Foliar Nitrogen Application Enhances Nitrogen Assimilation and Modulates Gene Expression in Spring Wheat Leaves

Abstract

Nitrogen (N) critically regulates wheat growth and grain quality, yet the molecular mechanisms underlying foliar nitrogen application remain unclear. This study evaluated the effects of foliar nitrogen application (12.25 kg ha⁻¹) on the growth, grain yield, and quality of spring wheat, as well as its molecular mechanisms. The results indicated that N was absorbed within 3 h post-application, with leaf nitrogen concentration peaking at 12 h. The N treatment increased whole-plant dry matter accumulation and grain protein content by 11.34% and 6.8%, respectively. Amino acid content peaked 24 h post-application, increasing by 25.3% compared to the control. RNA-sequencing analysis identified 4559 and 3455 differentially expressed genes at 3 h and 24 h after urea treatment, respectively, these DEGs being primarily involved in nitrogen metabolism, photosynthetic carbon fixation, amino acid biosynthesis, antioxidant systems, and nucleotide biosynthesis. Notably, the plastidic glutamine synthetase gene (GS2) is crucial in the initial phase of urea application (3 h post-treatment). The pronounced downregulation of GS2 initiates a reconfiguration of nitrogen assimilation pathways. This downregulation impedes glutamine synthesis, resulting in a transient accumulation of free ammonia. In response to ammonia toxicity, the leaves promptly activate the GDH (glutamate dehydrogenase) pathway to facilitate the temporary translocation of ammonium. This compensatory mechanism suggests that GS2 downregulation may be a key switch that redirects nitrogen metabolism from the GS/GOGAT cycle to the GDH bypass. Additionally, the upregulation of the purine and pyrimidine metabolic routes channels nitrogen resources towards nucleic acid synthesis, and thereby supporting growth. Amino acids are then transported to the seeds, culminating in enhanced seed protein content. This research elucidates the molecular mechanisms underlying the foliar response to urea application, offering significant insights for further investigation.

1. Introduction

Spring wheat (Triticum aestivum L.) is one of the most significant global staple crops [1]. Its grain yield and quality must be consistently improved to meet the escalating demands of a growing population and raised dietary standards [2]. However, during spring wheat production, the rather old-fashioned cultivation technology system and the scarcity of high-yield, high-quality, and multiple disease-resistant spring wheat varieties result in inferior yields and quality compared to winter wheat. The Qaidam Basin in Qinghai Province, China, is renowned for its high agricultural productivity, where spring wheat yields frequently exceed 12,000 kg ha⁻¹. Nonetheless, the region currently faces challenges such as low soil fertility, recurrent low-temperatures, and drought events in the spring, and low accumulated temperatures over the growing period. These issues often lead to decreases in spring wheat yields, compromising yield stability. Additionally, the lower temperatures during the growing season impede the uptake of nitrogen by wheat crops, resulting in reduced protein concentration in the grain and thereby lowering its quality. Consequently, the pressing challenge in these high-altitude regions of China is to devise effective measures to increase both the grain yield and grain protein concentration of spring wheat, achieving a synergistic advancement in both parameters.

Nitrogen, the yield-limiting element in many crops, is critically important for plant growth and development and plays a crucial role in the concentration of amino acids, proteins, chlorophyll, and several phytohormones; the appropriate application of nitrogen fertilizer in specific forms to the wheat crop can significantly improve both the grain yield and quality [3,4]. Wheat typically takes up nitrogen through its roots in the form of nitrate (NO₃⁻) or, to a lesser extent, ammonium (NH₄⁺) [5]. However, foliar urea application bypasses root uptake and directly delivers urea to the plant leaves [6]. Urea is hydrolyzed in the leaf tissues by the enzyme urease (EC 3.5.1.5) to generate ammonia (NH₃), which is subsequently incorporated into amino acids and proteins via the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle [7,8]. Foliar urea application is a form of nutrient management that is often used to provide a supplementary source of nitrogen during the grain development stage of the wheat growth cycle [9]. The foliar application of urea can increase grain protein concentration, grain starch concentration, flour sedimentation value, and grain yield [10,11]. Foliar application of a 2% urea solution post-anthesis can promote nitrogen uptake in winter wheat and the partitioning of nitrogen from temporary stores in vegetative organs to grains, thereby increasing grain protein concentration [12]. This method is particularly advantageous when soil nitrogen levels are insufficient or when environmental conditions, such as waterlogging, limit root function.

