The AMMONIUM TRANSPORTER (AMT) family comprises six isoforms in Arabidopsis thaliana. Here, we describe the complete functional organization of root-expressed AMTs for high-affinity ammonium uptake. High-affinity influx of 15 N-labeled ammonium in two transposon-tagged amt1;2 lines was reduced by 18 to 26% compared with wild-type plants. Enrichment of the AMT1;2 protein in the plasma membrane and localization of AMT1;2 promoter activity in the endodermis and root cortex indicated that AMT1;2 mediates the uptake of ammonium entering the root via the apoplasmic transport route. An amt1;1 amt1;2 amt1;3 amt2;1 quadruple mutant (qko) showed severe growth depression under ammonium supply and maintained only 5 to 10% of wild-type high-affinity ammonium uptake capacity. Transcriptional upregulation of AMT1;5 in nitrogen-deficient rhizodermal and root hair cells and the ability of AMT1;5 to transport ammonium in yeast suggested that AMT1;5 accounts for the remaining uptake capacity in qko. Triple and quadruple amt insertion lines revealed in vivo ammonium substrate affinities of 50, 234, 61, and 4.5 mM for AMT1;1, AMT1;2, AMT1;3, and AMT1;5, respectively, but no ammonium influx activity for AMT2;1. These data suggest that two principle means of achieving effective ammonium uptake in Arabidopsis roots are the spatial arrangement of AMT1-type ammonium transporters and the distribution of their transport capacities at different substrate affinities.

SummaryIn Arabidopsis four root-expressed AMT genes encode functional ammonium transporters, which raises the question of their role in primary ammonium uptake. After pre-culturing under nitrogen-deficiency conditions, we quantified the influx of 15 N-labeled ammonium in T-DNA insertion lines and observed that the loss of either AMT1;1 or AMT1;3 led to a decrease in the high-affinity ammonium influx of approximately 30%. Under nitrogen-sufficient conditions the ammonium influx was lower in Columbia glabra compared with Wassilewskija (WS), and AMT1;1 did not contribute significantly to the ammonium influx in Col-gl. Ectopic expression of AMT1;3 under the control of a 35S promoter in either of the insertion lines amt1;3-1 or amt1;1-1 increased the ammonium influx above the level of their corresponding wild types. In transgenic lines carrying AMT-promoter-GFP constructs, the promoter activities of AMT1;1 and AMT1;3 were both upregulated under nitrogen-deficiency conditions and were localized to the rhizodermis, including root hairs. AMT gene-GFP fusions that were stably expressed under the control of their own promoters were localized to the plasma membrane. The double insertion line amt1;1-1 amt1;3-1 showed a decreased sensitivity to the toxic ammonium analog methylammonium and a decrease in the ammonium influx of up to 70% relative to wildtype plants. These results suggest an additive contribution of AMT1;1 and AMT1;3 to the overall ammonium uptake capacity in Arabidopsis roots under nitrogen-deficiency conditions.

