Low-affinity Transport System Research Articles

Physiological, molecular, and environmental insights into plant nitrogen uptake, and metabolism under abiotic stresses.

Nitrogen (N) as an inorganic macronutrient is inevitable for plant growth, development, and biomass production. Many external factors and stresses, such as acidity, alkalinity, salinity, temperature, oxygen, and rainfall, affect N uptake and metabolism in plants. The uptake of ammonium (NH4 +) and nitrate (NO3 -) in plants mainly depends on soil properties. Under the sufficient availability of NO3 - (>1mM), low-affinity transport system is activated by gene network NRT1, and under low NO3 - availability (<1mM), high-affinity transport system starts functioning encoded by NRT2 family of genes. Further, under limited N supply due to edaphic and climatic factors, higher expression of the AtNRT2.4 and AtNRT2.5T genes of the NRT2 family occur and are considered as N remobilizing genes. The NH4 + ion is the final form of N assimilated by cells mediated through the key enzymes glutamine synthetase and glutamate synthase. The WRKY1 is a major transcription factor of the N regulation network in plants. However, the transcriptome and metabolite profiles show variations in N assimilation metabolites, including glycine, glutamine, and aspartate, under abiotic stresses. The overexpression of NO3 - transporters (OsNRT2.3a and OsNRT1.1b) can significantly improve the biomass and yield of various crops. Altering the expression levels of genes could be a valuable tool to improve N metabolism under the challenging conditions of soil and environment, such as unfavorable temperature, drought, salinity, heavy metals, and nutrient stress.

Identification and functional characterization of the sugarcane (Saccharum spp.) AMT2-type ammonium transporter ScAMT3;3 revealed a presumed role in shoot ammonium remobilization

Sugarcane (Saccharum spp.) is an important crop for sugar and bioethanol production worldwide. To maintain and increase sugarcane yields in marginal areas, the use of nitrogen (N) fertilizers is essential, but N overuse may result in the leaching of reactive N to the natural environment. Despite the importance of N in sugarcane production, little is known about the molecular mechanisms involved in N homeostasis in this crop, particularly regarding ammonium (NH4+), the sugarcane’s preferred source of N. Here, using a sugarcane bacterial artificial chromosome (BAC) library and a series of in silico analyses, we identified an AMMONIUM TRANSPORTER (AMT) from the AMT2 subfamily, sugarcane AMMONIUM TRANSPORTER 3;3 (ScAMT3;3), which is constitutively and highly expressed in young and mature leaves. To characterize its biochemical function, we ectopically expressed ScAMT3;3 in heterologous systems (Saccharomyces cerevisiae and Arabidopsis thaliana). The complementation of triple mep mutant yeast demonstrated that ScAMT3;3 is functional for NH3/H+ cotransport at high availability of NH4+ and under physiological pH conditions. The ectopic expression of ScAMT3;3 in the Arabidopsis quadruple AMT knockout mutant restored the transport capacity of 15N–NH4+ in roots and plant growth under specific N availability conditions, confirming the role of ScAMT3;3 in NH4+ transport in planta. Our results indicate that ScAMT3;3 belongs to the low-affinity transport system (Km 270.9 µM; Vmax 209.3 µmol g−1 root DW h−1). We were able to infer that ScAMT3;3 plays a presumed role in NH4+ source–sink remobilization in the shoots via phloem loading. These findings help to shed light on the functionality of a novel AMT2-type protein and provide bases for future research focusing on the improvement of sugarcane yield and N use efficiency.

Genome-wide identification and in silico analysis of NPF, NRT2, CLC and SLAC1/SLAH nitrate transporters in hexaploid wheat (Triticum aestivum)