In response to foliar urea application, wheat plants rapidly modulate their nitrogen metabolism to efficiently assimilate and redistribute nitrogen within the plant. Several key genes involved in nitrogen uptake, assimilation, and remobilization are upregulated in response to foliar urea. For example, urease (EC 3.5.1.5), a key enzyme responsible for hydrolyzing urea into ammonia and carbon dioxide, could enhance nitrogen mobilization from urea to ammonium [13]. Glutamine synthetase (GS, EC 6.3.1.2) and glutamate synthase (GOGAT, EC 1.4.1.13) are central to the assimilation of ammonium into amino acids. The upregulation of the expression of GS and GOGAT genes following foliar urea application supports efficient nitrogen incorporation into the plant’s metabolic pathways [14]. Expression of nitrate reductase (NR, EC 1.7.1.1) and nitrite reductase (NiR, EC 1.7.2.1) genes has been found to be co-regulated with urea assimilation genes in some wheat varieties, indicating potential crosstalk between nitrate and urea assimilation pathways [15,16]. However, the current research findings are primarily based on root response to N, and the molecular response to foliar urea application is unknown.

Transcriptome analysis is a pivotal tool for elucidating the molecular mechanisms underlying the response of crops to nitrogen fertilization [17]. Typically, the level of applied nitrogen fertilizer directly influences the expression of nitrogen metabolism-related genes in crops [18,19,20]. This study employed phenomic and transcriptomic analyses to investigate the effects of foliar urea application on the gene expression in spring wheat, with the aim of identifying key differentially expressed genes and regulatory pathways. This not only aids in enhancing the yield and quality of spring wheat but also provides a molecular basis for nitrogen fertilizer management in other crops.

2. Materials and Methods

2.1. Plant Material and Treatments

The experimental material used was the locally dominant spring wheat variety Gaoyuan 619, a medium flour strength variety, developed by the Northwest Plateau Institute of Biology, Chinese Academy of Sciences. The seeds were sown in March 2024, at the experimental station of Xiangride Farm, Dulan County, Haixi Prefecture, Qinghai Province, China. This experimental site is situated at an approximate altitude of 3000 m, where frequent cold and drought conditions prevail during spring, and the accumulated temperature during the growing period is relatively low. The soil at the experimental site was sandy loam, containing 18.1 g kg⁻¹ of organic matter, 1.26 g kg⁻¹ of total nitrogen, 89.7 mg kg⁻¹ of available phosphorus, and 177.2 mg kg⁻¹ of available potassium, with a pH value of 7.35. The experiment comprised two treatments arranged in a randomized block design. Prior to sowing, each plot was uniformly fertilized with 90 kg ha⁻¹ of nitrogen as the base fertilizer. Treatment 1 involved foliar spraying with a 2% urea (w/v) solution (20.4 g of urea l⁻¹ deionized water) (the N treatment) at the booting stage. Treatment 2 served as the no-urea control (CK), where deionized water was sprayed at the same stages as in Treatment 1. All spray solutions contained 0.01% (v/v) Tween-20 adhesion agent to prevent leaf burn. Spraying was conducted on windless, overcast days or during humid afternoons/evenings with low evaporation rates, specifically between 17:00 and 19:00. Each treatment was replicated independently three times. Each plot size was 15 m² (3 m × 5 m), with a sowing rate of 450 kg ha⁻¹, a sowing depth of 3–4 cm, and a row spacing of 15 cm. Other management practices followed local conventional field management protocols.

