Response of salt stress resistance in highland barley (Hordeum vulgare L. var. nudum) through phenylpropane metabolic pathway

ZhengLian Xue; BingSheng Wang; ChangYu Qu; MengDie Tao; Zhou Wang; GuoQiang Zhang; Ming Zhao; ShiGuang Zhao

doi:10.1371/journal.pone.0286957

Peer Review History

Original SubmissionFebruary 19, 2023
30 Mar 2023 Decision Letter - Mojtaba Kordrostami, Editor PONE-D-23-04873Response of stress resistance in highland barley ( Hordeum vulgare L. var. nudum ) through phenylpropane metabolic pathwayPLOS ONE Dear Dr. Zhao, 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. ============================== ACADEMIC EDITOR: Dear Authors, Three independent expert reviewers have finalized their revisions to the manuscript. Based on their comments, the manuscript cannot be accepted in its present form but needs extensive revisions. The main criticisms regard the Results and Discussion sections that should be rechecked before accepting. The Discussion must be facilitated with an adequate discussion of results avoiding speculation based on previous studies. The English language needs significant revision to meet the journal's standards. I encourage the authors to proceed with revisions and submit the revised version of the manuscript. ============================== Please submit your revised manuscript by May 14 2023 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. Please include the following items when submitting your revised manuscript: 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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In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability. "Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized. Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access. We will update your Data Availability statement to reflect the information you provide in your cover letter. [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? 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: Partly Reviewer #3: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: No Reviewer #3: Yes ****** 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: Yes Reviewer #2: Yes Reviewer #3: Yes ****** 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: Yes ****** 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: Why was gene expression of more important products of the phenylpropane pathway such as chavicol and methyl cinnamate not investigated in this research? What was the temperature of the intelligent artificial climate Chamber during the germination of the seeds? How many seeds were placed in each seedling boxes? Why was NaCLO used? Why was polyphenolic or percolation method not used for extracting? According to the results of the research, the discussion section should be expanded to better show the different aspects of the research topic. Reviewer #2: The manuscript "Response of stress resistance in highland barley ( Hordeum vulgare L. var. nudum ) through phenylpropane metabolic pathway" has been written well but needs minor revisions. The corrections has been highlighted in the manuscript file, please follow. The need to give ANOVA for significance of studied phenotypic traits. Reviewer #3: In title: type of stress? is not addressed. In introduction: in the end paragraph, authors findings for instance "Interestingly, the phenylpropane metabolic pathway was the most important pathway associated with the stress resistance of highland barley………" were represented that must transfer to section results. In materials and methods: -Recommended to use susceptible genotype for further understanding of salt tolerance mechanism as well as new pathway if there is. - The reason for selection of salinity level of 240 mmol/L? emphasizing on previous reports is not sufficient. Is there any data acquired by authors in this field? In Results: Number of biological replications related to RNA-seq experiment? ****** 6. 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 Reviewer #2: Yes: Dr. Qurban Ali Reviewer #3: 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. Attachments Attachment Submitted filename: PONE-D-23-04873 reviewed.pdf https://doi.org/10.1371/journal.pone.0286957.r001
Revision 1
