Disorders in brassinosteroids signal transduction triggers the profound molecular alterations in the crown tissue of barley under drought

Anetta Kuczyńska; Martyna Michałek; Piotr Ogrodowicz; Michał Kempa; Paweł Krajewski; Vladimiro Cardenia; Maria Teresa Rodriguez-Estrada; Marina Pérez-Llorca; Sergi Munné-Bosch; Krzysztof Mikołajczak

doi:10.1371/journal.pone.0318281

Peer Review History

Original SubmissionMay 24, 2024
12 Jul 2024 Decision Letter - Shailender Kumar Verma, Editor PONE-D-24-21000Disorders in brassinosteroids signal transduction triggers the profound molecular alterations in the crown tissue of barley under droughtPLOS ONE Dear Dr. Mikołajczak, 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 Aug 26 2024 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. 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If your ethics statement is written in any section besides the Methods, please move it to the Methods section and delete it from any other section. Please ensure that your ethics statement is included in your manuscript, as the ethics statement entered into the online submission form will not be published alongside your manuscript. [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: Partly Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't Know Reviewer #2: 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: No ****** 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: No Reviewer #2: No ****** 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: In “Disorders in brassinosteroids signal transduction triggers the profound molecular alterations in the crown tissue of barley under drought”, the authors describe a study examining the genomes, transcriptomes, and proteomes, and agronomic traits of two contrasting lines of barley, one of which encodes a mutation in the brassinosteroid receptor BRI1 (uzu1.a mutant). The manuscript includes extensive exploration of genes showing differential expression due to drought or genotype. Generally the study addresses understanding the roles of hormones in the major crop barley is valuable research. The bioinformatics for the transcriptome are appropriate. The supplementary data tables are transparent for evaluating the findings. Major comments I was unable to identify the figure legends in the submission. The motivation/relevance/justification for several of the experiments is difficult to identify, especially in the introduction and results sections. Most importantly, the authors examine the crown tissue of barley in the study. However, they do not provide information about whether or how brassinosteroids affect the crown (or tillering) in barley (or even other species) to motivate the omics approaches on this choice of tissue. As such, the examination of tillering genes was also poorly motivated in the results or introduction. Restructuring and more detail added to the introduction and results will likely address this. Further, the genomic comparison between lines requires more motivation—especially because the study was primarily transcriptomic and discussion of promoter SNPs was not discussed. Further, whether the SNPs are located in the regions that is supposed to be diverging between the NILs is not described. Providing Minor comments The conditions T1 and T2 have the same names as the agronomic traits measures (T1-T26). Please rename T1 and T2 to “3D” and “10D” to disambiguate the overlap and make it easier for the reader to follow the paper. Reviewer #2: Dear Authors, In this manuscript (Disorders in brassinosteroids signal transduction triggers the profound molecular alterations in the crown tissue of barley under drought) authors investigated how the response to drought stress in barley crown tissue occurs at the molecular level and were interested in how this response is primarily influenced by the brassinosteroid signalling pathway. The manuscript addresses a topical issue and is of interest to the scientific community, but unfortunately I cannot recommend it for publication in this form, as it contains flaws mainly in the methodological part. The major comments are listed below: 1.Keywords- do not correctly capture the meaning of the manuscript, should be corrected. 2.The section "Determination of phytohormone and sterol composition" should be rewritten and supplemented with relevant information. The citation of Dias et al. 2018, which is supposed to describe the LC-MS/MS methodology for the quantification of BRs, is not listed in the reference list, nor have I been able to locate such an article. So this methodology should be described in detail! 3. I assume that if 100 mg of plant material was used, this extract must have been purified somehow to remove ballast substances? Please provide details. 4. It is known from the available literature that 6-keto brassinosteroids (CS, 28-homoCS, norCS) occur in higher concentrations in cereals (wheat, barley), so it is interesting that you have quantified BL at relatively high levels (40-70 ng/g), this should be adequately discussed. Minor points: 1.There are a number of errors in citations throughout the text and many of the abbreviations used are not explained; this should be checked carefully. 2. The “Discussion” section is very long and the reader finds it difficult to navigate. In my opinion, it should be shortened. ****** 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: 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/. 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Revision 1
