Distinct expression and function of three ammonium transporter genes (OsAMT1;1-1;3) in rice

To study the regulation of ammonium uptake into rice roots, three ammonium transporter genes (OsAMT1;1, 1;2 and 1;3; Oryza sativa ammonium transporter) were isolated and examined. OsAMT1s belong to AMT1 family, containing 11 putative transmembrane-spanning domains. Southern blot analysis and screening of the rice genome database confirmed that with OsAMT1;1-1;3 the complete AMT1 family of rice had been isolated. Heterologous expression of OsAMT1s in the yeast Saccharomyces cerevisiae mutant 31019b showed that all three OsAMT1s exhibit ammonium transport activity. Northern blot analysis showed a distinct expression pattern for the three genes; more constitutive expression in shoots and roots for OsAMT1;1, root-specific and ammonium-inducible expression for OsAMT1;2, and root-specific and nitrogen-derepressible expression for OsAMT1;3. In situ mRNA detection revealed that OsAMT1;2 is expressed in the central cylinder and cell surface of root tips. This gene expression analysis revealed a distinct nitrogen-dependent regulation for AMTs in rice, differing from that in tomato or ARABIDOPSIS:     

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.     

Responses of rice cultivars with different nitrogen use efficiency to partial nitrate nutrition

BACKGROUND AND AIMS: There is increased evidence that partial nitrate (NO3-) nutrition (PNN) improves growth of rice (Oryza sativa), although the crop prefers ammonium (NH4+) to NO3- nutrition. It is not known whether the response to NO3- supply is related to nitrogen (N) use efficiency (NUE) in rice cultivars. Methods Solution culture experiments were carried out to study the response of two rice cultivars, Nanguang (High-NUE) and Elio (Low-NUE), to partial NO3- supply in terms of dry weight, N accumulation, grain yield, NH4+ uptake and ammonium transporter expression [real-time polymerase chain reaction (PCR)]. KEY RESULTS: A ratio of 75/25 NH4+ -N/NO3- -N increased dry weight, N accumulation and grain yield of 'Nanguang' by 30, 36 and 21 %, respectively, but no effect was found in 'Elio' when compared with those of 100/0 NH4+ -N/NO3- -N. Uptake experiments with 15N-NH4+ showed that NO3- increased NH4+ uptake efficiency in 'Nanguang' by increasing Vmax (14 %), but there was no effect on Km. This indicated that partial replacement of NH4+ by NO3- could increase the number of the ammonium transporters but did not affect the affinity of the transporters for NH4+. Real-time PCR showed that expression of OsAMT1s in 'Nanguang' was improved by PNN, while that in 'Elio' did not change, which is in accordance with the differing responses of these two cultivars to PNN. Conclusions Increased NUE by PNN can be attributed to improved N uptake. The rice cultivar with a higher NUE has a more positive response to PNN than that with a low NUE, suggesting that there might be a relationship between PNN and NUE.     

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.     

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.     

Effects of the fungal endophyte Phomopsis liquidambari on nitrogen uptake and metabolism in rice

Phomopsis liquidambari can establish a mutualistic symbiotic relationship with rice. It promotes the growth and yield of the host plant and reduces the amount of nitrogen (N) fertilizer required for plant growth. However, the mechanisms responsible for the effects of the fungal endophyte on N use in rice are largely unknown. We conducted a hydroponic experiment to investigate the effects of P. liquidambari on N uptake and N metabolism in rice plants. Rice plants were cultivated in the presence or absence of P. liquidambari under three N levels. Under the low-N treatment, fungal infection significantly increased the biomass, and the total N, soluble protein, free amino acid, free NH₄ ⁺, and chlorophyll contents of rice roots and shoots. The activities of nitrate reductase and glutamine synthetase were increased in infected rice plants. Some genes related to N uptake (OsAMT1;1, OsAMT1;3, OsAMT2;2, OsAMT3;2, OsAMT3;3, OsNRT2;1) and N metabolism (OsNR1, OsGS1, OsGS2, OsNADH-GOGAT) were also up-regulated in infected plants under the low-N treatment. However, these effects gradually weakened as the N level increased. The colonization rate of the endophyte substantially decreased with increasing N levels. Taken together, these results suggest that low-N fertilization induces a physiological state in rice that is favorable for the P. liquidambari symbiosis. The greater extent of P. liquidambari colonization under low-N conditions stimulated the expression of several genes involved in N uptake and N metabolism in rice, thereby enhancing N utilization. These results have implications for enhancing plant growth in low-input systems at nutrient-poor sites.     

