Effects of high NH+4 on K+ uptake, culm mechanical strength and grain filling in wheat

It is well established that a high external NH+4 concentration depresses many processes in plant development, but the underlying mechanisms are still not well understood. To determine whether the negative effects of high levels of NH+4 are related to competitive cation uptake, wheat was grown in a field with moderate (18 g N m−2) and high (30 g N m−2) supplies of NH+4 in the presence or absence of additional K+ (6 g K2O m−2) to examine culm mechanical strength, the main components of the vascular bundle, nitrogen (N) remobilization and the grain-filling rate. The results indicated that an excessive supply of NH+4 significantly decreased culm mechanical strength, the cellulose and lignin contents of vascular bundles, the N remobilization efficiency (NRE) and the grain-filling rate compared with a moderate level of NH+4. The additional provision of K+ considerably alleviated these negative effects of high NH+4, resulting in a 19.41–26.95% increase in culm mechanical strength during grain filling and a 34.59% increase in the NRE. An assay using the scanning ion-selective electrode technique (SIET) showed that the net rate of transmembrane K+ influx decreased by 84.62%, and measurements using flame photometry demonstrated that the K+ content decreased by 36.13% in wheat plants subjected to high NH+4. This study indicates that the effects of high NH+4 on culm mechanical strength, cellulose and lignin contents, the NRE and the grain-filling rate are probably associated with inhibition of K+ uptake in wheat.


INTRODUCTION
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 + , Mg 2+ and Ca 2+ (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 NH 3 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 .

PLANT MATERIALS
A field experiment was conducted at an experimental station ( The experiments were laid out in a split-plot design with three treatments and four replications. The treatments included moderate NH + 4 (18 g N m −2 ), high NH + 4 (30 g N m −2 ) and high NH + 4 plus additional K + (K + add ; 6 g K 2 O m −2 ). At sowing, 5 g N m −2 , 9 g P 2 O 5 m −2 , and 9 g K 2 O m −2 were applied per treatment as basal nutrition. At the first node stage (the end of tillering), 13 g N m −2 , 25 g N m −2 , or 25 g N m −2 plus 6 g K 2 O m −2 was top dressed in all three treatments, followed immediately by irrigation. N was supplied as NH 4 Cl. The field-grown plants were used for data collection unless otherwise stated.

MEASUREMENT OF CULM MECHANICAL STRENGTH
The culm mechanical strength of the middle point of the basal second internode without a leaf sheath was measured using a handmade device. The device contains two semicircular semicircular grooves (5 cm apart) with approximately the same diameter as the wheat culm. During measurement, the second internode was set on the grooves, and a pallet was hung at the center of the internode; fine sand was then gradually added to the pallet until the stem broke. The total weight of the added sand and the pallet was subsequently determined using a balance. Culm mechanical strength was directly expressed as the weight required to break the internode.

HISTOCHEMISTRY
For histochemical localization of lignin, Wiesner reactions were performed using the method of Speer (1987). Briefly, transverse sections of the wheat culm second internode were cut freehand with a razor blade. Fresh sections were then incubated for 3 min in a 2% phloroglucinol (w/v), 95% EtOH solution, followed by 3 min of incubation in 50% HCl and subsequent mounting in 50% glycerol, in which phloroglucinol produces a red-pink product under acidic conditions, primarily through reaction with lignin cinnamaldehyde groups. The plant sections were examined directly under a light microscope, and digital images were recorded using an AxioCam MRC camera (Zeiss Axioskop 40, Leica, Germany). The optical density of stained lignin was quantified using the Image-Pro Plus 6.0 software (Media Cybernetics, Silver Springs, MD) and expressed on a scale of 0-2.
For cellulose staining, second internodes were transverse sectioned and stained freehand with a 0.005% (w/v) solution of Fluorescent Brightener 28 (FB 28, Calcofluor White M2R; Sigma) for 5-10 min. The stained sections were observed under a fluorescence microscope (Zeiss Axioskop 40) using a BP 365 excitation filter, an FT 395 chromatic beam splitter and an LP 420 barrier filter. Digital images were recorded using an AxioCam MRC camera. The intensity of fluorescence was quantified with the Image-Pro Plus 6.0 software, and the cellulose content was expressed as the exponent optical density, ranging from 0 to 2.

