Optimizing Nutrition Protocols for Improved Rice Yield, Quality, and Nitrogen Use Efficiency in Coastal Saline Soils

Response to Reviewer X Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Must be improved

Are all the cited references relevant to the research?

Can be improved

Is the research design appropriate?

Must be improved

Are the methods adequately described?

Can be improved

Are the results clearly presented?

Yes

Are the conclusions supported by the results?

Yes

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: Title

The title should explicitly include "rice" to better reflect the study's focus. A suggested revision could be: "Optimizing nutrition protocols for improved rice yield in coastal saline soils."

Response 1: Optimizing nutrition protocols for improved rice yield, quality, and nitrogen use efficiency in coastal saline soils. Thank you for pointing this out. We agree with this comment. Therefore, we have revised the title accordingly to explicitly highlight rice and emphasize the scope of the study, which includes improving rice yield, quality, and nitrogen use efficiency in coastal saline soils. – Page 1, Lines 2-3.

Comments 2: Abstract

In line 21, the Apparent Nitrogen Use Efficiency (ANUE) calculation method should be briefly defined, as different approaches exist in literature. The Latin name of rice (Oryza sativa L.) should be included upon first mention.

Response 2:We agree with the reviewer’s suggestion to include the Latin name of rice (Oryza sativa L.) upon first mention and to define the calculation method for Agronomic Nitrogen Use Efficiency (ANUE). Accordingly, we have revised the abstract to emphasize these points.

Change 1: “This study evaluated the effects of one-time application of controlled-release fertilizer (CRF) on rice (Oryza sativa L.) grain yield…”. The Latin name of rice (Oryza sativa L.) has been added upon first mention in the abstract. - Page 1, Line 20.

Change 2: “…grain quality, and agronomic nitrogen use efficiency (ANUE, ANUE (kg/kg) = (Grain yield with N application - grain yield without N application) / N application amount)”. We have included the definition of the Agronomic Nitrogen Use Efficiency (ANUE) calculation method in the abstract, as suggested.- Page 1, Line 21-22.

Comments 3: Introduction

The introduction would benefit from additional references discussing the nitrogen use efficiency and seed yield relationships (doi:10.3390/horticulturae11030253; doi:10.1071/CP15069). The suggested references should be helpful for this purpose.

Additionally, rice physiological adaptations to alkaline soils should be better detailed (doi:10.2134/agronj2013.0017; doi:10.1007/s42729-023-01358-5).

Response 3: Nitrogen use efficiency (NUE) plays a pivotal role in achieving sustainable agricultural practices, as it directly influences yield, grain quality, and environmental sustainability. The relationship between NUE and yield has been extensively studied, with findings suggesting that optimized nitrogen management can enhance rice productivity while reducing environmental impacts such as nutrient leaching and greenhouse gas emissions [9,10]. Excessive urea application, commonly practiced to compensate for low soil fertility, often results in low NUE and suboptimal grain quality, posing further challenges in saline and alkaline soils [11,12]. Controlled-release fertilizer (CRF), which releases nitrogen gradually in synchrony with crop uptake patterns, has been proposed as a more efficient alternative to traditional nitrogen sources, offering improved NUE and environmental sustainability [13].  We agree with the reviewer’s suggestion to include additional references discussing the relationship between nitrogen use efficiency (NUE) and seed yield. Accordingly, we have incorporated the following references into the introduction (doi:10.3390/horticulturae11030253; doi:10.1071/CP15069). - Page 2, Line 63-74.

Physiologically, rice plants have developed several adaptive mechanisms to thrive in alkaline soils, such as the regulation of root architecture, ion transport, and the synthesis of osmotic regulators [7,8]. These adaptations allow rice to maintain nutrient uptake and tolerance under saline and alkaline stress, which are crucial for improving productivity in these challenging environments. We have provided a more detailed explanation of rice’s physiological adaptations to alkaline soils by incorporating the following references (doi:10.2134/agronj2013.0017; doi:10.1007/s42729-023-01358-5). - Page 2, Line 56-61.

Comments 4: Materials and Methods

Key methodological clarifications needed:

Site description (Line 92): Include geographic coordinates and elevation (meters above sea level)

Soil characterization:

Provide pre- and post-harvest soil properties for both growing cycles (tabular format recommended)

Report soil conductivity in dS/m alongside salinity percentage

Quantify sandy loam composition (percentages of sand, silt, clay)

Fertilizer composition (Line 102): Specify nitrogen forms (e.g., ammonium, nitrate, urea)

Seed source (Lines 106-108): Identify provider institution/company

Experimental design:

Include a summary table of nutrition protocols

Detail amylose and starch analysis methods or cite appropriate references

Specify ANOVA experimental factors and design

Response 4: We agree with the reviewer’s valuable suggestions to provide additional methodological details and clarifications in the Materials and Methods section. Accordingly, we have made the following changes:

4.1 Site description: Include geographic coordinates and elevation (meters above sea level).

Response 4.1: A field experiment was conducted in 2023 and 2024 at the Huanghai Breeding Farm in Dongtai, Yancheng, Jiangsu Province, China (32.0°N, 120.5°E, 6 m above sea level). We have included the geographic coordinates and elevation of the experimental site to better characterize the study location. - Page 3, Line 105.

