BACKGROUND
by  <dig>  the global population is expected to be 50% higher than at present and global grain demand is projected to double .
today the productivity of crops is based on the application of high amounts of industrially produced nitrogen  fertilizer, even though crop plants utilize only 30-40% of the applied n  <cit> .
as a consequence, the wide use of synthetic n fertilizer has led to negative impacts on the environment and on farmer economies.
in addition, the n use efficiency  of cereal crops has declined in the last 50 years  <cit> .
based on these considerations, crop yield needs to be improved in a more cost-effective and eco-compatible way.
this goal could be achieved by increasing the nue of cereals and optimizing the acquisition of naturally occurring and applied n. reducing the amount of fertilizers in maize culture will have economic and environmental benefits.
in particular combining reduced fertilizer application and breeding plants with better nue is one of the main goals of research in plant nutrition  <cit> .
urea is the most frequently used n fertilizer in the world, with annual amounts of over  <dig> million tons accounting for more than 50% of the world n-fertilizer consumption .
the great increase in urea-fertilizer use during the last decades is mainly due to its competitive price and the high n content , that allow reducing transport and distribution costs  <cit> .
besides the chemical input as fertilizer, urea is also a natural organic molecule synthesized by most organisms  <cit> .
in plants, urea represents an important metabolic intermediate produced during n-recycling  <cit> , while in mammals the urea production is associated with the detoxification of n compounds  <cit> .
although urea might be derived from both natural and chemical syntheses, in the soil it usually occurs only at micromolar concentrations .
also in soils of fertilized crop-plants, the urea concentration is maintained at low levels .
in part, this is due to the presence of microbial ureases in the soil solution, which rapidly hydrolyse urea into carbon dioxide and ammonia.
however, low concentrations of urea could remain in soils also after enzymatic degradation, since the microbial urease activity shows an affinity constant in the millimolar range  <cit> .
as evolutionary adaptation, plants might have developed strategies to use this diluted but available n source through high affinity urea uptake systems  <cit> .
only few studies have investigated the molecular basis of urea transporters in higher plants.
the first research was published by liu et al.
<cit>  reporting the cloning and characterization of a high affinity urea transporter of arabidopsis, called atdur <dig>  the coding sequence of atdur <dig> showed weak homology to an ortholog of saccharomyces cerevisiae , a member of the sodium-solute symporter  gene family, which is widespread in microorganisms, animals, and humans  <cit> .
members of the sss family have been described to transport a wide range of solutes, such as sugars, amino acids, nucleosides, inositols, vitamins, anions, and urea  <cit> .
atdur <dig> showed no significant homology to any other protein of arabidopsis  <cit> .
similarly, in the rice genome, osdur <dig> is the only gene that has significant homology to atdur <dig>  suggesting that plant dur <dig> proteins might represent a transporter subfamily consisting of only one member  <cit> .
to date, in higher plants only arabidopsis and rice dur <dig> have been characterized at the molecular and physiological level  <cit> .
the aim of the present work was to identify and functionally characterize the high affinity transport system involved in urea acquisition in maize.
to do this, the kinetic properties of urea uptake in intact maize roots were determined.
the putative urea transporter zmdur3-orf was isolated and its localization analysed using gfp-fusion proteins; its capability to transport urea was demonstrated by expression in heterologous systems, i.e.
dur <dig> saccharomyces cerevisiae and arabidopsis thaliana mutants.
RESULTS
urea acquisition in maize plants
to evaluate the capacity of maize roots to take up urea, a concentration dependent net-influx analysis was performed using 5-day-old plants grown in n-free nutrient solution.
before the uptake experiment, plants were exposed for 4 hours to a nutrient solution containing 1 mm urea as sole n source , or without n .
net uptake rates were determined measuring urea depletion from assay solutions, containing  <dig>  to 300 μm urea .figure  <dig>
kinetic assay of urea uptake by maize roots.
the concentration-dependent uptake was measured using 5-day-old maize plants exposed for  <dig> h to a nutrient solution supplied with  <dig> mm urea as a sole nitrogen source  or not .
subsequently roots were incubated for  <dig> minutes in the assay solution containing urea at different concentrations .
values are means ± sd .
in roots of control plants, the uptake rates of urea showed a typical saturation kinetic corresponding to the michaelis-menten model .
interestingly, the exposure of roots to 1 mm urea before the uptake assay modified the kinetic parameters .
indeed the net urea influx in roots of urea pre-treated plants was more than  <dig> fold higher compared to that measured in control plants, with vmax values of about  <dig> and 9 μmol urea g− <dig> fresh weight  h− <dig>  respectively.
the urea pre-treatment also affected the affinity, which decreased in pre-treated plants more than  <dig>  times with respect to control plants .
in order to independently verify the capacity of maize plants to acquire urea,  <dig> -labelled urea was supplied in the nutrient solution.
after 24 hours of treatment the accumulation of 15 n was  <dig>   mg 100 g− <dig> dry weight  in shoots and  <dig>   mg 100 g− <dig> dw in roots.
during the time span of the experiment, no detectable degradation of urea occurred in the nutrient solution .
in this way considering 15 n-data, maize plants took up around 25 μmol 100 mg− <dig> dw of urea from the external solution.
to investigate the contribution of urea taken up by roots in terms of intact molecule in the plants, the concentration of urea in roots and shoots of maize plants was analysed .
after 24 hours comparable amounts of urea were detected in urea- and control- treated plants.
nevertheless, the concentrations of urea within maize tissues, roots or shoots, were significantly different during the time span of the experiment.
after  <dig> and 8 hours, the urea concentration decreased in roots and increased in shoots of urea-treated plants.
this modulation in urea content might suggest a translocation of urea  even if a higher degradation in roots and a synthesis in shoots cannot be excluded.
in silico identification of a maize urea transporter
with the aim to identify a high affinity urea transporter from maize, an in-silico search was performed based on sequence similarity with atdur <dig>  using the blast algorithm on the aramemnon plant membrane protein database .
in the maize genome, only one predicted sequence coding for a dur <dig> homolog  was identified on chromosome  <dig> .
the expression of zmdur <dig> was confirmed by several est-sequences present in the nucleotide est database from genbank : bq <dig>  bq <dig>  fl <dig>  fl <dig>  dv <dig>  aw <dig>  bq <dig>  bq <dig> and fl <dig>  most ests covered the 3′-region of ac <dig> _fgt <dig> while only fl <dig> and fl <dig> aligned at the 5′-region.
we thus referred to this gene as zmdur <dig> .
when widening the search only a single predicted dur <dig> orf was found within each of the plant species analysed.
the phylogenetic analysis revealed that putative dur <dig> proteins are closely related among monocots, such as maize, rice, wheat, barley and millet , with more than 80% identity at the amino acid level.figure  <dig>
phylogenetic tree of dur <dig> urea transporters.
