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

Agriculture 2022, 12(11), 1891; https://doi.org/10.3390/agriculture12111891

by Rongrong Wang^1,2, Haiqi Wang^1,2, Guiying Jiang^1,2,*, Haojie Yin^1,2 and Ziqiang Che^1,2

Reviewer 1:

Mohamed Moussa

Reviewer 2:

Nawroz Tahir

Reviewer 3:

Nataša Ljubičić

Agriculture 2022, 12(11), 1891; https://doi.org/10.3390/agriculture12111891

Submission received: 9 October 2022 / Revised: 4 November 2022 / Accepted: 8 November 2022 / Published: 10 November 2022

(This article belongs to the Section Crop Production)

Round 1

Reviewer 1 Report

Comments to Authors:

The study is in the journal's scope and its title reflects the objectives of the work performed. The subject is of high scientific interest. However, the sections of the paper such as material and/or results i.e., are writing in confused. The English language should be revised, and some information should be completed.

Specific comments:

Title. It reflects the content of the study, but the English can be improved.
Title: suggest rewording to: " Effects of Nitrogen Application Strategy on Nitrogen Enzyme Activities and Protein Content in Spring Wheat Grain".

Abstract:

The abstract writing should be improved the conclusions and findings of the abstract are not clear, furthermore, the results are confusing. Need to rewrite it again.

Introduction:

The Nitrogen should be written the full name the first time and then written by the abbreviation.

“storyline” of the introduction needs to improve.

The hypothesizes at the end of the Introduction section are not clear.

M&M section:

Line 119 to 120 you mean to prevent the fertilizer and water leaching. Is it right?

The section (2.2. Experimental design and management) please revise and improve it.

Please mention the scientific name of wheat.

Check for complete information: Name of the instrument, Name of the company, city, and country.

Where is the soil analysis data before the cultivation of wheat plants?

- Could the authors explain why in Figures have used standard deviation while the Tables don’t have used it?

- Check the Figures caption write the full name of the abbreviation and complete it.

Results:

The whole Results section is written rather repetitively, and readers are challenged to keep on reading! You need to rewrite this section so that it is attractive to the reader. When describing your results, you nearly always use “relative comparisons”.

The quality of the figures should be improved! Special figures 6 and 7.

Discussion:

Line 341, write the full name first and then write the abbreviated the first time.

Lines 355 to 357, refer to figures or tables.

Somehow you fail to really interpret your treatment effects. So please rewrite the discussion section again and try writing sample sentences.

Conclusion:

Rather general and repeating aspects from the Results & Discussion sections!

L447 and 452: You do not show any result on “which N amount or treatment could be proper for wheat grain yield and more effectiveness on N enzymes and metabolism.

Author Response

Dear Editor and Reviewers:

On behalf of my co-authors, we are very grateful to you for giving us an opportunity to revise our manuscript. we appreciate you very much for your positive and constructive comments and suggestions on our manuscript entitled "Effects of nitrogen application strategy on key enzyme activities of nitrogen metabolism and protein content in drip-irrigated spring wheat grain" (ID: agriculture-1988846).

We have studied reviewers’ comments carefully and tried our best to revise our manuscript according to the comments. The following are the responses and revisions I have made in response to the reviewers' questions and suggestions on an item-by-item basis. Thanks again to the hard work of the editor and reviewer!

Reviewer #1

Comment No.1: Title: It reflects the content of the study, but the English can be improved.
Title: suggest rewording to: "Effects of Nitrogen Application Strategy on Nitrogen Enzyme Activities and Protein Content in Spring Wheat Grain".

Response: Thanks to reviewer for suggestions, we have revised the title in the manuscript.

Comment No.2: The abstract writing should be improved the conclusions and findings of the abstract are not clear, furthermore, the results are confusing. Need to rewrite it again.

Response: Thank you for your valuable comments. We have revised the abstract. After modification: “In order to determine the regulatory effect of different nitrogen (N) fertilizer application rates on the grain nitrogen 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. Under the nitrogen application levels of 300 (Nck), 285 (N5), 270 (N10), 255 (N15), 240 (N20), 225 (N25) and 0 (N0) kg hm^-2. The effects of nitrogen 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 increased first and then decreased with the decrease of nitrogen application. The response of the two varieties to nitrogen application was different. The indexes of XC 38 reached the maximum under N15 treatment, and the yield increased by 2.99%~81.45%. The yield of XC 49 was increased by 0.37%~71.29% under N25 treatment. Under the same nitrogen level, all indicators of XC38 were better than XC 49. Correlation analysis showed that the yield and protein yield were significantly positive correlated with NR, GS and GPT. The interaction of nitrogen fertilizer and varieties had significant effects on NR, GS, GPT, protein content, components and yield. These results showed that the protein content and yield of wheat grain could be improved by adjusting the nitrogen fertilizer application strategy reasonably.”

