Molecular and cellular characterisation of LjAMT2;1, an ammonium transporter from the model legume Lotus japonicus

Two related families of ammonium transporters have been identified and partially characterised in plants in the past; the AMT1 and AMT2 families. Most attention has focused on the larger of the two families, the AMT1 family, which contains members that are likely to fulfil different, possibly overlapping physiological roles in plants, including uptake of ammonium from the soil. The possible physiological functions of AMT2 proteins are less clear. Lack of data on cellular and tissue location of gene expression, and the intracellular location of proteins limit our understanding of the physiological role of all AMT proteins. We have cloned the first AMT2 family member from a legume, LjAMT2;1 of Lotus japonicus, and demonstrated that it functions as an ammonium transporter by complementing a yeast mutant defective in ammonium uptake. However, like AtAMT2 from Arabidopsis, and unlike AMT1 transporters from several plant species, LjAMT2;1 was unable to transport methylammonium. The LjAMT2;1 gene was found to be expressed constitutively throughout Lotus plants. In situ RNA hybridisation revealed LjAMT2;1 expression in all major tissues of nodules. Transient expression of LjAMT2;1-GFP fusion protein in plant cells indicated that the transporter is located on the plasma membrane. In view of the fact that nodules derive ammonium internally, rather than from the soil, the results implicate LjAMT2;1 in the recovery of ammonium lost from nodule cells by efflux. A similar role may be fulfilled in other organs, especially leaves, which liberate ammonium during normal metabolism.     

Regulation of NH4+ transport by essential cross talk between AMT monomers through the carboxyl tails

Ammonium transport across plant plasma membranes is facilitated by AMT/Rh-type ammonium transporters (AMTs), which also have homologs in most organisms. In the roots of the plant Arabidopsis (Arabidopsis thaliana), AMTs have been identified that function directly in the high-affinity NH4+ acquisition from soil. Here, we show that AtAMT1;2 has a distinct role, as it is located in the plasma membrane of the root endodermis. AtAMT1;2 functions as a comparatively low-affinity NH4+ transporter. Mutations at the highly conserved carboxyl terminus (C terminus) of AMTs, including one that mimics phosphorylation at a putative phosphorylation site, impair NH4+ transport activity. Coexpressing these mutants along with wild-type AtAMT1;2 substantially reduced the activity of the wild-type transporter. A molecular model of AtAMT1;2 provides a plausible explanation for the dominant inhibition, as the C terminus of one monomer directly contacts the neighboring subunit. It is suggested that part of the cytoplasmic C terminus of a single monomer can gate the AMT trimer. This regulatory mechanism for rapid and efficient inactivation of NH4+ transporters may apply to several AMT members to prevent excess influx of cytotoxic ammonium.     

Characterization of Three Ammonium Transporters of the Glomeromycotan Fungus Geosiphon pyriformis

Members of the Glomeromycota form the arbuscular mycorrhiza (AM) symbiosis. They supply plants with inorganic nutrients, including nitrogen, from the soil. To gain insight into transporters potentially facilitating nitrogen transport processes, ammonium transporters (AMTs) of Geosiphon pyriformis, a glomeromycotan fungus forming a symbiosis with cyanobacteria, were studied. Three AMT genes were identified, and all three were expressed in the symbiotic stage. The localization and functional characterization of the proteins in a heterologous yeast system revealed distinct characteristics for each of them. AMT1 of G. pyriformis (GpAMT1) and GpAMT2 were both plasma membrane localized, but only GpAMT1 transported ammonium. Neither protein transported the ammonium analogue methylammonium. Unexpectedly, GpAMT3 was localized in the vacuolar membrane, and it has as-yet-unknown transport characteristics. An unusual cysteine residue in the AMT signature of GpAMT2 and GpAMT3 was identified, and the corresponding residue was demonstrated to play an important role in ammonium transport. Surprisingly, each of the three AMTs of G. pyriformis had very distinct features. The localization of an AMT in the yeast vacuolar membrane is novel, as is the described amino acid residue that clearly influences ammonium transport. The AMT characteristics might reflect adaptations to the lifestyle of glomeromycotan fungi.     