a phylogentic analyses was performed using the dur <dig> amino acid sequences of saccharomyces cerevisiae , zea mays , oryza sativa , arabidopsis thaliana  and putative dur <dig> orthologs from aegilops tauschii , triticum urartu , hordeum vulgare , brachypodium distachyon , setaria italica , sorghum bicolor , cucumis sativus , vitis vinifera , populus trichocarpa , solanum lycopersicum , prunus persica , medicago truncatula , glycine max .
the tree was constructed by aligning the protein sequences by clustal-w and the evolutionary history was inferred using the neighbor-joining method.
the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test  are shown next to the branches.
the tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.
the evolutionary distances were computed using the poisson correction method and are in the units of the number of amino acid substitutions per site.
expression pattern of zmdur <dig> in maize tissues
as reported in figure  <dig>  real time rt-pcr data show the expression pattern of zmdur <dig> in maize plants up to 4 hours of root exposure to urea.
the highest gene expression level of zmdur <dig> was reached in roots while in leaves the transcript amount was at least an order of magnitude lower.figure  <dig>
transcriptional analyses of
zmdur3
in root and shoots of maize in response to urea treatment.
<dig> day-old plants were exposed for a maximum of  <dig> hours to nutrient solution without addition of any n source  or supplied with  <dig> mm urea .
gene mrna levels were normalized with respect to the mean transcript level of the housekeeping gene zmrps4; relative changes in gene transcript levels were calculated on the basis of the mean transcript level of zmrps <dig> in roots of control plants at  <dig> hour .
values are means ± sd of three independent experiments .
capital letters are referred to the statistical differences in the roots, while lower letters are referred to shoots.
up to 4 hours of urea treatment, the presence of the nitrogen source in the external solution induced a significant down regulation of the gene expression.
on the other hand, in urea and control leaves the expression levels were comparable and not significantly influenced by the treatment.
the coding sequence of zmdur <dig> was isolated from maize root mrna
using gene specific primers, a transcript from maize root was amplified by rt-assembly-pcr and cloned into the yeast expression vector pdr <dig>  <cit> .
the sequencing results showed an open reading frame of 2196-bp, zmdur3-orf , coding for  <dig> amino acids.
the alignment with the genomic sequence  revealed four exon regions of  <dig>   <dig>   <dig> and 1233 bp.
the length and the location of the exons were different from those predicted .
in addition, in comparison to the predicted cdna , the isolated zmdur3-orf contained three non-synonymous substitutions in the nucleotide sequence, modifying the following amino acids: k149n; a167v; q559h.
the nucleotide responsible for the q559h modification was also detected in a maize est sequence .
the region containing the other two substitutions was not covered by ests.
however, the presence of asparagine  and histidine  instead of lysine  and glutamine , respectively, was also found in the amino-acid sequence of rice osdur <dig>  <cit> .
blast analysis revealed that the zmdur <dig> cdna had a high similarity with osdur <dig> .
similar percentages were also observed at amino acid level with an identity of  <dig> and 75% to osdur <dig> and atdur <dig>  respectively .
zmdur <dig> comprises  <dig> amino acids containing fifteen predicted transmembrane spanning domains  with outside orientation of the n-terminus .
the comparison between zmdur <dig> and the rice ortholog osdur <dig>  revealed a similar predicted topology , especially with respect to the number of tmsds, and n- and c-terminus orientation.
functional characterization of zmdur3
the functional characterization was performed using different approaches in heterologous systems: i) functional complementation of a saccharomyces cerevisiae dur <dig> mutant, ii) subcellular localization of zmdur3/gfp  fusion proteins in nicotiana tabacum protoplasts and iii) 35scamv:: zmdur <dig> overexpression in the atdur <dig> mutant line of arabidopsis thaliana.
in order to verify the ability to transport urea, the zmdur3-orf was expressed in a dur3-mutant strain of s. cerevisiae, as described previously by liu et al.
<cit> .
the mutant ynvwi  is defective in urea uptake and cannot grow on less than 5 mm urea as sole n source  <cit> .
results showed that the dur <dig> mutant strain transformed with the vector pdr <dig> barely grew on a medium containing  <dig>   <dig> or 3 mm urea.
on the other hand, the heterologous expression of zmdur3-orf enabled ynvwi to grow well on urea medium .
moreover, since zmdur <dig> has a high gc-content , the level of heterologous expression in other organisms may be limited.
so, to reduce the gc content and favour the expression of zmdur <dig>   <dig> nucleotides in the first 216 nt of zmdur <dig> were modified.
these modifications are all synonymous substitutions occurring only at nucleotide level .
a great improvement in the yeast growth on urea medium was observed transforming ynvwi with a modified version of zmdur3-orf .figure  <dig>
zmdur <dig> mediates urea uptake in
s. cerevisiae
.
growth of the urea uptake-deficient strain ynvw <dig> expressing zmdur <dig> and zmdur3
mod.
the mutant ynvw <dig> transformed with the vector pdr <dig> , and pdr <dig> carrying orfs zmdur <dig>  or zmdur3
mod .
medium contained  <dig> % of ammonium sulphate  or urea at three different concentrations  as a sole nitrogen source.
pictures were taken after  <dig> days of incubation.
the ynvwi mutant expressing zmdur3-orfs  did not show any apparent growth difference on medium supplemented with  <dig> % ammonium sulphate, as n source.
when grown on selective plates supplemented with urea as a sole n source, growth differences between zmdur3- and zmdur3mod-transformants became apparent.
in particular, the size of the colonies of zmdur3mod-transformants was larger in comparison to those of the native zmdur3-orf, and this different growth was visible for all urea concentrations tested.
transient expression of zmdur3/gfp fusion proteins in tobacco protoplasts
functional complementation of the yeast mutant ynvwi by zmdur <dig> indicated that at least in a heterologous system the transporter is localized at the plasma membrane.
to confirm this subcellular localization, n- and c-terminal fusion proteins of zmdur <dig> and gfp  were transiently expressed in tobacco  protoplasts .
tobacco protoplasts were also transformed with atptr1-yfp  <cit>  or with free gfp, which were used as plasma membrane and cytosolic control, respectively .figure  <dig>
localization of zmdur3/gfp fusion proteins in tobacco protoplast.
co-localization of zmdur3-gfp and plasma membrane localized atptr1-yfp,  gfp-zmdur <dig> and atptr1-yfp, and  free gfp.
fluorescence was detected using a confocal laser-scanning microscope: bright-field images , chlorophyll fluorescence , gfp-fluorescence ; yfp-fluorescence  are shown.
in the last column, merged images show chlorophyll fluorescence , gfp-fluorescence  and yfp-fluorescence .
diameter of protoplasts was approximately  <dig> μm.
in free-gfp expressing protoplasts the fluorescent signal was localized in the cytoplasm .