Comment No.3: Introduction: The Nitrogen should be written the full name the first time and then written by the abbreviation “storyline” of the introduction needs to improve The hypothesizes at the end of the Introduction section are not clear.

Response: Thank you for your questions. The full name of nitrogen has been added at the first occurrence. The last paragraph in the Introduction has been revised. The revised content: “Several studies have reported positive effects of moderate reduced N fertilization on wheat growth and wheat grain protein content (Zörb et al., 2018; Ayadi et al., 2022). However, excessive nitrogen supplementation is still prevalent in the region, leading to reduced nitrogen use efficiency, thereby increasing production costs and contaminates soil and groundwater. Therefore, we conducted a two-year positioning experiment examining the effects of different N fertilisation strategies on drip-irrigated spring wheat yield in Xinjiang. The objectives were：(i) to investigate the response of drip-irrigated spring wheat yield, grain enzyme activity and protein content to N fertilizer application; (ii) to quantify the relationships between enzyme activity, protein content and yield; and (iii) to determine the optimal N regime for this region under drip conditions.”

Comment No.4: Line 119 to 120 you mean to prevent the fertilizer and water leaching. Is it right?

Response: Thank you very much for reading and giving us valuable comments on our manuscript. As you said, we buried an impermeable membrane with a depth of 1 m between the communities to prevent water and fertilizer from seeping out.

Comment No.5: The section (2.2. Experimental design and management) please revise and improve it.

Response: Thank you for your valuable comments. We have modified the section (2.2. Experimental design and management). The revised contents are as follows: 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 which 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%), as the widely cultivated wheat cultivars in Xinjiang province of China. Seven N fertilizer treatments were set, including Nck: normal N supply during the growing period (300 kg hm^-2is 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 2.

Table 2 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 length and 3 m width. 100 cm deep anti-seepage membrane shall be 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 whole wheat growth seasons., with a total of 9 times of irrigation. The irrigation amount in each period is accurately controlled through the 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 a way of "four rows per tube", with a 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 harvested on 10 July 2018 and 12 July 2018. Other field management was the same as field production.”

Comment No.6: Please mention the scientific name of wheat.

Response: Thank you for your suggestion. We have indicated the scientific name of wheat in the Experimental design and management. The specific contents are: “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.) varieties 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%), the main varieties in Xinjiang, China.”

Comment No.7: Check for complete information: Name of the instrument, Name of the company, city, and country.

Response: Thank you for your question. We have supplemented the instrument name, company name, city and country after the corresponding instrument. Specific contents are as follows: “Four protein fractions (albumins, globulins, gliadins and glutenins) were separated and analyzed according to the method of Liu et al. (2005). Grain protein content was calculated as nitrogen concentration multiplied by 5.7 , which was determined using Kjeldahl apparatus (K9840 Kjeldahl apparatus, Hanon, Shandong, China).”

Comment No.8: Where is the soil analysis data before the cultivation of wheat plants?

Response: Thank you very much for reading and making suggestions on our manuscript. We have put the soil analysis data before wheat planting in the supplementary document, and the specific contents are supplemented as follows:

Table. 1 Parameter of soil at 0–60 cm soil depth in the experimental plots.

Parameter	Year
Parameter	2018	2019
Clay (%)	18	19
Silt (%)	33	31
Sand (%)	40	39
pH	7.6	7.4
Organic matter (g kg^-1)	28.4	27.2
Alkaline-N (mg kg^-1)	7.3	6.9
Olsen-P (mg kg^-1)	15.2	14.9
Available K (mg kg^-1)	159.6	159.4

Comment No.9: Could the authors explain why in Figures have used standard deviation while the Tables don’t have used it?

Response: Thank you very much for your questions. We have added the standard deviation in the table, which is detailed in Table 4 and Table 5 in the manuscript.

Comment No.10: Check the Figures caption write the full name of the abbreviation and complete it.

Response: Thank you very much for your comments. We have supplemented the full name of the abbreviation in the Figures caption in the manuscript.

Comment No.11: The whole Results section is written rather repetitively, and readers are challenged to keep on reading! You need to rewrite this section so that it is attractive to the reader. When describing your results, you nearly always use “relative comparisons”.

Response: Thank you for your comments. We have made some modifications to the results and analysis. The revised contents are as follows:

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 trend 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 7th to 35th days after anthesis. With the decrease of N application, the NR activity in XC 38 grains decreased in the order N15 > N10 > N5 > Nck > N20 > N25 > N0 (Figure 2 a and 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 of N application, the NR activity of N25 treatment was the largest (Figure. 2 b and d), which was significantly higher than that of other treatments (P < 0.05), and the maximum value was 0.77 U·g^-¹. Under N25 treatment, the NR activity increased 1.19%~58.98% compared 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, while the maximum NR activity of the two varieties in 2019 was 10.03% and 8.70% higher than that in 2018. The interaction of year, variety and N treatment had significant effect on NR activity (Table 3).

