The industrial nitrogen (N) fertilizers application has contributed tremendously to the increase in global food production (Gong et al., 2011; Zhang et al., 2013). However, less than half of the applied N (30–40%) is utilized by crops in China to date (Ding et al., 2020). The N lost included run-off, volatilization, leaching, and denitrification, which together lead to a range of major ecological challenges and environmental problems (Chen et al., 2014). Unlike nitrate nitrogen (NN), ammonium (NH₄⁺) nitrogen (AN) can be assimilated into amino acid production by plants without further chemical reduction (Coskun et al., 2017). The NH₄⁺-based stabilized fertilizers application was considered to be a promising future strategy for improving agronomic N use efficiency (Ren et al., 2021). Unfortunately, most dryland crops, including wheat, can be markedly inhibited when the concentration of ammonium in soil is too high, whereas the comparable inhibitory effect does not occur for NN fertilization (Miller and Cramer, 2005; Coskun et al., 2017). In southern China, especially in red-yellow soil areas, approach 20 mM NH₄⁺ concentration in soil was recorded (Xu et al., 2012), much higher than the 0.1–0.5 mM optimal NH₄⁺ concentration for upland crops (Liu et al., 2013).

The membrane potential would be depolarized during the NH₄⁺ uptake and assimilation process, resulting in an increase in the net proton release, as well as external soil acidification (Zhu et al., 2012). Over the last few decades, the Ca²⁺, Mg²⁺, and K⁺ depletion and sulfur (S) and N atmospheric deposition have promoted soil acidification and reduced the soil’s buffering capacity, particularly in the red-yellow soil of southern China, which is located in tropical/subtropical regions; in this region, an average soil pH of 4.5–5.5 has been recorded (Guo et al., 2010). Soil acidification also produces adverse effects on cell metabolism (Van Den Berg et al., 2005). NH₄⁺ was assimilated into organic N compounds in cells primarily through the GS/GOGAT pathway (glutamine synthetase, GS, EC 6.3.1.2; glutamate synthase, GOGAT, EC 1.4.1.14) (El Omari et al., 2010; Xu et al., 2012). Plant species maintaining a low intracellular with higher GS activity are thought to be more tolerant to NH₄⁺ toxicity (Cruz et al., 2006). In addition, the increased carbon consumption caused by increased NH₄⁺ assimilation in roots also partially explain NH₄⁺-induced growth inhibition (Britto and Kronzucker, 2002). However, few studies have examined the relationship between NH₄⁺ stress and soil acidification or whether the NH₄⁺-induced growth inhibition is caused by an NH₄⁺ form source or soil acidification caused by NH₄⁺ absorption.

The understanding of plant growth in response to NH₄⁺ tolerance is mainly based on studies of mutants and transformants or some model plants such as rice, Arabidopsis et al. (Guo et al., 2007; Zhu et al., 2009; Li et al., 2010). Wheat is one of the most widely cultivated crops in the world, which is sensitive to NH₄⁺ toxicity (Wang et al., 2019). In many cases, wheat have to face excess NH₄⁺ in China, which is often the case in the field with the use of slow-release fertilizers and nitrification inhibitors, especially during fertilization stag. It is a very important and necessary object to study genetic variability of wheat adaptation to NH₄⁺ toxicity combined with soil acidification. Its ecotypes have been found show a wide range of habitats differing notably in soil richness. These natural variations indeed provide a framework to study the adaptation of different traits that define plant growth in relation to simultaneous genetic changes and environmental variations, while there is lack of studies on this topic In this study, we conducted hydroponic experiments using 31 wheat cultivars in media with different N forms (AN or NN) and pH values. The objectives of this study were to evaluate the inhibitory degree of NH₄⁺ form source and soil acidification on crop growth. The results would provide useful insight into the selection and breeding of crop cultivars with greater potential to adapt to NH₄⁺-form N source combined with soil acidification in the field.