2.2. Determination of N Concentrations

Following foliar application of urea during the booting stage, flag leaf samples were collected from both the N treatment group and the control group at intervals of 3 h, 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h. The collected leaves were rinsed thoroughly with water and then gently blotted dry with absorbent paper. One portion of each sample was snap-frozen in liquid nitrogen and subsequently stored at −80 °C for subsequent enzyme and gene analysis. The other portion was sealed in a self-locking bag and returned to the laboratory for drying at 75 °C. The dried samples were then cut into smaller pieces using scissors, thoroughly mixed within each treatment group, and subsequently pulverized using a grinding machine (Shanghai, China). Approximately 0.5 g of the pulverized sample was weighed and combined with 5 g of a mixed catalyst (K₂SO₄:CuSO₄·4H₂O = 20:1) and 10 mL of concentrated sulfuric acid, which was then subjected to complete digestion. The nitrogen content was subsequently determined using the Kjeldahl method.

2.3. Determination of Key Nitrogen Assimilation Enzyme Activities and Total Amino Acid Concentrations

After foliar application of urea (or water) at the booting stage, leaves from both the N treatment and control groups were harvested at 3 h, 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h after application. The collected leaves were thoroughly rinsed with distilled water, then patted dry, and 0.1 g samples were precisely weighed for subsequent analysis. The contents of glutamine synthetase (GS) and total amino acids (AA) in the leaf tissues were quantified using Glutamine Synthetase (GS) Activity Assay Kit (BC0915; Solarbio, Bijing, China) and Amino Acid (AA) Content Assay kit (BC1575; Solarbio, Bijing, China). The experiment was performed on three independent biological replicates for each of the two samples.

2.4. Transcriptomic Profiling Analysis

To analyze temporal effects of foliar urea on the wheat leaf transcriptome, we collected control and treated leaves at 3 h and 24 h post-application (three biological replicates each). RNA extraction and subsequent sequencing were conducted by Beijing Novogene Technology Co., Ltd. (Beijing, China). The raw sequencing data were subjected to stringent filtering to yield clean read segments. Reference genomes and gene model annotation files were procured from genomic databases. We employed HISAT2 (version 2.2.1) to construct an index of the reference genome and utilized the same software (HISAT2, available at https://daehwankimlab.github.io/hisat2/, accessed on 8 December 2024) to map the paired-end clean reads against the reference genome (ncbi_triticum_aestivum_gcf_018294505_1_iwgsc_cs_refseq_v2_1). The expression levels of the mapped reads were quantified using Fragments Per Kilobase per Million reads (FPKM) values. The average FPKM values from the three biological replicates were computed for differentially expressed gene (DEG) analysis, with DEGs determined between the 3 h N treatment and CK control replicates, as well as between the 24 h N and CK replicates, based on the criteria of false discovery rate (FDR) < 0.05, FPKM > 1, and |log2 fold change (FC)| > 0.5. These DEGs were mapped to the Gene Ontology (GO, http://geneontology.org/, accessed on 16 January 2025) and Kyoto Encyclopedia of Genes and Genomes (KEGG, https://www.genome.jp/kegg/, accessed on 16 January 2025 ) databases, and their enrichment in GO and KEGG pathways was analyzed using the clusterProfiler (v.4.0.0) software (https://bioconductor.org/packages/release/bioc/html/clusterProfiler.html, accessed on 18 January 2025). The principle behind the enrichment analysis is the hypergeometric distribution.

2.5. Quantitative Real-Time PCR (qPCR)

To validate the transcriptomic findings, we employed quantitative polymerase chain reaction (qPCR) analysis, which adhered to the same RNA extraction and reverse transcription protocols as those utilized in the transcriptomic profiling. The coding sequences (CDSs) of the genes of interest were derived from the transcriptomic data, and primers were designed using Primer5 (v.5.0.0) software, as detailed in Table S1, with actin serving as the housekeeping gene. Primer synthesis was outsourced to Shanghai Sangon Biotech (Shanghai, China). Using cDNA templates derived from the leaves of the different treatment groups, we conducted qPCR analysis on the Bio-Rad CFX system with the Hifair III One Step RT-qPCR SYBR Green Kit (Yeasen Biotechnology [Shanghai, China]). all qPCR data were derived from three biological replicates × three technical replicates per gene. The relative expression levels of the genes were calculated using the 2^−ΔΔCT method to provide a quantitative assessment of gene expression changes induced by foliar urea application.