Nitrogen transport is one of the most important processes in plants mediated by specialized transmembrane proteins. Plants have two main systems for nitrogen uptake from soil and its transport within the system—a low-affinity transport system and a high-affinity transport system. Nitrate transporters are of special interest in cereal crops because large amount of money is spent on N fertilizers every year to enhance the crop productivity. Till date four gene families of nitrate transporter proteins; NPF (nitrate transporter 1/peptide transporter family), NRT2 (nitrate transporter 2 family), the CLC (chloride channel family), and the SLAC/SLAH (slow anion channel-associated homologues) have been reported in plants. In our study, in silico mining of nitrate transporter genes along with their detailed structure, phylogenetic and expression analysis was carried out. A total of 412 nitrate transporter genes were identified in hexaploid wheat genome using HMMER based homology searches in IWGSC Refseq v2.0. Out of those twenty genes were root specific, 11 leaf/shoot specific and 17 genes were grain/spike specific. The identification of nitrate transporter genes in the close proximity to the previously identified 67 marker-traits associations associated with the nitrogen use efficiency related traits in nested synthetic hexaploid wheat introgression library indicated the robustness of the reported transporter genes. The detailed crosstalk between the genome and proteome and the validation of identified putative candidate genes through expression and gene editing studies may lay down the foundation to improve nitrogen use efficiency of cereal crops.

Anaerobic ATP synthesis pathways and inorganic phosphate transport and their possible roles in encystment in Acanthamoeba castellanii

Acanthamoeba castellanii is the etiological agent of amoebic keratitis and is present in the environment in trophozoite or cyst forms. Both forms can infect the vertebrate host and colonize different tissues. The high resistance of cysts to standard drugs used in clinics contributes to the lack of effective treatments. Therefore, in this context, studies have emerged to understand cyst physiology and metabolism. Phosphate transporters are proteins responsible for the uptake of extracellular inorganic phosphate and transport to the cytosol. This work aims to verify the relationship between Pi transport and energetic metabolism in cysts of A. castellanii. The phosphate uptake ratio was higher in cysts compared with trophozoites. Recently, three sequences related to phosphate transporters have been identified in the A. castellanii genome (AcPHS1, AcPHS2, and AcPHS3); the messenger RNA expression levels of which differ depending on the amoeba life form. Pi uptake in cysts displayed peak activity at alkaline pH, whereas Pi transport in trophozoites was not affected in the same pH ranges. Cysts harbor a low-affinity Pi transport system (K0,5 and Vmax values of 1.76 ± 0.26 mM and 104.6 ± 6.3 nmol Pi × h-1 × 106 cells) compared to the trophozoite phosphate transport system. Pi transport seems important for anaerobic adenosine triphosphate synthesis in cysts, which initially occurs through the glycolytic pathway and subsequently through the pyruvate ferredoxin oxidoreductase pathway. Altogether, these results suggest that contrary to that previously postulated, cysts are active metabolic forms, and, as noted in trophozoites, phosphate uptake is important for energetic metabolism.

Effects of Different Inorganic Nitrogen Sources of Iris pseudacorus and Iris japonica on Energy Distribution, Nitrogen, and Phosphorus Removal

High- and low-affinity transport systems are the main pathways for the transportation of NO3– and NH4+ across intracellular membranes. NO3− and NH4+ are assimilated through different metabolic pathways in plants. Fifteen ATP molecules are hydrolyzed in the metabolic process of NO3–; however, only five ATP molecules are hydrolyzed in that of NH4+. In this research, seedlings of Iris pseudacorus and Iris japonica were used as the experimental materials in the NO3–:NH4+ = 30:0, NO3–:NH4+ = 28:2, NO3–:NH4+ = 27:3, NO3–:NH4+ = 15:15, NO3–:NH4+ = 3:27, and NO3–:NH4+ = 0:30 treatments at the 7.5 mmol·L−1 the total nitrogen content (TN). The intracellular free energy was represented by physiological resistance (R) and physiological impedance (Z) according to the Nernst equation and could conveniently and comprehensively determine the cellular metabolic energy (GB). The maximum absorption rate (Vmax) and Michaelis constant (Km) for NH4+ and NO3– uptake were calculated according to the kinetic equation. The results showed that the cellular metabolic energy (GB) of I. pseudacorus was 1 to 1.5 times lower than that of I. japonica at each treatment on the 10th day. The GB values of I. pseudacorus and I. japonica seedlings increased with increasing NH4+ concentration. However, there was a turning point at the NO3–:NH4+ = 15:15 treatment for the cellular metabolic energy of I. pseudacorus and I. japonica. Correlation analysis showed that the value of cellular metabolic energy was negatively correlated with the Vmax and Km for NO3– uptake, whereas it was positively correlated with that for NH4+ uptake. These results demonstrate that the NO3–:NH4+ = 27:3 treatment level was the most suitable for I. pseudacorus and I. japonica. This indicates that the greater cellular metabolic energy is the most suitable for plant growth when the concentration of ammonium or nitrate had no significant difference at treatment. These results provide a simple and rapid solution for removal of nitrogen by determination of cellular metabolic energy.

