Effects of Nitrogen Application Strategy on Nitrogen Enzyme Activities and Protein Content in Spring Wheat Grain

Abstract

The aim of this study was to determine the regulatory effect of different nitrogen (N) fertilizer application rates on the grain N metabolism enzymes and protein content of drip-irrigated spring wheat under the climatic conditions in Xinjiang, China. A split plot experiment was conducted with strong gluten wheat Xinchun 38 (XC 38) and medium gluten wheat Xinchun 49 (XC 49) as experimental materials. We set up seven nitrogen treatments, in amounts of 300 (Nck), 285 (N5), 270 (N10), 255 (N15), 240 (N20), 225 (N25) and 0 (N0) kg hm⁻². The effects of N application rate on nitrate reductase (NR), glutamine synthetase (GS), glutamate-pyruvate aminotransferase (GPT), protein content, protein composition, and yield of wheat grain were studied. The results showed that NR, GS, GPT, protein content, albumin, globulin, glutenin, gliadin, and yield first increased and then decreased with the decrease in N application. Furthermore, different responses to different applications between different wheat varieties was also observed. The indexes of XC 38 reached the maximum in the N15 treatment, and the yield increased 2.99~81.45%. XC 49 showed the best indicators under the N25 treatment and the yield increased 0.37~71.29%. Under the same N level, all indicators of XC 38 were better than XC 49. Correlation analysis showed that the yield and protein yield were significantly positively correlated with NR, GS, and GPT. The interaction of N fertilizer and variety had significant effects on NR, GS, GPT, protein content, components, and yield. These results show that the protein content and yield of wheat grain can be improved by reasonably adjusting the N fertilizer application strategy.

1. Introduction

China is the largest wheat (Triticum aestivum L.) producer in the world [1]. Xinjiang province in China is one of the largest grain crop provinces, with a wheat planting area of more than 1,173,300 hm², accounting for 52% of the planting area of Xinjiang grain crops in 2017 [2]. Wheat flour milled from wheat is a staple food in the world. Steamed bread, noodles, and cakes made from wheat are especially popular. In addition, wheat is rich in complex carbohydrates (74~77%) and protein (11~15%), which are important sources of calories and plant protein, respectively. Nitrogen (N) is the main nutrient necessary for plant growth and development, and is also the main limiting factor for plant growth and development [3]. Applying N fertilizer is essential to ensuring high crop yields, but over-application of N fertilizer has been a widespread problem in agricultural production [4,5,6]. Excessive N residues and losses damage the long-term sustainability of the cropland environment and food production. In addition, N loss caused by leaching, runoff, denitrification, and ammonia volatilization leads to groundwater contamination, water eutrophication, and the greenhouse effect [7,8]. Therefore, reducing N fertilizer application is crucial to coordinate high crop yields with environmental stability and the improvement in crop production efficiency.

The process of N nutrition absorption, metabolism, and utilization in plants plays a vital role in the outcomes of yield and quality. Some studies have shown that the protein content of wheat grain increases with the amount of N applied in the field [9,10]. Rossmann et al. pointed out that it was necessary to consider the cultivar and N use efficiency in a study of the relation between protein content and N applied in the field [11]. N can affect the amount of protein in wheat grain, and N application can regulate the content of protein components and the processing quality of wheat grains. The effect of nitrogen on wheat grain protein content and protein components is closely related to wheat cultivars [12,13,14]. Nitrate reductase (NR), glutamine synthetase (GS), and glutamate-pyruvate aminotransferase (GPT) are key enzymes in N metabolism [15,16]. These three enzymes are indispensable, playing a vital role in regulating the metabolism of nitrogen, and they are also key enzymes in protein synthesis [17]. NR is a key enzyme for ammonia assimilation, which has important effects on crop photosynthesis and respiration. NR activity is closely related to N accumulation in the late growth stage of crops, and is positively related to protein content [18]. GS is a multifunctional enzyme in N metabolism, and is involved in protein synthesis and has a significant effect on wheat grain quality [19,20]. GPT is an important transaminase in plants; it catalyzes transamination to glutamic acid and is positively correlated with protein content [21,22].

The research on the application technology of N fertilizer in drip-irrigation wheat fields is extensive and detailed. The N fertilizer injected into the water directly acts on the root of the plant. The N fertilizer is sufficient and the soil moisture is effectively maintained, which greatly improves the N fertilizer utilization efficiency and reduces pollution. Previous studies focused on the effects of N fertilizer on wheat growth and grain protein content [23,24]. However, excessive N supplementation is still prevalent in the region, leading to reduced N use efficiency, and thereby increasing production costs and contaminating soil and groundwater. Therefore, we conducted a two-year positioning experiment examining the effects of different N fertilization strategies on the drip-irrigated spring wheat yield in Xinjiang. The objectives were to: (i) investigate the response of drip-irrigated spring wheat yield, grain enzyme activity, and protein content to N fertilizer application; (ii) quantify the relationships between enzyme activity, protein content, and yield; and (iii) determine the optimal N regime for this region under drip conditions.

