Ammonium Transport Research Articles

Molecular insights into the Darwin paradox of coral reefs from the sea anemone Aiptasia

Symbiotic cnidarians such as corals and anemones form highly productive and biodiverse coral reef ecosystems in nutrient-poor ocean environments, a phenomenon known as Darwin's paradox. Resolving this paradox requires elucidating the molecular bases of efficient nutrient distribution and recycling in the cnidarian-dinoflagellate symbiosis. Using the sea anemone Aiptasia, we show that during symbiosis, the increased availability of glucose and the presence of the algae jointly induce the coordinated up-regulation and relocalization of glucose and ammonium transporters. These molecular responses are critical to support symbiont functioning and organism-wide nitrogen assimilation through glutamine synthetase/glutamate synthase-mediated amino acid biosynthesis. Our results reveal crucial aspects of the molecular mechanisms underlying nitrogen conservation and recycling in these organisms that allow them to thrive in the nitrogen-poor ocean environments.

Hiat1 as a new transporter involved in ammonia regulation

The orphan transporter hippocampus-abundant transcript 1 (Hiat1) was first identified in the mammalian brain. Its specific substrate specificity, however, has not been investigated to date. Here, we identified and analyzed Hiat1 in a crustacean, the green crab Carcinus maenas. Our phylogenetic analysis showed that Hiat1 protein is conserved at a considerable level between mammals and this invertebrate (ca. 78% identical and conserved amino acids). Functional expression of Carcinus maenas Hiat1 in Xenopus laevis oocytes demonstrated the capability to transport ammonia (likely NH4+) in a sodium-dependent manner. Furthermore, applying quantitative polymerase chain reaction, our results indicated a physiological role for Carcinus maenas Hiat1 in ammonia homeostasis, as mRNA abundance increased in posterior gills in response to elevated circulating hemolymph ammonia upon exposure to high environmental ammonia. Its ubiquitous mRNA expression pattern also suggests an essential role in general cellular detoxification of ammonia. Overall, our results introduce a new ubiquitously expressed ammonia transporter, consequently demanding revision of our understanding of ammonia handling in key model systems from mammalian kidneys to crustacean and fish gills.

Molecular mechanism of adsorbing triclocarban by the activated sludge in wastewater treatment systems

The ubiquitous use of antimicrobial triclocarban (TCC) has raised widespread concern about the potential impacts on wastewater treatment systems, the environment, and the human population. As important constituents of activated sludge secretions, extracellular polymeric substances (EPS) play essential roles in removing micropollutants during wastewater treatment. Here, the adsorption of TCC by sludge and EPS in a multistage biological process was investigated. The EPS was enriched upon TCC stress, and the adsorption by sludge and EPS showed multilayer heterogeneity. The adsorption constant Kf (6.3794–7.6061) of EPS was over 10 times higher than that of sludge (0.5661–0.6200) in all stages, potentially owing to the loose multilayer structure and substantial functional groups (tryptophan-protein as the main binding site) in EPS. TCC significantly inhibited the biological removal of ammonia nitrogen. Notably, TCC once reaching the cell surface, formed a stable complex with ammonia transporter (Amt, binding energy: −73.1 ± 16.6 kJ·mol−1) and bound to the selectivity filter vestibule of Amt (tryptophan 144 and serine 227), inhibiting the transport of ammonia. Our work drew molecular-level insights into the interaction between TCC and activated sludge, and evaluated the adverse impacts on treatment performance through an in silico approach, providing new information on the fate and risk of TCC in wastewater treatment systems.

Insights into Adaptive Regulation of the Leaf-Petiole System: Strategies for Survival of Water Lily Plants under Salt Stress.

