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Plant Physiol. Apr 2004; 134(4): 1763–1774.
PMCID: PMC419849

Characterization of Three Functional High-Affinity Ammonium Transporters in Lotus japonicus with Differential Transcriptional Regulation and Spatial Expression1

Abstract

Ammonium is a primary source of nitrogen for plants. In legume plants ammonium can also be obtained by symbiotic nitrogen fixation, and equation M1 is also a regulator of early and late symbiotic interaction steps. Ammonium transporters are likely to play important roles in the control of nodule formation as well as in nitrogen assimilation. Two new genes, LjAMT1;2 and LjAMT1;3, were cloned from Lotus japonicus. Both were able to complement the growth defect of a yeast (Saccharomyces cerevisiae) ammonium transport mutant. Measurement of [14C]methylammonium uptake rates and competition experiments revealed that each transporter had a high affinity for equation M2. The Ki for ammonium was 1.7, 3, and 15 μm for LjAMT1;1, 1;2, and 1;3, respectively. Real-time PCR revealed higher expression of LjAMT1;1, 1;2, and 1;3 genes in leaves than in roots and nodule, with expression levels decreasing in the order LjAMT1;1 > 1;2 > 1;3 except in flowers, in which LjAMT1;3 was expressed at higher level than in leaves, and LjAMT1;1 showed the lowest level of expression. Expression of LjAMT1;1 and 1;2 in roots was induced by nitrogen deprivation. Expression of LjAMT1;1 was repressed in leaves exposed to elevated CO2 concentrations, which also suppress photorespiration. Tissue and cellular localization of LjAMT1 genes expression, using promoter-β-glucuronidase and in situ RNA hybridization approaches, revealed distinct cellular spatial localization in different organs, including nodules, suggesting differential roles in the nitrogen metabolism of these organs.

Plants can extract and use various forms of nitrogen (N) from soils. Ammonium (equation M3) is a primary source of inorganic N, as its assimilation requires little energy compared to that of equation M4, for instance. The competition for this nutrient is usually quite intense, and various strategies have been developed by plants to increase their equation M5 acquisition capacity (von Wiren et al., 2000a; Howitt and Udvardi, 2000). For example, multiple forms of ammonium transporters in higher plants allow a great regulatory flexibility and organelle, cell, tissue, or organ-specialization, in addition to enabling cells to take up equation M6 over a wide range of concentrations. Physiological experiments have revealed two types of equation M7 transport systems in plant roots. The first is a low-affinity channel-like transporter, which operates in the millimolar concentration range and is nonsaturable. The second system, high-affinity channel-like transporters (HATS), is based on high-affinity transporters, which operate in the submillimolar concentration range. The HATS exhibits saturation kinetics, energy dependence, and leads to depolarization of the plasma membrane electrical potential (Ludewig et al., 2002). A family of HATS (AMT1) has been identified spanning most living organisms (Marini et al., 1997), and six AMT1 homologs are encoded in the genome of Arabidopsis. Three of the Arabidopsis ammonium transporters have been characterized in yeast (Saccharomyces cerevisiae), and they vary in their affinities for substrates such as methylammonium and ammonium, suggesting that they perform different yet complementary roles in equation M8 uptake (Gazzarrini et al., 1999). Different capacities (Vmax) to transport equation M9 could also contribute to specialization among ammonium transporters. More recently, a second family of HATS (AMT2), with distinct biochemical features, has been identified in Arabidopsis, Lotus japonicus, and rice (Oryza sativa; Sohlenkamp et al., 2000; Simon-Rosin et al., 2003; Suenaga et al., 2003), which is distantly related to plant AMT proteins but more closely related to some bacterial ammonium transporters.

In eukaryotes, the prototypical AMT1 protein contains around 500 to 530 amino acids and is highly hydrophobic with 11 putative membrane-spanning regions (von Wiren et al., 2000a). The plant members of the AMT1 family are preferentially expressed in roots (with the exception of LeAMT1;3; von Wiren et al., 2000b). Only AtAMT1;1 is transcribed in all major organs of Arabidopsis plants, although it maintains the highest level of expression in roots. AtAMT1;1 and LeAMT1;1 exhibit strong N-dependent transcriptional regulation in roots of Arabidopsis and tomato (Lycopersicon esculentum), respectively (Gazzarrini et al., 1999; von Wiren et al., 2000b). The induction of AtAMT1;1 in N deficiency conditions appears to be predominantly dependent on the local status of the roots (Gansel et al., 2001). It has been shown that changes in AtAMT1;1 transcript levels parallel those of HATS activity following changes in N supply to plants (Gazzarrini et al., 1999; Shelden et al., 2001), indicating that AtAMT1;1 in roots is primarily responsible for equation M10 uptake from the soil. Recently, this correlation was directly supported by the characterization of the first Arabidopsis AtAMT1;1 knockout mutant (Kaiser et al., 2002). In the Arabidopsis AMT1;1:T-DNA mutant, removal of N decreased high-affinity equation M11 influx by 30% compared with wild-type plants. Further analysis of the AtAMT1;1 knockout mutant revealed different regulation of some of the other Arabidopsis HATS, with root AtAMT1;3 and AtAMT2;1 mRNA levels significantly increased compared to wild-type plants. This indicates that disruption of AtAMT1;1 was compensated for by overexpression of two other members of the AMT family (Kaiser et al., 2002).

Although ammonium transporters are preferentially expressed in roots, where they probably play an important role in equation M12 uptake and/or retrieval from the soil, several lines of evidence indicate that transport of equation M13 might be of particular importance for N nutrition and metabolism in leaves. First, equation M14 can be imported from the atmosphere into leaf cells through stomata (Husted and Schjoerring, 1996). Second, significant concentrations of equation M15 have been measured in the xylem (Rawat et al., 1999), indicating that considerable amounts of N are translocated to shoots in this form. Third, the photorespiratory nitrogen cycle generates a large amount of equation M16 in leaf mitochondria that is subsequently transported to chloroplasts for reassimilation by Gln synthetase. Thus, the expression of AMT genes can be important to ensure the recycling of equation M17 during photorespiration.