Characterization of Imcrop, a Mutator-like MITE family in the rice genome

Sequence comparisons of ammonium transporter 1?C2 genes (OsAMT1-2) in different rice accessions revealed a MITE insertion in the upstream region of the gene. The 391-bp MITE, classified as a Mutator superfamily member and named Imcrop, included terminal inverted repeat (TIR) and 9-bp target site duplication (TSD) sequences. We identified 151 Imcrop elements dispersed on 12 chromosomes of the japonica reference genome. Of these, 12.6% were found in genic regions and 33.1% were located within 1.5 kb of annotated rice genes. We constructed comparative insertion maps with 111 and 102 intact Imcrop elements in the japonica and indica reference genomes, respectively. The Imcrop elements showed relatively even distribution across all chromosomes although their frequency was higher on chromosomes 1, 3, and 4 in both genomes. Seventy seven Imcrop elements were detected in both subspecies, whereas 34 and 25 insertions were found only in the japonica or indica genome, respectively. We compared insertion polymorphisms of 19 Imcrop elements found inside genes in 48 Korean rice cultivars, consisting of 42 japonica and six Tongil-types (indica-japonica cross). Thirteen insertions were common to all cultivars indicating these elements were present before indica-japonica divergence. The six other elements showed insertion polymorphisms among accessions, showing their recent insertion history or no critical positive effect of their insertion on the rice genome.     

Assimilation of ammonium ions and reutilization of nitrogen in rice (Oryza sativa L.)

A major source of inorganic nitrogen for rice plants grown in paddy soil is ammonium ions. The ammonium ions are actively taken up by the roots via ammonium transporters and subsequently assimilated into the amide residue of glutamine (Gln) by the reaction of glutamine synthetase (GS) in the roots. The Gln is converted into glutamate (Glu), which is a central amino acid for the synthesis of a number of amino acids, by the reaction of glutamate synthase (GOGAT). Although a small gene family for both GS and GOGAT is present in rice, ammonium-dependent and cell type-specific expression suggest that cytosolic GS1;2 and plastidic NADH-GOGAT1 are responsible for the primary assimilation of ammonium ions in the roots. In the plant top, approximately 80% of the total nitrogen in the panicle is remobilized through the phloem from senescing organs. Since the major form of nitrogen in the phloem sap is Gln, GS in the senescing organs and GOGAT in developing organs are important for nitrogen remobilization and reutilization, respectively. Recent work with a knock-out mutant of rice clearly showed that GS1;1 is responsible for this process. Overexpression studies together with age- and cell type-specific expression strongly suggest that NADH-GOGAT1 is important for the reutilization of transported Gln in developing organs. The overall process of nitrogen utilization within the plant is discussed.     

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.     

Gene structure and expression of the high-affinity nitrate transport system in rice roots

Rice has a preference for uptake of ammonium over nitrate and can use ammonium-N efficiently. Consequently, transporters mediating ammonium uptake have been extensively studied, but nitrate transporters have been largely ignored. Recently,some reports have shown that rice also has high capacity to acquire nitrate from growth medium, so understanding the nitrate transport system in rice roots is very important for improving N use efficiency in rice. The present study identified four putative NRT2 and two putative NAR2 genes that encode components of the high-affinity nitrate transport system (HATS) in the rice (Oryza sativa L. subsp, japonica cv. Nipponbare) genome. OsNRT2.1 and OsNRT2.2 share an identical coding region sequence, and their deduced proteins are closely related to those from monocotyledonous plants. The two NAR2 proteins are closely related to those from mono-cotyledonous plants as well. However, OsNRT2.3 and OsNRT2.4 are more closely related to Arabidopsis NRT2 proteins. Relative quantitative reverse tranecdption-polymerase chain reaction analysis showed that all of the six genes were rapidly upregulated and then downregulated in the roots of N-starved rice plants after they were re-supplied with 0.2 mM nitrate, but the response to nitrate differed among gene members.The results from phylogenetic tree, gene structure and expression analysis implied the divergent roles for the individual members of the rice NRT2 and NAR2 families. High-affinity nitrate influx rates associated with nitrate induction in rice roots were investigated and were found to be regulated by external pH. Compared with the nitrate influx rates at pH 6.5, alkaline pH (pH 8.0) inhibited nitrate Influx, and acidic pH (pH 5.0) enhanced the nitrate influx In I h nitrate induced roots, but did not significantly affect that in 4 to 8 h nitrate induced roots.