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 at the 7th day after anthesis (Figure 3). The change trend of each treatment in 2018 and 2019 is similar, and N fertilizer had a significant impact on GS activity. XC 38 reached the maximum value (0.52 U·g^-¹) at 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) at N25, which was significantly different from other treatments and 1.45%~68.66% higher than other treatments (Figure 3 b and 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, while the maximum GS activity of the two varieties in 2019 was 18.10% and 11.26% higher than that in 2018. The interaction of year, variety and N treatment had a significant effect on GS activity (Table 3).

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 4 a and c). The difference between N15 and other treatments was significant (P<0.05). The GPT activity of XC 38 under N15 treatment were 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 4 b and d). At N25 treatment, the GPT activity of XC 49 were 0.70 U·g^-¹ (2018) and 0.77 U·g^-¹ (2019), respectively. 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 3).

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 were 12.29 % (2018) and 14.89 % (2019), respectively. The changes of spring wheat grain protein content in the two years decreased in the order N15>N10>N5> Nck>N20>N25>N0 (Figure 5 a and c). At 35th days 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 other treatments. The change trend of XC 49 was N25>N20>N15>N10>N5>Nck>N0 (Figure 5 b and d). At 35th day after anthesis, the GPT activity of XC 49 under N25 treatment were 11.07% (2018) and 12.24% (2019), respectively. N25 treatment significantly increased 3.63%~22.32% compared with other treatments.

The maximum value of XC 38 was 21.67% higher than that of XC 49. Compared with 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 significant effect on protein content (Table 3)

Comment No.12: The quality of the figures should be improved! Special figures 6 and 7.

Response: Thank the reviewers for their suggestions on our manuscript. We have replaced the figures in the manuscript with higher resolution and pixel. See Figures 2-7 in the manuscript for details.

Comment No.13: Line 341, write the full name first and then write the abbreviated the first time.

Response: Thank you for this comment and question. The full names of three nitrogen metabolizing enzymes have been added.

Comment No.14: Lines 355 to 357, refer to figures or tables.

Response: Thank the review experts for reminding. The corresponding content has been filed. The specific content “Strong gluten wheat XC 38 reached the maximum at N15 treatment, while medium gluten wheat reached the best at N25 treatment. NR, GS and GPT activities in XC 38 grain were 4.74%, 8.56% and 21.28% higher than XC 49 respectively (Fig. 2-4).”

Comment No.15: Somehow you fail to really interpret your treatment effects. So please rewrite the discussion section again and try writing sample sentences.

Response: Thank you for your comments. We have modified the discussion section. The revised contents are as follows: “

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 (Gonzalez-Dugo et al., 2010; Li et al., 2017). 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 (Ma et al., 2018). Studies have shown that nitrate reductase (NR), glutamine synthetase (GS), and glutamate-pyruvate aminotransferase (GPT) are key enzymes in the nitrogen metabolism process. Increasing their enzyme activities can promote nitrogen metabolism in plants and promote the synthesis and transformation of proteins (Chen et al., 2022). NR is the starting factor and rate limiting enzyme in the nitrogen metabolism, which can regulate the transformation of inorganic nitrogen absorbed by plants into organic nitrogen. Its activity is positively correlated with nitrogen accumulation and protein content (Tikhomirova et al., 1985; Thaluarn et al., 1988; Hou et al., 2019). In higher plants, about 95% of NH₄⁺is assimilated to form amino acids through 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 (Giunta et al., 2021). In this study, with the decrease of nitrogen application, the nitrogen metabolizing enzymes activities in the grain of the two varieties increased first and then decreased. Proper nitrogen reduction significantly increased the NR, GS and GPT activities, which was consistent with the results of Effah et al. (2022). However, we observed that XC 38 and XC 49 had different responses to the grain nitrogen metabolizing enzymes activities of nitrogen fertilizer. Strong gluten wheat XC 38 reached the maximum value at N15 treatment, while medium gluten wheat reached the optimal value at 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 (Fig. 2-4). This finding indicated that different wheat varieties had different responses to nitrogen fertilizer, and there were significant differences in the degree of nitrogen 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. (2019).

4.2. Effects of fertilizer management on protein content and protein components in spring wheat grain

Nitrogen accumulation is the basis for the formation of wheat protein (Fischer et al., 1993; Luo et al., 2018; Zhang et al., 2022). Protein is the material formed by the decomposition of nitrogen in wheat vegetative organs into amino acids, then transferred to grain, and then converted into other types of amino acids (Godfrey et al., 2010). The research showed that the protein content presented a "high-low-high" change with the growth period. Grain protein content increased with the increase of nitrogen application in the field, and showed a very significant positive correlation (Wu et al., 2019). When the nitrogen 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 at N15 treatment, and XC49 reached the peak value at N25 treatment. The main purpose of traditional agricultural production is to increase grain yield. Equally important is that the protein content in grain increases significantly, thus improving their nutritional value (Zhang et al., 2016, Liu et al., 2016). Tuener et al. (2004) suggested that the protein content in grain should be higher than 12.5%, so it is particularly important to determine the optimal amount of nitrogen application. In this experiment, under N15 treatment, the grain protein content of XC 38 was about 12.5% at 35th days after anthesis, while that of XC 49 was lower than 12.5%. This indicated that the response of grain protein content changes to nitrogen was obviously different among different varieties. High protein content and more sensitive to nitrogen application were one of the characteristics of strong gluten wheat, which was one of the reasons why XC 38 was higher than XC 49 in protein content and protein components (Reznick et al., 2021).