Seed characterization and early nitrogen metabolism performance of seedlings from Altiplano and coastal ecotypes of Quinoa

BackgroundEarly seed germination and a functional root system development during establishment are crucial attributes contributing to nutrient competence under marginal nutrient soil conditions. Chenopodium quinoa Willd (Chenopodiaceae) is a rustic crop, able to grow in marginal areas. Altiplano and Coastal/Lowlands are two representative zones of quinoa cultivation in South America with contrasting soil fertility and edaphoclimatic conditions.In the present work, we hypothesize that the ecotypes of Quinoa from Altiplano (landrace Socaire) and from Coastal/Lowland (landrace Faro) have developed differential adaptive responses in order to survive under conditions of low availability of N in their respective climatic zones of Altiplano and Lowlands. In order to understand intrinsic differences for N competence between landraces, seed metabolite profile and germinative capacity were studied. Additionally, in order to elucidate the mechanisms of N uptake and assimilation at limiting N conditions during establishment, germinated seeds of both landraces were grown at either sufficient nitrate (HN) or low nitrate (LN) supply. We studied the photosynthetic performance, protein storage, root morphometrical parameters, activity and expression of N-assimilating enzymes, and the expression of nitrate transporters of roots in plants submitted to the different treatments.ResultsSeeds from Socaire landrace presented higher content of free N-related metabolites and faster seed germination rate compared to Faro landrace. Seedlings of both ecotypes presented similar physiological performance at HN supply, but at LN supply their differences were exalted. At LN, Socaire plants showed an increased root biomass (including a higher number and total length of lateral roots), a differential regulation of a nitrate transporter (a NPF6.3-like homologue) belonging to the Low Affinity Transport System (LATS), and an upregulation of a nitrate transporter (a NRT2.1-like homologue) belonging to the High Affinity nitrate Transport System (HATS) compared to Faro. These responses as a whole could be linked to a higher amount of stored proteins in leaves, associated to an enhanced photochemical performance in Altiplano plants, in comparison to Lowland quinoa plants.ConclusionsThese differential characteristics of Socaire over Faro plants could involve an adaptation to enhanced nitrate uptake under the brutal unfavorable climate conditions of Altiplano.

Root architecture traits variation and nitrate-influx responses in diverse wheat genotypes under different external nitrogen concentrations

In order to identify the genetic variations in root system architecture traits and their probable association with high- and low-affinity nitrate transport system, we performed several experiments on a genetically diverse set of wheat genotypes grown under two external nitrogen levels (optimum and limited nitrate conditions) at two growth points of the seedling stage. Further, we also examined the nitrate uptake and its transport under different combinations of nitrate availability in the external media using 15N-labelled N-source (15NO3−), and gene expression pattern of different high- and low-affinity nitrate transporters. We observed that nitrate starvation invariably increases the total root size in all genotypes. However, the variation of component traits of total root size under nitrate starvation is genotype-specific at both stages. Further, we also observed genotypic variation in both nitrate uptake and translocation depending on the growth stage, external nitrate concentration and growing conditions. The expression of the TaNRT2.1 gene was invariably up-regulated under low external nitrate concentration; however, it gets reduced after a longer period (21 days) of starvation than the early stage (14 days). Among the four NRT1.1 orthologs, TaNPF6.3 and TaNPF6.4 consistently showed higher expression than TaNPF6.1 and TaNPF6.2 at higher nitrate concentration at both the growth stages. TaNPF6.3 and TaNPF6.4 apparently showed a feature of typical low-affinity nitrate transporter gene at higher external nitrate concentration at 14 and 21 days growth stages, respectively. The present study reveals the complex root system of wheat that has genotype-specific N-foraging along with highly coordinated high- and low-affinity nitrate transport systems for nitrate uptake and transport.

