Molecular and physiological interactions of urea and nitrate uptake in plants

Abstract

While nitrate acquisition has been extensively studied, less information is available on transport systems of urea. Furthermore, the reciprocal influence of the two sources has not been clarified, so far. In this review, we will discuss recent developments on plant response to urea and nitrate nutrition. Experimental evidence suggests that, when urea and nitrate are available in the external solution, the induction of the uptake systems of each nitrogen (N) source is limited, while plant growth and N utilization is promoted. This physiological behavior might reflect cooperation among acquisition processes, where the activation of different N assimilatory pathways (cytosolic and plastidic pathways), allow a better control on the nutrient uptake. Based on physiological and molecular evidence, plants might increase (N) metabolism promoting a more efficient assimilation of taken-up nitrogen. The beneficial effect of urea and nitrate nutrition might contribute to develop new agronomical approaches to increase the (N) use efficiency in crops.

Abbreviations

Asn

asparagine

DUR3

urea transporter

N

Nitrogen

NAR

nitrate transport accessory protein

NIP

nodulin 26-like intrinsic protein

NUE

Nitrogen use efficiency

NRT

nitrate transporter

Gln

glutamine

GS

glutamine synthetase

GOGAT

glutamine oxoglutarate aminotransferase

TIP

tonoplast Intrinsic Protein

PIP

plasma-membrane intrinsic protein.

It is well known that nitrogen (N) is one of the key nutrients limiting plant growth. This is especially true for cereals, mainly because of their high demand but low N-Use Efficiency (NUE). Actually a common agronomic practice to improve the cereal yield is to apply high amount of N fertilizers. Especially if losses of cereal cropping area continues at the rate of the past 20 years (−0.33% per year) and the NUE of cereals will not be increased substantially, in the future the cereal demand will lead to a 60% increase of the global N use.¹ Logical consequence of a wide use of chemical inputs leads to negative impacts at ecological and also economical level. Based on these considerations, a goal of modern agriculture is to improve the capability of crops to use N sources, both native and applied ones (for a review see^2,3).

Three main N forms are present in soil as native sources or as applied fertilizers (ammonium, nitrate and urea) and, at least for a short time, plant roots are exposed to a combination of all these three N sources.⁴ Nevertheless, their relative amount and bioavailability under field conditions are obviously influenced by biogeochemical reactions.⁵ To improve the comprehension and the optimization of N nutrition in plants, a research topic of great relevance is the study of the interactions among different N-sources on the root uptake; however up to date only few works have investigated this topic.^4,6-8 The present review focuses on recent evidence about the acquisition of two major N-forms, urea and nitrate, and on the reciprocal influence of these two sources on their root uptake systems.

Soil Availability

It is well known that plants are able to absorb and assimilate different N forms, such as nitrate (NO₃⁻), ammonium (NH₄⁺), urea(CO(NH₂)₂) and even some amino acids.^9,10 However the relative contribution of a single source to plant nutrition is strictly dependent to its availability in the external solution. However, due to the ready use of nitrate by plants and microorganisms and its leachability, nitrate concentration in the soil is fluctuating widely (typically range between 0.5 and 10 mM).¹¹ Thus, in the modern agriculture the massive crop production is sustained by high inputs of N fertilizers. The most commonly used fertilizers include a different collection of compounds with N in form of nitrate, urea or a combination of both. Principally due to the low price and the high N content, today urea is the most used N fertilizer in the world accounting for more than 50 % of the N fertilizer consumption. Urea can be a direct source of N for plant,¹² however most of it is rapidly hydrolyzed by soil ureases in ammonium, which in turn can be converted into the highly mobile nitrate in aerobic soils.⁵ Urea concentration in natural environment is estimated to be between 0.1 and 3 µM), but in agricultural soils urea can reach higher concentration, up to 70 µM.^13-15

Physiological and Molecular Characterization of Nitrate Acquisition

In agricultural soil, nitrate is the main N source absorbed by plants and the mechanisms for its acquisition have been extensively studied (for a review see^16,17). From soil solution, nitrate is actively transported across plasma membranes of epidermal and cortical cells of roots by specific transporters. Among higher plants, two different families of nitrate transporter have been identified, NRT1 and NRT2 which mediate the acquisition of the anion in symport with protons exhibiting low and high affinity for the anion, respectively.¹⁸

Beside these transport systems, the acquisition of nitrate is even regulated by an efflux component operated by NAXT-1 proteins¹⁹ and by changes occurring at transcriptional and post-translational level.