FOURIER TRANSFORM INFRARED (FTIR) SPECTROSCOPY
Freehand sections (c. 40 μm) were oven-dried at 60 • C, and the vascular bundles were removed using a razor blade under an anatomical microscope before FTIR analysis. The FTIR spectra were recorded using an FTIR spectrometer (Magna-IR 750, Thermo Nicolet, Kanagawa, Japan) equipped with a Mercury-Cadmium-Telluride detector. The spectra of each sample were obtained in the range of 4000-400 cm −1 at a resolution of 4 cm −1 with 128 co-added interferograms and were normalized to obtain the relative absorbance.

DETERMINATION OF SHOOT N CONTENTS
Entire wheat shoots were dried at 70 • C to constant weight and ground to pass through a 1-mm sieve. The samples (1 g of dry weight) were then placed in a Kjeldahl flask, and 20 ml of concentrated H 2 SO 4 was added. After digestion, the solutions were cooled and diluted with deionized water to the specified volume. The total shoot N content was determined using the standard Kjeldahl procedure (Watkins et al., 1987). N remobilization is defined as the difference in the amount of shoot N between anthesis and the harvest stage. The NRE was calculated as the ratio between remobilized N and the total shoot N content at anthesis.

GRAIN FILLING
Based on grain development, five growth stages were designated for measurement: 0 days after anthesis (DAA) (end of anthesis); 8 DAA (milk development stage); 16 DAA (soft dough development stage); 24 DAA (hard dough development stage); and 32 DAA (ripening stage). The ears were collected at each stage and dried at 70 • C for 48 h to a constant mass. The samples were then manually threshed, and the grains were weighed. The data were averaged from three replicates, each with 60 ears.

MEASUREMENT OF K + CONTENTS
Fifteen germinated wheat seeds were transplanted into plastic basins (25 cm high and 20 cm in diameter) containing sterilized wet sand. The plants were then grown at 20-22 • C under an 18h photoperiod (white fluorescent light; 400 μmol m −2 s −1 ). The plants were watered daily with full-strength Hoagland's nutrient solution (HNS) as a control; with HNS supplemented with 10 mM NH 4 Cl as the high NH + 4 treatment; or with HNS supplemented with 10 mM NH 4 Cl and 6 mM KCl as the high NH + 4 plus additional K + treatment. For the K + analysis, entire 40-day-old wheat plants were collected, washed with DD H 2 O and ovendried at 70 • C for 48 h. The dried material was finely powdered and then subjected to wet digestion with HNO 3 :HClO 4 (4:1) under shaking for 20 min at 200 rpm. The samples were filtered through Whatman No. 2 filter paper. The resulting solutions were appropriately diluted, and the K + content was measured using a flame photometer (FP640, Shanghai, China).

K + FLUX ANALYSIS USING THE SCANNING ION-SELECTIVE ELECTRODE TECHNIQUE (SIET)
For SIET, wheat seeds were surface sterilized in an aqueous solution of 1% NaClO for 5 min. The seeds were then washed several times with sterilized water, placed on wet filter paper in Petri dishes and incubated in distilled water. After germination, the seeds were transferred to larger plastic vessels and cultured hydroponically in full-strength HNS. The net K + fluxes into the root epidermal cells were measured noninvasively in 15-dayold seedlings using SIET (BIO-001A SIET system; Younger USA Sci. & Tech. Corp., Amherst, MA, USA; Applicable Electronics Inc., Forestdale, MA, USA and ScienceWares Inc., East Falmouth, MA, USA). Recordings of steady-state K + fluxes were performed as described by Sun et al. (2009). Prior to the measurements, the probes were calibrated in a solution (0.05 mM NH 4 NO 3 , 0.05 mM KCl, 0.1 mM CaCl 2 , and 0.3 mM MES, pH 6.0) for 10 min. The steady fluxes were assayed in measuring solution (0.1 mM NH 4 NO 3 , 0.1 mM KCl, 0.1 mM CaCl 2 , and 0.3 mM MES, pH 6.0) containing 10 mM NH 4 Cl for approximately 10 min to verify that a steady-state condition was reached. Then, the transient K + kinetics in the root epidermal cells 14 mm from the apex were measured for an additional 30 min (Figures 6A,B). As a control, the K + flux was measured in measuring solution not containing 10 mM NH 4 Cl.

