Overexpression of MEKK18 from Arabidopsis pumila in rice significantly enhances stress resistance at the early stage

Junrong Li; Yilin Yang; Zhanglun Sun; Edwin Afriyie Owusu; Leiming Liu; Tianrun Mei; Qingli Zhang; Xianzhong Huang

doi:10.1371/journal.pone.0325550

Peer Review History

Original SubmissionJanuary 15, 2025
Decision Letter - Koppolu Raja Rajesh Kumar, Editor Dear Dr. Huang, 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. ============================== I have now received feedback from the reviewers. The reviewers have raised some very relevant points that need to be addressed before a decision can be made on the manuscript. I request you to respond to the reviewers comments and revise the manuscript accordingly. Please ensure that all concerns are adequately addressed in your response. . ============================== Please submit your revised manuscript by May 05 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'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols . We look forward to receiving your revised manuscript. Kind regards, Koppolu Raja Rajesh Kumar, PhD Academic Editor PLOS ONE Journal requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf . 2. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section. 3. Thank you for stating the following financial disclosure: [This work was supported by the National Natural Science Foundation of China (32270385), the Excellent Scientific Research and Innovation team of the Education Department of Anhui Province (2022AH010087), the Science and technology innovation team of Anhui Sciences and Technology University (2023KJCXTD001), the Talent Introduction Start-up Fund Project of Anhui Science and Technology University (NXYJ202001), the Construction Funds for Crop Science of Anhui Science and Technology University (XK-XJGF001), and the National Innovation and Entrepreneurship Training Program for College Students (202310879009).]. Please state what role the funders took in the study. If the funders had no role, please state: ""The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."" If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. 4. Thank you for stating the following in the Acknowledgments Section of your manuscript: [This work was supported by the National Natural Science Foundation of China (32270385), the Excellent Scientific Research and Innovation team of the Education Department of Anhui Province (2022AH010087), the Science and technology innovation team of Anhui Sciences and Technology University (2023KJCXTD001), the Talent Introduction Start-up Fund Project of Anhui Science and Technology University (NXYJ202001), the Construction Funds for Crop Science of Anhui Science and Technology University (XK-XJGF001), and the National Innovation and Entrepreneurship Training Program for College Students (202310879009).]. We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: [This work was supported by the National Natural Science Foundation of China (32270385), the Excellent Scientific Research and Innovation team of the Education Department of Anhui Province (2022AH010087), the Science and technology innovation team of Anhui Sciences and Technology University (2023KJCXTD001), the Talent Introduction Start-up Fund Project of Anhui Science and Technology University (NXYJ202001), the Construction Funds for Crop Science of Anhui Science and Technology University (XK-XJGF001), and the National Innovation and Entrepreneurship Training Program for College Students (202310879009).]. Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 5. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. [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: Yes Reviewer #2: Yes ****** Reviewer #1: This manuscript describes the overexpression of ApMEKK18 from Arabidopsis pumila increases tolerances to osmotic, salinity, and ABA treatment. The work is OK and informative. However, I have several concerns requiring a revision before this manuscript can be accepted. (1) MEKK is a large gene family in plants. Does rice have its own MEKK18? Please add a phylogenetic analysis for ApMEKK18 in Arabidopsis pumila, Arabidopsis thaliana, and rice. (2) Please provide the statistical data such as survival ratio or ratio of dead leaves for Figure 6E-G. (3) The author found that the ApMEKK18 was mainly located in nucleus. However, MAPKKK (MEKK) is commonly the first step of MAPK pathway. How it works in nucleus. Authors should discuss this. (4) Authors suggested the ApMEKK18 enhanced tolerances by through ABA pathway. Please test the expression of some genes associated with ABA metabolism and signal transduction in WT and overexpression lines. (5) As the most stress tolerances were measured in germination and seedling stages, I recommend the title as “Overexpression of MEKK18 from Arabidopsis pumila in rice significantly enhances stress resistance at the early stage”. (6) How ApMEKK18 enhances the productivity in rice? This is not clear. Please add some sentences to discuss this. Reviewer #2: As the authors emphasized in Abstract and conclusion, the results of this study provide a basis for further research on the function of ApMEKK18 in stress response. To improve the implication of current study, the phenotype of the transgenic rice plants under drought stress should be analyzed and shown. To understand at least a hint behind the role of ApMEKK18 