Nitrogen 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, but previous conclusions are not consistent. Zhao et al. (2009) found that with the increase of nitrogen 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 of nitrogen application, the albumin and glutenin content in grains decreased, while the globulin and gliadin content increased (Johansson et al., 2001). However, some studies showed that the content of protein components increased significantly with the increase of nitrogen application. In our study, within a certain range, high nitrogen application rate had a significant effect on improving the albumin, globulin, gliadin and glutenin content in wheat grains. The protein components content was strong gluten wheat (XC 38) > medium gluten wheat (XC 49). The protein content and component content of the two types of wheat grains reached the highest value at N15 and N25 treatments, respectively. The grain protein content was significantly related with albumin, globulin, gliadin and glutenin. This indicated that the influence of nitrogen applied on the content of each protein component was also one of the reasons for the change of total protein content (Luo et al., 2019).

4.3. Effects of fertilizer management on grain yield and protein yield in spring wheat grain

Applying nitrogen 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 of yield and protein yield (Giunta et al., 2019). Some studies believed that the wheat grain yield was mostly influenced by the grain number per spike and the 1000-grain weight (Zhang et al., 2016). The other experiments showed that the yield of wheat was mainly determined by the grain number per spike and the spike number, the grain number per spike is mainly determined by the genetic characteristics of varieties (Huang et al., 2020). While the amount of nitrogen applied was one of the reasons for increasing the grain number per spike and the spike number (Koppensteiner et al., 2022). 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 increased first and then decreased with the increase of nitrogen application (Lan et al., 2021). In our study, the response of grain yield and protein yield to the amount of nitrogen application conforms to a quadratic curve, and the amount of nitrogen application could determine 98.13% and 99.89% of the grain yield of XC 38 and XC 49, as well as 66.07% and 65.54% of the protein yield (Fig. 7). According to the regression curve, the highest yield and protein yield were acquired at 276 and 229 kg hm^-2. Similar to the results of Abad et al. (2004), we found that the grain yield achieved a gentle level when the nitrogen application rate was low compared with the protein content. Therefore, under certain circumstances, suitable nitrogen level was an effective way to boost grain protein content without causing yield decline. Compared with the conventional nitrogen 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 strong gluten wheat > medium gluten wheat, which indicated that strong gluten varieties were more sensitive to nitrogen fertilizer and more effective in increasing yield.”

Comment No.16: Rather general and repeating aspects from the Results & Discussion sections!

Response: Thank you for pointing out our problem. We have deleted the duplicate part in the manuscript and apologize again for our mistakes.

Comment No.17: L447 and 452: You do not show any result on “which N amount or treatment could be proper for wheat grain yield and more effectiveness on N enzymes and metabolism.

Response: Thank you very much for your valuable comments. As you said, our conclusions were not written strictly, and we did not clearly give the best treatment, So we change it to the following: "Our results showed that chemical nitrogen reduction can improve three key enzymes (NR, GS and GPT) of what nitrogen metropolis, protein content and yield. Strong glue (XC 38) and medium glue (XC 49) what showed the best performance at 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 when XC38 were higher than those of medium gluten when XC 49.” There were great differences among varieties and different responses to different nitrogen fertilizer treatments. Therefore, in this experiment, the best nitrogen application rate was found for the two varieties.

Reviewer 2 Report

Dear Authors:

The following changes are necessary for the manuscript:

Abstract

In general, this section is poorly written and arranged.

L10-11 (In order to explore the appropriate amount of nitrogen application to improve the efficient 10 production and quality of drip-irrigated wheat): This sentence needs to be modified because it is unclear and makes no sense.

The authors should describe the type of experimental design, components, and levels they utilized after stating the purpose of the study.

L20: The word application should add to the word nitrogen

The authors should discuss the impact of each factor and how they interact in this section with regard to the enzymes, protein content, and protein components.

L22 (While XC 49 had the best indicators under N25 treatment) What does this statement mean exactly?

The abstract should be finished with a succinct conclusion.

Keywords

It is inappropriate to mention the words that make up the manuscript's title here.

Introduction

L47-53 (currently …… Xinjiang.): This sentence should remove

The authors could include some details on wheat and its significance in terms of nutrition value in this section.

L67-76: These sentences ought to be deleted because they make no sense.

The authors should also include some details on the significance of drip irrigation and its connection to N application in this part.