12 Sep 2024 Author Response We would like to thank you for the suggestions and comments, which have been useful for improving the quality of the manuscript entitled “Disorders in brassinosteroids signal transduction triggers the profound molecular alterations in the crown tissue of barley under drought” by Anetta Kuczyńska, Martyna Michałek, Piotr Ogrodowicz, Michał Kempa, Paweł Krajewski, Vladimiro Cardenia, Maria Teresa Rodriguez-Estrada, Marina Pérez-Llorca, Sergi Munné-Bosch and Krzysztof Mikołajczak. An itemized list has been prepared for the Reviewers, stating how each point was addressed and modified according to the suggestions. We believe that all objections were properly faced and a suitable answer/modification was provided. Reviewer #1: In “Disorders in brassinosteroids signal transduction triggers the profound molecular alterations in the crown tissue of barley under drought”, the authors describe a study examining the genomes, transcriptomes, and proteomes, and agronomic traits of two contrasting lines of barley, one of which encodes a mutation in the brassinosteroid receptor BRI1 (uzu1.a mutant). The manuscript includes extensive exploration of genes showing differential expression due to drought or genotype. Generally the study addresses understanding the roles of hormones in the major crop barley is valuable research. The bioinformatics for the transcriptome are appropriate. The supplementary data tables are transparent for evaluating the findings. Major comments I was unable to identify the figure legends in the submission. Figure captions have been placed directly after the paragraph in which they are first cited in the text, according to the journal's guidelines. According to the reviewer's suggestion, the figure legend has also appeared before the Supporting Information. The motivation/relevance/justification for several of the experiments is difficult to identify, especially in the introduction and results sections. Most importantly, the authors examine the crown tissue of barley in the study. However, they do not provide information about whether or how brassinosteroids affect the crown (or tillering) in barley (or even other species) to motivate the omics approaches on this choice of tissue. As such, the examination of tillering genes was also poorly motivated in the results or introduction. Restructuring and more detail added to the introduction and results will likely address this. We thank the Reviewer for the suggestion. The examination of the main tillering genes was removed from the manuscript, because their behavior did not change significantly in our study. Moreover, this also allowed the reduction of the paper length as requested by Reviewer 2. We have also enriched the manuscript with additional information related to connection of brassinosteroid and tillering (lines 121-132 in the Revised Manuscript with Track Changes): It appears that since BRs promote cell division, a higher level of BRs in the crown should facilitate tillering. It has been established that the overexpression of miR397 activates the BR response and leads to an increase in tillering in rice [29]. It was also found that BRs promotes bud outgrowth in tomato, and BR signaling integrates multiple pathways that control shoot branching [30]. Noteworthy, the growth-promoting phytohormones BRs have distinct roles in regulating tillering in rice and rosette branching in Arabidopsis, respectively. In Arabidopsis, BRs do not regulate the primary branch number [31]; however, in rice, BRs significantly promote tillering [32]. Given that the relationship between BRs and tillering is understood, it should be noted that the functioning of BRs in the crown has not been previously investigated, so multiomic studies were designed in the crown of genotypes differentiated by their efficiency in BR signaling mediated by the BRI1 receptor. Further, the genomic comparison between lines requires more motivation—especially because the study was primarily transcriptomic and discussion of promoter SNPs was not discussed. Further, whether the SNPs are located in the regions that is supposed to be diverging between the NILs is not described. Providing As the genetic background difference for the near isogenic lines are clear we used SNP genotyping to identify the range of the polymorphism between Bowman and BW885 line. We proved that they were genetically more polymorphic than it has been commonly