Transcriptome analysis of Kentucky bluegrass subject to drought and ethephon treatment

Jiahang Zhang; Yanan Gao; Lixin Xu; Liebao Han

doi:10.1371/journal.pone.0261472

Peer Review History

Original SubmissionMay 20, 2021
1 Oct 2021 Decision Letter - Anil Kumar Singh, Editor PONE-D-21-16354Transcriptome analysis of Kentucky bluegrass subject to drought and ethephon treatmentPLOS ONE Dear Dr. Xu, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Nov 14 2021 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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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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No Reviewer #2: No ****** 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 ****** 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: 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: In this manuscript, the authors have investigated how ethephon pretreatment could help to improve the drought tolerance of Kentucky bluegrass by carrying out a comparative transcriptomic studies on three different treatment groups i.e. control plants grown under well-watered conditions (CK), plants subjected to drought treatment without ethephon pretreatment (Drought) and plants subjected to drought treatment with ethephon pretreatment (ETH_D). The conclusion appears to be that the ethephon pretreatment might improve the drought tolerance of Kentucky bluegrass by modulating the expression of several genes associated with protein degradation and stabilization, phytohormones, intracellular transport, antioxidant system and the glyoxalase pathway, cell wall and cuticular wax, signal transduction pathway, fatty acid unsaturation, photosynthesis and defense and osmoregulation. The scientific content of the present study design is useful for understanding how ethephon pretreatment can help in improving the drought tolerance in Kentucky bluegrass. This study will also help to identify the potential candidate genes that can be genetically engineered to enhance drought tolerance in plants. However, the manuscript needs extensive revision for typographical errors and grammar. Reviewer #2: The manuscript titled “Transcriptome analysis of Kentucky bluegrass subject to drought and ethephon treatment”, describe a technically sound piece of scientific research where Illumina RNAseq analysis has been performed on drought and ethephon treated Kentucky bluegrass and the relative water content and malondialdehyde content were measured, with validation of few differentially expressed genes. The authors made all data underlying the findings in their manuscript fully available. The language in submitted articles is clear, correct, and unambiguous. However, some major concerns need to be addressed • Most conclusions are exaggerated and are drawn based on RNAseq data with qRT-PCR validation of randomly selected eight differentially expressed genes. • The statistical analysis has not been performed for any of the experiments especially for Relative water content (RWC) and Malondialdehyde (MDA) content. Even though statement significantly increased and decreased is used without performing the statistical test. Based on MDA and RWC content, we cannot conclude that ethephon could improve the drought tolerance of Kentucky bluegrass. Few more assays need to be performed before drawing any of the conclusions. • Authors need to show representative images of plants before and after drought stress with ethephon treatment, to understand any physiological changes in control, drought and ethephon treated plants. • Authors have claimed that changes in the cell wall and cuticular wax along with proline, antioxidant enzymes and unsaturated fatty acids levels due to ethephon treatment has improved the Kentucky bluegrass drought tolerance, based on expression levels. Authors need to further support their findings and claims by performing assays and anatomical studies. • Authors need to improve the quality of the figures provided. In Fig.6, provide the name of genes instead of gene I.D’s, as authors have not mentioned which gene I.D represent which gene. Also, need to discuss two downregulated genes (c117236 and c119413) in fig.6. • The objective of this study was to investigate the effect of ethephon on drought tolerance of Kentucky bluegrass and to understand the underlying mechanism by analyzing and identifying genes involved in ethephon mediated drought tolerance improvement, which authors had not provide any mechanism or tentative figure which assimilates the whole study into hypothesis and randomly selected eight differentially expressed genes does not prove or show any mechanism as such. This study is a more theoretical approach than experimental in revealing the mechanism for how ethephon regulates drought response and improves drought tolerance of Kentucky bluegrass. Overall, authors need to perform few more assays with proper statistical analysis which can support the conclusions drawn by authors and submit high-quality figures. ****** 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: Yes:** Monika Bhuria 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. Attachments Attachment Submitted filename: Comments for PONE-D-21-16354.docx Attachment Submitted filename: Reviewer comments.docx https://doi.org/10.1371/journal.pone.0261472.r001
Revision 1