Materials and methods

L96: The minimum and maximum temperature averages should be added.

L101: Why didn't the researchers choose a plant with a lower protein content?

Section 2.3 and 2.5: The number of plants utilized in the measurement should be mentioned by the authors.

Section 2.4: The age of the plants or samples used to determine the protein content should be mentioned by the authors.

L206 (2.6. Statistical analysis): Because there are differences between Two-Way ANOVA and Split plot design, the authors should identify the name of the experimental design they employed in their study.

Because this experiment repeated in two consecutive years, the authors should use the combined analysis.

Results

As seen in Table 1 and Figures 1–5, the authors chose to use factorial analyses rather than split analysis. To better comprehend the type of analysis utilized in their study, the authors ought to include the entire ANOVA table.

The authors used One-way ANOVA for the assessment of the impact of N treatments on the enzyme activities during various anthesis periods, as shown in the ANOVA in Figures 2–5. The authors should therefore confirm the procedure followed for the statistical data analysis.

The researchers only displayed the interactions between Variety X N applications and Year x Varities in Tables 1 and 2, respectively. The authors ought to display the outcomes of the interactions between Year x N applications as well.

Discussion

The organization of this section is poor. It should be improved by the authors. The mechanism of boosting enzyme activity and protein component by application of N should be described by the authors.

Best regards

Author Response

Reviewer #2

Comment No.1: L10-11 (In order to explore the appropriate amount of nitrogen application to improve the efficient 10 production and quality of drip-irrigated wheat): This sentence needs to be modified because it is unclear and makes no sense.

Response: Thank you very much for the suggestions of the evaluation experts. We revised this sentence to read "In order to determine the regulatory effect of different nitrogen fertilizer application rates on the grain nitrogen metabolism enzymes and protein content of drip-irrigated spring wheat under the climatic conditions in Xinjiang, China."

Comment No.2: The authors should describe the type of experimental design, components, and levels they utilized after stating the purpose of the study.

Response: Thank you very much for your suggestions. The Abstract is modified as " In order 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 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 increased first and then decreased with the decrease of N application. Furthermore, different respond to different application between different wheat varieties was also observed. The indexes of XC 38 reached the maximum at N15 treatment, and the yield increased 2.99%~81.45%. XC 49 showed the greatest indicators under N25 treatment and 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 positive correlated with NR, GS and GPT. The interaction of N fertilizer and varieties had significant effects on NR, GS, GPT, protein content, components and yield. These results showed that the protein content and yield of wheat grain could be improved by adjusting the N fertilizer application strategy reasonably."

Comment No.3: L20: The word application should add to the word nitrogen

Response: Thank you for your careful revision of our manuscript. We have made changes in corresponding positions. "The response of the two varieties to nitrogen application was different."

Comment No.4: The authors should discuss the impact of each factor and how they interact in this section with regard to the enzymes, protein content, and protein components.

Response: Thank you for your comment. Our manuscript studied the effects of different nitrogen fertilizer treatments on grain nitrogen metabolism enzyme activity, protein content, protein composition and yield. Nitrate reductase (NR), glutamine synthetase (GS), glutamate-pyruvate aminotransferase (GPT), protein content, albumin, globulin, glutenin, gliadin and yield all increased first and then decreased with the decrease of nitrogen application. The correlation analysis showed that there was significant correlation between each index (p<0.05). Revised summary: "In order to determine the regulatory effect of different nitrogen fertilizer application rates on the grain nitrogen 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. Under the nitrogen application levels of 300 (Nck), 285 (N5), 270 (N10), 255 (N15), 240 (N20), 225 (N25) and 0 (N0) kg hm^-2. The effects of nitrogen 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 increased first and then decreased with the decrease of nitrogen application. The response of the two varieties to nitrogen application was different. The indexes of XC 38 reached the maximum under N15 treatment, and the yield increased by 2.99%~81.45%. The yield of XC 49 was increased by 0.37%~71.29% under N25 treatment. Under the same nitrogen level, all indicators of XC38 were better than XC 49. Correlation analysis showed that the yield and protein yield were significantly positive correlated with NR, GS and GPT. The interaction of nitrogen fertilizer and varieties had significant effects on NR, GS, GPT, protein content, components and yield. These results showed that the protein content and yield of wheat grain could be improved by adjusting the nitrogen fertilizer application strategy reasonably."

Comment No.5: L22 (While XC 49 had the best indicators under N25 treatment) What does this statement mean exactly?

Response: Thank you for carefully reading our manuscript. We selected two different varieties for the experiment, both of which are the main varieties in Xinjiang, China. One of the two varieties is strong gluten wheat XC 38, and the other is medium gluten wheat XC 49. The responses of two wheat varieties to nitrogen fertilizer were different. The indexes of XC 38 (NR, GS, GPT, protein content, protein composition and yield) reached the maximum value when the nitrogen application amount was N15 (255 kg hm^-2), while XC 49 reached the maximum value at N25 (225 kg hm^-2).

