to GmN70), some of which are expressed in other organs besides the nodule. Whether these are responsible for some of the anion/nitrate transport activities documented on other plant membranes remains to be determined. Since these plant proteins appear to represent a structurally and functionally related family of anion transporters, we suggest a name for this family of "nodulin-like anion transport proteins" (NLAT; Fig. 8).
The transport properties of the symbiosome membrane have attracted considerable interest given the pivotal role that this plant-derived membrane plays in nitrogen-fixing symbioses (Udvardi and Day, 1997 ; Day et al., 2001). The membrane is energized by an ATPase pump that generates an electrical and pH gradient by transporting protons into the lumen of the symbiosome (Udvardi and Day, 1989). This gradient has been proposed to play an important role in driving the uptake of dicarboxylates as an energy source to support nitrogen fixation by the bacteroid (Ou Yang et al., 1990) and also is proposed to regulate the release of fixed NH4+ through an inwardly rectified voltage-dependent cation channel (Tyerman et al., 1995; Roberts and Tyerman, 2002). Additionally, the pH of the lumen is thought to be an important factor controlling pH-sensitive hydrolytic activities within the symbiosome, which is considered to be a lytic organelle (Brewin, 1991). By using the potential-sensitive dye oxonol V, it was previously shown that the   generated by the H+-ATPase in isolated intact soybean symbiosomes can be collapsed by the addition of anion salts, suggesting an anion transporter on the symbiosome membrane (Udvardi and Day, 1989; Udvardi et al., 1991). Interestingly, the effectiveness of the various anion salts to dissipate the soybean symbiosome membrane is similar to the permeability profile of the GmN70 that we report here (nitrate > nitrite >> chloride >>> acetate = dicarboxylates). The finding of GmN70 on symbiosome membranes supports the hypothesis that this protein is responsible for this activity measured with isolated symbiosomes.
What potential function would an anion/nitrate transporter play on the symbiosome membrane? While the functional significance of this activity is unclear, several potential roles are possible. First, the
The selectivity for nitrate is also interesting in light of the fact that nitrogen-fixing symbioses are induced under nitrogen-poor conditions (i.e. low-soil nitrate). Indeed, in the presence of nitrate (a preferred source of nitrogen), nodule development is suppressed, nitrogen fixation of existing nodules is inhibited, and senescence is promoted (Streeter, 1988
Nucleic Acid and Molecular Cloning Techniques
Nodulated soybean (Glycine max) cv Essex plants infected with Bradyrhizobium japonicum strain USDA 110 were grown as previously described (Weaver et al., 1991 The cDNA fragment encoding part of the GmN70 ORF was cloned into the TOPO-TA vector following the manufacturer's protocol (Invitrogen). Upstream 5' regions of the ORF were obtained by using total nodule cDNA and 5' RACE (TakaRa Biomedicals, Madison, WI). The 3' end was identified from analysis of a soybean EST library (accession no. TC142224; www.tigr.org). From these sequences, a contiguous cDNA containing the full ORF was obtained by PCR of nodulin cDNA using the following primer set: F-5'-ATGGTAGTTGGAGGTTCGAATACC, R-5'-TTACTTCTGAGTTGGCATCACATG.
BglII restriction sites were engineered into the 5' end of the primers to facilitate cloning into the pX Automated DNA sequencing of both strands of all constructs was performed on a Perkin-Elmer Applied Biosystems (Foster City, CA ) 373 DNA sequencer at the University of Tennessee Molecular Biology Research Facility (Knoxville, TN) using M13 reverse and forward primer sites. Sequence alignments were done by using ClustalW multiple sequence alignment program (version 1.7, June 1997) and the Blosum62 algorithm (Seqtools software package, www.seqtools.dk). Phylogenetic trees were constructed using the TreeView program of the same software package. The DNA and protein sequence information for GmN70 has been deposited (GenBank accession no. AY726670).
