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First published online May 28, 2008; 10.1104/pp.108.119339 Plant Physiology 147:1225-1238 (2008) © 2008 American Society of Plant Biologists
Physiological and Transcriptomic Aspects of Urea Uptake and Assimilation in Arabidopsis Plants1,[W]INRA, Jean-Pierre Bourgin Institute, Unité de Nutrition Azotée des Plantes, F–78000 Versailles, France (P.M., M.L., C.M., F.D.-V.); INRA, Unité Mixte de Recherche en Génomique Végétale, F–91057 Evry, France (F.B., J.-P.R.); and BiotechMarine BP 65, 22260 Pontrieux, France (X.B.)
Urea is the major nitrogen (N) form supplied as fertilizer in agriculture, but it is also an important N metabolite in plants. Urea transport and assimilation were investigated in Arabidopsis (Arabidopsis thaliana). Uptake studies using 15N-labeled urea demonstrated the capacity of Arabidopsis to absorb urea and that the urea uptake was regulated by the initial N status of the plants. Urea uptake was stimulated by urea but was reduced by the presence of ammonium nitrate in the growth medium. N deficiency in plants did not affect urea uptake. Urea exerted a repressive effect on nitrate influx, whereas urea enhanced ammonium uptake. The use of [15N]urea and [15N]ammonium tracers allowed us to show that urea and ammonium assimilation pathways were similar. Finally, urea uptake was less efficient than nitrate uptake, and urea grown-plants presented signs of N starvation. We also report the first analysis, to our knowledge, of Arabidopsis gene expression profiling in response to urea. Our transcriptomic approach revealed that nitrate and ammonium transporters were transcriptionally regulated by urea as well as key enzymes of the glutamine synthetase-glutamate synthase pathway. AtDUR3, a high-affinity urea transporter in Arabidopsis, was strongly up-regulated by urea. Moreover, our transcriptomic data suggest that other genes are also involved in urea influx.
Urea [CO(NH2)2] is the major nitrogen (N) form supplied as fertilizer in agricultural plant production, ahead of nitrate and ammonium (http://www.fertilizer.org/ifa/statistics/STATSIND/). Urea in soils is hydrolyzed by urease, a nickel-dependent enzyme produced by soil microorganisms (Watson et al., 1994  ), into ammonium (NH4+), with often a concurrent nitrification to nitrate (NO3–). Thus, fertilization with urea may result in plant roots being simultaneously exposed to urea, ammonium, and nitrate, at least for short periods of time.
The uptake of urea in plant roots and leaves before hydrolysis into carbon dioxide and ammonium has been observed in several independent experiments (Hine and Spent, 1988
Little is known about the interactions between urea and ammonium or nitrate uptake and assimilation. Such interactions have been suggested, since it has been shown that the initial N status of plants and/or the presence of other N metabolites alters the uptake and reduction of nitrate (Crawford and Glass, 1998
Besides being the predominant N form used for fertilizer application to crops plants, urea is also an important N metabolite in plants. Two major biochemical pathways, Arg degradation and ureide catabolism, together with root and leaf applications of urea, are the three major sources of urea in plants. Since urea accumulates in source leaves of older plants and in germinating seeds (Zonia et al., 1995 The objectives of the work reported here were (1) to characterize urea uptake and assimilation by Arabidopsis seedlings by following N metabolites of seedlings after exposure to different N fertilization regimes; (2) to examine possible interactions between urea, ammonium, and nitrate on N transport systems by 15N influx measurements; and (3) to better understand the molecular basis for urea uptake and assimilation mechanisms using a transcriptomic approach.
Growth and N Status
We previously demonstrated that in our hydroponic culture conditions, urea is not hydrolyzed in the nutrient solution (Merigout et al., 2008
The analysis of the accumulated dry biomass revealed two groups: the first one corresponded to AN and AN+U treatments, the second to U and S treatments (Fig. 1B). The first group (AN and AN+U) was characterized by a slightly lower root fresh weight and a higher shoot fresh weight (mean 25% increase) than the second group (U and S; Fig. 1B). This difference in plant growth might reflect differences in N assimilation or uptake related to the N regime. We thus measured the total N content in the two groups of plants. We observed a lower root and shoot N content in the plants grown with urea as the sole N source (Fig. 1C) compared with AN- and AN+U-grown plants, which showed no difference in N content. As expected, this decrease was even more pronounced in N-starved plants (Fig. 1C). Biomass accumulation of Arabidopsis plants on the different N regimes seemed to be correlated with total N content. In order to better characterize the plant N status, we then measured nitrate, ammonium, urea, and amino acid contents.
