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Summary Serendipita vermifera ssp. bescii, hereafter referred to as S. bescii, is a root‐associated fungus that promotes plant growth in both its native switchgrass host and a variety of monocots and dicots. Winter wheat (Triticum aestivum L.), a dual‐purpose crop, used for both forage and grain production, significantly contributes to the agricultural economies of the Southern Great Plains, USA. In this study, we investigated the influence of S. bescii on growth and transcriptome regulation of nitrogen (N) and phosphorus (P) metabolism in winter wheat. Serendipita bescii significantly improved lateral root growth and forage biomass under a limited N or P regime. Further, S. bescii activated sets of host genes regulating N and P starvation responses. These genes include, root‐specific auxin transport, strigolactone and gibberellin biosynthesis, degradation of phospholipids and biosynthesis of glycerolipid, downregulation of ammonium transport and nitrate assimilation, restriction of protein degradation by autophagy and subsequent N remobilization. All these genes are hypothesized to regulate acquisition, assimilation and remobilization of N and P. Based on transcriptional level gene regulation and physiological responses to N or P limitation, we suggest S. bescii plays a critical role in modulating stress imposed by limitation of these two critical nutrients in winter wheat.

It is crucial for the growth and development of an organism whether ammonium is transported across its membranes in a form of NH4+ or NH3. The transport of both molecules follows different pH-dependent gradients across membranes and transport of both substrates differentially affects the internal and external pH. As a consequence, they directly influence the physiology and organism development. CaMep2 from Candida albicans shows a dual transceptor function in ammonium transport and sensing. CaMep2 senses low ammonium availability and induces filamentous growth. CaMep1, by contrast, is only active in transport, but not involved in ammonium signaling. Here, both proteins were heterologously expressed in Xenopus laevis oocytes. This study identified electrogenic NH4+ transport by CaMep1 and electroneutral NH3 transport by CaMep2, which might be a prerequisite for the induction of pseudohyphal growth.

Conventional wheat production utilizes fertilizers of various nitrogen forms. Sole ammonium nutrition has been shown to improve grain quality, despite the potential toxic effects of ammonium at elevated concentrations. We therefore investigated the responses of young seedlings of winter wheat to different nitrogen sources (NH4 NO3 = NN, NH4 Cl = NNH4+ and KNO3 = NNO3- ). Growth with ammonium-nitrate was superior. However, an elevated concentration of sole ammonium caused severe toxicity symptoms and significant decreases in biomass accumulation. We addressed the molecular background of the ammonium uptake by gathering an overview of the ammonium transporter (AMT) of wheat (Triticum aestivum) and characterized the putative high-affinity TaAMT1 transporters. TaAMT1;1 and TaAMT1;2 were both active in yeast and Xenopus laevis oocytes and showed saturating high-affinity ammonium transport characteristics. Interestingly, nitrogen starvation, as well as ammonium resupply to starved seedlings triggered an increase in the expression of the TaAMT1s. The presence of nitrate seamlessly repressed their expression. We conclude that wheat showed the ability to respond robustly to sole ammonium supply by adopting distinct physiological and transcriptional responses.

Protein kinase-mediated phosphorylation modulates the absorption of many nutrients in plants. CALCIUM-DEPENDENT PROTEIN KINASES (CPKs) are key players in plant signaling to translate calcium signals into diverse physiological responses. However, the regulatory role of CPKs in ammonium uptake remains largely unknown. Here, using methylammonium (MeA) toxicity screening, CPK32 was identified as a positive regulator of ammonium uptake in roots. CPK32 specifically interacted with AMMONIUM TRANSPORTER 1;1 (AMT1;1) and phosphorylated AMT1;1 at the non-conserved serine residue Ser450 in the C-terminal domain. Functional analysis in Xenopus oocytes showed that co-expression of CPK32 and AMT1;1 significantly enhanced the AMT1;1-mediated inward ammonium currents. In transgenic plants, the phosphomimic variant AMT1;1S450E, but not the non-phosphorylatable variant AMT1;1S450A, fully complemented the MeA insensitivity and restored high-affinity 15NH4+ uptake in both amt1;1 and cpk32 mutants. Moreover, in the CPK32 knockout background, AMT1;1 lost its ammonium transport activity entirely. These results indicate that CPK32 is a crucial positive regulator of ammonium uptake in roots and the ammonium transport activity of AMT1;1 is dependent on CPK32-mediated phosphorylation.