2. Materials and Methods

2.1. Test Site Overview

The field experiments were established during the 2018 and 2019 growing seasons for spring wheat at the Shihezi University Agronomy Experiment Station (44°26.5′ N, 86°01′ E), Xinjiang Province, China. This area is characterized by a continental climate. The precipitation amounts during the wheat growing season (April to July) were 109.2 mm in 2018 and 103.6 mm in 2019. The average maximum temperatures in the growth period were 27 °C (2018) and 26 °C (2019), and the average minimum temperatures were 13 °C (2018) and 14 °C (2019). The soil is classified as a gray desert soil. The physicochemical properties of the soil at the test site were similar over the two years (Table S1).

2.2. Experimental Design and Management

The experiment was a split plot design with nitrogen as the main plot and varieties as the sub-plot. The tested wheat (Triticum aestivum L.) cultivars were strong gluten wheat Xinchun 38 (cv. XC 38, protein content 15.04%) and medium gluten wheat Xinchun 49 (cv. XC 49, protein content 12.89%), which are the widely cultivated wheat cultivars in Xinjiang province of China. Seven N fertilizer treatments were used: Nck: normal N supply during the growing period (300 kg hm⁻² is the conventional N fertilizer application in local management practices); N0: no N application-control; N5: reduction N 5% (285 kg hm⁻²); N10: reduction N 10% (270 kg hm⁻²); N15: reduction N 15% (255 kg hm⁻²); N20: reduction N 20% (240 kg hm⁻²); N25: reduction N 25% (225 kg hm⁻²). The ratio of base to topdressing of N fertilizer was 2:8, and the specific amount of N fertilizer applied in each growth period is shown in

Table 1. Amount of nitrogen fertilizer in different treatments in 2018 and 2019.

Treatment	Pure Nitrogen (kg hm−2)	Base Fertilizer (20%)	Top Dressing (80%)	Two-Leaf One-Hearted Period (10%)	Tillering Period (10%)	Jointing Period:5 Leaf Age (40%)	Booting Period (20%)	Flowering Period (15%)	Milk Ripening Period (5%)
Nck	300	60	240	24	24	96	48	36	12
N5	285	57	228	22.8	22.8	91.2	45.6	34.2	11.4
N10	270	54	216	21.6	21.6	86.4	43.2	32.4	10.8
N15	255	51	204	20.4	20.4	81.6	40.8	30.6	10.2
N20	240	48	192	19.2	19.2	76.8	38.4	28.8	9.6
N25	225	45	180	18	18	72	36	27	9
N0	0	0	0	0	0	0	0	0	0

.

Each treatment was replicated three times, and the planting area in the plot was 4 m long and 3 m wide. A 100 cm deep anti-seepage membrane was embedded between each plot to prevent the fertilizer from moving out. Before sowing, 120 kg hm⁻² P₂O₅ (calcium superphosphate) was used as base fertilizer in each plot to plow the soil. The N fertilizer applied during the growth period was urea (N = 46%). The total irrigation amount was 600 mm during the entire wheat growth seasons, and irrigation was applied 9 times. The irrigation amount in each period was accurately controlled through a water meter.

The seeds were sown on 6 April 2018 and 8 April 2019, with a seeding amount of 345 kg hm⁻². The wide and narrow rows were planted in the manner of “four rows per tube”, with row spacing of 12.5 + 20 + 12.5 + 15 cm (Figure 1). The drip irrigation belt (pipe diameter 16 mm, drip head spacing 30 cm, flow rate 2.6 L·h⁻¹) was placed in a 20 cm wide row, and harvesting occurred on 10 July 2018 and 12 July 2018. Other field management steps were the same as for field production.

2.3. Key Enzyme Activities of Nitrogen Metabolism in Spring Wheat Grain: The Nitrate Reductase (NR), Glutamine Synthetase (GS), and Glutamate-Pyruvate Aminotransferase (GPT) in Spring Wheat Grain

For each treatment, single stem tags with the same flowering date and normal growth, and basically the same growth trend, were selected as the sampling observation materials. Thirty wheat spikes of each treatment were taken at 7, 14, 21, 28, 35 days after anthesis, immediately placed in liquid N for quick freezing for 30 min, and stored at a temperature of −80 °C to prevent enzyme inactivation. Separation of wheat grain was manually performed and grided with liquid N. Then, the extract was obtained by low temperature centrifugation at 13,000 r/min, which was used to determine the key enzyme activity of N metabolism.

For determination of nitrate reductase activity (NR) in grain, refer to the method of Cruz et al. [25]. Glutamine synthetase (GS) activity in grains was determined according to the method of Cruz et al. [25]. For determination of glutamate-pyruvate aminotransferase (GPT) activity in grains, refer to the method of Baars et al. [26].

2.4. Protein Content and Its Fraction Content

Twenty wheat spikes of uniform growth were selected from each treatment at 7, 14, 21, 28, and 35 days after anthesis and placed in paper bags. This was replicated three times. Samples were immediately placed in an oven at 105 °C for 30 min and dried at 75 °C to a constant weight. The constant weight samples were crushed through a 100-mesh sieve and the N content was determined using Kjeldahl apparatus (K9840 Kjeldahl apparatus, Hanon, Shandong, China). Grain protein content was calculated as nitrogen concentration multiplied by 5.7. Four protein fractions (albumins, globulins, gliadins, and glutenins) were separated and analyzed according to the method of Liu et al. [27].