Comparative transcriptomics analysis of potassium uptake pathways mediated cesium accumulation differences and related molecular mechanisms in Brassica juncea and Vicia faba

To analyze the differences between high- and low-accumulation plants in cesium (Cs) uptake and its related mechanism, Brassica juncea (a hyperaccumulation plant for Cs) and Vicia faba (a low-accumulation plant for Cs) were selected as comparative experimental materials. The contributions to Cs uptake of a K-transporter-mediated high-affinity transport system and a K-channel-mediated low-affinity transport system in the two plants were compared and analyzed. The difference between the two plants in the mechanism of Cs uptake was further analyzed using transcription sequence technology. The results show that the transfer characteristics of Cs in the two plants had a similar distribution relationship with K. The contribution rate of the K-channel pathway to Cs uptake was 32.00% in the V. faba seedling roots, which was significantly higher than for B. juncea (9.81%) (P < 0.01); the contribution rate of the K-transporter pathway to Cs uptake of the B. juncea seedlings was 32.08%, which was significantly higher than that of the V. faba seedlings (17.13%)(P < 0.05). Other uptake pathways also mediated the uptake of Cs by roots in B. juncea and V. faba (contribution rate: 54.92-60.09% and 42.18-59.73%, respectively). The transcriptome sequencing results confirmed that Cs-induced treatment significantly inhibited the expression of the K-transporter protein and K-channel protein-related genes in the V. faba roots, but it had no significant effect on the expression of related genes in the B. juncea roots. Thus, one reason for the significant difference between the two plant in the accumulation of Cs is that Cs inhibited the expression of related transporter protein genes in the V. faba roots.

Developmental and physiological responses of Brachypodium distachyon to fluctuating nitrogen availability

The Nitrogen Use Efficiency (NUE) of grain cereals depends on nitrate (NO3−) uptake from the soil, translocation to the aerial parts, nitrogen (N) assimilation and remobilization to the grains. Brachypodium distachyon has been proposed as a model species to identify the molecular players and mechanisms that affects these processes, for the improvement of temperate C3 cereals. We report on the developmental, physiological and grain-characteristic responses of the Bd21-3 accession of Brachypodium to variations in NO3− availability. As previously described in wheat and barley, we show that vegetative growth, shoot/root ratio, tiller formation, spike development, tissue NO3− and N contents, grain number per plant, grain yield and grain N content are sensitive to pre- and/or post-anthesis NO3− supply. We subsequently described constitutive and NO3−-inducible components of both High and Low Affinity Transport Systems (HATS and LATS) for root NO3− uptake, and BdNRT2/3 candidate genes potentially involved in the HATS. Taken together, our data validate Brachypodium Bd21-3 as a model to decipher cereal N nutrition. Apparent specificities such as high grain N content, strong post-anthesis NO3− uptake and efficient constitutive HATS, further identify Brachypodium as a direct source of knowledge for crop improvement.

A new conceptual framework for plant responses to soil metals based on metal transporter kinetic parameters.

Based on a review of the literature, we have developed a functional conceptual framework of plant metal uptake in relation to plant available metal concentration in the soil. This framework applies to all plant parts and plant available metal levels in soils, and was validated using independent datasets from field surveys and the literature. This is the first framework based on metal transporter kinetic parameters and combining Michaelis-Menten (hyperbolic) kinetics facilitated by the High Affinity Transport System (HATS) for soil concentrations below the transition concentration between transport systems, and linear metal uptake facilitated by the Low Affinity Transport System (LATS) for higher soil available metal concentrations. We propose a new terminology for metal tolerant plants, i.e. metal tolerators, based on this framework. Depending on the plant available metal levels in the soil, tolerator responses to metals can be described best by either Vmax and Km for soil concentrations below the transition concentration between metal transport systems (HATS), or by the slope for greater soil concentrations (LATS). This conceptual framework may be a useful tool for selecting suitable metal tolerators for specific phytoremediation purposes, and may be also applied to non-metal elements or ions.