It is an integral property of sorghum (Sorghum bicolor L.) to extensively release biological nitrification inhibitors (BNIs) under NH4+ nutrition, in comparison to NO3− nutrition. Our previous research indicated that plasma membrane (PM) H+-ATPase activity was stimulated by NH4+ and low rhizosphere pH, which in turn provided the driving force for BNIs release from sorghum roots. However, the regulatory mechanism of PM H+-ATPase itself in this regard is not fully elucidated. The present study thus aims at post-translational regulation of PM H+-ATPase via phosphorylation in response to NH4+ nutrition and its functional link to the release of BNIs from sorghum roots. A hydroponic system is used to grow sorghum with 1 mM NH4+ or NO3− as N source at pH 3.0 or pH 7.0 in root medium for the analysis of PM H+-ATPase and BNIs release. The effect of NH4+ on the regulation of PM H+-ATPase was further evaluated by the treatment of NO3−cultivated sorghum roots with different NH4+ concentrations (0.1~1 mM). In addition, fusicoccin (a stimulator of PM H+-ATPase) and vanadate (an inhibitor of PM H+-ATPase) were added to check the effect of PM H+-ATPase phosphorylation on BNIs release. Further, methionine sulphoximine (MSX), which inhibits glutamine synthetase, is used to analyze the effect of ammonium transport/assimilation process on the PM H+-ATPase and BNIs release. Microsomal membrane protein isolated from these roots was used for the test of PM H+-ATPase phosphorylation level by western blot technique. Meanwhile, the root exudates were collected for the analysis of BNIs. Higher amount of PM H+-ATPase protein with higher phosphorylation level were detected in sorghum roots in response to NH4+ and low rhizosphere pH, as compared to NO3− and high pH. Further, PM H+-ATPase protein amount and phosporylation level were dependent on the local supplement of NH4+ (from 0.1 ~ 1 mM) to roots. Nevertheless, the enhanced posphorylation level under all of these treatments was significantly higher than the enhanced protein level of PM H+ ATPase. Unlike protein level, phosphorylation level is closely correlated to the release of BNIs from sorghum roots. In addition, phosphorylation level of PM H+-ATPase adjusted by fusicoccin or vanadate directly affected the release of BNIs, irrespective of the protein level. In addition, ammonium assimilation inhibitor MSX caused decreased phosphorylation level of PM H+-ATPase without affecting the protein level, meanwhile inhibited the release of BNIs from sorghum roots. Our research suggests that phosphorylation of PM H+-ATPase is one of the important regulation mechanisms involved in the release of BNIs from sorghum roots. NH4+ stimulated PM H+-ATPase phosphorylation via excessive H+ generated by NH4+ assimilation in cytoplasm. The up regulation of PM H+-ATPase at post-translational level thus activated the H+ pumping activity to provide the driving force for BNIs release. A new hypothesis is proposed to elucidate the interplay of these functionally inter-linked processes involving ammonium-uptake, −assimilation, and H+-pumps activation in PM on the release of BNIs from sorghum roots.

The controversial arguments on the true substrate in nitritation kinetics might be due to the cells' dual substrate-transport system. Our experiments revealed that, under ammonia-rich environments, it diffused into the membrane (ammonia was the direct substrate); but, under oligotrophic, ammonium ion was actively transported (ammonium was the direct substrate). Facilitating this change-over, the bacterial composition in the sludge was altered, although the predominant was Nitrosomonas eutropha in most of the six chemostats. Also, the substrate affinity constant (Ks) fell resulting in partial compensation for the reduced availability of substrate. When the environmental ammonia concentration was lower than the cytoplasmic one, a backward diffusion appeared to take place, which probably had the cells accelerate its energy-consuming ammonium transport. The % ammonium oxidizing bacteria (AOB) to the total number of bacteria in the sludge remarkably decreased when cells were grown under oligotrophic environments. This could be evidence of the cellular energy dissipation caused by ammonia loss and recovery. Intracellular total ammonium nitrogen (TAN) accumulations were observed, which gradually increased from a basal value of ∼1 M (for AOB grown under copious environments) to much higher values (grown under oligotrophic environment). It did not affect the reaction kinetics but potentially served as a reserve against famine.