2.5. Yield and Protein Yield of Spring Wheat Grain

At the harvest stage of wheat (10 July 2018 and 12 July 2019), plants were randomly harvested manually from an area of 1 m² of each plot to measure spike number, grain number per spike, 1000-grain weight, and grain yield. The grains were threshed by a threshing machine and then dried in an oven at 80 °C for at least 72 h. The grain yield of wheat was expressed by dry weight. Protein yield of spring wheat grain was calculated as the grain yield multiplied by the protein content.

2.6. Statistical Analysis

All statistical analyses were conducted using SPSS 26.0 (SPSS Inc., Chicago, IL, USA) for Windows. One-way analysis of variance (ANOVA) and subsequent Duncan’s multiple range tests at the 0.05 significance level were used to determine differences in the indicators of the same wheat variety at the same stage in different N treatments. The statistical analysis of the split plot design was repeated three times with nitrogen as the main plot and varieties as the sub-plot. Pearson correlation analysis was used to analyze the correlation between indicators.

3. Results

3.1. Nitrate Reductase (NR) Activity of Spring Wheat Grain

In the two-year experiment, the effect of N application on the NR activity of the grain was significant, and the trends of NR activity in XC 38 and XC 49 were similar (Figure 2). The maximum value of wheat NR activity appeared at the 7th day after anthesis and then gradually decreased from the 7th to the 35th days after anthesis. With the decrease in N application, the NR activity in XC 38 grains decreased in the order N15 > N10 > N5 > Nck > N20 > N25 > N0 (Figure 2a,c). Compared with other treatments, N15 had the highest NR activity, and the maximum NR activity of XC 38 was 0.81 U·g⁻¹. The NR activity under N15 treatment was 1.25~53.11% higher than that of other treatments. However, for medium gluten wheat XC 49, with the reduction in N application, the NR activity of N25 treatment was the largest (Figure 2b,d), and was significantly higher than that of other treatments (p < 0.05), with a maximum value of 0.77 U·g⁻¹. Under N25 treatment, the NR activity increased 1.19~58.98% compared to other treatments.

In the two-year experiment, the NR activity of XC 38 was always higher than that of XC 49, and the maximum NR activity of XC 38 was 4.74% higher than that of XC 49. Furthermore, the maximum NR activity of the two varieties in 2019 was 10.03% and 8.70% higher, respectively, than that in 2018. The interaction of year, variety, and N treatment had a significant effect on NR activity (

Table 2. ANOVA of wheat grain physiological indicators under variety and nitrogen fertilizer interaction.

Source of Variation	Dependent Variable	SS	DF	MS	F	Sig.
Y	protein	15.28	1	15.28	12,272.12	**
	NR	0.15	1	0.15	8700.14	**
	GS	0.06	1	0.06	2155.40	**
	GPT	0.14	1	0.14	8724.61	**
V	protein	35.87	1	35.87	28,819.49	**
	NR	0.00	1	0.00	265.28	**
	GS	0.01	1	0.01	349.38	**
	GPT	0.09	1	0.09	5361.47	**
T	protein	26.25	6	4.37	3513.90	**
	NR	0.13	6	0.02	1260.98	**
	GS	0.05	6	0.01	286.57	**
	GPT	0.13	6	0.02	1360.32	**
Y × V	protein	1.00	1	1.00	800.81	**
	NR	0.00	1	0.00	57.23	**
	GS	0.00	1	0.00	1.61	ns
	GPT	0.01	1	0.01	357.64	**
Y × N	protein	2.57	6	0.43	343.76	**
	NR	0.00	6	0.00	22.25	**
	GS	0.01	6	0.00	31.18	**
	GPT	0.01	6	0.00	119.87	**
V × N	protein	13.25	6	2.21	1773.73	**
	NR	0.07	6	0.01	700.52	**
	GS	0.03	6	0.00	157.18	**
	GPT	0.09	6	0.01	880.63	**
Y × V × N	protein	1.10	6	0.18	147.07	**
	NR	0.00	6	0.00	5.45	**
	GS	0.00	6	0.00	8.71	**
	GPT	0.00	6	0.00	19.58	**

V: Variety, N: Nitrogen, Y: Year. ** represents significant differences at 0.01 level; ns indicates insignificant differences.

).

3.2. Glutamine Synthetase (GS) Activity of Spring Wheat Grain

GS activity decreased gradually from the 7th day to the 35th day after anthesis, and reached the highest level at the 7th day after anthesis (Figure 3). The change trend of each treatment in 2018 and 2019 was similar, and N fertilizer had a significant impact on GS activity. XC 38 reached the maximum value (0.52 U·g⁻¹) under N15 treatment, which was significantly different from other treatments (p < 0.05), but there was no significant difference between Nck and N5 treatment. XC 49 reached the maximum (0.48 U·g⁻¹) under N25, which was significantly different from other treatments and 1.45~68.66% higher than other treatments (Figure 3b,d).