17 May 2023 Author Response Editor's Comments to Author: Comment 1: Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. Answer 1: Thank you very much for your suggestions. We have revised our manuscript according to the PLOS ONE's style requirements. Comment 2: In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. Answer 2: Thank you very much for your suggestions. We apologize for the lack of information for this part. We have supplemented the information of our study’s minimal underlying data (please see lines 218-220 and Table S2) in the revised-manuscript. Reviewer #1 Comment 1: Why was gene expression of more important products of the phenylpropane pathway such as chavicol and methyl cinnamate not investigated in this research? Answer 1: Thank you very much for your comment. Because of that phenylpropane metabolic pathway products such as polyphenols, flavonoids, and lignin play an important role in enhancing plant stress resistance, the above product synthesis pathways were just investigated in this research. KEGG enrichment analysis also showed that DEGs in highland barley cells under salt stress were significantly enriched in the above product synthesis pathways. Certainly, lacking of informations for other products of the phenylpropane pathway could not make the data fully the conclusions. We apologize for the lack of results for this part. Therefore, we have added the methyl cinnamate synthesis pathway in this research and screened its DEG (Figure 5 and Figure 6). The expression level of related enzymes in chavicol synthesis pathway did not significantly change under salt stress. We have added the discussion (please see lines 298) in the revised-manuscript. Comment 2: What was the temperature of the intelligent artificial climate Chamber during the germination of the seeds? Answer 2: Thank you very much for your comment. We apologize for the lack of information for this part. We choose 25℃ as the culture temperature of the intelligent artificial climate chamber during the germination of the seeds, because the highland barley can tolerate cold in the whole growth period, and generally can grow well in the area where the annual average temperature is above 3℃. It was found that the germination rate, germination potential and germination index of highland barley seeds reached the highest at 25℃. We have added this information (please see lines 98) in the revised-manuscript. Comment 3: How many seeds were placed in each seedling boxes? Answer 3: Thank you very much for your comment. We apologize for the lack of information for this part. About 20 g of seeds are placed in each box to ensure that there is space between each seed and that it does not pile up, thus ensuring germination rate and growth. We have added this information (please see lines 96-97) in the revised-manuscript. Comment 4: Why was NaCLO used Answer 4: Thank you very much for your comment. As a typical example of growing in extreme environmental conditions, highland barley has its unique tolerance to adversity and is very suitable for growing on saline soil as a food crop. We analyzed the response mechanism of barley under NaCl condition by transcriptomics and qRT-PCR, which laid the foundation for improving the utilization rate of saline soil. We have added the discussion (please see lines 98-100) in the revised-manuscript. Comment 5: Why was polyphenolic or percolation method not used for extracting? Answer 5: Thank you very much for your comment. The ultrasonic assisted extraction method adopted in this experiment is to extract the effective components in highland barley by increasing the motion velocity and penetration of medium molecules by using the mechanical effect, cavitation effect and thermal effect of ultrasonic. Ultrasonic wave can dissolve polyphenols, flavonoids and other substances contained in plants in methanol as completely as possible. The method has the advantages of short time, low temperature, wide adaptability, less impurity and simple extraction. We have added the Preparation of Highland Barley Extract Solution (please see lines 103-105) in the revised-manuscript. Comment 6: According to the results of the research, the discussion section should be expanded to better show the different aspects of the research topic Answer 6: Thank you very much for your comment. We apologize for the imperfect discussiion section in our manuscript. We have rewritten the discussiion section and the revised parts were marked in red in the revised-manuscript. Reviewer #2 Comment 1: The corrections has been highlighted in the manuscript file, please follow. Answer 1: Thank you very much for reviewing this manuscript and revising the questions. Revised portions were marked in red in the revised manuscript. Comment 2: The need to give ANOVA for significance of studied phenotypic traits. Answer 2: Thank you very much for your comment. We have supplement ANOVA for significance of the content of antioxidants.We have added these results (please see lines 185 and Table S1) in the revised-Supplementary information. Table S1 The content of antioxidants Day Content of total phenolic（mg·g-1DW） Content of total flavonoid（mg·g-1DW） Content of total ferulic acid（μg·g-1DW） Content of total caffeic acid（μg·g-1DW） non-salt salt non-salt salt non-salt salt non-salt salt 0d 2.15±0.25d 3.46±0.26e 0.98±0.11c 1.01±0.01d 2.01±0.14e 2.35±0.05e 0.21±0.034e 