Hypoxic conditions, in addition to effects on the CO2 and O2 transport, may have an impact on various functions of red blood cells (RBCs) including ion and metabolic regulation, oxidative stress induction, and transport of nitrites, ammonia, urea, etc. We used laser diffraction, spectrometry, and flow cytometry for evaluation of responses of RBCs of lower vertebrates and human RBCs to hypoosmotic conditions, ammonia, and oxidative stress in normoxia and hypoxia. Under hypoosmotic conditions RBCs of lower vertebrates swell and immediately initiate the regulatory volume decrease (RVD) reaction. In our experiments, the RVD reaction in RBCs of Carassius carassius and Rana temporaria was inhibited by hypoxic conditions and restored by reoxygenation. Transport of ammonia/ammonium in human RBCs is facilitated via functional interaction of ammonium (RhAG) and anion (AE1) transporters. Ammonium transport in human RBCs was inhibited in hypoxic conditions, whereas the switch of hemoglobin from R- to T-state in hypoxia restored the rate of ammonia/ammonium transport. Oxidative stress inhibits esterase activity, triggers hemoglobin oxidation to ferri-forms, Fe(III), Fe(IV), phosphatidylserine externalization, AE1 clusterization, and microparticles formation. In the conditions of oxidative stress, hypoxia prevents cell death via apoptosis by inhibiting phosphatidylserine externalization, AE1 clusterization, and microparticle formation. The obtained results indicate that hemoglobin conformation plays a significant role in the process of volume regulation not only in higher but in lower vertebrates as well. In human RBCs transport of CO2 ($${\text{HCO}}_{3}^{ - }$$) and NH3 ($${\text{NH}}_{4}^{ + }$$) is dependent on the AE1 activity, which is regulated by hemoglobin conformational changes. Hypoxic conditions prevent apoptosis of RBCs by preserving them from oxidative stress. Thus, our data clearly show that hypoxic conditions have beneficial effects on the RBC long-term storage.

First large-scale experiences of bioelectrochemical systems (BES) are underway. However, there is still little knowledge on how the different elements that integrate a BES behave in near real-life conditions. This paper aims at assessing the impact of long-term operation on the cation exchange membrane and on the anodic biofilm of two 16 L Microbial Electrolysis Cells (MEC) designed for hydrogen production and ammonia recovery from pig slurry. Membrane deterioration was examined by physical, chemical and microscopy techniques at different locations, revealing a strong attachment of microorganisms and a significant decay in membrane properties such as ion exchange capacity and thermal stability. Anode microbial communities did not show a dramatic shift in the eubacteria composition at different sampling areas, although the relative abundance of some bacterial groups showed a clear vertical stratification. After 100 days of continuous operation, MEC performance did not declined significantly maintaining ammonium transport rates and H2 production rates of 15.3 gN d−1 m−2 and 0.2 LH2·L−1reactor·d−1 respectively.

The Tibetan Plateau is the world's largest plateau that is characterized by low temperature, low oxygen, and strong ultraviolet radiation. Colonized species that invade the plateau must change their characteristics to adapt to the harsh environment. However, research on the adaptation of introduced species on plateaus is limited. In this study, brown trout (Salmo trutta fario) was studied as a colonized species of the Tibetan Plateau, and a de novo transcriptome assembly was conducted to explore the genetic basis of plateau adaptation. A total of 38,606 unigenes were assembled, with over 90% of the unigenes annotated. Using comparative transcriptomics analysis, we identified 129 positively selected genes and 126 species‐specific gene families in colonized brown trout that were associated with energy metabolism, angiogenesis, heart development, ammonium transport, and DNA damage and repair, which may play essential roles in the adaptation to high altitude of colonized brown trout compared to other inland Salmonidae species. Our results provide an integrated and comprehensive transcriptomic resource for brown trout colonized in Tibet. These results potentially contribute to future studies aiming to identify candidate genes and pathways underlying the genetic basis of adaptation in the Tibetan plateau and assist in the population protection and aquaculture of brown trout in Tibet.

