Identification of a high affinity NH4+ transporter from plants (original) (raw)
Related papers
Functional characterization of an ammonium transporter gene from Lotus japonicus
Gene, 2001
1 is the main product of symbiotic nitrogen ®xation and the external concentration of combined nitrogen plays a key regulatory role in all the different step of plant-rhizobia interaction. We report the cloning and characterization of the ®rst member of the ammonium transporter family, LjAMT1;1 from a leguminous plant, Lotus japonicus. Sequence analysis reveals a close relationship to plant transporters of the AMT1 family. The wild type and two mutated versions of LjAMT1;1 were expressed and functionally characterized in yeast. LjAMT1;1 is transcribed in roots, leaves and nodules of L. japonicus plants grown under low nitrogen conditions, consistent with a role in uptake of NH 4 1 by the plant cells. q
THE PLANT CELL ONLINE, 1999
Ammonium and nitrate are the prevalent nitrogen sources for growth and development of higher plants. 15 N-uptake studies demonstrated that ammonium is preferred up to 20-fold over nitrate by Arabidopsis plants. To study the regulation and complex kinetics of ammonium uptake, we isolated two new ammonium transporter (AMT) genes and showed that they functionally complemented an ammonium uptake-deficient yeast mutant. Uptake studies with 14 C-methylammonium and inhibition by ammonium yielded distinct substrate affinities between Յ 0.5 and 40 M. Correlation of gene expression with 15 NH 4 ؉ uptake into plant roots showed that nitrogen supply and time of day differentially regulated the individual carriers. Transcript levels of AtAMT1;1 , which possesses an affinity in the nanomolar range, steeply increased with ammonium uptake in roots when nitrogen nutrition became limiting, whereas those of AtAMT1;3 increased slightly, with AtAMT1;2 being more constitutively expressed. All three ammonium transporters showed diurnal variation in expression, but AtAMT1;3 transcript levels peaked with ammonium uptake at the end of the light period, suggesting that AtAMT1;3 provides a link between nitrogen assimilation and carbon provision in roots. Our results show that highaffinity ammonium uptake in roots is regulated in relation to the physiological status of the plant at the transcriptional level and by substrate affinities of individual members of the AMT1 gene family.
Different Transport Mechanisms in Plant and Human AMT/Rh-type Ammonium Transporters
The Journal of General Physiology, 2006
The conserved family of AMT/Rh proteins facilitates ammonium transport across animal, plant, and microbial membranes. A bacterial homologue, AmtB, forms a channel-like structure and appears to function as an NH3 gas channel. To evaluate the function of eukaryotic homologues, the human RhCG glycoprotein and the tomato plant ammonium transporter LeAMT1;2 were expressed and compared in Xenopus oocytes and yeast. RhCG mediated the electroneutral transport of methylammonium (MeA), which saturated with Km = 3.8 mM at pHo 7.5. Uptake was strongly favored by increasing the pHo and was inhibited by ammonium. Ammonium induced rapid cytosolic alkalinization in RhCG-expressing oocytes. Additionally, RhCG expression was associated with an alkali-cation conductance, which was not significantly permeable to NH4+ and was apparently uncoupled from the ammonium transport. In contrast, expression of the homologous LeAMT1;2 induced pHo-independent MeA+ uptake and specific NH4+ and MeA+ currents that we...