The water lily (Nymphaea tetragona) is an ancient angiosperm that belongs to the Nymphaeaceae family. As a rooted floating-leaf plant, water lilies are generally cultivated in fresh water, therefore, little is known about their survival strategies under salt stress. Long-term salt stress causes morphological changes, such as the rapid regeneration of floating leaves and a significant decrease in leaf number and surface area. We demonstrate that salt stress induces toxicity soon after treatment, but plants can adapt by regenerating floating leaves that are photosynthetically active. Transcriptome profiling revealed that ion binding was one of the most-enriched GO terms in leaf-petiole systems under salt stress. Sodium-transporter-related genes were downregulated, whereas K+ transporter genes were both up- and downregulated. These results suggest that restricting intracellular Na+ importing while maintaining balanced K+ homeostasis is an adaptive strategy for tolerating long-term salt stress. ICP-MS analysis identified the petioles and leaves as Na-hyperaccumulators, with a maximum content of over 80 g kg-1 DW under salt stress. Mapping of the Na-hyperaccumulation trait onto the phylogenetic relationships revealed that water lily plants might have a long evolutionary history from ancient marine plants, or may have undergone historical ecological events from salt to fresh water. Ammonium transporter genes involved in nitrogen metabolism were downregulated, whereas NO3--related transporters were upregulated in both the leaves and petioles, suggesting a selective bias toward NO3- uptake under salt stress. The morphological changes we observed may be due to the reduced expression of genes related to auxin signal transduction. In conclusion, the floating leaves and submerged petioles of the water lily use a series of adaptive strategies to survive salt stress. These include the absorption and transport of ions and nutrients from the surrounding environments, and the ability to hyperaccumulate Na+. These adaptations may serve as the physiological basis for salt tolerance in water lily plants.

Genomic and metabolic properties of Staphylococcus gallinarum FCW1 MCC4687 isolated from naturally fermented coconut water towards GRAS assessment

Staphylococcus gallinarum FCW1 was isolated from naturally fermented coconut-water and identified by biochemical and molecular methods. Probiotic characterization and safety assessment were conducted through a series of in vitro tests. A high survival rate was observed when the strain was tested for resistance to bile, lysozyme, simulated gastric and intestinal fluid, phenol, and different temperature and salt concentrations. The strain showed antagonism against some pathogens, was susceptible to all antibiotics tested except penicillin, and showed no hemolytic and DNase activity. Hydrophobicity, autoaggregation, biofilm formation, and antioxidation tests indicated that the strain possessed a high adhesive and antioxidant ability. Enzymatic activity was used to evaluate the metabolic capacities of the strain. In-vivo experiment on zebrafish was performed to check its safety status. The whole-genome sequencing indicated that the genome contained 2,880,305 bp with a GC content of 33.23%. The genome annotation confirmed the presence of probiotic-associated genes and genes for oxalate degradation, sulfate reduction, acetate metabolism, and ammonium transport in the FCW1 strain, adding to the theory that this strain may be helpful in treating kidney stones. This study revealed that the strain FCW1 might be an excellent potential probiotic in developing fermented coconut beverages and treating and preventing kidney stone disease.

Effects of Carbon-Based Fertilizer on Maize Root Morphology, Root Bleeding Rate and Components in Northeast China