STATISTICAL ANALYSIS
All of the data were subjected to analysis of variance (ANOVA) using the Data Processing System (DPS) statistical software (v.14.10, Refine Information Tech. Co., Ltd., Hangzhou, Zhejiang, China) (Tang and Zhang, 2013). The data are presented as the mean ± standard deviation. The treatment means were compared using the least significant difference (LSD) test at P < 0.05.

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Because lodging often occurred from the base of the plants, we determined the culm mechanical strength of the basal second internode of the wheat plants. Figure 1 shows that under field conditions, culm mechanical strength decreased continuously from anthesis to ripening. The application of high NH + 4 (30 g N m −2 ) led to significantly lower culm mechanical strengths, which were reduced by 29.20, 26.37, and 20.88% at 0, 15 and 30 DAA, respectively, compared with the treatment with a moderate level of NH + 4 (18 g N m −2 ). Under high NH + 4 conditions, K + add improved culm mechanical strength by 26.95, 19.41, and 23.46% at 0, 15 and 30 DAA, respectively, compared with the treatment without K + add . A decrease in culm mechanical strength under high NH + 4 (HNS containing 10 mM NH + 4 ) ( Figure S1B) and an improvement in the K + add treatment ( Figure S1C) were also observed in wheat plants in a sand culture system. However, the adverse effect of high NH + 4 was only partially reversed by elevated K + .

DIFFERENTIAL EFFECTS OF HIGH NH + 4 AND K + add ON CELLULOSE AND LIGNIN CONTENTS
To determine whether high NH + 4 affects the contents of lignin and cellulose and whether elevated K + modifies their biosynthesis and localization, culm cross-sections from the different treatments were stained with Calcofluor, to visualize cellulose, or with Wiesner reagents, to visualize lignin (Figures 2, 3). As shown in the obtained images and through analysis with Image-pro Plus 6.0 software, the cellulose fluorescence intensity, particularly in the vascular bundles, was weaker in high-NH + 4treated wheat (Figures 2B,E,G) than in moderate-NH + 4 -treated wheat (Figures 2A,D,G). When the wheat plants were exposed to high NH + 4 , K + add significantly promoted cellulose deposition in the vascular bundles (Figures 2C,F,G). Similarly, the amount of lignin decreased in the internodes of the wheat plants under high NH + 4 , as indicated by the weaker red-pink color observed (Figures 3B,E,G) and the measurements of optical density performed using the Image-Pro Plus 6.0 software, compared with the moderate NH + 4 treatment (Figures 3A,D,G). Additionally, K + significantly relieved the reduction of the lignin content detected under high NH + 4 (Figures 3C,F,G). Figure 5A illustrates the typical FTIR spectra obtained from the vascular bundles of the basal second internode of wheat plants at 16 DAA; the data point shown in each spectrum is an average of four measurements. In all three treatments, peaks occurred at wavelengths of approximately 2920 and 2850 cm −1 , which were ascribed to the aliphatic saturated C-H stretching vibration that takes place in polysaccharides, particularly cellulose and hemicelluloses (Lichtfouse et al., 1998;Kaushik et al., 2010). A prominent peak at 1248 cm −1 and a shoulder peak at 1130 cm −1 represent the C-H, O-H, or CH 2 bending frequencies (Himmelsbach et al., 2002) and are indicators of lignin (Kaparaju and Felby, 2010). An indistinct band at 897 cm −1 indicates the typical structure of cellulose (due to the β-glycosidic linkages of the glucose ring of cellulose) (Gañán et al., 2004).
To quantitatively distinguish the changes in the cellulose and lignin contents of the vascular bundles, difference spectra were generated through digital subtraction of the spectra of wheat plants treated with high levels of NH + 4 from those of plants treated with moderate NH + 4 or through digital subtraction of the spectra of K + add treatment from those without additional K + under high NH + 4 . In the different spectra recorded, distinct peaks appeared at 2920, 2850, 1248, 1130, and 897 cm −1 . The decreased absorbance intensity due to a high level of NH + 4 indicated that the cellulose and lignin contents decreased, whereas the positive values due to elevated K + treatment under high NH + 4 conditions suggested that the cellulose and lignin contents increased in the vascular bundles of wheat.

