Benjamin Neuhäuser

A Mycorrhizal-Specific Ammonium Transporter from Lotus japonicus Acquires Nitrogen Released by Arbuscular Mycorrhizal Fungi

In mycorrhizal associations, the fungal partner assists its plant host by providing nitrogen (N) in addition to phosphate. Arbuscular mycorrhizal (AM) fungi have access to inorganic or organic forms of N and translocate them via arginine from the extra- to the intraradical mycelium, where the N is transferred to the plant without any carbon skeleton. However, the molecular form in which N is transferred, as well as the involved mechanisms, is still under debate. NH4+ seems to be the preferential transferred molecule, but no plant ammonium transporter (AMT) has been identified so far. Here, we offer evidence of a plant AMT that is involved in N uptake during mycorrhiza symbiosis. The gene LjAMT2;2, which has been shown to be the highest up-regulated gene in a transcriptomic analysis of Lotus japonicus roots upon colonization with Gigaspora margarita, has been characterized as a high-affinity AMT belonging to the AMT2 subfamily. It is exclusively expressed in the mycorrhizal roots, but not in the nodules, and transcripts have preferentially been located in the arbusculated cells. Yeast (Saccharomyces cerevisiae) mutant complementation has confirmed its functionality and revealed its dependency on acidic pH. The transport experiments using Xenopus laevis oocytes indicated that, unlike other plant AMTs, LjAMT2;2 transports NH3 instead of NH4+. Our results suggest that the transporter binds charged ammonium in the apoplastic interfacial compartment and releases the uncharged NH3 into the plant cytoplasm. The implications of such a finding are discussed in the context of AM functioning and plant phosphorus 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.

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.

Channel-like NH3 flux by ammonium transporter AtAMT2

Prokaryotes, plants and animals control ammonium fluxes by the regulated expression of ammonium transporters (AMTs) and/or the related Rhesus (Rh) proteins. Plant AMTs were previously reported to mediate electrogenic transport. Functional analysis of AtAMT2 from Arabidopsis in yeast and oocytes suggests that NH 4 + is the recruited substrate, but the uncharged form NH3 is conducted. AtAMT2 partially co‐localized with electrogenic AMTs and conducted methylamine with low affinity. This transport mechanism may apply to other plant ammonium transporters and explains the different capacities of AMTs to accumulate ammonium in the plant cell.

Role of the Npr1 kinase in ammonium transport and signaling by the ammonium permease Mep2 in Candida albicans.

ABSTRACT The ammonium permease Mep2 induces a switch from unicellular yeast to filamentous growth in response to nitrogen limitation in Saccharomyces cerevisiae and Candida albicans. In S. cerevisiae, the function of Mep2 and other ammonium permeases depends on the protein kinase Npr1. Mutants lacking NPR1 cannot grow on low concentrations of ammonium and do not filament under limiting nitrogen conditions. A G349C mutation in Mep2 renders the protein independent of Npr1 and results in increased ammonium transport and hyperfilamentous growth, suggesting that the signaling activity of Mep2 directly correlates with its ammonium transport activity. In this study, we investigated the role of Npr1 in ammonium transport and Mep2-mediated filamentation in C. albicans. We found that the two ammonium permeases Mep1 and Mep2 of C. albicans differ in their dependency on Npr1. While Mep1 could function well in the absence of the Npr1 kinase, ammonium transport by Mep2 was virtually abolished in npr1Δ mutants. However, the dependence of Mep2 activity on Npr1 was relieved at higher temperatures (37°C), and Mep2 could efficiently induce filamentous growth under limiting nitrogen conditions in npr1Δ mutants. Like in S. cerevisiae, mutation of the conserved glycine at position 343 in Mep2 of C. albicans to cysteine resulted in Npr1-independent ammonium uptake. In striking contrast, however, the mutation abolished the ability of Mep2 to induce filamentous growth both in the wild type and in npr1Δ mutants. Therefore, a mutation that improves ammonium transport by Mep2 under nonpermissible conditions eliminates its signaling activity in C. albicans.

Uncoupling of Ionic Currents from Substrate Transport in the Plant Ammonium Transporter AtAMT1;2

Background: The substrate transported by AMT/Rh proteins (NH3/NH4+/NH3+H+) is strongly disputed. Results: In the net NH4+ transporter AtAMT1;2 mutations led to a change in coupling of NH3 and H+ transport. Conclusion: AMT/Rh proteins might share a general mechanism of NH3 transport with different H+ coupling ratios. Significance: Different NH3/H+ coupling ratios in AMT/Rh proteins can explain the contrasting results for their transported substrate (NH3/NH4+/NH3+H+). The ammonium flux across prokaryotic, plant, and animal membranes is regulated by structurally related ammonium transporters (AMT) and/or related Rhesus (Rh) glycoproteins. Several plant AMT homologs, such as AtAMT1;2 from Arabidopsis, elicit ionic, ammonium-dependent currents when expressed in oocytes. By contrast, functional evidence for the transport of NH3 and the lack of coupled ionic currents has been provided for many Rh proteins. Furthermore, despite high resolution structures the transported substrate in many bacterial homologs, such as AmtB from Escherichia coli, is still unclear. In a heterologous genetic screen in yeast, AtAMT1;2 mutants with reduced transport activity were identified based on the resistance of yeast to the toxic transport analog methylamine. When expressed in oocytes, the reduced transport capacity was confirmed for either of the mutants Q67K, M72I,and W145S. Structural alignments suggest that these mutations were dispersed at subunit contact sites of trimeric AMTs, without direct contact to the pore lumen. Surprisingly, and in contrast to the wild type AtAMT1;2 transporter, ionic currents were not associated with the substrate transport in these mutants. Whether these data suggest that the wild type AtAMT1;2 functions as H+/NH3 co-transporter, as well as how the strict substrate coupling with protons is lost by the mutations, is discussed.

Protein Dynamics in Young Maize Root Hairs in Response to Macro- and Micronutrient Deprivation.

Plants increase their root surface with root hairs to improve the acquisition of nutrients from the soil. The unicellular character of root hairs and their position at the root surface make them an attractive system to investigate adaptive processes of rhizodermal cells that are in direct contact with the soil solution. In young maize seedlings, roots are densely covered with root hairs, although nutrient reserves in the seed are sufficient to support seedling growth rates for a few days. We used a label-free quantitative proteomics approach to study protein abundance adjustments in 4 day old root hairs grown in aeroponic culture in the presence and absence of several macro- and micronutrients. Compared to the proteome of root hairs developed under full nutrition, protein abundance changes were observed in pathways related to macronutrient (N, P, K, and Mg) deficiencies. For example, lack of N in the medium repressed the primary N metabolism pathway, increased amino acid synthesis, but repressed their degradation, and affected the primary carbon metabolism, such as glycolysis. Glycolysis was similarly affected by K and P deprivation, but the glycolytic pathway was negatively regulated by the absence of the micronutrients Fe and Zn. In contrast, the deprivation of Mn had almost no affect on the root hair proteome. Our results indicate either that the metabolism of very young root hairs adjusts to cellular nutrient deficiencies that have been already experienced or that root hairs sense the external lack of specific nutrients in the nutrient solution and adjust their metabolism accordingly.

Auxin-modulated root growth inhibition in Arabidopsis thaliana seedlings with ammonium as the sole nitrogen source

Ammonium is the major nitrogen source in some plant ecosystems but is toxic at high concentrations, especially when available as the exclusive nitrogen source. Ammonium stress rapidly leads to various metabolic and hormonal imbalances that ultimately inhibit root and shoot growth in many plant species, including Arabidopsis thaliana (L.) Heynh. To identify molecular and genetic factors involved in seedling survival with prolonged exclusive NH4+ nutrition, a transcriptomic analysis with microarrays was used. Substantial transcriptional differences were most pronounced in (NH4)2SO4-grown seedlings, compared with plants grown on KNO3 or NH4NO3. Consistent with previous physiological analyses, major differences in the expression modules of photosynthesis-related genes, an altered mitochondrial metabolism, differential expression of the primary NH4+ assimilation, alteration of transporter gene expression and crucial changes in cell wall biosynthesis were found. A major difference in plant hormone responses, particularly of auxin but not cytokinin, was striking. The activity of the DR5::GUS reporter revealed a dramatically decreased auxin response in (NH4)2SO4-grown primary roots. The impaired root growth on (NH4)2SO4 was partially rescued by exogenous auxin or in specific mutants in the auxin pathway. The data suggest that NH4+-induced nutritional and metabolic imbalances can be partially overcome by elevated auxin levels.

Regulation of length and density of Arabidopsis root hairs by ammonium and nitrate

Root hairs expand the effective root surface to increase the uptake of nutrients and water from the soil. Here the local effects of the two major nitrogen sources, ammonium and nitrate, on root hairs were investigated using split plates. In three contrasting accessions of A. thaliana, namely Col-0, Tsu-0 and Sha, root hairs were differentially affected by the nitrogen forms and their concentration. Root hairs in Sha were short in the absence of nitrate. In Col-0, hair length was moderately decreased with increasing nitrate or ammonium. In all accessions, the root hair density was insensitive to 1,000-fold changes in the ammonium concentrations, when supplied locally as the exclusive nitrogen form. In contrast, the root hair density generally increased with nitrate as the exclusive local nitrogen source. The nitrate sensitivity was reduced at mM concentrations in a loss-of-function mutant of the nitrate transporter and sensor gene NRT1;1 (NPF6.3). Little differences with respect to ammonium were found in a mutant lacking four high affinity AMT-type ammonium transporters, but interestingly, the response to high nitrate was reduced and may indicate a general defect in nitrogen signaling in that mutant. Genetic diversity and the presence of the nitrogen transceptor NRT1;1 explain heterogeneity in the responses of root hairs to different nitrogen forms in Arabidopsis accessions.

Switching substrate specificity of AMT/MEP/ Rh proteins.

In organisms from all kingdoms of life, ammonia and its conjugated ion ammonium are transported across membranes by proteins of the AMT/Rh family. Efficient and successful growth often depends on sufficient ammonium nutrition. The proteins mediating this transport, the so called Ammonium Transporter (AMT) or Rhesus like (Rh) proteins, share a very similar trimeric overall structure and a high sequence similarity even throughout the kingdoms. Even though structural components of the transport mechanism, like an external substrate recruitment site, an essential twin histidine pore motif, a phenylalanine gate and the hydrophobic pore are strongly conserved and have been analyzed in detail by molecular dynamic simulations and mutational studies, the substrate(s), which pass the central pores of the AMT/Rh subunits, NH4+, NH3 + H+, NH4+ + H+ or NH3, are still a matter of debate for most proteins, including the best characterized AmtB protein from Escherichia coli. The lack of a robust expression system for functional analysis has hampered proof of structural and mutational studies, although the NH3 transport function for Rh-like proteins is rarely disputed. In plant transporters belonging to the subfamily AMT1, transport is associated with electrical currents, while some plant transporters, notably of the AMT2 type, were suggested to transport NH3 across the membrane, without associated ionic currents. Here we summarize data in favor of each substrate for the distinct AMT/Rh classes, discuss mutants and how they differ in structure and functionality. A common mechanism with deprotonation and subsequent NH3 transport through the central subunit pore is suggested.