Several studies have provided a general description of plant responses to changes in the AN environment (Britto and Kronzucker, 2013; Wang et al., 2019); however, most of them have considered only a small number of genotypes, in which the effects of soil acidification caused by NH₄⁺ absorption is neglected, thus only a partial view of the phenomena given. Since wheat ecotypes have been found in a wide range of habitats differing notably in NH₄⁺ conditions in our previous studies (Wang et al., 2016, 2019, 2020), few studies have reported that using this natural variation among ecotypes as an alternative way to search genes related to NH₄⁺ tolerance. In the present studies, the responses of wheat to different N forms and pH values was studied by measuring morphological traits and metabolic traits in 31 wheat genotypes. Result showed that cultivar variance explained the largest proportion of total variance in shoot dry weight, plant dry weight, leaf area, nodal root number, total root surface area, total root volume and the free NH₄⁺ concentration in shoot when wheat seedlings were planted in the solution as NN-6.5, AN-6.5, NN-5.0 or AN-5.0. Previous studies found wheat was sensitive to increased NH₄⁺ concentration in soil (Cao et al., 2009; Ariz et al., 2010; Zhu et al., 2012; Setien et al., 2013), furtherly, the present results clearly indicated that NH₄⁺ nutrition and soil acidification significantly reduced biomass accumulation and leaf area in wheat plants. The dry weight of roots was considerably reduced by AN treatment, not only due to limitations in root emergence but also because of a reduction in root length (reduced by 22.3 and 21.7% in maximum seminal root length and nodal root length, respectively). This NH₄⁺-induced inhibition is related to root growth and development also has been observed by some previous studies (Cramer and Lewis, 1993; Britto and Kronzucker, 2002; Zhang and Wang, 2012). Notably, in the present study, the decline was even greater at lower pH levels under AN conditions, but N form still accounted for the largest proportion of variance in root length (Table 2). Li et al. (2010, 2013) found that nearly 70% of the NH₄⁺-induced root elongation inhibition was accounted by the cell expansion reduction, which root elongation and development, was mediated by reduced auxin by NH₄⁺ condition (Li et al., 2011). In the present study, the nodal root number was maintained or increased under AN but decreased at lower pH. The nodal root number was more strongly affected by variation in pH than by variation in N form (Table 2), which might be explanted by a higher accumulation of cytokinin in NH₄⁺-fed roots (Wang et al., 2019) in our previous study.

As a typical symptom of “ammoniacal syndrome,” free NH₄⁺ concentration in plant cells was significantly higher under AN conditions than under NN. This may be associated with the ability of root system to absorb NH₄⁺ (Zhu et al., 2009), the rate of NH₄⁺ efflux from root inside (Li et al., 2011), and also NH₄⁺ assimilation in root cell (Setien et al., 2013). The free NH₄⁺ concentration increased under AN condition but decreased with lower pH in roots, but N forms was the crucial factor as Table 4 shown. The uptake of NH₄⁺ depolarizes the plasma membrane, increasing the net proton (H⁺) release from the cell inside to rhizosphere soil (Zhu et al., 2009), furtherly leading to rhizosphere acidification. The H⁺-ATPase in plasma membrane made a decisive contribution to maintain the balance of pH inside and outside cells, which hydrolyzed ATP to pump H⁺ out; and an electrochemical gradient generated which could drive the free NH₄⁺ ions active transport crossing the plasma membrane (Zhu et al., 2012). At a low medium root pH, the activity of H⁺-ATPase must be smartly enhanced to pump H⁺ outside to adapt to an increased electrochemical gradient, to ensure NH₄⁺ uptake (Zhu et al., 2009). Therefore, the low medium pH may inhibit NH₄⁺ absorption while high pH may facilitate NH₄⁺ uptake (Brix et al., 2002). Our results also showed that, in wheat root, the N form was the main reason for the changes in the free NH₄⁺ concentration which was consistent with the previous studies; however, interestingly, free NH₄⁺ in wheat shoot varied most by cultivar, which had not been reported before.

Apart from the ability of plant root to absorb NH₄⁺, the allocation of NH₄⁺ also depends on the N metabolism process. NH₄⁺ absorbed by roots is mostly assimilated into amino acids in root cells (Masclaux-Daubresse et al., 2010). In the present study, AN resulted in a remarkedly increase in the free amino acid concentration in shoots and roots, confirming a metabolic adaptation to AN condition, as previously discussed by Lothier et al. (2011). The enhanced N compound pool was probably due to both high NH₄⁺ absorption and increased N assimilation to cope with having solely AN to maintain plant growth (Wang et al., 2020). The negative correlation between free amino acid concentration and soluble sugar concentration in roots and shoots was detected, and AN markedly reduced the soluble sugar/free amino acid ratio, indicating that more N-assimilation occurred in roots. Thus, a sufficient carbon skeleton supply for NH₄⁺ assimilation is required under AN condition. A higher photosynthetic capacity increases plant NH₄⁺ assimilation and tolerance, providing C skeletons to fuel the cell respiratory action and translocate more carbohydrates downward to maintain root growth (Ariz et al., 2011). In the present study, a distinct decrease in soluble sugar concentration accrued under AN condition (as the carbon skeleton for NH₄⁺ assimilation) in 31 wheat cultivars, particularly in the roots (Figure 3); however, this decrease was lower at pH 6.5 than at pH 5.0, possibly due to the higher photosynthetic capacity at pH 6.5 (Ariz et al., 2011, 2013), and also the inhibited capacity of NH₄⁺ assimilation. If the capacity of NH₄⁺ assimilation is depressed by low pH, more NH₄⁺ may accumulate in root and then be transported upward to the shoot, which is proven to be more sensitive to NH₄⁺ stress (Britto and Kronzucker, 2002; Cruz et al., 2006); this leads to inhibition of photosynthesis and CO₂ fixation (Guo et al., 2007), resulting in a further reduction in C assimilation.

