Nitrogen (N) is the most important nutrient for plant growth, productivity, and grain quality (Hussain et al., 2020). Optimal use of N fertilizers is essential to improve yield, increase profitability, and minimize environmental consequences from nitrate leaching and nitrous oxide (N₂O, a greenhouse gas) emissions from denitrification by soil bacteria (Lazcano et al., 2021). The global use of N fertilizer is expected to reach 240 million tons by 2050 (Zhou et al., 2018; Randive et al., 2021). However, plants only use about 40% of the fertilizer N, with the rest escaping into the atmosphere as nitrogenous emissions or getting into groundwater via leaching (Nations, 2015).

During crop growth, the plant remobilizes a part of the stored N from the vegetative stem and leaves to reproductive organs such as panicles and grains (Yan et al., 2018; Broberg et al., 2021). N remobilization is genetically controlled (Alpuerto et al., 2021) but could also be influenced by environmental factors (Zheng et al., 2020) and N fertilizer application. The accumulation and relocation of N are considered very significant processes in determining grain yield and grain quality (Gaju et al., 2011; Prey et al., 2019).

The use of ¹⁵N stable isotope labeling is an excellent method that enables a less biased and more precise estimation of N uptake after anthesis and remobilization of N accumulated during the pre-anthesis period in various organs. N isotope discrimination is controlled by the link between N supply and demand when the N environment is held at a steady-state similar to how carbon isotopes are discriminated during photosynthesis (Dong et al., 2017; Hu and Guy, 2020; Qian et al., 2021). The employment of specialized methodologies, especially for tracking aboveground N dynamics in plants, is required for the precise quantification of N cycling in plant-soil systems (Shan et al., 2012; Meng et al., 2017; Zheng et al., 2018; Xu et al., 2019). Fertilizers can be isotopically labeled with ¹⁵N to follow its evolution movement in soil-plant systems. Under field conditions, labeling with ¹⁵N shows promise in analyzing N dynamics from crop residues, which are influenced by the chemical composition and quantity of the crop residue (Berg et al., 1991; Tahir et al., 2018). Handley et al. (2020) defined nitrogen fluxes and ¹⁵N remobilization during plant development. During the post-anthesis period, the partitioning of the ¹⁵N assimilated during the pre-anthesis period and remobilized after showed that leaves, stalks, ear, chaff, and sheath serve successively as N sinks and sources. Along with physiological data obtained from model plants (Masclaux-Daubresse and Chardon, 2011; Hawkesford, 2017), N remobilization was shown to be involved in N fluxes from leaf to leaf during the vegetative phase (Wendler, 2018) and from leaves to grains during the reproductive phase. Maintaining yield and profit while minimizing environmental effects requires better fertilizer N management that harmonizes N supply with crop demand (Cui et al., 2010).

Studies have shown that there is a possible interaction between grain isotope discrimination in the aboveground dry matter and grain yield; yet studies on this interaction are few, especially during the crop’s main growth stages (Liang et al., 2013; Chen et al., 2016; Sun et al., 2019). As a result, further information about N isotope discrimination during N uptake and absorption is needed to effectively track the remobilization of N that had been accumulated during the early crop growth stages and the post-anthesis phase under field conditions. However, little has been done in terms of using ¹⁵N tracing under field conditions to study the effect of N fertilizers on N accumulation and partitioning in spring wheat in the study area. Fewer research efforts have been conducted on how N fertilization affects plant ¹⁵N (Wang et al., 2016), with previous studies yielding contradicting results (Khalil et al., 2020; Agisho and Hairat, 2021) along with the comparison of N sources being emphasized more frequently (Asif et al., 2020). We hypothesized that the long-term application of inorganic nitrogen fertilizers would impact the relationship between N supply and crop N uptake, and thereby N use efficiency and grain yield. To test this hypothesis, we conducted a study with ¹⁵N-labeled fertilizer applied to microplots within a long-term trial with five N fertilization rates to study N remobilization, uptake, use efficiency, yield, and post-anthesis losses in spring wheat under rainfed conditions in the semi-arid loess plateau. For determining N remobilization, ¹⁵N labeling has proven to be a reliable and quantitative method.

The N distribution in different plant parts, grain yield as well as N uptake of spring wheat fertilized with different amounts of N were studied in this work. Application of N fertilizer increased the ¹⁵N content of the various aboveground component of spring wheat cultivar under field growing conditions. During the grain filling stage and maturity, there were considerable differences in ¹⁵N excess among wheat aboveground components; the most significant difference, between N1 and N5, was 57.67% for upper leaf and 34.29% for grain (Figure 2). These findings imply that N fertilization was more important for grain filling in wheat plants during the grain filling stage. Our findings are consistent with that of Ba et al. (2020) who reported that the redistribution of labeled N from source organs (flag leaves and stem) to sink organs (ears) was more pronounced in high N wheat plants. These differences were caused by the fact that N remobilization begins earlier in plants under low N fertilization conditions than it does under high N fertilization conditions (Aranjuelo et al., 2013). In spring wheat, the ¹⁵N enrichment of shoots and ears varied according to developmental stages.

In our present study, when compared to other aboveground components, spring wheat ears accumulate more ¹⁵N during the grain filling stage (14 DAA) (Figure 2G). The concentration of ¹⁵N in the ear did not decrease much with time (14 DAA-maturity) as it did in stem and leaves, except under N1 and N2 (Figure 2). Similar effects have been observed in sorghum (He et al., 2022). The ears serve as a strong N sink, impacting N accumulation in the ears throughout grain filling, whereas the shoots provide nutrients to the ears (Aranjuelo et al., 2013; Zhou et al., 2016; Sun et al., 2018).