Economic and nature-friendly amendments are needed to increase fertilizer efficiency and crop yield for sustainable agriculture in coarse-textured soil. In this investigation, the effects of different application rates (2%, 4% and 8%) of vermiculite, nanoclay and zeolite amendments on adsorption and transport of ammonium (NH4-N) in sandy loam soil were studied using batch and column experiments. The results of batch experiment indicated that the maximum distribution coefficient (Kd) was obtained in soil amended with zeolite 8%, while minimum Kd was achieved in the control soil. Column experiments revealed that the application of inorganic amendments decreased the leaching of NH4-N from 89.4% in control soil to 77.6%, 70.2%, 52.5%, 85.3%, 60.8%, 38.5%, 12.6% and 2.1% in soil columns amended with vermiculite 2%, 4%, 8%, nanoclay 2%, 4% and zeolite 2%, 4%, 8%, respectively. On the basis of the application rate of inorganic amendments required to retain 40% of injected NH4-N, the effectiveness of zeolite is about 2 and 4 times that of nanoclay and vermiculite, respectively. The HYDRUS-1D program was used to simulate the leaching of NH4-N using the advection–dispersion equation model (ADE) along with batch and fitted Kd. The simulation results indicated that the ADE model with fitted Kd described the behavior of NH4-N transport more suitable than the ADE model using batch Kd (root-mean-square error, RMSE

Several studies have analyzed gene expression profiles in the substantia nigra to better understand the pathological mechanisms causing Parkinson’s disease (PD). However, the concordance between the identified gene signatures in these individual studies was generally low. This might be caused by a change in cell type composition as loss of dopaminergic neurons in the substantia nigra pars compacta is a hallmark of PD. Through an extensive meta-analysis of nine previously published microarray studies, we demonstrated that a big proportion of the detected differentially expressed genes was indeed caused by cyto-architectural alterations due to the heterogeneity in the neurodegenerative stage and/or technical artifacts. After correcting for cell composition, we identified a common signature that deregulated the previously unreported ammonium transport, as well as known biological processes including bioenergetic pathways, response to proteotoxic stress, and immune response. By integrating with protein-interaction data, we shortlisted a set of key genes, such as LRRK2, PINK1, and PRKN known to be related to PD; others with compelling evidence for their role in neurodegeneration, such as GSK3β, WWOX, and VPC; as well as novel potential players in the PD pathogenesis, including NTRK1, TRIM25, ELAVL1. Together, these data showed the importance of accounting for cyto-architecture in these analyses and highlight the contribution of multiple cell types and novel processes to PD pathology providing potential new targets for drug development.Significance StatementThe exploration of the transcriptomic landscape in PD is pivotal for the understanding of the pathological mechanisms of this disease. Nonetheless, little attention has been paid to the influence of cell composition on the transcriptome even though it is known that cyto-architecture undergoes major alterations in neurodegenerative diseases such as PD. Our study signifies that changes in cellular architecture of human substantia nigra in PD have a strong effect on the set of detected differentially expressed genes. By reanalyzing the data and accounting for cell composition, we provide an updated description of deregulated biological processes in PD and nominate a shortlist of PD-associated genes for further investigations.

In plants high affinity transport proteins mediate the essential transport of ammonium across membranes. In Arabidopsis thaliana six of these AMmonium Transporters (AMTs) are encoded by the genome. All of them show a unique expression pattern. While most AMTs are highly expressed in the root, AtAMT1;4 expression is limited to the pollen grains and the pollen tube. Here, we addressed the transport characteristics of AtAMT1;4 in the heterologous Xenopus laevis oocytes system. The transport saturated and showed high affinity for ammonium with a Km value lower than 10 µM. Based on our electrophysiological analysis, we classified AtAMT1;4 as a high affinity ammonium transporter.

