Nitrogen (N) is the most abundant mineral nutrient element in plants and plays an essential role in plant growth and development. Nitrate (NO₃^–-N) and ammonium (NH₄⁺-N) are predominant forms of inorganic N in soil, both of which can be absorbed by plants directly (Marschner, 2011). The preferred N form varies among plant species and among genotypes of individual plant species, environmental conditions, and developmental stages (Cramer and Lewis, 1993; Zhao et al., 2013). Maize usually grows in well-aerated dryland soils, where NO₃^– is the major source of N due to soil nitrification (Fang et al., 2006). Some studies showed the enhanced growth of maize under NO₃^– nutrition compared with NH₄⁺ nutrition (Cramer and Lewis, 1993; Schortemeyer et al., 1997). Others showed that NH₄⁺ uptake may be an important strategy for maize plants to take up sufficient quantities of N for growth compared with NO₃^– nutrition (Gu et al., 2013; George et al., 2016; Wang et al., 2019; Zhang H. Q. et al., 2019). For most plants, a mixture of both NO₃^– and NH₄⁺ is superior to either NO₃^– or NH₄⁺ sources alone, possibly due to improved cytokinin transportation from root to shoot, stimulated leaf growth, maintained rhizosphere pH, and increased nutrient uptake rates, such as N, phosphorus (P), copper (Cu), and iron (Fe) (Gentry and Below, 1993; Marschner, 2011; Cao et al., 2018; Wang et al., 2019).

Zinc (Zn) is an essential microelement for plant growth. Zinc plays a vital role in many essential cellular functions and metabolic pathways and is important to the normal health and reproduction of plants, animals, and humans (Welch and Graham, 2004). It is estimated that more than 30% of the agricultural soils in the world are prone to Zn deficiency (Alloway, 2008). According to a national soil survey, China has nearly 0.63 billion hm² of soil, accounting for 45.7% of the farmland, is subject to Zn deficiency, primarily calcareous soil in Northern China (Liu, 1996). Although Zn is an essential micronutrient for plant growth, Zn input has received much less attention than N, P, or irrigation during the Green Revolution (Tilman et al., 2002; Mueller et al., 2012). Therefore, the application of Zn fertilizers is necessary in such soils to ensure cereal yield and grain Zn concentration (Cakmak, 2008).

Maize is one of the world’s major crops and is expected to contribute increasingly to human and animal nutrition and to energy production. Maize is sensitive to Zn deficiency (Wang and Jin, 2007). Zn-deficient symptoms, such as stunted and chlorotic plants, are often observed in maize plants growing on calcareous soil in the field (Alloway, 2008). Many studies have demonstrated that grain yield of maize was increased by the application of Zn fertilizer to Zn-deficient soils. The positive effects of Zn supply are related to the increased photosynthesis rates, chlorophyll content in leaves, and increased kernel number and weight in the apical benefited from increased pollen viability at the tasseling stage under field conditions (Potarzycki, 2010; Wang et al., 2012; Liu et al., 2016, 2020).

Previous results from greenhouse conditions showed that increasing N supply did not improve Zn accumulation in grain and shoot of wheat in case of low soil Zn availability. Otherwise, increasing N supply substantially improved Zn concentration and accumulation of both grain and shoot in wheat when Zn supply was not limited, showing a synergistic effect of N fertilization on Zn accumulation (Kutman et al., 2010, 2011a,b). Furthermore, our results obtained from field conditions also showed positive effects of N application on the root uptake, root-to-shoot translocation, and remobilization of Zn in wheat and maize grown in Zn-sufficient soils (Xue et al., 2012, 2014, 2019). However, little information is available about the effects of different mineral N sources combined with the presence or absence of Zn on the growth of both shoot and root and nutrient uptake.

This study aimed to determine how the different N forms affected the growth of shoot and root, root morphological traits, and nutrient uptake of maize seedlings grown with and without Zn supply under greenhouse conditions. This information may be useful for enabling integrated N management of Zn-deficient and Zn-sufficient soils and increasing plant and grain production in the future.