In legume plants, an alternative source of combined N is equation M18 production via symbiotic nitrogen fixation (von Wiren et al., 2000a; Howitt and Udvardi, 2000). Given the prime importance of equation M19 in nodule metabolism and whole-plant N nutrition, it is interesting to explore the roles that ammonium transporters play in the development and functioning of nodules and how they contribute to integration of the additional equation M20 supplied by the nodule organ in the general frame of partitioning of nitrogenous solutes to the whole plant. In addition to possible roles in intracellular and intercellular transport of equation M21, ammonium transporters may play a role in sensing equation M22 released by bacteroids in nodules, which triggers pathways for ammonium assimilation. Ammonium transporters in uninfected roots may also report on the N availability in the soil and either promote or block the establishment of nitrogen fixing symbiosis.

We reported previously the isolation and partial characterization of the first AMT1 protein from a legume (Salvemini et al., 2001). To clarify the roles that different AMT1 proteins play in the control of equation M23 fluxes in different organs and/or in the N sensing during symbiosis, we isolated and characterized two new members of the L. japonicus AMT1 family. The biochemical properties of LjAMT1;2 and LjAMT1;3 were determined following expression in yeast. This, together with a detailed analysis of the temporal and spatial patterns of LjAMT1 gene expression in response to developmental and environmental cues enables us to postulate different roles for LjAMT1 proteins in L. japonicus.

RESULTS

Gene Isolation and Structural Analysis of LjAMT1;2 and LjAMT1;3

To identify new members of the L. japonicus AMT1 family, we used a PCR-based approach and the same degenerate oligonucleotides that were used for the isolation of the LjAMT1;1 gene (Salvemini et al., 2001). The resulting 400-bp fragment amplicons were cloned into the pCR2.1 vector (Invitrogen, Carlsbad, CA) and sequenced. Analysis of 30 randomly selected clones revealed a high level of homology with AMT1 proteins in 90% of the cases (27 out of 30). Among the amplicons sequenced, five clones (16%) carried sequences identical to the LjAMT1;1 gene, whereas the remaining 22 clones represented two different genes, named LjAMT1;2 and LjAMT1;3. Southern-blot analysis under high stringency conditions with genomic DNA digested with three restriction enzymes confirmed the presence of three different loci with different hybridization patterns (data not shown). 5′ and 3′ RACE PCR were used to isolate two full-length cDNAs for LjAMT1;2 and LjAMT1;3 (see “Material and Methods”). The colinearity of the RACE-amplified fragments and the originally isolated amplicons was confirmed by PCRs using different combination of oligonucleotides and by Southern-blot analysis (data not shown).

The complete open reading frames of LjAMT1;2 and LjAMT1;3 encode 57.6- and 56.2-kD polypeptides of 519 and 507 amino acid residues, respectively (GenBank accession nos. AJ135020 and AJ575588). LjAMT1;1 and LjAMT1;2 are more closely related with 76% identity at the amino acid level, whereas LjAMT1;3 is more distantly related with 73% and 71% similarity to LjAMT1;1 and LjAMT1;2, respectively.

Hydrophobicity analysis predicted that each protein contains 11 putative membrane-spanning regions (Tusnàdy and Simon, 1998; Krogh et al., 2001), which is in agreement with the predicted prototype of the ammonium transporter proteins. Analysis of the N-terminal hydrophobic region of the three AMT1 proteins (Nielsen et al., 1997) showed a weakly predicted signal sequence for AMT1;1 (cleavage site between residues 23 and 24), whereas AMT1;2 and AMT1;3 have a stronger predicted signal sequence (between residues 27 and 28 and 26 and 27 for AMT1;2 and AMT1;3, respectively). Finally, the prediction for N-glycosylation sites revealed the presence of a conserved putative glycosylated site (NAT) before the proposed cleavage site in the three LjAMT1 sequences (positions 19, 20, and 21 for LjAMT1;1, 1;2, and 1;3, respectively). A similar site, which is indeed glycosylated, is present in the yeast Mep2 protein (Marini and Andrè, 2000).

Functional Expression of LjAMT1;1, LjAMT1;2, and LjAMT1;3 in a Yeast Mutant Defective in High-Affinity equation M24 Uptake

The yeast strain 31019b is defective in three endogenous equation M25 transporters (Mep1, Mep2, and Mep3) and is unable to grow on medium containing less than 5 mm equation M26 as the sole N source (Marini et al., 1997). Transformation of the 31019b strain with the yeast vector p426 expressing LjAMT1;2 or LjAMT1;3 under the control of the Met-25 promoter resulted in growth of 31019b down to 1 mm equation M27 as the sole N source (Fig. 1A). Thus, these two genes encode functional equation M28 transporters. To determine differences in substrate affinities between the L. japonicus AMT1 proteins, we used 14C-labeled methylammonium as a substrate analog and measured short-term uptake in transformed yeast strains. Expression of the LjAMT1 proteins conferred the ability to take up 14C-labeled methylammonium in a range of 0.025 to 2 mm (Fig. 1B), whereas 31019b transformed with the vector alone did not take up significant amounts of 14C-labeled methylammonium (data not shown). Kinetic parameters were calculated from Lineweaver-Burk plots (Fig. 1C). LjAMT1;1 displayed the highest affinity for methylammonium (Km = 0.16 mm), followed closely by LjAMT1;3 (Km = 0.47 mm) and finally by LjAMT1;2, whose affinity was considerably lower (Km = 1.72 mm; Fig. 1D). The corresponding Vmax values were 6.5, 73, and 24 nmol methylammonium min−1 mg−1 protein for AMT1;1, AMT1;2, and AMT1;3, respectively (Fig. 1D). The results of the methylammonium sensitivity tests show a clear intoxication of the yeast cells transformed with LjAMT1;2 and LjAMT1;1, while the cells transformed with LjAMT1;3 do not exhibit the same phenotype (Fig. 1A). A similar differential behavior also has been observed in yeast cells, in which Mep1 expression is essentially associated to the methylammonium sensitivity (Dubois and Grenson, 1979). Since the affinities for methylammonium don't necessarily reflect the affinity for ammonium, we performed competition studies with varying equation M29 concentrations. Ki values were deduced from Dixon plots, and a 50% inhibition was observed at 1.7, 3, and 15 μm equation M30 for AMT1;1, AMT1;2, and AMT1;3, respectively (Fig. 1D). Thus, the competition studies indicated that the three equation M31 transporters possess distinct substrate affinities, with a much higher affinity for equation M32 than for methylammonium.

