Superior glucose metabolism supports NH4+ assimilation in wheat to improve ammonium tolerance

Abstract

The use of slow-release fertilizers and seed-fertilizers cause localized high-ammonium (NH₄ ⁺) environments in agricultural fields, adversely affecting wheat growth and development and delaying its yield. Thus, it is important to investigate the physiological responses of wheat and its tolerance to NH₄ ⁺ stress to improve the adaptation of wheat to high NH₄ ⁺ environments. In this study, the physiological mechanisms of ammonium tolerance in wheat (Triticum aestivum) were investigated in depth by comparative analysis of two cultivars: NH₄ ⁺-tolerant Xumai25 and NH₄ ⁺-sensitive Yangmai20. Cultivation under hydroponic conditions with high NH₄ ⁺ (5 mM NH₄ ⁺, AN) and nitrate (5 mM NO₃ ^-, NN), as control, provided insights into the nuanced responses of both cultivars. Compared to Yangmai20, Xumai25 displayed a comparatively lesser sensitivity to NH₄ ⁺ stress, as evident by a less pronounced reduction in dry plant biomass and a milder adverse impact on root morphology. Despite similarities in NH₄ ⁺ efflux and the expression levels of TaAMT1.1 and TaAMT1.2 between the two cultivars, Xumai25 exhibited higher NH₄ ⁺ influx, while maintaining a lower free NH₄ ⁺ concentration in the roots. Furthermore, Xumai25 showed a more pronounced increase in the levels of free amino acids, including asparagine, glutamine, and aspartate, suggesting a superior NH₄ ⁺ assimilation capacity under NH₄ ⁺ stress compared to Yangmai20. Additionally, the enhanced transcriptional regulation of vacuolar glucose transporter and glucose metabolism under NH₄ ⁺ stress in Xumai25 contributed to an enhanced carbon skeleton supply, particularly of 2-oxoglutarate and pyruvate. Taken together, our results demonstrate that the NH₄ ⁺ tolerance of Xumai25 is intricately linked to enhanced glucose metabolism and optimized glucose transport, which contributes to the robust NH₄ ⁺ assimilation capacity.

Keywords: ammonium assimilation; ammonium stress; ammonium tolerance; glucose metabolism; wheat.

Figure 1

Effect of ammonium stress on biomass accumulation of two different ammonium-sensitive cultivars. (A) plant dry weight; (B) shoot dry weight; (C) root dry weight. Data are given as means of three biological replicates, and error bars indicate SD. NN, nitrate conditions; AN, ammonium stress conditions. YM, NH₄ ⁺-sensitive cultivar Yangmai20; XM, NH₄ ⁺-tolerant cultivar Xumai25. C, T, and C×T represent the F-value of cultivar, treatment, and the interaction between cultivar and treatment, respectively. The symbols * and ** indicate significant differences at the 0.05 and 0.01 levels, respectively, while ns refers to no significant difference.

Figure 2

Effects of ammonium stress on root free NH₄ ⁺ concentration of wheat seedlings after 1, 3, 5, 10, and 20 days. Data are given as means of three biological replicates, and error bars indicate SD. NN, nitrate conditions; AN, ammonium stress conditions. YM, NH₄ ⁺-sensitive cultivar Yangmai20; XM, NH₄ ⁺-tolerant cultivar Xumai25.

Figure 3

Effects of ammonium stress on root NH₄ ⁺ influx and efflux of wheat seedlings. (A) NH₄ ⁺ influx; (B) NH₄ ⁺ efflux. Data are given as means of 8 replicates. Error bar labels with different letters indicate significant differences (P < 0.05) between cultivars. YM20, NH₄ ⁺-sensitive cultivar Yangmai20; XM25, NH₄ ⁺-tolerant cultivar Xumai25.

Figure 4

Effects of ammonium stress on nitrogen status of wheat seedlings at 3 and 20 days after treatment. (A) Plant nitrogen accumulation; (B) Root free amino acid concentration; (C) Root asparagine concentration; (D) Root glutamine concentration; (E) Root aspartate concentration; (F) Root glutamate concentration. Data are provided as means of three biological replicates and error bar labels with different letters indicate significant differences (P < 0.05) between cultivars and treatment. NN, nitrate conditions; AN, ammonium stress conditions. YM, NH₄ ⁺-sensitive cultivar Yangmai20; XM, NH₄ ⁺-tolerant cultivar Xumai25.