in protoplasts expressing zmdur3-gfp  and gfp-zmdur <dig>  plasma membrane localization could not be unequivocally demonstrated, since the green fluorescence was mostly confined to internal membranes.
the functionality of zmdur3mod/gfp constructs was verified in dur3-yeast mutant.
overexpression of zmdur <dig> in arabidopsis mutant line atdur3-3
in order to test the activity of zmdur <dig> in planta, zmdur3mod was overexpressed in a dur <dig> mutant line of arabidopsis.
the atdur3- <dig> mutant is defective in the endogenous urea transporter atdur <dig> and showed impaired growth on a medium with urea  as sole n source  <cit> .
in particular the mutant line showed a slow development and chlorotic leaves at  <dig>  and 1 mm urea  <cit> , suggesting a condition of n deficiency.
three independent 35scamv: zmdur3mod-overexpressing lines were tested: line-a, line-b and line-c. plants were grown for 16 days on sterile half strength ms medium without any additional n, or supplemented with urea at three different concentrations  or  <dig>  mm ammonium nitrate.
the complementation assay demonstrated that in all three overexpression lines the capacity to grow on a medium supplemented with  <dig>  mm and 1 mm urea was restored .
on agar plates without n supply, all plants showed a poor development of shoots and roots and symptoms of n deficiency appeared.
on medium containing  <dig>  mm urea, wild type shoots developed slightly better than dur <dig> shoots, as previously described by kojima et al.
<cit> .
at  <dig>  mm urea, the zmdur3mod-overexpressing lines grew better than wild type plants with a good development of shoots and with a higher root proliferation .
it is interesting to note that on agar plates supplemented with  <dig>  mm urea, overexpression lines showed a higher biomass production with a significantly higher fresh weights than wild type or atdur3- <dig> mutant plants .
no detectable differences were observed among all arabidopsis lines tested when plants were grown on 3 mm urea or on  <dig>  mm ammonium nitrate .figure  <dig>
growth of
zmdur3
mod
-expressed in the
dur3- <dig> arabidopsis
mutant.
the arabidopsis dur <dig> mutant, atdur3- <dig>  <cit> , was transformed with zmdur3
mod-orf under the control of the camv 35s-promoter.
growth of the wild type col- <dig> , atdur3- <dig> mutant line and three zmdur3
mod-overexpressing lines  on sterile half strength ms medium supplied with  <dig> μm nicl <dig> and  <dig> μm no3
− and different concentrations of urea or  <dig>  mm ammonium nitrate  as a sole n source.
effect of urea treatment on root morphology in arabidopsis plants grown with  <dig>  mm urea.
plants were grown for  <dig> days on nutrient agar-medium.
effect of urea treatment on biomass production of
arabidopsis
plants grown on  <dig>  mm urea.
arabidopsis plants were grown on sterile half strength ms medium supplemented with  <dig> μm nicl <dig> and  <dig> μm no3
− plus  <dig>  mm urea as sole n sources .
the fresh weights of  <dig> plants were measured after  <dig> days.
data are mean ±sd of three independent experiments and different letters above the bars indicate statistically significant differences .
phenotyping results were validated by 15-urea influx assay using 6-weeks-old arabidopsis plants.
col- <dig>  atdur3- <dig> and atdur3-3 + zmdur3-a, −b, −c overexpression lines were grown in hydroponic culture in a complete nutrient solution containing 1 mm ammonium nitrate for 38 days before being transferred for 4 days in a n-free nutrient solution.
at the time of the experiment, no phenotypical differences in root architectures were visible between different arabidopsis lines under these growth conditions.
when 100 μm 15-urea was supplied to roots, all three zmdur3-overexpressing lines were able to take up urea, restoring the wild-type transport rates .
in particular, the highest urea uptake rates were found in line b of the atdur3-3 + zmdur <dig> overexpression line, while line -a and -c showed levels of urea uptake comparable to those in wild type plants.figure  <dig>
15
-urea influx in
arabidopsis
plants.
urea uptake into roots was determined using 6-weeks-old plants of wild type col- <dig> , atdur3- <dig> mutant line and three zmdur3
mod-transformed lines  grown in a complete nutrient solution containing nitrogen as  <dig> mm ammonium nitrate.
<dig> days before the experiment, plants were transferred to n-free medium.
for the assay,  <dig> μm 15-urea was supplied to the medium for  <dig> min.
data are mean ±sd of three independent experiments and different letters above the bars indicate statistically significant differences .
discussion
although urea is the most used n fertilizer worldwide, little is known on the capacity of crop plants to use urea per se as an n source.
maize is one of the crops supplied with huge amount of urea fertilizers and it is known that urea sustains n nutrition.
however, it is not clear how much urea is directly taken up  <cit> .
therefore in this work, the high affinity urea uptake by maize roots was characterized and a high affinity urea transporter  identified and functionally characterized.
among higher plants, the kinetic characterization of urea uptake was previously described only in arabidopsis and rice  <cit> .
in the present work, intact maize roots exposed to urea up to 300 μm, showed saturable kinetics of urea transport fitting into the michaelis-menten model .
this behaviour is compatible with the presence of a high-affinity transport system for urea in maize roots, with kinetic features similar to those already characterized in other higher plants  <cit> .
the kinetic assay in maize roots revealed an important aspect of urea uptake that has not been previously described in higher plants.
data showed that when maize plants were supplied with 1 mm urea for 4 hours, the affinity and capacity to take up this n source in the high-affinity concentration range  increased in comparison to plants without urea pre-treatment .
thus, urea pre-treatment increases its own uptake, causing a modification of the kinetic parameters, which is very similar to the well-described physiological induction by substrate of the inducible high-affinity-nitrate transport system   <cit> .
on the other hand, concerning the low-affinity transport system, the up-regulation of urea uptake by pre-treatment with urea was previously reported in arabidopsis  <cit> .
results were inferred from influx assays performed by exposing plants to a high concentration of urea, 10 mm 15 n-urea .
the influx capacity of urea-fed plants  was higher than in n-starved plants or plants fed with ammonium nitrate or ammonium nitrate plus urea, which showed values around 200 μmol urea g− <dig> dw h− <dig>  thus, these data suggest that in arabidopsis  <cit>  and maize , roots are able to induce urea uptake when urea is available in the external medium.
moreover, as observed in the present work, the induction of hats in maize roots might reflect an efficient response of plants by increasing the capacity of urea acquisition especially when this n source occurs at micromolar levels in the soil solution.
although after 24 hours high amount of external urea are taken up by the roots, the total concentration of urea as an intact molecule within maize plants did not increase .
so, the urea treatment seemed to have no effect on urea content in maize, similar results were also reported by mérigout et al.