The Plant Journal, 2000
To elucidate the role of NH 4 + transporters in N nutrition of tomato, two new NH 4 + transporter genes were isolated from cDNA libraries of root hairs or leaves of tomato. While LeAMT1;2 is closely related to LeAMT1;1 (75.6% amino acid identity), LeAMT1;3 is more distantly related (62.8% identity) and possesses two short upstream open reading frames in the 5¢ end of the mRNA and a particularly short N-terminus of the protein as unique features. When expressed in yeast mutants defective in NH 4 + uptake, all three genes complemented NH 4 + uptake. In roots of hydroponically grown plants, transcript levels of LeAMT1;2 increased after NH 4 + or NO 3 ± supply, while LeAMT1;1 was induced by N de®ciency coinciding with low glutamine concentrations, and LeAMT1;3 was not detected. In aeroponic culture, expression of LeAMT1;1 and LeAMT1;2 was higher in root hairs than in the remaining root fraction. Growth of plants at elevated CO 2 slightly decreased expression of LeAMT1;2 and LeAMT1;3 in leaves, but strongly repressed transcript levels of chloroplast glutamine synthetase and photorespiratory serine hydroxymethyl-transferase. Expression of LeAMT1;2 and LeAMT1;3 showed a reciprocal diurnal regulation with highest transcript levels of LeAMT1;3 in darkness and highest levels of LeAMT1;2 after onset of light. These results indicate that in tomato at least two high-af®nity NH 4 + transporters, LeAMT1;1 and LeAMT1;2, are differentially regulated by N and contribute to root hair-mediated NH 4 + acquisition from the rhizosphere. In leaves, the reciprocally expressed transporters LeAMT1;2 and LeAMT1;3 are supposed to play different roles in N metabolism, NH 4 + uptake and/or NH 3 retrieval during photorespiration.
Plant, Cell and Environment, 2003
In order to investigate the molecular basis of high-affinity ammonium absorption by roots of rice plants ( Oryza sativa subspecies indica ) the expression patterns of three members of the AMT1 family of genes in rice seedling roots in response to altered nitrogen provision and diurnal changes in irradiance were examined. The 13 NH 4 influx is differentially regulated at the transcriptional level through the expression of three members of the OsAMT1 family of genes in roots of rice seedlings in response to changes of N status and daily irradiance. In general, these findings are in agreement with earlier observations in Arabidopsis and tomato.
PLANT PHYSIOLOGY, 2002
ϩ acquisition by plant roots is thought to involve members of the NH 4 ϩ transporter family (AMT) found in plants, yeast, bacteria, and mammals. In Arabidopsis, there are six AMT genes of which AtAMT1;1 demonstrates the highest affinity for NH 4 ϩ . Ammonium influx into roots and AtAMT1;1 mRNA expression levels are highly correlated diurnally and when plant nitrogen (N) status is varied. To further investigate the involvement of AtAMT1;1 in high-affinity NH 4 ϩ influx, we identified a homozygous T-DNA mutant with disrupted AtAMT1;1 activity. Contrary to expectation, high-affinity 13 NH 4 ϩ influx in the amt1;1:T-DNA mutant was similar to the wild type when grown with adequate N. Removal of N to increase AtAMT1;1 expression decreased high-affinity 13 NH 4 ϩ influx in the mutant by 30% compared with wild-type plants, whereas lowaffinity 13 NH 4 ϩ influx (250 m-10 mm NH 4 ϩ ) exceeded that of wild-type plants. In these N-deprived plants, mRNA copy numbers of root AtAMT1;3 and AtAMT2;1 mRNA were significantly more increased in the mutant than in wild-type plants. Under most growth conditions, amt1;1:T-DNA plants were indistinguishable from the wild type, however, leaf morphology was altered. However, when grown with NH 4 ϩ and sucrose, the mutant grew poorly and died. Our results are the first in planta evidence that AtAMT1;1 is a root NH 4 ϩ transporter and that redundancies within the AMT family may allow compensation for the loss of AtAMT1;1fax 604 -822-6089.