Previous studies have shown that co-provision of NO₃^– and NH₄⁺ stimulates plant growth beyond that observed with either N source provided individually in several crops (Gentry and Below, 1993; Marschner, 2011; Hachiya and Sakakibara, 2017; Cao et al., 2018; Wang et al., 2019). Furthermore, fertilizer Zn supply also improves shoot biomass accumulation and the final grain yield of maize (Sarwar et al., 2017; Zhang J. Y. et al., 2019; Liu et al., 2020) and wheat (Liu et al., 2019). However, the interaction between N forms and Zn nutritional status on plant growth has received considerably less attention. In this study, our results suggested that N regimes (including N levels and N forms), Zn supply, and their interactions exerted a significant effect on the growth of maize seedlings.

The extent of the effects of Zn supply on the DW of both shoot and root and N uptake depended on N availability in the growth medium. When N was deficient in solution, Zn supply had little effects on the growth and N absorption of maize seedlings, in agreement with others (Kutman et al., 2011b). However, when N was applied at a recommended rate, the positive effects of Zn supply on the growth and N absorption of maize seedlings were dependent on N forms. Generally, the growth and N absorption of maize seedlings supplied with NH₄⁺ supply were superior to either NO₃^– or mixed-N nutrition under Zn-deficient conditions. However, the growth and N absorption of maize seedlings supplied with mixed-N supply were superior to independent NH₄⁺ and NO₃^– nutrition under Zn-sufficient conditions. Furthermore, the positive effects of mixed-N nutrition and Zn supply on the growth of maize seedlings were more than additive. For example, with zero Zn supply, NH₄⁺ supply resulted in 374.2% of increase in shoot DW, and with zero N supply, Zn supply increased the shoot DW by 22.4%, but the combined effects of mixed-N nutrition and Zn supply were over 700% (Table 1). Similar results were also found for N absorption in the shoot, root, and whole plant (Figure 4D). Similarly, a previous study also reported that the positive effects of high N and Zn supply on the grain yield of wheat were more than additive under greenhouse conditions (Kutman et al., 2011a). For example, at low Zn supply, high N supply resulted in a 73% increase in grain yield, and at low N supply, high Zn supply increased the grain yield by 35%, but the combined effect of high N–high Zn treatment was over 350%. Previous results obtained from greenhouse conditions showed Zn and N are synergistic in their effects on increasing plant Zn concentration, and their levels in growth medium should be at enough levels to achieve the synergistic effect of N on wheat root Zn uptake (Kutman et al., 2010, 2011a,b). Here, our results showed that compared with no N supply, a substantial increase in DW of shoot and root by N supply resulted in a significant decrease in Zn concentration (a so-called dilution effect) with the exception of Zn concentration in shoots of plants receiving both NH₄⁺ and Zn supply (Table 1 and Figure 4C). Finally, the total Zn accumulation, including shoot and root, was significantly increased by NH₄⁺ supply in the presentence of Zn supply, but not significantly affected by N rates and forms in the absence of Zn supply. These results suggested that the combined applications of NH₄⁺ and Zn fertilizers have beneficial effects on plant Zn nutrition. The NH₄⁺-induced acidification and a decline in apoplastic pH may play an important role in increasing Zn availability in solution for root uptake. The abundance of root Zn uptake transporters in the plasma membrane of root cells, including ZIPs such as IRT1 and other unknown proteins (Ishimaru et al., 2005; Palmer and Guerinot, 2009; Tiong et al., 2014), may also be enhanced by NH₄⁺ supply.

