Ammonium (NH⁺₄) is an important source of inorganic N and can be used by plants as the sole N source. However, high levels of ambient NH⁺₄ can be toxic to plant growth, resulting in many symptoms, including stunted root growth, yield reduction and leaf chlorosis (Britto and Kronzucker, 2002; Balkos et al., 2010; Li et al., 2010; Ariz et al., 2011; Chen et al., 2013). In rice (Oryza sativa L.), the anatomical traits of the culm are altered when high N is provided, leading to a reduction in culm mechanical strength and an increase in lodging scores (Yang et al., 2009). When larger quantities of urea (transformed to NH⁺₄ through urease hydrolysis) are supplied, the efficiency of silicon in imparting rigidity in rice plants at low N doses is greatly reduced (Idris et al., 1975). In cereal crop production, lodging resulting from low mechanical strength severely damages the vascular bundles (Kashiwagi et al., 2008), thereby affecting the transport of water, nutrients and reserves contained in vegetative organs to the developing grain and decreasing grain yield and quality. Little information is currently available concerning the effects of NH⁺₄ on culm mechanical strength and reserve transport in wheat. However, field observations and experience show that when wheat is grown at a high N rate, there appears to be an increased occurrence of lodging compared with growth at a moderate N rate, indicating that culm strength is low under high N levels. In Arabidopsis thaliana, when excessive N is supplied, the N concentration in the biomass increases significantly, whereas the N remobilization efficiency (NRE) decreases compared with moderate N application (Masclaux-Daubresse and Chardon, 2011). In a previous study, we found that application of excessive urea decreases the export of flag leaf-stored protein to the developing grains (Kong et al., 2012). Therefore, improving the NRE is a good strategy for achieving a high grain yield and crop quality under high N conditions.

The majority of studies investigating the toxicity of NH⁺₄ at high concentrations have been associated with NH⁺₄ assimilation and ion imbalances due to the decreased uptake of essential cations, such as K⁺, Mg²⁺ and Ca²⁺ (Barker et al., 1967; Roosta and Schjoerring, 2008; ten Hoopen et al., 2010), and they are often associated with the availability of K⁺ in particular (Yang et al., 2009). Because NH⁺₄ can be transported through plant K⁺ transporters and channels, NH⁺₄ toxicity may be attributed to unregulated NH⁺₄ uptake via these transporters, especially at low K⁺ levels (ten Hoopen et al., 2010). It was recently proposed that NH⁺₄ toxicity in NH⁺₄-fed plants originates from NH₃ uptake by plants through one component of the low-affinity transport system (LATS) for NH⁺₄ and from interference with K⁺ transport through the second component (Ariz et al., 2011). In Arabidopsis, stimulation of the NH⁺₄ efflux in the elongation zone following treatment with elevated NH⁺₄ is linked to root growth inhibition by NH⁺₄ (Li et al., 2010). The NH⁺₄ efflux significantly enhances futile and energy-costly NH⁺₄ cycling at the plasma membrane in rice (Chen et al., 2013). Although a variety of hypotheses have been proposed to explain the mechanisms underlying NH⁺₄ toxicity, no single convincing mechanism has yet been able to fully account for this toxicity (Roosta and Schjoerring, 2008; Chen et al., 2013).

Under field conditions, we have often observed the adverse effects of high NH⁺₄ provision on wheat. Therefore, in this study, experiments were conducted in both the field and laboratory to examine the cellulose and lignin contents of the vascular bundle, culm mechanical strength and the NRE under treatment with moderate or high NH⁺₄, or with high NH⁺₄ combined with an elevated K⁺ supply. The main objectives of this study were to investigate the effects of excessive external NH⁺₄ on wheat growth and to determine whether elevated K⁺ concentrations can alleviate these adverse effects of high NH⁺₄.

Previous studies have shown that high N application leads to thinner stems and significant increases in lodging, the lodging angle and the lodging score in wheat (Tripathi et al., 2003) and rice (Yang et al., 2009). Under a high N level (240 kg ha⁻¹, supplied as urea), the culm and root strengths of wheat were found to be 20% and 17% weaker, respectively, compared with the application of 160 kg N ha⁻¹ (Crook and Ennos, 1995), suggesting that high N application led to a lower culm strength and an increased occurrence of lodging. However, these investigations did not provide information explaining the mechanisms underlying these results. Because the basal part of the culm plays an important role in lodging resistance, as it provides a lever to hold the plant upright (Neenan and Spencer-Smith, 1975), and because culm breakage usually occurs at lower internodes (Kashiwagi et al., 2008), we determined the culm mechanical strength of the basal second internode. The results showed that the application of high NH⁺₄ significantly decreased culm mechanical strength.