In the two-year experiment, the GS activity of XC 38 was always higher than that of XC 49, and the maximum GS activity of XC 38 was 8.56% higher than that of XC 49; the maximum GS activity of the two varieties in 2019 was 18.10% and 11.26% higher, respectively, than that in 2018. The interaction of year, variety, and N treatment had a significant effect on GS activity (Table 2).

3.3. Glutamate-Pyruvate Aminotransferase (GPT) Activity of Spring Wheat Grain

The change trend of GPT activity was the same as that of NR and GS activities, which reached the maximum value on the 7th day after anthesis, and then gradually decreased with the growth period (Figure 4). In different N reduction treatments, the GPT activity XC 38 decreased in the order N15 > N10 > N5 > Nck > N20 > N25 > N0 (Figure 4a,c). The difference between N15 and other treatments was significant (p < 0.05). The GPT activity of XC 38 under N15 treatment was 0.82 U·g⁻¹ (2018) and 0.94 U·g⁻² (2019). N15 treatment increased 1.79~50.89% compared with other treatments. The GPT activity of XC 49 decreased in the order N25 > N20 > N15 > N10 > N5 > Nck > N0 (Figure 4b,d). Under N25 treatment, the GPT activity of XC 49 was 0.70 U·g⁻¹ (2018) and 0.77 U·g⁻¹ (2019). N25 treatment increased 2.35~44.32% compared with other treatments.

The maximum value of XC 38 was 21.28% higher than that of XC 49. Compared with different years, the maximum value of the two varieties in 2019 increased 14.98% and 10.31%, respectively, compared with that in 2018. The interaction of year, variety, and N had a significant effect on GPT activity (Table 2).

3.4. Protein Content of Spring Wheat Grain

The results showed that grain protein content first decreased and then increased gradually after the 28th day after anthesis, showing a “V”-shape change pattern with time after anthesis (Figure 5). Under different fertilization management, the optimal value of grain protein content of XC 38 was 12.29% (2018) and 14.89% (2019). The changes in spring wheat grain protein content in the two years decreased in the order N15 > N10 > N5 > Nck > N20 > N25 > N0 (Figure 5a,c). At the 35th day after anthesis (mature stage), XC 38 under N15 treatment had significant differences compared with other treatments. The grain protein content was 6.16~28.78% higher than that in other treatments. The change trend of XC 49 was N25 > N20 > N15 > N10 > N > Nck > N0 (Figure 5b,d). At the 35th day after anthesis, the GPT activity of XC 49 under N25 treatment was 11.07% (2018) and 12.24% (2019). N25 treatment significantly increased, by 3.63~22.32%, compared with other treatments.

The maximum value of XC 38 was 21.67% higher than that of XC 49. Comparing the different years, the maximum value of the two varieties in 2019 increased 21.16% and 10.52%, respectively, compared with that in 2018. The interaction of year, variety, and N treatment had a significant effect on protein content (Table 2).

3.5. Protein Components of Spring Wheat Grain

Different N treatments had significant effects on the content of protein components in wheat grains (

Table 3. Averages of protein components in 2018 and 2019.

Year (Y)	Varieties (V)	Treatment (T)	Content of Albumin (%)	Content of Globulin (%)	Content of Gliadin (%)	Content of Glutelin (%)
2018	XC 38	Nck	2.28 ± 0.12 ab	1.34 ± 0.07 ab	2.74 ± 0.06 b	4.33 ± 0.13 a
		N5	2.29 ± 0.12 ab	1.35 ± 0.04 ab	2.75 ± 0.03 ab	4.34 ± 0.07 a
		N10	2.39 ± 0.16 a	1.42 ± 0.08 a	2.88 ± 0.04 a	4.43 ± 0.06 a
		N15	2.42 ± 0.11 a	1.43 ± 0.08 a	2.89 ± 0.08 a	4.44 ± 0.09 a
		N20	2.24 ± 0.11 ab	1.31 ± 0.05 bc	2.71 ± 0.07 bc	4.32 ± 0.12 a
		N25	2.24 ± 0.1 ab	1.29 ± 0.04 bc	2.68 ± 0.11 bc	4.28 ± 0.08 b
		N0	2.13 ± 0.14 b	1.21 ± 0.01 c	2.59 ± 0.1 c	4.28 ± 0.1 b
	XC 49	Nck	1.83 ± 0.03 b	1.14 ± 0.03 bc	2.6 ± 0.02 bc	3.49 ± 0.11 bc
		N5	1.86 ± 0.01 b	1.16 ± 0.09 abc	2.65 ± 0.04 b	3.5 ± 0.07 bc
		N10	1.87 ± 0.02 b	1.18 ± 0.03 abc	2.67 ± 0.02 b	3.53 ± 0.01 bc
		N15	1.88 ± 0.02 b	1.19 ± 0.1 abc	2.68 ± 0.02 b	3.56 ± 0.07 ab
		N20	1.98 ± 0.03 a	1.27 ± 0.02 ab	2.8 ± 0.09 a	3.67 ± 0.04 a
		N25	1.99 ± 0.03 a	1.28 ± 0.12 a	2.81 ± 0.02 a	3.68 ± 0.02 a
		N0	1.78 ± 0.05 c	1.11 ± 0.03 c	2.54 ± 0.03 c	3.41 ± 0.09 c
2019	XC 38	Nck	2.54 ± 0.09 bc	1.53 ± 0.1 b	3.11 ± 0.13 bc	4.75 ± 0.03 bc
		N5	2.63 ± 0.07 b	1.6 ± 0.05 b	3.18 ± 0.08 bc	4.86 ± 0.06 b
		N10	2.66 ± 0.09 b	1.64 ± 0.17 b	3.2 ± 0.1 b	4.88 ± 0.09 b
		N15	2.97 ± 0.11 a	1.81 ± 0.1 a	3.54 ± 0.07 a	5.41 ± 0.03 a
		N20	2.43 ± 0.09 cd	1.46 ± 0.06 bc	2.99 ± 0.15 cd	4.61 ± 0.14 c
		N25	2.34 ± 0.08 d	1.33 ± 0.07 cd	2.84 ± 0.08 d	4.43 ± 0.09 d
		N0	2.17 ± 0.11 e	1.27 ± 0.08 d	2.6 ± 0.1 e	4.22 ± 0.09 e
	XC 49	Nck	1.96 ± 0.08 d	1.22 ± 0.09 bc	2.75 ± 0.12 cd	3.6 ± 0.12 cd
		N5	1.2 ± 0.07 cd	1.27 ± 0.11 bc	2.95 ± 0.13 bc	3.76 ± 0.09 bc
		N10	2.12 ± 0.14 bc	1.34 ± 0.13 b	3.05 ± 0.1 b	3.9 ± 0.14 b
		N15	2.18 ± 0.13 b	1.36 ± 0.07 b	3.06 ± 0.12 b	3.93 ± 0.13 b
		N20	2.2 ± 0.13 b	1.37 ± 0.11 b	3.12 ± 0.11 b	3.94 ± 0.06 b
		N25	2.52 ± 0.11 a	1.56 ± 0.05 a	3.38 ± 0.11 a	4.52 ± 0.08 a
		N0	1.82 ± 0.06 e	1.14 ± 0.06 c	2.57 ± 0.10 d	3.46 ± 0.09 d
F		Y	**	**	**	**
		V	**	**	**	**
		T	**	**	**	**
		Y × V	**	ns	ns	ns
		Y × N	**	ns	**	**
		V × N	**	**	**	**
		Y × V × N	**	ns	**	**