2.6. Determination of Grain Protein Concentration and Grain Physical Parameters

At the maturity stage, grains were harvested from randomly selected 1 m² areas within each replicate experimental plot and naturally air-dried until the moisture content reached 13%. The thousand-grain weight (TGW), mean grain length, and mean grain width were measured using an intelligent seed counter (Tianyi510S-07IRB; Hangzhou, China). The grain protein concentration was determined using the Kjeldahl method, converting total nitrogen concentration to protein concentration.

2.7. Data Analysis and Visualization

Data analysis was meticulously performed utilizing Excel 2019 and SPSS 22.0 software (IBM, Armonk, NY, USA). The general linear model was strategically employed, incorporating fixed effects for treatment group categories, temporal variables, and their interactive influences, while also factoring in random intercepts at the individual level. This methodological approach was adopted to provide a holistic evaluation of the dynamic variations in treatment effects and to ascertain their statistical significance with precision. Graphical representation of the variance in grain protein content and grain weight across different treatment groups was achieved through the creation of bar charts with Origin 2021 (OriginLab, Northampton, MA, USA). Adobe Illustrator 2023 (Adobe Inc., San Jose, CA, USA) was employed to generate pathway diagrams. For visualizing data, volcano plots, Venn diagrams, and heatmaps of gene expression were crafted utilizing the cloud-based tools on the Novogene Magic-Plus platform (https://magic-plus.novogene.com/#/tool/list, accessed on 24 January 2025).

3. Results

3.1. Nitrogen Assimilation Following Foliar Urea Application to Spring Wheat

The foliar application of urea at the booting stage was followed by leaf sampling at seven time points from 3 h to 72 h after application for subsequent nitrogen content analysis. The results indicated that at 3 h, the nitrogen content in Treatment 1 (N) was 53.62 g kg⁻¹, which represented a 3.37% increase compared with Treatment 2, the control group (51.87 g kg⁻¹). The leaf nitrogen concentration in Treatment 1 reached its peak at 12 h, with a value of 56.69 g kg⁻¹, after which it gradually approached the level in the unsprayed treatment, Treatment 2 (Figure 1, Table S2). These findings suggest that the leaves absorbed the urea within 3 h, leading to an increase in leaf nitrogen content, but over time, nitrogen utilization gradually returned to initial levels. Amino acids, as terminal products of nitrogen assimilation, exhibited a peak concentration 24 h post-urea application, surpassing the control group by 25.3%. Additionally, the urea treatment was observed to slow the rate of amino acid concentration decline in later stages; for instance, 72 h after urea application, the concentration in the treated group was still 13.7% higher than that of the control group (Figure 1b, Table S3). These findings indicate that foliar application of urea can effectively enhance amino acid synthesis.

At maturity, the accumulation of dry matter per plant, individual plant yield, and protein content were quantified. The results demonstrated that foliar application of urea significantly increased the dry matter accumulation from 22.75 g (control) to 25.33 g per plant (urea-treated plants), an improvement of 11.34% (p < 0.05, Figure 1c), and significantly increased grain protein content (GPC), from 14.93% in the control (CK) to 15.95% in the urea-treated plants, representing an increase of 6.8% (p < 0.05, Figure 1c), with the grain plumpness of the urea-treated samples visibly superior to that of the control group (Figure 1d). However, there was no significant change in yield per plant (Figure 1e). This indicates that foliar spraying of 12.25 kg ha⁻¹ of a 2% urea solution during the booting stage significantly contributes to enhancing the protein content in spring wheat grains.

3.2. Transcriptomics Overview

To investigate the molecular mechanisms underlying the response of wheat leaves to foliar urea application, we sampled leaves at two critical time points for RNA sequencing: 3 h post-application (when urea absorption occurs) and 24 h post-application (during the peak accumulation of amino acids in the leaves). A total of 79.31 GB of clean data was obtained from the 12 samples (2 treatments × 2 time points × 3 replicates; Table S4). In total, 4559 DEGs were identified between the N treatment and control samples 3 h after foliar urea application, with 1885 genes upregulated and 2674 genes downregulated (Figure 2a). At 24 h, 3455 DEGs were detected in the leaves, with 2079 genes upregulated and 1376 genes downregulated (Figure 2b). A total of 278 genes were commonly differentially expressed in the leaves at both 3 h and 24 h in response to the N treatment (Figure 2c), of which 140 were upregulated (Figure 2d) and 43 were downregulated (Figure 2e). The results indicate that the number of upregulated genes increased over time following CK or N treatment in spring wheat, suggesting that specific responses may be associated with nitrogen transport in wheat leaves.