Figure 1.
Complementation analysis and biochemical characterization of LjAMT1;1, 1;2, and 1;3. A, Growth test on minimal medium containing 3 and 1 mm equation M71 as the sole nitrogen source and methylammonium sensitivity test on minimal Pro (0.1%) medium containing 75 and ...

Organ-Dependent Expression and Influence of High CO2 and N Deficiency

To gain insight into the possible physiological roles of the LjAMT1 family in L. japonicus and a possible involvement in the regulation of different stages of the symbiotic interaction with rhizobia, we first measured the transcript abundance of the three genes in different plant organs, including nodules. RNA was extracted from roots, leaves, and mature nodules (5 weeks) of plants grown in medium with no N supply and inoculated with Mesorhizobium loti (Fig. 2A). Since LjAMT1;3 could not be detected by northern hybridization, we decided to investigate the LjAMT1 genes expression profile by quantitative real-time reverse transcription (RT)-PCR to obtain a complete comparative picture of the relative abundance of transcript of each gene. In all the reported experiments, we used as internal standard the 18S ribosomal RNA primers/competimers (Ambion, Austin, TX) in a ratio of 1:9. The primers specific for LjAMT1;1 bracket the 1-kb intronic sequence (Salvemini et al., 2001), and the 1.3-kb amplified fragment obtained on genomic DNA was never obtained with the cDNA samples. As shown in Figure 2A, the three AMT1 members showed preferential expression in leaves of the inoculated plants grown in N deficiency conditions (P < 0.0004). For LjAMT1;1, the observed relative level of expression in different organs was consistent with the previously reported results from RNAse protection assays (Salvemini et al., 2001). Abundance of transcript decreased in the order LjAMT1;1 > LjAMT1;2 > LjAMT1;3 in leaves, roots, and nodules (P = 0.001 for leaves and <0.0001 for roots and nodules, respectively). As shown in Figure 2A, the analysis of expression was also performed in flowers, in which RNA was extracted by mature plants grown in soil. Interestingly, in this organ we found the highest level of LjAMT1;3 expression, whereas LjAMT1;1 showed the lowest level of expression (P < 0.0001).

Figure 2.
Organ-dependent expression of the three LjAMT1 genes revealed by quantitative real-time RT-PCR. A, RNAs were prepared by leaves, roots, and nodules of Lotus plants 5 weeks postinoculation and by flowers of mature plants grown in soil. B, RNAs extracted ...

To test whether preferential gene expression in leaves may reflect an involvement of the LjAMT1 proteins in the recycling of the equation M33 produced during photorespiration, we analyzed the RNA levels of the three LjAMT1 genes in leaves of plants that were exposed to high CO2 in the atmosphere (0.7%, v/v). Plants were grown in vermiculite under high CO2 for 45 d before being transferred into hydroponic culture under the same conditions for 5 further d. Finally, some of the plants were shifted to air for 34 h and RNAs extracted from leaves. The high CO2 treatment resulted in 52% reduction in the LjAMT1;1 transcript in leaves (P = 0.002), whereas LjAMT1;2 and 1;3 level was not affected by this treatment (Fig. 2B).

Since sensing of N deficiency plays a crucial role in initiating the symbiotic interaction, we studied the effect of N deficiency on the LjAMT1 transcript abundance in roots of mature plants. L. japonicus plants were first grown on B5 solid substrate in the presence of 5 mm NH4NO3 as sole N source for 5 weeks. The plants were then transferred to hydroponic conditions while maintaining the same N regime. After 1 more week, plants were transferred to fresh nutrient solution containing no N and RNA extracted from roots at time 0, 8, and 32 h after the shift (T0, T1, and T2 in Fig. 2C). LjAMT1;1 LjAMT1;2, and LjAMT1;3 were measured by real-time PCR. At T0 (high N condition), the level of expression of LjAMT1;2 was slightly higher than that of LjAMT1;1, while transcript from LjAMT1;3 was merely detectable. The shift from 5 mm NH4NO3 to −N solution resulted in a rapid increase in LjAMT1;1 transcript level, which increased 9.5- and 16-fold after 8 and 32 h relative to that at T0 (P = 0.0017). A 2-fold increase in LjAMT1;2 transcript was observed by 32 h (P = 0.0053), but no enhancement was observed for LjAMT1;3. These results indicate that N deficiency strongly induced the transporter exhibiting the highest substrate affinity, suggesting that the AMT1;1 protein may be primarily responsible for equation M34 influx into roots under N deficiency.

Isolation of 5′ AMT Gene Regulatory Regions and in Situ Localization of Gene Expression

To isolate the promoter (5′) regions of L. japonicus AMT1s, genome walking PCR was performed on genomic DNA with gene-specific primers located immediately downstream of the start codon of the LjAMT1;1, LjAMT1;2, and LjAMT1;3 sequences and the adaptor primer AP1 from the Universal Genome Walker kit (CLONTECH, Palo Alto, CA). The resulting PCR products were reamplified with nested gene-specific primers and the adaptor primer AP2, which produced fragments of 720, 636, and 715 bp for LjAMT1;1 LjAMT1;2, and LjAMT1;3, respectively. These fragments were then subcloned in pCR2.1 (Invitrogen) and sequenced (Fig. 3).