XC 38: Xinchun 38; XC 49: Xinchun 49. Nck: 300 kg hm⁻²; N5: 285 kg hm⁻²; N10: 270 kg hm⁻²; N15: 255 kg hm⁻², N20: 240 kg hm⁻², N25: 225 kg hm⁻², N0: 0 kg hm⁻². V: Variety, N: Nitrogen treatments, and Y: Year. “**” indicates significant difference at 0.01 level; ns indicates insignificant. Different lowercase letters indicate significant differences at the 0.05 level.

). The two-year experiment showed that the change trend of protein component content was similar under different nitrogen treatments, but the response of varieties to N fertilizer was different. With the decrease in N application, the protein components of XC 38 reached the maximum under the N15 treatment. The albumin, globulin, gliadin, and glutenin increased 6.61~25.26%, 5.90~30.41%, 5.62~23.65%, and 5.72~15.92%, respectively, compared with other treatments. However, the protein components of XC 49 decreased in the order N25 > N20 > N15 > N10 > N5 > Nck > N0. The N25 treatment showed a significant difference compared with the other treatments (2019). Under the N25 treatment, the albumin, globulin, gliadin, and glutenin of XC 49 increased 7.86~47.37%, 7.27~26.13%, 4.67~21.43%, 7.85~19.50% on average, respectively, compared with the other treatments.

The protein component content of XC 38 under the optimal treatment increased 17.87%, 16.11%, 4.65%, and 19.64%, respectively, compared with XC 49. The maximum protein component content of XC 38 (22.87%, 26.45%, 22.72%, 22.00%) and XC 49 (26.71%, 21.46%, 20.30%, 22.90%) in 2019 increased compared with that in 2018. Year, variety, and nitrogen treatment had significant effects on protein components. The interaction between variety and nitrogen treatment had significant effects on protein components, while the interaction of year, variety, and treatment had significant effects on albumin, gliadin, and glutenin, but no significant effect on globulin.

3.6. Yield, Yield Components, and Protein Yield of Spring Wheat

It can be seen from

Table 4. Averages of yield of spring wheat under drip irrigation in 2018 and 2019.