Objectives The purpose of this study was to investigate the association and impact of TMEM50A on RH genes activity and function. Background SMP1 is located on chromosome 1p36.11 in the RH gene locus, between the RHD and RHCE gene, where its position may be linked to RH haplotypes and contribute to selective pressures regarding certain RH haplotypes. TMEM50A is encoded by the SMP1 located in the intergenic region of RH, its influence on the function of the RH genes remains unclear. Methods The expression of TMEM50A was regulated by transfection of plasmid and siRNA in K562 cell model. Western blot and real-time PCR were used to detect possible expression changes in the RH. The ammonium transport function of cells was monitored using pH-sensitive dye, while transcriptome sequencing was used to predict the potential function of TMEM50A. Results The overexpression of TMEM50A significantly up-regulated RHCE gene activity (63.56%). The inhibition of TMEM50A resulted in significantly decreased RHCE (41.82%) and RHD expression (27.35%). Compared to control group, there was no significant change in the NH4 + transport function of cells in the overexpressed TMEM50A group. Transcriptome analysis showed that TMEM50A not only affected the transcription of target gene through splicing activities, but also played a role in the development of embryonic nervous system. Conclusions TMEM50A may regulate the expression of RH gene by affecting the stability of RH mRNA through splicing function. It speculates that TMEM50A may play an important role in the development of embryonic nervous system.

SummaryAmmonium uptake at plant roots is regulated at the transcriptional, post-transcriptional and post-translational levels. Phosphorylation by the protein kinase CIPK23 transiently inactivates the ammonium transporters (AMT1s) but the phosphatases activating AMT1s remain unknown. Here, we have identified the PP2C phosphatase ABI1 as an activator of AMTs in Arabidopsis thaliana. We show that high external ammonium concentrations elevate the stress phytohormone abscisic acid (ABA) by de-glycosylation. Active ABA is sensed by ABI1-PYL complexes followed by the inactivation of ABI1 activating CIPK23. Under favourable growth conditions, ABI1 reduces AMT1 phosphorylation, both by binding and inactivating CIPK23, and by the direct dephosphorylation of AMT1s. Thus, ABI1 is a positive regulator of ammonium uptake, coupling nutrient acquisition to abiotic stress signalling. Elevated ABA reduces ammonium uptake during stress situations, such as ammonium toxicity, whereas ABI1 reactivates AMT1s under favourable growth conditions.

Listeria monocytogenes is a fastidious bacterial pathogen that can utilize only a limited number of nitrogen sources for growth. Both glutamine and ammonium are common nitrogen sources used in listerial defined growth media, but little is known about the regulation of their uptake or utilization. The functional role of L. monocytogenes GlnR, the transcriptional regulator of nitrogen metabolism genes in low-G+C Gram-positive bacteria, was determined using transcriptome sequencing and real-time reverse transcription-PCR experiments. The GlnR regulon included transcriptional units involved in ammonium transport (amtB glnK) and biosynthesis of glutamine (glnRA) and glutamate (gdhA) from ammonium. As in other bacteria, GlnR proved to be an autoregulatory repressor of the glnRA operon. Unexpectedly, GlnR was most active during growth with ammonium as the nitrogen source and less active in the glutamine medium, apparently because listerial cells perceive growth with glutamine as a nitrogen-limiting condition. Therefore, paradoxically, expression of the glnA gene, encoding glutamine synthetase, was highest in the glutamine medium. For the amtB glnK operon, GlnR served as both a negative regulator in the presence of ammonium and a positive regulator in the glutamine medium. The gdhA gene was subject to a third mode of regulation that apparently required an elevated level of GlnR for repression. Finally, activity of glutamate dehydrogenase encoded by the gdhA gene appeared to correlate inversely with expression of gltAB, the operon that encodes the other major glutamate-synthesizing enzyme, glutamate synthase. Both gdhA and amtB were also regulated, in a negative manner, by the global transcriptional regulator CodY. IMPORTANCEL. monocytogenes is a widespread foodborne pathogen. Nitrogen-containing compounds, such as the glutamate-containing tripeptide, glutathione, and glutamine, have been shown to be important for expression of L. monocytogenes virulence genes. In this work, we showed that a transcriptional regulator, GlnR, controls expression of critical listerial genes of nitrogen metabolism that are involved in ammonium uptake and biosynthesis of glutamine and glutamate. A different mode of GlnR-mediated regulation was found for each of these three pathways.

