Dear Dr. Jiang,

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Xi Liang, Ph.D.

Academic Editor

PLOS ONE

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the Natural Science Foundation of Heilongjiang Province(No. LH2024D020)

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: This is an informative manuscript, and I appreciate the authors’ efforts in the experimental work and data analysis. However, several aspects of the manuscript would benefit from clarification and revision to improve scientific rigor, language clarity, and overall presentation.

Please refer to the specific comments provided in the manuscript for detailed suggestions.

Reviewer #2: The submitted manuscript addresses the timely and agriculturally significant topic of low-temperature stress (LTS) in japonica rice, with a particular focus on stage-specific physiological responses and yield outcomes in cold regions of China. The experimental design covers key developmental stages (tillering, booting, and heading), and the authors utilize a wide range of physiological, biochemical, and ultrastructural indicators to evaluate rice response under simulated field-like low-temperature conditions. However, several critical issues must be addressed before this manuscript is suitable for publication. These include: (1) insufficient mechanistic depth in key findings such as non-stomatal limitation and pollen sterility; (2) no quantitative definition of stress thresholds; (3) absence of yield-physiology correlation analysis; and (4) substantial language editing needed throughout the manuscript. Moreover, the manuscript would benefit from expanded discussion on practical implications and comparison with other studies.

Overall, I recommend major revision. If the authors can address the methodological and interpretive shortcomings and revise the manuscript thoroughly for language and statistical clarity, the work has potential for publication and could make a meaningful contribution to the field of crop stress physiology.

General Comments

Strengths:

� Field-relevant simulation of LTS

The authors designed a temperature-controlled phytotron experiment that closely mimics real-world diurnal temperature patterns in cold rice-growing regions. This increases the ecological and practical relevance of the findings compared to conventional constant-temperature studies.

� Stage-specific stress comparison

The manuscript thoroughly investigates the impact of LTS at three critical growth stages (tillering, booting, heading), enabling a clear understanding of developmental stage sensitivity. The identification of the booting stage as the most susceptible period is consistent with physiological expectations and provides actionable insights.

� Potential as a reference for future LTS modeling or breeding work

This dataset could serve as a valuable reference for future efforts in modeling LTS responses or identifying and breeding cold-tolerant rice genotypes.

The minor points are:

Abstract:

1. The abstract should accurately reflect the full scope of the study, including the background, objectives, methods, key results (with quantitative data where possible), and main conclusions or novelty.

2. All abbreviations should be spelled out in full when first mentioned in the abstract, in accordance with journal style. Please ensure consistency throughout the manuscript as well.

3. It is recommended that key quantitative results be explicitly stated in the abstract—for example, the percentage reduction in net photosynthetic rate (Pn), yield loss, and pollen viability decline. This will enhance the informativeness and scientific rigor of the abstract.

4. The term "non-stomatal limiting factor" is somewhat vague. Please clarify whether it refers to inhibition of photosynthetic enzymes, damage to chloroplast ultrastructure, or other specific mechanisms.

5. The abstract appears to report data from one experimental year, while the main text presents two years of data. Please verify and revise for consistency.

6. Consider adding a closing sentence to the abstract that summarizes the practical implications of the findings, such as their relevance to cold stress modeling, rice cultivation strategies, or breeding for cold tolerance.

Introduction:

1. Please consider revising the opening sentence of the Introduction by changing “The rice plant” to “Rice”, which is more concise and conventional in scientific writing. In addition, the manuscript would benefit from thorough English language editing to improve clarity, grammar, and readability.

2. Expand the literature review to include recent insights into cold stress responses in rice, especially on hormonal signaling (e.g., ABA, JA, SA) and regulatory networks.

3. Introduce more clearly the cold tolerance background of the two rice cultivar.

4. Explicitly state the scientific novelty of this study compared to existing research.

5. Strengthen the articulation of the study‘s aims by emphasizing its relevance to rice cold stress physiology and breeding.

6. To strengthen the scientific context and support the discussion, I suggest citing additional relevant studies. The following references may be particularly useful for comparison or background: [DOI: 10.3389/fpls.2023.1134308], [DOI: 10.3390/ijms21041284], [DOI: 10.3969/j.issn.1000-6362.2018.12.005], [DOI: 10.1093/jxb/erae452]

Materials and methods:

1. Lack of replication details: The number of biological replicates and technical replicates per treatment is not clearly stated. For example, were measurements such as Pn or enzyme activity based on three or four replicates? Please specify the replication scheme for each experimental component.

2. Incomplete description of greenhouse temperature control: It remains unclear whether the temperature treatments were constant or simulated by daily temperature fluctuations to reflect natural field conditions (i.e., diurnal variation vs. fixed temperatures). Does Figure 1 show actual temperature fluctuation curves? This should be clearly described in the text.