suggested so far. Thus, to reduce the influence of multiple introgressions, defined as regions containing SNPs, on gene expression analysis the polymorphic DEGs were filtered out from the biological interpretation; only those located in the introgressed region around the uzu1.a locus remained. Overall, such approach did not affect substantially our interpretation of differential gene expression analysis, since only about 5% of all DEGs contained SNPs (most of them were not of HIGH translation effect according to VEP tool). In a consequence, only few genes were removed from the discussion, namely, 1 DEG related to brassinosteroids, 1 DEG of ABC transporter, 1 DEG of oxidative stress); however, polymorphic DEGs are available in the supplementary data as the obtained result. Meanwhile, it should be bear in mind that some removed DEGs can play important regulatory functions – this also has been added to the text. The relevant fragments were added to the manuscript: - „…including 136 polymorphic genes between genotypes.” (lines 307-308 in the Revised Manuscript with Track Changes) - “Thus, to reduce the influence of multiple introgressions, defined as regions containing SNPs, on gene expression analysis the polymorphic DEGs were filtered out from the biological interpretation; only those located in the introgressed region around the uzu1.a locus remained. This region was extrapolated roughly based on Dockter et al. study using the HvBRI1 (uzu1.a) gene position (464,679,001-464,682,900; Ensembl Plants), namely, it was the interval 460,879,001-467,372,900 of chromosome 3H (Ensembl Plants); in total seven DEGs were identified there. Nonetheless, such approach did not substantially affect our interpretation of differential gene expression analysis, since only about 5% of all DEGs contained SNPs. Meanwhile, it should be bear in mind that some removed DEGs can play important regulatory functions. For instance, two DEGs containing SNPs in the “upstream” region of the gene (in promoter sequence putatively) were annotated to heat shock factors (HSFs) regulatory network; in fact, they are suggested to participate in plant growth and stress response [51].” (lines 684 – 696 in Revised Manuscript with Track Changes) The small number of polymorphic DEGs between genotypes, 136 (ca. 5% of the total), prompted us to remove them from the interpretation of the results, which is now described in the text. This approach means that we do not further consider the effects of other polymorphic regions between genotypes including the analysis of promoter polymorphism. Nevertheless, on the suggestion of the reviewer, we isolated those genes (17) that had a homozygous SNP in the "upstream region" i.e. potentially related to the gene promoter, and only four of them were DEGs. This convinced us that elimination of polymorphic DEGs from the results interpretation was the suitable approach. The relevant fragment was added to the manuscript; “There were 22 homozygous SNPs (50k Chip) located in ‘upstream gene variant’ of 17 genes with ‘MODIFIER’ effect (Supplementary Table S1).” (lines 282-283 in the Revised Manuscript with Track Changes) The consideration about the relationship between SNPs polymorphism and differential gene expression analysis was removed; therefore, Results and Discussion sections were shortened accordingly. Minor comments The conditions T1 and T2 have the same names as the agronomic traits measures (T1-T26). Please rename T1 and T2 to “3D” and “10D” to disambiguate the overlap and make it easier for the reader to follow the paper. As suggested by the Reviewer we have changed throughout the publication T1 -> 3D and T2 -> 10D. Reviewer #2: Dear Authors, In this manuscript (Disorders in brassinosteroids signal transduction triggers the profound molecular alterations in the crown tissue of barley under drought) authors investigated how the response to drought stress in barley crown tissue occurs at the molecular level and were interested in how this response is primarily influenced by the brassinosteroid signalling pathway. The manuscript addresses a topical issue and is of interest to the scientific community, but unfortunately I cannot recommend it for publication in this form, as it contains flaws mainly in the methodological part. The major comments are listed below: 1.Keywords- do not correctly capture the meaning of the manuscript, should be corrected. Thank you for this suggestion. Keywords have been changed as follows: abiotic stress, crown, functional annotation, Hordeum vulgare L., phytohormones, stress-induced proteins, transcriptomics, uzu1.a 2.The section "Determination of phytohormone and sterol composition" should be rewritten and supplemented with relevant information. The citation of Dias et al. 2018, which is supposed to describe the LC-MS/MS methodology for the quantification of BRs, is not listed in the reference list, nor have I been able to locate such an article. So this methodology should be described