Selective removal and resource recovery of ammonium nitrogen (NH4+-N) from high-strength ammonium waste streams is of practical importance for biological wastewater treatment and environmental protection. In this study, we demonstrate the simultaneous removal and reclamation of ammonium from synthetic digested sludge centrate via a novel osmosis-distillation hybrid membrane (ODHM) process. Using NaHCO3 as the draw solute, ammonium diffuses from the synthetic centrate to the draw solution by utilizing the bidirectional cation transport nature of the thin film composite (TFC) membrane. Then, NH4+ is converted to gaseous NH3 at 60 °C and recovered by a sweeping gas membrane distillation (SGMD) process. Herein, the bidirectional transport of monovalent cations in the osmotic process, selectivity of TFC membranes for different cations, and recovery of the draw solution following the extraction of ammonia through the SGMD process were systematically investigated. The removal of NH4+-N from the synthetic centrate achieved 21.34% during a 6-h continuous operation of the ODHM system, with ammonium fluxes through the TFC and SGMD membranes at 1.39 and 0.57 mol m-2 h-1, respectively. A secondary interfacial polymerization was proposed to further enhance ammonium transport through the TFC membrane. Results reported here highlight the potential of the ODHM process for the selective removal and reclamation of ammonium from ammonium-rich waste streams.

Mungbean (Vigna radiata (L.) R. Wilzeck var. radiata) is a protein-rich short-duration legume that fits well as a rotation crop into major cereal production systems of East and South-East Asia. Salinity stress in arid areas affects mungbean, being more of a glycophyte than cereals. A significant portion of the global arable land is either salt or sodium affected. Thus, studies to understand and improve salt-stress tolerance are imminent. Here, we conducted a genome-wide association study (GWAS) to mine genomic loci underlying salt-stress tolerance during seed germination of mungbean. The World Vegetable Center (WorldVeg) mungbean minicore collection representing the diversity of mungbean germplasm was utilized as the study panel and variation for salt stress tolerance was found in this germplasm collection. The germplasm panel was classed into two agro-climatic groups and showed significant differences in their germination abilities under salt stress. A total of 5288 SNP markers obtained through genotyping-by-sequencing (GBS) were used to mine alleles associated with salt stress tolerance. Associated SNPs were identified on chromosomes 7 and 9. The associated region at chromosome 7 (position 2,696,072 to 2,809,200 bp) contains the gene Vradi07g01630, which was annotated as the ammonium transport protein (AMT). The associated region in chromosome 9 (position 19,390,227 bp to 20,321,817 bp) contained the genes Vradi09g09510 and Vradi09g09600, annotated as OsGrx_S16-glutaredoxin subgroup II and dnaJ domain proteins respectively. These proteins were reported to have functions related to salt-stress tolerance.

The transport of charged molecules across biological membranes faces the dual problem of accommodating charges in a highly hydrophobic environment while maintaining selective substrate translocation. This has been the subject of a particular controversy for the exchange of ammonium across cellular membranes, an essential process in all domains of life. Ammonium transport is mediated by the ubiquitous Amt/Mep/Rh transporters that includes the human Rhesus factors. Here, using a combination of electrophysiology, yeast functional complementation and extended molecular dynamics simulations, we reveal a unique two-lane pathway for electrogenic NH4+ transport in two archetypal members of the family, the transporters AmtB from Escherichia coli and Rh50 from Nitrosomonas europaea. The pathway underpins a mechanism by which charged H+ and neutral NH3 are carried separately across the membrane after NH4+ deprotonation. This mechanism defines a new principle of achieving transport selectivity against competing ions in a biological transport process., SCOPUS: ar.j, info:eu-repo/semantics/published

The biggest challenge to apply Anammox to treat wastewater with elevated salt content is the inhibitory effect of salinity on freshwater Anammox bacteria (FAB). Most of the research into salinity inhibition has focused on the osmotic pressure effect, while the inhibitory effect and its mechanisms induced by ion composition are poorly understood. In this study, the individual and combined effect of NaCl, KCl and Na2SO4 on FAB (>99% belonging to Ca. Brocadia genera) were systematically investigated by batch tests. The corresponding responses of mRNA abundance of three functional genes (including nitrite reductase gene (nirS), hydrazine synthase gene (hzsB) and hydrazine dehydrogenase gene (hdh)) under different salt conditions were analyzed. The results indicated that NaCl, KCl and Na2SO4 have different inhibition effects, with the 50% inhibition at 0.106, 0.096 and 0.063 M, respectively. The combined inhibition of NaCl+KCl and NaCl+Na2SO4 on FAB were both synergistic; while the combined inhibition of NaCl+KCl+Na2SO4 was additive. The responses of mRNA (of genes: nirS, hzsB and hdh) suggested NaCl inhibited the transport of ammonium; Na2SO4 inhibited both nitrite and ammonium transport; high salinity inhibited functional enzyme activity. These results suggest both ionic stress and ion composition contributed to the observed inhibition.

