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Plant Growth Regulators Enhance Maize (Zea mays L.) Yield under High Density by Optimizing Canopy Structure and Delaying Leaf Senescence

Agronomy 2024, 14(6), 1262; https://doi.org/10.3390/agronomy14061262

by Tong Xu¹, Dan Wang¹, Yu Si¹, Yuanyuan Kong¹, Xiwen Shao¹, Yanqiu Geng¹

, Yanjie Lv^1,2,* and Yongjun Wang^1,2,*

Reviewer 1:

Abil Dermail

Reviewer 2: Anonymous

Reviewer 3: Anonymous

Agronomy 2024, 14(6), 1262; https://doi.org/10.3390/agronomy14061262

Submission received: 10 May 2024 / Revised: 2 June 2024 / Accepted: 7 June 2024 / Published: 11 June 2024

(This article belongs to the Special Issue Cropping Systems and Agronomic Management Practices of Field Crops—2nd Edition)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The researchers investigated the effects of different application timing of PGR containing ethephon under two different maize plant densities on maize yield, yield components, canopy, and physiology traits. They found that the application of PGR at the 15th leaf stage can enhance the yield of maize under high plant density. The manuscript can be accepted after addressing my below comments.

Title

· The title can convey the main topics of the study but a bit wordy, considering reducing some words to make it succinct.

Abstract

· The abstract covers justification, methods, and key findings, but it lacks research objectives.

· Provide the full names of each abbreviation mentioned in the abstract when they first appear. For example: ….superoxide dismutase activity (SOD)…..

Keywords

· The keywords are informative and cover the overall work of the study.

Introduction:

· In the first paragraph (line 36-53), authors have provided sufficient information regarding the importance of maize, increased planting density, and the strategy to increase maize yields.

· In the second paragraph (line 54-67), authors have briefly described the plant growth regulators and hormones. Perhaps authors could add more justification for the importance of plant growth regulators, and extensive research in this area needs to conduct.

· In the third paragraph (line 68-96), authors have provided sufficient information regarding a plant growth regulator EDAH including the timing of application, but there is no description regarding the planting density and its effect on maize yields. Please add them.

· This part lacks expected outcomes. Please add them in the last paragraph.

Materials and Methods:

· Experimental design: Is it enough to involve 3 × 2 treatment combinations to address the hypothesis? If so, please explain.

· Pollen density: How effective this method was used? Please provide the references if it is possible.

Results:

· Did D2 treatment (higher planting density) result in smaller ears? Either shorter ear or smaller ear diameter?

· ANOVA (Table 1, Table 2, Table 3): Please provide the coefficient of variation (cv) for each trait observed.

· The image resolution of Figure 8 needs to be improved.

Discussions:

· Please provide the discussion regarding the assimilate partitioning between the male and female reproductive organs. As the author mentioned, “the increase in pollen density, in turn, results in a higher number of kernels and ultimately contributes to an enhancement in corn yield”. To my best understanding, the larger tassel size will reduce the ear size.

· Provide an additional sub-section to discuss comprehensive mechanisms of maize responses to plant growth regulator and planting density involving multiple traits (morpho-physio-yield).

Conclusion:

· Conclusion can address all research objectives mentioned in the manuscript.

References

· All citations are listed in the references, update, and relevant to the research topic.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Minor editing of English language required.

Author Response

Thank you very much for your and the reviewers’ comments and professional advice concerning our manuscript entitled “Plant growth regulator application favors yield improvement of maize (Zea mays L.) under high planting density by optimizing canopy structure and delaying leaf senescence” (ID: agronomy-3028529). These opinions help us to improve academic rigor of the manuscript. Based on your suggestions and requests, the manuscript has been thoroughly and very carefully revised. We have improved the description of the materials and methods. In addition, we also revised the text to make it more logical and readable. Thanks again for giving us this opportunity to improve our manuscript.

Sincerely,

Yongjun Wang

Furthermore, we would like to show the details as follows:

Title：

The title can convey the main topics of the study but a bit wordy, considering reducing some words to make it succinct.

Response: Thank you for the reviewer's suggestions. We have rewritten the title named it as “Plant growth regulators enhance maize (Zea mays L.) yield under high density by optimizing canopy structure and delaying leaf senescence”.

Abstract:

The abstract covers justification, methods, and key findings, but it lacks research objectives.