Maize (Zea mays L.) is the largest grain crop in Heilongjiang Province. Carbon-based fertilizer is a mixed fertilizer produced by adding a certain proportion of chemical fertilizer with biochar as the loading substrate. In this study, the effects of carbon-based fertilizer on the rhizosphere soil microenvironment and maize root system were discussed. Two maize varieties, Xianyu 335 and Jingke 968, were selected and six treatments were set as follows, including no fertilization (CK1), conventional fertilizer (CK2) and the amount of carbon-based fertilizer, which were 3 t/hm2, 3.75 t/hm2, 4.5 t/hm2 and 5.25 t/hm2, respectively. The results showed that carbon-based fertilizer increased the total root length, root volume, root area and root tip number of maize, and the root length, root volume, root area and root tip number of 4.5 t treatment performed better at all stages, which was significantly higher than that of chemical fertilizer. On 16 August (early filling stage), most of the root color changed from milky white to dark brown, the root clarity decreased, the number of roots decreased, the root volume significantly decreased and the root began to age, while the number and volume of roots treated with the carbon-based fertilizer remained stable, the root color was milky white, the morphological structure was clear and there was basically no aging. The carbon-based fertilizer treatment significantly increased the root biomass of 0–15 cm above the plant, 15–30 cm and 30–45 cm between the plants and 0–15 cm between the ridges, forming a wide and deep high-yield root system. The carbon-based fertilizer significantly increased the bleeding rate. On 8 July (jointing stage), Xianyu 335 and Jingke 968 reached the maximum value at the 3 t and 3.75 t treatments, respectively. The carbon-based fertilizer treatment had no significant effect on the amino acid content, but significantly increased the amino acid transport rate on 8 July (jointing stage) and 16 August (early filling stage). The transport rate of inorganic phosphorus gradually decreased with the advancement of the growth process. On 8 July (jointing stage), the ammonium nitrogen content and transport rate of the two varieties reached the maximum value at the treatment of 4.5 t and 3.75 t, which was significantly higher than the treatment of chemical fertilizer and no fertilizer, and showed a gradual downward trend with the advancement of the growth process. The soluble sugar content was relatively low in the early stage and increased rapidly on 4 September (waxy ripening stage). Both varieties reached the maximum value at 4.5 t treatment, and the transport rate reached the maximum value at 3.75 t treatment, which was significantly higher than that of the chemical fertilizer treatment. In conclusion, the carbon-based fertilizer significantly increased the yield of maize, and the yield of maize under the 4.5 t treatment reached the maximum, which was 15.02% and 18.24% higher than that of the chemical fertilizer treatment, respectively.

Genome-wide identification, structural and evolutionary characteristics, and expression analysis of aquaporin gene family members in Mercenaria mercenaria

Aquaporins (AQPs) are highly-selective transmembrane water transporters that are involved in the adaptation to environmental challenges. However, the structure, function, and evolution of AQPs in bivalves remain largely unknown. In this study, AQP gene family members were identified in nine bivalve species, and their abundance rangs from 7 to 15. Nine AQPs (MmAQPs) were identified in the genome of hard clam (Mercenaria mercenaria), which is a euryhaline bivalve that has evolved sophisticated osmoregulatory mechanisms and salinity adaptation. Structurally, all MmAQPs contain 6 or 12 transmembrane α-helices, a major intrinsic protein (MIP) domain, and 2 asparagine-proline-alanine (NPA) motifs. MmAQPs were classified into three subfamilies based on phylogenetic analysis: AQP1-like, AQP3-like, and AQP8-like. No AQP11-like subfamily member was identified in the genome of hard clam. Tandem duplication resulted in a lineage-specific expansion in AQP8-like subfamily in hard clams. MmAQP8 genes showed different expression sensitivity to different environmental stressors. The gene expression patterns of three MmAQP8 were similar under heat, hypoxia, and air exposure stress, but differed greatly under salinity stress, indicating that tandem duplication events may accelerate the functional divergence of AQP8 genes in hard clams. AQP3-like members may have undergone gene loss during evolution, resulting in weakened glycerol and urea penetration in hard clams. Three orthologs of MmAQPs were detected in the genomes of Cyclina sinensis and Archivesica marissinica through synteny analyses. Tissue expression profiles showed that MmAQP genes were highly expressed in the foot and hepatopancreas. Under environmental stress, the expression levels of most of the MmAQP genes changed significantly to maintain metabolic homeostasis. Several MmAQP genes were downregulated to reduce water permeability under salinity and air exposure stress. Several MmAQP genes were significantly upregulated to promote the transmembrane transport of ammonia and reactive oxygen species and activate anti-apoptotic responses to resist stress. This study provides a comprehensive understanding of the AQP gene family in hard clams, and lays a foundation for further studies to explore the functions of AQPs in bivalves.

Genome-Wide Identification and Characterization of Ammonium Transporter (AMT) Genes in Rapeseed (Brassica napus L.)

