1. Introduction
Nitrogen (N) is essential for life sustainability. Some morphological, developmental and reproductive phenomena such as flowering, growth, senescence, oxidation, reduction and allocation of photosynthates in a plant are regulated by the availability of N [
1]. Application of N fertilizers has both positive and negative impacts; it has increased the supply of food, feed and several biobased products remarkably on one hand, but also deteriorated the quality of the environment and caused huge economic losses by depleting the fossil-fuel reserves, on the other. Various forms of N released into the environment pose a serious threat to the health of humans, plants and animals [
2]. Excessive use of non-sustainable fossil fuels results in heavy emission of greenhouse gases, causing depletion of the ozone layer, global warming and other serious environmental threats. The rapid increase in human population demands more agricultural production, which is achievable by using heavy nitrogenous fertilizers. However, agricultural crops, particularly rice, maize and wheat, have an N-utilization efficiency of only 30–40%, leaving behind 60–70% N unused, which severely deteriorates the environmental health [
3]. It is assessed that the anthropogenic contribution to greenhouse gas production from agricultural fields is around 10–12% of the total greenhouse gas (GHG) emissions resulting from human activities [
4]. The main source responsible for agricultural releases results from the manufacture and excessive application of nitrogenous fertilizers to cultivable land. Synthetic production of N fertilizers employing the Haber–Bosch reaction accounts for about fifty percent of the total energy expenditure in agriculture [
5]. Moreover, application of nitrogenous fertilizers to agricultural fields is associated with heavy production of nitrous oxide. The global warming effect of this greenhouse gas is approximately 300 times greater than that of carbon dioxide [
6] and constitutes about forty percent of GHG emissions directly from agricultural soils [
4]. It is, therefore, important to limit the application of nitrogenous fertilizers without affecting the crop yield [
3]. Hence, a clear rationale has to be defined, emphasizing on the reduction of the excessive utilization of N fertilizers in the agriculture sector.
The targets for future research include development of a highly productive agriculture to increase crop productivity, coupled with a reduction in the use of N fertilizers [
7]. An appraisal by the FAO has shown that there is a need to increase agricultural production by 60% in 2030–2050 over the production levels in 2005–2007 [
8]. In India, maize is the third most important food crop after rice and wheat, and consumes large quantities of nitrogenous fertilizers [
9]. The average nitrogen-use-efficiency (NUE) is far less than 50% and, therefore, enhancing the NUE is the best approach in majority of crops, specifically in those that require huge quantities of N fertilizers for a maximum yield [
10]. Few agronomic methods such as the slow release of fertilizers, nitrification inhibitors and split application of N are used to get the maximum benefits of applied fertilizers. The conventional breeding is also used to select the most appropriate traits, but this practice provides no information on the molecular basis of enhancement in NUE. Various studies, including the whole plant physiology, agronomy and molecular genetics, have been undertaken over the last two decades for characterizing the switches that regulate the NUE. Some genomic studies have indicated that overexpression of transcription factor
DOF1 under low-N conditions results in increased plant growth and N content [
11]. All such studies have been based on a particular gene, protein or trait; however, the NUE may depend on the interaction of a network of genes. Therefore, a holistic approach that considers all the genes and their associated pathways for improving NUE is the need of the hour.
To address the root causes that regulate the NUE, some studies have used metabolomic, transcriptomic and proteomic approaches [
12,
13,
14]. The physiological status of a cell, tissue and organ is recorded through the ‘omics’ procedure at different developmental stages of the plant [
15]. This offers a complete overview of alterations in the concentration of metabolites (primary and secondary), gene transcripts and proteins [
16,
17]. An untargeted metabolite profiling, covering the entire range of metabolites, must be helpful in developing strategies to improve NUE and in collecting information about gene products or expression of new genes [
18]. Metabolomics provides complete information about what is happening inside the tissue, organ or a cellular compartment of plant under various stresses caused by adverse factors, physiological adaption to dietary change or environmental perturbations [
19,
20,
21,
22,
23,
24]. It has also been applied to studies related to shortage in nutrients [
25]. Earlier studies on metabolic responses of maize to N generally concentrated at the vegetative and maturation stages of leaves only [
26]. These studies did not include the resupply condition, which is important to validate whether the effect is due to the factor under observation or to some other reasons. Further, metabolomic data of low-N tolerant and low-N sensitive maize, involving leaf, roots and the effect of resupply of N at different time intervals still remain uncovered. Given this, the present investigation was conducted to analyze the non-targeted metabolic profiling of shoot and root of two contrasting maize genotypes (low-N tolerant and low-N sensitive) in response to N-deficiency and resupply of N. This could help in explaining the key role of master switches/regulators, responsible for the diverse N responsiveness of different genotypes of the same species.