Response: Thank you for the reviewer's suggestions. We have added new content: “To explore the effects of PGR application time on maize canopy structure, leaf senescence characteristics and yield.” (L16-17, Page 1)

Provide the full names of each abbreviation mentioned in the abstract when they first appear. For example: ….superoxide dismutase activity (SOD)….

Response: We thank the reviewer for pointing this out. We have improved these descriptions, “Furthermore, relative green leaf area at maturity (RGLAM) showed positive correlations with chlorophyll b, superoxide dismutase activity (SOD), peroxidase activity (POD), catalase activity (CAT) and soluble protein content (p <0.01), while displaying a negative correlation with malondialdehyde content (MDA) (p<0.01).” (Line28-31, Page 1)

Keywords:

The keywords are informative and cover the overall work of the study.

Response: We thank the reviewer for the appreciation of our work.

Introduction:

In the first paragraph (line 36-53), authors have provided sufficient information regarding the importance of maize, increased planting density, and the strategy to increase maize yields.

Response: We thank the reviewer for the appreciation of our work.

In the second paragraph (line 54-67), authors have briefly described the plant growth regulators and hormones. Perhaps authors could add more justification for the importance of plant growth regulators, and extensive research in this area needs to conduct.

Response: Thank you for the reviewer's suggestions. We have added new content: “Several reports had shown that jasmonates played an important role in signaling drought-induced antioxidant responses. Jasmonic acid can enhance the antioxidant system of soybean and photosynthesis to alleviate combined heat and drought stress effects[1,2]. Exogenous IAA sprayed on plants during drought or heat stresses mitigated the adverse stress effects on pollen viability, spikelet fertility and yield components[3]. Foliar Gibberellic acid (GA₃) application also increased leaf area, leaf fresh weight, leaf dry weight, chlorophyll content, the level of chlorophyll a and b, individual fruit weight, and number of fertile seeds of the blueberry[4]. A significantly improved ROS plant defense system was noted to increase enzymatic antioxidant activities and yield characters due to the exogenous application of ABA under drought stress[5].” (Line 70-79, Page 2)

In the third paragraph (line 68-96), authors have provided sufficient information regarding a plant growth regulator EDAH including the timing of application, but there is no description regarding the planting density and its effect on maize yields. Please add them.

Response: Thank you for the reviewer's suggestions. We have added new content: “Achieving efficient yield increase is a crucial objective in maize research, and increasing planting density is one of the most direct and effective measures for increasing yield[20]. Maize population yield and leaf area index increase with the increase of planting density. However, when the planting density reaches a certain range, it can cause photosynthetic obstacles and early leaf senescence in maize. Intense competition occurs between plants, and the yield and leaf area index no longer increase[21,22]. Excessive accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA) in the leaves, along with the gradual decrease in the activity of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), accelerates the aging of maize leaves, thereby affecting maize yield. Therefore, exploring agricultural measures that improve photosynthetic efficiency and leaf physiology under high-density planting is of great significance for establishing high-yielding and efficient maize populations and promoting intensive and sustainable agricultural production[23,24].” (Line 80-92, Page 2)

[20] Lan, T.; Du, L.; Wang, X.; Zhan, X.; Liu, Q.; Wei, G.; Lyu, C.; Liu, F.; Gao, J.; Feng, D.; et al. Synergistic Effects of Planting Density and Nitrogen Fertilization on Chlorophyll Degradation and Leaf Senescence after Silking in Maize. The Crop Journal 2024, 12, 605–613, doi:10.1016/j.cj.2024.02.006.

[21] Liu, X.; Gu, W.; Li, C.; Li, J.; Wei, S. Effects of Nitrogen Fertilizer and Chemical Regulation on Spring Maize Lodging Characteristics, Grain Filling and Yield Formation under High Planting Density in Heilongjiang Province, China. Journal of Integrative Agriculture 2021, 20, 511–526, doi:10.1016/S2095-3119(20)63403-7.

[22] Li, Y.; Ming, B.; Fan, P.; Liu, Y.; Wang, K.; Hou, P.; Xue, J.; Li, S.; Xie, R. Quantifying Contributions of Leaf Area and Longevity to Leaf Area Duration under Increased Planting Density and Nitrogen Input Regimens during Maize Yield Improvement. Field Crops Research 2022, 283, 108551, doi:10.1016/j.fcr.2022.108551.