Comment No.6: The abstract should be finished with a succinct conclusion.

Response: Thank you for your comments. We change the conclusion to a more concise expression. The modified content is "These results showed that the protein content and yield of wheat grain could be improved by adjusting the nitrogen fertilizer application strategy reasonably."

Comment No.7: Keywords: It is inappropriate to mention the words that make up the manuscript's title here.

Response: Thank you for your suggestion. We have modified the key words. Modified keywords: Triticum aestivum L.; Nutrient; Nitrogen metabolism; Yield.

Comment No.8: L47-53 (currently …… Xinjiang.): This sentence should remove.

Response: Thank you for your valuable comments. We have removed the relevant content based on your suggestion.

Comment No.9: The authors could include some details on wheat and its significance in terms of nutrition value in this section.

Response: Thank you very much for your comments. We have added the importance of wheat and its nutritional value in the foreword. Specific contents are as follows: “Wheat flour milled from wheat is a staple food in the world. Steamed bread, noodles and cakes made from it are especially popular. In addition, wheat is rich in complex carbohydrates (74%~77%) and protein (11%~15%), which is an important source of calories and plant protein.”

Comment No.10: L67-76: These sentences ought to be deleted because they make no sense.

Response: Thank you for your valuable comments. We have removed the relevant content based on your suggestion.

Comment No.11: The authors should also include some details on the significance of drip irrigation and its connection to N application in this part.

Response: Thank you for your valuable suggestions. We have added relevant contents to the manuscript, as follows: “The research on the application technology of N fertilizer in drip-irrigation wheat field is extensive and in-depth. 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 greatly maintained, which greatly improves the N fertilizer utilization efficiency and reduces the pollution.”

Comment No.12: L96: The minimum and maximum temperature averages should be added.

Response: Thank you for your question. We have added the average maximum temperature and average minimum temperature during the growth period of spring wheat from 2018 to 2019 in the manuscript. The specific contents are as follows: “The average maximum temperature in the growth period were 27 ℃ (2018) and 26 ℃ (2019), and the average minimum temperature were 13 ℃ (2018) and 14 ℃ (2019).”

Comment No.13: L101: Why didn't the researchers choose a plant with a lower protein content?

Response: Thank you very much for your question. We selected two wheat varieties with different gluten types, strong gluten wheat XC 38 and medium gluten wheat XC 49. These two varieties are the most widely planted wheat varieties in Xinjiang, China. The main reason is the dietary habits in Xinjiang. The wheat varieties with high grain protein content are the main raw materials for making noodles such as Xinjiang noodles and naan (some traditional Xinjiang cuisine). Therefore, we first selected two wheat varieties with relatively high grain protein content in our experiment.

Comment No.14: Section 2.3 and 2.5: The number of plants utilized in the measurement should be mentioned by the authors.

Response: Thank you for reading our manuscript and making valuable comments. Based on your suggestions, we have added relevant content. The specific modifications are as follows: “For each treatment, single stem tags with the same flowering date, normal growth and basically the same growth trend were selected as the sampling observation materials. Take 30 wheat spikes of each treatment at 7, 14, 21, 28, 35 days after anthesis, immediately place them in liquid N for quick freezing for 30 minutes, and store them in - 80 ℃ refrigerator to prevent enzyme inactivation. Wheat grain were artificially separated and ground with liquid N, and then the extract was obtained by low temperature centrifugation at 13000r/min, which was used to determine the key enzyme activity of nitrogen metabolism.”

Comment No.15: Section 2.4: The age of the plants or samples used to determine the protein content should be mentioned by the authors.

Response: Thank you for your question. Based on your suggestions, we have added relevant content. The specific modifications are as follows: “Twenty wheat spikes of uniform growth were selected from each treatment at 7, 14, 21, 28 and 35 days after anthesis, placed in paper bags and replicated three times, immediately placed in an oven at 105°C for 30 minutes 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. (2005).”

Comment No.16: L206 (2.6. Statistical analysis): Because there are differences between Two-Way ANOVA and Split plot design, the authors should identify the name of the experimental design they employed in their study.

Response: Thank you for pointing out our mistakes. We are sorry that there is a problem in the statistical analysis due to our carelessness. We have made the following modifications in the manuscript: “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 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.”

Comment No.17: Because this experiment repeated in two consecutive years, the authors should use the combined analysis

Response: Thank you for your suggestions on data analysis. When we analyze the data, we use the ANOVA of split plot design, and the specific analysis results are marked in the upper right corner of the figure and the table. In addition, according to your suggestion, we have also added a split ANOVA table to the manuscript. We haven't covered the combined analysis you mentioned yet. Because our experimental design adopts split design, we also adopted the ANOVA of split design in data analysis. In our future work, we will take the combination analysis as the learning goal. Thank you again for your suggestions.

Comment No.18: As seen in Table 1 and Figures 1–5, the authors chose to use factorial analyses rather than split analysis. To better comprehend the type of analysis utilized in their study, the authors ought to include the entire ANOVA table.

