ROS-induced ATP synthase mRNA degradation and metabolism dysfunction reveals the mechanism of artificial deteriorated cotton seeds

Ci Song; Junming Zhang; Zhenzhen Xing; Fengxu Gu; Junying Chen

doi:10.1371/journal.pone.0339977

Peer Review History

Original SubmissionSeptember 9, 2025
1 Oct 2025 Decision Letter - Haitao Shi, Editor Dear Dr. Chen, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Nov 15 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. 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If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. [Note: HTML markup is below. Please do not edit.] 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: Partly Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes Reviewer #2: Yes ****** 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 ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: No Reviewer #2: Yes ****** Reviewer #1: I have given my deep reading to the manuscript tilted as ROS production and loss of ATP synthase subunit mRNA integrity as a mechanism of artificial deteriorated cotton seedshave the following concerns regarding the manuscript 1) Can you please reconsider the title with a unique word as it seems so simple and less attractive than the audience 2) Abstract and conclusion are pessimistic as very simple language is used no novelty or significance of work is properly mentioned. I am always very curious about conclusion as it provided crux of whole documents please refine your English in this section. 3) In continuation to my previous comment Please double check grammar as overall language of the paper needs to verify. You can get help through software. 4) Please rewrite your introduction or add constructive information to make it attractive for readers 5) Can you please add graphical/ general picture of your work along abstract to make it clear graphical or tabular representation to make finding easy t access for readers and it usually enhances beauty of the paper. I do like to check these first whenever reading a paper. That’s what I believe, maybe other reviewers think differently. 6) Graphs are very small and very hard to understand. Please make them suitable size, font and titles 7) Surprisingly results are acceptable form but please add more justification in your discussion part and add new findings relevant to you work 8) In Reference sections please add all required details. Make sure you are following the journal pattern overall. 9) Good luck Reviewer #2: The manuscript addresses an important and underexplored aspect of cotton seed deterioration by linking ROS accumulation with ATP synthase subunit mRNA damage. The study is strengthened by its integration of physiological, biochemical, and ultrastructural analyses, providing a multifaceted view of seed aging. The correlation between ROS levels and mRNA integrity is particularly novel and has potential implications for seed quality preservation. However, the manuscript cannot be accepted in its present form. The authors should address the following points. 1. The rationale for choosing the cultivar "Xinluzao 74" has been briefly stated, but its relevance to broader cotton varieties worldwide could be explained more clearly. 2. Please provide the justification for selecting 45°C and 100% RH could be better supported with references or comparative discussion. 3. In case of Figures 1–3, the figure legends could be expanded to ensure they stand alone without the need to refer to the text. 4. The intensity of TTC staining could be quantified by using image J or similar software if possible. 5. Conductivity data should be discussed for the involvement of any possible non-aging factors influencing conductivity (e.g., seed coat permeability differences). 6. The ROS measurements show the initial decline in H₂O₂ after 5 days of aging should be more carefully interpreted, as alternative explanations beyond ROS scavenging could exist. 7. Regarding the results of respiration and ATP synthase,more explanation is needed regarding whether the observed decreases are cause or consequence of mRNA damage. 8. The discussion section occasionally reiterates results rather than offering deeper mechanistic insights, particularly in the first two paragraphs. The discussion section should be improved please. 9. Minor language issues (e.g., “seriously damaged” on p.6) could be revised for precision and to maintain a formal scientific tone throughout. ****** 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: Yes: Muhammad Amjad Ali ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0339977.r001
Revision 1
22 Nov 2025 Author Response We sincerely thank the editor and reviewers for their careful reading and constructive comments. We have carefully revised our manuscript according to all suggestions. Below, we provide detailed responses point by point, with modifications highlighted in the revised manuscript and summarized with artificial line numbers. Response to Reviewer 1 Comment 1: Can you please reconsider the title with a unique word as it seems so simple and less attractive than the audience. Response: Thank you for your valuable suggestion. We agree that a more specific and engaging title will help attract readers’ attention. We have revised the title to better emphasize the mechanistic focus and novelty of our findings. Modification: Before (Title-L1): ROS production and loss of ATP synthase subunit mRNA integrity as a mechanism of artificial deteriorated cotton seeds. After: ROS-induced ATP synthase mRNA degradation and metabolism dysfunction reveals the mechanism of artificial deteriorated cotton seeds Comment 2: Abstract and conclusion are too simple; no novelty or significance is highlighted. Response: We appreciate the reviewer’s advice. We have rewritten the abstract and conclusion to highlight the novelty, emphasizing the mechanistic link between ROS accumulation and ATP synthase mRNA integrity loss, and its implications for seed vigor and germplasm preservation. Comment 3: Please double-check grammar; overall language needs verification. Response: We have carefully revised the entire manuscript for grammar, tense consistency, and scientific style. Comment 4: Please rewrite the introduction or add constructive information to make it attractive. Response: We have enriched the Introduction by emphasizing the gap in understanding mRNA oxidation during cotton seed aging. Comment 5: Add graphical/pictorial representation of your work along the abstract. Response: We have prepared a Graphical Abstract illustrating the sequence: ROS accumulation → mRNA oxidative damage → impaired ATP synthesis → Mitochondrial dysfunction. The figure provides an intuitive summary of our findings. (A figure are provided below this document.) Comment 6: Graphs are too small and fonts unclear. Response: All figures have been resized to improve readability(Results section, Figs. 1–10). Comment 7: Add more justification in discussion and include relevant new findings. Response: We have expanded the Discussion to better connect ROS