Figure 5

Effects of ammonium stress on root carbon skeleton supply of wheat seedlings at 3 and 20 days after treatment (DAT). (A) Sucrose concentration; (B) Fructose concentration; (C) Glucose concentration; (D) Pyruvate concentration; (E) 2-Oxoglutarate concentration; (F) Oxaloacetate concentration. Data are supplied as means of six biological replicates. Error bar labels with different letters indicate significant differences (P < 0.05) between cultivars and treatment. NN, nitrate conditions; AN, ammonium stress conditions. YM, NH₄ ⁺-sensitive cultivar Yangmai20; XM, NH₄ ⁺-tolerant cultivar Xumai25.

Figure 6

Effects of ammonium stress on the activity of sugar metabolizing enzyme in wheat seedlings at 3 and 20 days after treatment (DAT). (A) Hexokinase (HXK) activity; (B) Phosphofructokinase (PFK) activity; (C) Pyruvate kinase (PK) activity; (D) Phosphoenolpyruvate carboxylase (PEPc) activity. Data are expressed as means of three biological replicates. Error bar labels with different letters indicate significant differences (P < 0.05) between cultivars and treatment. NN, nitrate conditions; AN, ammonium stress conditions. YM, NH₄ ⁺-sensitive cultivar Yangmai20; XM, NH₄ ⁺-tolerant cultivar Xumai25.

Figure 7

Effects of ammonium stress on the activity of ammonium metalizing in wheat seedlings at 3 and 20 days after treatment (DAT). (A) Glutamine synthetase activity; (B) Glutamate synthase activity; (C) NADH-GDH activity; (D) NAD⁺-GDH activity. Data are expressed as means of three biological replicates. Error bar labels with different letters indicate significant differences (P < 0.05) between cultivars and treatment. NN, nitrate conditions; AN, ammonium stress conditions. YM, NH₄ ⁺-sensitive cultivar Yangmai20; XM, NH₄ ⁺-tolerant cultivar Xumai25.

Figure 8

Effects of ammonium stress on relative gene expression in the root of wheat seedlings at 6 and 120 hours after treatment. (A) TaAMT1.1; (B) TaAMT1.2; (C) TaAMT2.1; (D) TaPFK; (E) TaPK; (F) TaHXK; (G) TaERDL; (H) TaTST. Data are expressed as means of three biological replicates. Error bar labels with different letters indicate significant differences (P < 0.05) between cultivars. NN, nitrate conditions; AN, ammonium stress conditions. YM, NH₄ ⁺-sensitive cultivar Yangmai20; XM, NH₄ ⁺-tolerant cultivar Xumai25.

Figure 9

Physiological mechanisms of the enhanced NH₄ ⁺ assimilation in NH₄ ⁺-tolerant wheat cultivar under NH₄ ⁺ stress. Under NH₄ ⁺ stress, the NH₄ ⁺-tolerant cultivar has an increased NH₄ ⁺ uptake, its superior glucose metabolism and transport capacity contributed to the acquisition of more C skeletons, which improved NH₄ ⁺ assimilation and reduced the accumulation of free NH₄ ⁺ in the root, thus effectively alleviating the inhibitory effects of NH₄ ⁺ stress. Red and green, respectively, indicate inhibition/reduction, and activation/increase under NH₄ ⁺ stress. Solid red arrows indicate enhanced metabolism of the NH₄ ⁺-tolerant cultivar, compared to the NH₄ ⁺-sensitive cultivar. AMTs, ammonium transporters; Asn, asparagine; Asp, aspartate; ERDL, tonoplast H+/glucose symporter; GDH, glutamate dehydrogenase; GS, glutamine synthetase; GOGAT, glutamate synthase; Gln, glutamine; Glu, glutamate; HXK, hexokinase; OAA, oxaloacetate; PEPc, phosphoenolpyruvate carboxylase; PFK, phosphofructokinase; PK, pyruvate kinase; TST, tonoplast sugar transporter; 2-OG, 2-oxoglutarate.