Constitutive expression of a novel-type ammonium transporter OsAMT2 in rice plants

To characterize ammonium transport pathways in rice, two cDNAs with high homology to MEP/AMT2-type ammonium transporters, OsAMT2;1 and OsAMT3;1, were isolated. Expression of OsAMT2;1 in an ammonium-uptake-defective yeast mutant showed that this gene encodes functional ammonium transporters. OsAMT2;1 was constitutively expressed in both roots and shoots irrespective of the supply of inorganic nitrogen to the medium, whereas OsAMT3;1 expression was relatively weak. A database search with the amino acid sequence of OsAMT2;1 showed that there are 10 putative OsAMT genes in rice, i.e. three each for OsAMT1, OsAMT2 and OsAMT3, respectively, and one for OsAMT4.     

Understanding the differential nitrogen sensing mechanism in rice genotypes through expression analysis of high and low affinity ammonium transporter genes

Two rice genotypes, Kalanamak 3119 (KN3119) and Pusa Basmati 1(PB1) differing in their optimum nitrogen requirements (30 and 120 kg/ha, respectively) were undertaken to study the expression of both high and low affinity ammonium transporter genes responsible for ammonium uptake. Exposing the roots of the seedlings of both the genotypes to increasing (NH₄)₂SO₄ concentrations revealed that all the three families of rice AMT genes are expressed, some of which get altered in a genotype and concentration specific manner. This indicates that individual ammonium transporter genes have defined contributions for ammonium uptake and plant growth. Interestingly, in response to increasing nitrogen concentrations, a root specific high affinity gene, AMT1;3, was repressed in the roots of KN3119 but not in PB1 indicating the existence of a differential ammonium sensing mechanism. This also indicates that not only AMT1;3 is involved not only in ammonium uptake but may also in ammonium sensing. Further, if it can differentiate and could be used as a biomarker for nitrogen responsiveness. Expression analysis of low affinity AMT genes showed that, both AMT2;1 and AMT2;2 have high levels of expression in both roots and shoots and in KN3119 are induced at low ammonium concentrations. Expressions of AMT3 family genes were higher shoots than in the roots indicating that these genes are probably involved in the translocation and distribution of ammonium ions in leaves. The expression of the only high affinity AMT gene, AMT1;1, along with six low affinity AMT genes in the shoots suggests that low affinity AMTs in the shoots leaves are involved in supporting AMT1;1 to carry out its activities/function efficiently.     

The high-affinity poplar ammonium importer PttAMT1.2 and its role in ectomycorrhizal symbiosis

One way to elucidate whether ammonium could act as a nitrogen (N) source delivered by the fungus in ectomycorrhizal symbiosis is to investigate plant ammonium importers. Expression analysis of a high-affinity ammonium importer from Populus tremulax tremuloides (PttAMT1.2) and of known members of the AMT1 gene family from Populus trichocarpa was performed. In addition, PttAMT1.2 function was studied in detail by heterologous expression in yeast. PttAMT1.2 expression proved to be root-specific, affected by N nutrition, and strongly increased in a N-independent manner upon ectomycorrhiza formation. The corresponding protein had a K(M) value for ammonium of c. 52 microm. From the seven members of the AMT1 gene family, one gene was exclusively expressed in roots while four genes were detectable in all poplar organs but with varying degrees of expression. Ectomycorrhiza formation resulted in a strong upregulation of three of these genes. Our results indicate an increased ammonium uptake capacity of mycorrhized poplar roots and suggest, together with the expression of putative ammonium exporter genes in the ectomycorrhizal fungus Amanita muscaria, that ammonium could be a major N source delivered from the fungus towards the plant in symbiosis.     

Influence of different nitrogen inputs on the members of ammonium transporter and glutamine synthetase genes in two rice genotypes having differential responsiveness to nitrogen

Two aromatic rice genotypes, Pusa Basmati 1 (PB1) and Kalanamak 3119 (KN3119) having 120 and 30 kg/ha optimum nitrogen requirement respectively, to produce optimal yield, were chosen to understand their differential nitrogen responsiveness. Both the genotypes grown under increasing nitrogen inputs showed differences in seed/panicle, 1,000 seed weight, %nitrogen in the biomass and protein content in the seeds. All these parameters in PB1 were found to be in the increasing order in contrast to KN3119 which showed declined response on increasing nitrogen dose exceeding the normal dose indicating that both the genotypes respond differentially to the nitrogen inputs. Gene expression analysis of members of ammonium transporter gene family in flag leaves during active grain filling stage revealed that all the three members of OsAMT3 family genes (OsAMT1;1-3), only one member of OsAMT2 family i.e., OsAMT2;3 and the high affinity OsAMT1;1 were differentially expressed and were affected by different doses of nitrogen. In both the genotypes, both increase and decline in seed protein contents matched with the expressions levels of OsAMT1;1, OsGS1;1 and OsGS1;2 in the flag leaves during grain filling stage indicating that high nitrogen nutrition in KN3119 probably causes the repression of these genes which might be important during grain filling.     