4.2 Soil characterization:

4.2.1 Provide pre- and post-harvest soil properties for both growing cycles (tabular format recommended).

Response 4.2.1: Table 1 presents the soil nutrient status of the field prior to rice transplantation.

Thank you very much for your valuable suggestion. As the focus of this study is primarily on the impact of fertilization on rice quality, the investigation of soil properties was not the main objective of this research. Therefore, we only collected pre-harvest soil properties and did not conduct post-harvest soil sampling. We have now included the pre-harvest soil data in a table, as requested. We plan to include post-harvest soil measurements in future studies to provide a more comprehensive understanding of the impact of fertilization on soil health. We truly appreciate your understanding and your constructive feedback! - Page 3, Table 1.

4.2.2 Report soil conductivity in dS/m alongside salinity percentage

Response 4.2.2: Prior to rice transplantation, the topsoil (plow layer) had an electrical conductivity of 4.6-4.9 dS/m. Thank you for pointing this out. We agree with this comment. Therefore, we have now included the soil conductivity (in dS/m) alongside the salinity percentage in the revised manuscript. This additional data will help provide a clearer understanding of the soil salinity levels in relation to the overall soil properties. - Page 3, Line 112.

4.2.3 Quantify sandy loam composition (percentages of sand, silt, clay).

Response 4.2.3: The preceding crop was wheat, and the soil was classified as Fluvisols according to the World Reference Base for Soil Resources (WRB), with a composition of approximately 68% sand, 30% silt, and 12% clay. We believe this provides a clearer and more precise description of the soil characteristics in our study. - Page 3, Line 106-109.

4.3 Fertilizer composition (Line 102): Specify nitrogen forms (e.g., ammonium, nitrate, urea).

Response 4.3: All nitrogen fertilizers used in this study were in the form of urea. This included two types of polymer-coated controlled-release fertilizers (CRF): an 80-day CRF (42% N, Shandong Maoshi Fertilizer Co., Ltd.) and a 120-day CRF (30% N, Heilongjiang Jiucheng Agricultural Technology Co., Ltd.), in addition to conventional urea (46% N). Thank you for your valuable comment. In response, we have revised the sentence to clearly specify the nitrogen forms used in the study. This clarification has been added to the Materials and Methods section. - Page 3, Line 116-119.

4.4 Seed source: Identify provider institution/company.

Response 4.4: Two rice cultivars were provided by Jiangsu Jindadi Seed Industry Co., Ltd. in the experiment: Nanjing 5718, a medium-maturity japonica inbred variety, and Yongyou 4953, a medium-maturity indica-japonica hybrid variety.We have identified the provider company for the seeds used in the experiment to offer transparency. - Page 3, Line 121-123.

4.5 Experimental design:

4.5.1 Include a summary table of nutrition protocols

Response 4.5.1: Table 2 summarizes the experimental design and the nitrogen application treatments used in this study. The treatments included different nitrogen fertilizer types, application rates, and timing methods, all of which were applied in a completely randomized block design.

Thank you very much for your insightful suggestion. We have now included a summary table (Table 2) of the nutrition protocols used in this study. The table clearly outlines the different nitrogen fertilization treatments, application rates, and timing methods. We believe this addition will enhance the clarity and presentation of the experimental design in our manuscript. - Page 4, Table 2.

4.5.2 Detail amylose and starch analysis methods or cite appropriate references

Response 4.5.2: Amylose content and starch concentration were measured from rice flour. The amylose content in the rice flour was determined using the iodine colorimetric method. For this, 0.1 g of rice flour was dispersed in 100 mL of distilled water. Iodine solution (I₂/KI) was then added to the mixture, and the absorbance was measured at 620 nm. The amylose content was calculated based on a calibration curve prepared with known concentrations of amylose standards. Starch concentration was determined using a spectrophotometric method. First, a known weight of rice flour was extracted with 80% ethanol, and then the starch was digested with a sodium hydroxide (NaOH) solution to break it down into glucose. The glucose concentration was subsequently measured using the 3,5-dinitrosalicylic acid (DNS) method, where the color development was observed spectrophotometrically at 540 nm. The starch concentration was calculated by comparing the absorbance to a standard glucose curve. Thank you for your valuable feedback. In response to your suggestion, we have now provided a detailed description of the amylose and starch analysis methods used in the study. - Page 3-4, Line 157-168.

4.5.3 Specify ANOVA experimental factors and design

Response 4.5.3: Analyses of variance (ANOVA) were conducted to determine the effects of year, fertilization treatment, and rice variety (as independent variables), as well as their interaction effects, on yield and quality-related traits of rice (as dependent variables), with statistical significance assessed at the 5% and 1% levels. Thank you for your valuable feedback. In response to your suggestion, we have updated the Statistical Analysis section of the manuscript. - Page 5, Line 184-188.

