Despite the fact that urea is a ubiquitous nitrogen source in soils and the most widespread form of nitrogen fertilizer used in agricultural plant production, membrane transporters that might contribute to the uptake of urea in plant roots have so far been characterized only in heterologous systems. Two T-DNA insertion lines, atdur3-1 and atdur3-3, that showed impaired growth on urea as a sole nitrogen source were used to investigate a role of the H+/urea co-transporter AtDUR3 in nitrogen nutrition in Arabidopsis. In transgenic lines expressing AtDUR3-promoter:GFP constructs, promoter activity was upregulated under nitrogen deficiency and localized to the rhizodermis, including root hairs, as well as to the cortex in more basal root zones. Protein gel blot analysis of two-phase partitioned root membrane fractions and whole-mount immunolocalization in root hairs revealed the plasma membrane to be enriched in AtDUR3 protein. Expression of the AtDUR3 gene in nitrogen-deficient roots was repressed by ammonium and nitrate but induced after supply of urea. Higher accumulation of urea in roots of wild-type plants relative to atdur3-1 and atdur3-3 confirmed that urea was the substrate transported by AtDUR3. Influx of 15N-labeled urea in atdur3-1 and atdur3-3 showed a linear concentration dependency up to 200 lM external urea, whereas influx in wild-type roots followed saturation kinetics with an apparent Km of 4 lM. The results indicate that AtDUR3 is the major transporter for high-affinity urea uptake in Arabidopsis roots and suggest that the high substrate affinity of AtDUR3 reflects an adaptation to the low urea levels usually found in unfertilized soils.

In 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 15N-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 pl asmamembrane. 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.

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 15N-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.

Pollen represents an important nitrogen sink in fl owers to ensure pollen viability. Since pollen cells are symplasmically isolated during maturation and germination, membrane transporters are required for nitrogen import across t hepollen plasma membrane. This study describes the characterization of the ammonium transporter AtAMT1;4, a so far uncharacterized member of the Arabidopsis AMT1 family, which is suggested to be involved in transporting ammonium into pollen. The AtAMT1;4 gene encodes a functional ammonium transporter when heterologously expressed in yeast or when overexpressed in Arabidopsis roots. Concentration-dependent analysis of 15 N-labeled ammonium infl ux into roots of AtAMT1;4 –transformed plants allowed characterization of AtAMT1;4 as a highaffi nity transporter with a K m of 17 μ M. RNA and protein gel blot analysis showed expression of AtAMT1;4 in fl owers, and promoter–gene fusions to the green fl uorescent protein (GFP) further defi ned its exclusive expression in pollen grains and pollen tubes. The AtAMT1;4 protein appeared to be localized to the plasma membrane as indicated by protein gel blot analysis of plasma membraneenriched membrane fractions and by visualization of GFP-tagged AtAMT1;4 protein in pollen grains and pollen tubes. However, no phenotype related to pollen function could be observed in a transposon-tagged line, in which AtAMT1;4 expression is disrupted. These results suggest that AtAMT1;4 mediates ammonium uptake across the plasma membrane of pollen to contribute to nitrogen nutrition of pollen via ammonium uptake or retrieval.

Ammonium transporter (AMT) proteins of the AMT family mediate the transport of ammonium across plasma membranes. To investigate whether AMTs are regulated at the posttranscriptional level, a gene construct consisting of the cauliflower mosaic virus 35S promoter driving the Arabidopsis (Arabidopsis thaliana) AMT1;1 gene was introduced into tobacco (Nicotiana tabacum). Ectopic expression of AtAMT1;1 in transgenic tobacco lines led to high transcript levels and protein levels at the plasma membrane and translated into an approximately 30% increase in root uptake capacity for 15N-labeled ammonium in hydroponically grown transgenic plants. When ammonium was supplied as the major nitrogen (N) form but at limiting amounts to soil-grown plants, transgenic lines overexpressing AtAMT1;1 did not show enhanced growth or N acquisition relative to wild-type plants. Surprisingly, steady-state transcript levels of AtAMT1;1 accumulated to higher levels in N-deficient roots and shoots of transgenic tobacco plants in spite of expression being controlled by the constitutive 35S promoter. Moreover, steady-state transcript levels were decreased after addition of ammonium or nitrate in N-deficient roots, suggesting a role for N availability in regulating AtAMT1;1 transcript abundance. Nitrogen deficiency-dependent accumulation of AtAMT1;1 mRNA was also observed in 35S:AtAMT1;1-transformed Arabidopsis shoots but not in roots. Evidence for a regulatory role of the 3#-untranslated region of AtAMT1;1 alone in N-dependent transcript accumulation was not found. However, transcript levels of AtAMT1;3 did not accumulate in a N-dependent manner, even though the same T-DNA insertion line atamt1;1-1 was used for 35S:AtAMT1;3 expression. These results show that the accumulation of AtAMT1;1 transcripts is regulated in a N- and organ-dependent manner and suggest mRNA turnover as an additional mechanism for the regulation of AtAMT1;1 in response to the N nutritional status of plants.

Boron (B) is essential for plants but toxic when present in excess. Arabidopsis thaliana BOR1 is a B exporter for xylem loading and is essential for efficient B translocation from roots to shoots under B limitation. B translocation to shoots was enhanced under B limitation in WT but not in bor1-1 mutant plants. The enhanced translocation was suppressed upon resupply of high levels of B within several hours. Unlike a number of transporters for essential mineral nutrients, BOR1 mRNA accumulation was not strongly affected by B conditions. However, accumulation of a constitutively expressed BOR1-GFP fusion protein was elevated under conditions of limited B supply. Upon resupply of high levels of B, BOR1-GFP was degraded within several hours. These findings demonstrate that posttranscriptional mechanisms play a major role in regulation of BOR1 accumulation. Confocal laser scanning microscopy of root tip cells showed that BOR1-GFP is localized to the plasma membrane under B limitation. Shortly after B application, the protein was observed in dot-like structures in the cytoplasm before degradation. Colocalization studies of the fusion protein with an endocytic tracer FM4-64 and an endosomal Rab-GTPase Ara7 fused to monomeric red fluorescent protein suggested that BOR1 is transferred from the plasma membrane via the endosomes to the vacuole for degradation. These results establish that endocytosis and degradation of BOR1 are regulated by B availability, to avoid accumulation of toxic levels of B in shoots under high-B supply, while protecting the shoot from B deficiency under B limitation.