A nitrogen-dependent switch in the high affinity ammonium transport in Medicago truncatula

Ammonium transporters (AMTs) are crucial for the high affinity primary uptake and translocation of ammonium in plants. In the model legume Medicago truncatula, the genomic set of AMT-type ammonium transporters comprises eight members. Only four genes were abundantly expressed in young seedlings, both in roots and shoots. While the expression of all AMTs in the shoot was not affected by the nitrogen availability, the dominating MtAMT1;1 gene was repressed by nitrogen in roots, despite that cellular nitrogen concentrations were far above deficiency levels. A contrasting de-repression by nitrogen was observed for MtAMT1;4 and MtAMT2;1, which were both expressed at intermediate level. Weak expression was found for MtAMT1;2 and MtAMT2;3, while the other AMTs were not detected in young seedlings. When expressed from their endogenous promoters, translational fusion proteins of MtAMT1;1 and MtAMT2;1 with green fluorescent protein were co-localized in the plasma membrane of rhizodermal cells, but also detected in cortical root layers. Both transporter proteins similarly functionally complemented a yeast strain that is deficient in high affinity ammonium transport, both at acidic and neutral pH. The uptake into yeast mediated by these transporters saturated with K_{m AMT1;1} = 89 µM and K_{m AMT2;1} = 123 µM, respectively. When expressed in oocytes, MtAMT1;1 mediated much larger ¹⁵N-ammonium uptake than MtAMT2;1, but NH₄ ⁺ currents were only recorded for MtAMT1;1. These currents saturated with a voltage-dependent K_m = 90 µM at ?80 mV. The cellular localization and regulation of the AMTs suggests that MtAMT1;1 encodes the major high affinity ammonium transporter gene in low nitrogen grown young M. truncatula roots and despite the similar localization and substrate affinity, MtAMT2;1 appears functionally distinct and more important at higher nitrogen supply.     

Additive contribution of AMT1;1 and AMT1;3 to high-affinity ammonium uptake across the plasma membrane of nitrogen-deficient Arabidopsis roots

In Arabidopsis four root-expressed AMT genes encode functional ammonium transporters, which raises the question of their role in primary ammonium uptake. After pre-culturing under nitrogen-deficiency conditions, we quantified the influx of (15)N-labeled ammonium in T-DNA insertion lines and observed that the loss of either AMT1;1 or AMT1;3 led to a decrease in the high-affinity ammonium influx of approximately 30%. Under nitrogen-sufficient conditions the ammonium influx was lower in Columbia glabra compared with Wassilewskija (WS), and AMT1;1 did not contribute significantly to the ammonium influx in Col-gl. Ectopic expression of AMT1;3 under the control of a 35S promoter in either of the insertion lines amt1;3-1 or amt1;1-1 increased the ammonium influx above the level of their corresponding wild types. In transgenic lines carrying AMT-promoter-GFP constructs, the promoter activities of AMT1;1 and AMT1;3 were both upregulated under nitrogen-deficiency conditions and were localized to the rhizodermis, including root hairs. AMT gene-GFP fusions that were stably expressed under the control of their own promoters were localized to the plasma membrane. The double insertion line amt1;1-1amt1;3-1 showed a decreased sensitivity to the toxic ammonium analog methylammonium and a decrease in the ammonium influx of up to 70% relative to wild-type plants. These results suggest an additive contribution of AMT1;1 and AMT1;3 to the overall ammonium uptake capacity in Arabidopsis roots under nitrogen-deficiency conditions.     