CHANGES IN SHOOT N CONCENTRATIONS
As expected, the total shoot N concentration (%) in wheat during grain filling was consistently higher in the excessive compared with the insufficient NH + 4 treatment, suggesting that high NH + 4 promoted N uptake by the plants (Table 1). At 0 DAA, the greater total shoot N concentration caused by a high NH + 4 supply was further increased by the K + add treatment. The shoot N concentration gradually decreased during the grain-filling period in all three treatments. However, the shoot N concentration decreased more sharply under moderate NH + 4 than at high NH + 4 ; as a result, high NH + 4 led to a lower NRE compared with the moderate NH + 4 treatment. Under high NH + 4 conditions, the shoot N concentration was greater from 0 to 16 DAA in the K + add treatment than in the treatment without additional K + , but the concentrations in these treatments were similar at maturity (32 DAA); consequently, the K + add treatment resulted in a greater NRE. These data strongly indicate that the K + add treatment might increase the N-use efficiency under high NH + 4 conditions.

GRAIN-FILLING RATE
The grain dry mass decreased when the wheat plants were treated with high NH + 4 , especially during the early stages of grain filling (8, 16, and 24 DAA), compared with the application of moderate NH + 4 . Under high NH + 4 conditions, additional K + increased the grain dry mass throughout the grain-filling period ( Table 2); i.e., the elevated K + supply increased the grain-filling rate when wheat was subjected to high NH + 4 .

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A sand culture experiment was conducted to evaluate K + uptake by the wheat plants using flame photometry. The high NH + 4 treatment significantly decreased the K + concentration in whole wheat plants compared with the moderate NH + 4 supply (P < 0.01). As expected, under high NH + 4 conditions, the K + add treatment increased the K + content and improved culm mechanical strength (Figure 1). However, K + add only partly relieved the significant reduction in K + contents caused by high NH + 4 ( Figure 5).

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To further evaluate whether high NH + 4 influences K + uptake in wheat seedlings, we measured the net K + flux responses in root epidermal cells using SIET (Figure 6A). In the roots of wheat plants that were not treated with 10 mM NH 4 Cl, the net K + influx into the root epidermal cells was determined ( Figure 6B). SIET analyses showed that the average influx rate was approximately 17.88 pmol cm −2 s −1 (Figure 6C). However, adding 10 mM NH 4 Cl to the measuring solution resulted in a remarkable decrease in the K + influx rate, which dropped to an average of 2.75 pmol cm −2 s −1 (Figure 6C).

DISCUSSION
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 (18 g N m −2 ; N m ), high NH + 4 (30 g N m −2 ; N h ) and high NH + 4 combined with K + add (6 g K 2 O m −2 ; N h + K + add ). The spectra are separated for comparison. (B) Different spectra generated through digital subtraction of the spectrum of the vascular bundles of wheat treated with high NH + 4 from that of wheat treated with moderate NH + 4 (N h -N m ), or subtraction of the spectrum of vascular bundles of wheat treated with high NH + 4 and K + add from that of wheat treated with high NH + 4 without an additional K + supply [(N h + K + add ) −N m ].
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 . The values are the means ± SD of five replicates. The different letters indicated above each column refer to significant differences at the P < 0.05 level. 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