Stable isotope fractionation of zinc and cadmium in soil-plant system: A review

Stable isotope fractionation provides a useful tool for tracing the transfer of zinc (Zn) and cadmium (Cd) in the soil-plant system. Isotope fractionation processes include equilibrium fractionation, e.g. dissolution, complexation, precipitation, and kinetic fractionation, e.g. diffusion and evaporation. Therefore, isotope fractionation of metallic element can be used to study the mechanisms of metal translocation in biogeochemical cycling. With the development of multiple collector inductively coupled plasma mass spectrometry (MC-ICP-MS), Zn and Cd isotopic composition in soil and plant samples can be accurately measured. The determination of Zn and Cd stable isotope ratios usually includes sample digestion, purification and mass discrimination correction. In the soil-plant system, plants are generally enriched with heavy Zn isotopes in comparison to soils, while roots are enriched with heavy Zn isotopes in comparison to shoots. For Cd, shoots are likely to be isotopically heavier than roots. The transport processes of Zn/Cd in the soil-plant system mainly involve transport in rhizosphere, root uptake and transport within plants. The adsorption and desorption of Zn/Cd by soil minerals can affect the metal mobility and the isotope fractionation between soil solid phases and soil solution. Heavy Zn isotopes are likely to be complexed, while light Zn isotopes are more mobile in soil solution. Conversely, soil minerals tend to adsorb light Cd isotopes, thereby resulting in an enrichment with heavy isotopes in the soil solution. Among the rhizospheric processes, the secretion of root exudates and the changes in pH also have an impact on the speciation and translocation of heavy metals, which lead to the release of heavy Zn isotopes into soil solution. Root uptake process is of pivotal importance for the determination of the isotopic composition of the whole plant. The diffusion of Zn2+ from the soil solution to the root surface leads to a light isotopes enrichment in plant. Heavy Zn isotopes are preferentially chelated or precipitated in the root apoplast. During the transport across the root cell membranes, the low-affinity transport leads to an enrichment with light isotopes in plant, while the high-affinity transport generates little or no isotope fractionation. Specific transporter of Cd in roots has never been found yet. Therefore, Cd may be took up mainly through low-affinity transport systems and thus be enriched in light isotopes in plants. The isotope fractionation between roots and shoots is related to the sequestration of Zn/Cd in roots and the xylem transport processes. The functional groups containing oxygen and nitrogen of organic acids in plant roots tend to chelate heavy Zn isotopes, with subsequent compartmentation in root vacuoles. The light Zn isotopes could be then loaded by the xylem and transferred to the aboveground parts. Interestingly, the intensity of isotope fractionation is mainly related to the transport distance in the xylem. Contrary to Zn, light Cd isotopes mainly bind to the thiol groups in roots, leading to the enrichment in heavy isotopes in shoots. The different behaviors between Zn and Cd in isotope fractionation in the soil-plant system could reflect the distinct accumulation and tolerance mechanisms of plants in response to Zn or Cd stress.

Morphological and kinetic parameters of the uptake of nitrogen forms in clonal peach rootstocks

Peach (Prunus persica L.) rootstock cultivars are typically selected for scion compatibility, ease of propagation, vigor, development, flowering season, yield, low need for cold temperatures, resistance to diseases, effects on the physical-chemical characteristics of the fruit, plant longevity and adaptation to adverse edaphoclimatic conditions. However, kinetic parameters related to nutrient uptake efficiency are usually not considered, such as those of nitrate (NO3−) and ammonium (NH4+). N is the nutrient that most impacts growth and yield. The objective of this study was to show the importance of the kinetic parameters of NO3− and NH4+ uptake as additional criteria for selecting peach rootstocks. The experiment was conducted in a greenhouse. Three rootstock (‘Aldrighi’, ‘Tsukuba1’ and ‘Clone 15′) were grown for 30 days in a pot containing 0.1 mol L-1 CaSO4 solution to reduce internal reserves of N. Afterwards, the plants were placed in Hoagland nutrient solution, where periodic collections of the nutrient solution were carried out for three days and the concentrations of NO3− and NH4+ were determined. After the third day of collecting the solution, the plants were collected and then separated into leaves, roots and stems. Dry matter and total N content were assessed. The kinetic parameters related to NO3− and NH4+ uptake (maximum uptake rate - Vmax, affinity constant - Km, Minimum concentration - Cmin, Influx - I) were calculated using Cinética software. The most efficient rootstock for NO3− and NH4+ uptake was ‘Tsukuba1’, as it showed the lowest values ​​of Cmin and Km and the highest values ​​of Vmax and Imax for NO3− and NH4+. NO3− uptake in ‘Tsukuba1’ and ‘Aldrighi’ showed a two-phase uptake pattern, suggesting the presence of low and high affinity transport systems. On the other hand, NH4+ uptake in the three cultivars apparently followed a one-phase uptake pattern, suggesting the presence of a high affinity transport system. The kinetic parameters of NO3− and NH4+ uptake are additional criteria that can be used in selecting peach rootstocks, as they directly influence shoot and root dry matter production and N accumulation in leaves.