in detail! We thank the Reviewer for bringing this to our attention. According to the suggestion, the citation of Dias et al. 2018 has been added and the section “Determination of phytohormone and sterol composition” has been modified as follows (lines 233-239 in the Revised Manuscript with Track Changes): Briefly, extracts were obtained by vortexing, ultrasonication for 30 min and centrifugation (9500 g, 15 min, 4°C) of the mixture of plant material powder and extraction solvent (methanol and 1% glacial acetic acid) containing labelled standards (d3 – BL, d3 – CS, d3 – CT). The extracts were then filtered with 0.22 µm PTFE filters (Phenomenex, Torrance, CA, USA) prior to injection into the UHPLC-MS/MS system. The measurements were conducted by multiple reaction monitoring (MRM) in positive ion mode; technical details are reported in Setsungnern et al. [43]. 3. I assume that if 100 mg of plant material was used, this extract must have been purified somehow to remove ballast substances? Please provide details. The missing details have been placed in the lines 236-238 in the Revised Manuscript with Track Changes: The extracts were then filtered with 0.22 µm PTFE filters (Phenomenex, Torrance, CA, USA) prior to injection into the UHPLC-MS/MS system. 4. It is known from the available literature that 6-keto brassinosteroids (CS, 28-homoCS, norCS) occur in higher concentrations in cereals (wheat, barley), so it is interesting that you have quantified BL at relatively high levels (40-70 ng/g), this should be adequately discussed. As suggested by the Reviewer the high level of brassinolide obtained in the study has been discussed (lines 950-956 in the Revised Manuscript with Track Changes): Generally, castasterone is suggested to show the highest biological activity among BRs hormones in monocots including barley [57, 80]; however, this finding is primarily ground on the leaves-based study. Apparently, in the crown, brassinolide may act more stimulatively than castasterone, thus facilitating the development of lateral shoots. Consequently, the higher concentration of brassinolide in the crown, where the branching is controlled, may lead to lower levels of castasterone, as brassinolide promotes the branching of shoots at the expense of castasterone activity; however, this aspect requires further investigation. Minor points: 1.There are a number of errors in citations throughout the text and many of the abbreviations used are not explained; this should be checked carefully. We thank the Reviewer for the suggestion. As recommended, we have carefully checked the entire text and the citations are corrected and the abbreviations are explained. 2. The “Discussion” section is very long and the reader finds it difficult to navigate. In my opinion, it should be shortened. The discussion has been shortened based on the results along with changes suggested by the Reviewers. The consideration about the relationship between SNPs polymorphism and differential gene expression analysis was removed; therefore, Results and Discussion sections were shortened accordingly, and three tables were deleted. Also, some fragments concerning less interesting observations were deleted to reduce the length of the manuscript (e.g. lines 697-713, 732-750, 763-773, 848-858, 883-896 in the Revised Manuscript with Track Changes). Additionally, to facilitate the readability and navigation through the discussion few subheadings were introduced into the Discussion section. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0318281.r002
29 Oct 2024 Decision Letter - Shailender Kumar Verma, Editor PONE-D-24-21000R1Disorders in brassinosteroids signal transduction triggers the profound molecular alterations in the crown tissue of barley under droughtPLOS ONE Dear Dr. Mikołajczak, 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 Dec 13 2024 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'. 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, Shailender Kumar Verma, Ph.D. Academic Editor PLOS ONE [Note: HTML markup is below. Please do not edit.] 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: (No Response) ******** 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: No ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: 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: No ****** 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 ****** 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: Dear authors, Dias et al. 2018 and Setsungnern et al. 2020 do not provide validation parameters for the UHPLC-MS/MS method; if this method has been properly validated, please provide the appropriate citation where these data can be found. If not, I strongly recommend including the corresponding chromatograms and spectral data in the Supplements. Did you made sure that matrix effects, when working with 100 mg of plant material without using SPE or derivatization , is not disturbing your quantification? ****** 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 ******** [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.0318281.r003