The addition of urea in AN nutrient solution did not significantly affect the NO3– content of plants (Table I
). In contrast, in plants fed for 1 week with either urea as the sole N source or without any N source, NO3– almost disappeared from roots and only a small amount of NO3– was still present in shoots. In both treatments, chloride contents increased while sulfate and phosphate contents remain stable (Supplemental Fig. S1). These results show that the duration of the treatment was sufficient to provoke changes in the plant N status. Plants grown on the ureic nutrition contained the highest free NH4+ concentration in both roots and shoots, whereas the N-starved plants had low amounts of NH4+. The two other treatments (AN and AN+U) revealed comparable and intermediate amounts of NH4+ (Table I). With the exception of N-starved plants, in which, as described previously (Kojima et al., 2007
In order to determine which amino acids were responsible for the strong accumulation of total soluble amino acids observed in plants fed with urea as the sole N source, we analyzed the amino acid composition of these plants (Fig. 2 ). Gln and Ala were 5- and 4-fold more abundant, respectively, in roots of urea-treated plants than in control plants grown on AN solution (Fig. 2A). Similar high Gln and Ala levels were also found in plants fertilized with 1 mM (NH4)2SO4 (data not shown). Gly, Ser, Asn, and  -aminobutyric acid (GABA) levels were also increased. N-starved plants always had lower concentrations of amino acids, whereas plants grown on AN+U gave similar results to those for AN-grown plants. In the shoots, the concentration of Gln was much higher than in roots, except for N-starved plants, but, unlike in roots, U-grown plants showed only a modest increase in Gln concentration (Fig. 2B). Only Ser levels seemed to augment similarly in roots and shoots when plants were grown on urea as the sole N source and conserved the same ratio between AN and U conditions (Fig. 2B). Mere traces of Orn and citrulline, intermediate products of the urea cycle, could be detected in both plant parts (data not shown).
15N Influx and Pattern of 15N Labeling in Amino Acids To study urea absorption by root cells, we determined urea influx using 15N-labeled urea. Arabidopsis plants grown on AN, AN+U, U, or S nutrient solutions were incubated with labeled urea for 5 min. N influxes using 15NO3– and 15NH4+ were also measured in order to detect possible effects of urea nutrition on the nitrate and ammonium transport systems. Labeled urea, nitrate, and ammonium were used at high concentrations (10, 6, and 6 mM, respectively) to measure whole uptake processes involving both high- and low-affinity systems. Urea influx in roots was detected regardless of the N regime of the plants (Fig. 3A ). The urea influx reached the highest level when urea was present as the sole N source for 1 week (Fig. 3A). The uptake of nitrate was not affected when urea was used in combination with ammonium nitrate (AN or AN+U conditions) but was reduced in the urea-grown and N-starved plants. Conversely, the influx of ammonium in N-starved plants was 2-fold higher than in AN or AN+U plants. While ammonium and urea seemed to be assimilated directly in roots, nitrate was rapidly (5 min) translocated to the shoots, as the value of the shoot-root 15N ratio was about 1 for the AN and AN+U plants (Fig. 3B). Interestingly, it seems that the presence of nitrate is required during the growth period to induce a high level of nitrate transport to the aerial parts. Indeed, very little labeled nitrate was found in shoots of urea-grown plants and even less in N-starved leaves at the end of the 5-min labeling period (Fig. 3B).
Incorporation of 15N into amino acid in roots was followed by gas chromatography coupled to mass spectrometry (GC-MS) after 5 min of labeling. Feeding the plants with 15NO3 resulted in a very low proportion of labeled amino acids (less than 2%), with a peak for the amide [Gln(5N)] group of Gln after N starvation (Fig. 4A). The low 15N enrichment in 15NO3–-fed plants can be explained by (1) an ineffective reduction of nitrate to ammonium in roots in the 5-min labeling time, (2) a storage of nitrate in root vacuoles, or (3) an export of nitrate in aerial parts via the xylem vessels, as shown in Figure 3B. Conversely, when plants were labeled using 15NH4 or CO(15NH2), the relative level of labeled amino acids was higher, especially for Gln (Fig. 4, B and C ). Indeed, around 70% and 25% of the Gln pool in N-starved plants was labeled in 15NH4+- and CO(15NH2)2-fed plants, respectively. This suggests that the Gln pool in roots of N-starved plants is mainly synthesized during the 5-min labeling period and that N is quickly transferred from 15NH4+ or CO(15NH2)2 to Gln. Since urea first needs to be hydrolyzed into 15NH4,+ the rate of Gln synthesis by the GS enzyme may be lower in urea-labeled plants. The ratio of labeled Gln is significantly reduced in roots of plants grown in the presence of nitrate and ammonium and even lower in urea-grown plants when fed with 15NH4+ or CO(15NH2)2. This could be due to a higher Gln concentration at the start of the 15N labeling period, particularly in roots of urea-grown plants (Fig. 2A).
