AtDUR3 encodes a new type of high-affinity urea/H+ symporter in Arabidopsis

Urea is the major nitrogen form supplied as fertilizer in agricultural plant production but also an important nitrogen metabolite in plants. We report the cloning and functional characterization of AtDUR3, a high-affinity urea transporter in plants. AtDUR3 contains 14 putative transmembrane-spanning domains and represents an individual member in Arabidopsis that belongs to a superfamily of sodium-solute symporters. Heterologous expression in urea uptake-defective yeast as well as two-electrode voltage clamp and uptake studies using (14)C-labeled urea in AtDUR3-expressing oocytes demonstrated that AtDUR3 mediates urea transport. In both heterologous systems, urea transport was stimulated at low pH. In oocytes, inward currents indicated that urea is cotransported with protons. By contrast, a supply of Na(+) ions could not stimulate urea transport. Transport of (14)C-labeled urea by AtDUR3 in oocytes exhibited saturation kinetics with a K(m) of approximately 3 micro M. AtDUR3 was expressed in shoots and roots and upregulated during early germination and under nitrogen deficiency in roots. We propose a role of AtDUR3 in urea uptake by plant cells at low external urea concentrations.     

Inadequate root uptake may represent a major component limiting rice to use urea as sole nitrogen source for growth

Background and aims

Urea represents over 50 % of nitrogen fertilizers applied worldwide to crop production, however one-third of nitrogen fertilized could be recovered by crops. Previous studies have provided useful knowledge of urea-related plant nitrogen-nutrition, whereas information about crop growth-response to urea without its external degradation seems limiting. We thus assess the ability of rice seedlings to use urea at a physiological level.

Methods

Rice growth on urea versus other nitrogen regimes was tested under aseptic conditions. Activity of urease and GS was analyzed; urea, ammonium, total nitrogen and expression of a nitrogen limitation-responsive gene OsDUR3 were examined.

Results

Growth phenotyping revealed urea-dose-dependent growth improvement but significant growth reduction associated with nitrogen-deficiency of plants compared to those on other nitrogen-sources, indicating a physiological impediment of effective urea utilization by rice. Enzymatic assay showed that activities of urease and GS were well expressed in plants supplied with urea. Low concentrations of urea and ammonium were detected in rice (particularly in roots) on 1 mM urea or other nitrogen-forms, and a less nitrogen-content was determined in urea-fed plants. Additionally, the strongest OsDUR3-expression occurred in seedlings on no-nitrogen or 1 mM urea.

Conclusions

We suggest that insufficient urea-absorption but not assimilation represents likely a factor contraining rice to use urea as sole nitrogen-source.     

AtKuP1: a dual-affinity K+ transporter from Arabidopsis.

Plant roots contain both high- and low-affinity transport systems for uptake of K+ from the soil. In this study, we characterize a K+ transporter that functions in both high- and low-affinity uptake. Using yeast complementation analysis, we isolated a cDNA for a functional K+ transporter from Arabidopsis (referred to as AtKUP1 for Arabidopsis thaliana K+ uptake). When expressed in a yeast mutant, AtKUP1 dramatically increased K+ uptake capacity at both a low and high [K+] range. Kinetic analyses showed that AtKUP1-mediated K+ uptake displays a "biphasic" pattern similar to that observed in plant roots. The transition from the high-affinity phase (K(m) of 44 microM) to the low-affinity phase (K(m) of 11 mM) occurred at 100 to 200 microM external K+. Both low- and high-affinity K+ uptake via AtKUP1 were inhibited by 5 mM or higher concentrations of NaCl. In addition, AtKUP1-mediated K+ uptake was inhibited by K+ channel blockers, including tetraethylammonium, Cs+, and Ba2+. Consistent with a possible function in K+ uptake from the soil, the AtKUP1 gene is primarily expressed in roots. We conclude that the AtKUP1 gene product may function as a K+ transporter in Arabidopsis roots over a broad range of [K+] in the soil.     

TRH1 encodes a potassium transporter required for tip growth in Arabidopsis root hairs

Root hair initiation involves the formation of a bulge at the basal end of the trichoblast by localized diffuse growth. Tip growth occurs subsequently at this initiation site and is accompanied by the establishment of a polarized cytoplasmic organization. Arabidopsis plants homozygous for a complete loss-of-function tiny root hair 1 (trh1) mutation were generated by means of the T-DNA-tagging method. Trichoblasts of trh1 plants form initiation sites but fail to undergo tip growth. A predicted primary structure of TRH1 indicates that it belongs to the AtKT/AtKUP/HAK K(+) transporter family. The proposed function of TRH1 as a K(+) transporter was confirmed in (86)Rb uptake experiments, which demonstrated that trh1 plants are partially impaired in K(+) transport. In line with these results, TRH1 was able to complement the trk1 potassium transporter mutant of Saccharomyces, which is defective in high-affinity K(+) uptake. Surprisingly, the trh1 phenotype was not restored when mutant seedlings were grown at high external potassium concentrations. These data demonstrate that TRH1 mediates K(+) transport in Arabidopsis roots and is responsible for specific K(+) translocation, which is essential for root hair elongation.     