Understanding the differential nitrogen sensing mechanism in rice genotypes through expression analysis of high and low affinity ammonium transporter genes

Two rice genotypes, Kalanamak 3119 (KN3119) and Pusa Basmati 1(PB1) differing in their optimum nitrogen requirements (30 and 120 kg/ha, respectively) were undertaken to study the expression of both high and low affinity ammonium transporter genes responsible for ammonium uptake. Exposing the roots of the seedlings of both the genotypes to increasing (NH₄)₂SO₄ concentrations revealed that all the three families of rice AMT genes are expressed, some of which get altered in a genotype and concentration specific manner. This indicates that individual ammonium transporter genes have defined contributions for ammonium uptake and plant growth. Interestingly, in response to increasing nitrogen concentrations, a root specific high affinity gene, AMT1;3, was repressed in the roots of KN3119 but not in PB1 indicating the existence of a differential ammonium sensing mechanism. This also indicates that not only AMT1;3 is involved not only in ammonium uptake but may also in ammonium sensing. Further, if it can differentiate and could be used as a biomarker for nitrogen responsiveness. Expression analysis of low affinity AMT genes showed that, both AMT2;1 and AMT2;2 have high levels of expression in both roots and shoots and in KN3119 are induced at low ammonium concentrations. Expressions of AMT3 family genes were higher shoots than in the roots indicating that these genes are probably involved in the translocation and distribution of ammonium ions in leaves. The expression of the only high affinity AMT gene, AMT1;1, along with six low affinity AMT genes in the shoots suggests that low affinity AMTs in the shoots leaves are involved in supporting AMT1;1 to carry out its activities/function efficiently.     

N-terminal cysteines affect oligomer stability of the allosterically regulated ammonium transporter LeAMT1;1

AMMONIUM TRANSPORTER (AMT) proteins are conserved in all domains of life and mediate the transport of ammonium or ammonia across cell membranes. AMTs form trimers and use intermolecular interaction between subunits to regulate activity. So far, binding forces that stabilize AMT protein complexes are not well characterized. High temperature or reducing agents released mono- and dimeric forms from trimeric complexes formed by AMT1;1 from Arabidopsis and tomato. However, in the paralogue LeAMT1;3, trimeric complexes were not detected. LeAMT1;3 differs from the other AMTs by an unusually short N-terminus, suggesting a role for the N-terminus in oligomer stability. Truncation of the N-terminus in LeAMT1;1 destabilized the trimer and led to loss of functionality when expressed in yeast. Swapping of the N-terminus between LeAMT1;1 and LeAMT1;3 showed that sequences in the N-terminus of LeAMT1;1 are necessary and sufficient for stabilization of the interaction among the subunits. Two N-terminal cysteine residues are highly conserved among AMT1 transporters in plants but are lacking in LeAMT1;3. C3S or C27S variants of LeAMT1;1 showed reduced complex stability, which coincided with lower transport capacity for the substrate analogue methylammonium. Both cysteine-substituted LeAMT1;1 variants showed weaker interactions with the wildtype as determined by a quantitative analysis of the complex stability using the mating-based split-ubiquitin assay. These data indicate that the binding affinity of AMT1 subunits is stabilized by cysteines in the N-terminus and suggest a role for disulphide bridge formation via apoplastic N-terminal cysteine residues.     

Characterization of Arabidopsis AtAMT2, a novel ammonium transporter in plants

We have cloned and characterized the first member of a novel family of ammonium transporters in plants: AtAMT2 from Arabidopsis thaliana. AtAMT2 is more closely related to bacterial ammonium transporters than to plant transporters of the AMT1 family. The protein was expressed and functionally characterized in yeast. AtAMT2 transported ammonium in an energy-dependent manner. In contrast to transporters of the AMT1 family, however, AtAMT2 did not transport the ammonium analogue, methylammonium. AtAMT2 was expressed more highly in shoots than roots and was subject to nitrogen regulation.     