Concerning the CO(15NH2)2 assimilation, the highest 15N labeling was detected as [15N]urea regardless of the N nutrition. This result suggests that most of the urea taken up was not metabolized in roots. The labeled urea pool reached 50% of total 15N in urea-grown plants but was high regardless of the nutrition regime (Fig. 4C). This result was consistent with the measurements of urea influx. Labeled urea was detected in aerial organs (data not shown), suggesting that at least part of the [15N]urea absorbed by roots was transferred to shoots before its hydrolysis and assimilation (see above; Table I). [5-15N]Gln and then [2-15N]Gln were the second and third major sinks, respectively, for 15N from urea. This shows that urea molecules can be taken up and quickly assimilated and/or translocated by Arabidopsis roots. Finally, there was a low incorporation of 15N from urea or ammonium in Gly, Glu, pyro-Glu, Asn, GABA, Asp, and Ala (Fig. 4, B and C). These labeling patterns correspond to those seen in other labeling experiments (Masclaux-Daubresse et al., 2006 ).
As demonstrated above, the N treatments led to major changes in plant physiology at the levels of both N source uptake and enzymatic activities related to N assimilation. These changes might be linked to differences in gene expression, although global analysis could also reveal other urea-regulated processes. To determine the genomic response of Arabidopsis roots and shoots to urea N nutrition, transcriptome profiling was performed on 42-d-old U- versus AN-grown seedlings with CATMA arrays carrying gene-specific tags (GSTs; Crowe et al., 2003 Based on the statistical analysis, 960 (about 4% of total GSTs) and 474 (about 2% of total GSTs) genes were found differentially expressed between U- and AN-grown plants, respectively, in root and shoot parts. Fewer genes were differentially expressed for the comparison between AN+U and AN: 120 (about 0.5% of total GSTs) and 131 in roots and shoots, respectively. These genes are listed in the supplemental data (Supplemental Tables S1, S2, S4, and S5). Figure 5 shows the proportion of up- and down-regulated genes for each comparison. The ratios between up- and down-regulated genes were identical in roots for both comparisons. In contrast, more genes were down-regulated in shoots when ammonium nitrate was mixed with urea (Fig. 5 ).
The lists of the urea differentially regulated root and shoot genes are available as Supplemental Tables S1 and S2, respectively. Likewise, the lists of the AN+U differentially regulated root and shoot genes are available as Supplemental Tables S4 and S5, respectively. Genes differentially expressed were classified in functional categories according to the MapMan tool (Thimm et al., 2003 ; Supplemental Tables S3 and S6).
Urea versus Ammonium Nitrate In shoots, the most represented category, the RNA machinery (58 genes), was the same as that for roots. However, a similar number of genes were up- and down-regulated. With a difference of only 10 genes, protein (synthesis and degradation) was the second functional group, and more genes of this class were induced than repressed. Other genes found to respond to urea belonged to hormone metabolism, amino acid metabolism, transport, and stress. Ninety-one percent of genes of the amino acid metabolism class were up-regulated. For hormone metabolism, stress, and transport (aquaporins), the majority of genes were repressed.
Ammonium Nitrate + Urea versus Ammonium Nitrate
The comparison of results for the AN+U and U conditions with the AN reference allowed us to distinguish genes for which transcription was specifically regulated by urea. We found 58 and 32 common genes differentially regulated in roots and shoots, respectively, between the AN+U and U groups compared with the reference AN group. These genes are listed in Tables II and III. The patterns of regulation of gene expression in root for U and AN+U-grown plants were perfectly identical (Table II). This was less true for shoots (Table III), but globally, the regulation profiles were similar for the majority of the genes. In roots, 46 of the common genes were induced, and only 12 were repressed. These 58 genes were mainly distributed between transcription and transport categories. Two MIPs, At2g29870 and At2g34390, were included in the 46 induced genes (Table II). In shoots, 17 and 15 genes were up- and down-regulated, respectively. We found genes implicated in N and amino acid metabolism. While the transcription of a putative GS (At5g37600) was induced, genes coding for nitrite reductase (At2g15620) and for the Asn synthetase ASN2 (At5g65010) were negatively regulated (Table III).