[23] Wu, P.; Liu, F.; Chen, G.; Wang, J.; Huang, F.; Cai, T.; Zhang, P.; Jia, Z. Can Deep Fertilizer Application Enhance Maize Productivity by Delaying Leaf Senescence and Decreasing Nitrate Residue Levels? Field Crops Research 2022, 277, 108417, doi:10.1016/j.fcr.2021.108417.

[24] Antonietta, M.; Fanello, D.D.; Acciaresi, H.A.; Guiamet, J.J. Senescence and Yield Responses to Plant Density in Stay Green and Earlier-Senescing Maize Hybrids from Argentina. Field Crops Research 2014, 155, 111–119, doi:10.1016/j.fcr.2013.09.016.

This part lacks expected outcomes. Please add them in the last paragraph.

Response: We thank the reviewer for pointing this out. We have made additions, and the new sentence reads as follows: “We expect the experiment to produce the following results: (1) Spraying plant growth regulators (PGRs) at the 15th leaf stage will optimize the canopy structure of maize and enhance the yield. (2) Spraying plant growth regulators (PGRs) at the 15th leaf stage will increase pollen dnsity of the corn. (3) Spraying plant growth regulators (PGRs) at the 15th leaf stage will delay the senescence and prolong functional period of the leaves.” (Line 133-138, Page 3)

Materials and Methods:

Experimental design: Is it enough to involve 3 × 2 treatment combinations to address the hypothesis? If so, please explain.

Response: We thank the reviewers for their questions. These two periods are critical for both the differentiation of maize ears and the elongation and development of stalks. Applying plant growth regulators during these periods is essential for controlling plant height, reducing the center of gravity, and minimizing the impact on ear differentiation, which is crucial for the accumulation of dry matter and the formation of yield in the later stages. At the same time, the experimental design was also obtained based on the previous research foundation of our research group and the reference of relevant literature.

[1] Huang, S.; Gao, Y.; Li, Y.; Xu, L.; Tao, H.; Wang, P. Influence of Plant Architecture on Maize Physiology and Yield in the Heilonggang River Valley. The Crop Journal 2017, 5, 52–62, doi: 10.1016/j.cj.2016.06.018.

[2] Zhao, Y. Shortening Internodes Near Ear: An Alternative to Raise Maize Yield. Journal of Plant Growth Regulation 2022, 41, 628–638, doi: 10.1007/s00344-021-10326-1.

[3] Zhao, Y.; Zhang, S.; Lv, Y.; Ning, F.; Cao, Y.; Liao, S.; Wang, P.; Huang, S. Optimizing Ear-Plant Height Ratio to Improve Kernel Number and Lodging Resistance in Maize (Zea Mays L.). Field Crops Research 2022, 276, 108376, doi: 10.1016/j.fcr.2021.108376.

10 Pollen density: How effective this method was used? Please provide the references if it is possible.

Response: We thank the reviewers for their questions. Before carrying out this experiment, we checked the method of determining the pollen density of Larix kaempferi and Cunninghamia lanceolata, which has certain feasibility. (Line 197, Page 5)

[48] Zhuowen, Z. Pollen Dispersal and Its Spatial Distribution in Seed Orchards of Cunninghamia Lanceolata (LAMB.) Hook. Silvae Genetica 2002, 51, 237–241.

[49] Zhuowen, Z.; Juanjuan, S.; Li, M.; Changqing, S. Pollen Dispersal and Its Spatial Distribution in a Seed Orchard of Larix Kaempferi (Lamb.) Carr. Silvae Genetica 2008, 57, 256–261, doi:10.1515/sg-2008-0039.

Results:

11 Did D2 treatment (higher planting density) result in smaller ears? Either shorter ear or smaller ear diameter?

Response: We thank the reviewer for pointing this out. Table 2 shows that planting density has an impact on ear length, and the increase of planting density will lead to shorter ear length of maize.

12 ANOVA (Table 1, Table 2, Table 3): Please provide the coefficient of variation (cv) for each trait observed.

Response: We thank the reviewer for pointing this out. We have added the coefficient variation in Table 1, Table 2 and Table 3.

13 The image resolution of Figure 8 needs to be improved.

Response: We thank the reviewers for their questions. We have replaced Figure 8 with a higher resolution image.

Discussions:

14 Please provide the discussion regarding the assimilate partitioning between the male and female reproductive organs. As the author mentioned, “the increase in pollen density, in turn, results in a higher number of kernels and ultimately contributes to an enhancement in corn yield”. To my best understanding, the larger tassel size will reduce the ear size.