To identify potential pathways involving the DEGs underlying the response of wheat leaves to urea, GO and KEGG enrichment analyses were performed. At 3 h, 1041 GO pathways were enriched, such as photosynthesis, oxidative stress response, monocarboxylic acid biosynthesis, membrane-associated structures, and enzyme activities (such as transferases and oxidoreductases) (Figure S1A). This indicates that during the early stage following foliar urea application, wheat rapidly initiates photosynthesis and antioxidant mechanisms, while activating various enzyme activities to cope with the rapid uptake and metabolism of nitrogen. At 24 h, 946 GO pathways were enriched, such as amino acid catabolism, cell wall macromolecule metabolism, and lyase activities (Figure S1B). A total of 779 enriched GO processes were shared at both of the two points, including glutamine metabolism, aspartate family amino acid metabolism, glycosaminoglycan metabolism, response to oxidative stress, and G protein-coupled receptor-signaling pathways, among others (Table S5). These results highlight the dynamic and multifaceted molecular responses of wheat leaves to urea treatment.

Based on KEGG enrichment analysis, at 3 h post-application, a total of 4559 DEGs were enriched in 121 metabolic pathways. These pathways primarily included photosynthesis, phenylpropanoid biosynthesis, glutathione metabolism, nitrogen metabolism, starch and sucrose metabolism, and ABC transporters (Figure S1C). At 24 h post-application, 3455 DEGs were enriched in 116 pathways, which were particularly involved in the synthesis of amino acids and nucleotides, secondary metabolism, as well as intracellular substance transport and degradation (Figure S1D). Among these, 278 DEGs were co-expressed at both time points, which were enriched in 44 pathways, including cysteine and methionine metabolism, galactose metabolism, peroxisomes, purine metabolism, and ABC transporters (Table S6). These findings indicate that the foliar application of urea leads to the adjustment of multiple metabolic pathways in the leaves to adapt and respond to the additional supply of nitrogen.

3.3. Effects of Foliar Urea Application on Metabolic Pathways and Associated DEGs

The foliar application of urea significantly influenced leaf metabolism and signaling pathways. Among the DEGs, 32 key metabolic processes were identified (Figure 3a), including N-glycan biosynthesis, the TCA cycle, oxidative phosphorylation, biosynthesis of amino acids (such as phenylalanine, tyrosine, and tryptophan), fatty acid biosynthesis, antioxidant systems (e.g., glutathione metabolism), signal transduction (e.g., phosphatidylinositol-signaling system), and cell structural substance synthesis (e.g., flavonoid biosynthesis). Notably, these pathways exhibited a higher number of upregulated DEGs than downregulated DEGs (Figure S2). In contrast, DEGs related to photosynthesis, nitrogen metabolism, and energy storage (e.g., starch and sucrose metabolism) were predominantly downregulated (Figure S3). These findings indicate that foliar urea application coordinates gene expression to reprogram metabolic flux from carbon fixation towards nitrogen assimilation. Further analysis of KEGG pathway enrichment of the DEGs revealed significant enrichment in the pentose phosphate pathway, nitrogen metabolism, arginine and proline metabolism, purine metabolism, pyrimidine metabolism, and starch and sucrose metabolism in the urea-treated wheat leaves.