Arabidopsis Plants Absorb Urea as an External N Source To our knowledge, this study is the first to associate physiological and molecular analyses of Arabidopsis plants treated with different N supplies including urea. Hydroponic culture conditions were chosen in order to circumvent any conversion of the N forms supplied during the course of the experiments. Indeed, our results demonstrate that, under our conditions, urea is directly taken up by root cells prior to hydrolysis.
Our study on CO(15NH2)2 influx, measured after 5 min, provides direct evidence that (1) urea is able to pass through the root cell membrane as an intact molecule regardless of the initial N status of the plants, (2) this uptake is stimulated when plants are grown on urea as the sole N source compared with AN, and (3) this uptake is inhibited by ammonium or nitrate (Fig. 3A). The concentration of urea in influx experiments (10 mM) was five times higher than that in the hydroponic urea treatment (2 mM). At this high concentration, a combination of high- and low-affinity urea transport systems could operate. At least four Arabidopsis genes coding for TIPs are able to complement a Saccharomyces cerevisiae mutant unable to use urea as the sole N source. Among them, AtTIP2.1 expression in Xenopus laevis oocytes also promotes urea accumulation even when urea concentrations are raised to 30 mM, suggesting a putative role as a low-affinity transporter (Liu et al., 2003b
The detection of labeled urea in roots after urea influx using mass spectrometry (Fig. 4C) provides the definitive proof of intact urea uptake by root cells. Urea uptake via the AtDUR3 high-affinity transporter was already described in Arabidopsis (Liu et al., 2003a
The stable protein content and the much higher levels of ammonium and total amino acids in roots after exposure to the urea solution compared with other N treatments (Table I) indicate efficient assimilation of urea N in roots. We assume, therefore, that urea, like ammonium but in contrast to nitrate (Loqué and von Wirén, 2004
In order to dissect the assimilation pathways of urea, we examined the individual amino acids in the different N-treated plants (Fig. 2), and we followed the route of 15N-radiolabeled urea by GC-MS in comparison with 15N from enriched nitrate and ammonium (Fig. 4). After 1 week of exposure to the urea solution, plant roots contained high levels of Gln and Ala. This suggests that in the root tissue, urea is first degraded by cytosolic ureases and then ammonium is incorporated via the GS-GOGAT cycle (Miflin and Lea, 1980
Some genes of the GS-GOGAT cycle were differentially expressed in plants grown with urea (Supplemental Tables S1 and S2), and their regulation correlates with the proposed pathway for urea N assimilation (see above). At1g66200, At5g37600 (GS1 putative), and At2g41220 (GLU2) were up-regulated by urea nutrition. GDH1 (At5g18170) was negatively regulated by urea treatment. Some genes involved in primary ammonium assimilation into amino acids were also regulated by the N status of plants. The expression levels of Asp aminotransferase 2 (At5g19550), Ala aminotransferase (At1g17290), as well as Asn synthetase 1 (At3g47340; ASN1) were increased with urea. These increases were clearly consistent with the increase of Asp, Ala, and Asn in roots supplied with urea. In contrast to ASN1, ASN2 (At5g65010) was down-regulated by urea, but it is already know that ASN1 and ASN2 are regulated in a reciprocal fashion by carbon or metabolites like amino acids (Lam et al., 1998 ), which is in agreement with our transcriptome results. As far as the enzymes of the urea cycle were concerned, no transcriptional regulation by urea was observed for urease, arginase, Orn carbamyl transferase, arginosuccinate synthetase, or arginosuccinate lyase. Only a urease accessory protein, UREG (At2g34470), was up-regulated by urea in roots of urea-grown plants. AtUREG, along with AtURED and AtUREF, has been demonstrated to be required for urease activation in Arabidopsis (Witte et al., 2005).
Both N-minus treatment and urea as the sole N source stimulated ammonium uptake. The gene AMT1.1 makes a major contribution to this process in roots (Loqué and von Wirén, 2004
If the supply of urea in conjunction with ammonium nitrate did not affect the growth and the N status of plants, plants treated with urea as the sole N source showed symptoms of N starvation, such as a severe drop of nitrate. After 7 d, nitrate was nearly undetectable in the roots and in very low abundance in the shoots. The root development of the urea-grown plants was also favored at the expense of the shoots, as in plants starved for N (Fig. 1B). Nevertheless, total N contents were statistically different in roots and shoots between urea-grown and N-starved plants (Fig. 1B). Thus, symptoms of N starvation after an exposure to the urea solution seem to reflect a less efficient but still active uptake and/or assimilation of the N in the ureic form compared with ammonium or nitrate by Arabidopsis. Comparable results were previously reported for a number of other species (Kirkby and Mengel, 1967 In conclusion, the data presented here demonstrate the capacity of Arabidopsis to absorb and assimilate urea as the sole N source. Urea uptake was regulated by urea itself and ammonium nitrate, but not by N starvation, as is the case for nitrate or ammonium uptake. Urea uptake was less efficient than nitrate uptake, and urea-grown plants presented signs of N starvation. Urea assimilation, however, was similar to the ammonium assimilation pathway. When mixed in equal proportion with ammonium nitrate, urea did not affect plant growth or N uptake, and nitrate content in plants tended to be lower. Thus, fertilization with urea in combination with ammonium nitrate fertilizers represents an alternative to reduce nitrate in crops. A comprehensive physiological and molecular investigation of urea transport and assimilation in planta is required not only to better understand the importance of urea for plants but also to improve its utilization as a root- and leaf-applied N fertilizer in agricultural crop production.