Distinct expression and function of three ammonium transporter genes (OsAMT1;1-1;3) in rice

To study the regulation of ammonium uptake into rice roots, three ammonium transporter genes (OsAMT1;1, 1;2 and 1;3; Oryza sativa ammonium transporter) were isolated and examined. OsAMT1s belong to AMT1 family, containing 11 putative transmembrane-spanning domains. Southern blot analysis and screening of the rice genome database confirmed that with OsAMT1;1-1;3 the complete AMT1 family of rice had been isolated. Heterologous expression of OsAMT1s in the yeast Saccharomyces cerevisiae mutant 31019b showed that all three OsAMT1s exhibit ammonium transport activity. Northern blot analysis showed a distinct expression pattern for the three genes; more constitutive expression in shoots and roots for OsAMT1;1, root-specific and ammonium-inducible expression for OsAMT1;2, and root-specific and nitrogen-derepressible expression for OsAMT1;3. In situ mRNA detection revealed that OsAMT1;2 is expressed in the central cylinder and cell surface of root tips. This gene expression analysis revealed a distinct nitrogen-dependent regulation for AMTs in rice, differing from that in tomato or ARABIDOPSIS:     

A proteomic and phosphoproteomic analysis of Oryza sativa plasma membrane and vacuolar membrane

Proteomic and phosphoproteomic analyses of rice shoot and root tonoplast-enriched and plasma membrane-enriched membrane fractions were carried out to look at tissue-specific expression, and to identify putative regulatory sites of membrane transport proteins. Around 90 unique membrane proteins were identified, which included primary and secondary transporters, ion channels and aquaporins. Primary H(+) pumps from the AHA family showed little isoform specificity in their tissue expression pattern, whereas specific isoforms of the Ca(2+) pump ECA/ACA family were expressed in root and shoot tissues. Several ABC transporters were detected, particularly from the MDR and PDR subfamilies, which often showed expression in either roots or shoots. Ammonium transporters were expressed in root, but not shoot, tissue. Large numbers of sugar transporters were expressed, particularly in green tissue. The occurrence of phosphorylation sites in rice transporters such as AMT1;1 and PIP2;6 agrees with those previously described in other species, pointing to conserved regulatory mechanisms. New phosphosites were found in many transporters, including H(+) pumps and H(+):cation antiporters, often at residues that are well conserved across gene families. Comparison of root and shoot tissue showed that phosphorylation of AMT1;1 and several further transporters may be tissue dependent.     

Homo- and hetero-oligomerization of ammonium transporter-1 NH4 uniporters

In most organisms, high affinity ammonium uptake is catalyzed by members of the ammonium transporter family (AMT/MEP/Rh). A single point mutation (G458D) in the cytosolic C terminus of the plasma membrane transporter LeAMT1;1 from tomato leads to loss of function, although mutant and wild type proteins show similar localization when expressed in yeast or plant protoplasts. Co-expression of LeAMT1;1 and mutant in Xenopus oocytes inhibited ammonium transport in a dominant negative manner, suggesting homo-oligomerization. In vivo interaction between LeAMT1;1 proteins was confirmed by the split ubiquitin yeast two-hybrid system. LeAMT1;1 is isolated from root membranes as a high molecular mass oligomer, converted to a approximately 35-kDa polypeptide by denaturation. To investigate interactions with the LeAMT1;2 paralog, co-localizing with LeAMT1;1 in root hairs, LeAMT1;2 was characterized as a lower affinity NH4+ uniporter. Co-expression of wild types with the respective G458D/G465D mutants inhibited ammonium transport in a dominant negative manner, supporting the formation of heteromeric complexes in oocytes. Thus, in yeast, oocytes, and plants, ammonium transporters are able to oligomerize, which may be relevant for regulation of ammonium uptake.     

Molecular mechanisms of ammonium transport and accumulation in plants

The integral membrane proteins of the ammonium transporter (AMT/Rh) family provide the major route for shuttling ammonium (NH(4)(+)/NH(3)) across bacterial, archaeal, fungal and plant membranes. These proteins are distantly related to the Rh (rhesus) glycoproteins, which are absent in higher plants, but are present in many species, including bacteria and mammals. It appears that the large nitrogen requirement of plants resulted in unique strategies to acquire, capture and/or release ammonium. The biological function of plant ammonium transporters will be discussed and compared to other AMT/Rh proteins.