高等植物尿素代谢及转运的分子机理

尿素广泛存在于自然界中, 是易于被许多生物(如植物)利用的生长氮源。该文通过概述尿素在不同生命系统中存在的基础生理意义及各类型尿素转运蛋白, 讨论了植物细胞中尿素合成与分解的各种途径及尿素在植物氮营养、代谢和运输中的生理作用。迄今为止, 在植物中已发现了2类转运尿素的膜蛋白, 即MIPs和DUR3, 它们分别在低亲和力、高亲和力尿素运输中发挥潜在作用。异源表达结果表明, MIPs介导了尿素的被动迁移; 而AtDUR3则参与拟南芥根系对尿素的吸收。对MIPs和DUR3转运尿素的酶学特征、亚细胞作用位点和表达调控状况等的研究表明: 它们的分子生物学功能与植物的氮营养及氮素再分配和利用相关。     

高等植物尿素代谢及转运的分子机理

尿素广泛存在于自然界中,是易于被许多生物（如植物）利用的生长氮源。该文通过概述尿素在不同生命系统中存在的基础生理意义及各类型尿素转运蛋白,讨论了植物细胞中尿素合成与分解的各种途径及尿素在植物氮营养、代谢和运输中的生理作用。迄今为止,在植物中已发现了2类转运尿素的膜蛋白,即MIPs和DUR3,它们分别在低亲和力、高亲和力尿素运输中发挥潜在作用。异源表达结果表明MIPs介导了尿素的被动迁移：而AtDUR3则参与拟南芥根系对尿素的吸收。对MIPs和DUR3转运尿素的酶学特征、亚细胞作用位点和表达调控状况等的研究表明：它们的分子生物学功能与植物的氮营养及氮素再分配和利用相关。     

Characterization of urea transport in Bufo arenarum oocytes

Xenopus laevis oocytes have been extensively used for expression cloning, structure/function relationships, and regulation analysis of transporter proteins. Urea transporters have been expressed in Xenopus oocytes and their properties have been described. In order to establish an alternative system in which urea transporters could be efficiently expressed and studied, we determined the urea transport properties of ovarian oocytes from Bufo arenarum, a toad species common in Argentina. Bufo oocytes presented a high urea permeability of 22.3 x 10(-6) cm/s, which was significantly inhibited by the incubation with phloretin. The urea uptake in these oocytes was also inhibited by mercurial reagents, and high-affinity urea analogues. The urea uptake was not sodium dependent. The activation energy was 3.2 Kcal/mol, suggesting that urea movement across membrane oocytes may be through a facilitated urea transporter. In contrast, Bufo oocytes showed a low permeability for mannitol and glycerol. From these results, we propose that one or several specific urea transporters are present in ovarian oocytes from Bufo arenarum. Therefore, these oocytes cannot be used in expression studies of foreign urea transporters. The importance of Bufo urea transporter is not known but could be implicated in osmotic regulation during the laying of eggs in water.     

Kinetic independence between red cell anion exchange and urea transport

Urea equilibrium exchange fluxes were measured in human red cells under conditions which recruit the anion transporter into an outward-facing or an inward-facing state (with respect to the anion transport site). Regardless of these conditions, urea transport always occurred at the same rate: 41 +/- 2 mol.(kg cell solids.min)-1 with 1.5 M urea at 0 degrees C. These data suggest that the pathway on the band-3 protein which mediates anion transport is kinetically uncoupled from urea transport and is probably not involved in the transport of urea across the red cell membrane.     

AtDUR3 represents the major transporter for high-affinity urea transport across the plasma membrane of nitrogen-deficient Arabidopsis roots

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 microM external urea, whereas influx in wild-type roots followed saturation kinetics with an apparent Km of 4 microM. 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.     

Multiple roles of nitrate transport accessory protein NAR2 in plants

Two component high affinity nitrate transport system, NAR2/NRT2, has been defined in several plant species. In Arabidopsis, AtNAR2.1 has a role in the targeting of AtNRT2.1 to the plasma membrane. The gene knock out mutant atnar2.1 lacks inducible high-affinity transport system (IHATS) activity, it also shows the same inhibition of lateral root (LR) initiation on the newly developed primary roots as the atnrt2.1 mutant in response to low nitrate supply. In rice, OsNAR2.1 interacts with OsNRT2.1, OsNRT2.2 and OsNRT2.3a to provide nitrate uptake over high and low concentration ranges. In rice roots OsNAR2.1 and its partner NRT2s show some expression differences in both tissue specificity and abundance. It can be predicted that NAR2 plays multiple roles in addition to being an IHATS component in plants.Key words: NAR2, NRT2, nitrate transporter, root     

Phloem-localizing sulfate transporter,Sultr1;3, mediates re-distribution of sulfur from source to sink organs in Arabidopsis

For the effective recycling of nutrients, vascular plants transport pooled inorganic ions and metabolites through the sieve tube. A novel sulfate transporter gene, Sultr1;3, was identified as an essential member contributing to this process for redistribution of sulfur source in Arabidopsis. Sultr1;3 belonged to the family of high-affinity sulfate transporters, and was able to complement the yeast sulfate transporter mutant. The fusion protein of Sultr1;3 and green fluorescent protein was expressed by the Sultr1;3 promoter in transgenic plants, which revealed phloem-specific expression of Sultr1;3 in Arabidopsis. Sultr1;3-green fluorescent protein was found in the sieve element-companion cell complexes of the phloem in cotyledons and roots. Limitation of external sulfate caused accumulation of Sultr1;3 mRNA both in leaves and roots. Movement of (35)S-labeled sulfate from cotyledons to the sink organs was restricted in the T-DNA insertion mutant of Sultr1;3. These results provide evidence that Sultr1;3 transporter plays an important role in loading of sulfate to the sieve tube, initiating the source-to-sink translocation of sulfur nutrient in Arabidopsis.