Response: We thank the reviewer for pointing this out. We have made additions, and the new sentences read as follows: “Previous studies suggested that increasing planting density could result in smaller branches, fewer male ears of maize, degradation of florets, reduced total floret number, and pollen amount, ultimately affecting pollination and fruit setting, leading to a higher female ear abortion rate and lower yield[63]. This study also confirmed this point. However, following the application of plant growth regulators at the 15th leaf stage, changes in canopy structure resulted in an increase in pollen density. This led to a higher number of the pollen scattered on the silk, enhanced fertilization ability of silk, increased grain yield per ear, and reduced the rate of abortion.”. (Line 542-549, Page 16)

[63]. Zhang, M.; Chen, T.; Latifmanesh, H.; Feng, X.; Cao, T.; Qian, C.; Deng, A.; Song, Z.; Zhang, W. How Plant Density Affects Maize Spike Differentiation, Kernel Set, and Grain Yield Formation in Northeast China? Journal of Integrative Agriculture 2018, 17, 1745–1757, doi:10.1016/S2095-3119(17)61877-X.

The study found that planting density had a significant impact on the total number of florets and the length of ears of maize. As planting density increased, both the total number of florets and the length of ears decreased. However, spraying plant growth regulator did not have any effect on the number of florets in maize.

15 Provide an additional sub-section to discuss comprehensive mechanisms of maize responses to plant growth regulator and planting density involving multiple traits (morphophysio-yield).

Response: We thank the reviewers for their questions. We have added new content: “Increasing planting density is a crucial cultural practice for optimizing corn production potential, while the application of plant growth regulators serves as a technical method to enhance corn density tolerance[41]. In this study, it was found that spraying plant growth regulators under low planting density did not significantly alter plant morphology, improve canopy structure, or affect light conditions, resulting in minimal changes in corn yield and yield components. This can be attributed to the larger internal space within the plant group at low planting density, making corn plants less responsive to plant growth regulators[39]. The distribution of light energy to each leaf layer of the group remains relatively stable, allowing for steady growth. High light transmittance benefits the middle leaves, enhancing photosynthesis in later stages of corn growth. Conversely, at high density, poor light conditions within the plant population are observed. Application of plant growth regulators improves plant morphology, alters light conditions for middle leaves, impacting photosynthesis and ultimately leading to increased yield[40].” (Line 485-498, Page 15)

Conclusion:

16 Conclusion can address all research objectives mentioned in the manuscript.

Response: We thank the reviewer for the appreciation of our work.

References

17 All citations are listed in the references, update, and relevant to the research topic.

Response: We thank the reviewer for the appreciation of our work.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript entitled, “Plant growth regulator application favors yield improvement of maize (Zea mays L.) under high planting density by optimizing canopy structure and delaying leaf senescence” fits well within the scope of Agronomy. The paper is well written but needs minor modification in the introduction, methods, results and discussion before acceptance.

Abstract;

· The authors should adopt the word maize instead of corn throughout the manuscript.

· The location of the experiment should be indicated.

· What do the authors mean by the term RGLAM?

Introduction;

· In Lines 55-66, avoid providing long paragraphs without references.

· The introduction has no information on the influence of plant densities on yield of maize.

· Also include previous studies conducted with PGRS applied at various rates, indicating their effects on growth and yield of crops. Specifically, by what percentage increment in yield is observed on application of PGRs?

· In line 97, what do the authors mean by “previous agricultural research”? This should be corrected.

Method;

· Provide rationale for applying PGR at 10-leaf stag.

Results;

· The correlation heat map is not clear, thus can’t be read.

Discussion;

· The discussion should be improved while explain how your results are consistent with other studies and providing scientific evidences for such observations. Merely, saying yield increment due to PGRs addition is congruent with previous studies is not sufficient.

· We also expect that the parameters e.g., yield be discussed in line with plant density as well as the interactions between the factors be clearly explained. In the current version, discussion is focusing on the PGRS neglecting the role of plant densities in influencing plant growth and yield.

………………………End……………………..

Author Response

Sincerely,

Yongjun Wang

Furthermore, we would like to show the details as follows:

Abstract:

The authors should adopt the word maize instead of corn throughout the manuscript.

Response: We thank the reviewer for pointing this out. We have replaced corn with maize in this article.