Figure 3.
Sequence of the LjAMT1;1, LjAMT1;2, and LjAMT1;3 5′ promoter regions. Numbers on the left indicate the nucleotide position relative to the putative ATG translational start codon. The putative TATA and CAAT boxes are indicated in bold. The 5′-GAT(A/T)A-3′ ...

The 5′ upstream sequences were analyzed for the presence of plant conserved regulatory motifs (Shahmuradov et al., 2003), and our analysis was extended to the 5′ upstream regions of the characterized genes of the Arabidopsis AMT1 and AMT2 families. No clear-cut conserved motifs were revealed. However, we noticed the presence of two TGACTT motifs in the 5′ upstream region of LjAMT1;1 (Fig. 3). The same motif was also found once in the regulatory regions of AtAMT1;1 (data not shown). The TGACTT sequence is recognized and bound by TGA1 related proteins in tobacco (Nicotiana tabacum; Fromm et al., 1991). We also looked for the presence of multiple copies of the 5′-GAT(A/T)A-3′ sequence motif, which is important for N control of gene expression in fungi. Multiple copies of this motif have been identified in the 5′ upstream region of two plant genes involved in N metabolism, the tomato NIA gene that encodes nitrate reductase and the Arabidopsis AMT2;1 gene (Howitt and Udvardi, 2000). Interestingly, the 5′ upstream regions of the LjAMT1;1 and LjAMT1;2 contained two and three conserved 5′-GAT(A/T)A-3′ sequence motifs, respectively, whereas the 5′ regulatory region of LjAMT1;3 contained 11 such motifs (Fig. 3). Finally, analysis of the 5′ regulatory sequences revealed in the LjAMT1;1 upstream region, several CTCTT repeats, and in the region from −215 to −194, the sequence AAAGTT-N11-CTCTT, which partially matches the consensus of a cis-acting element that controls the expression of several nodulin genes (Gallusci et al., 1991; Stougaard, 2000). The transcription initiation site of LjAMT1;1 shown in Figure 3 was experimentally determined using an RNAse protection assay with a specific fragment spanning over the 5′ of the gene as a probe (data not shown).

To analyze the promoter-dependent spatial regulation of the LjAMT1 genes, PCR-amplified fragments obtained with gene-specific oligonucleotides that introduced different restriction sites into the PCR products were cloned in the pBI101 binary vector to obtain translational fusions with the gusA reporter gene (Jefferson, 1987). L. japonicus composite plants obtained upon transformation with Agrobacterium rhizogenes (Martirani et al., 1999) were used to analyze the expression of the translational pLjAMT1-gusA fusions in a transgenic root system. As shown in Figure 4, β-glucuronidase (GUS) staining activity was detected in different regions of the root tissue. LjAMT1;1 and 1;2 showed an overlapping pattern of GUS expression in root tissues with AMT1;1 being more highly expressed, consistent with the real-time PCR analysis (Fig. 2A). The two fusions were primarily expressed in the root cap cells of primary as well as emerging and mature lateral roots (Fig. 4A). GUS staining was observed in peripheral and columella root cap cells, and in some cases GUS activity was also extended to the very tip of the root. Strong GUS staining was also observed in the regions where lateral roots emerged from the parent root and also in epidermal cells at the site of emergence (Fig. 4C). Cross sections in Figure 4, G and H, show GUS activity in the epidermis and in the pericycle cell layers of transgenic roots obtained with both pLjAMT1;1-gusA and pLjAMT1;2-gusA fusions. Faint staining was also observed in the cortical cell layers (Fig. 4, G and H). A difference between the pattern of pAMT1;1 and pAMT1;2 GUS activity was observed in root hairs, which often showed pAMT1;1-gusA activity (Fig. 4F) but never pAMT1;2-gusA activity. Low LjAMT1;3-gusA activity was observed in the region of the vascular cylinder of the root (Fig. 4K), consistent with the low level of expression detected by real-time PCR (Fig. 2A). In the case of the pLjAMT1;1-gusA fusion, stable transformants were also obtained upon transformation with A. tumefaciens (Lombari et al., 2003), and the patterns of expression in roots were consistent with those observed with composite plants. Analysis of the pAMT1;1-gusA fusion in four independent stable transformants (T2 lines) showed the highest level of GUS activity in transgenic leaves and in particular in the leaves vascular system (Fig. 4I), consistent with the real-time PCR result shown in Figure 2A. The cross section of a transgenic leaf in Figure 4J shows blue staining also in the mesophyll cells.

Figure 4.
Pattern of expression of the LjAMT1 genes in different tissues. A, Whole mount staining of a root tip of a Lotus plant transformed with the pLjAMT1;1-gusA construct. GUS activity is present in the peripheral root cap cells and in the columella cells. ...

To determine whether the 492-bp 5′ region of the LjAMT1;1 gene contained all the regulatory sequences needed to report correctly its spatial expression in roots, we analyzed the localization of the LjAMT1;1 mRNA in root tissues by in situ hybridization and found an excellent agreement between these results and the reporter gene expression (Fig. 4, B, D, and F).