Revision 2
15 Nov 2024 Author Response Dear Reviewer, To address your comment regarding the methodology for phytohormone determination, we have significantly expanded the description in the Materials and Methods section (lines 228-242). Additionally, we have introduced a supplementary table (Supplementary Table S1) presenting the optimized MRM parameters, and, as per your suggestion, we have included a supplementary figure (Figure S1) featuring the extracted ion chromatograms (XIC) of MRM and enhanced product ion (EPI) spectra of the brassinosteroids analyzed in the barley samples. These revisions have resulted in a renumbering of the supplementary materials. The citation Dias et al. 2018 has been removed that resulted in renumbering of cited literature within the manuscript. Did you made sure that matrix effects, when working with 100 mg of plant material without using SPE or derivatization, is not disturbing your quantification? Response: Matrix effects are certainly reducing a little bit the sensitivity of the MS/MS but it is not disturbing the quantification. Peaks were in some cases small but clear enough to allow a proper quantification (see newly submitted suppl. file). Many thanks for all your comments and criticisms raised, which have served to improve our work. We hope these changes comprehensively address your concerns and enhance the clarity and robustness of the manuscript. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0318281.r004
30 Dec 2024 Decision Letter - Nguyen Hoai Nguyen, Editor PONE-D-24-21000R2Disorders in brassinosteroids signal transduction triggers the profound molecular alterations in the crown tissue of barley under droughtPLOS ONE Dear Dr. Mikołajczak, 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 Feb 13 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. 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'. 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, Nguyen Hoai Nguyen 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 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 ******** 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: (No Response) ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: (No Response) ****** 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: (No Response) ****** 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: (No Response) ****** 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: The authors have added the required information regarding LC-MS analysis.I agree to accept this manuscript. ****** 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 ******** [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.0318281.r005
Revision 3
12 Jan 2025 Author Response We would like to thank you for the suggestions and comments, which have been useful for improving the quality of the manuscript entitled “Disorders in brassinosteroids signal transduction triggers the profound molecular alterations in the crown tissue of barley under drought” by Anetta Kuczyńska, Martyna Michałek, Piotr Ogrodowicz, Michał Kempa, Paweł Krajewski, Vladimiro Cardenia, Maria Teresa Rodriguez-Estrada, Marina Pérez-Llorca, Sergi Munné-Bosch, and Krzysztof Mikołajczak. Noteworthy, in this round of the revision no additional reviewers’ comments required addressing. However, the Editor suggested via email message (the original message bellow) to double check the manuscript to make sure there are no typos, which has been carefully done. The file Manuscript with Tracking Changes.doc reflects these corrections. Additionally, the funding information has been removed from the main text, as requested by the editorial office. We hope these changes comprehensively addresses Editor’s concerns. “Dear Dr. Krzysztof Mikołajczak, Please double check your manuscript once again to make sure there are no typos in the manuscript. Please note that the microRNA names should be italicized as well. After that, please resubmit the manuscript via the PLOS ONE system. Thank you very much. Best regards, Nguyen” https://doi.org/10.1371/journal.pone.0318281.r006
14 Jan 2025 Decision Letter - Nguyen Hoai Nguyen, Editor Disorders in brassinosteroids signal transduction triggers the profound molecular alterations in the crown tissue of barley under drought PONE-D-24-21000R3 Dear Dr. Mikołajczak, 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, Nguyen Hoai Nguyen Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0318281.r007
Formally Accepted
17 Jan 2025 Acceptance Letter - Nguyen Hoai Nguyen, Editor PONE-D-24-21000R3 PLOS ONE Dear Dr. Mikołajczak, 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 If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks 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. Nguyen Hoai Nguyen Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0318281.r008

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