Low affinity uniporter carrier proteins can increase net substrate uptake rate by reducing efflux

Many organisms have several similar transporters with different affinities for the same substrate. Typically, high-affinity transporters are expressed when substrate is scarce and low-affinity ones when it is abundant. The benefit of using low instead of high-affinity transporters remains unclear, especially when additional nutrient sensors are present. Here, we investigate two hypotheses. It was previously hypothesized that there is a trade-off between the affinity and the catalytic efficiency of transporters, and we find some but no definitive support for it. Additionally, we propose that for uptake by facilitated diffusion, at saturating substrate concentrations, lowering the affinity enhances the net uptake rate by reducing substrate efflux. As a consequence, there exists an optimal, external-substrate-concentration dependent transporter affinity. A computational model of Saccharomyces cerevisiae glycolysis shows that using the low affinity HXT3 transporter instead of the high affinity HXT6 enhances the steady-state flux by 36%. We tried to test this hypothesis with yeast strains expressing a single glucose transporter modified to have either a high or a low affinity. However, due to the intimate link between glucose perception and metabolism, direct experimental proof for this hypothesis remained inconclusive. Still, our theoretical results provide a novel reason for the presence of low-affinity transport systems.

Nickel hyperaccumulation mechanisms: a review on the current state of knowledge

Hyperaccumulator plants are unusual plants that accumulate particular metals or metalloids, such as nickel, zinc, cadmium and arsenic, in their living tissues to concentrations that are hundreds to thousands of times greater than what is normal for most plants. The hyperaccumulation phenomenon is rare (exhibited by less than 0.2% of all angiosperms), with most of the ~500 hyperaccumulator species known globally for nickel. This review highlights the contemporary understanding of nickel hyperaccumulation processes, which include root uptake and sequestration, xylem loading and transport, leaf compartmentation and phloem translocation processes. Hyperaccumulator plants have evolved highly efficient physiological mechanisms for taking up nickel in their roots followed by rapid translocation and sequestration into the aerial shoots. The uptake of nickel is mainly involved with low affinity transport systems, presumably from the ZIP family. The presence of high concentrations of histidine prevents nickel sequestration in roots. Nickel is efficiently loaded into the xylem, where it mainly presents as Ni2+. The leaf is the main storage organ, which sequestrates nickel in non-active sites, e.g. vacuoles and apoplast. Recent studies show that phloem translocates high levels of nickel, which has a strong impact on nickel accumulation in young growing tissues.

Uptake and reduction of Se(IV) in two heterotrophic aerobic Pseudomonads strains isolated from boreal bog environment.

Selenite (Se(IV), SeO32−) uptake and the effect of selenite supplement on protein synthesis was investigated in two Pseudomonas sp. strains isolated from a boreal bog. These aerobic bacteria efficiently reduced Se(IV) with intracellular reduced Se0 observed in the cytoplasm under dark aerobic conditions. The proteome analysis of Se(IV) supplement and temperature responses by SDS-PAGE gel electrophoresis showed variations in the protein expression on the 40–60 kDa regions following these stress factors, probably through enzymes associated to oxidative stress or temperature adaptation. NO3−/NO2−/SO42− addition enhanced Se(IV) uptake in both bacteria, but Se(IV) uptake sustained also under sulphur and nitrogen starvation. Our findings suggest two different transport mechanisms for Se(IV) uptake in these Pseudomonas sp. strains; a low affinity transport system up-regulated by NO3−/NO2−/SO42− and a distinct Se(IV)O32− regulated transport system. Following transport, Se(IV) is reduced in the cytoplasm, forming Se0 granules, visible in TEM and verified using EDX.