Diversity and selective sweep in the OsAMT1;1 genomic region of rice

Background  

Ammonium is one of the major forms in which nitrogen is available for plant growth. OsAMT1;1 is a high-affinity ammonium transporter in rice (Oryza sativa L.), responsible for ammonium uptake at low nitrogen concentration. The expression pattern of the gene has been reported. However, variations in its nucleotides and the evolutionary pathway of its descent from wild progenitors are yet to be elucidated. In this study, nucleotide diversity of the gene OsAMT1;1 and the diversity pattern of seven gene fragments spanning a genomic region approximately 150 kb long surrounding the gene were surveyed by sequencing a panel of 216 rice accessions including both cultivated rice and wild relatives.     

Expression of OsAMT1 (1.1-1.3) in rice varieties differing in nitrogen accumulation

OsAMT is a high-affinity ammonium transporter responsible for NH ₄ ⁺ uptake by rice plants. To investigate the expression patterns of OsAMT in different genotypes in relation to nitrogen accumulation, we measured the expression of OsAMT1.1, OsAMT1.2, and OsAMT1.3 using Real-Time PCR (RT-PCR) in GD (higher N accumulation) and NG (lower N accumulation) seedlings of the Oryza sativa L. cultivar treated with 0.1 mM NH₄NO₃ and 2 mM NH₄NO₃. We found that the expression level of OsAMT1.1 was significantly higher than those of OsAMT1.2 and OsAMT1.3 in the roots treated with 0.1 mM NH₄NO₃, suggesting that OsAMT1.1 contributed the most to N accumulation among the three genes. In GD root, OsAMT1.1 had significantly higher expression levels when it was up-regulated by 0.1 mM NH₄NO₃ than when down-regulated by 2 mM NH₄NO₃. OsAMT1.1 was mainly found in GD roots treated with 0.1 mM NH₄NO₃. We conclude that the OsAMT1.1 in GD roots, which was significantly up-regulated by low N and down-regulated by high N, was the dominating factor in determining the higher N acquisition in GD than in NG at 0.1 mM NH₄NO₃.     

The OsAMT1.1 gene functions in ammonium uptake and ammoniumepotassium homeostasis over low and high ammonium concentration ranges

Rice(Oryza sativa) grown in paddy fields is an ammonium(NH~+_4)-preferring crop;however,its AMT-type NH~+_4transporters that mediate root N acquisition have not been well characterized yet.In this study,we analyzed the expression pattern and physiological function of the OsAMT1.1 gene of the AMT1 subfamily in rice.Os AMT1.1 is located in the plasma membrane and is mainly expressed in the root epidermis,stele and mesophyll cells.Disruption of the Os AMT1.1 gene decreased the uptake of NH~+_4,and the growth of roots and shoots under both low NH~+_4and high NH~+_4conditions.Os AMT1.1 contributed to the short-term(5 min)~(15)NH~+_4influx rate by approximately one-quarter,irrespective of the NH~+_4concentration.Knockout of Os AMT1.1 significantly decreased the total N transport from roots to shoots under low NH~+_4conditions.Moreover,compared with the wild type,the osamt1.1 mutant showed an increase in the potassium(K)absorption rate under high NH~+_4conditions and a decrease under low NH~+_4conditions.The mutants contained a significantly high concentration of K in both the roots and shoots at a limited K(0.1 mmol/L)supply when NH~+_4was replete.Taken together,the results indicated that OsAMT1.1 significantly contributes to the NH~+_4uptake under both low and high NH~+_4conditions and plays an important role in Ne K homeostasis in rice.     