<cit> .
this result may be explained by the high activity of the cytosolic urease enzyme, ubiquitously present in plant tissues, which has been shown to efficiently hydrolyse urea within the plant tissues  <cit> .
nevertheless, data here presented showed a transient modulation of urea content within the tissues suggesting a translocation of urea from roots to shoots.
among higher plants, urea transporters have been identified only as orthologs of scdur <dig>  an urea transporter of s. cerevisiae.
up to date, only atdur <dig> and osdur <dig>  of arabidopsis and rice, respectively have been functionally characterized, while in other monocots and dicots putative dur3-orthologs were predicted by bioinformatics .
in arabidopsis, atdur <dig> has been described to be a major component of the high-affinity transport system, suggesting that also in other plants, the dur3-orthologs might play a crucial role in urea acquisition.
the expression level of dur <dig> orthologs has been shown to be increased by the nitrogen deficiency in arabidopsis and rice plants  <cit> .
as reported for the orthologous gene in rice  <cit> , the expression level of zmdur <dig> coding for the putative urea transporter in maize is different among the tissues .
the higher expression of the gene coding for dur <dig> in the radical tissue might reflect its involvement in the mechanisms of urea acquisition from the root external medium.
roots of n-deficient plants treated with nitrogen sources exhibits divergent expression level of dur <dig> orthologs: in rice, osdur <dig> is weakly induced after 3 hours of treatments with 1 mm urea  <cit> , in arabidopsis, 1 mm urea represses atdur <dig> expression at  <dig> and 6 hours and induced it at  <dig> and 24 hours  <cit> .
in maize plants, during the timespan when 1 mm urea induced an increase in the root capacity to take up urea, the expression level was decreasing  similarly to the variations found by kojima et al.
<cit> .
therefore in the short term, the modulation in the root capacity to take up urea is not related to changes in the expression level of the gene zmdur <dig>  suggesting the involvement of regulation mechanisms that do not operate at transcriptional level.
expression of zmdur <dig> in a dur3-s. cerevisiae mutant demonstrated a functional urea transport .
as zmdur3-transformants grew very slowly, a zmdur3-orf was prepared with a lower gc content and therefore an optimized codon usage for s. cerevisiae.
therefore in the first part  of the orf, g and c in the third codon position were replaced with a or t generating codons which are more frequently used in yeast.
interestingly the zmdur3mod-transformants grew slightly faster than yeast mutants transformed with the unmodified zmdur3-orf .
since the two constructs differed only at nucleotide level, the slow growth rate of zmdur3-orf-expressing cells might be the consequence of a lower accumulation of zmdur <dig> protein possibly deriving from a lower transcription/translation of the native maize transgene in comparison to the zmdur3mod-transformed yeast.
these results highlight that especially for plant species with a high gc content, the orf-optimization strategy may be a valid method to improve the expression of transgenes in heterologous systems like yeast or also in other model organisms allowing an easier molecular characterization of plant proteins.
the yeast complementation assay demonstrated that zmdur <dig> can mediate urea uptake from the external medium into the cells.
with the aim to clarify the subcellular localization of zmdur <dig>  tobacco protoplasts were transiently transformed with zmdur3mod-orf fused with gfp.
results showed that the fluorescent signal was mostly detected in internal membranes , although the localization of a minor fraction of zmdur3-gfp on plasma membrane would be compatible with the observed signal.
these localization results are comparable to those previously reported in arabidopsis protoplasts for the orthologs of rice and arabidopsis, osdur <dig> and atdur <dig>  <cit> .
for these proteins, the fluorescent signals were not uniformly distributed at the periphery of protoplasts, indicating that the protein might be localized not only at the plasma membrane, but also in internal membranes.
besides gfp-localization, further experimental evidences suggested that dur <dig> might not exclusively be targeted to the plasma membrane.
in particular, for atdur <dig> the plasma membrane localization in arabidopsis root cells was previously described by two immunological approaches.
kojima et al.
<cit>  used polyclonal antibodies against atdur <dig> in two independent analyses: a protein gel-blot analysis of membrane-protein fraction from arabidopsis roots and an immunohistochemical assay on whole-mount root samples.
both immunological techniques gave the same results: although atdur <dig> localized at the plasma membrane, a fraction of the protein appeared to be localized in the cytoplasm.
the authors suggested that a fraction of atdur <dig> might reside in endomembrane compartments, reflecting proteins that were moving to or from the plasma membrane  <cit> .
interestingly, in root cells, the subcellular-localization of another high affinity transporter  was found to be mainly localized in the early endosomes  <cit>  while at the plasma membrane the abundance of irt <dig> was low and tightly regulated by an ubiquitin-dependent trafficking and turnover.
the turnover of the irt <dig> protein was investigated and the localization of irt <dig> was explained by the authors as a result of a “rapid endocytosis and slower recycling to the plasma membrane, where it likely performs iron uptake from the soil, and is addressed to the lytic vacuole for turnover”  <cit> .
the authors concluded that the internal traffic controls the amounts of irt <dig> protein at the plasma membrane and therefore participates in the tight regulation of the nutrient uptake.
these considerations about irt <dig> suggest that the presence of zmdur <dig> in internal membranes may reflect a similar situation where the abundance of the protein at the plasma membrane is under control of a trafficking/recycling pathway.
this hypothesis is further supported by the fact that the higher root uptake capacity of urea  was not accompanied by an overexpression of zmdur <dig> .
to provide more detailed assessment of the molecular and physiological role of this maize transporter in planta, the overexpression of zmdur3mod in a dur <dig> mutant line of arabidopsis was performed.
all three overexpression lines were able to phenotypically recover the dur3-mutant  and produced significantly higher plant biomass and root proliferation than dur <dig> mutant and wild type .
this result might reflect a possible overexpression of the transgene in all the tissues of lines a, b and c, determining an improvement on the utilization of urea  within the plants.
in short term 100 μm 15-urea influx experiment , all three lines complement the mutant phenotype, reaching the highest uptake rates in line b. the differences in the uptake rates might be due to a different expression level of the transgene zmdur <dig> in the three independent lines.
moreover the influx experiment was performed at a micromolar concentration suggesting the capacity of zmdur <dig> to operate in the high affinity range.
in conclusion, these evidences demonstrated the complementation of the mutant phenotype by zmdur <dig> and confirmed the physiological role of this protein as a high-affinity transporter of urea from soil into plants.
CONCLUSIONS
for the first time, we report a physiological characterization of urea uptake in roots of intact maize plants.
results indicated that at micromolar urea concentrations , maize roots are able to take up this n source using a high affinity transport system characterized by saturable kinetics.
moreover, the pre-treatment of plants with urea increases their capacity to take up urea, showing that high-affinity uptake of urea is inducible by the substrate.
the capability of the identified zmdur <dig> to phenotypically complement dur <dig> yeast and arabidopsis mutants further demonstrates that zmdur <dig> encodes a high-affinity urea uptake system in maize.