Several genome-wide data have focused on the transcriptional response induced by nitrate. In Arabidopsis, as well in maize and other plants, the presence of nitrate in the external solution induced the expression of those genes involved in the transport and assimilation. Thus NRT transporters, NRT accessory proteins (NAR), nitrate reductase, nitrite reductase, and genes involved in the GS-GOGAT cycle are well known to be nitrate- induced in plants.^8,20-22 Moreover nitrate regulates a wide variety of processes, including glycolysis, amino acid synthesis, organic acid metabolism, production of cofactors (Uroporphyrinogen III methyltransferase) for the reduction processes, synthesis of reducing equivalent (Ferredoxin, Ferredoxin-NADP reductase) and activation of the oxidative penthose phosphate pathway.^8,22-26

In this way, when nitrate is present in the soil solution, plant roots are able to tightly regulate the absorption of the anion as well its assimilation. Depending on nitrate concentration in the external solution and on the plant species, the maximum uptake capacity of nitrate is reached after few hours or days after root exposure to the anion.²² Usually the induction of high affinity nitrate transport system leads to overshoot the plant demand for N uptake and after the initial exposure to nitrate, the uptake system is rapidly down regulated by a feedback control of downstream N metabolites, as ammonium and amino acids.^27,28 Thus it has been suggested that while nitrate is responsible for inducing gene expression,²⁹ nitrate assimilation products might be responsible for the down regulation of nitrate acquisition.^30,31

Physiological and Molecular Characterization of Urea Acquisition

Similarly to nitrate, even urea represents a source of N available for plant nutrition and in the recent years different mechanisms for its direct acquisition have been described. Immediately after fertilization, the external urea concentration can reach high concentrations for a short time. Under these conditions, the root acquisition of external urea may be operated by aquaporins, which mediate a low affinity permeation of the source.¹⁵ This feature has been described for many members belonging to different aquaporins family Tonoplast Intrinsic Protein (TIP) family, Plasma-membrane Intrinsic Protein (PIP) and Nodulin 26-like Intrinsic Protein (NIP-like) in many species (Arabidopsis, AtTIP1.1, AtTIP1.2, AtTIP2.1 and AtTIP4.1; tobacco, NtTIPa; zucchini, CpNIP1; maize, ZmPIP1.5, ZmNIP2.1, ZmNIP2.4 and ZmTIP4.4).^32-36 However for most of them, the subcellular localization and their physiological contribution to overall urea transport remains to be clarified. Similarly to nitrate, a high affinity transport system for urea acquisition has been identified in higher plants, which is mediated by a secondary active symport with protons. Up to date, a single gene coding for an urea transporter DUR3 has been identified in sequenced plant genomes: being characterized in Arabidopsis, rice and maize,¹² and predicted in grapevine, poplar and tomato. Experimental evidence suggests that DUR3 is the main component of the high affinity transport system for urea acquisition in roots and the expression of DUR3 gene appears to be induced under N starvation.^12,37,38

Data reported in Arabidopsis and maize have revealed that the urea acquisition is induced by the presence in the external solution of the substrate itself.^8,39 In particular in maize roots, the high affinity transport system of urea appeared to be inducible by urea itself, retro-regulated and dependent on the external urea concentration and on the duration of root exposure to the N source.⁸ Despite this physiological response to urea, transcriptional changes in plants are rather limited in this condition. In Arabidopsis and maize, transcriptomic studies revealed that the presence of external urea induced the expression of a gene coding for an asparagine synthetase, which seems to participate in the urea assimilation pathway.^8,39 Moreover the activity of this enzyme and the plant content of its metabolic products seem to have a crucial role in the regulation of urea acquisition mechanisms.

Physiological and Transcriptional Changes Occurring Under Urea and Nitrate

To date only a limited number of studies have focused on the reciprocal influence of urea and nitrate uptake.^4,6-8,39 Several authors have demonstrated that the root exposure to a combination of different N sources, led to positive effects on the nutritional status of crop plants. ^6,7,40,41 In long term, the presence of both urea and nitrate enhanced plant growth ^6,8 and the relative use of each N-source ⁷ as compared to nitrate or, especially, to urea, when provided alone. The mechanisms behind this reciprocal influence remain mostly unknown.

In short term experiments (up to 24 hours) it was demonstrated that the induction of urea transport system was much reduced in plants treated with nitrate and urea in comparison to plants exposed to urea alone^8,39 and the same held true for nitrate uptake when urea was supplied in conjunction with nitrate.⁸ This might indicate that root N acquisition is regulated depending on the form of N available in the soil solution.