Response: Thank you for your question. Our analysis in Table 1 and Figure 1-5 is mainly to show whether different nitrogen fertilizer treatments have significant effects on each indicator in each period. We have marked the impact of year, treatment, variety and their interaction on this indicator in Table 1, and the corresponding results are also marked in the upper right corner of Figure 1-5. Because the results are scattered, we added an ANOVA table according to your suggestions. The details are as follows:

Table 3 ANOVA of wheat grain physiological indicators under variety and nitrogen fertilizer interaction.

Source of variation	protein	NR	GS	GPT
Y	12272.12**	8700.14**	2155.4**	8724.61**
V	28819.49**	265.28**	349.38**	5361.47**
T	3513.9**	1260.98**	286.57**	1360.32**
Y×V	800.81**	57.23**	1.61ns	357.64**
Y×N	343.76**	22.25**	31.18**	119.87**
V×N	1773.73**	700.52**	157.18**	880.63**
Y×V×N	147.07**	5.45**	8.71**	19.58**

Comment No.19: The authors used One-way ANOVA for the assessment of the impact of N treatments on the enzyme activities during various anthesis periods, as shown in the ANOVA in Figures 2–5. The authors should therefore confirm the procedure followed for the statistical data analysis.

Response: Thank you for your suggestions. Our experimental design is a split-zone design. The ANOVA of split-zone design is marked in the upper right corner of each figure. According to your suggestion, we have also added an ANOVA table for all indicators in the manuscript.

Comment No.20: The researchers only displayed the interactions between Variety X N applications and Year x Varities in Tables 1 and 2, respectively. The authors ought to display the outcomes of the interactions between Year x N applications as well.

Response: Thank you for your valuable comments. We supplemented the results of interaction analysis in Table 4 and Table 5.

Comment No.21: Discussion: The organization of this section is poor. It should be improved by the authors. The mechanism of boosting enzyme activity and protein component by application of N should be described by the authors.

Response: Thank you for your suggestion. We have already added the corresponding content. The details are as follows: Nitrogen (N) is one of the macronutrients necessary for plant growth and development (Gonzalez-Dugo et al., 2010; Li et al., 2017). 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 (Ma et al., 2018). Studies have shown that nitrate reductase (NR), glutamine synthetase (GS), and glutamate-pyruvate aminotransferase (GPT) are key enzymes in the nitrogen metabolism process. Increasing their enzyme activities can promote nitrogen metabolism in plants and promote the synthesis and transformation of proteins (Chen et al., 2022). NR is the starting factor and rate limiting enzyme in the nitrogen metabolism, which can regulate the transformation of inorganic nitrogen absorbed by plants into organic nitrogen. Its activity is positively correlated with nitrogen accumulation and protein content (Tikhomirova et al., 1985; Thaluarn et al., 1988; Hou et al., 2019). In higher plants, about 95% of NH₄⁺is assimilated to form amino acids through 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 (Giunta et al., 2021). In this study, with the decrease of nitrogen application, the nitrogen metabolizing enzymes activities in the grain of the two varieties increased first and then decreased. Proper nitrogen reduction significantly increased the NR, GS and GPT activities, which was consistent with the results of Effah et al. (2022). However, we observed that XC 38 and XC 49 had different responses to the grain nitrogen metabolizing enzymes activities of nitrogen fertilizer. Strong gluten wheat XC 38 reached the maximum value at N15 treatment, while medium gluten wheat reached the optimal value at 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 (Fig. 2-4). This finding indicated that different wheat varieties had different responses to nitrogen fertilizer, and there were significant differences in the degree of nitrogen 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 similar to that observed by Ma et al. (2019).

Reviewer 3 Report

Dear appreciated authors,

The manuscript “Effects of nitrogen application strategy on key enzyme activities of nitrogen metabolism and protein content in drip-irrigated spring wheat grain ” was found interesting, appears to be scientifically sound and the topic gives important information to improve the nitrogen fertilizer application strategies in spring wheat in drip-irrigated conditions. However, to improve the quality of the manuscript, I propose the Minor revisions and the following:

Line 10, 11, 12, 13 and 14 - I suggest change in sentence such as: In order to explore the appropriate amount of nitrogen application to improve the production and quality of drip-irrigated wheat, a two-year field experiment was conducted. It was determined the effect of nitrogen fertilizer application on the activities of enzymes and protein related to nitrogen metabolism in two different varieties of spring wheat (Triticum aestivum L.) under drip irrigation.

Row 16: Instead "which were", sound better: "in amounts 300...

Line 17, 18, 19:
The two-year experiment showed that the nitrogen metabolizing enzymes of wheat, such as nitrate reductase (NR), glutamine synthetase (GS), glutamate-pyruvate aminotransferase (GPT), protein content, protein components and yield, varied with the nitrogen application strategy. Furthermore, different respond to different application between different wheat varieties was also observed.