3. Discussion
In this study, an effort was made to profile metabolites of leaves and roots of two contrasting genotypes, PHEM-2 (low-N tolerant, LNT) and HM-4 (low-N sensitive, LNS) of maize, growing under low-N and restoration conditions. The study has provided a comprehensive and comparative analysis of the metabolite composition in leaves and roots of contrasting maize genotypes under conditions of low-N and restoration of N supply. When data from LNT and LNS genotypes were combined, leaves and roots of LNT had the most similar metabolite content to the associated control (4.5 mM N), whereas in the case of the LNS genotype, significant variations were detected within treatments, i.e., low-N 0.05 mM N and its control 4.5 mM N, and between genotypes. Out of 130 detected peaks, 94 metabolites were putatively identified as known compounds based on the mass spectra library NIST, Wiley Registry, Golm and Fiehn database. Most of the amino acids except glutamine, asparagine, glycine and the N-containing metabolites were significantly reduced under low-N conditions. Serine was significantly reduced under low-N conditions, as was reported earlier by Rossella et al. [
27]. This might be due to the reduction in the ATP-sulphurylase and O-acetylserine sulphydrylase activities under N-deprivation. Proline and alanine were also reduced significantly under low-N conditions. An increase in the content of proline and alanine could serve as an indicator of an imbalance in N nutrition [
28]. Aspartic acid and glutamate were also reduced under low-N conditions, as they may be involved in the formation of oxalacetate, an important intermediate of the tricarboxylic acid cycle, as assimilation of NH
4+ into amides and amino acids requires carbon skeletons from the tricarboxylic acid cycle [
29]. This could be why there is a general decrease in the organic acid content, as there is a shortage in the amount of precursor molecule. The main regulators of the amino acid biosynthesis are 2-oxoglutarate and glutamate. The level of these metabolites increased, following a transfer to the N-sufficient medium [
30]. Our study substantiated these findings. There was a general reduction in the levels of various amino acids under low-N condition. There was a contrast between γ-amino butyric acid contents of leaves and roots in LNS and LNT genotypes; it accumulated in the LNS genotype and declined in the LNT genotype under low-N conditions. The level of this amino acid may control the interaction between assimilatory pathways of N and C and photorespiration [
31]. Accumulation of GABA was seen in our study under the low-N condition. Synchronized regulation of GABA in plants has also been reported earlier [
32]. Other amino acids that accumulated under low-N conditions include asparagine, glutamine and glycine. Accumulation of glutamine and asparagine could be related to the remobilization of assimilated N. Glutamine is not only used for N transport but also serves as an amino donor to other amino acids. Previous studies have shown that N-containing macromolecules and C reserve compounds like carbohydrates and fats are accumulated under N-starvation conditions [
33,
34]. Most of the N-containing metabolites, such as urea, citrulline, celapanine, sapropterin, anhydrotetracycline, 2-aminoanthraquinone and dehydrocarpaine I, were reduced under low-N conditions, possibly to conserve nitrogen for important developmental processes. It has been suggested earlier that the reduced concentration of urea might owe to the slow catabolism of arginine in the urea cycle in the N-deficient plants [
35]. Our study also revealed a reduced concentration of allantoin under low-N conditions. Allantoin is involved in storage, translocation and signaling of N [
36,
37]. That plants save nitrogen by reducing the N-containing metabolite levels was also proposed by Lu and Zhang [
38]. It has been reported that there is conservation of N by the lessening synthesis of proteins and chlorophyll in low-N grown plants. Since, N deficiency in plants alter mainly nitrogen and carbon metabolism, we attempted to integrate different primary and secondary metabolic pathways at important metabolic switches. These metabolic switches function as precursors and regulate the development and yield of crops. Therefore, giving an integrative picture of the metabolic network affected by nutrition deficiency may pave the way for future studies to enhance nutrient use efficiency and yield of crops (
Figure 5). Apart from the decreases in various N-containing metabolites and peptides, the level of glutathione, a tri-peptide, was increased significantly in leaves and roots of the LNS genotype under low-N conditions, while no significant change observed in the LNT genotype (
Figure 5). Glutathione protects the plasma membranes by maintaining the level of α-tocopherol and zeaxanthin. Since, membranes are more vulnerable to environmental stresses, maintenance of the proper structure of biomembranes is an essential requirement.