in seed yield and/or stress tolerance, the potential target genes involved in ABA, salt, and drought response should be analyzed. Figure 1; the expression patterns of MpMEKK18 seem to follow circadian rhythm. To rule out the circadian effects, its expression level should be measured at different time points under normal conditions. In Figure legend, the developmental stage of the plant material used should be described. Figure 2; “Chlorophyll indicated by chloroplast autofluorescence” in figure legend is wrong; the red signals should be chlorophyll autofluorescence. Figure S5 is an important supporting data, which should be moved to main Figure 5. Figure 6; only germination rates were measured under salt stress conditions. To further support the role of MpMEKK18 in salt tolerance, other parameters, such as survival rate and chlorophyll content, should be measured. Line 265-265; ApMEKK18 gene expression levels in 13 tissue types from A. pumila were analyzed using qRT-PCR. Flowers exhibited the highest expression, followed by the pedicel, flower buds, and young roots (S1 Fig), which is wrong. Figure S4 should be S1. Subsequent Supplementary figure numbers are also wrong. ****** 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 ******** [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.0325550.r001
Revision 1
14 Apr 2025 Author Response Dear editors, Thank you very much for taking the time to review our manuscript entitled “Overexpression of MEKK18 from Arabidopsis pumila in rice significantly enhances stress resistance” (Submission ID: PONE-D-25-02616). We sincerely appreciate the reviewers for their valuable comments and suggestions on our manuscript. These comments are very helpful in revising and improving our manuscript, and providing important guidance for our future research work. According to the comments of editors and reviews, we have made a careful correction in the revised manuscript. We hope this meets the requirements for a publication. All the revised sections of the text were highlighted using track changes. Our specific responses to each question are as follows: We ensure that we provided the correct grant numbers, and amended the ‘Acknowledgments section’ to ‘Funding Information’, and we confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Also, we provided the original uncropped and unadjusted images of S4 Fig in the revised manuscript. Reviewer #1: This manuscript describes the overexpression of ApMEKK18 from Arabidopsis pumila increases tolerances to osmotic, salinity, and ABA treatment. The work is OK and informative. However, I have several concerns requiring a revision before this manuscript can be accepted. (1) MEKK is a large gene family in plants. Does rice have its own MEKK18? Please add a phylogenetic analysis for ApMEKK18 in Arabidopsis pumila, Arabidopsis thaliana, and rice. Response: Thank you for your suggestion. According to your suggestion, we used the amino acid sequence of ApMEKK18 as a query to blast in the rice database, downloaded the OsMEKK18 partial homologous protein of rice together with the AtMEKK18 partial homologous protein reported in Arabidopsis, and constructed a phylogenetic tree of 11 MEKK18 proteins, as a supplementary figure S1 Fig. Accordingly, the original S1 Fig was renamed S2 fig, and so on. The results showed that ApMEKK18 was closest to AtMEKK18, but farthest from OsMEKK18. (2) Please provide the statistical data such as survival ratio or ratio of dead leaves for Figure 6E-G. Response: Thanks! We provided the detailed statistical data on the survival rate of rice seedlings in Figure 6E-D. The corresponding statistical data can be found in Table S2. (3) The author found that the ApMEKK18 was mainly located in nucleus. However, MAPKKK (MEKK) is commonly the first step of MAPK pathway. How it works in nucleus. Authors should discuss this. Response: Thank you for your very good reviews! There are many MAPKKK gene family members in plants. The subcellular localizations of MAPKKK proteins in plants are jointly regulated by signal stimuli, protein interactions and the characteristics of family members. Their dynamic changes are an important basis for accurate signal transduction. Different members may present different characteristics of subcellular localization due to differences in N-terminal regulatory domains and their interacting proteins (Tajdel-Zielińska et al., 2024). For example, Arabidopsis AtMEKK18 is mainly localized in the nucleus and plays an important role in the ABA signaling pathway (Mitula et al., 2015). In the discussion part of the revised manuscript, relevant discussions are supplemented. The references are as follows: [48] Tajdel-Zielińska M, Janicki M, Marczak M, Ludwików A. Arabidopsis HECT and RING-type E3 Ligases Promote MAPKKK18 Degradation to Regulate Abscisic Acid Signaling. Plant & Cell Physiology. 2024; 65(3): 390-404. https://doi.org/ 10.1093/pcp/pcad165. PMID: 38153765. [49] Mitula F, Tajdel M, Cieśla A, Kasprowicz-Maluśki A, Kulik A, Babula-Skowrońska D, Michalak M, Dobrowolska G, Sadowski J, Ludwików A. Arabidopsis ABA-Activated Kinase MAPKKK18 is Regulated by Protein Phosphatase 2C ABI1 and the Ubiquitin-Proteasome Pathway. Plant & Cell Physiology. 