We subsequently focused on the DEGs associated with carbohydrate metabolism, energy production, and secondary metabolism. Our findings revealed that seven DEGs involved in the metabolic pathway of polysaccharide degradation to monosaccharides were downregulated in response to urea treatment. These DEGs included β-fructofuranosidase, β-glucosidase, fructose-1,6-bisphosphatase, triosephosphate isomerase, phosphoglucomutase, fructose-1,6-bisphosphate aldolase, and phosphoglycerate kinase. Concurrently, the synthesis and degradation of starch were inhibited, as indicated by the downregulation of α-glucan phosphorylase, α-amylase 3, and β-amylase 3 (Figure 3b). DEGs associated with photosynthesis were also downregulated, including the cytochrome b6 complex (Cytb6), plastocyanin, which is integral to the photosynthetic electron transport chain, and ferredoxin-NADP reductase. Similarly, the key enzyme responsible for ATP synthesis from ADP and inorganic phosphate (Pi), the F-type ATP synthase, was downregulated, while proteins involved in the stability and assembly of Photosystem I and Photosystem II were downregulated in response to foliar urea application (Figure 3c).

As a nitrogen source, urea directly influenced the synthesis pathways of secondary metabolites. Glutamine synthetase and ferredoxin-dependent glutamate synthase were downregulated, while 12 DEGs involved in antioxidant systems, including phenylalanine ammonia-lyase, peroxidase, and glutathione transferase, were upregulated, with phenylalanine ammonia-lyase showing the highest level of upregulation under urea treatment (Figure 3d). Furthermore, the DEGs associated with purine and pyrimidine metabolism pathways (which are crucial for nucleic acid synthesis), energy metabolism, antioxidant activity, and signal transduction were upregulated, including the following enzymes: phosphoribosylformylglycinamidine synthase, adenine phosphoribosyltransferase, guanosine deaminase, urate oxidase, nucleoside diphosphate kinase, invertase, aspartate carbamoyltransferase, nucleotide pyrophosphatase, and inositol-tetrakisphosphate 1-kinase. Of these, guanosine deaminase exhibited the highest fold-increase in expression (Figure 3e).

We randomly selected 11 differentially expressed genes (DEGs), identified by transcriptomic analysis, which were validated through qPCR (Figure 3f). The results demonstrated that the qPCR expression trends of these 11 genes were consistent with those observed from the RNA-sequencing analysis, thereby substantiating the reliability of the transcriptomic data.

3.4. Dynamic Model of Nitrogen Metabolism in Response to Foliar Application of Urea+

To further clarify the changes in response to the foliar application of urea to spring wheat in a high-altitude region of China, a potential dynamic model was constructed based on transcriptomic analysis (Figure 4a). After foliar urea application, urea rapidly decomposes in the leaf tissue, generating ammonia, which is then absorbed and engaged in nitrogen assimilation pathways. In response to application at the booting stage, wheat suppresses the expression of glutamine synthetase (GS2) genes (|log2 fold change (FC)| = −1.26), preventing excessive nitrogen consumption of energy. Concurrently, the elevated activity of glutamate dehydrogenase (GDH) facilitated the direct conversion of ammonia to glutamate via the dehydrogenase pathway. As nitrogen metabolism dynamically adjusts, multiple genes associated with purine nucleotide biosynthesis, degradation, and purine metabolite conversion are significantly upregulated (Figure S4), including inosine monophosphate synthase (|log2 FC| = 0.84), 5′-nucleotidase SurE-like gene (0.97), xanthine dehydrogenase (0.57), and uricase (1.21). In addition, genes involved in de novo pyrimidine nucleotide synthesis and metabolism, such as carbamoyl phosphate synthetase (0.54), uridine-5′-monophosphate synthase (0.56), nucleoside diphosphate kinase (0.82), and CTP synthase (0.81), are also significantly upregulated (Figure S5). This triggers an increase in purine/pyrimidine synthesis flux, providing a nitrogen source for nucleotide biosynthesis, promoting the biosynthesis and transformation of nucleotides, and supplying sufficient precursors for DNA and RNA synthesis, thereby supporting cell growth and division. Furthermore, the upregulation of the amino acid synthesis pathways achieves the synergy of nitrogen storage and osmotic regulation. For instance, the expression levels of aspartate aminotransferase (|log2 FC| = 0.52) and alanine aminotransferase (0.90) are elevated, facilitating the synthesis of aspartate and alanine, respectively (Figure S6). This appears to enhance cellular protein synthesis capacity while helping to maintain nitrogen balance and energy metabolism stability. These metabolic changes may ultimately contribute to improved wheat grain quality, associated with an increase in grain protein content (by approximately 6.8%). Subsequently, the activity of glutamine synthetase, as detected, exhibits dynamic fluctuations (Figure 4c): the urea-treated group shows slightly reduced activity compared to the control at 3 h but maintains elevated activity from 12 to 72 h. Both groups display synchronized oscillations—rising from 3 to 12 h, declining from 12 to 24 h, and rebounding from 24 to 36 h, followed by a continuous decline after 36 h. This indicates a 3 h lag phase in urea absorption, and the fluctuation in activity reflects the diurnal rhythm regulated by the photoperiod in the leaves (a 12 h cycle that synchronizes with light responses). The synchronized decline after 36 h suggests the initiation of metabolite feedback inhibition. This mechanism reveals the physiological basis by which foliar urea application optimizes nitrogen use efficiency and enhances seed protein accumulation.