Ammonium transporters (AMTs) are plasma membrane proteins mediating ammonium uptake and transport. As such, AMTs play vital roles in ammonium acquisition and mobilization, plant growth and development, and stress and pathogen defense responses. Identification of favorable AMT genotypes is a prime target for crop improvement. However, to date, systematic identification and expression analysis of AMT gene family members has not yet been reported for rapeseed (Brassica napus L.). In this study, 20 AMT genes were identified in a comprehensive search of the B. napus genome, 14 members of AMT1 and 6 members of AMT2. Tissue expression analyses revealed that the 14 AMT genes were primarily expressed in vegetative organs, suggesting that different BnaAMT genes might function in specific tissues at the different development stages. Meanwhile, qRT-PCR analysis found that several BnaAMTs strongly respond to the exogenous N conditions, implying the functional roles of AMT genes in ammonium absorption in rapeseed. Moreover, the rapeseed AMT genes were found to be differentially regulated by N, P, and K deficiency, indicating that crosstalk might exist in response to different stresses. Additionally, the subcellular localization of several BnaAMT proteins was confirmed in Arabidopsis protoplasts, and their functions were studied in detail by heterologous expression in yeast. In summary, our studies revealed the potential roles of BnaAMT genes in N acquisition or transportation and abiotic stress response and could provide valuable resources for revealing the functionality of AMTs in rapeseed.

Transcriptomics reveals the effect of ammonia nitrogen concentration on Pseudomonas stutzeri F2 assimilation and the analysis of amtB function

The biological treatment of wastewater with high concentrations of ammonia nitrogen has become a hot research issue, but there are limited reports on the mechanism of ammonia nitrogen utilization by microorganisms. In this paper, a transcriptomic approach was used to investigate the differences in gene expression at 500.0 mg/L (Amo 500) and 100.0 mg/L (Amo 100) ammonium concentrations to reveal the mechanism of ammonia nitrogen removal from water by Pseudomonas stutzeri F2. The transcriptome data showed 1015 (459 up-regulated and 556 down-regulated) differentially expressed genes with functional gene annotation related to nitrogen source metabolism, glycolysis, tricarboxylic acid cycle, extracellular polysaccharide synthesis, energy conversion and transmembrane transport, revealing the metabolic process of ammonium nitrogen conversion to biological nitrogen in P. stutzeri F2 through assimilation. To verify the effect of ammonium transporter protein (AmtB) of cell membrane on assimilation, a P. stutzeri F2-ΔamtB mutant strain was obtained by constructing a knockout plasmid (pK18mobsacB-ΔamtB), and it was found that the growth characteristics and ammonium removal rate of the mutant strain were significantly reduced at high ammonium concentration. The carbon source components and dissolved oxygen conditions were optimized after analyzing the transcriptome data, and the ammonium removal rate was increased from 41.23% to 94.92% with 500.0 mg/L ammonium concentration. The study of P. stutzeri F2 transcript level reveals the mechanism of ammonia nitrogen influence on microbial assimilation process and improvement strategy, which provides a new strategy for the treatment of ammonia nitrogen wastewater.

Distinct influence of conventional and biodegradable microplastics on microbe-driving nitrogen cycling processes in soils and plastispheres as evaluated by metagenomic analysis

Plastic mulching is one of the large contributors to microplastic (MP) accumulation in agricultural landscapes. However, the effects of conventional (PE-MPs) and biodegradable MPs (BMPs) on microbial functional and genomic information encoding nitrogen (N) cycling have yet to be addressed. Here, a soil microcosmic experiment was conducted by adding PE-MPs and BMPs to a Mollisol at dosage of 5% (w/w) followed by incubation for 90 days. The soils and MPs were examined by metagenomics and genome binning methods. The results revealed that BMPs harbored rougher surfaces and induced stronger alterations in microbial functional and taxonomic profiles in the soil and plastisphere than PE-MPs. In comparison to their respective soils, the plastispheres of PE-MPs and BMPs stimulated the processes of N fixation, N degradation and assimilatory nitrate reduction (ANRA) and reduced the gene abundances encoding nitrification and denitrification, in which BMPs induced stronger influences than PE-MPs. Ramlibacter mainly drove the differences in N cycling processes between the soils containing two types of MPs and was further enriched in the BMP plastisphere. Three high-quality genomes were identified as Ramlibacter stains with higher abundances in the plastisphere of BMP than that of PE-MP. These Ramlibacter strains had the metabolic capacities of N fixation, N degradation, ANRA and ammonium transport, which were potentially attributed to their biosynthesis and the accumulation of soil NH4+-N. Taken together, our results highlight the genetic mechanisms of soil N bioavailability in the presence of biodegradable MPs, which have important implications for maintaining sustainable agriculture and controlling microplastic risk.