 by the global population is expected to be 50% higher than at present and global grain demand is projected to double. today the productivity of crops is based on the application of high amounts of industrially produced nitrogen fertilizer, even though crop plants utilize only 30-40% of the applied n. as a consequence, the wide use of synthetic n fertilizer has led to negative impacts on the environment and on farmer economies. in addition, the n use efficiency of cereal crops has declined in the last 50 years. based on these considerations, crop yield needs to be improved in a more cost-effective and eco-compatible way. this goal could be achieved by increasing the nue of cereals and optimizing the acquisition of naturally occurring and applied n. reducing the amount of fertilizers in maize culture will have economic and environmental benefits. in particular combining reduced fertilizer application and breeding plants with better nue is one of the main goals of research in plant nutrition. urea is the most frequently used n fertilizer in the world, with annual amounts of over million tons accounting for more than 50% of the world n-fertilizer consumption. the great increase in urea-fertilizer use during the last decades is mainly due to its competitive price and the high n content, that allow reducing transport and distribution costs. besides the chemical input as fertilizer, urea is also a natural organic molecule synthesized by most organisms. in plants, urea represents an important metabolic intermediate produced during n-recycling, while in mammals the urea production is associated with the detoxification of n compounds. although urea might be derived from both natural and chemical syntheses, in the soil it usually occurs only at micromolar concentrations. also in soils of fertilized crop-plants, the urea concentration is maintained at low levels. in part, this is due to the presence of microbial ureases in the soil solution, which rapidly hydrolyse urea into carbon dioxide and ammonia. however, low concentrations of urea could remain in soils also after enzymatic degradation, since the microbial urease activity shows an affinity constant in the millimolar range. as evolutionary adaptation, plants might have developed strategies to use this diluted but available n source through high affinity urea uptake systems. only few studies have investigated the molecular basis of urea transporters in higher plants. the first research was published by liu et al. reporting the cloning and characterization of a high affinity urea transporter of arabidopsis, called atdur the coding sequence of atdur showed weak homology to an ortholog of saccharomyces cerevisiae, a member of the sodium-solute symporter gene family, which is widespread in microorganisms, animals, and humans. members of the sss family have been described to transport a wide range of solutes, such as sugars, amino acids, nucleosides, inositols, vitamins, anions, and urea. atdur showed no significant homology to any other protein of arabidopsis. similarly, in the rice genome, osdur is the only gene that has significant homology to atdur suggesting that plant dur proteins might represent a transporter subfamily consisting of only one member. to date, in higher plants only arabidopsis and rice dur have been characterized at the molecular and physiological level. the aim of the present work was to identify and functionally characterize the high affinity transport system involved in urea acquisition in maize. to do this, the kinetic properties of urea uptake in intact maize roots were determined. the putative urea transporter zmdur3-orf was isolated and its localization analysed using gfp-fusion proteins; its capability to transport urea was demonstrated by expression in heterologous systems, i.e. dur saccharomyces cerevisiae and arabidopsis thaliana mutants.  urea acquisition in maize plants to evaluate the capacity of maize roots to take up urea, a concentration dependent net-influx analysis was performed using 5-day-old plants grown in n-free nutrient solution. before the uptake experiment, plants were exposed for 4 hours to a nutrient solution containing 1 mm urea as sole n source, or without n. net uptake rates were determined measuring urea depletion from assay solutions, containing to 300 μm urea.figure kinetic assay of urea uptake by maize roots. the concentration-dependent uptake was measured using 5-day-old maize plants exposed for h to a nutrient solution supplied with mm urea as a sole nitrogen source or not. subsequently roots were incubated for minutes in the assay solution containing urea at different concentrations. values are means ± sd. in roots of control plants, the uptake rates of urea showed a typical saturation kinetic corresponding to the michaelis-menten model. interestingly, the exposure of roots to 1 mm urea before the uptake assay modified the kinetic parameters. indeed the net urea influx in roots of urea pre-treated plants was more than fold higher compared to that measured in control plants, with vmax values of about and 9 μmol urea g− fresh weight h− respectively. the urea pre-treatment also affected the affinity, which decreased in pre-treated plants more than times with respect to control plants. in order to independently verify the capacity of maize plants to acquire urea, -labelled urea was supplied in the nutrient solution. after 24 hours of treatment the accumulation of 15 n was mg 100 g− dry weight in shoots and mg 100 g− dw in roots. during the time span of the experiment, no detectable degradation of urea occurred in the nutrient solution. in this way considering 15 n-data, maize plants took up around 25 μmol 100 mg− dw of urea from the external solution. to investigate the contribution of urea taken up by roots in terms of intact molecule in the plants, the concentration of urea in roots and shoots of maize plants was analysed. after 24 hours comparable amounts of urea were detected in urea- and control- treated plants. nevertheless, the concentrations of urea within maize tissues, roots or shoots, were significantly different during the time span of the experiment. after and 8 hours, the urea concentration decreased in roots and increased in shoots of urea-treated plants. this modulation in urea content might suggest a translocation of urea even if a higher degradation in roots and a synthesis in shoots cannot be excluded. in silico identification of a maize urea transporter with the aim to identify a high affinity urea transporter from maize, an in-silico search was performed based on sequence similarity with atdur using the blast algorithm on the aramemnon plant membrane protein database. in the maize genome, only one predicted sequence coding for a dur homolog was identified on chromosome. the expression of zmdur was confirmed by several est-sequences present in the nucleotide est database from genbank: bq bq fl fl dv aw bq bq and fl most ests covered the 3′-region of ac _fgt while only fl and fl aligned at the 5′-region. we thus referred to this gene as zmdur. when widening the search only a single predicted dur orf was found within each of the plant species analysed. the phylogenetic analysis revealed that putative dur proteins are closely related among monocots, such as maize, rice, wheat, barley and millet, with more than 80% identity at the amino acid level.figure phylogenetic tree of dur urea transporters. a phylogentic analyses was performed using the dur amino acid sequences of saccharomyces cerevisiae, zea mays, oryza sativa, arabidopsis thaliana and putative dur orthologs from aegilops tauschii, triticum urartu, hordeum vulgare, brachypodium distachyon, setaria italica, sorghum bicolor, cucumis sativus, vitis vinifera, populus trichocarpa, solanum lycopersicum, prunus persica, medicago truncatula, glycine max. the tree was constructed by aligning the protein sequences by clustal-w and the evolutionary history was inferred using the neighbor-joining method. the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test are shown next to the branches. the tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. the evolutionary distances were computed using the poisson correction method and are in the units of the number of amino acid substitutions per site. expression pattern of zmdur in maize tissues as reported in figure  real time rt-pcr data show the expression pattern of zmdur in