accumulation, mitochondrial dysfunction, and ATP synthase mRNA damage. We also referenced recent literature supporting the role of mRNA oxidation in post-transcriptional regulation during seed aging. Comment 8: Ensure references follow journal pattern. Response: All references have been reformatted according to PLOS ONE guidelines, including DOI addition and consistent author-year style. Response to Reviewer 2 Comment 1: Explain why cultivar “Xinluzao 74” was chosen. Response: We have clarified this in Introduction “Xinluzao 74” is a major high-yield, disease-resistant cultivar widely cultivated in Xinjiang, representing typical seed storage behavior of upland cotton. Its characteristics make it an ideal model for artificial aging studies with global relevance. Comment 2: Justify 45°C and 100% RH conditions. Response: We have added in Method part: “These conditions follow Tesnier et al. (2002) and are widely used to simulate accelerated seed aging, providing reproducible deterioration kinetics comparable across oilseed species.” Comment 3: Expand figure legends for stand-alone clarity. Response: Revised all figure legends (Figs. 1–10) to include species, treatment, replicates, and statistical test information, ensuring they can be understood independently. Comment 4: Quantify TTC staining intensity. Response: We appreciate the reviewer's suggestion to quantify the TTC staining intensity. While we agree that quantification can be valuable, the visual difference in staining between the experimental and control groups is consistently clear and striking across all replicates, as shown in Figure 2. Furthermore, we would like to highlight that the dehydrogenase activity assay (presented in Figure 3 provides a precise and quantitative measurement of the same biological process that the TTC staining visually represents. We believe that this enzymatic data robustly supports our qualitative TTC observations. Comment 5: Discuss conductivity—possible non-aging effects. Response: While conductivity increase mainly reflects membrane leakage from aging, minor variation due to seed coat permeability cannot be excluded and warrants further study, so we have added clarification in case any possible misunderstanding. Comment 6: Interpret initial H₂O₂ decline more carefully. Response: The transient drop in H₂O₂ after 5 days might also result from peroxidase-mediated conversion or ROS-induced degradation, rather than exclusive scavenging, so we have added clarification in the Discussion part. Comment 7: Clarify whether ATP synthase and respiration decline are cause or consequence of mRNA damage. Response: The observed reduction in ATP synthase activity is likely a downstream effect of mRNA integrity loss, suggesting that ROS-induced mRNA oxidation may precede metabolic inhibition, so we added mechanistic clarification in Discussion part: Comment 8: Improve discussion—avoid repetition. Response: We revised the first two Discussion paragraphs to merge redundant result descriptions and focus on mechanistic interpretation and broader biological implications. Comment 9: Minor English issues. Response: Thanks for your comments, we have rephrased all informal expressions. Attachments Attachment Submitted filename: Response_to_Reviewers.docx https://doi.org/10.1371/journal.pone.0339977.r002
16 Dec 2025 Decision Letter - Smita Kumar, Editor ROS-induced ATP synthase mRNA degradation and metabolism dysfunction reveals the mechanism of artificial deteriorated cotton seeds PONE-D-25-49082R1 Dear Dr. Chen, 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. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact billing support . If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Smita Kumar, 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: Response to Reviewer 1 Comment 1: Can you please reconsider the title with a unique word as it seems so simple and less attractive than the audience. Response: Thank you for your valuable suggestion. We agree that a more specific and engaging title will help attract readers’ attention. We have revised the title to better emphasize the mechanistic focus and novelty of our findings. Modification: Before (Title-L1): ROS production and loss of ATP synthase subunit mRNA integrity as a mechanism of artificial deteriorated cotton seeds. After: ROS-induced ATP synthase mRNA degradation and metabolism dysfunction reveals the mechanism of artificial deteriorated cotton seeds Comment 2: Abstract and conclusion are too simple; no novelty or significance is highlighted. Response: We appreciate the reviewer’s advice. We have rewritten the abstract and conclusion to highlight the novelty, emphasizing the mechanistic link between ROS accumulation and ATP synthase mRNA integrity loss, and its implications for seed vigor and germplasm preservation. Comment 3: Please double-check grammar; overall language needs verification. Response: We have carefully revised the entire manuscript for grammar, tense consistency, and scientific style. Comment 4: Please rewrite the introduction or add constructive information to make it attractive. Response: We have enriched the Introduction by emphasizing the gap in understanding mRNA oxidation during cotton seed aging. Comment 5: Add graphical/pictorial representation of your work along the abstract. Response: We have prepared a Graphical Abstract illustrating the sequence: ROS accumulation → mRNA oxidative damage → impaired ATP synthesis → Mitochondrial dysfunction. The figure provides an intuitive summary of our findings. (A figure are provided below this document.) Comment 6: Graphs are too small and fonts unclear. Response: All figures have been resized to improve readability(Results section, Figs. 1–10). Comment 7: Add more justification in discussion and include relevant new findings. Response: We have expanded the Discussion to better connect ROS accumulation, mitochondrial dysfunction, and ATP synthase mRNA damage. We also referenced recent literature supporting the role of mRNA oxidation in post-transcriptional regulation during seed aging. Comment 8: Ensure references follow journal pattern. Response: All references have been reformatted according to PLOS ONE guidelines, including DOI addition and consistent author-year style ALL COMMENTS HAVE BEEN PROPERLY ADDRESSED IN CURRENT MANUSCRIPT GOOD LUCK Reviewer #2: The authors have significantly improved the manuscript and addressed all comments, the paper should be accepted. ****** 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: Yes: Dr. Beenish Afzal Reviewer #2: Yes: Muhammad Amjad Ali ******** https://doi.org/10.1371/journal.pone.0339977.r003
Formally Accepted
Acceptance Letter - Smita Kumar, Editor PONE-D-25-49082R1 PLOS One Dear Dr. Chen, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS One. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. You will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Smita Kumar Academic Editor PLOS One https://doi.org/10.1371/journal.pone.0339977.r004

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