Zinc is an essential micro-nutrient for plants. As expected, Zn-deficient stress resulted in a 21.1–55.7% of decrease in shoot DW and 0.5–47.9% of decrease in root DW irrespective of N forms. These results revealed that shoots were more sensitive to Zn deficiency than the roots, in agreement with previous studies on Zn deficiency in maize (Wang and Jin, 2005; Zhang J. Y. et al., 2019). Zn deficiency causes visible symptoms when Zn content is below 15–20 mg kg^–1 in plants (Marschner, 2011; Zhang J. Y. et al., 2019). In this study, under Zn-deficient conditions, shoot Zn concentration significantly decreased from 14.6 mg kg^–1 to around 8.7 mg kg^–1 with N supply (Figure 4C). These values were below the critical Zn-deficient range of 15–20 mg kg^–1 (Marschner, 2011), indicating a Zn-deficient status although severe Zn-deficient symptoms did not occur in maize seedlings (Supplementary Figure 2). The reduced shoot Zn concentration by N supply without causing severe Zn-deficient symptoms in maize seedlings may also indicate that adequate N application improves Zn mobility and physiological availability at the cellular level by affecting the level of Zn-chelating compounds, such as amino acids, peptides, or nicotianamine (Kutman et al., 2010). Alternatively, Denghai605 may be a highly Zn-efficient cultivar with ZE values of shoot ad root being 53.9 and 69.5%. However, the corresponding ZE values of shoot and root were only 36.8 and 50.0% for Zhengdan958 grown in Zn-free nutrient solution, which showed severely Zn-deficient symptoms 15 days after treatment (Zhang J. Y. et al., 2019). According to the criteria suggested by Farquhar and Sharkey (1982), when the P_n decreases along with an increase in C_i, photosynthesis is mainly limited by non-stomatal factors. In this study, compared with Zn supply, Zn deficiency resulted in a slight decrease in P_n, whereas C_i was significantly increased with g_s and T_r not being affected regardless of N forms. Therefore, the reduction in P_n caused by Zn deficiency was mainly due to non-stomatal factors. The decreased P_n resulting from Zn deficiency may be due to the decline in the activities of carbonic anhydrase (Ohki, 1978), ribulose 1,5-bisphosphate carboxylase/oxygenase and fructose-1,6-bisphosphase (Hacisalihoglu, 2003; Marschner, 2011), and the dramatic damages of chloroplast structure and functions (Chen et al., 2010; Zhang J. Y. et al., 2019) as shown by the decreasing leaf SPAD values (Figure 1A).

Under Zn-deficient stress, the DW of both shoot and root was the highest with NH₄⁺ nutrition among the three N forms. Furthermore, ZE of both shoot and root was significantly higher with NH₄⁺ nutrition compared with NO₃^– and mixed-N supply (Table 1). These results suggested that NH₄⁺ nutrition enhanced the tolerance of maize seedlings to Zn-deficient stress. Similarly, other studies also showed the preference of growth and N uptake for NH₄⁺ over NO₃^– in maize (Gu et al., 2013; George et al., 2016; Zhang H. Q. et al., 2019). Several possible reasons are suggested for the growth preference of maize supplied by NH₄⁺ over either NO₃^– or mixed-N nutrition under Zn-deficient conditions. First, NH₄⁺ supply produced the highest FRL, MRL, and thick roots and finally resulted in the highest TRL and total root surface area among the three N forms in the absence of Zn supply (Figure 3). The root proliferation with NH₄⁺ supply was beneficial to promote the root nutrient uptake such as N, P, K, and especially Fe, Mn, Mg, and Ca accumulation (Figures 4, 6) and thus to improve crop growth. Similarly, a previous study also reported that the root density and extension of maize seedlings were greater in nutrient solutions containing NH₄⁺ than in those containing NO₃^– nutrition due to more rapid cell division in the root apical meristem of maize under NH₄⁺ nutrition (Bloom et al., 2002). Results from field experiments also showed that the localized application of NH₄-N + P significantly improved maize root biomass, the TRL, and lateral root proliferation at the seedling stage compared with localized application of NO₃-N + P and urea +P, which could greatly contribute to improved nutrient uptake and biomass accumulation, and thus improve grain yield of maize grown in the calcareous soil with a critical Zn deficiency (DTPA-extractable Zn was 0.65 mg kg^–1) (Ma et al., 2014). Furthermore, the NH₄⁺-induced acidification and a decline in apoplastic pH cannot be ruled out as playing a role in increasing nutrient availability (and hence uptake), especially for Fe, Mn, Mg, and Ca uptake even though the pH of the nutrient solution was maintained at 6.0. More importantly, the uptake and assimilation of NH₄⁺ by maize is a more energy-efficient process than that of NO₃^– because the reduction of each nitrate molecule to ammonium consumes about 15 ATP molecules, which are not consumed when ammonium is supplied (Salsac et al., 1987). Less energy is needed for roots to take up and assimilate NH₄⁺ may be more adaptive under Zn-deficient stress. Additionally, the leaf protein content with NH₄⁺ nutrition was significantly improved by Zn deficiency compared with Zn supply and was also slightly higher when compared to NO₃^– and mixed-N nutrition under Zn-deficient conditions (Figure 1B), which can prevent the dehydration of cells and enhanced the structure and function of cell membranes in adverse environments (Andrews et al., 2005; Cao et al., 2018), such as in Zn-deficient stress. Other studies reported that the supply of NH₄⁺ nutrition significantly enhanced the drought tolerance of rice seedlings compared with the application of NO₃^–, and this effect seems to be closely related to the larger root tips and surface area, higher chlorophyll and Rubisco contents, and higher Rubisco activity due to the higher distribution of N absorbed to Rubisco (Guo et al., 2007; Gao et al., 2010; Ding et al., 2015). This information may be useful for enabling integrated management of Zn-deficient soils and increasing plant and grain production in the future.

Under Zn-sufficient conditions, the DW of shoot, root, and whole plant tended to increase in the order NH₄⁺ < NO₃^– < mixed-N nutrition. Compared with NH₄⁺ nutrition alone, mixed-N supply resulted in a 27.4 and 28.1% increase in leaf P_n and g_s, which further resulted in a 35.7 and 33.5% increase in shoot C accumulation and shoot DW, respectively. Furthermore, mixed-N supply resulted in 19.7% of higher shoot C/N ratio vs. NH₄⁺ nutrition alone, which means a higher shoot biomass accumulation because of a significant positive correlation between shoot C/N ratio and shoot DW (Figure 5C). Additionally, compared with the mixed-N supply, the negative effect of NH₄⁺ nutrition on the root DW was larger than that on the TRL due to insufficient C supply from shoot to root, resulting in an overall increase in the specific root length (the ratio of TRL to root DW) (Table 1 and Figure 3A). Previous studies suggested that an increase in specific root length is an adaptive response to insufficient carbohydrate (Ostonen et al., 2007; Xie et al., 2007). Wang et al. (2019) also showed that compared with NH₄⁺ nutrition alone, mixed-N nutrition resulted in a significantly increased shoot and root biomass mainly due to increased shoot ATP content and greater leaf area with similar or even lower P_n under lower and higher planting densities. Other studies also reported that mixed-N nutrition tended to increase both the accumulation of whole-shoot DW and the proportion partitioned to reproductive tissues and finally to increase the grain yield of maize, compared to NO₃^– nutrition only (Gentry and Below, 1993). Another field experiment showed that maize grain and straw yields were highest when fertilized with calcium ammonium nitrate, followed by ammonium sulfate, whereas urea exhibited the lowest yields (Abbasi et al., 2013).

Irrespective of N forms, compared with Zn supply, Zn deficiency resulted in a substantial decrease in both shoot and root DW, with the magnitude of decrease consistently being the highest with mixed-N nutrition, followed by NO₃^– nutrition whereas NH₄⁺ nutrition exhibited the lowest decrease. Furthermore, ZE of both shoot and root was significantly higher with NH₄⁺ nutrition compared with NO₃^– and mixed-N supply. These results suggested that NH₄⁺ nutrition enhanced the tolerance of maize seedlings to Zn-deficient stress. Under Zn-sufficient conditions, the DW of the shoot, root, and whole plant tended to increase in the order NH₄⁺ < NO₃^– < mixed-N nutrition. Furthermore, the positive effects of mixed-N nutrition and Zn supply on the growth of maize seedlings were more than additive. The combined applications of mixed-N nutrition and Zn fertilizer can promote root growth (including the increased root DW, root length in different diameter, TRL, and total root surface area), synchronously improve root absorption capacity of N, Fe, Cu, Mn, Mg, and Ca, increase leaf SPAD values and photosynthetic rate, and thus maximize the aboveground plant dry matter accumulation. In the case of total Zn uptake, the positive effect of NH₄⁺ nutrition with Zn supply was more pronounced. This information may be useful for enabling integrated N management of Zn-deficient and Zn-sufficient soils and increasing plant and grain production in the future.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.

KL, ZL, and YX conceived and designed the experiments, analyzed the data, and wrote the manuscript. WY, YG, HZ, LJ, XQ, ZC, CZ, SL, and HW performed the experiments. All the 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.

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