Plant Materials and Growth Conditions
Seed stocks of Arabidopsis (Arabidopsis thaliana) from the Columbia ecotype were used for all experiments. Plants were grown under hydroponic culture conditions in a growth chamber with an 8-h-light/16-h-dark cycle at 21°C/17°C, respectively, 80% relative humidity, and 150 µmol m–2 s–1 irradiation. Seeds were sterilized and stratified at 4°C for 5 d. Plant growth in hydroponic conditions was performed as described earlier (Orsel et al., 2004
An aliquot of the powder was weighed (50 mg fresh weight) and extracted with a two-step ethanol-water procedure, and supernatants were analyzed for anion concentration by HPLC on a DX-120 apparatus (Dionex) as described by Loudet et al. (2003)
Free amino acids were determined after extraction of an aliquot of powder (100 mg fresh weight) in 1 mL of a 2% (w/v) solution of sulfosalicylic acid. Supernatants were assayed for total amino acid determination by ninhydrin (Rosen, 1957
For urea extraction (method according to Killingsbaeck, 1975 For urea measurements, 1 mL of color development reagent was added to 30 µL of the formic acid extract in a microcentrifuge tube. The color development reagent consisted of 1:1:1 mixed acid reagent (20% [v/v] H2SO4 and 0.06% [v/v] 74 mM ferric chloride hexahydrate in 9% [v/v] ortho-phosphoric acid), mixed color reagent (7% [v/v] 0.2 M diacetylmonoxime and 7% [v/v] 0.05 M thiosemicarbazide), and water. Tubes were incubated for 15 min at 99°C. The samples were cooled for 5 min on ice, and the A540 was determined with a spectrophotometer.
N influxes were performed by 15N labeling as described by Orsel et al. (2004)
Both the evolution of amino acid contents and respective 15N enrichments were examined by GC-MS. Amino acids and ammonium from an aliquot of the root and shoot powder (about 150 mg fresh weight) were extracted with 1 mL of 2% (w/v) sulfosalicylic acid. Extracts were centrifuged at 17,500g for 20 min to eliminate cellular debris. To purify amino acids, extracts were applied to a column (AG 50W-X8 resin, 100–200 mesh, H+ form, 5 x 0.5 cm; Bio-Rad Laboratories), washed with 2 mL of water, and eluted with 2.5 mL of 6 M NH4OH. The amino acid fraction was frozen, evaporated, and rinsed twice with 500 µL of water, then evaporated. Amino acid samples were resuspended in 0.1 N HCl, dried under N2, and derivatized with N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide in acetonitrile at 75°C for 30 min (Chaves Das Neves and Vasconcelos, 1987
Statistical analyses were performed with the ANOVA function in XLStat-Pro 7.5 (Addinsoft).
Microarray analysis was carried out at the Research Unit in Plant Genomics using the CATMA array (Crowe et al., 2003
Statistical Analysis of Microarray Data Microarray data from this article were deposited at Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/; accession no.GSE9258) and at CATdb (http://urgv.evry.inra.fr/CATdb/; project RA05-09_UREA) according to the Minimum Information about a Microarray Experiment standards.
The following materials are available in the online version of this article.
The help of S. Boutet and M.-T. Leydecker with amino acid and nitrate analyses, respectively, is gratefully acknowledged. We thank Hoai-Nam Truong for assistance with MapMan and Helen North for carefully rereading the manuscript. We are grateful to Joël Talbotec and François Gosse for taking care of plant materials. Received March 18, 2008; accepted May 16, 2008; published May 28, 2008.
1 This work was supported by BiotechMarine.  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Françoise Daniel-Vedele (vedele{at}versailles.inra.fr).
[W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.108.119339 * Corresponding author; e-mail vedele{at}versailles.inra.fr.
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