Comments 5: Results

The results presentation could be enhanced by:

1.Adding standard deviation/error values to all tables

2.Including separate means for years and genotypes where interaction was not significant

3.Revising Figure 3:

Justify the two-panel format or consider merging

Explore additional trait correlations if biologically relevant

4..Expanding subsection 3.8 with brief commentary on key correlations

The title should explicitly include "rice" to better reflect the study's focus. A suggested revision could be: "Optimizing nutrition protocols for improved rice yield in coastal saline soils."

Response 5: We agree with the reviewer’s suggestions to enhance the Results section by adding more details and clarifying certain aspects. Accordingly, we have made the following revisions:

5.1.Adding standard deviation/error values to all tables.

Response 5.1: We have included standard deviation (SD) values in all relevant tables to provide a more accurate representation of the data’s variability. - Throughout Tables in the Results section.

5.2 Including separate means for years and genotypes where interaction was not significant. Response 5.2: We have added separate means for each year and genotype in cases where the interaction was not significant, as per the reviewer’s suggestion. - Throughout Tables in the Results section.

5.3.Revising Figure 3:

5.3.1 Justify the two-panel format or consider merging

Response 5.3.1: Thank you for your valuable feedback. In response to your suggestion, we have now merged the panels in Figure 4 for a more streamlined presentation. The revised figure provides a clearer and more concise view of the correlation matrix. - Page 13, Figure 4.

Figure 4. Correlation of starch fractions, protein, and soluble sugar content and rice viscosity characteristics (RVA parameters) with taste quality. *** indicats significance at p ≤ 0.001, ** indicats significance at p ≤ 0.01, * indicats significance at p ≤ 0.05.

5.3.2 Explore additional trait correlations if biologically relevant

Response 5.3.2: Furthermore, yield was positively correlated with protein content, soluble sugar content, and peak viscosity, suggesting that higher yield was associated with increased protein and sugar levels, which in turn may have enhanced peak viscosity. ANUE exhibited a highly significant positive correlation with yield, indicating that improved nitrogen use efficiency directly contributes to higher productivity. Thank you for your insightful suggestion. In response, we expanded the analysis to include additional biologically relevant correlations related to rice yield and agronomic nitrogen use efficiency (ANUE). The revised Figure 3 now includes these traits. - Page 12, Line 299-303.

5.4 Expanding subsection 3.8 with brief commentary on key correlations.

Response 5.4: Starch content, amylose content, protein content, and soluble sugar content were negatively correlated to appearance, stickiness, balance degree, and taste value. This suggests that higher starch, amylose, and protein content might result in rice with less desirable sensory properties, such as poorer texture and taste. This negative relationship is likely due to the impact of higher starch and protein content on the rice's cooking properties, which can affect its mouthfeel and overall acceptability. Branched-chain starch content showed a highly significant positive correlation with appearance, viscosity, balance degree, and taste value while demonstrating a highly significant negative correlation with hardness. The positive correlation between branched-chain starch content and taste value suggests that this type of starch plays an important role in enhancing rice quality, likely by improving its texture and mouthfeel. Peak viscosity was positively correlated with appearance, stickiness, balance degree, and taste value, whereas trough viscosity and final viscosity were negatively correlated with taste value. These results highlight the importance of peak viscosity as an indicator of rice's cooking and sensory qualities. High peak viscosity generally indicates better cooking characteristics, such as softness and less stickiness. In contrast, lower final viscosity and trough viscosity are associated with harder rice and undesirable texture. Additionally, peak viscosity and breakdown were negatively correlated with the hardness of rice. This suggests that rice varieties with higher peak viscosity and lower breakdown tend to have softer, more desirable textures. These findings underline the importance of controlling viscosity parameters for optimizing the texture and overall quality of rice (Figure 3). Furthermore, yield was positively correlated with protein content, soluble sugar content, and peak viscosity, suggesting that higher yield was associated with increased protein and sugar levels, which in turn may have enhanced peak viscosity. ANUE exhibited a highly significant positive correlation with yield, indicating that improved nitrogen use efficiency directly contributes to higher productivity.

Thank you for your valuable suggestion. In response to your comment, we have expanded subsection 3.8 with brief commentary on the key correlations. We have provided additional context to explain the biological significance of the correlations between starch content, amylose content, protein content, soluble sugar content, and sensory quality parameters such as appearance, stickiness, balance degree, and taste value. We also elaborated on the positive correlations between branched-chain starch content and sensory properties like appearance, viscosity, and taste value, as well as the relationships between viscosity parameters and rice hardness. - Page 12, Line 278-299.

Response to Reviewer X Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files. [This is only a recommended summary. Please feel free to adjust it. We do suggest maintaining a neutral tone and thanking the reviewers for their contribution although the comments may be negative or off-target. If you disagree with the reviewer's comments please include any concerns you may have in the letter to the Academic Editor.]

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes

Are all the cited references relevant to the research?

Yes

Is the research design appropriate?

Can be improved

Are the methods adequately described?

Yes

Are the results clearly presented?

Yes

Are the conclusions supported by the results?

Yes

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: Line 69: Authors should write; “Most commercially available CRF are designed to…”

Response 1: Thank you for your suggestion. We have revised the sentence as recommended. The sentence has been updated to: “Most commercially available CRF are designed to…”. - Page 2, Line 84.