These physiological responses would be accompanied by changes occurring at both transcriptional and posttranscriptional level. Indeed, in comparison to plants treated with only nitrate, the presence of external urea in conjunction with nitrate determined in the roots a reduced expression of NRT2.1. Moreover roots did not induce the expression of NAR, an accessory protein involved in the targeting and activation of NRT proteins. Thus, at least in maize, the low abundance of ZmNRT2.1 transcripts and the lack of those coding for NAR proteins might explain the low capacity of plants to take up nitrate when urea is also present in the external solution.⁸ On the other hand, the effect of nitrate to limit urea uptake was sustained by a down regulation of urea transporter DUR3 when inorganic N source, nitrate or ammonium nitrate, were supplied along with urea.^8,39,42

Further transcriptional changes of the assimilation pathways were identified when both sources were applied in the external solution. Microarray data in Arabidopsis and maize, revealed that urea and nitrate treatment in comparison to nitrate alone increased the up-regulation of nitrate-responsive genes, in particular of those involved in the uptake and assimilation of nitrate. Beside the induction of plastidial GS2-GOGAT cycle, a putative cytosolic pathway (involving a Gln synthetase I and an Asn synthetase) for the assimilation of urea-derived ammonium was found to be induced only in the presence of both urea and nitrate.^8,39 This transcriptional modulation is further sustained by metabolomic data. In wheat, when nitrate was supplied along with urea, Gln and Asn contents increased significantly in comparison to plants treated with only one N source.⁶ In turn, we can hypothesize that the increase of primary assimilation might play a crucial role in determining a better use of the two N-sources when they are provided in conjunction⁷ (Fig. 1).

Figure 1.

Proposed pathway for urea and nitrate acquisition in root cells. Comparison of 3 treatments containing nitrogen in the form of: (A), urea alone (orange dots); (B), nitrate alone (green dots); or (C), urea plus nitrate. Blue rectangles, transporters belonging to HATS (i.e. DUR3, NRT2s); blue ovals, transporters belonging to LATS (i.e., PIPs, NIPs, NRT1s); line arrows, enzymatic reactions for N-assimilation; dashed arrows, feedback regulation on acquisition systems by N-sources itself or by metabolic products; bold arrows, activation of an assimilatory pathway; blue triangle, translocation of amino acids to the sink organs (A, B): putative response of root cells when a single N source (urea or nitrate) is available in the external solution. The acquisition of urea (or nitrate) is induced by N source and the source is rapidly accumulated in the cytosol. To overcome cytotoxic levels of urea (or nitrate), a feedback regulation on transport systems are activated by substrate itself or by N-metabolic products. Concerning the feedback regulation of urea-HATS, it might involve a putative post-transcriptional modification or re-targeting of DUR3 protein using a trafficking pathway (such as the trans-Golgi network, TGN).³⁸ C: when both N-sources are present, root cells are more efficient in the acquisition of urea and nitrate through the enhancement and cooperation between assimilatory pathways, cytosolic and plastidial ones. This mechanisms might even allow a better control on the homeostasis of urea and nitrate in the cytosol, with no need for a feedback regulation on the uptake. Drawing of plant has been modified from http://udl.concord.org/artwork/plant_34/pl_34_index.html.

The beneficial effect of urea plus nitrate nutrition might be due to an enhancement of N assimilation mediated by both plastidial and cytosolic pathways. In this way plants ensure an internal homeostasis of urea and nitrate and no feedback control on the mechanisms of acquisition are needed. This hypothesis might suggest that more than transport, the assimilation of N sources might be the limiting step in the acquisition of the sources. Based on the reciprocal interaction of urea and nitrate on their mechanisms of acquisition, new agronomical practice might be considered to improve the use efficiency of fertilizers in crops.

Concluding Remarks and Future Prospective

Study of the reciprocal interactions between nitrate and urea can provide a basis for understanding use of N sources and optimize their acquisition. Accurate metabolomic and proteomic studies are needed to shed light on fate of absorbed N and to investigate on regulatory aspects of these processes in plants. Furthermore, considering the great agronomical relevance of these sources, new experimental approaches closest to the real condition of cultivated soils should be used. In the recent years chemical inputs in agriculture (e.g. fertilizers, urease and nitrification inhibitors) have been widely used with negative impacts on environment. Based on these consideration, new research focusing on the interaction among N forms conceivably present in the soil may lead to a more sustainable and efficient use of fertilizer procedures.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

This work has been funded by the Department of Agricultural and environmental Sciences (DISA), University of Udine (UNICO Project -2014).