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Ammonium (NH 4 + ) represents a primary nitrogen source for many plants, its effective transport into and between tissues and further assimilation in cells determine greatly plant nitrogen use efficiency. However, biological components involved in NH 4 + movement in woody plants are unclear. Here, we report kinetic evidence for cotton NH 4 + uptake and molecular identification of certain NH 4 + transporters (AMTs) from cotton (Gossypium hirustum). A substrate-influx assay using 15 N-isotope revealed that cotton possessed a high-affinity transport system with a K m of 58 μM for NH 4 + . Sequence analysis showed that GhAMT1.1-1.3 encoded respectively a membrane protein containing 485, 509 or 499 amino acids. Heterologous functionality test demonstrated that GhAMT1.1-1.3 expression mediated NH 4 + permeation across the plasma membrane (PM) of yeast and/or Arabidopsis qko-mutant cells, allowing a growth restoration of both mutants on NH 4 + . Quantitative PCR measurement showed that GhAMT1.3 was expressed in roots and leaves and markedly up-regulated by N-starvation, repressed by NH 4 + resupply and regulated diurnally and age-dependently, suggesting that GhAMT1.3 should be a N-responsive gene. Importantly, GhAMT1.3 expression in Arabidopsis improved plant growth on NH 4 + and enhanced total nitrogen accumulation (∼50% more), conforming with the observation of 2-fold more NH 4 + absorption by GhAMT1.3-transformed qko plant roots during a 1-h root influx period. Together with its targeting to the PM and saturated transport kinetics with a K m of 72 μM for NH 4 + , GhAMT1.3 is suggested to be a high-affinity NH 4 + permease that may play a significant role in cotton NH 4 + acquisition and utilization, adding a new member in the plant AMT family., (© 2018 Scandinavian Plant Physiology Society.)

• Despite the great agricultural and ecological importance of efficient use of urea-containing nitrogen fertilizers by crops, molecular and physiological identities of urea transport in higher plants have been investigated only in Arabidopsis. • We performed short-time urea-influx assays which have identified a low-affinity and high-affinity (K(m) of 7.55 μM) transport system for urea-uptake by rice roots (Oryza sativa). • A high-affinity urea transporter OsDUR3 from rice was functionally characterized here for the first time among crops. OsDUR3 encodes an integral membrane-protein with 721 amino acid residues and 15 predicted transmembrane domains. Heterologous expression demonstrated that OsDUR3 restored yeast dur3-mutant growth on urea and facilitated urea import with a K(m) of c. 10 μM in Xenopus oocytes. • Quantitative reverse-transcription polymerase chain reaction (qPCR) analysis revealed upregulation of OsDUR3 in rice roots under nitrogen-deficiency and urea-resupply after nitrogen-starvation. Importantly, overexpression of OsDUR3 complemented the Arabidopsis atdur3-1 mutant, improving growth on low urea and increasing root urea-uptake markedly. Together with its plasma membrane localization detected by green fluorescent protein (GFP)-tagging and with findings that disruption of OsDUR3 by T-DNA reduces rice growth on urea and urea uptake, we suggest that OsDUR3 is an active urea transporter that plays a significant role in effective urea acquisition and utilisation in rice., (© 2011 The Authors. New Phytologist © 2011 New Phytologist Trust.)

Rice (Oryza sativa) production relies strongly on nitrogen (N) fertilization with urea, but the proteins involved in rice urea metabolism have not yet been characterized. Coding sequences for rice arginase, urease, and the urease accessory proteins D (UreD), F (UreF), and G (UreG) involved in urease activation were identified and cloned. The functionality of urease and the urease accessory proteins was demonstrated by complementing corresponding Arabidopsis (Arabidopsis thaliana) mutants and by multiple transient coexpression of the rice proteins in Nicotiana benthamiana. Secondary structure models of rice (plant) UreD and UreF proteins revealed a possible functional conservation to bacterial orthologs, especially for UreF. Using amino-terminally StrepII-tagged urease accessory proteins, an interaction between rice UreD and urease could be shown. Prokaryotic and eukaryotic urease activation complexes seem conserved despite limited protein sequence conservation for UreF and UreD. In plant metabolism, urea is generated by the arginase reaction. Rice arginase was transiently expressed as a carboxyl-terminally StrepII-tagged fusion protein in N. benthamiana, purified, and biochemically characterized (K(m) = 67 mm, k(cat) = 490 s(-1)). The activity depended on the presence of manganese (K(d) = 1.3 microm). In physiological experiments, urease and arginase activities were not influenced by the external N source, but sole urea nutrition imbalanced the plant amino acid profile, leading to the accumulation of asparagine and glutamine in the roots. Our data indicate that reduced plant performance with urea as N source is not a direct result of insufficient urea metabolism but may in part be caused by an imbalance of N distribution.