PONE-D-25-60666Abscisic acid positively regulates rice spikelet closurePLOS One

Dear Dr. Huang,

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PLOS One

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“Xiaochun Zeng（XCZ）the National Natural Science Foundation Project (31360297) National Natural Science Foundation of China https://www.nsfc.gov.cn/”

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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?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

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

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Overall Recommendation:Major Revision

General Comments:

This study investigates the role of abscisic acid (ABA) in regulating rice spikelet closure using physiological, molecular, and field application approaches. The topic is of significant agronomic importance, especially for hybrid rice seed production. The experimental design is generally sound, and the combination of hormone treatments, RNA-seq, and field trials provides a comprehensive approach. However, several issues need to be addressed before the manuscript can be considered for publication.

Major Comments:

Lack of Statistical Details:

While the manuscript mentions statistical tests (e.g., F-tests, multiple comparisons), it does not provide sufficient details on the statistical methods used, such as the specific type of ANOVA, post-hoc tests, or sample sizes for each experiment. Please include these details in the Methods section and ensure that all figures include clear indications of statistical significance (e.g., asterisks with defined p-values).

RNA-Seq Data Availability:

The manuscript states that "all relevant data are within the manuscript and its Supporting Information files." However, RNA-seq data are not typically included in the main text or supplementary files. The raw or processed RNA-seq data should be deposited in a public repository (e.g., NCBI GEO or SRA), and the accession number must be provided.

Hormonal Interactions:

The study focuses on ABA but also involves MeJA. However, the interaction between ABA and other hormones (e.g., IAA, GA) is only briefly discussed. Given the complexity of hormonal regulation, more discussion or data on how ABA interacts with other hormones during spikelet closure would strengthen the manuscript.

Mechanistic Insight:

While RNA-seq identified DEGs related to ABA synthesis and signaling, functional validation (e.g., mutant analysis, transgenic plants) is lacking. The authors should acknowledge this limitation and discuss how future studies could address it.

Field Application Data:

The field trial results are promising, but the sample size and replication are not clearly described. Please specify the number of biological and technical replicates for the field experiments and whether the trials were repeated across multiple seasons or locations.

Minor Comments:

Language and Clarity:

The manuscript would benefit from professional English editing to improve clarity and flow. Some sentences are overly long or awkwardly phrased.

Figure and Table Quality:

Some figures (e.g., Fig. 1–4) are difficult to interpret due to overlapping labels and unclear legends. Please improve the resolution and organization of figures.

Table 3 (primers) is in Chinese. All content should be in English for international readability.

Introduction and Discussion:

The introduction could better contextualize the study within the broader literature on ABA and spikelet dynamics.

The discussion should more directly link the molecular findings (e.g., up-regulation of PYL4, down-regulation of PP2C) to the physiological observations.

Reference Formatting:

Some references are incomplete or inconsistently formatted (e.g., missing titles, journal names not abbreviated). Please ensure all references follow PLOS ONE guidelines.

Conclusion:This manuscript presents valuable insights into the role of ABA in rice spikelet closure and has potential applications in hybrid seed production. With revisions addressing the above points, it would be suitable for publication in PLOS ONE.

Reviewer #2: The manuscript "Abscisic acid positively regulates rice spikelet closure " is a well thought research reaching to a conclusion that reflects solution to the concern raised on spikelet closure. However, there are several issues that must be taken care:

1.I struggled to find in the introduction section of the manuscript why the authors chose to use combination of ABA and MeJA used for spraying. Ultimately I found the answer in the methodology section (L197), which in fact should have been stated in the introduction section.

2. if both Jasmonic acid (JA) and Methyl Jasmonate (MeJA) can promote the opening of mature rice spikelets, why only MeJA has been considered for the study?

3. The authors have considered 3 fertile and 3 rice varieties were considered, but the results obtained for them have not been discussed with regard to any difference the closure of stomata among them. Thus the reason of considering 3 rice varieties in both the sterile and fertile group remained unaddressed.

4. All the figures in their right panel need to state in the caption the 'alphabets' used against the top of each column.

5. The discussion section is without citation of any figures. Besides, the discussion section does not seem to interpret the results depicted in all the figures. It appears the discussion is independent of the results.

Reviewer #3: This manuscript addresses the role of abscisic acid (ABA) in regulating rice spikelet closure and explores its potential application in hybrid rice seed production. To address this question, the authors applied exogenous ABA and the ABA biosynthesis inhibitor fluoridone, analyzed transcriptome changes in lodicules, examined the agronomic effects of combined ABA and methyl jasmonate treatment. The transcriptome analysis suggests coordinated regulation of ABA biosynthesis and signaling genes during spikelet movement, and the combined hormone treatment is reported to enhance spikelet closure and hybrid seed yield.

While the topic is agronomically relevant, and the authors have generated a substantial amount of physiological, hormonal, and transcriptomic data, the manuscript in its current form contains fundamental issues in structure, rigor, and presentation. The biological question is not clearly framed, the Results section lacks synthesis and clarity, and key methodological and statistical details are insufficiently described. Addressing these problems would require a comprehensive reworking of the manuscript rather than a standard revision. I therefore recommend rejection, with the suggestion that the authors substantially revise and reorganize the manuscript before considering resubmission to an appropriate journal.

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Reviewer #1: No

Reviewer #2: Yes: Birendra Prasad Shaw

Reviewer #3: No

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Abscisic acid positively regulates rice spikelet closure

PONE-D-25-60666R1

Dear Dr. Huang,

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.

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Kind regards,

Keqiang Wu, Ph.D

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

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

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

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

Reviewer #1: Yes

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

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

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

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: You have responded to all the questions and made appropriate modifications on the vast majority of non principled issues. The manuscript has significantly improved in terms of language, statistical methods, charts, literature, introduction, and discussion structure and logic. RNA seq data is not publicly available. This is something that must be resolved. The author needs to immediately prepare to upload the data to public databases such as GEO or SRA. If insisting on not disclosing, extremely special and sufficient reasons need to be given in the response (such as involving unpublished patent applications or ethical/legal restrictions), but "incomplete analysis" and "confidentiality stage" are usually not accepted.

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7. PLOS authors have the option to publish the peer review history of their article (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

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