A field experiment was conducted at the State Key Laboratory of Crop Biology and the experimental farm of Shandong Agricultural University, China (36°10′N, 117°04′E, 151 m altitude) in 2012 and 2013. The region has a temperate continental monsoon climate. The effective accumulated temperatures of maize growth periods in 2012 and 2013 were 1710.9°C d and 1740.5°C d, respectively. The mean total precipitation values during maize growth periods in 2012 and 2013 were 350.0 mm and 348.5 mm, respectively. The soil type was sandy loam, and soil pH was 8.25 (Cambisols; FAO/EC/ISRIC 2003). The plowed soil (0–20 cm) before the experiment contained 10.2 g kg^-1 of organic matter, with total mounts of N: 0.9 g kg^-1, rapidly available phosphorus (P): 50.3 mg kg^-1, and rapidly available potassium (K): 85.4 mg kg^-1.

Each plot was 4 m × 4 m and separated by 4 m × 2.3 m polyvinyl chloride (PVC) boards as water barriers. Every PVC board was buried 2.0 m below the surface, with remaining 0.3 m aboveground. Experimental treatments matched different waterlogging stages: the third leaf stage (V3; V3-W), the sixth leaf stage (V6; V6-W), and the 10th day after the tasseling stage (10VT; 10VT-W), and no waterlogging (CK). In the CK, soil moisture was kept optimum during the whole growth period. We selected the maize hybrids DengHai605 (DH605) and ZhengDan958 (ZD958) as experimental materials, because they are the most commonly planted varieties in China. Maize was sown on June 16 in both years, at a plant density of 67,500 plants ha^-1. The water in waterlogged pools was maintained at 2–3 cm above the soil surface for 6 d. Each treatment was replicated three times, in a completely randomized block design. 300 kg ha^-1 urea (N 46%), 857 kg ha^-1 calcium superphosphate (P₂O₅ 17%), and 400 kg ha^-1 muriate of potash (K₂O 60%) were applied. Prior to seeding, P, K compound fertilizer was applied one-off to prepare the soil for sowing, 40% N compound fertilizer was applied at V6, and 60% N compound fertilizer was applied at V12. Disease, weeds, and pests were well controlled in each treatment.

Five representative plant samples were obtained from each plot at V6, the twelfth leaf stage (V12), the tasseling stage (VT), the milk stage (R3), and the physiological maturity stage (R6), according to Ritchie and Hanway (1982). Samples were preserved after being separated into stem and leaf at V6, V12, and VT, and separated into stem, leaf, and ear at R3 and R6. Samples were dried at 80°C in a force-draft oven (DHG-9420A, Bilon Instruments Co., Ltd, Shanghai, China) to a constant weight and weighed separately. Total N was measured using the Kjeldahl method (Hibbard, 2002):

The functional leaves from five plant samples were obtained from the center of each plot at the next day after the end of waterlogging treatments. NR activity was estimated using the method of Aslam et al. (2001). The samples (0.5 g) were placed in 10 mL of incubation medium, which was 0.1 M potassium phosphate buffer (pH 7.5) containing 0.1 M KNO₃ with 1% (v/v) propanol. Prior to the assay, the buffer solution was purged with N₂ gas for 30 min to remove dissolved oxygen, and samples then were vacuum-infiltrated (two times), and incubated in a water bath at 30°C for 30 min in the dark. 1 mL sample was withdrawn for the color reaction, and initiated by adding 2 mL aminobenzenesulfonic acid and 2 mL a-naphthylamine. After 20 min of incubation, the amount of nitrite (NO₂^-) was determined by absorbance at 520 nm using a standard curve.

The functional leaves from five plant samples were obtained from the center of each plot at the next day after the end of waterlogging treatments. To extract enzymes, 0.5 g of leaf tissue was homogenized with 10 mM Tris-HCl buffer (pH 7.6, containing 1mM MgCl₂, 1 mM EDTA, and 1mM 2-mercaptoethanol) in a chilled pestle and mortar. The homogenate was centrifuged at 15000 × g for 30 min at 4°C. The supernatant was used to determine enzyme activities.

Glutamine was assayed according to Mohanty and Fletcher (1980). The reaction mixture contained in a final volume of 1 mL, 80 μmol Tris-HCl buffer, 40 μmol L-glutamic acid, 8 μmol ATP, 24 μmol MgSO₄, and 16 μmol NH₂OH. The final pH was 8.0. The reaction was initiated by the addition of the enzyme extract. After incubation for 30 min at 30°C, the reaction was stopped by adding 2 mL 2.5% (w/v) FeCl₃ and 5% (w/v) trichloroacetic acid in 1.5 M HCl. After centrifugation at 3000 × g for 10 min, the absorbance of the supernatant was read at 540 nm. GS activity was expressed as 1 μmol L-glutamate γ-monohydroxamate (GHA) formed g^-1 FW h^-1, with μmol GHA g^-1 FM h^-1 said.

