The acquisition of nutrients requires tight regulation to ensure optimal supply while preventing accumulation to toxic levels. Ammonium transporter/methylamine permease/rhesus (AMT/Mep/Rh) transporters are responsible for ammonium acquisition in bacteria, fungi, and plants. The ammonium transporter AMT1;1 from Arabidopsis thaliana uses a novel regulatory mechanism requiring the productive interaction between a trimer of subunits for function. Allosteric regulation is mediated by a cytosolic C-terminal trans-activation domain, which carries a conserved Thr (T460) in a critical position in the hinge region of the C terminus. When expressed in yeast, mutation of T460 leads to inactivation of the trimeric complex. This study shows that phosphorylation of T460 is triggered by ammonium in a time-and concentration-dependent manner. Neither Gln nor L-methionine sulfoximine-induced ammonium accumulation were effective in inducing phosphorylation, suggesting that roots use either the ammonium transporter itself or another extracellular sensor to measure ammonium concentrations in the rhizosphere. Phosphorylation of T460 in response to an increase in external ammonium correlates with inhibition of ammonium uptake into Arabidopsis roots. Thus, phosphorylation appears to function in a feedback loop restricting ammonium uptake. This novel autoregulatory mechanism is capable of tuning uptake capacity over a wide range of supply levels using an extracellular sensory system, potentially mediated by a transceptor (i.e., transporter and receptor).

SummaryDespite 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 15 N-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 K m 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.

Urea is a soil nitrogen form available to plant roots and a secondary nitrogen metabolite liberated in plant cells. Based on growth complementation of yeast mutants and "in-silico analysis", two plant families have been identified and partially characterized that mediate membrane transport of urea in heterologous expression systems. AtDUR3 is a single Arabidopsis gene belonging to the sodium solute symporter family that cotransports urea with protons at high affinity, while members of the tonoplast intrinsic protein (TIP) subfamily of aquaporins transport urea in a channel-like manner. The following review summarizes current knowledge on the membrane localization, energetization and regulation of these two types of urea transporters and discusses their possible physiological roles in planta.

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In plants, urea derives either from root uptake or protein degradation. Although large quantities of urea are released during senescence, urea is mainly seen as a short-lived nitrogen (N) catabolite serving urease-mediated hydrolysis to ammonium. Here, we investigated the roles of DUR3 and of urea in N remobilization. During natural leaf senescence urea concentrations and DUR3 transcript levels showed a parallel increase with senescence markers like ORE1 in a plant age- and leaf age-dependent manner. Deletion of DUR3 decreased urea accumulation in leaves, whereas the fraction of urea lost to the leaf apoplast was enhanced. Under natural and N deficiency-induced senescence DUR3 promoter activity was highest in the vasculature, but was also found in surrounding bundle sheath and mesophyll cells. An analysis of petiole exudates from wild-type leaves revealed that N from urea accounted for >13% of amino acid N. Urea export from senescent leaves further increased in ureG-2 deletion mutants lacking urease activity. In the dur3 ureG double insertion line the absence of DUR3 reduced urea export from leaf petioles. These results indicate that urea can serve as an early metabolic marker for leaf senescence, and that DUR3-mediated urea retrieval contributes to the retranslocation of N from urea during leaf senescence.