2015; 56(12): 2351-2367. https://doi.org/10.1093/pcp/pcv146. PMID: 26443375. (4) Authors suggested the ApMEKK18 enhanced tolerances by through ABA pathway. Please test the expression of some genes associated with ABA metabolism and signal transduction in WT and overexpression lines. Response: Thank you for your suggestion. We analyzed the expression levels of three ABA synthesis genes, 9-Cis-Epoxycarotenoid Dioxygenase (OsNCED1), OsNCED2, and OsNCED4, and one ABA metabolism gene, ABA 8′-Hydroxylase (OsABA8ox1) in the control (NP) and ApMEKK18-OE8 plants. qRT PCR results showed that compared with NP seedlings, the expression levels of OsNCED1 and OsNCED4 genes were significantly reduced in ApMEKK18-OE8 seedlings, but OsNCED2 was up-regulated in ApMEKK18-OE8 seedling, while OsABA8ox1 was significantly down regulated in ApMEKK18-OE8 (S6 Fig.). (5) As the most stress tolerances were measured in germination and seedling stages, I recommend the title as “Overexpression of MEKK18 from Arabidopsis pumila in rice significantly enhances stress resistance at the early stage”. Response: Thanks. According to your suggestion, we amended the title to “Overexpression of MEKK18 from Arabidopsis pumila in rice significantly enhances stress resistance at the early stage”. (6) How ApMEKK18 enhances the productivity in rice? This is not clear. Please add some sentences to discuss this. Response: Thanks. MEKK is an upstream regulatory factor of the MAPK cascade signaling pathway, which regulates cell division and differentiation, participates in plant growth and development, stress response, and metabolic regulation by activating MKK and MAPK. In rice, overexpression of OsMEKK17 gene regulated ABA stress response, optimized stomatal development and water use efficiency, improved drought tolerance, significantly enhanced plant adaptation to drought, and maintained high yields under drought conditions (Cui et al., 2024). ZmMEKK indirectly enhanced drought resistance and yield potential in maize by promoting root development and optimizing root shoot ratio, enhancing water absorption capacity (Shi et al., 2022). In the discussion of the revised manuscript, relevant discussions were added. The references are as follows: [50] Cui L, Song Y, Zhao Y, Gao R, Wang Y, Lin Q, Jiang J, Xie H, Cai Q, Zhu Y, Xie H, Zhang J. Nei 6 You 7075, a hybrid rice cultivar, exhibits enhanced disease resistance and drought tolerance traits. BMC Plant Biology. 2024; 24(1): 1252. https://doi.org/10.1186/s12870-024-05998-2. PMID: 39725902; PMCID: PMC11670435. [51] Shi Z, Zhao B, Song W, Liu Y, Zhou M, Wang J, Zhao J, Ren W. Genome-wide identification and characterization of the MAPKKK, MKK, and MPK families in Chinese elite maize inbred line Huangzaosi. Plant Genome. 2022; 15(3): e20216. https://doi.org/10.1002/tpg2.20216. PMID: 35535627. Reviewer #2: As the authors emphasized in Abstract and conclusion, the results of this study provide a basis for further research on the function of ApMEKK18 in stress response. To improve the implication of current study, the phenotype of the transgenic rice plants under drought stress should be analyzed and shown. To understand at least a hint behind the role of ApMEKK18 in seed yield and/or stress tolerance, the potential target genes involved in ABA, salt, and drought response should be analyzed. Figure 1; the expression patterns of MpMEKK18 seem to follow circadian rhythm. To rule out the circadian effects, its expression level should be measured at different time points under normal conditions. In Figure legend, the developmental stage of the plant material used should be described. Response: Thank you for your insightful reviews. Figure 1 showed the expression profiles of the ApMEKK18 gene under four abiotic stresses within 12 hours. Gene expression exhibited different characteristics of change, and the expression pattern did not follow the regulated expression pattern. Moreover, the samples were all grown under sunlight conditions. Our previous research found that during 48 hours of continuous high salt stress, the expression of the ApMEKK18 gene continued to upregulate, without showing a rhythmic pattern (Yang et al., 2018). In the caption, we have added four time point descriptions. Yang LF, Jin YH, Huang W, Sun Q, Liu F, Huang XZ. Full-length transcriptome sequences of ephemeral plant Arabidopsis pumila provides insight into gene expression dynamics during continuous salt stress. BMC Genomics. 2018; 19(1): 717. doi: 10.1186/s12864-018-5106-y. PMID: 30261913; PMCID: PMC6161380. Figure 2; “Chlorophyll indicated by chloroplast autofluorescence” in figure legend is wrong; the red signals should be chlorophyll autofluorescence. Response: Thanks. We apologized for the mistake and corrected it in the revised manuscript. Figure S5 is an important supporting data, which should be moved to main Figure 5. Response: Thanks. According to your suggestion, S5 Fig. was moved to main text as Fig 6 and the corresponding description was modified. Correspondingly, Fig 6 was renamed Fig 7. Figure 6; only germination rates were measured under salt stress conditions. To further support the role of MpMEKK18 in salt tolerance, other parameters, such as survival rate and chlorophyll content, should be measured. Response: Thanks. Based on your and Reviewer 1's suggestions, in the revised manuscript we added the statistical data on survival rates (S2 Table). Line 265-265; ApMEKK18 gene expression levels in 13 tissue types from A. pumila were analyzed using qRT-PCR. Flowers exhibited the highest expression, followed by the pedicel, flower buds, and young roots (S1 Fig), which is wrong. Figure S4 should be S1. Subsequent Supplementary figure numbers are also wrong. Response: Thank you for your careful reviews. We carefully checked the numbering of the supplementary figures and made corrections to ensure accurate naming of all the figures. Attachments Attachment Submitted filename: response to reviewers.docx https://doi.org/10.1371/journal.pone.0325550.r002