Nitrogen and potassium are two macroelements that are essential for plant growth. Although plants exhibit a wide variety of transport systems for the acquisition of these elements, competition for uptake between plants still exists and has become one of the main topics of studies conducted by biologists and agronomists. Rice growth is negatively affected by elevated NH⁺₄, particularly under low K⁺ levels, and NH⁺₄ toxicity could be relieved by elevated K⁺ (Balkos et al., 2010). Similar findings have been reported in Arabidopsis (Li et al., 2010). Therefore, we expected that elevated K⁺ could compensate for the reduction in culm mechanical strength observed under high NH⁺₄ conditions. Indeed, in this study, we found that K⁺_add alleviated the negative effects of high NH⁺₄ on culm strength (Figure 1 and Figure S1). The data provided herein suggest that the plant K⁺ status might be involved in the high NH⁺₄-induced reduction in culm mechanical strength. These results are highly consistent with those of previous studies. In a field trial with maize (Zea mays L.), the application of K⁺ was shown to significantly affect stem strength and stalk breakage (Melis and Farina, 1984). In rice, the K⁺ culm content is closely correlated with culm mechanical strength because proper K⁺ nutrition is associated with the lignification of sclerenchyma cells and vascular bundles, thereby strengthening the culms and increasing lodging resistance (De Datta and Mikkelsen, 1985; Zhang et al., 2010). To date, there is no any direct evidence to support that K⁺ strengthens cellulose and lignin deposition. However, it is widely accepted that K⁺ play a key role in photosynthesis and metabolism of the resulting carbohydrates in plants (White and Karley, 2010; Hafsi et al., 2014). Considering that both cellulose and lignin are carbohydrates or it's derivate and several pieces of indirect evidence presented herein are supportive, we could postulate that K⁺ is involved in the cellulose and lignin deposition and thus in culm mechanical strength.

Mechanical strength is largely dependent on the chemical and biochemical components of the cell wall (Kashiwagi and Ishimaru, 2004; Kashiwagi et al., 2008). Generally, lignin and cellulose, which are the main biochemical components of plant tissues, particularly in the vascular bundles, are closely associated with culm mechanical strength (Yang et al., 2009). Cellulose usually constitutes 20–30% or 40–90% of the dry weight of primary or secondary walls, respectively, varying with the cell type (Taylor et al., 1999). Moreover, lignin can be incorporated into the cell wall to enhance its mechanical strength. In the rice mutant brittleculm1 (bc1), altered biosynthesis of cellulose, hemicellulose and lignin in the culms reduces secondary cell wall thickness and mechanical strength (Li et al., 2003). In wheat, the expression of COMT, a gene involved in lignin biosynthesis in the developing culm, is associated with culm rigidity and lodging traits (Ma et al., 2002). The TaCAD1 gene is also responsible for lignin synthesis, and the roles of lignin in maintaining stem strength and lodging resistance were further confirmed in maize (Halpin et al., 1998) and sorghum (Sorghum vulgare Pers.) (Sattler et al., 2009) using CAD mutants. In the present study, through FTIR, histochemistry and Image-Pro Plus software analyses, we found that the application of high NH⁺₄ decreased the cellulose and lignin contents in the vascular bundles of the second internode (Figures 2–4). In maize, K⁺ stimulates rapid expression of phenylalanine ammonia-lyase and enhances the activities of tyrosine ammonia-lyase, cinnamyl alcohol dehydrogenase and phenoloxidase, thereby increasing lignin biosynthesis (Liu et al., 2007). Considering that culm strength is correlated with the contents of cellulose and lignin and, more importantly, with K⁺ status, we speculate that an elevated K⁺ supply likely alleviates the negative effect of NH⁺₄ on the deposition of cellulose and lignin in vascular bundles. Indeed, using FTIR and histochemistry, we observed that the K⁺_add treatment increased the contents of both of these cell wall components (Figures 2–4).

It is widely known that as major components, cellulose and lignin are mainly deposited in the walls of certain specialized cells, such as the tracheary elements, sclerenchyma and phloem fibers (also shown in the present study; Figures 2, 3). Considering that cellulose and lignin impart rigidity and structural support to the wall and strongly assist in the transport of water and nutrients within xylem tissue by decreasing the permeability of the cell wall (Ma et al., 2002), we decided to investigate the transport of shoot reserves to developing organs. In this study, we found that high NH⁺₄ nutrition influenced N transport from vegetative organs to the developing grains (Table 1) and decreased the grain-filling rate (Table 2). Given that carbohydrates account for the majority of the wheat grain composition and that more significant differences in the grain-filling rate than in the NRE are observed between different treatments (Tables 1, 2), the decrease in the grain-filling rate may suggest that the nutrient translocation efficiency is not only determined by the lower permeability of the cell wall, but by the tissue C/N balance as well. Interestingly, elevated K⁺ also alleviates the negative effects of high NH⁺₄ on N remobilization and grain filling. These findings are consistent with previous studies in Arabidopsis thaliana in which the NRE was shown to be greater under low N than under a high N supply (Masclaux-Daubresse and Chardon, 2011), and the toxicity of high NH⁺₄ can be alleviated by K⁺ supplementation (Cao et al., 1993).