Synonymous and Nonsynonymous Substitutions in Dictyostelium discoideum Ammonium Transporter amtA Are Necessary for Functional Complementation in Saccharomyces cerevisiae.

Ammonium transporters are present in all three domains of life. They have undergone extensive horizontal gene transfer (HGT), gene duplication, and functional diversification and therefore offer an excellent paradigm to study protein evolution. We attempted to complement a mep1Δmep2Δmep3Δ strain of Saccharomyces cerevisiae (triple-deletion strain), which otherwise cannot grow on ammonium as a sole nitrogen source at concentrations of <3 mM, with amtA of Dictyostelium discoideum, an orthologue of S. cerevisiae MEP2. We observed that amtA did not complement the triple-deletion strain of S. cerevisiae for growth on low-ammonium medium. We isolated two mutant derivatives of amtA (amtA M1 and amtA M2) from a PCR-generated mutant plasmid library that complemented the triple-deletion strain of S. cerevisiae. amtA M1 bears three nonsynonymous and two synonymous substitutions, which are necessary for its functionality. amtA M2 bears two nonsynonymous substitutions and one synonymous substitution, all of which are necessary for functionality. Interestingly, AmtA M1 transports ammonium but does not confer methylamine toxicity, while AmtA M2 transports ammonium and confers methylamine toxicity, demonstrating functional diversification. Preliminary biochemical analyses indicated that the mutants differ in their conformations as well as their mechanisms of ammonium transport. These intriguing results clearly point out that protein evolution cannot be fathomed by studying nonsynonymous and synonymous substitutions in isolation. The above-described observations have significant implications for various facets of biological processes and are discussed in detail. IMPORTANCE Functional diversification following gene duplication is one of the major driving forces of protein evolution. While the role of nonsynonymous substitutions in the functional diversification of proteins is well recognized, knowledge of the role of synonymous substitutions in protein evolution is in its infancy. Using functional complementation, we isolated two functional alleles of the D. discoideum ammonium transporter gene (amtA), which otherwise does not function in S. cerevisiae as an ammonium transporters. One of them is an ammonium transporter, while the other is an ammonium transporter that also confers methylammonium (ammonium analogue) toxicity, suggesting functional diversification. Surprisingly, both alleles require a combination of synonymous and nonsynonymous substitutions for their functionality. These results bring out a hitherto-unknown pathway of protein evolution and pave the way for not only understanding protein evolution but also interpreting single nucleotide polymorphisms (SNPs).

Genome-wide identification, expression profiling, and functional analysis of ammonium transporter 2 (AMT2) gene family in cassava (Manihot esculenta crantz).