13 Nov 2021 Author Response Response to Reviewer #1: 1. In the sub section 2.1, authors have discussed about the well-watered control plants with ethephon pre-treatment (ETH). However, authors did not provide any data related to this group. Response: Discussions related to well-watered control plants with ethephon pre-treatment (ETH) was deleted. In this study, we want to investigate the effect of ethephon on drought tolerance of Kentucky bluegrass by comparing and analyzing DEGs between (ETH_D vs CK) and (Drought vs CK). Therefore, we decided to delete the corresponding part related to well-watered control plants with ethephon pre-treatment (ETH). 2. In the sub section 2.3, authors should provide manufacturer’s country for the TRIzol kit used for RNA isolation. Response: The manufacturer’s country for the TRIzol kit used for RNA isolation has been added. 3. In the sub section 2.6, authors should provide manufacturer’s name and country for qPCR kit used for two-step qRT-PCR detection. Response: The manufacturer’s name and country for qPCR kit used for two-step qRT-PCR detection have been added. 4. In the sub section 2.6, authors should mention the citation for evaluating the relative quantitative data using the 2-ΔΔ CT method. Response: The citation for evaluating the relative quantitative data using the 2-ΔΔ CT method has been added. 5. In the sub-section 3.3, please properly mention the different sub-figures of Fig. 3 in the text while explaining the figure. Response: Revised. 6. In the sub-section 3.5, please correct the sentence by adding ‘regulation of’ in the line “with drought by mediating the regulation of fewer genes in response to drought”. Response: Corrected. 7. In the sub-section 3.5.1, please mention the plant in which ERF113 have been characterized. Response: Revised. 8. The manuscript needs to be proofread. Please correctly frame the sentences. e.g. “Type II PI(4,5)P2 phosphatase (Transmembrane protein 55A, 4.7 fold) identified………..transduction pathways”. “Ras superfamily of small GTP-binding proteins (GTPases) the involvement of ARFs in …………….stresses in plant species”. “Transgenic Arabidopsis plants overexpressing heterologous remorin gene………..including dehydration and salinity”. “They are among the plant proteases that are increased in their activity following stress”. “Abiotic stresses drive …………including salt, anoxia, hypoxia, heat, mechanical damages, drought, cold, and waterlogging”. “During drought…………………..internal turgor pressure is important”. “In addition, pectin may ………………by modulation of stomata movement”. Response: We sincerely appreciated the carefulness and suggestions from the reviewer and revised accordingly. 9. The acronym for ADP-ribosylation factor-like protein is ARL and please correct the line as “ARF and ADP-ribosylation factor-like protein (ARL) belong to Ras”. Response: We have corrected it as suggested. 10. Please italicize the scientific name of plants in the entire manuscript. e.g. “Spartina alterniflora Loisel” “Arabidopsis”, Oryza sativa Response: Revised as suggested. 11. Proofread the entire manuscript for typographical and grammatical errors. e.g. “Tanscript of a remorin gene…..” correct the spelling of Transcript. “also serves as a precursor for the syntheses of Methionine (Met), glutathione” correct the spelling of synthesis.” “Plastocyanin like protein (3.1 fold) is evolved in electron transport and…..” correct the spelling of involved. “Plant defensins are small, highly stable, cysteine-rich peptides constitute a part of the innate…… pathogens”. “ETH treatment caused up-regulation of genes under drought associated with protein degradation and stabilization.” “Transport across intracellular membranes………………….mediated by membrane amino acid transporters generally differ in substrate selectivity and affinity”. “The VGAT is initially……………….. know as the amino acid/auxin permease superfamily”. “Sulfite exporter TauE/SafE gene were also up-regulated” “While the mechanisms remains elucidated, one possible role….” “Photosystem II and electron transport were found up regulated by drought in ETH treated plants……..” “ETH treatment might help plants maintain higher O2 evolution…..”. Response: We proofread the entire manuscript and revised accordingly. We really appreciated the reviewer for the time spent on this manuscript. 12. In the line, “Proline dehydrogenase (-2.3 and -2.6) catalyzes the”, Please mention what – 2.3 and – 2.6 are specifying and mention the gene name given in the RNA seq data. Response: Revised. Should be “Two proline dehydrogenase unigene (-2.3 and -2.6) catalyzes......”. 13. In the line, “CER3 (c90612_g1, 2.5 fold) and WAX2 (c135869_g1, Inf)” what does Inf specify? Response: If the normalized readcounts of one particular gene in one sample is 0 and not 0 in another sample, fold change would be Inf or -Inf. 14. Fig. 1A is not mentioned anywhere in the text. Response: Fig 1A (Fig 1 in revised manuscript) was mentioned the sub section 2.1 for methods explanation. 15. Please mention the supplementary data where it is discussed in the text. Response: We have mentioned supplementary data in the text where it has been discussed. 16. In Fig. 6, perform the statistical analysis to determine the significance for the differences in the RNA seq and qRT-PCR. Response: Instead of carrying out a statistical analysis by LSD value for the same gene between RNA-Seq results and qRT-PCR expression, we performed a correlation statistical analysis. The correlation between RNA-Seq results and qRT-PCR expression was analyzed to get Pearson’s correlation coefficients as describe in sub section 3.4 (Pearson’s r = 0.98 and P 0.001). 17. Author should follow authors guidelines of PLoS One for writing references. Response: We have revised the references accordingly. 