maize plants up to 4 hours of root exposure to urea. the highest gene expression level of zmdur was reached in roots while in leaves the transcript amount was at least an order of magnitude lower.figure transcriptional analyses of zmdur3 in root and shoots of maize in response to urea treatment. day-old plants were exposed for a maximum of hours to nutrient solution without addition of any n source or supplied with mm urea. gene mrna levels were normalized with respect to the mean transcript level of the housekeeping gene zmrps4; relative changes in gene transcript levels were calculated on the basis of the mean transcript level of zmrps in roots of control plants at hour. values are means ± sd of three independent experiments. capital letters are referred to the statistical differences in the roots, while lower letters are referred to shoots. up to 4 hours of urea treatment, the presence of the nitrogen source in the external solution induced a significant down regulation of the gene expression. on the other hand, in urea and control leaves the expression levels were comparable and not significantly influenced by the treatment. the coding sequence of zmdur was isolated from maize root mrna using gene specific primers, a transcript from maize root was amplified by rt-assembly-pcr and cloned into the yeast expression vector pdr. the sequencing results showed an open reading frame of 2196-bp, zmdur3-orf, coding for amino acids. the alignment with the genomic sequence revealed four exon regions of and 1233 bp. the length and the location of the exons were different from those predicted. in addition, in comparison to the predicted cdna, the isolated zmdur3-orf contained three non-synonymous substitutions in the nucleotide sequence, modifying the following amino acids: k149n; a167v; q559h. the nucleotide responsible for the q559h modification was also detected in a maize est sequence. the region containing the other two substitutions was not covered by ests. however, the presence of asparagine and histidine instead of lysine and glutamine, respectively, was also found in the amino-acid sequence of rice osdur. blast analysis revealed that the zmdur cdna had a high similarity with osdur. similar percentages were also observed at amino acid level with an identity of and 75% to osdur and atdur respectively. zmdur comprises amino acids containing fifteen predicted transmembrane spanning domains with outside orientation of the n-terminus. the comparison between zmdur and the rice ortholog osdur revealed a similar predicted topology, especially with respect to the number of tmsds, and n- and c-terminus orientation. functional characterization of zmdur3 the functional characterization was performed using different approaches in heterologous systems: i) functional complementation of a saccharomyces cerevisiae dur mutant, ii) subcellular localization of zmdur3/gfp fusion proteins in nicotiana tabacum protoplasts and iii) 35scamv:: zmdur overexpression in the atdur mutant line of arabidopsis thaliana. in order to verify the ability to transport urea, the zmdur3-orf was expressed in a dur3-mutant strain of s. cerevisiae, as described previously by liu et al.. the mutant ynvwi is defective in urea uptake and cannot grow on less than 5 mm urea as sole n source. results showed that the dur mutant strain transformed with the vector pdr barely grew on a medium containing or 3 mm urea. on the other hand, the heterologous expression of zmdur3-orf enabled ynvwi to grow well on urea medium. moreover, since zmdur has a high gc-content, the level of heterologous expression in other organisms may be limited. so, to reduce the gc content and favour the expression of zmdur nucleotides in the first 216 nt of zmdur were modified. these modifications are all synonymous substitutions occurring only at nucleotide level. a great improvement in the yeast growth on urea medium was observed transforming ynvwi with a modified version of zmdur3-orf.figure zmdur mediates urea uptake in s. cerevisiae. growth of the urea uptake-deficient strain ynvw expressing zmdur and zmdur3 mod. the mutant ynvw transformed with the vector pdr, and pdr carrying orfs zmdur or zmdur3 mod. medium contained % of ammonium sulphate or urea at three different concentrations as a sole nitrogen source. pictures were taken after days of incubation. the ynvwi mutant expressing zmdur3-orfs did not show any apparent growth difference on medium supplemented with % ammonium sulphate, as n source. when grown on selective plates supplemented with urea as a sole n source, growth differences between zmdur3- and zmdur3mod-transformants became apparent. in particular, the size of the colonies of zmdur3mod-transformants was larger in comparison to those of the native zmdur3-orf, and this different growth was visible for all urea concentrations tested. transient expression of zmdur3/gfp fusion proteins in tobacco protoplasts functional complementation of the yeast mutant ynvwi by zmdur indicated that at least in a heterologous system the transporter is localized at the plasma membrane. to confirm this subcellular localization, n- and c-terminal fusion proteins of zmdur and gfp were transiently expressed in tobacco protoplasts. tobacco protoplasts were also transformed with atptr1-yfp or with free gfp, which were used as plasma membrane and cytosolic control, respectively.figure localization of zmdur3/gfp fusion proteins in tobacco protoplast. co-localization of zmdur3-gfp and plasma membrane localized atptr1-yfp, gfp-zmdur and atptr1-yfp, and free gfp. fluorescence was detected using a confocal laser-scanning microscope: bright-field images, chlorophyll fluorescence, gfp-fluorescence; yfp-fluorescence are shown. in the last column, merged images show chlorophyll fluorescence, gfp-fluorescence and yfp-fluorescence. diameter of protoplasts was approximately μm. in free-gfp expressing protoplasts the fluorescent signal was localized in the cytoplasm. in protoplasts expressing zmdur3-gfp and gfp-zmdur plasma membrane localization could not be unequivocally demonstrated, since the green fluorescence was mostly confined to internal membranes. the functionality of zmdur3mod/gfp constructs was verified in dur3-yeast mutant. overexpression of zmdur in arabidopsis mutant line atdur3-3 in order to test the activity of zmdur in planta, zmdur3mod was overexpressed in a dur mutant line of arabidopsis. the atdur3- mutant is defective in the endogenous urea transporter atdur and showed impaired growth on a medium with urea as sole n source. in particular the mutant line showed a slow development and chlorotic leaves at and 1 mm urea, suggesting a condition of n deficiency. three independent 35scamv: zmdur3mod-overexpressing lines were tested: line-a, line-b and line-c. plants were grown for 16 days on sterile half strength ms medium without any additional n, or supplemented with urea at three different concentrations or  mm ammonium nitrate. the complementation assay demonstrated that in all three overexpression lines the capacity to grow on a medium supplemented with  mm and 1 mm urea was restored. on agar plates without n supply, all plants showed a poor development of shoots and roots and symptoms of n deficiency appeared. on medium containing  mm urea, wild type shoots developed slightly better than dur shoots, as previously described by kojima et al.. at  mm urea, the zmdur3mod-overexpressing lines grew better than wild type plants with a good development of shoots and with a higher root proliferation. it is interesting to note that on agar plates supplemented with  mm urea, overexpression lines showed a higher biomass production with a significantly higher fresh weights than wild type or atdur3- mutant plants. no detectable differences were observed among all arabidopsis lines tested when plants were grown on 3 mm urea or on  mm ammonium nitrate.figure growth of zmdur3 mod -expressed in the dur3- arabidopsis mutant. the arabidopsis dur mutant, atdur3-, was transformed with zmdur3 mod-orf under the control of the camv 35s-promoter. growth of the wild type col-, atdur3- mutant line and three zmdur3 mod-overexpressing lines on sterile half strength ms medium supplied with μm nicl and μm no3 − and different concentrations of urea or mm ammonium nitrate as a sole n source. effect of urea treatment on root morphology in arabidopsis plants grown with mm urea. plants were grown for days