Real-time PCR analysis revealed differential expression of the LjAMT1 genes in mature nodules. To gain insight into the possible roles played by these genes in nodule development and/or functioning, we analyzed the pattern of AMT1 promoters-GUS expression in mature nodules. This analysis was first performed on 4-week-old nodules of composite plants obtained upon infection with A. rhizogenes equipped with the different gusA fusion T-DNA constructs. As shown in Figure 5, A to D, pLjAMT1;1-gusA and pLjAMT1;2-gusA gave different pattern of nodular expression, while LjAMT1;3 promoter activity was not detectable. LjAMT1;2 was expressed mainly in the noninfected zone of the nodule, in the outer cortical cells, and in the very peripheral cell layers of the central zone (Fig. 5, A and B). By contrast, the pLjAMT1;1gusA fusion produced GUS activity in the central zone of the nodule (Fig. 5, C and D) and in the vascular bundles (data not shown). The 60-μm sections shown in Figure 5, C and D, indicate a localized GUS activity in the infected cells of the central zone of the nodule. This pattern of activity was detectable only in 25% of the composite plants analyzed (17 out of 70; on average six independent transgenic roots arose from the wounded site, and 75% of these are cotransformed with the gusA and pRi-born T-DNAs; Martirani et al., 1999). However, GUS activity was never detected during the initial stages of M. loti infection. To confirm the GUS localization data, we performed a set of in situ mRNA localization experiments for LjAMT1;1. A leghemoglobin specific RNA probe was used as control for invaded cells of the central tissue. The leghemoglobin protein is present at a high concentration in the cytosol of invaded host cells, where it ensures a continuous supply of oxygen for respiratory metabolism at very low free oxygen concentrations. Leghemoglobin mRNA was found in 3- and 4-week-old nodules in invaded cells of root nodules from L. japonicus (Fig. 5, E and F). The in situ pattern obtained with the AMT1;1 probe was similar to that obtained with the leghemoglobin probe. Transcripts of LjAMT1;1 were detected in infected cells but also in cells of the vascular bundle of 4-week-old nodules (Fig. 5, G and H). The detection method displays a dark staining in the bright-field images and a pink staining of the cells in the dark-field images when compared to the sense control (data not shown). Taken together, these results indicate that the 496-bp 5′ fragment of the LjAMT1;1 promoter contains all the information needed to ensure the correct spatial pattern of expression of this gene.

Figure 5.
Analysis of the expression in nodular tissues. A, Whole mount staining of a nodulated root of a Lotus plant transformed with the pLjAMT1;2-gusA construct. B, Longitudinal section of a transgenic nodule transformed with the pLjAMT1;2-gusA construct. GUS ...

In conclusion, the LjAMT1;1 promoter activity shows a very precise spatial and temporal pattern of activity during the nodule development.

DISCUSSION

Higher plants possess small multigene families encoding AMT1-like proteins. For instance, on the basis of the analysis of their genome databases, Arabidopsis contains six and rice three AMT1 genes (Kumar et al., 2003; Sonoda et al., 2003), which presumably carry out discrete, though perhaps partially overlapping, roles in the plant. We have expanded the number of known AMT1 proteins in L. japonicus to three and have presented evidence that indicates some degree of specialization. The approach we followed to isolate the AMT1 genes was based on PCR amplification on genomic DNA using degenerated oligonucleotides. This probably permitted the isolation of the LjAMT1;3 gene that was expressed at a very low level in all the organs and tested conditions.

Determination of affinity constants for 14C-methylammonium uptake by LjAMT1;1, LjAMT1;2, and LjAMT1;3 in yeast and transport inhibition by equation M35 (Fig. 1) revealed that all three proteins are high-affinity ammonium transporters (transport affinities range from 1.7–15 μm) adapted to transport external equation M36 in a very restricted low range of concentrations. A similar range of affinities for equation M37 was reported for the Arabidopsis AMT1 transporters (Gazzarrini et al., 1999). Interestingly, in contrast to the Arabidopsis AMT1 members, all three LjAMT1 proteins exhibited a high selectivity for ammonium compared to methylammonium when expressed in yeast.

The analysis of the pattern of expression of the LjAMT1 genes in different organs revealed organ specificity and differential regulation of LjAMT1;1, LjAMT1;2, and LjAMT1;3. The three members were preferentially expressed in leaves compared to roots or nodules of plants grown in N starvation conditions and inoculated with M. loti, with a gene expression level decreased in the order LjAMT1;1 > LjAMT1;2 > LjAMT1;3 (Fig. 2A). This hierarchy was reversed only in flowers of plants grown in soil, in which AMT1;2 and AMT1;3 showed a higher level of expression than AMT1;1 (Fig. 2A). LjAMT1;3 was the only gene of the family showing an expression level higher in flowers than in leaves, suggesting differential roles of the three proteins in the equation M38 translocation and utilization in sink and source tissues. Higher levels of expression in leaves were observed also in hydroponic conditions with both high and low N supply (data not shown). Such a pattern of expression is uncommon among AMT1 genes in plants and has only been observed for the LeAMT1;3 and AtAMT2 genes in nonlegume plants (von Wiren et al., 2000b; Sohlenkamp et al., 2002). Analysis of promoter-gusA fusion activity in plants performed for LjAMT1;1 confirmed a stronger expression in this organ compared to the root (Fig. 4I). GUS activity was observed in the vascular system of leaves and in the mesophyll cells (Fig. 4, I and J). The apoplast surrounding leaf cells has been shown to contain substantial quantities of equation M39 (Nielsen and Schjoerring, 1998), which may originate from the xylem stream, from the photorespiratory cycle (as well as protein catabolism), or directly from the atmosphere (Cowling and Lockyer, 1981). The pattern of expression of the pLjAMT1;1-gusA fusion in Figure 4I, with prevalent GUS activity in the mid- and secondary veins of the leaves, is consistent with a role of LjAMT1;1 in uptake of equation M40 from the xylem stream. However, the analysis of AMT1 transcripts abundance performed on leaves of plants grown in air conditions versus higher CO2 concentrations showed a strong (60%) decline of the LjAMT1;1 transcript in the latter condition (Fig. 2B), indicating a possible additional role of LjAMT1;1 in the retrieval of photorespiratory NH3/equation M41 in the mesophyll cells, where photosynthesis takes place. It will be of interest to test whether the LjAMT1;1 gene expression is also altered in a L. japonicus mutant affected in a plastidic GS isoform that is viable in high CO2 and accumulates equation M42 (Orea et al., 2002). A slight reduction of transcription in leaves of plants growing in high CO2 conditions has also been reported for the LeAMT1;2, LeAMT1;3, and AtAMT2 genes (von Wiren et al., 2000b; Sohlenkamp et al., 2002). LjAMT1;1 could be involved in the retrieval of equation M43 lost to the apoplastic space surrounding mesophyll cells. Such a role would be consistent with a preliminary analysis carried out with a LjAMT1;1-green fluorescent protein fusion, which indicates a plasma membrane location for this transporter (data not shown).