NH⁺₄ nutrition dramatically affects cation uptake by plants, leading to a reduction of cation contents (Szczerba et al., 2006, 2008). The mechanism underlying this effect is unknown, but it is commonly considered to result from direct competition between NH⁺₄ and other cations for the transmembrane through common pathways (ten Hoopen et al., 2010). In particular, K⁺ and NH⁺₄ may use the same channels because these cations are highly similar regarding their charge, size and hydration energy, which are characteristics that are important for membrane transport (Wang et al., 1996; White, 1996; Szczerba et al., 2008). K⁺ channels are an important component of the LATS for NH⁺₄ (ten Hoopen et al., 2010). Therefore, it has been speculated that these negative effects of high NH⁺₄ on wheat might be related to the modification of K⁺ flux in root cells.

In the present study, a high NH⁺₄ supply decreased the K⁺ content in wheat seedlings (Figure 5), indicating that high NH⁺₄ suppressed K⁺ uptake by the plants. Furthermore, we examined the net K⁺ flux across the root epidermal cells in SIET experiments and further confirmed that high NH⁺₄ decreased the K⁺ influx into the root epidermal cells (Figure 6). The results presented herein strongly suggest that uptake competition for NH⁺₄ over K⁺ mediates K⁺ transport under high NH⁺₄ conditions. This speculation is strongly supported by studies using intact barley seedlings, in which K⁺ fluxes into the root were shown to be much lower in seedlings grown using 10 mM NH⁺₄ compared with seedlings grown using 10 mM nitrate (NO⁻₃), and elevated K⁺ was able to ameliorate NH⁺₄ toxicity (Kronzucker et al., 2003; Szczerba et al., 2006). The authors proposed that this protection may be associated with the restoration of a moderate K⁺ status to the plant, a process that ultimately depends on K⁺ fluxes into the roots and its subsequent translocation to the shoots (Kronzucker et al., 2003; Szczerba et al., 2006). The suppression of the K⁺ influx at the plasma membrane may be due to the inhibitory action of high NH⁺₄ on high-affinity KUP/HAK/KT transporters (Spalding et al., 1999).

K⁺ and NH⁺₄ exhibit numerous similarities, including their size and charge, and are poorly distinguished by some channels and transporters (Wang et al., 1996; White, 1996; Szczerba et al., 2008; Ariz et al., 2011). One hypothesis to explain how K⁺ ameliorates the toxicity of NH⁺₄ is that K⁺ decreases the uptake of NH⁺₄. Indeed, suppression of the NH⁺₄ influx by K⁺ within minutes of increasing the K⁺ supply was observed in rice cultured in climate-controlled growth chambers under fluorescent lights (Balkos et al., 2010). Therefore, we examined this effect in wheat seedlings through SIET analyses and found that a high K⁺ rate did not alter NH⁺₄ flux but stimulated NO⁻₃ influx (data not shown) and thus high K⁺ treatment increased the total N uptake. This effect was also revealed by higher shoot N concentration in wheat plants under high K⁺ (Table 1). In field, plants grown under a slightly high NH⁺₄ or NO⁻₃ conditions show no toxicity on canopy growth but usually develop a weak culm mechanical strength as indicated by the higher lodging when strong wind occurs during grain filling. Given that the high K⁺ do not suppress the N uptake, the positive effects of K⁺ on cellulose and lignin deposition might be contributed to the additional K⁺ treatment in this study. Inhibition of K⁺ uptake by NH⁺₄ has also been observed in Arabidopsis by Cao et al. (1993); these authors reported that the protective effect of K⁺ was not due to inhibition of NH⁺₄ uptake. Additionally, as an intermediate of N metabolism and the most abundant amino acid in plants grown on NH⁺₄-containing media (Hachiya et al., 2012), glutamine may be involved in the NH⁺₄ toxicity. Therefore, experiments are required to investigate the relationships among glutamine content and K⁺ uptake and there effects on cellulose and lignin deposition and culm mechanical strength under different levels of K⁺ supply and to elucidate the mechanisms of the morphogenesis of cellulose and lignin phenotypes.

In summary, based on the data provided herein, we conclude that at high external concentrations, NH⁺₄ decreases culm mechanical strength, cellulose and lignin contents, N remobilization from vegetative organs to the grain, the grain-filling rate and K⁺ uptake by wheat plants. These effects can be partially reversed by providing an additional K⁺ supply, most likely via competition with NH⁺₄ uptake and translocation. Thus, an understanding of the roles of interaction between NH⁺₄ and K⁺ in the regulation of culm mechanical strength and grain filling will be necessary to improve lodging resistance and productivity in wheat.