Background: Nitrogen (N), absorbed primarily as ammonium (NH4 +) from soil by plant, is a necessary macronutrient in plant growth and development. Ammonium transporter (AMT) plays a vital role in the absorption and transport of ammonium (NH4 +). Cassava (Manihot esculenta Crantz) has a strong adaptability to nitrogen deprivation. However, little is known about the functions of ammonium transporter AMT2 in cassava. Methods: The cassava AMT2-type genes were identified and their characteristics were analyzed using bioinformatic techniques. The spatial expression patterns were analyzed based on the public RNA-seq data and their expression profiles under low ammonium treatment were studied using Real-time quantitative PCR (RT-qPCR) method. The cassava AMT2 genes were transformed into yeast mutant strain TM31019b by PEG/LiAc method to investigate their functions. Results: Seven AMT2-type genes (MeAMT2.1-2.7) were identified in cassava and they were distributed on 6 chromosomes and included two segmental duplication events (MeAMT2.2/MeAMT2.4 and MeAMT2.3/MeAMT2.5). Based on their amino acid sequences, seven MeAMT2 were further divided into four subgroups, and each subgroup contained similar motif constitution and protein structure. Synteny analysis showed that two and four MeAMT2 genes in cassava were collinear with those in the Arabidopsis and soybean genomes, respectively. Sixteen types of cis-elements were identified in the MeAMT2 promoters, and they were related to light-, hormone-, stress-, and plant growth and development-responsive elements, respectively. Most of the MeAMT2 genes displayed tissue-specific expression patterns according to the RNA-seq data, of them, three MeAMT2 (MeAMT2.3, MeAMT2.5, and MeATM2.6) expressions were up-regulated under ammonium deficiency. Complementation experiments showed that yeast mutant strain TM31019b transformed with MeAMT2.3, MeAMT2.5, or MeATM2.6 grew better than untransgenic yeast cells under ammonium deficiency, suggesting that MeAMT2.3, MeAMT2.5, and MeATM2.6 might be the main contributors in response to ammonium deficiency in cassava. Conclusion: This study provides a basis for further study of nitrogen efficient utilization in cassava.

Genome-Wide Identification and Expression Analysis of the Ammonium Transporter Family Genes in Soybean.

Ammonium transporters (AMTs) are responsible for ammonium absorption and utilization in plants. As a high-nitrogen-demand crop and a legume, soybean can also obtain ammonium from symbiotic root nodules in which nitrogen-fixing rhizobia convert atmospheric nitrogen (N2) into ammonium. Although increasing evidence implicates vital roles of ammonium transport in soybean, no systematic analyses of AMTs in soybean (named GmAMTs) or functional analyses of GmAMTs are available. In this study, we aimed to identify all GmAMT family genes and gain a better understanding of the characteristics of GmAMT genes in soybean. Here, due to the improved genome assembly and annotation of soybean, we tried to generate a phylogenetic tree of 16 GmAMTs based on new information. Consistent with reported data, GmAMT family members can be divided into two subfamilies of GmAMT1 (6 genes) and GmAMT2 (10 genes). Interestingly, unlike Arabidopsis, which has only one AMT2, soybean has substantially increased the number of GmAMT2s, suggesting enhanced demand for ammonium transport. These genes were distributed on nine chromosomes, of which GmAMT1.3, GmAMT1.4, and GmAMT1.5 were three tandem repeat genes. The gene structures and conserved protein motifs of the GmAMT1 and GmAMT2 subfamilies were different. All the GmAMTs were membrane proteins with varying numbers of transmembrane domains ranging from 4 to 11. Promoter analysis found that these GmAMT genes have phytohormone-, circadian control-, and organ expression-related cis-elements in their promoters, and notably, there were nodulation-specific and nitrogen-responsive elements in the promoters of the GmAMT1 and GmAMT2 genes. Further expression data showed that these GmAMT family genes exhibited different spatiotemporal expression patterns across tissues and organs. In addition, GmAMT1.1, GmAMT1.2, GmAMT2.2, and GmAMT2.3 were responsive to nitrogen treatment, while GmAMT1.2, GmAMT1.3, GmAMT1.4, GmAMT1.5, GmAMT1.6, GmAMT2.1, GmAMT2.2, GmAMT2.3, GmAMT3.1, and GmAMT4.6 showed circadian rhythms in transcription. RT-qPCR validated the expression patterns of GmAMTs in response to different forms of nitrogen and exogenous ABA treatments. Gene expression analysis also confirmed that GmAMTs are regulated by key nodulation gene GmNINa, indicating a role of GmAMTs in symbiosis. Together, these data indicate that GmAMTs may differentially and/or redundantly regulate ammonium transport during plant development and in response to environmental factors. These findings provide a basis for future research on the functions of GmAMTs and the mechanisms through which GmAMTs regulate ammonium metabolism and nodulation in soybean.