4. Discussion

Foliar nitrogen application is a rapid and efficient fertilization method in crops [21]. Based on its excellent hygroscopic properties, urea is widely regarded as the preferred foliar nitrogen fertilizer [22]. The research on foliar urea application has been conducted on various crops, including maize [23], winter wheat [24], cotton [25], and fruits and vegetables [26,27]. However, most prior studies have primarily focused on the application effects, with limited research into the molecular mechanisms involved. Furthermore, studies on foliar nitrogen application in spring wheat regions of China have been relatively limited, leaving the effects and underlying mechanisms largely unexplored. Chen et al. demonstrated that post-anthesis spraying of a 2% urea solution could enhance nitrogen uptake and its redistribution to grains in winter wheat, thereby improving nitrogen-use efficiency [28]. In the current study, a foliar nitrogen application rate of 12.25 kg ha⁻¹ was employed, with a 2% urea solution sprayed on spring wheat during the booting stage, as the spike develops. The foliar application of urea significantly increased dry matter accumulation by 11.34% (p < 0.05) and markedly enhanced grain plumpness. Furthermore, the urea treatment significantly increased grain protein concentration by 6.8% (p < 0.05). These findings suggest that the foliar application of urea has a significant positive impact on wheat quality.

Previous studies have shown that the foliar application of urea can be detected by leaf nitrogen absorption within a few hours, with urea applied to maize leaves beginning to induce effects within 6 h [28]. In the present study, leaves absorbed urea within a short period of application (3 h) and increased nitrogen content by 3.37%, peaking at 12 h (at 56.69 g kg⁻¹). However, after 12 h, the nitrogen concentration in the urea-treated leaves began to decrease, although it remained significantly higher than that of the control group. This suggested that the absorption and utilization of nitrogen by the leaves is a dynamic process, during which the absorbed nitrogen is gradually redistributed to other tissues or utilized for metabolic activities. Compared with the control group, the nitrogen concentration in the leaves following foliar urea application consistently remained elevated. This increase in nitrogen concentration may positively influence wheat growth, photosynthetic efficiency, and other metabolic processes. Consequently, optimizing the timing and dosage of urea application could be a critical strategy for further improving wheat grain yield and quality.