GintAMT3 - a Low-Affinity Ammonium Transporter of the Arbuscular Mycorrhizal Rhizophagus irregularis.

Nutrient acquisition and transfer are essential steps in the arbuscular mycorrhizal (AM) symbiosis, which is formed by the majority of land plants. Mineral nutrients are taken up by AM fungi from the soil and transferred to the plant partner. Within the cortical plant root cells the fungal hyphae form tree-like structures (arbuscules) where the nutrients are released to the plant-fungal interface, i.e., to the periarbuscular space, before being taken up by the plant. In exchange, the AM fungi receive carbohydrates from the plant host. Besides the well-studied uptake of phosphorus (P), the uptake and transfer of nitrogen (N) plays a crucial role in this mutualistic interaction. In the AM fungus Rhizophagus irregularis (formerly called Glomus intraradices), two ammonium transporters (AMT) were previously described, namely GintAMT1 and GintAMT2. Here, we report the identification and characterization of a newly identified R. irregularis AMT, GintAMT3. Phylogenetic analyses revealed high sequence similarity to previously identified AM fungal AMTs and a clear separation from other fungal AMTs. Topological analysis indicated GintAMT3 to be a membrane bound pore forming protein, and GFP tagging showed it to be highly expressed in the intraradical mycelium of a fully established AM symbiosis. Expression of GintAMT3 in yeast successfully complemented the yeast AMT triple deletion mutant (MATa ura3 mep1Δ mep2Δ::LEU2 mep3Δ::KanMX2). GintAMT3 is characterized as a low affinity transport system with an apparent Km of 1.8 mM and a Vmax of 240 nmol-1 min-1 108 cells-1, which is regulated by substrate concentration and carbon supply.

Salt-induced NO&lt;sub&gt;3&lt;/sub&gt;&lt;sup&gt;-&lt;/sup&gt; uptake inhibition in cowpea roots is dependent on the ionic composition of the salt and its osmotic effect

Salinity remarkably inhibits NO3 - uptake but the mechanisms are not well understood. This study was addressed to elucidate the role of ionic and osmotic components of salinity on NO3 - influx and efflux employing classic kinetics involving a low affinity transport system (LATS) and a high affinity transport system (HATS). In the presence of KCl, NaCl, and Na2SO4 at 100 mM concentrations, in both LATS and HATS, Michaelis constant (Km) was similar for the three salts and maximum rate (Vmax) decreased as follows: KCl > NaCl > Na2SO4, compared to control indicating a non-competitive interaction with NO3 -. Unexpectedly, iso-osmotic solutions (osmotic potential Ψπ = -0.450) of polyethylene glycol (PEG, 17.84 %, v/v) and mannitol (100 mM) remarkably increased Km in both the LATS and the HATS, but Vmax did not change indicating a competitive inhibition. Under the PEG and mannitol treatments, Km and Vmax were higher than under the salt treatments. The salts increased slightly NO3 - efflux in the following order KCl > NaCl > Na2SO4. In contrast, mannitol strongly stimulated and the PEG inhibited NO3 - efflux. The obtained data reveal that salinity effects were not dependent on the anion type (Cl- versus SO4 2-) indicating a non-competitive inhibition mechanism between Cl- and NO3 -. In contrast, the cation types (K+ versus Na+) had a pronounced effect. The osmotic component is important to net NO3 - uptake affecting remarkably the influx in both LATS and HATS components of cowpea roots.

Molecular Mechanism Underlying the Plant NRT1.1 Dual-Affinity Nitrate Transporter.