Very recently, the first experimental evidence for a direct role of an AMT1 member in equation M44 uptake into plant roots was reported (Kaiser et al., 2002). The AMT1;1 knockout mutant isolated by T-DNA tagging showed a 30% reduction in high-affinity equation M45 uptake. In addition, the AtAMT1;1 mutant showed altered leaf morphology and a lethal phenotype due to interactions between C supply (Suc) and equation M46 transport. The transcriptional up-regulation of LjAMT1;1 in roots observed after the transfer to N-free nutrient solution suggests that the cognate protein has a primary role in the uptake of equation M47 under N deficiency. This correlates with the observation that LjAMT1;1 had the highest affinity for equation M48 among the three AMT1s tested in yeast (Fig. 1C). A strong induction (at least 5-fold) by N deficiency was also reported for AtAMT1;1, LeAMT1;1, and OsAMT1;3 (Gazzarrini et al., 1999; von Wiren et al., 2000b; Sonoda et al., 2003). Among the three LjAMT1 genes, LjAMT1;2 had a 2-fold increase in N deficiency and showed the highest level of expression in high N conditions, while LjAMT1;3 showed the most stable low expression levels in high and low N. The observed pattern of transcriptional regulation in different N conditions was unexpected on the basis of the features of their 5′ regulatory sequences with the 11 5′-GAT(A/T)A-3′ binding motifs found in the upstream region of LjAMT1;3. The meaning of these motifs remains to be investigated, but these could be related to other environmental conditions controlling the LjAMT1;3 gene expression (Crespo et al., 2001; Teakle et al., 2002). However, the organ-dependent expression of the three members of the L. japonicus family as well as the differential effect of N deficiency on their transcript abundance match close the differential regulation of three tomato AMT1 members reported previously (von Wiren et al., 2000b).

The analysis of promoter-gusA fusion activity in transgenic roots was performed for each of the LjAMT1 genes, obtaining for the first time, to our knowledge, a complete map of the spatial expression of different members of an AMT1 family in the roots cells. LjAMT1;1 and LjAMT1;2 showed a similar pattern of GUS expression in root tissues. The GUS activity observed in the root cap cells and in the secondary root emergence zones may reflect a role of the corresponding transporters in equation M49 retrieval function. In fact, in both these root regions an abundant leaking of organic and inorganic material can take place due to the sloughed-off cells of the root cap structure or to the breaking of the epidermal cell layer. The slight promoter activity detected in the cortex and pericycle cells observed with GUS fusions of the LjAMT1;1 and 1;2 members (Fig. 4, G and H) can be related to the cycling between roots and shoots of the equation M50 that is not completely assimilated in the root and shoot cells or simply to the recover of equation M51 in the root apoplast that isn't taken up by epidermal cells. A similar role can be postulated for LjAMT1;3, whose weak promoter activity was detected only in the region of the primary root central stele in the tested conditions, consistently with the very low level of expression observed by real-time PCR. An important discriminating feature in the pattern of GUS activity obtained with the three AMT1 promoter fusions was the blue staining observed in root hairs with LjAMT1;1 only (Fig. 4E). This activity was weak and observed only in 30% of the composite plants analyzed, which may reflect copy number of the T-DNA in the transformed roots. Nevertheless, GUS activity was never observed in root hairs with the LjAMT1;2-gusA and LjAMT1;3-gusA constructs. The presence of abundant transcripts in root hairs has been reported for LeAMT1;1 and LeAMT1;2 (Lauter et al., 1996) and is consistent with the expression of genes involved in equation M52 assimilation observed in the root hairs of bean and rice (Watson and Cullimore, 1996; Ishiyama et al., 1998). However, in legume plants this specific location is of particular interest since root hairs represent the initial site of interaction between legume roots and M. loti bacteria living in the soil. In yeast, Rhodobacter capsulatus, and more recently in Heleboma cylindrosporum and Tuber borchii (Lorenz and Heitman 1998; Yakunin and Hallenbeck, 2002; Javelle et al., 2003), AMT proteins have been implicated in sensing equation M53 in the external medium, which is linked to specific developmental programs. Thus, it is interesting to speculate on a possible role of AMT1;1 in N signaling and nodule organogenesis program. The pattern of LjAMT1;1 transcription, especially its regulation by N availability and its expression in root epidermis and root hairs, is consistent with a possible role of the protein in signaling. Isolation of a plant mutant deficient in the LjAMT1;1 expression will be essential to test this hypothesis.

The legume root nodule is an organ specialized in de novo synthesis of equation M54 from N2. Nitrogen-fixing bacteroids in the infected central cells of nodules are the prime source of equation M55 for mature nodule cells. Interestingly, promoter-GUS and in situ RNA hybridization revealed distinct expression patterns for AMT1;1 and AMT1;2 in L. japonicus nodules (Fig. 5, B and D–F). LjAMT1;2 expression was confined to the outer cortex and to the periphery of the central tissue (Fig. 5, C and D), while LjAMT1;1 was expressed in the vascular bundles and cells of the central infected zone of nodules (Fig. 5, C–F). Thus, LjAMT1;1 and LjAMT1;2 likely play complementary, nonoverlapping roles in nodules. Bearing in mind that LjAMT1;1, as other high-affinity ammonium transporters, could be localized in the plasma membrane of plant cells (Sohlenkamp et al., 2002; Simon-Rosin et al., 2003), LjAMT1;1 and LjAMT1;2 likely play primary roles in retrieving equation M56 lost from nodule cells in the different tissues during normal metabolism. Additionally, LjAMT1;2 may play a role in scavenging equation M57 from the soil, although this is unlikely to be a major source of N for nodule cells during symbiotic nitrogen fixation.

Furthermore, the pattern of GUS activity observed with the LjAMT1;1-gusA construct in mature nodules (Fig. 5, C and D) and the in situ analysis (Fig. 5, E–H) indicate that the supposed AMT1;1 retrieval function is confined to the infected cells of the central tissue and hence must be controlled by a signal that discriminates between infected and uninfected cells. A possible speculation is that the intracellular production of equation M58, due to the nitrogenase activity in the symbiosomes, creates the difference between intracellular and extracellular environment of infected and uninfected cells, although we cannot exclude different developmental signals.