on nutrient agar-medium. effect of urea treatment on biomass production of arabidopsis plants grown on mm urea. arabidopsis plants were grown on sterile half strength ms medium supplemented with μm nicl and μm no3 − plus mm urea as sole n sources. the fresh weights of plants were measured after days. data are mean ±sd of three independent experiments and different letters above the bars indicate statistically significant differences. phenotyping results were validated by 15-urea influx assay using 6-weeks-old arabidopsis plants. col- atdur3- and atdur3-3 + zmdur3-a, −b, −c overexpression lines were grown in hydroponic culture in a complete nutrient solution containing 1 mm ammonium nitrate for 38 days before being transferred for 4 days in a n-free nutrient solution. at the time of the experiment, no phenotypical differences in root architectures were visible between different arabidopsis lines under these growth conditions. when 100 μm 15-urea was supplied to roots, all three zmdur3-overexpressing lines were able to take up urea, restoring the wild-type transport rates. in particular, the highest urea uptake rates were found in line b of the atdur3-3 + zmdur overexpression line, while line -a and -c showed levels of urea uptake comparable to those in wild type plants.figure 15 -urea influx in arabidopsis plants. urea uptake into roots was determined using 6-weeks-old plants of wild type col-, atdur3- mutant line and three zmdur3 mod-transformed lines grown in a complete nutrient solution containing nitrogen as mm ammonium nitrate. days before the experiment, plants were transferred to n-free medium. for the assay, μm 15-urea was supplied to the medium for min. data are mean ±sd of three independent experiments and different letters above the bars indicate statistically significant differences. discussion although urea is the most used n fertilizer worldwide, little is known on the capacity of crop plants to use urea per se as an n source. maize is one of the crops supplied with huge amount of urea fertilizers and it is known that urea sustains n nutrition. however, it is not clear how much urea is directly taken up. therefore in this work, the high affinity urea uptake by maize roots was characterized and a high affinity urea transporter identified and functionally characterized. among higher plants, the kinetic characterization of urea uptake was previously described only in arabidopsis and rice. in the present work, intact maize roots exposed to urea up to 300 μm, showed saturable kinetics of urea transport fitting into the michaelis-menten model. this behaviour is compatible with the presence of a high-affinity transport system for urea in maize roots, with kinetic features similar to those already characterized in other higher plants. the kinetic assay in maize roots revealed an important aspect of urea uptake that has not been previously described in higher plants. data showed that when maize plants were supplied with 1 mm urea for 4 hours, the affinity and capacity to take up this n source in the high-affinity concentration range increased in comparison to plants without urea pre-treatment. thus, urea pre-treatment increases its own uptake, causing a modification of the kinetic parameters, which is very similar to the well-described physiological induction by substrate of the inducible high-affinity-nitrate transport system. on the other hand, concerning the low-affinity transport system, the up-regulation of urea uptake by pre-treatment with urea was previously reported in arabidopsis. results were inferred from influx assays performed by exposing plants to a high concentration of urea, 10 mm 15 n-urea. the influx capacity of urea-fed plants was higher than in n-starved plants or plants fed with ammonium nitrate or ammonium nitrate plus urea, which showed values around 200 μmol urea g− dw h− thus, these data suggest that in arabidopsis and maize, roots are able to induce urea uptake when urea is available in the external medium. moreover, as observed in the present work, the induction of hats in maize roots might reflect an efficient response of plants by increasing the capacity of urea acquisition especially when this n source occurs at micromolar levels in the soil solution. although after 24 hours high amount of external urea are taken up by the roots, the total concentration of urea as an intact molecule within maize plants did not increase. so, the urea treatment seemed to have no effect on urea content in maize, similar results were also reported by mérigout et al.. this result may be explained by the high activity of the cytosolic urease enzyme, ubiquitously present in plant tissues, which has been shown to efficiently hydrolyse urea within the plant tissues. nevertheless, data here presented showed a transient modulation of urea content within the tissues suggesting a translocation of urea from roots to shoots. among higher plants, urea transporters have been identified only as orthologs of scdur an urea transporter of s. cerevisiae. up to date, only atdur and osdur of arabidopsis and rice, respectively have been functionally characterized, while in other monocots and dicots putative dur3-orthologs were predicted by bioinformatics. in arabidopsis, atdur has been described to be a major component of the high-affinity transport system, suggesting that also in other plants, the dur3-orthologs might play a crucial role in urea acquisition. the expression level of dur orthologs has been shown to be increased by the nitrogen deficiency in arabidopsis and rice plants. as reported for the orthologous gene in rice, the expression level of zmdur coding for the putative urea transporter in maize is different among the tissues. the higher expression of the gene coding for dur in the radical tissue might reflect its involvement in the mechanisms of urea acquisition from the root external medium. roots of n-deficient plants treated with nitrogen sources exhibits divergent expression level of dur orthologs: in rice, osdur is weakly induced after 3 hours of treatments with 1 mm urea, in arabidopsis, 1 mm urea represses atdur expression at and 6 hours and induced it at and 24 hours. in maize plants, during the timespan when 1 mm urea induced an increase in the root capacity to take up urea, the expression level was decreasing similarly to the variations found by kojima et al.. therefore in the short term, the modulation in the root capacity to take up urea is not related to changes in the expression level of the gene zmdur suggesting the involvement of regulation mechanisms that do not operate at transcriptional level. expression of zmdur in a dur3-s. cerevisiae mutant demonstrated a functional urea transport. as zmdur3-transformants grew very slowly, a zmdur3-orf was prepared with a lower gc content and therefore an optimized codon usage for s. cerevisiae. therefore in the first part of the orf, g and c in the third codon position were replaced with a or t generating codons which are more frequently used in yeast. interestingly the zmdur3mod-transformants grew slightly faster than yeast mutants transformed with the unmodified zmdur3-orf. since the two constructs differed only at nucleotide level, the slow growth rate of zmdur3-orf-expressing cells might be the consequence of a lower accumulation of zmdur protein possibly deriving from a lower transcription/translation of the native maize transgene in comparison to the zmdur3mod-transformed yeast. these results highlight that especially for plant species with a high gc content, the orf-optimization strategy may be a valid method to improve the expression of transgenes in heterologous systems like yeast or also in other model organisms allowing an easier molecular characterization of plant proteins. the yeast complementation assay demonstrated that zmdur can mediate urea uptake from the external medium into the cells. with the aim to clarify the subcellular localization of zmdur tobacco protoplasts were transiently transformed with zmdur3mod-orf fused with gfp. results showed that the fluorescent signal was mostly detected in internal membranes, although the localization of a minor fraction of zmdur3-gfp on plasma membrane would be compatible with the observed signal. these localization results are comparable to those previously reported in arabidopsis protoplasts for the orthologs of rice and arabidopsis, osdur and atdur. for these