2 The location of the experiment should be indicated.

Response: We thank the reviewer for pointing this out. We have added new content: “at the Gongzhuling Experimental Station of the Jilin Academy of Agricultural Sciences.” (Line18-19, page1)

3 What do the authors mean by the term RGLAM?

Response: We thank the reviewers for their questions. RGLAM means relative green leaf area at maturity. We have improved these descriptions: “Furthermore, relative green leaf area at maturity (RGLAM).” (Line28, page1)

Introduction;

4 In Lines 55-66, avoid providing long paragraphs without references.

Response: We thank the reviewer for pointing this out. We have added the new references. (Line 64 and line 69)

[12] Anfang, M.; Shani, E. Transport Mechanisms of Plant Hormones. Current Opinion in Plant Biology 2021, 63, 102055, doi:10.1016/j.pbi.2021.102055.

[13] Huang, Y.; Ji, Z.; Zhang, S.; Li, S. Function of Hormone Signaling in Regulating Nitrogen-Use Efficiency in Plants. Journal of Plant Physiology 2024, 294, 154191, doi:10.1016/j.jplph.2024.154191.

[14] Yang, D.-L.; Yang, Y.; He, Z. Roles of Plant Hormones and Their Interplay in Rice Immunity. Molecular Plant 2013, 6, 675–685, doi:10.1093/mp/sst056.

5 The introduction has no information on the influence of plant densities on yield of maize.

Response: Thank you for the reviewer's suggestions. We have added new content: “Achieving efficient yield increase is a crucial objective in corn research, and increasing planting density is one of the most direct and effective measures for increasing yield[6]. Corn population yield and leaf area index increase with the increase of planting density. However, when the planting density reaches a certain range, it can cause photosynthetic obstacles and early leaf senescence in corn. Intense competition occurs between plants, and the yield and leaf area index no longer increase[7,8]. Excessive accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA) in the leaves, along with the gradual decrease in the activity of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), accelerates the aging of corn leaves, thereby affecting corn yield. Therefore, exploring agricultural measures that improve photosynthetic efficiency and leaf physiology under high-density planting is of great significance for establishing high-yielding and efficient corn populations and promoting intensive and sustainable agricultural production[9,10].” (Line 80-92, Page 2)

6 Also include previous studies conducted with PGRS applied at various rates, indicating their effects on growth and yield of crops. Specifically, by what percentage increment in yield is observed on application of PGRs?

Response: We thank the reviewer for pointing this out. We have added new content: “Additionally, EDAH significantly increased grain yield by 14.3% by increasing kernel number. (Line 109-110, Page 2); thereby increasing corn yield by 5%. (Line 108-109, line 117-118, Page 3)”

7 In line 97, what do the authors mean by “previous agricultural research”? This should be corrected.

Response: We thank the reviewer for pointing this out. The description of “Previous agricultural research” is not appropriate, we have replaced it with "based on previous research within the research group and added the references ". (Line 123, Page 3; line 127, Page 3).

[28] Zhao, Y.; Zhang, S.; Lv, Y.; Ning, F.; Cao, Y.; Liao, S.; Wang, P.; Huang, S. Optimizing Ear-Plant Height Ratio to Improve Kernel Number and Lodging Resistance in Maize (Zea Mays L.). Field Crops Research 2022, 276, 108376, doi:10.1016/j.fcr.2021.108376.

[45] Zhao, Y.; Lv, Y.; Zhang, S.; Ning, F.; Cao, Y.; Liao, S.; Wang, P.; Huang, S. Shortening Internodes Near Ear: An Alternative to Raise Maize Yield. Journal of Plant Growth Regulation 2022, 41, 628–638, doi:10.1007/s00344-021-10326-1.

Method:

8 Provide rationale for applying PGR at 10-leaf stag.

Response: We thank the reviewers for their questions. These two periods are critical for both the differentiation of corn ears and the elongation and development of stalks. Applying plant growth regulators during these periods is essential for controlling plant height, reducing the center of gravity, and minimizing the impact on ear differentiation, which is crucial for the accumulation of dry matter and the formation of yield in the later stages. At the same time, the experimental design was also obtained based on the previous research foundation of our research group and the reference of relevant literature.

[2] Zhao, Y. Shortening Internodes Near Ear: An Alternative to Raise Maize Yield. Journal of Plant Growth Regulation 2022, 41, 628–638, doi: 10.1007/s00344-021-10326-1.

Results:

9 The correlation heat map is not clear, thus can’t be read.

Response: We thank the reviewers for their questions. We have replaced Figure 8 with a higher resolution image.