Glutamate synthase activity was measured based on the method described by Singh and Srivastava (1986), in units of μmol NADH g^-1 FM min^-1. The assay mixture contained 0.4 mL 20 mM L-glutamine, 0.05 mL 0.1 M 2-oxoglutarate, 0.1 mL 10 mM KCI, 0.2 mL 3 mM NADH, and 0.5 mL of the enzyme extract in a final volume of 3 mL, prepared with 25 mM Tris-HCl buffer (pH 7.6). The reaction was initiated by adding L-glutamine immediately following the enzyme preparation. The decrease in absorbance was recorded for 3 min at 340 nm.

Glutamate dehydrogenase activity was estimated by using the method of Lin and Kao (1996), in unites of μmol NADH g^-1 FM min^-1. The assay mixture contained 0.3 mL 0.1 M 2-oxoglutarate, 0.3 mL 1 M NH₄Cl, 0.2 mL 3 mM NADH and 1 mL of the enzyme extract in a final volume of 3 mL, prepared with 0.2 M Tris-HCl buffer (pH 8.0). The reaction was initiated by adding the enzyme extract. The decrease in absorbance was recorded for 3 min at 340 nm.

At R6, 30 ears harvested from three rows at the center of each plot were used to determine yield and ear traits including length, width, weight, row number, kernels per row, bald tip length, cob weight, and cob width. All kernels were air-dried to determine yield, and grain yield was expressed at 14% moisture content, according to the standard moisture content of maize for storage or sale is 14% in China (GB/T 29890-2013).

The data were subjected to three-way analysis of variance (ANOVA). Growing season, blocks, and block interactions were included as random effects. Waterlogging treatment and hybrids were included as fixed effects. In case of significant treatment effects, comparison of means was performed by means of LSD at a significance level of 0.05. LSD was used to compare adjacent means arranged in order of magnitude. ANOVA and the LSD test were conducted using the SPSS17.0 software program (Ver. 17.0, SPSS, Chicago, IL, United States). Figures were prepared using a SigmaPlot 10.0 program.

Waterlogging significantly affected N uptake and translocation in maize. The V3 stage was most susceptible to waterlogging, followed by V6 and 10VT stages. Waterlogging significantly decreased total N accumulation in maize, compared to CK. The total N accumulation levels of DH605 in treatments V3-W, V6-W, and 10VT-W were 39, 36, and 19% lower than that of CK across hybrids and years (Figure 1). There were no significant year × hybrid × waterlogging treatment interaction effects on N accumulation and distribution at the R6 stage (Table 1). The greatest reduction sin stem, leaf, cob, and grain N accumulation were found in the V3-W treatment at 25, 18, 34, and 46% across hybrids and years, compared to those in CK. Additionally, waterlogging increased N distribution rate in leaf, stem, and cob, whereas grain N distribution rate decreased significantly after waterlogging, compared to CK. Waterlogging significantly decreased NHI of maize, with the most significant reduction (approximately 10%) in the V3-W treatment across hybrids and years, compared to that of CK (Table 1).

Waterlogging significantly decreased the activities of key N metabolism enzymes, compared to CK, with no significant hybrid × waterlogging treatment interaction effects (Table 2). The greatest reduction in the activities of NR (approximately 60%), GS (approximately 50%), GDH (approximately 33%), and GOGAT (approximately 26%) took place when waterlogging occurred at V3, whereas waterlogging at V6 produced activity reductions of 37, 47, 25, and 20%, and waterlogging at 10VT produced activity reductions of 11, 29, 17, and 16% for these key N metabolism enzymes across hybrids and years.

Effects of waterlogging on dry matter accumulation were similar between the two hybrids (Figure 2). Total dry matter accumulation was significantly reduced by waterlogging at different stages, with the most significant reduction (approximately 34%) in the V3-W treatment across hybrids and years. There were no significant year × hybrid × waterlogging treatment interaction effects on dry matter accumulation or distribution at the R6 stage (Table 3). The greatest reduction in dry matter weight in stem (approximately 22%), leaf (approximately 27%), cob (approximately 21%), and grain (approximately 42%) occurred in the V3-W treatment. Waterlogging increased dry matter distribution rate in stem, leaf, and cob, whereas that in grain was significantly reduced after waterlogging. Waterlogging also significantly decreased the HI, with the most significant reduction (approximately 11%) in V3-W across hybrids and years, compared to that of CK (Table 3).

Waterlogging resulted in a significant yield reduction in maize (Table 4). The greatest yield reduction (approximately 38%) occurred in the V3, whereas waterlogging at V6 and 10VT produced yield reductions of 30 and 15% across hybrids and years, respectively. Waterlogging also decreased grain number per ear and 1000-grain weight, with the most significant reduction in the V3-W treatment with decreases of 22 and 16% across hybrids and years.