For northern-blot analysis, total RNA (40 µg) was resolved by electrophoresis on 1.2% (w/v) agarose gels in the presence of 5.6% (w/v) formaldehyde (Sambrook et al., 1989
Peptide antigens corresponding to the large central loop region of GmN70 (CD-18 peptide with the sequence CDTRWWENVFSPPARGED) or nodulin 26 (C-loop peptide with the sequence CMGNHDQFSGTVPNGT) were prepared synthetically (Invitrogen). Peptides were coupled to maleimide-activated keyhole limpet hemocyanin (Pierce-Endogen Chemicals, Rockford, IL) through amino-terminal Cys residues as described previously (Guenther et al., 2003
Western immunoblots of proteins resolved by SDS-polyacrylamide electrophoresis were done as described in Weaver et al. (1991) For immunolocalization, soybean nodules were cut into 1- to 2-mm slices with a razor blade and were immersed in 4% (w/v) paraformaldehyde in phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 9.6 mM NaH2PO4, 1.5 mM K2HPO4, pH 7.3) at 4°C for 16 h. Tissue samples were washed twice in PBS for 1 h and then were cryoprotected in 20% (w/v) Suc for 20 h at 4°C. Samples were then transferred to optimal cutting temperature compound (OCT; Fisher Scientific, Suwanee, GA) for 6 h at room temperature. The samples were then placed in embedding moulds with fresh OCT compound, were flash frozen in liquid nitrogen, and were stored at –80°C. Embedded tissue blocks were equilibrated at –20°C for 4 h, and 6-micron sections were cut on a vibratome cryostat (Fisher Scientific), transferred to poly-L-lysine-coated coverslips, and stored at –20°C. For immunostaining, sections were washed in PBS to remove OCT compound and were incubated in blocking buffer (5% [v/v] normal goat serum, 2% [w/v] bovine serum albumin, 0.05% [w/v] Triton X-100 in PBS) for 45 min at room temperature. The sections were then incubated with antibodies or preimmunization sera controls in blocking buffer at 37°C for 2 h. The sections were washed 3 times in PBS at room temperature and were then incubated at 37°C for 1 h in Alexa 647-conjugated goat anti-rabbit IgG (Molecular Probes, Eugene, OR) diluted 1:100 in blocking buffer. The stained sections were washed three times in PBS and were mounted in anti-fade (Molecular Probes). Staining was visualized with an Axioplan microscope (Zeiss, Jena, Germany) equipped with an HBO 100-W mercury lamp for epifluorescence and with a scientific-grade cooled charge-coupled device. Visualization was done using a far-red (685 nm) filter. Grayscale digital images were collected, pseudocolored, and merged using the Metamorph Software (Universal Imaging, West Chester, PA).
The GmN70 insert was subcloned into the Bgl II site of pX
Oocyte recordings were done by two-electrode voltage clamp by using an Oocyte Clamp Amplifier model OC-725C (Warner Instruments, Hamden, CT). The microelectrodes were filled with 3 M KCl and tipped with 2% agarose-3M KCl to reduce KCl leakage (electrode resistances < 1.5 M
In experiments testing nitrate conductance at pH 5.0, 5 mM HEPES was replaced with 5 mM MES. In experiments in which the concentration of nitrate was varied, the osmolarity was adjusted to that of the standard buffer by using sodium gluconate. Steady-state membrane currents (I) were fit to the following equation:
Permeability comparisons for the various anions were done by substitution of the test anion for gluconate in the bath and evaluation of the induced currents. The permeability ratio PNO3/PCl was determined by the difference in reversal potentials between recordings of identical oocytes under two bath conditions with differing anion concentrations: condition 1 contained 100 mM NO3– and 16 mM Cl–, and condition 2 contained 116 mM Cl–. The concentrations of cations and buffer salts were identical for both conditions. It was assumed that the internal oocyte Cl– concentration is 33 mM and that the internal nitrate concentration is negligible (Dascal, 1987
Erev is the difference in reversal potential between condition 1 and condition 2, R is the gas constant, T is the absolute temperature, and F is Faraday's constant. Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession numbers AY726670, TC235634, TC238235, NP937656, NP889385, TC217196, TC244401, TC222019, At2g39210-AAL31925, At2g28120-AAL14413, LjN70-AAC3950, OxlT1-Q51330, MCT1-P53986, and GmN70-AY726670.
We thank Dr. Stephen D. Tyerman, Adelaide University, and Dr. C. David Weaver, Vanderbilt University, for constructive comments during the preparation of this manuscript. Received August 19, 2004; returned for revision October 11, 2004; accepted October 14, 2004.
1 This work was supported by the National Science Foundation (grant no. MCB–0237219 to D.M.R.).  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.051953. * Corresponding author; e-mail drobert2{at}utk.edu; fax 865–974–6306.
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ABSTRACT
RESULTS
-helices (represented by gray-shaded boxes above the sequence) were identified from a hydropathy plot (shown below the aligned sequences) by using the Kyte-Doolittle algorithm (Kyte and Doolittle, 1982
ski et al., 2002
G-ev1 expression vector (Preston et al., 1992
). The standard recording bath solution consisted of 2 mM KCl, 5 mM MgCl2, 6 mM CaCl2, 5 mM HEPES-NaOH, pH 7.6, containing 100 mM of the sodium salt of the test anion conductant (final solution osmolarity = 215 mosm/kg). Recordings were performed using a step-wise voltage protocol in which the oocyte Vm was clamped from +80 to –80 mV in 20-mV increments. Each potential was maintained for 1 s with a 0.5-s recovery period at a holding potential of –35 mV between each voltage pulse. Voltage pulses were controlled with the Labscribe program suite version 1.6 (iWork; CB Sciences, Dover, NH). Membrane current (Im) output was filtered at 1 kHz with a 4-pole Bessel Filter, was digitized via the iWork/118 analog to digital converter hardware, and was analyzed using the Labscribe software. Current convention was such that outward current refers to the movement of cations toward the bath and anions into the oocyte. 