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., Author summary Fungal Mep2-type ammonium transport proteins of the conserved Mep-Amt-Rh family that includes human Rhesus factors are specifically required to allow filamentation in response to ammonium limitation. These proteins were therefore assigned a receptor role while the underlying mechanism of signal transduction remains poorly understood. The “transceptor” property has subsequently been proposed to concern transporters of all kind of micro- and macro- nutrients in eukaryotes, from fungi to human. However, bringing the firm demonstration of their existence remains challenging as variants with full uncoupling of transport and receptor functions are difficult to obtain. Our data question the involvement of the C-terminal extremity of Saccharomyces cerevisiae Mep2 in the signal mediation leading to filamentation. If signaling partners exist, they should also bind to cytosolic loops and/or membrane-embedded domains. The capacity of Mep2 to enable filamentation is closely intertwined to the mechanism of substrate translocation through the pore of the hydrophobic core of the protein. In Xenopus oocytes, the transport activity of non-signaling Mep1 is electrogenic while it is electroneutral for Mep2, the latter likely translocating the weak base NH3, but not the proton released after NH4+ recognition and depronotation. We propose that given consequences of a Mep2-specific transport process, such as an intracellular pH modification, could be the underlying cause of the filamentation signal ensured by Mep2-type proteins.

Ammonium transporters (AMT), methylamine permeases (Mep), and the more distantly related rhesus factors (Rh) are trimeric membrane proteins present in all domains of life. AMT/Mep/Rhs are highly selective membrane proteins required for ammonium uptake or release, and they efficiently exclude the similarly sized K(+) ion. Previously reported crystal structures have revealed that each transporter subunit contains a unique hydrophobic but occluded central pore, but it is unclear whether the base (NH(3)) or NH(3) coupled with an H(+) are transported. Here, using expression of two plant AMTs (AtAMT1;2 and AMT2) in budding yeast, we found that systematic replacements in the conserved twin-histidine motif, a hallmark of most AMT/Mep/Rh, alter substrate recognition, transport capacities, N isotope selection, and selectivity against K(+). AMT-specific differences were found for histidine variants. Variants that completely lost ammonium N isotope selection, a feature likely associated with NH(4)(+) deprotonation during passage, substantially transported K(+) in addition to NH(4)(+). Of note, the twin-histidine motif was not essential for ammonium transport. However, it conferred key AMT features, such as high substrate affinity and selectivity against alkali cations via an NH(4)(+) deprotonation mechanism. Our findings indicate that the twin-His motif is the core structure responsible for substrate deprotonation and isotopic preferences in AMT pores and that decreased deprotonation capacity is associated with reduced selectivity against K(+). We conclude that optimization for ammonium transport in plant AMT represents a compromise between substrate deprotonation for optimal selectivity and high substrate affinity and transport rates.

The nutrient loss caused by soil erosion is the main reason for soil degradation and environmental pollution, and polyacrylamide (PAM) as a common soil amendment has a great influence on runoff and erosion processes at the slope. In order to investigate the mechanism of nutrient transport with runoff, a field experiment was conducted and a simple mathematical model was developed in this study. Four PAM application rates (0, 1, 2, and 4 g·m−2) and two rainfall intensities (50 and 80 mm·h−1) were applied in the field experiment. The results revealed that runoff rate of 2 g·m−2 PAM application treatments decreased by 5.3%-10.6% compared with the control groups, but it increased by10.9%-18.7% at 4 g·m−2 PAM application treatments. Polyacrylamide application reduced ammonium nitrogen concentrations of runoff by 10.0% to 44.3% relative to the control groups. The best performance with correlation coefficient (R2) and Nash–Sutcliffe efficiency (NSE) showed that the ammonium transport with runoff could be well described by the proposed model. Furthermore, the model parameter of the depth of the mixing layer (hm) linearly increased with an increase in flow velocity, but exponentially decreased with an increase in PAM application rate.