Functional characterization of PsAMT1.1 from Populus simonii in ammonium transport and its role in nitrogen uptake and metabolism

Ammonium (NH4+) is an important inorganic nitrogen (N) form essential for plant growth. The role of ammonium transporters (AMTs) in plant growth and how AMTs mediate NH4+ uptake and regulate N metabolism remain vague. To explore the role of AMT1.1 in poplar, PsAMT1.1 was cloned from Populus simonii and was functionally characterized in NH4+ uptake, metabolism and poplar growth. PsAMT1.1 was highly expressed in fine roots and old leaves of P. simonii and PsAMT1.1 protein was located on the plasma membrane. PsAMT1.1 mediates NH4+ uptake in yeast mutants and restores growth with a high-affinity ammonium transporter for NH4+ (Km=79.27 ± 9.5 μM). Transgenic poplar overexpressing PsAMT1.1 showed higher plant height, increased stem diameter, net photosynthetic rate and total biomass than the wild type (WT) (Populus tremula × Populus alba INRA 717-IB4 genotype) after 96 days' of low nitrogen stress. The NH4+ concentration, glutamine concentration, total amino acid and total N content were markedly raised in the roots of PsAMT1.1 overexpression poplar in comparison to WT under low N stress. At the same time, compared with WT, the activities of NR, NiR, GS and GOGAT in both roots and leaves of transgenic poplar overexpressing PsAMT1.1 were significantly increased when the external N was very low. Most of the investigated N-metabolism related genes were upregulated in the roots and leaves of PsAMT1.1-overexpression poplar when N-limitation conditions. In short, PsAMT1.1 has a vital role on nitrogen absorption and metabolism and could increase poplar growth.

Effects of demand-feeding and dietary protein level on nitrogen metabolism and symbiont dinitrogen gas production of common carp (Cyprinus carpio, L.).

Ammonia accumulation is a major challenge in intensive aquaculture, where fish are fed protein-rich diets in large rations, resulting in increased ammonia production when amino acids are metabolized as energy source. Ammonia is primarily excreted via the gills, which have been found to harbor nitrogen-cycle bacteria that convert ammonia into dinitrogen gas (N2) and therefore present a potential in situ detoxifying mechanism. Here, we determined the impact of feeding strategies (demand-feeding and batch-feeding) with two dietary protein levels on growth, nitrogen excretion, and nitrogen metabolism in common carp (Cyprinus carpio, L.) in a 3-week feeding experiment. Demand-fed fish exhibited significantly higher growth rates, though with lower feed efficiency. When corrected for feed intake, nitrogen excretion was not impacted by feeding strategy or dietary protein, but demand-fed fish had significantly more nitrogen unaccounted for in the nitrogen balance and less retained nitrogen. N2 production of individual fish was measured in all experimental groups, and production rates were in the same order of magnitude as the amount of nitrogen unaccounted for, thus potentially explaining the missing nitrogen in the balance. N2 production by carp was also observed when groups of fish were kept in metabolic chambers. Demand feeding furthermore caused a significant increase in hepatic glutamate dehydrogenase activities, indicating elevated ammonia production. However, branchial ammonia transporter expression levels in these animals were stable or decreased. Together, our results suggest that feeding strategy impacts fish growth and nitrogen metabolism, and that conversion of ammonia to N2 by nitrogen cycle bacteria in the gills may explain the unaccounted nitrogen in the balance.

Phase state of lipid bilayers modulates ammonia and water permeability through AtTip2;1.