Some studies have found that the relative assimilation enzyme activity in the GS/GOGAT cycle plays an important role for crops in adaptation to AN condition (El Omari et al., 2010). In the present study, GS activity was much increased, but NR activity was decreased by AN (Figure 4); pH level had the strongest effect on shoot GS activity and N form had the greatest effect on root GS activity. In different tissues of cells, GS exists as isoforms GS1 (found in the cell cytosol) and GS2 (in plastids) (Singh and Ghosh, 2013). GS1 is the predominant enzyme in non-photosynthetic tissues or root, but slightly presents in green tissues like young stem or leaf (Simon and Sengupta-Gopalan, 2010), which metabolic role includes assimilating NH₄⁺ into amino acids (glutamine) for N transport and distribution (Bernard et al., 2008). AN treatment increased the GS activity, especially the root GS activity (the predominant isoform GS1). The increased capacity of NH₄⁺assimilation by GS1 could prevent NH₄⁺ accumulation in the roots and transport to the shoots (Setien et al., 2013), but this higher GS activity was not inhibited by the low medium pH significantly, indicating GS1 was insensitive to low pH. GS2 played important roles in NH₄⁺ re-assimilation during photorespiration and the part of NH₄⁺, which was derived from nitrate reduction (Singh and Ghosh, 2013), mostly located in the chloroplasts stroma, and its physiological importance has been demonstrated in transgenic plants overexpressing GS2 in leaves (Bernard et al., 2008). Increased GS2 activity had an enhanced capacity for photorespiration, also retained more than 90% of their photosystem II activity to fight against osmotic stress (Bernard and Habash, 2009). In the present study, a relative increase in shoot GS activity (GS2) may maintain photorespiratory capacity while improving the cultivar’s tolerance to NH₄⁺ stress, but unlike GS1, its activity decreased sharply under low pH, and pH level had the strongest effect on it, indicating GS2 was more susceptible to soil acidification stress.

In conclusion, we studied the variation in N assimilation capacity of 31 wheat seedlings with respect to plant resistance to NH₄⁺ stress and rhizosphere acidification (the model as shown in Figure 5). The growth of wheat seedling was seriously inhibited by AN, and this inhibitory effect was enhanced by soil acidification stress. Under AN, crops accumulated more NH₄⁺ in tissues and more efficiently converted NH₄⁺ into amino acids, and the N form accounted for the largest proportion of the variance in this process compared to the cultivar and pH level in root, but pH level had the strongest effect in shoot. To select and breed crop cultivars with a greater capacity to adapt to NH₄⁺-N nutrition combined with soil acidification stress in the future, a higher capacity for N assimilation in both shoot and root and the ability to maintain high levels of C, must be a key focus.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

FW: experiments implementation, writing–original draft, resources, data curation, methodology, formal analysis, and visualization. JM: supervision and funding acquisition. QW, QY, JYe, JG, and HL: writing–original draft, data curation, methodology, formal analysis, and visualization. JYo and XH: conceptualization and writing–review and editing. YY, XL, and HK: investigation. All authors contributed to the article and approved the submitted version.

We acknowledge generous financial support from the “Zhejiang Provincial Key Research and Development Program” (2020C02030 and 2021C02035); Project of leading talents of scientific and technological innovation in Zhejiang Province (2021R52045); Construction Project of Agricultural Green Development Pilot Support System of Huangyan District (HY202001); and Project of Unification of Water and Soil Science for One Healthy funded by the Zhejiang Academy of Agricultural Sciences.

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.

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