As discussed above, a role in equation M59 retrieval from the apoplast can be also envisaged for LjAMT1;1 protein in leaf mesophyll cells (Fig. 4J), where equation M60 is produced and potentially lost as result of photorespiration. By comparison with the situation observed in the nodule organ, the production of intracellular equation M61 in the mitocondria could be crucial for the LjAMT1;1 expression in mesophyll cells.

In conclusion, the data presented here on the biochemical properties of three LjAMT1 proteins and the spatial expression patterns and regulation of the corresponding genes indicate that each transporter plays a distinct role or set of roles in L. japonicus. The data provide a sound foundation for future reverse-genetics experiments that will help to elucidate the specific roles of each transporter.

MATERIAL AND METHODS

Plant and Bacterial Material

Lotus japonicus GIFU B-129-S9 seeds were used. Mesorhizobium loti strain R7A was kindly provided by Dr. Clive Ronson (University of Otago, New Zealand) and grown to mid-log phase in liquid tryptone-yeast extract medium.

Plant Growth

L. japonicus seeds were germinated on water agar, and after a week seedlings were transferred on B5 solid medium (Duchefa, Haarlem, The Netherlands) and grown for 2 more weeks. Then, plants were transferred on solid medium with the same composition as the B5, except that (NH4)2SO4 and KNO3 were omitted and replaced by 5 mm NH4NO3 and 10 mm KCl, respectively. After a week the plants were transferred for 10 d in hydroponic sterile conditions, in vessels harboring eight plants in about 100 mL of the 5 mm NH4NO3 B5 medium. In the nitrogen-free solution, the NH4NO3 was omitted. The described media contained 1% Suc and vitamins, and the pH was adjusted to 5.7 with MES. To avoid depletion, the nutrient solution was renewed every 3 d during the 10 d of growth in hydroponic cultures. The pH of the medium was checked daily, and it was maintained within close limits (5.8–5.6) in all the conditions of hydroponics growth. Plants were cultivated in a growth chamber with a light intensity of 200 μmol m−2 s−1 at 23°C with a 16 h/8 h day/night cycle.

Plant Transformation

We followed the procedures described by Lombari et al. (2003) and by Martirani et al. (1999) for the Agrobacterium tumefaciens and A. rhizogenes mediated plant transformations.

Nucleic Acid Manipulations, Plasmid Construction, and gusA Fusions

To obtain pLjAMT1;1-gusA (pED11), a PCR-amplified fragment was obtained on genomic DNA with two specific oligonucleotides, 5′-ACCTAGGCAGTGTTAAAAAG-3′ and 5′-ACGCGTCGACATGCCACATGAGGGGCAGCAAG-3′ (containing the BamHI site). The fragment was cloned into the pCR2.1 vector (Invitrogen) to obtain pED3. pED3 was double digested with BamHI and SalI and ligated in the pBI101.3 binary vector to obtain a translational fusion with the gusA reporter gene (Jefferson, 1987).

To obtain pLjAMT1;2-gusA (pED5), the two oligonucleotides were 5′-TACATCCGATGACCCATTTG-3′ and 5′-TCTAGAAGCGAGGTCTGTGGCTGA-3′ (including a XbaI site). The amplified fragment was cloned into the pCR2.1 vector (Invitrogen) to obtain pED1. pED1 was double digested with HindIII and XbaI and ligated into pBI101.1 to obtain the translational fusion pED5.

To obtain pLjAMT1;3-gusA (pED15), the two oligonucleotides were 5′-TCTATTGTTATTGATAGACTC-3′ and 5′-ACTCTAGAGTCGGCGGCAGAGCAAGTGAA-3′ (including a XbaI site). The amplified fragment was cloned into the pCR2.1 vector (Invitrogen) to obtain pED6. PED6 was double digested with HindIII and XbaI and ligated into pBI101.1 to obtain the translational fusion pED15.

Yeast Complementing Plasmids

To obtain plasmids expressing LjAMT1;2 and LjAMT1;3, we first amplified genomic DNA with primers AMT1;2-A and AMT1;2-B (5′-TCTGTCTGCACACTCACAAGT-3′ and 5′-ACCCCATGGCATCTCATAAT-3′) or AMT1;3-A and AMT1;3-B (5′-AGTACGCGGGACTTGTTATTCT-3′ and 5′-ACGTGATCTTTACAAAGTAACACA-3′). PCR products of the expected size (1.7 kb) were gel eluted, sequenced, and subcloned in the pCR2.1 vector (Invitrogen) to obtain pAB1 and pAB2. Plasmid DNAs were digested with EcoRV and HindIII (a partial HindIII digestion was performed in the case of LjAMT1;3), ligated into the yeast vector p426 MET25, and SmaI and HindIII double digested to obtain pAB4 (LjAMT1;2) and pAB5 (LjAMT1;3), respectively.