The regulation of nitrate and ammonium transport systems in plants
Journal of Experimental Botany, 2002
Inorganic nitrogen concentrations in soil solutions vary across several orders of magnitude among different soils and as a result of seasonal changes. In order to respond to this heterogeneity, plants have evolved mechanisms to regulate NO À 3 and NH q 4 influx. In addition, efflux analysis using 13 N has revealed that there is a co-ordinated regulation of all component fluxes within the root, including biochemical fluxes. Physiological studies have demonstrated the presence of two high-affinity transporter systems (HATS) for NO À 3 and one HATS for NH q 4 in roots of higher plants. By contrast, in Arabidopsis thaliana there exist seven members of the NRT2 family encoding putative HATS for NO À 3 and five members of the AMT1 family encoding putative HATS for NH q 4 . The induction of high-affinity NO À 3 transport and Nrt2.1 and Nrt2.2 expression occur in response to the provision of NO À 3 , while down-regulation of these genes appear to be due to the effects of glutamine. High-affinity NH q 4 transport and AMT1.1 expression also appear to be subject to down-regulation by glutamine. In addition, there is evidence that accumulated NO À 3 and NH q 4 may act post-transcriptionally on transporter function. The present challenge is to resolve the functions of all of these genes. In Aspergillus nidulans and Chlamydomonas reinhardtii there are but two high-affinity NO À 3 transporters and these appear to have undergone kinetic differentiation that permits a greater efficiency of NO À 3 absorption over the wide range of concentration normally found in nature. Such kinetic differentiation may also have occurred among higher plant transporters. The characterization of transporter function in higher plants is currently being inferred from patterns of gene expression in roots and shoots, as well as through studies of heterologous expression systems and knockout mutants.
FEBS Letters, 2006
Ammonium is a primary source of N for plants, so knowing how it is transported, stored, and assimilated in plant cells is important for rational approaches to optimise N-use in agriculture. Electrophysiological studies of Arabidopsis AtAMT1;1 expressed in oocytes revealed passive, Deltapsi-driven transport of NH(4)(+) through this protein. Expression of AtAMT1;1 in a novel yeast mutant defective in endogenous ammonium transport and vacuolar acidification supported the above mechanism for AtAMT1;1 and revealed a central role for acid vacuoles in storage and retention of ammonia in cells. These results highlight the mechanistic differences between plant AMT proteins and related transporters in bacteria and animal cells, and suggest novel strategies to enhance nitrogen use efficiency in agriculture.
Methylammonium as a Transport Analog for Ammonium in Tomato (Lycopersicon esculentum L.)
Plant physiology, 1994
Methylammonium (CH3NH3+) has been widely used as an analog of ammonium (NH4+) for examining transport in bacteria and fungi. We compared the kinetics of root CH3NH3+ and NH4+ uptake from solution culture in intact tomato (Lycopersicon esculentum cv T5) plants. Efflux of NH4+ and CH3NH3+ was negligible. The apparent maximum rate of absorption (apparent Vmax) was similar for NH4+ and CH3NH3+, but the apparent affinity (apparent Km) was about 10-fold greater for NH4+ than for CH3NH3+. In characterizing the interaction between NH4+ and CH3NH3+ transport, we used [15N]NH4+ and [14C]CH3NH3+ as well as improved methods for analysis of nonisotopic CH3NH3+ and NH4+. CH3NH3+ acted as an inhibitor of NH4+ influx. Relatively low concentrations of NH4+ strongly inhibited CH3NH3+ influx. Treatments with 1 mM methionine sulfoximine that blocked NH4+ assimilation had little influence on NH4+ inhibition of CH3NH3+ influx. These results suggest that the two ions share a common transport system in tom...
Characterization of Arabidopsis AtAMT2, a High-Affinity Ammonium Transporter of the Plasma Membrane
Plant Physiology, 2002
AtAMT2 is an ammonium transporter that is only distantly related to the five members of the AtAMT1 family of high-affinity ammonium transporters in Arabidopsis. The short-lived radioactive ion13NH4 + was used to show that AtAMT2, expressed in yeast (Saccharomyces cerevisiae), is a high-affinity transporter with a K m for ammonium of about 20 μm. Changes in external pH between 5.0 and 7.5 had little effect on the K m for ammonium, indicating that NH4 +, not NH3, is the substrate for AtAMT2. TheAtAMT2 gene was expressed in all organs of Arabidopsis and was subject to nitrogen (N) regulation, at least in roots where expression was partially repressed by high concentrations of ammonium nitrate and derepressed in the absence of external N. Although expression of AtAMT2 in shoots responded little to changes in root N status, transcript levels in leaves declined under high CO2 conditions. Transient expression of an AtAMT2-green fluorescent protein fusion protein in Arabidopsis leaf epi...