Lipid-protein interactions determine the structure, stability and function of transmembrane proteins. Protein conformation can be influenced by local interactions at the lipid-protein interface or global interactions such as the lateral pressure profile of the membrane, which in turn is influenced by the lipid composition of the membrane. However, despite its methodological importance for in vitro protein characterization and its biological implications the influence of lipid bilayer fluidity on membrane protein activity is largely unknown. Reconstituting the aqua-ammonia channel AtTip2;1, from Arabidopsis thaliana, into OSPC containing large unilamellar vesicles we modulate its unitary water and ammonia permeability switching between the liquid-disordered state and the gel phase state of the bilayer. Quantitative activity characterization utilizing fluorescence correlation spectroscopy, stopped-flow methodology, and dynamic light scattering is complemented by molecular dynamics simulations of AtTIP2;1 in OSPC bilayers. Our in vitro experiments revealed a unitary water permeability in the liquid-disordered phase of 2.5·10−13 cm³s−1. On the contrary, selective transport of ammonia is an order of magnitude lower questioning if ammonia transport is the main function of AtTIP2;1. Moreover, results obtained from our MD simulations show, similar to our in vitro efforts, that AtTIP2;1 exhibits lower activity in the gel state as compared to its activity in the liquid-disordered state. This finding highlights the crucial importance of the lipid matrix in in vitro and in silico protein characterization and is expected to trigger a wealth of future investigations on permeability and regulation of AQPs and other membrane proteins.

Necessary and sufficient molecular determinants of quaternary ammonium antiseptic export by small multidrug resistance transporters.

The small multidrug resistance (SMR) family of transporters are exceptional models for studying polyspecific drug transport. The family is functionally split between two major clusters: the selective Guanidinium exporters (Gdx) that selectively transport the metabolite, guanidinium, and the polyspecific quaternary ammonium compound (Qac) drug exporters that transport a range of man-made compounds with different structural properties. Members from each cluster are small, structurally similar, and only differ in what substrates they transport which makes them ideal models for investigating the molecular determinants required for multidrug transport. Using a combination of directed evolution, high throughput mutagenesis, and next generation sequencing, we identified six mutations that are necessary and sufficient to convert a selective Gdx into a polyselective quaternary ammonium transporter. Biochemical, biophysical, and structural characterization of this transporter will reveal the mechanistic basis for promiscuous transport in the SMR family.

Morpho-Physiological and Transcriptional Regulation of Root System under Saline Conditions in Nymphaea Plants

Water lilies (Nymphaea L.) are ancient angiosperms that can be cultivated in both fresh and brackish water. Water lily plants have adapted morphologically and physiologically to the aqueous environment. Nonetheless, little is known about the regulatory mechanisms that enable water lily to acclimate to saline conditions, restricting its production and distribution. To illustrate the role of roots in water lily salinity tolerance, we investigated the adaptive regulation of the water lily root system under high salinity. Aspects of its root architecture, including root length, surface area, volume, and tip number, were significantly reduced by salt stress. Transcriptome sequencing showed that 120 genes were upregulated and 1214 genes were downregulated under salt stress. The differentially expressed genes were mainly enriched in oxidoreductase activity, structural molecule activity, and transmembrane transporter activity. Most ion transporter genes were downregulated, suggesting that water lily may partially close ion channels and/or transporters to avoid excessive ion accumulation or ion imbalance under long-term salt stress. Genes related to NO3− transport were both up- and downregulated, whereas genes related to ammonium transport were uniformly downregulated, suggesting that transcriptional changes may play a role in balancing nitrogen metabolism under long-term saline conditions. The roots showed relatively high concentrations of Na+ and had the ability to hyper-accumulate Na+ under salt stress. These findings provide insight into the regulatory mechanisms that enable water lily roots to tolerate salinity and lay a foundation for the breeding of salt-tolerant cultivars.

The wheat AMT2 (AMmonium Transporter) family, possible functions in ammonium uptake and pathogenic/symbiotic interactions

AbstractAmmonium uptake into wheat roots relies primarily on two AMmonium Transporters of subfamily one, while the wheat genome comprises 4 to 6 AMT2 type transporters. Plant AMT2s generally show functions in root‐to‐shoot translocation or pathogenic and symbiotic plant–microorganism interactions. We addressed the activity of TaAMT2s in ammonium transport. Nitrogen‐dependent expression implicated a physiological function in ammonium uptake for TaAMT2;1 and in ammonium distribution for TaAMT2;2‐6.

Ammonium Uptake, Mediated by Ammonium Transporters, Mitigates Manganese Toxicity in Duckweed, Spirodela polyrhiza.

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.