Line 22: Variety XC 49 showed the greatest indicators under N25 treatment and yield increased 0.37% - 71.29 % compared with other treatment.

Line 35: Xinjiang province in China is one of the largest grain crop province, with wheat planting area more than 1,173,300 hm2, accounting for 52 % of the planting area of Xinjiang grain crops in 2017 (Zhang et al., 2018).

Line 54-57: The process of nitrogen nutrition absorption, metabolism and utilization in plants play a vital role in the grain yield formation and yield quality, while the nitrate reductase (NR), glutamine synthetase (GS), and glutamate-pyruvate aminotransferase (GPT), are key enzymes in nitrogen metabolism (Zhang et al, 2017; Hou et al., 2019).

Line 77: Certain researches have reported the effects of reduced...

Line 81: Drip irrigation can provide the benefits of water and fertiliser and facilitate fertilisation menagment. Therefore, this paper deals with two specific aims: (1) To estimate the effects of different nitrogen treatments on enzyme activity, protein and yield of spring wheat grain under drip irrigation and (2) to evaluate the relationship between grain enzyme activity, protein content and yield under drip irrigation. The main aim of the present study is to assess the performance of two different wheat cultivars in a fertilizer management experiment, to determine a novel fertilization method and select a suitable wheat cultivar for local drip-irrigated wheat production.

Line 93: Station (44°26.5′N, -Please put one space between: Station (44°26.5′N,

Line 101-103: The wheat cultivars which were tested were Xinchun 38 (cv. XC38, Protein 101 content is 15.04%) and Xinchun 49 (cv. XC49, Protein content is 12.89%), as the widely cultivated wheat cultivars in Xinjiang province of China.

Line 104- 109: The experiment trial was a nitrogen reduction experiment with seven treatments. The treatments were: 1. Nck - presents normal nitrogen supply during the growing period (300 kg hm-2 is the conventional nitrogen fertilizer application in local management practices), 2. N0: no nitrogen application - control (0 % N, 0 kg hm-2 N), 3. N5: presents 5 % nitrogen reduction (95 % N, 285 kg hm-2 N), 4. N10: Nitrogen reduction 10% (90% N, 270 kg hm-2 N), 5. N15: Nitrogen 108 reduction 15% (85% N, 255 kg hm-2 N), 6. N20: Nitrogen reduction 20% (80% N, 240 kg hm- 109 2 N), 7. N25 treatment - presents nitrogen reduction 25% (75% N, 225 kg hm-2 N).

Line 109: 4m lenght and 3 m width.

Line 122: in Figure 1.

Line 126: Drip irrigation was consist from the first irrigation after sowing and then once every ten days after the wheat emerged. The amount was 6000 m3 hm-2 during the whole wheat growth seasons.

Line 137: at temperature of - 80 C.

Line 138: . Separation of wheat grains was manually done and grided with liquid N.

Line 230 - 231: 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).

Line 362: which was in accordance with results reported by Ma et al. (2019).

Line 453: Since that the experiment deals with the two specific research questions, it can be conclude that treatment N15
and variety XC38 were the optimal combination of fertilization treatment and cultivar respectively for drip irrigated spring wheat production in northern Xinjiang.

Kind regards,

Comments for author File: Comments.pdf

Author Response

Dear Editor and Reviewers:

Reviewer #3

Comment No.1: Line 10, 11, 12, 13 and 14 - I suggest change in sentence such as: In order to explore the appropriate amount of nitrogen application to improve the production and quality of drip-irrigated wheat, a two-year field experiment was conducted. It was determined the effect of nitrogen fertilizer application on the activities of enzymes and protein related to nitrogen metabolism in two different varieties of spring wheat (Triticum aestivum L.) under drip irrigation.

Response: Thank you very much for your careful revision of our manuscript. Because we have modified the content of the Abstract, but we have modified the unchanged content according to your modifications.

Comment No.2: Row 16: Instead "which were", sound better: "in amounts 300...

Response: Thank you for your suggestion. We have modified the corresponding content.

Comment No.3: Line 17, 18, 19: The two-year experiment showed that the nitrogen metabolizing enzymes of wheat, such as nitrate reductase (NR), glutamine synthetase (GS), glutamate-pyruvate aminotransferase (GPT), protein content, protein components and yield, varied with the nitrogen application strategy. Furthermore, different respond to different application between different wheat varieties was also observed.

Response: Thank you for your suggestion. We have modified the corresponding content.

Comment No.4: Line 22: Variety XC 49 showed the greatest indicators under N25 treatment and yield increased 0.37% - 71.29 % compared with other treatment.

Response: Thank you for your suggestion. We have modified the corresponding content.

Comment No.5: Line 35: Xinjiang province in China is one of the largest grain crop province, with wheat planting area more than 1,173,300 hm2, accounting for 52 % of the planting area of Xinjiang grain crops in 2017 (Zhang et al., 2018).