5′ RACE and 3′ RACE

5′ RACE and 3′ RACE were performed with total RNA extracted from freshly harvested roots using the RNeasy kit from Qiagen (Darmstadt, Germany) and following the protocol supplied with the Marathon cDNA amplification kit (CLONTECH). Oligo(dT)-primed double-stranded cDNA was synthesized using procedures and reagents from the Marathon RACE cDNA amplification kit; the cDNA was ligated to Marathon adapters (CLONTECH). 3′ and 5′ RACE products were generated by long PCR using gene-specific primers and the AP1 primer (5′-CCATCCTAATACGACTCACTATAGGGC-3′; CLONTECH). To increase the specificity of the procedure, a second PCR was carried out using nested gene-specific primers and the nested AP2 adaptor primer (5′-ACTCACTATAGGGCTCGAGCGGC-3′; CLONTECH). PCRs were performed according to the Marathon protocol using Platinum Taq antibody (Invitrogen) and 35 cycles of 95°C for 30 s, 60°C for 30 s for the first PCR and 62°C for the inner PCR, 72°C for 1 min 30 s, and a final extension at 72°C for 10 min. RACE products were electrophoresed, cloned in pCR2.1 (TOPO cloning kit; Invitrogen), and analyzed by DNA sequencing. The sequences of gene-specific primers used are as follows: for 5′ RACE, AMT1;2 outer primer, 5′-GTG CGA GAC GAC CGG GTA GAC AAA-3′; AMT1;2 inner primer, 5′-GAG TAG ATG AGG TAG GCG ACG AAT-3′; AMT1;3 outer primer, 5′-CAC AAC ACC AGA GCC AGC AAA ATC-3′; AMT1;3 inner primer, 5′-CCA AAC CAG TCA AGA ACG AAG AGT-3′; and for 3′ RACE, AMT1;2 outer primer, 5′-GCA CCA CTG GCA GCT TAC TAT TC-3′; AMT1;2 inner primer, 5′-CAG TGC GTC CCT AGT TGT CTC TC-3′; AMT1;3 outer primer, 5′-ATT GAT TTT GCT GGC TCT GGT GT-3′; and AMT1;3 inner primer, 5′-CAT TGA GGG GAC ACA GTG GAA CA-3′.

Isolation of the 5′ Regulatory Sequences

Manufacturer's instructions were followed (Universal Genome Walker PT3042–1; CLONTECH). Five different blunt-end digestions on genomic DNA were performed with DraI, EcoRV, ScaI, StuI, and PvuII restriction enzymes. The primers of the three AMT1 genes were the following: AMT1;1-out, 5′-AACTGGTCGCAGATGAATCCGGCGACTGC-3′; AMT1;1-in, 5′-AGCGCCGCCATCGTTTTGGGGTAGTG-3′; AMT1;2-out, 5′-TGTATCGAGCTGGTTGCAGAGGTAGGTGGC-3′; AMT1;2-in, 5′-AGAGTGGAGCGAGGTCTGTGGCTGAG-3′; AMT1;3-out, 5′-CTCTGTTCTTGCAAAAAAAGGGGAAC-3′; and AMT1;3-in, 5′-AGAGCAAGTGAACGCCGCAGCCATGG-3′.

Quantitative Real-Time RT-PCR

Total RNA was prepared from plants grown in different conditions using the RNeasy plant mini kit (CLONTECH). To remove contaminating DNA, the samples were treated with DNAse I. A total of 1.5 μg of total RNA was annealed to random decamer and reverse transcribed using RT (Ambion) to obtain cDNA. Real-time PCR was performed with a DNA Engine Opticon 2 system (MJ Research, Boston), using SYBR to monitor dsDNA synthesis. As internal standard, the 18S ribosomal RNA primers/competimers (Ambion) in a ratio of 1:9 were used. Every reaction was set up in three replicates. The program used was as follows: 95°C for 2 min and 35 cycles of 95°C for 20 s, 60°C for 15 s, and 72°C for 20 s. Data were analyzed using Opticon Monitor Analysis Software version 2.01 (MJ Research). The relative level of expression was calculated with the following formula: relative expression ratio of the gene of interest is 2ΔCT with ΔCT = Ctgene − CT18S. The AMT1 fragments PCR amplified from total cDNA were gel purified and sequenced to assure accuracy and specificity. The sequences of the gene-specific oligonucleotides designed in the nonconserved 3′ regions of the genes and used for real-time RT-PCR are the following: 1;1-RTA, 5′-AGCGCCTATGATTCAGGCAAC-3′; 1;1-RTB, 5′-TACTCCTCCTTGGCGAATAGC-3′; 1;2-RTA, 5′-AAAGCCTACGGTAACAACGGC-3′; 1;2-RTB, 5′-ATTAACAACCCGTACGGCATC-3′; 1;3-RTA, 5′-ATGGTGAGAGTGGTTCTGGT-3′; and 1;3-RTB, 5′-AGCCCATATGGCCTATCAGGA-3′.

Statistical Analysis

Statistical analyses were performed with ANOVA program available at the following Web site: http://faculty.vassar.edu/lowry/VassarStats.html.

[14C]Methylammonium Uptake Assay

Rates of [14C]methylammonium uptake and competition studies were measured as previously described (Marini et al., 1997), with yeast cells grown on minimal Pro medium.

Histochemical GUS Analysis

Histochemical staining of whole plant material was performed as described by Jefferson (1987). For sections, after staining, whole roots were fixed with 4% paraformaldehyde, 0.25% glutaraldehyde in 50 mm KPO4 buffer, 5 mm EGTA, 10 mm dithiothreitol, pH 7.2, and stored at 4°C. The tissues were then washed with 50 mm KPO4 buffer, pH 7.2. The tissue segments were embedded in 4% agar and 60-μm sections obtained with vibratome (Leica VT1000S; Wetzlar, Germany). The sections were analyzed with a light microscope by means of dark- and bright-field optics.

Acknowledgments

We thank Rita Vito and Sandra Lecomte for technical assistance and A. Aliperti for help with the manuscript. We also thank Biagio Giordano and the gardeners of the Royal Botanical Garden of Naples for their excellent work with the plant care.

Notes

1This work was supported by the European Union (LOTUS: HPRN–CT–2000–00086); Ministry of Education, University and Research (Fondo per gli Investimenti della Ricerca di Base RBNE01KZE7_001); MURST legge 488/92 cluster 02; and Ministro delle Politiche Agricole e Forestali (Progetto Speciale; Risorse genetiche di organismi utili per il miglioramento di specie di interesse agrario e per un'agricoltura sostenibile). H.E.A., M.B., and A.B. were supported by a European Union fellowship (LOTUS: HPRN–CT–2000–00086). A.M. acknowledges project BM C2001–3162 (Spain). A.M.M. is a Chercheur Qualifiè du Le Fonds National de la Recherche Scientifique.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.034322.

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