In our study, the grain yield of the spring wheat cultivar was 11.52–24.13% greater with the N treatment (N2–N5) than with the control (N1). N treatments, on the other hand, had no significant effect on grain yield (Figure 3). When the N rate was increased from 105 to 210 kg ha^–1, wheat grain yield did not differ significantly (Figure 3). This could be explained by the fact that the study site has been fertilized continuously for a long time. In some studies, N fertilizer application has proven to boost crop yield (Abad et al., 2004), while excessive and long-term N fertilizer use has resulted in yield reduction (Garrido-Lestache et al., 2005; Wang et al., 2011; Ierna et al., 2015; Agegnehu et al., 2016).

Since water availability is the major factor limiting grain yield, the overall biomass yield in 2020 was higher than in 2021, a result that was linked to less rainfall throughout the growing period in 2021 (Figure 1; Selles and Zentner, 2001). The current study found that increasing the N rate enhanced grain, straw, and aboveground biomass of spring wheat (P < 0.001), as found by other researchers (Wang et al., 2010; Zhao et al., 2014). The spring wheat cultivar had a lower HI in the control. However, there were no significant differences between the N treatments. This may be attributed to the fact that increased N-rate-enhanced wheat development in the early stages than the control treatment (Mahler et al., 1994). Our results appeared to agree with those of Mahjourimajd et al. (2016), who observed that a different N rate supply did not influence HI in an Australian wheat mapping population.

Increasing N application rates improved both grain (17.91–19.66 g kg^–1) and straw (3.12–5.29 g kg^–1) N concentration of spring wheat in our present field experiment, with the best effect observed in N3 in both growing seasons (Table 3). Chen et al. (2016) reported a similar pattern that while increasing the N application rate from 60 to 240 kg ha^–1, the N concentration increased from 2 to 4 and 15 to 22 g kg^–1 for straw and grain, respectively. Post-anthesis nitrogen uptake is an important parameter for identifying higher-yield wheat varieties (Monaghan et al., 2001), which allowed researchers to distinguish the contrasting behavior between wheat varieties under varying N supply. Results from our study showed that the N fertilizer rate had a significant effect on N uptake by grain and straw. This may be attributed to enhanced biomass yield and N concentration of the N treatments when compared to the control (Table 3). The lower NUE values recorded at maturity (Table 2) in our present study is an indication that excessive and continuous application of N fertilizer does not only promote vegetative growth but also reduces plants’ ability to utilize nutrients under field conditions. According to the “law of diminishing returns,” a high N application rate indicates a low NUE under normal conditions. Crop N uptake, in addition to fertilizer N rate, is another direct factor that affects NUE (Zhang et al., 2021).

Our findings revealed that, when the rate of applied N increased, the amount of early stored ¹⁵N remobilized to both ears and grain increased as also observed by previous research efforts (Palta and Fillery, 1995; Zhou et al., 2018). They suggested that the rate of applied N increased the demand for early stored N, probably because the increased early availability of N increased the size of the sink. The rate at which N was applied affected the loss of pre-anthesis stored nitrogen during post-anthesis. Between 31 and 38% of the ¹⁵N in the crop at the grain filling stage (14 DAA) had been lost by maturity at N1–N5, and the entire excess N that remained in the crop was remobilized to the grain, resulting in an absolute increase in pre-anthesis stored N transferred to the grain. Another important finding of this study was that increasing the rate of applied N increased post-anthesis N losses in spring wheat (Table 4). However, N3 compared to the other treatments (N2, N4, and N5) recorded the least N loss, indicating that at this rate the plant can remobilize a greater portion of the applied N (Table 5). Shivay et al. (2020) observed that N application rate and year have an effect on N losses in spring wheat and suggested ammonia volatilization from the aerial parts of the plants as the major source. Higher N losses from the plant are attributable to higher N expenditure during the remobilization process (O’Deen, 1989; Reining et al., 1995; Sra et al., 2004). Losses from the crop need to be explored further, and attention must be drawn to the crop as more than simply a sink for N. The N recovery for wheat in the Middle and Lower Yangtze River Region was reported to be between 33.0 and 49.0% by some researchers (Zhao et al., 2009; Shi et al., 2012). Xiao-Tang et al. (2007) found that the N recovery in wheat was 54% at 120 kg N ha^–1 and 32% at 360 kg N ha^–1, and it is affected by wheat cultivars, soil fertility, and climate conditions (Wang et al., 2011), a result that is similar to our present study.

In conclusion, this study found that NUE strength in the study area is relatively low and that it has to be improved in spring wheat cultivation by using appropriate fertilization strategies. Our results showed that soil with low fertility (N1 and N2) treatments had the lowest grain yield and straw and grain dry matter which is an indication that N fertilization affects yield and its attributes. Long-term and excessive N application (210 kg ha^–1) reduces grain yield and increases N loss in a wheat crop under the conditions of this study. The upper portions of leaves and stem were more strongly enriched with ¹⁵N than the lower (aged) ones at both 14 DAA and maturity which is an indication that the upper portion of the leaves, in particular, maybe a great sink and storage for N in the plant during development, as well as a major sink for N freshly taken up post-anthesis. The importance of the ear as a sink for nitrogen is shown by the fact that a greater percentage of total plant nitrogen at 14 DAA was found in the ear, which accounted for a chunk of total dry matter. The results from this study offer useful insights for N application in spring-cultivated wheat in an attempt to reduce the quantity of N fertilizer inputs, thereby costs, and in producing wheat in an environmentally sustainable manner.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

LL: funding acquisition, conceptualization, and supervision. JW: resources and project administration. ZE: investigation and writing—original draft. JX: methodology. ZE, JW, and MZ: data collection. ZE, SB, and BK: formal analysis. LL, BK, and JP: writing—review and editing. All authors read and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.