The molecular physiology of ammonium uptake and retrieval

Plants are able to take up ammonium from the soil, or through symbiotic interactions with microorganisms, via the root system. Using functional complementation of yeast mutants, it has been possible to identify a new class of membrane proteins, the ammonium transporter/methylammonium permease (AMT/MEP) family, that mediate secondary active ammonium uptake in eukaryotic and prokaryotic organisms. In plants, the AMT gene family can be subdivided according to their amino-acid sequences into three subfamilies: a large subfamily of AMT1 genes and two additional subfamilies each with single members (LeAMT1;3 from tomato and AtAMT2;1 from Arabidopsis thaliana). These transporters vary especially in their kinetic properties and regulatory mechanism. High-affinity transporters are induced in nitrogen-starved roots, whereas other transporters may be considered as the 'work horses' that are active when conditions are conducive to ammonium assimilation. The expression of several AMTs in root hairs further supports a role in nutrient acquisition. These studies provide basic information that will be needed for the dissection of nitrogen uptake by plants at the molecular level and for determining the role of individual AMTs in nutrient uptake and potentially in nutrient efficiency.     

Molecular and cellular characterisation of LjAMT2;1, an ammonium transporter from the model legume Lotus japonicus

Two related families of ammonium transporters have been identified and partially characterised in plants in the past; the AMT1 and AMT2 families. Most attention has focused on the larger of the two families, the AMT1 family, which contains members that are likely to fulfil different, possibly overlapping physiological roles in plants, including uptake of ammonium from the soil. The possible physiological functions of AMT2 proteins are less clear. Lack of data on cellular and tissue location of gene expression, and the intracellular location of proteins limit our understanding of the physiological role of all AMT proteins. We have cloned the first AMT2 family member from a legume, LjAMT2;1 of Lotus japonicus, and demonstrated that it functions as an ammonium transporter by complementing a yeast mutant defective in ammonium uptake. However, like AtAMT2 from Arabidopsis, and unlike AMT1 transporters from several plant species, LjAMT2;1 was unable to transport methylammonium. The LjAMT2;1 gene was found to be expressed constitutively throughout Lotus plants. In situ RNA hybridisation revealed LjAMT2;1 expression in all major tissues of nodules. Transient expression of LjAMT2;1-GFP fusion protein in plant cells indicated that the transporter is located on the plasma membrane. In view of the fact that nodules derive ammonium internally, rather than from the soil, the results implicate LjAMT2;1 in the recovery of ammonium lost from nodule cells by efflux. A similar role may be fulfilled in other organs, especially leaves, which liberate ammonium during normal metabolism.     

Cloning of a cDNA for a constitutive NRT1 transporter from soybean and comparison of gene expression of soybean NRT1 transporters

We have isolated a cDNA for a putative transporter, named GmNRT1-3, in the NRT1 family from soybean. It was predicted to have a similar topological structure not only to both GmNRT1-1 and GmNRT1-2 reported previously, but also to other members of the family. Two other cDNAs isolated have parts of the sequence for putative NRT1 transporters, GmNRT1-4 and GmNRT1-5, suggesting that at least five NRT1 transporters occur in soybean. These GmNRT1 genes and the GmNRT2 gene, encoding a soybean NRT2 nitrate transporter, showed different expression patterns to each other under various nitrogen conditions. Specifically, GmNRT1-3 was constitutively expressed in both roots and leaves, while GmNRT1-2 was gradually expressed as the roots developed in the presence of ammonium as a nitrogen source, but not in the presence of both ammonium and nitrate. Based on these results, we discussed the possible regulation in the expression and role of these transporters in nitrate uptake.     

Concurrent activation of OsAMT1;2 and OsGOGAT1 in rice leads to enhanced nitrogen use efficiency under nitrogen limitation

Nitrogen (N) is a major factor for plant development and productivity. However, the application of nitrogenous fertilizers generates environmental and economic problems. To cope with the increasing global food demand, the development of rice varieties with high nitrogen use efficiency (NUE) is indispensable for reducing environmental issues and achieving sustainable agriculture. Here, we report that the concomitant activation of the rice (Oryza sativa) Ammonium transporter 1;2 (OsAMT1;2) and Glutamate synthetase 1 (OsGOGAT1) genes leads to increased tolerance to nitrogen limitation and to better ammonium uptake and N remobilization at the whole plant level. We show that the double activation of OsAMT1;2 and OsGOGAT1 increases plant performance in agriculture, providing better N grain filling without yield penalty under paddy field conditions, as well as better grain yield and N content when plants are grown under N llimitations in field conditions. Combining OsAMT1;2 and OsGOGAT1 activation provides a good breeding strategy for improving plant growth, nitrogen use efficiency and grain productivity, especially under nitrogen limitation, through the enhancement of both nitrogen uptake and assimilation.