Glutamine synthetase (GS; EC 6.3.1.2) is a key enzyme of nitrogen assimilation, catalyzing the synthesis of glutamine from ammonium and glutamate. In Arabidopsis, cytosolic GS (GS1) was accumulated in roots when plants were excessively supplied with ammonium; however, the GS activity was controlled at a constant level. The discrepancy between the protein content and enzyme activity of GS1 was attributable to the kinetic properties and expression of four distinct isoenzymes encoded by GLN1;1, GLN1;2, GLN1;3 and GLN1;4, genes that function complementary to each other in Arabidopsis roots. GLN1;2 was the only isoenzyme significantly up-regulated by ammonium, which correlated with the rapid increase in total GS1 protein. GLN1;2 was localized in the vasculature and exhibited low affinities to ammonium (K m ‫؍‬ 2450 ؎ 150 M) and glutamate (K m ‫؍‬ 3.8 ؎ 0.2 mM). The expression of the counterpart vascular tissue-localizing low affinity isoenzyme, GLN1;3, was not stimulated by ammonium; however, the enzyme activity of GLN1;3 was significantly inhibited by a high concentration of glutamate. By contrast, the high affinity isoenzyme, GLN1;1 (K m for ammonium < 10 M; K m for glutamate ‫؍‬ 1.1 ؎ 0.4 mM) was abundantly accumulated in the surface layers of roots during nitrogen limitation and was down-regulated by ammonium excess. GLN1;4 was another high affinity-type GS1 expressed in nitrogen-starved plants but was 10-fold less abundant than GLN1;1. These results suggested that dynamic regulations of high and low affinity GS1 isoenzymes at the levels of mRNA and enzyme activities are dependent on nitrogen availabilities and may contribute to the homeostatic control of glutamine synthesis in Arabidopsis roots.Glutamine synthetase (GS 1 ; EC 6.3.1.2) is responsible for the primary assimilation of ammonium in higher plants (1-4). Ammonium is assimilated into glutamine and glutamate through a consecutive reaction of GS and glutamate synthase (GOGAT), the so-called GS/GOGAT cycle. Plants have two types of GS isoenzymes that localize in different compartments: one located in the cytosol (GS1) and the other in the plastid/chloroplasts (GS2) (1-4). GS1 is the major form of GS in plant roots, and the ammonium taken up from the soil is directly converted to Gln by its reaction. Molecular biological studies have identified a number of genes encoding GS1 from various plant species (5-9). The presence of multiple GS1 isoenzymes complicates the overall understanding of their physiological functions. The isoenzymes of GS1 show organ-and cell-specific patterns of expression and are developmentally regulated (10 -17). In addition, the expression of GS1 is metabolically regulated by the availability of nitrogen and carbon sources (18 -22, 24).In the roots of legumes, GS1 is regulated by ammonium supplied from the environment or the symbiotic nitrogen fixation (18 -21). Transgenic studies with the soybean GS1 promoter suggested that ammonium-dependent regulation is specific for nitrogen assimilation in leguminous plants; the soybean-derived GS1...

SummaryRice (Oryza sativa L.) plants possess three homologous but distinct genes for cytosolic glutamine synthetase (GS1): these are OsGS1;1, OsGS1;2, and OsGS1;3. OsGS1;1 was expressed in all organs tested with higher expression in leaf blades, while OsGS1;2, and OsGS1;3 were expressed mainly in roots and spikelets, respectively. We characterized knockout mutants caused by insertion of endogenous retrotransposon Tos17 into the exon-8 (lines ND8037 and ND9801) or the exon-10 (line NC2327) of OsGS1;1. Mendelian segregation occurred in each progeny. Homozygously inserted mutants showed severe retardation in growth rate and grain filling when grown at normal nitrogen concentrations. Abnormal mRNA for GS1;1 was transcribed, and the GS1 protein and its activity in the leaf blades were barely detectable in these mutants. The glutamine pool in the roots and leaf blades of the mutants was lower than that of the wild type. Re-introduction of OsGS1;1 cDNA under the control of its own promoter into the mutants successfully complemented these phenotypes. Progeny where Tos17 was heterozygously inserted or deleted during segregation showed normal phenotypes. The results indicate that GS1;1 is important for normal growth and grain filling in rice; GS1;2 and GS1;3 were not able to compensate for GS1;1 function.

Prolamine and glutelin RNAs are localized to two subdomains of the cortical endoplasmic reticulum (ER), the protein body ER and the cisternal ER, in developing rice seeds. The addition of nearly full-length prolamine sequences at the 3 untranslated region of a reporter RNA redirects its localization from the cisternal ER to the protein body ER. Deletion analysis of prolamine RNA sequences indicates the presence of two partially redundant cis elements required for protein body ER targeting. The addition of glutelin 3 untranslated region to protein body ER cis sequences, however, redirects RNA localization to the cisternal ER. These results indicate that there are at least two regulated RNA transport pathways as well as a constitutive pathway to the cortical ER.