Year (Y)	Variety (V)	Nitrogen (N)	1000-Grain Weight (g)	Spike Number (×104·hm−2)	Grain Number per Spike	Actual Yield (kg·hm−2)	Protein Yield (kg·hm−2)
2018	XC 38	Nck	42.51 ± 0.87 bc	437.28 ± 9.87 bc	39.15 ± 0.93 a	6915.10 ± 9.52 d	729.78 ± 1.00 d
		N5	42.81 ± 1.33 abc	444.61 ± 13.47 ab	39.34 ± 2.29 a	7035.70 ± 5.08 c	742.51 ± 0.54 c
		N10	44.15 ± 0.69 ab	455.13 ± 10.47 a	40.41 ± 1.12 a	7108.20 ± 8.05 b	750.16 ± 0.85 b
		N15	45.36 ± 1.42 a	455.85 ± 3.07 a	40.33 ± 0.58 a	7355.70 ± 15.24 a	776.28 ± 1.61 a
		N20	41.51 ± 1.9 bc	432.18 ± 7.03 bc	38.98 ± 1.51 a	6892.40 ± 14.31 e	727.39 ± 1.51 e
		N25	41.32 ± 1.82 c	431.98 ± 5.74 bc	38.09 ± 2.13 a	6862.50 ± 5.29 f	724.23 ± 0.56 f
		N0	40.65 ± 1.56 c	426.42 ± 1.81 c	37.61 ± 1.00 a	4047.40 ± 5.55 g	427.14 ± 0.59 g
	XC 49	Nck	37.03 ± 1.5 bc	386.12 ± 12.26 ab	36.14 ± 1.12 ab	6537.90 ± 7.12 e	689.98 ± 0.75 e
		N5	37.23 ± 0.49 bc	386.85 ± 6.05 ab	37.06 ± 2.4 ab	6587.50 ± 17.69 d	695.21 ± 1.87 d
		N10	37.32 ± 0.92 bc	387.45 ± 11.87 ab	37.57 ± 1.14 ab	6678.20 ± 13.24 c	704.78 ± 1.40 c
		N15	37.51 ± 0.87 bc	397.86 ± 6.60 a	38.14 ± 2.35 ab	6787.80 ± 12.96 b	716.35 ± 1.37 b
		N20	39.02 ± 1.76 ab	398.25 ± 10.59 a	39.54 ± 2.22 a	6795.10 ± 10.21 b	717.12 ± 1.08 b
		N25	39.94 ± 1.41 a	398.54 ± 8.14 a	39.12 ± 2.31 ab	6822.60 ± 8.32 a	720.02 ± 0.88 a
		N0	36.57 ± 1.03 c	375.65 ± 8.61 b	35.56 ± 1.13 b	3984.20 ± 18.40 f	420.47 ± 1.94 f
2019	XC 38	Nck	42.39 ± 0.61 ab	422.10 ± 8.38 abc	37.39 ± 2.85 a	6999.71 ± 13.48 d	912.88 ± 1.76 b
		N5	42.77 ± 2.01 ab	423.75 ± 3.42 abc	37.88 ± 2.67 a	7147.39 ± 7.32 c	932.14 ± 0.96 b
		N10	44.39 ± 0.88 ab	432.52 ± 4.66 ab	39.46 ± 4.01 a	7216.03 ± 6.72 b	941.09 ± 0.88 a
		N15	45.22 ± 1.69 a	437.90 ± 3.77 a	39.11 ± 3.78 a	7397.39 ± 8.03 a	964.74 ± 1.05 a
		N20	42.05 ± 2.84 ab	420.78 ± 3.57 abc	37.25 ± 1.33 a	6931.4 ± 8.65 e	903.97 ± 1.13 c
		N25	41.56 ± 2.22 b	420.39 ± 15.79 bc	36.83 ± 1.55 a	6882.17 ± 6.49 f	897.55 ± 0.85 c
		N0	38.24 ± 1.31 c	414.93 ± 13.33 c	35.21 ± 0.93 a	4083.30 ± 19.74 g	532.53 ± 2.57 d
	XC 49	Nck	40.13 ± 2.07 ab	399.26 ± 10.67 a	36.62 ± 1.74 ab	6567.50 ± 4.97 d	856.51 ± 0.65 d
		N5	40.54 ± 2.18 ab	399.46 ± 10.26 a	37.24 ± 0.86 ab	6724.60 ± 8.31 c	877.00 ± 1.08 c
		N10	40.94 ± 2.41 ab	401.94 ± 10.66 a	37.27 ± 2.29 ab	6755.50 ± 4.28 b	881.03 ± 0.56 b
		N15	41.13 ± 1.58 ab	403.78 ± 5.40 a	37.48 ± 2.85 ab	6778.87 ± 16.13 a	884.08 ± 2.10 a
		N20	42.48 ± 2.99 ab	409.54 ± 6.84 a	38.79 ± 0.69 a	6821.09 ± 13.36 e	889.58 ± 1.74 e
		N25	43.73 ± 2.97 a	414.68 ± 10.68 a	38.09 ± 1.77 ab	6844.40 ± 8.86 f	892.62 ± 1.15 f
		N0	37.44 ± 3.61 b	397.29 ± 15.19 a	34.82 ± 1.46 b	3994.50 ± 10.47 g	520.95 ± 1.37 g
F		Y	**	ns	*	**	**
		V	**	**	*	**	**
		T	**	**	**	**	**
		Y × V	**	**	ns	**	**
		Y × N	ns	ns	ns	**	**
		V × N	**	**	ns	**	**
		Y × V × N	ns	ns	ns	**	**

XC 38: Xinchun 38; XC 49: Xinchun 49. Nck: 300 kg hm⁻²; N5: 285 kg hm⁻²; N10: 270 kg hm⁻²; N15: 255 kg hm⁻², N20: 240 kg hm⁻², N25: 225 kg hm⁻², N0: 0 kg hm⁻². V: variety, N: Nitrogen treatments. “*” and “**” indicate significant difference at 0.05 and 0.01 levels; ns indicates insignificant. Different lowercase letters indicate significant differences at the 0.05 level.