Nitrate () is one of the most important sources of mineral nitrogen, which also serves as a key signaling molecule for plant growth and development. To cope with nitrate fluctuation in soil that varies by up to four orders of magnitude, plants have evolved high- and low-affinity nitrate transporter systems, consisting of distinct families of transporters. Interestingly, the first cloned nitrate transporter in Arabidopsis, NRT1.1 functions as a dual-affinity transporter, which can change its affinity for nitrate in response to substrate availability. Phosphorylation of a threonine residue, Thr101, switches NRT1.1 from low- to high-affinity state. Recent structural studies have unveiled that the unmodified NRT1.1 transporter works as homodimers with Thr101 located in close proximity to the dimer interface. Modification on the Thr101 residue is shown to not only decouple the dimer configuration, but also increase structural flexibility, thereby, altering the substrate affinity of NRT1.1. The structure of NRT1.1 helps establish a novel paradigm in which protein oligomerzation and posttranslational modification can synergistically expand the functional capacity of the major facilitator superfamily (MFS) transporters.

Molecular and functional characterization of choline transporter in the human trophoblastic cell line JEG-3 cells

IntroductionCholine is essential for the synthesis of the major membrane phospholipid phosphatidylcholine (PC), the methyl donor betaine and the neurotransmitter acetylcholine (ACh), which is involved in several vital biological functions that play key roles in fetal development. In this study, we examined the molecular and functional characteristics of choline uptake in the human trophoblastic cell line JEG-3. MethodsWe examined [3H]choline uptake in the human trophoblastic cell line JEG-3. The expression of CTL1 and CTL2 was evaluated by quantitative real-time PCR, western blotting and immunocytochemistry. ResultsWe demonstrated that JEG-3 cells take up [3H] choline by a saturable process that is mediated by a Na+-independent and pH-dependent transport system. The cells have two different [3H] choline transport systems, high- and low-affinity, with Km values of 28.4 ± 5.0 μM and 210.6 ± 55.1 μM, respectively. Cationic compounds and hemicholinium-3 (HC-3) inhibited choline uptake. Choline transporter-like protein 1 (CTL1) and CTL2 mRNA and protein were highly expressed in JEG-3 cells and were localized to the plasma membrane. DiscussionThe present results suggest that choline is mainly transported via a high-affinity choline transport system (CTL1) and a low-affinity choline transport system (CTL2) in human trophoblastic JEG-3 cells. These transporters play an important role in the growth of the fetus.

Dynamics of Zn in an urban wetland soil–plant system: Coupling isotopic and EXAFS approaches

Plants play a key role in the stabilization of metals in contaminated environments. Studies have been performed on Zn uptake and storage mechanisms, mainly for Zn hyperaccumulating plants, though less is known about Zn stabilization in the rhizosphere of non-accumulating plants. This study was focused on the dynamics of Zn in a whole soil–litter–plant system and the processes controlling Zn mobilization and stabilization. The site studied was an infiltration basin receiving urban stormwater, in which Phalaris arundinacea (reed canary grass) developed spontaneously. A combination of chemical extractions (CaCl2, DTPA), EXAFS spectroscopy and Zn stable isotope measurements was applied for the water inlet, soil, plant organs and decaying biomass. Zn speciation changed from the water inlet to the soil. In the soil, Zn was present as Zn-layered double hydroxide (Zn-LDH), tetrahedral and octahedral sorbed Zn species. The formation of Zn-LDH participates in Zn stabilization. Tetrahedral Zn species, which were partly DTPA exchangeable, were enriched in heavy isotopes, whereas octahedral Zn (Zn-LDH and sorbed species) were enriched in light isotopes. Based on a linear model between δ66Zn and Zn speciation, δ66Zn for pure tetrahedral and octahedral end-members were estimated at ca. 0.33‰ and 0.04‰, respectively. In the plant, a mixture of octahedral Zn (attributed to aqueous Zn-organic acid complexes present in the symplasm), and tetrahedral Zn (attributed to apoplasmic Zn-cell wall complexes) was observed in all organs. Large enrichment in light isotopes from the soil to the plant Δ66Zn (of ca. −0.6‰) was observed. The stem was enriched in light isotopes versus roots and, to a lesser extent, versus leaves. The results suggest that Zn was taken up via a low-affinity transport system and that Zn was sequestrated in the stem symplasm after transit through leaves. Finally, intense Zn exchanges were observed between the decaying biomass and the soil, with the sorption of heavy Zn from the soil to cell wall remains and release of light Zn to the soil. Overall, this study provides a complete overview of Zn cycling in an urban wetland soil–plant system, and describes several changes in Zn speciation with Zn isotopic fractionation processes in a complex system.