3. Missing agronomic management details: Important background parameters are lacking, such as fertilizer application rates, irrigation methods, pest and disease control measures, and whether such factors were standardized across treatments. These are essential for reproducibility and should be included.

4. The Materials and Methods section (Page 3, Line 135) cited references incorrectly. The citation of reference (No. 15) needs to be removed.

5. The Materials and Methods section (Page 5, Line 188) mentioned the yield and its composition. However, the method for calculating the yield composition should be provided.

6. What is the basis for choosing Longgeng31 as the experimental variety? How is the cultivation situation of this variety in the cold regions of China?

7. In line 192, "hr" should be changed to "h". Please carefully check the spelling of the entire text.

8. The preparation of chloroplast electron microscope samples requires supplementary references.

Results:

1. Inconsistent significance annotations in figures: The use of lettering (e.g., a/b/c) to indicate significant differences is inconsistent and at times confusing across multiple treatment groups. Please standardize the notation and provide clear explanations in the figure legends.

2. Lack of integrative data analysis: The results section would benefit from additional correlation or regression analyses, such as between Pn and yield, or MDA content and seed setting rate. These would strengthen the biological relevance of the findings.

3. Limited physiological interpretation of differences: The manuscript often states that differences are “significant” without discussing their physiological implications. Please elaborate on what these differences suggest in terms of stress response or metabolic disruption.

4. Lack of a quantitative threshold for "critical stress". The manuscript mentions a “critical threshold” at 7 days of LTS, but the physiological basis or criteria for this threshold (e.g., ROS peak, Pn decline percentage) is not well-defined. Please include a quantitative definition.

5. Language and grammar issues: Some terms are grammatically incorrect or colloquial, such as “memorably increased” or “spanking reduced.” These should be replaced with scientifically appropriate expressions like “significantly increased” or “markedly decreased.”

6. Chloroplast ultrastructure results lack quantification. While transmission electron microscopy (TEM) images are useful, there is no quantification of structural changes (e.g., number of grana stacks, chloroplast area, membrane disorganization). Consider including image-based quantification to strengthen this section.

7. Supplement the legend of Figure 16.

8. The significant figures retained in Table 2 are consistent.

Discussion:

1. Insufficient mechanistic interpretation: The discussion lacks depth regarding the mechanisms of non-stomatal limitations. Please consider whether reduced photosynthesis is related to decreased Rubisco activity, PSII inhibition, or impaired ATP synthesis, and support the interpretation with relevant literature.

2. Limited discussion on pollen viability reduction: The manuscript does not adequately explore the physiological basis of reduced pollen viability. It is recommended to discuss potential disruptions in nutrient transport, another development, or hormonal regulation during cold stress.

3. Lack of comparison with existing studies: The discussion would benefit from comparisons with related research on japonica rice in cold regions, indica varieties, or known cold-tolerant cultivars, in order to contextualize the findings within the broader scientific literature.

4. Missing implications for agricultural practices: Consider adding a paragraph that summarizes the practical significance of the findings. This could include recommendations for field management under LTS (e.g., adjustment of sowing dates, fertilization strategies, water layer control), or suggest screening indicators for breeding cold-tolerant japonica rice.

5. Why are the booting and heading stages more sensitive to LTS? What are the differences in the physiological characteristics of rice at these two stages?

6. The discussion section (Page 17, Paragraph1) focuses on the alterations in the ultrastructure of rice chloroplasts under LTS. A comparison with existing studies is needed.

Recommendation:

Accept after Major Revisions.

While the manuscript addresses a scientifically significant and practically relevant topic-namely, the physiological and yield-related impacts of LTS at different growth stages of rice-it currently requires major revision before it can be considered for publication.

The study is well-conceived and includes a comprehensive set of physiological, biochemical, and agronomic measurements. However, several important issues must be resolved. These include clarification of the experimental design and replication, deeper mechanistic interpretation (especially regarding non-stomatal limitations and pollen viability), improvements in statistical presentation, and thorough language editing. Additionally, the authors are encouraged to strengthen the discussion on practical implications and integrate more quantitative and comparative analyses.

Provided that the authors make these substantial revisions, the manuscript has the potential to contribute meaningfully to the field of crop stress physiology and cold-region rice production.

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what does this mean? ). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

**********

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Attachments

Attachment

Submitted filename: PONE-D-25-20271.pdf

Effects of Low-Temperature Stress at Different Growth Stages on Rice Physiology, Pollen Viability and Yield in China’s Cold Region

PONE-D-25-20271R1

Dear Dr. Lixia Jiang,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Xi Liang, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

**********

Reviewer #1: Thank you for your detailed and thoughtful responses and revisions. The manuscript has been considerably improved and is now suitable for publication in its present form.

Reviewer #2: (No Response)

**********

what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy

Reviewer #1: No

Reviewer #2: No

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