that with the reduction in N application, the yield, yield composition, and protein yield all increased first and then decreased. The 1000-grain weight and spike number of XC 38 reached the maximum value under the N15 treatment, increasing 2.30~14.82% and 0.69~6.23%, respectively, compared with the other treatments. The grain number per spike reached the maximum value under the N10 treatment, and was 0.54~9.67% higher than that in the other treatments. The nitrogen treatment had a significant impact on the yield and protein yield. The yield and protein yield of XC 38 reached the maximum under the N15 treatment, and there was a significant difference with other treatments, increasing 2.99~81.45% and 2.94~81.42%, respectively. In the two years, under the N15 treatment, the yield and protein yield in 2019 increased 0.57% and 24.28%, respectively, compared with those in 2018.

The 1000-grain weight and spike number of XC 49 reached the peak under the N25 treatment. Compared with the other treatments, the 1000-grain weight and spike number increased 2.66~13.04% and 0.67~5.21% on average, respectively. The grain number per spike reached the maximum under the N20 treatment, and was 1.46~11.31% higher than that in the other treatments. Nitrogen fertilizer had a significant impact on the yield and protein yield. Among the N fertilizer treatments, the yield and protein yield under the N25 treatment reached the maximum, and were 0.37~71.29% and 0.37~71.30%, respectively.

Nitrogen treatment had no significant effect on the grain number per spike in 2018, or the spike number and grain number per spike in 2019. The interaction of varieties and N fertilizer had a significant effect on the 1000-grain weight, the spike number, the yield, and the protein yield in 2018, and had a significant effect on the yield and protein yield in 2019.

3.7. Correlation between Grain Indexes of Spring Wheat

The N application rate had a significant effect on the nitrogen metabolism and protein content of spring wheat grains, and the correlation between each index is shown in Figure 6. The activities of nitrogen-metabolizing enzymes (NR, GS, GPT) were significantly correlated with grain protein content and protein components (albumin, globulin, gliadin, glutenin). The yield and protein yield were significantly correlated with the activities of nitrogen-metabolizing enzymes and protein content.

In the range of 0~300 kg hm⁻² of nitrogen application, the grain yield and protein yield of the two wheat varieties increased with the increase in nitrogen application. After reaching a certain threshold, they did not continue to increase, but tended to decrease. The relationship between grain yield, protein yield, and nitrogen application of the two wheat varieties could be simulated by quadratic parabolic equation, and the fitting effect reached a very significant level (Figure 7). Through the regression equation, the theoretical nitrogen application amount for the two varieties to obtain the highest grain yield was calculated: for XC 38 it was 276 kg hm⁻², and for XC 49 it was 229 kg hm⁻². The theoretical nitrogen application amount for the two varieties to obtain the highest grain protein yield was 276 kg hm⁻² for XC 38 and 228 kg hm⁻² for XC 49. The grain yield of XC 38 was higher than that of XC 49, indicating that XC 38 was more sensitive to nitrogen fertilizer.

4. Discussion

4.1. Effects of Fertilizer Management on Activities of Key Enzymes for Nitrogen Metabolism in Spring Wheat Grain

Nitrogen (N) is one of the macronutrients necessary for plant growth and development [28,29]. After absorbing nitrogen, plants need to go through a series of metabolic processes to synthesize the nutrients they need, such as protein, nucleic acid, and other nitrogen compounds [30]. Studies have shown that nitrate reductase (NR), glutamine synthetase (GS), and glutamate-pyruvate aminotransferase (GPT) are key enzymes in the N metabolism process. Increasing their enzyme activities can promote N metabolism in plants and promote the synthesis and transformation of proteins [31]. NR is the starting factor and rate-limiting enzyme in the N metabolism, and can regulate the transformation of inorganic nitrogen absorbed by plants into organic nitrogen. Its activity is positively correlated with N accumulation and protein content [32]. In higher plants, about 95% of NH₄⁺ is assimilated to form amino acids through the GS/GOGAT cycle. Amino acids are the main form of nitrogen in plants and transport. GS is one of the key enzymes in this cycle [33]. In this study, with the decrease in N application, the N-metabolizing enzymes’ activities in the grain of the two varieties first increased and then decreased. Proper N reduction significantly increased the NR, GS, and GPT activities, which was consistent with the results of Effah et al. [34]. However, we observed that XC 38 and XC 49 had different responses to the grain N-metabolizing enzymes’ activities of nitrogen fertilizer. Strong gluten wheat XC 38 reached the maximum value under the N15 treatment, while medium gluten wheat reached the optimal value under the N25 treatment. The NR, GS, and GPT activities of XC 38 were 4.74%, 8.56%, and 21.28% higher than those of XC 49, respectively (Figure 2, Figure 3 and Figure 4). This finding indicates that different wheat varieties had different responses to N fertilizer, and there were significant differences in the degree of N metabolism. In actual production, the best amount of nitrogen should be determined according to different varieties. Related analysis indicated that there was a strong association between GS, NR, and GPT activities and grain yield, which was in accordance with results reported by Ma et al. [35].