0.26±0.06e 1d 2.76±0.21b 5.53±0.47d 1.01±0.13c 1.05±0.02d 3.83±0.12c 7.31±0.06c 1.26±0.15d 1.61±0.19d 3d 3.09±0.26a 8.38±0.36a 1.62±0.15a 1.98±0.08a 8.97±0.06a 12.22±0.35d 2.89±0.16a 4.69±0.24a 5d 2.31±0.14c 7.97±0.23b 1.20±0.11b 1.48±0.04b 4.59±0.14b 8.53±0.23b 1.98±0.14b 2.71±0.14b 7d 2.19±0.18cd 7.38±0.28c 1.02±0.09c 1.20±0.05c 2.94±0.1d 4.34±0.32d 1.42±0.07c 2.12±0.05c Note: The data are the mean ± standard deviation of 3 independent experiments There was statistical significance for different characters (p < 0.05). Reviewer #3 Comment 1: In title: type of stress? is not addressed Answer 1: Thank you very much for your comment. We have revised the title (please see lines 2) in the revised-manuscript. Comment 2: In introduction:in the end paragraph, authors findings for instance "Interestingly, the phenylpropane metabolic pathway was the most important pathway associated with the stress resistance of highland barley………" were represented that must transfer to section results. Answer 2: Thank you very much for your advice. We have transferred "Interestingly, the phenylpropane metabolic pathway was the most important pathway associated with the stress resistance of highland barley………" from the introduction to the section results (please see lines 286-290) in the revised-manuscript. Comment 3: Recommended to use susceptible genotype for further understanding of salt tolerance mechanism as well as new pathway if there is. Answer 3: Thank you very much for your suggestion.We plan to analyze the salt tolerance of different varieties of highland barley in the next step, and then screen out susceptible plants for further understanding of salt tolerance mechanism as well as new pathway(please see lines 405-408). Comment 4: The reason for selection of salinity level of 240 mmol/L? emphasizing on previous reports is not sufficient. Is there any data acquired by authors in this field? Answer 4: Thank you very much for your comment. We previously determined the contents of total polyphenols and total flavonoids in highland barley under different NaCl concentrations, and found that the contents of total polyphenols and flavonoids reached the maximum under the condition of 240 mmol/L. Therefore, 240 mmol/L NaCl was selected as the salt stress concentration. We have added these results (please see lines 181-183 and Figure S2) in the revised-Supplementary information. Figure S2. Content analysis of highland barley seeds under different salt concentration. (A) The content of total polyphenols under different salt concentration. (B) The contents of total flavonoids changes under different salt concentration Comment 5: Number of biological replications related to RNA-seq experiment? Answer 4: Thank you very much for your question. We apologize for the lack of information for this part. There were 3 biological replicates for each group of RNA-seq data. We have added this information (please see lines 156) in the revised-manuscript. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0286957.r002
26 May 2023 Decision Letter - Mojtaba Kordrostami, Editor Response of salt stress resistance in highland barley ( Hordeum vulgare L. var. nudum ) through phenylpropane metabolic pathway PONE-D-23-04873R1 Dear Dr. Zhao, 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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, 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, Mojtaba Kordrostami, 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 #2: All comments have been addressed Reviewer #3: All comments have been addressed ******** 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 #2: Yes Reviewer #3: Partly ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: Yes Reviewer #3: Yes ****** 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 #2: Yes Reviewer #3: Yes ****** 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 #2: Yes Reviewer #3: Yes ****** 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 #2: (No Response) Reviewer #3: Dear chief editor Based on reviewing results, the manuscript can be published in this journal. Best regards ****** 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 #2: No Reviewer #3: Yes: Hamid Hatami Maleki ******** https://doi.org/10.1371/journal.pone.0286957.r003
Formally Accepted
2 Jun 2023 Acceptance Letter - Mojtaba Kordrostami, Editor PONE-D-23-04873R1 Response of salt stress resistance in highland barley (Hordeum vulgare L. var. nudum) through phenylpropane metabolic pathway Dear Dr. Zhao: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. 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 plosone@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. Mojtaba Kordrostami Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0286957.r004

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