18. Authors should maintain uniformity while writing the references Response: References are revised. Response to Reviewer #2: 1. Most conclusions are exaggerated and are drawn based on RNAseq data with qRT-PCR validation of randomly selected eight differentially expressed genes. Response: Conclusions from our study are drawn based on a verified RNAseq data set combined with analysis from various published data as discussed in the manuscript. 2. The statistical analysis has not been performed for any of the experiments especially for Relative water content (RWC) and Malondialdehyde (MDA) content. Even though statement significantly increased and decreased is used without performing the statistical test. Based on MDA and RWC content, we cannot conclude that ethephon could improve the drought tolerance of Kentucky bluegrass. Few more assays need to be performed before drawing any of the conclusions. Response: The statistical analysis result of Relative water content (RWC) and Malondialdehyde (MDA) content has been added and noted in the corresponding figure. As suggested, we provided additional images to support our physiological data. For Kentucky bluegrass, RWC and MDA are two key and confirmed indicators for drought tolerance and they are used and investigated in many turfgrass studies for drought tolerance comparison and evaluation. The relevant studied have been mentioned in the corresponding results and discussion. 3. Authors need to show representative images of plants before and after drought stress with ethephon treatment, to understand any physiological changes in control, drought and ethephon treated plants. Response: The representative images of Kentucky bluegrass plants in well-watered conditions, drought-stressed conditions and ethephon-pretreated plants under drought-stressed conditions have been added accordingly. 4. Authors have claimed that changes in the cell wall and cuticular wax along with proline, antioxidant enzymes and unsaturated fatty acids levels due to ethephon treatment has improved the Kentucky bluegrass drought tolerance, based on expression levels. Authors need to further support their findings and claims by performing assays and anatomical studies. Response: We agree that with corresponding assays and anatomical analysis, the conclusions of this study would be more consolidated and thoroughly confirmed. Meanwhile, this study could still provide meaningful gene regulation information to understand the mechanism underlying ethephon-promoted drought tolerance of Kentucky bluegrass. To date, there is few research on this particular aspect. We hope this study could lay a basic and informative foundation for more and more detailed and experimental research in the future. 5. Authors need to improve the quality of the figures provided. In Fig.6, provide the name of genes instead of gene I.D’s, as authors have not mentioned which gene I.D represent which gene. Also, need to discuss two downregulated genes (c117236 and c119413) in fig.6. Response: Genes in Fig 6 (Fig 8 in the revised manuscript) were selected randomly to verify the effectiveness of RNA-Seq data for further expression level analysis. Genes that we discussed in the text were selected based on the comparison of DEGs between different sampling groups. Gene I.Ds and corresponding names were listed in the Supplementary Data 6 and 7, and were highlighted accordingly. 6. The objective of this study was to investigate the effect of ethephon on drought tolerance of Kentucky bluegrass and to understand the underlying mechanism by analyzing and identifying genes involved in ethephon mediated drought tolerance improvement, which authors had not provide any mechanism or tentative figure which assimilates the whole study into hypothesis and randomly selected eight differentially expressed genes does not prove or show any mechanism as such. This study is a more theoretical approach than experimental in revealing the mechanism for how ethephon regulates drought response and improves drought tolerance of Kentucky bluegrass. Response: We appreciated the reviewer’s suggestion. The idea to generalize the tentative mechanism into figure is really helpful in improving our manuscript. As suggested, Fig 9 was added to provide a mechanism hypothesis based on results from the whole study. Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0261472.r002
3 Dec 2021 Decision Letter - Anil Kumar Singh, Editor Transcriptome analysis of Kentucky bluegrass subject to drought and ethephon treatment PONE-D-21-16354R1 Dear Dr. Xu, 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, Anil Kumar Singh, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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 #1: 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 #1: 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 #1: Authors have substantially revised the manuscript and addressed all the raised concerns. The manuscript can be accepted in its current form. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: Yes:** Monika Bhuria https://doi.org/10.1371/journal.pone.0261472.r003
Formally Accepted
7 Dec 2021 Acceptance Letter - Anil Kumar Singh, Editor PONE-D-21-16354R1 Transcriptome analysis of Kentucky bluegrass subject to drought and ethephon treatment Dear Dr. Xu: 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. Anil Kumar Singh Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0261472.r004

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