Lipids contents, e.g., linolenic acid, linoseaure 16:2-Glc-Campesterol, PC (O-6:0/2:0)[U] and sitosteryl acetate, increased significantly under low-N conditions, possibly because plants tend to accumulate lipids as an alternate source of energy. Metabolism slows down under low-N conditions, as lipids are slowly broken to release energy. Earlier, accumulation of carbon metabolites as lipids was reported under nitrogen deficient conditions in algal cells [
39]. Sterols are also the important components of biomembranes; any alteration in their composition will produce drastic effects in the development of the plant. This study has revealed that lipid and sterol, like 16:2-Glc-Campesterol and sitosteryl acetate increased in the LNT genotype; while opposite was the case in the LNS genotype under low-N conditions (
Figure 5), because the LNS genotype was not able to acclimatize to low-N conditions. In general, plant sterols, such as stigmosterol, accumulated in response to low-N conditions. Current evidence indicates that plant sterols are proficient to modify the activity of the plasma membrane H
+-ATPases [
40]. It was shown that the sterol modulation of the plasma membrane H
+-ATPase activity depends on both the concentration and molecular species of sterol. One of the functions of ATPase is the production of the proton motive force across the plasma membrane, which is essential for the transport of ions and metabolites. The reduced biomass observed in the case of the LNS genotype under low-N and restoration confirmed the effect of a change of H
+-ATPase activity (cell growth). Altered signaling of auxin and ethylene shown by
hyd mutants could be elucidated by such effects [
41]. As the alteration in auxin and ethylene signaling will affect the exudation of carbohydrates, amino acids and organic acids, it is an important strategy plants utilize for obtaining the locked nutrients from the soil under nutrient-limiting conditions, because these organic acids solubilize the rhizosphere in order to release elemental nutrients from bound forms. Consistently increased exudation of organic acids has been reported earlier under the conditions of low P and low Fe [
42,
43]; however, a lower root exudation of carboxylates, sugars and amino acids has been detected in N-deficient bean plants [
44]. The regulatory switches behind this strategy remain unclear and maybe plant growth regulators, for instance indol-acetic acid (IAA) and zeatin, play a role. Comparative study of the H
+-ATPase activities at the cell membrane under various nutrient regimes may shed further light on the significance of retrieval mechanisms for the net exudate discharge from roots. Cholesterol has been reported to act as a signaling molecule in the animal system, without the transformation into steroid hormones [
45]. Such a function can also be assumed for plant sterols. Under low-N conditions, in our study, monoacyl and tri-acyl glycerols such as PC (O-6:0/2:0)[U], PC [O-18:0/O-2:1(1E)], Cer(d14:2(4E,6E)/16:0) and PC (O-10:0/O-10:0)[U] accumulated. These results endorse earlier findings with
Chlamydomonas reinhardtii that builds up both starch and TAGs in response to various types of stresses including N deprivation [
46,
47,
48,
49,
50,
51,
52,
53,
54,
55,
56].
Under low-N conditions, there was a dramatic reduction in the levels of major organic acids of the tricarboxylic acid cycle, particularly ketoglutaric acid, succinate, isocitric acid and malic acid. The findings were in accordance with those of Scheible et al. [
57] who reported that the limited N supply lead to large decreases in 2-oxoglutarate, isocitrate and malate. Organic acids are the preferred source of carbon under nutrient-limiting conditions. Additionally, phosphate-containing sugars (glucose-PO
4 and fructose-PO
4) are significantly reduced under low-N conditions, revealing that there was a low accumulation of some intermediate products of glycolytic pathway or sucrose biosynthesis. A number of other compounds, like erythritol among others, which act as a source of precursors for carotenoids, are reduced in quantity, i.e., their biosynthesis may be modified [
58]. The reduced levels of glucose, fructose, lactose, mannose and mannitol are indicative of altered metabolic and signaling function [
59]. Similarly, changes in the relative levels of phenylpropanoids such as dihydroxycoumarin and phenmetrazine suggest that biosynthesis of lignin was rehabilitated [
60] under low-N conditions. Metabolites showing increased response to low-N conditions in our study include raffinose, maltose, trehalose, galactinol and mannitol; this is in line with the previously described results from N-deficient
Arabidopsis [
17]. The possible reason for their increase can be explained on the basis of precursor molecules fructose and glucose, as these were detected in low concentration in plants growing under low-N conditions. The picture provided here by metabolomic profiling will serve as an important source in explaining the regulating switches of N metabolism.
Levels of flavones such as brosimacutin C and deoxymiroestrol increased under low-N conditions. One of the possible reasons for increased flavonoid synthesis under nitrogen limitation is that enhanced PAL activity will free nitrogen for amino acid metabolism, whereas carbon products are shunted via 4-coumaroyl-CoA into the flavonoid biosynthetic pathway [
61]. Some peptides detected as containing N, for example, Asp Thr Gly Cys, Glu Ser Gly Cys, Ala Ala Cys Cys, Glu Trp Thr Gln, Asp Pro Gln and Trp, are highly reduced under low-N conditions in order to conserve nitrogen.
The limitation of this study is that the metabolites were identified putatively. Validation of these metabolites is required in the future.