Discussion:

10 The discussion should be improved while explain how your results are consistent with other studies and providing scientific evidences for such observations. Merely, saying yield increment due to PGRs addition is congruent with previous studies is not sufficient.

Response: We thank the reviewers for their questions. We have added new content: “ Kernel number formation mainly depends on the potential kernel number that is established in the early vegetative growth period (i.e., floret initiation period), approximately the 8- to 13-leaf stages, depending on varieties[56]. Ethephon applied at this period suppressed or damaged floret initiation, thus reducing ear size and potential[57]. This was clearly confirmed by literature and experimental results in the present study.”. (Line523-528, Page 16)

[56] Gonzalez, V.H.; Lee, E.A.; Lukens, L.N.; Swanton, C.J. The Relationship between Floret Number and Plant Dry Matter Accumulation Varies with Early Season Stress in Maize (Zea Mays L.). Field Crops Research 2019, 238, 129–138, doi:10.1016/j.fcr.2019.05.003.

[57] Schluttenhofer, C.M.; Massa, G.D.; Mitchell, C.A. Use of Uniconazole to Control Plant Height for an Industrial/Pharmaceutical Maize Platform. 2011, doi:10.1016/j.indcrop.2011.01.009.

11 We also expect that the parameters e.g., yield be discussed in line with plant density as well as the interactions between the factors be clearly explained. In the current version, discussion is focusing on the PGRS neglecting the role of plant densities in influencing plant growth and yield.

Response: We thank the reviewers for their questions. We have added new content: “Increasing planting density is a crucial cultural practice for optimizing corn production potential, while the application of plant growth regulators serves as a technical method to enhance corn density tolerance[38]. In this study, it was found that spraying plant growth regulators under low planting density did not significantly alter plant morphology, improve canopy structure, or affect light to the larger internal space within the plant group at low planting density, making corn plants less responsive to plant growth regulators[36]. The distribution of light energy to each leaf layer of the group remains relatively stable, allowing for steady growth. High light transmittance benefits the middle leaves, enhancing photosynthesis in later stages of corn growth. Conversely, at high density, poor light conditions within the plant population are observed. Application of plant growth regulators improves plant morphology, alters light conditions for middle leaves, impacting photosynthesis and ultimately leading to increased yield[37].” (Line 485-498, Page 15)

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript evaluates the effect of PGR containing ethephon as the active ingredient for maize. The experiment was structured with two plant densities of 60.000 and 90.000 plants ha⁻¹, and three distinct PGR application protocols. The authors emphasize that spraying plant growth regulators at the 15-leaf stage under high density can effectively enhance the top canopy structure of the maize, optimizes light resource utilization, and enhances the maize yield. The results were statistically interpreted and presented in tables and graphs.

However, there are some shortcomings:

- - what were the working methods for determining Soil physiochemical properties;

- include more details for the varieties used in the experiment.

Author Response

Sincerely,

Yongjun Wang

Furthermore, we would like to show the details as follows:

1 What were the working methods for determining Soil physiochemical properties;

Response: We thank the reviewer for pointing this out. We have added the methods of the soil physiochemical properties: “2.3.9 Soil physiochemical properties Soil organic carbon (SOC) content was measured by oxidation with potassium dichromate. Soil alkali-hydrolyzable nitrogen (AN) content was determined by the alkali diffusion method. Available phosphorus (AP) was measured by the molybdenum blue method using an ultraviolet spectrophotometer. Available potassium (AK) was measured by flame photometry method. Soil pH was measured in soil–water (1:2.5) with a glass electrodemeter [50].” (L264-270, Page 7)

[50] Zhang, Y.-W.; Peng, Z.-C.; Chang, S.-H.; Wang, Z.-F.; Li, D.-C.; An, Y.-F.; Hou, F.-J.; Ren, J.-Z. Long-Term Grazing Improved Soil Chemical Properties and Benefited Community Traits under Climatic Influence in an Alpine Typical Steppe. Journal of Environmental Management 2023, 348, 119184, doi:10.1016/j.jenvman.2023.119184.

2 Include more details for the varieties used in the experiment.

Response: We thank the reviewer for pointing this out. The Fumin985 is one of the main maize cultivars grown in the region (L165-166, Page 4). Therefore, we chose it as a research material to support the generalizability of the results.

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Plant Growth Regulators Enhance Maize (Zea mays L.) Yield under High Density by Optimizing Canopy Structure and Delaying Leaf Senescence

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