4.2. Effects of Fertilizer Management on Protein Content and Protein Components in Spring Wheat Grain

N accumulation is the basis for the formation of wheat protein [36,37,38]. Protein is the material formed by the decomposition of nitrogen in wheat vegetative organs into amino acids, then transferred to grain, and converted into other types of amino acids [39]. The research showed that the protein content presented a “high-low-high” change with the growth period. Grain protein content increased with the increase in N application in the field, and showed a very significant positive correlation [40]. When the N application rate increased from 0 to 300 kg hm⁻², the grain protein content first increased and then decreased. XC 38 reached the maximum value under the N15 treatment, and XC 49 reached the peak under the N25 treatment. The main purpose of traditional agricultural production is to improve the grain yield. Equally important is that the protein content in grain increases significantly, thus improving its nutritional value [41,42]. Tuener et al. [43] suggested that the protein content in grain should be higher than 12.5%, so it is particularly important to determine the optimal amount of N application. In this experiment, under the N15 treatment, the grain protein content of XC 38 was about 12.5% at the 35th day after anthesis, while that of XC 49 was less than 12.5%. This indicates that the response of grain protein content changes to nitrogen was obviously different among different varieties. High protein content and more sensitivity to N application are among the characteristics of strong gluten wheat, and among the reasons why the protein content and protein components of XC 38 are higher than those of XC 49 [44].

N not only affects the quantity of grain protein, but also has a significant regulatory effect on the quality of protein; that is, it has a regulatory effect on the proportion and content of protein components in grains. However, previous conclusions are not consistent. Zhao et al. [45] found that with the increase in N application, gliadin and glutenin in wheat grain increased to varying degrees, but albumin and globulin were not sensitive to nitrogen. Some studies also showed that with the increase in nitrogen application, the albumin and glutenin content in grain decreased, while the globulin and gliadin content increased [46]. However, some studies showed that the content of protein components increased significantly with the increase in N application. In our study, within a certain range, a high N application rate had a significant effect on improving the albumin, globulin, gliadin, and glutenin content in wheat grain. The protein component content was in the order of strong gluten wheat (XC 38) > medium gluten wheat (XC 49). The protein content and component content of the two types of wheat grain reached the highest value under N15 and N25 treatments, respectively. The grain protein content was significantly related to albumin, globulin, gliadin, and glutenin. This indicates that the influence of nitrogen applied on the content of each protein component was also one of the reasons for the change in total protein content [47].

4.3. Effects of Fertilizer Management on Grain Yield and Protein Yield in Spring Wheat Grain

Applying N fertilizer is one of the effective ways to rapidly increase crop yield. Reasonable application of nitrogen fertilizer can improve the population structure of crops and promote the increase in yield and protein yield [48]. Some studies found that the wheat grain yield was mostly influenced by the grain number per spike and the 1000-grain weight [49]. Other experiments showed that the yield of wheat is mainly determined by the grain number per spike and the spike number, and that the grain number per spike is mainly determined by the genetic characteristics of varieties [50]. Furthermore, the amount of nitrogen applied was found to be one of the reasons for increasing the grain number per spike and the spike number [51]. In this experiment, the 1000-grain weight and spike number of XC 38 and XC 49 reached the maximum under N15 and N25 treatments, respectively. The grain yield and protein yield of different types of wheat first increased and then decreased with the increase in N application [52]. In our study, the response of grain yield and protein yield to the amount of N application conforms to a quadratic curve, and the amount of N application determined 98.13% and 99.89% of the grain yield of XC 38 and XC 49, respectively, and 66.07% and 65.54%, respectively, of the protein yield (Figure 7). According to the regression curve, the highest yield and protein yield were acquired at 276 and 229 kg hm⁻², respectively. Similarly to the results of Abad et al. [53], we found that the grain yield achieved a gentle level when the N application rate was low compared with the protein content. Therefore, under certain circumstances, a suitable nitrogen level is an effective way to boost grain protein content without causing a decline in yield. Compared with the conventional N application rate, the grain yield and protein yield of different quality types of wheat performed better under the condition of moderately reducing the nitrogen application rate. Specifically, the ranking was strong gluten wheat > medium gluten wheat, which indicates that strong gluten varieties were more sensitive to nitrogen fertilizer and more effective in increasing yield.

5. Conclusions

Our results show that chemical nitrogen reduction can improve three key enzymes (NR, GS, and GPT) of wheat nitrogen metabolism, protein content, and yield. Strong gluten (XC 38) and medium gluten (XC 49) wheat showed the best performance under N15 (255 kg hm⁻²) and N25 (225 kg hm⁻²) treatments, respectively, with the highest enzyme activity, protein content, and wheat grain yield. The enzyme activity, protein content, and yield of strong gluten wheat XC38 were higher than those of medium gluten wheat XC 49.

Because the experiment deals with the three specific research questions, it can be concluded that treatment N15 and XC38 were the optimal fertilization treatment and cultivar, respectively, for drip-irrigated spring wheat production in northern Xinjiang. Future research needs to be undertaken to further determine the economic and environmental benefits of this work, and to assess whether the studied approach can be widely applied in local wheat production and similar arid and semi-arid regions.