proteins, the fluorescent signals were not uniformly distributed at the periphery of protoplasts, indicating that the protein might be localized not only at the plasma membrane, but also in internal membranes. besides gfp-localization, further experimental evidences suggested that dur might not exclusively be targeted to the plasma membrane. in particular, for atdur the plasma membrane localization in arabidopsis root cells was previously described by two immunological approaches. kojima et al. used polyclonal antibodies against atdur in two independent analyses: a protein gel-blot analysis of membrane-protein fraction from arabidopsis roots and an immunohistochemical assay on whole-mount root samples. both immunological techniques gave the same results: although atdur localized at the plasma membrane, a fraction of the protein appeared to be localized in the cytoplasm. the authors suggested that a fraction of atdur might reside in endomembrane compartments, reflecting proteins that were moving to or from the plasma membrane. interestingly, in root cells, the subcellular-localization of another high affinity transporter was found to be mainly localized in the early endosomes while at the plasma membrane the abundance of irt was low and tightly regulated by an ubiquitin-dependent trafficking and turnover. the turnover of the irt protein was investigated and the localization of irt was explained by the authors as a result of a “rapid endocytosis and slower recycling to the plasma membrane, where it likely performs iron uptake from the soil, and is addressed to the lytic vacuole for turnover”. the authors concluded that the internal traffic controls the amounts of irt protein at the plasma membrane and therefore participates in the tight regulation of the nutrient uptake. these considerations about irt suggest that the presence of zmdur in internal membranes may reflect a similar situation where the abundance of the protein at the plasma membrane is under control of a trafficking/recycling pathway. this hypothesis is further supported by the fact that the higher root uptake capacity of urea was not accompanied by an overexpression of zmdur. to provide more detailed assessment of the molecular and physiological role of this maize transporter in planta, the overexpression of zmdur3mod in a dur mutant line of arabidopsis was performed. all three overexpression lines were able to phenotypically recover the dur3-mutant and produced significantly higher plant biomass and root proliferation than dur mutant and wild type. this result might reflect a possible overexpression of the transgene in all the tissues of lines a, b and c, determining an improvement on the utilization of urea within the plants. in short term 100 μm 15-urea influx experiment, all three lines complement the mutant phenotype, reaching the highest uptake rates in line b. the differences in the uptake rates might be due to a different expression level of the transgene zmdur in the three independent lines. moreover the influx experiment was performed at a micromolar concentration suggesting the capacity of zmdur to operate in the high affinity range. in conclusion, these evidences demonstrated the complementation of the mutant phenotype by zmdur and confirmed the physiological role of this protein as a high-affinity transporter of urea from soil into plants.  for the first time, we report a physiological characterization of urea uptake in roots of intact maize plants. results indicated that at micromolar urea concentrations, maize roots are able to take up this n source using a high affinity transport system characterized by saturable kinetics. moreover, the pre-treatment of plants with urea increases their capacity to take up urea, showing that high-affinity uptake of urea is inducible by the substrate. the capability of the identified zmdur to phenotypically complement dur yeast and arabidopsis mutants further demonstrates that zmdur encodes a high-affinity urea uptake system in maize. 
kinetic characterization of urea uptake demonstrated the presence in maize roots of a high-affinity and saturable transport system; this system is inducible by urea itself showing higher vmax and km upon induction. the expression of the isolated sequence, zmdur3-orf, in dur yeast mutant demonstrated the ability of the encoded protein to mediate urea uptake into cells. at molecular level, the orf sequence coding for the urea transporter, zmdur was isolated and functionally characterized using different heterologous systems: a dur yeast mutant strain, tobacco protoplasts and a dur arabidopsis mutant. the subcellular targeting of dur3/gfp fusion proteins in tobacco protoplasts gave results comparable to the localization of the orthologous transporters of arabidopsis and rice, suggesting a partial localization at the plasma membrane. with respect to maize, a worldwide-cultivated crop requiring high amounts of nitrogen fertilizer, the mechanisms involved in the transport of urea have not yet been identified. the aim of the present work was to characterize the high-affinity urea transport system in maize roots and to identify the high affinity urea transporter. despite its extensive use as a nitrogen fertilizer, the role of urea as a directly accessible nitrogen source for crop plants is still poorly understood. data may provide a key to better understand the mechanisms involved in urea acquisition and contribute to deepen the knowledge on the overall nitrogen-use efficiency in crop plants. so far, the physiological and molecular aspects of urea acquisition have been investigated only in few plant species highlighting the importance of a high-affinity transport system. these data provide a clear evidence in planta for a role of zmdur in urea acquisition from an extra-radical solution. this work highlights the capability of maize plants to take up urea via an inducible and high-affinity transport system. zmdur is a high-affinity urea transporter mediating the uptake of this molecule into roots.
 despite its extensive use as a nitrogen fertilizer, the role of urea as a directly accessible nitrogen source for crop plants is still poorly understood. so far, the physiological and molecular aspects of urea acquisition have been investigated only in few plant species highlighting the importance of a high-affinity transport system. with respect to maize, a worldwide-cultivated crop requiring high amounts of nitrogen fertilizer, the mechanisms involved in the transport of urea have not yet been identified. the aim of the present work was to characterize the high-affinity urea transport system in maize roots and to identify the high affinity urea transporter.  kinetic characterization of urea uptake demonstrated the presence in maize roots of a high-affinity and saturable transport system; this system is inducible by urea itself showing higher vmax and km upon induction. at molecular level, the orf sequence coding for the urea transporter, zmdur was isolated and functionally characterized using different heterologous systems: a dur yeast mutant strain, tobacco protoplasts and a dur arabidopsis mutant. the expression of the isolated sequence, zmdur3-orf, in dur yeast mutant demonstrated the ability of the encoded protein to mediate urea uptake into cells. the subcellular targeting of dur3/gfp fusion proteins in tobacco protoplasts gave results comparable to the localization of the orthologous transporters of arabidopsis and rice, suggesting a partial localization at the plasma membrane. moreover, the overexpression of zmdur in the atdur3- arabidopsis mutant showed to complement the phenotype, since different zmdur3-overexpressing lines showed either comparable or enhanced 15-urea influx than wild-type plants. these data provide a clear evidence in planta for a role of zmdur in urea acquisition from an extra-radical solution.  this work highlights the capability of maize plants to take up urea via an inducible and high-affinity transport system. zmdur is a high-affinity urea transporter mediating the uptake of this molecule into roots. data may provide a key to better understand the mechanisms involved in urea acquisition and contribute to deepen the knowledge on the overall nitrogen-use efficiency in crop plants. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords cornhigh affinity transport systemdur3maizenitrogen rootureaissue-copyright-statement© the author 2014 