Nitrogen is a crucial element for plant growth and development, playing a key role in protein synthesis processes [29]. Urea, as an efficient nitrogen source, can be rapidly absorbed by plants and converted into amino acids, thereby promoting protein synthesis [30]. The transcriptome analysis revealed that urea treatment significantly regulated dynamic changes in gene expression and metabolic pathways in leaves. Early (within 3 h of foliar urea application) responses primarily involved the rapid activation of photosynthesis, antioxidant mechanisms, and enzyme activities to cope with the rapid absorption and metabolism of nitrogen. After 24 h, changes in metabolic pathways were observed, primarily concerning adjustments in amino acid degradation, cell wall metabolism, and secondary metabolism. When urea is sprayed on leaves, urease in plant cells typically decomposes urea into ammonia (NH₃) and carbon dioxide (CO₂) [31]. Ammonia entering the cell is usually converted into glutamine (Gln) by glutamine synthetase (GS), a critical step in nitrogen metabolism in the roots of plants [32,33]. However, this study found that in the early stage after urea application (within 3 h), the activity of GS decreased and the expression of the GS2 gene was downregulated, indicating that GS2 is likely a negative regulator in foliar nitrogen response. This may be related to the rapid increase in nitrogen supply after foliar application of urea, where wheat initially reduces nitrogen assimilation by inhibiting the expression of the GS2 gene to avoid excessive energy consumption. At the same time, we found that although the downregulation of the GS2 gene hinders the traditional GS/GOGAT pathway, GDH (despite being a lncRNA) may activate alternative ammonium assimilation pathways by competitively binding regulatory elements or ceRNA mechanisms to facilitate the temporary transfer of ammonia. This mode is conserved with the response to nitrogen starvation recovery in rice: Shin et al., (2018) found that in rice, the downregulation of OsGS2 is accompanied by the upregulation of OsGDH2, but the regulation involves species-specific transcription factors such as the rice-specific OsNAC5, reflecting species-specific differences [34]. Notably, our study found that urea treatment inhibited the expression of genes related to starch synthesis and photosynthesis, indicating a preferential allocation of nitrogen to protein synthesis rather than energy storage. This finding may explain why a single application of urea at a rate of 12.25 kg ha⁻¹ during the booting stage led to a significant increase in protein concentration without a corresponding increase in grain weight. Additionally, it was found that this metabolic adjustment caused by nitrogen supply triggers an increase in the flux of purine/pyrimidine synthesis, providing a nitrogen source for nucleotide biosynthesis, and achieves coordinated nitrogen storage and osmotic regulation by upregulating the arginine/proline synthesis pathway. Ultimately, this leads to an increase in grain protein content. By 24 h, the chloroplastic GS2 is completely silenced, accompanied by a compensatory upregulation of the cytosolic glutamine synthetase gene, indicating a shift in the site of nitrogen assimilation from the chloroplast to the cytosol, forming a “two-stage regulatory model” —initially relying on GDH-mediated emergency metabolism and nucleotide synthesis, and later the sustained supply of glutamine through the cytosolic GS system, ultimately driving grain protein accumulation through increased amino acid flux, which may also be the reason why the amino acid content peaks at 24 h. In contrast to studies on barley, Wang et al. (2023) found that nitrogen-efficient barley varieties maintain nitrogen homeostasis by upregulating nitrogen transporter genes (such as AMT), while wheat relies more on the GDH bypass for foliar nitrogen application, reflecting the divergence in nitrogen utilization strategies among Poaceae crops [35]. These findings indicate that whereas urea treatment affects certain aspects of nitrogen metabolism, it effectively promotes wheat growth and improves grain quality through the coordination of metabolic pathways. These insights provide a crucial molecular basis and theoretical support for optimizing nitrogen management strategies.

In summary, this study revealed multiple mechanisms by which the foliar application of urea enhances spring wheat protein content through transcriptomic analysis. A single foliar application of urea at a nitrogen level of 12.25 kg ha⁻¹ during the booting stage significantly enhanced grain quality by regulating nitrogen metabolism, carbon metabolism, and antioxidant systems. This research provides new theoretical insights for the efficient utilization of urea in crop production and offers scientific guidance for optimizing fertilizer application strategies.

5. Conclusions

This study elucidates the molecular mechanisms by which the foliar application of urea enhances grain protein concentration and grain yield in spring wheat. A single urea application equivalent to 12.25 kg ha⁻¹ during the booting stage significantly increases vegetative dry matter accumulation, protein concentration, and total amino acid concentration. Initially, nitrogen assimilation is moderated by downregulating glutamine synthetase expression, prioritizing structural development and antioxidant mechanisms. Concurrently, accumulated nitrogen activates amino acid and nucleotide metabolism, notably upregulating the biosynthetic pathways of alanine, aspartate, arginine, and proline. Enhanced purine and pyrimidine metabolism facilitate DNA and RNA synthesis, ultimately boosting grain protein concentration. These findings provide a molecular foundation for optimizing foliar urea application in spring wheat production.