Decision Letter - Koppolu Raja Rajesh Kumar, Editor Dear Dr. Huang, 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. The reviewers have evaluated your revised manuscript. I am pleased to inform you that the manuscript can be accepted for publication once the comments from Reviewer 2 are fully addressed and incorporated into the manuscript. I look forward to receiving your revised version. Please submit your revised manuscript by Jun 19 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'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols . We look forward to receiving your revised manuscript. Kind regards, Koppolu Raja Rajesh Kumar, PhD Academic Editor PLOS ONE Journal Requirements: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author Reviewer #1: All comments have been addressed Reviewer #2: (No Response) ******** 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: Good job! This manuscript is much improved since the last revision. I am satisfied with it and have no further concerns. Reviewer #2: The authors addressed all of my comments and suggestions with additional experimental data, which improves and clarifies the manuscript. However, a couple of points should be further considered. Fig. S6 showing the expression levels of ABA-related genes is crucial for supporting the role of ApMEKK18 in ABA response. First, the description in line 368-369 “while ABA 8′-Hydroxylase (OsABA8ox1) was significantly down regulated in ApMEKK18-OE8 (S6 Fig)” is wrong; its level was upregulated in the transgenic lines. Moreover, it is necessary to analyze the expression levels of these genes in other transgenic line (OE 7 line) to further support the conclusion. In addition, it should be discussed in Discussion section that how the up- and down-regulation of these genes is associated with the ABA-responsive phenotype of the plants. ****** 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 ******** [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 https://doi.org/10.1371/journal.pone.0325550.r003
Revision 2
12 May 2025 Author Response Dear editors, Thank you very much for taking the time to review our manuscript entitled “Overexpression of MEKK18 from Arabidopsis pumila in rice significantly enhances stress resistance” (Submission ID: PONE-D-25-02616) again. According to the comments of Reviewer #2, we have made a careful correction in the revised manuscript. We hope this meets the requirements for a publication. All the revised sections of the text were highlighted using track changes. Our specific responses to each question are as follows: We ensure that our references are complete and correct, and we have not cited papers that have been retracted. Reviewer #2: The authors addressed all of my comments and suggestions with additional experimental data, which improves and clarifies the manuscript. However, a couple of points should be further considered. Fig. S6 showing the expression levels of ABA-related genes is crucial for supporting the role of ApMEKK18 in ABA response. First, the description in line 368-369 “while ABA 8′-Hydroxylase (OsABA8ox1) was significantly down regulated in ApMEKK18-OE8 (S6 Fig)” is wrong; its level was upregulated in the transgenic lines. Moreover, it is necessary to analyze the expression levels of these genes in other transgenic line (OE 7 line) to further support the conclusion. In addition, it should be discussed in Discussion section that how the up- and down-regulation of these genes is associated with the ABA-responsive phenotype of the plants. Response: Thank you for your careful reviews. We apologized for the mistake and corrected it in the revised manuscript. We have analyzed the expression levels of these genes in ApMEKK18-OE7 line, and revised the S6 Fig. in the revised manuscript. Furthermore, we added the corresponding descriptions of the implications of changes in gene expression levels in Discussion section. Attachments Attachment Submitted filename: Response_to_reviewers_auresp_2.docx https://doi.org/10.1371/journal.pone.0325550.r004
Decision Letter - Koppolu Raja Rajesh Kumar, Editor Overexpression of MEKK18 from Arabidopsis pumila in rice significantly enhances stress resistance at the early stage PONE-D-25-02616R2 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. 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. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org. 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, Koppolu Raja Rajesh Kumar, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0325550.r005
Formally Accepted
Acceptance Letter - Koppolu Raja Rajesh Kumar, Editor PONE-D-25-02616R2 PLOS ONE Dear Dr. Huang, 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. 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. Koppolu Raja Rajesh Kumar Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0325550.r006

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .