Superior glucose metabolism supports NH4+ assimilation in wheat to improve ammonium tolerance

The use of slow-release fertilizers and seed-fertilizers cause localized high-ammonium (NH4+) environments in agricultural fields, adversely affecting wheat growth and development and delaying its yield. Thus, it is important to investigate the physiological responses of wheat and its tolerance to NH4+ stress to improve the adaptation of wheat to high NH4+ environments. In this study, the physiological mechanisms of ammonium tolerance in wheat (Triticum aestivum) were investigated in depth by comparative analysis of two cultivars: NH4+-tolerant Xumai25 and NH4+-sensitive Yangmai20. Cultivation under hydroponic conditions with high NH4+ (5 mM NH4+, AN) and nitrate (5 mM NO3-, NN), as control, provided insights into the nuanced responses of both cultivars. Compared to Yangmai20, Xumai25 displayed a comparatively lesser sensitivity to NH4+ stress, as evident by a less pronounced reduction in dry plant biomass and a milder adverse impact on root morphology. Despite similarities in NH4+ efflux and the expression levels of TaAMT1.1 and TaAMT1.2 between the two cultivars, Xumai25 exhibited higher NH4+ influx, while maintaining a lower free NH4+ concentration in the roots. Furthermore, Xumai25 showed a more pronounced increase in the levels of free amino acids, including asparagine, glutamine, and aspartate, suggesting a superior NH4+ assimilation capacity under NH4+ stress compared to Yangmai20. Additionally, the enhanced transcriptional regulation of vacuolar glucose transporter and glucose metabolism under NH4+ stress in Xumai25 contributed to an enhanced carbon skeleton supply, particularly of 2-oxoglutarate and pyruvate. Taken together, our results demonstrate that the NH4+ tolerance of Xumai25 is intricately linked to enhanced glucose metabolism and optimized glucose transport, which contributes to the robust NH4+ assimilation capacity

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

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 NH(3) 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

Regulation of NH(4)(+) 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 NH(4)(+) 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 NH(4)(+) transporter. Mutations at the highly conserved carboxyl terminus (C terminus) of AMTs, including one that mimics phosphorylation at a putative phosphorylation site, impair NH(4)(+) 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 NH(4)(+) transporters may apply to several AMT members to prevent excess influx of cytotoxic ammonium

A Mycorrhizal-Specific Ammonium Transporter from Lotus japonicus Acquires Nitrogen Released by Arbuscular Mycorrhizal Fungi1

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

Yeast filamentation signaling is connected to a specific substrate translocation mechanism of the Mep2 transceptor.

The dimorphic transition from the yeast to the filamentous form of growth allows cells to explore their environment for more suitable niches and is often crucial for the virulence of pathogenic fungi. In contrast to their Mep1/3 paralogues, fungal Mep2-type ammonium transport proteins of the conserved Mep-Amt-Rh family have been assigned an additional receptor role required to trigger the filamentation signal in response to ammonium scarcity. Here, genetic, kinetic and structure-function analyses were used to shed light on the poorly characterized signaling role of Saccharomyces cerevisiae Mep2. We show that Mep2 variants lacking the C-terminal tail conserve the ability to induce filamentation, revealing that signaling can proceed in the absence of exclusive binding of a putative partner to the largest cytosolic domain of the protein. Our data support that filamentation signaling requires the conformational changes accompanying substrate translocation through the pore crossing the hydrophobic core of Mep2. pHluorin reporter assays show that the transport activity of Mep2 and of non-signaling Mep1 differently affect yeast cytosolic pH in vivo, and that the unique pore variant Mep2H194E, with apparent uncoupling of transport and signaling functions, acquires increased ability of acidification. Functional characterization in Xenopus oocytes reveals that Mep2 mediates electroneutral substrate translocation while Mep1 performs electrogenic transport. Our findings highlight that the Mep2-dependent filamentation induction is connected to its specific transport mechanism, suggesting a role of pH in signal mediation. Finally, we show that the signaling process is conserved for the Mep2 protein from the human pathogen Candida albicans

Functional and physiological evidence for a rhesus-type ammonia transporter in Nitrosomonas europaea.

Ammonium transporters form a conserved family of transport proteins and are widely distributed among all domains of life. The genome of Nitrosomonas europaea codes for a single gene (rh1) that belongs to the family of the AMT/Rh ammonium transporters. For the first time, this study provides functional and physiological evidence for a rhesus-type ammonia transporter in bacteria (N. europaea). The methylammonium (MA) transport activity of N. europaea correlated with the Rh1 expression. The K(m) value for the MA uptake of N. europaea was 1.8+/-0.2 mM (pH 7.25), and the uptake was competitively inhibited by ammonium [K(i)(NH(4) (+)) 0.3+/-0.1 mM at pH 7.25]. The MA uptake rate was pH dependent, indicating that the uncharged form of MA is transported by Rh1. An effect of the glutamine synthetase on the MA uptake was not observed. When expressed in Saccharomyces cerevisiae, the function of Rh1 from N. europaea as an ammonia/MA transporter was confirmed. The results suggest that Rh1 equilibrates the uncharged substrate species. A low pH value in the periplasmic space during ammonia oxidation seems to be responsible for the ammonium accumulation functioning as an acid NH(4) (+) trap

Ammonium Uptake, Mediated by Ammonium Transporters, Mitigates Manganese Toxicity in Duckweed, <i>Spirodela polyrhiza</i>

Nitrogen is an essential nutrient that affects all aspects of the growth, development and metabolic responses of plants. Here we investigated the influence of the two major sources of inorganic nitrogen, nitrate and ammonium, on the toxicity caused by excess of Mn in great duckweed, Spirodela polyrhiza. The revealed alleviating effect of ammonium on Mn-mediated toxicity, was complemented by detailed molecular, biochemical and evolutionary characterization of the species ammonium transporters (AMTs). Four genes encoding AMTs in S. polyrhiza, were classified as SpAMT1;1, SpAMT1;2, SpAMT1;3 and SpAMT2. Functional testing of the expressed proteins in yeast and Xenopus oocytes clearly demonstrated activity of SpAMT1;1 and SpAMT1;3 in transporting ammonium. Transcripts of all SpAMT genes were detected in duckweed fronds grown in cultivation medium, containing a physiological or 50-fold elevated concentration of Mn at the background of nitrogen or a mixture of nitrate and ammonium. Each gene demonstrated an individual expression pattern, revealed by RT-qPCR. Revealing the mitigating effect of ammonium uptake on manganese toxicity in aquatic duckweed S. polyrhiza, the study presents a comprehensive analysis of the transporters involved in the uptake of ammonium, shedding a new light on the interactions between the mechanisms of heavy metal toxicity and the regulation of the plant nitrogen metabolism

New insights into HcPTR2A and HcPTR2B, two high-affinity peptide transporters from the ectomycorrhizal model fungus Hebeloma cylindrosporum

International audienceWhile plants mainly rely on the use of inorganic nitrogen sources like ammonium and nitrate, soil-borne microorganisms like the ectomycorrhizal fungus Hebeloma cylindrosporum can also take up soil organic N in the form of amino acids and peptides that they use as nitrogen and carbon sources. Following the previous identification and functional expression in yeast of two PTR-like peptide transporters, the present study details the functions and substrates of HcPTR2A and HcPTR2B by analysing their transport kinetics in Xenopus laevis oocytes. While both transporters mediated high-affinity di- and tripeptide transport, HcPTR2A also showed low-affinity transport of several amino acids-mostly hydrophobic ones with large side chains