Transcriptome-wide identification and expression profiling of the ERF gene family suggest roles as transcriptional activators and repressors of fruit ripening in durian

Gholamreza Khaksar; Supaart Sirikantaramas

doi:10.1371/journal.pone.0252367

Peer Review History

Original SubmissionMay 12, 2021
17 Jun 2021 Decision Letter - Takaya Moriguchi, Editor PONE-D-21-15671 Transcriptome-wide identification and expression profiling of the ERF gene family suggest roles as transcriptional activators and repressors of fruit ripening in durian PLOS ONE Dear Dr. Sirikantaramas, 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. Your manuscript was reviewed by two scientists with expertise in the subject area of your study. As you can see from their reviews, both referees positively evaluated your manuscript, but still had several concerns especially on your experimental design including statistical method. In view of the referees’ reviews and recommendations, the current manuscript could be accepted after major revision. I hope that the reviews will aid you and colleagues in considering the additional studies. From handling editor: Please submit your revised manuscript by Aug 01 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. In Fig. 2, explanation for motifs 3-10 should be added in the legend. Resolution for some Figures are quite low, provide good ones. In Fig.7D, the expression of DzERF9 in the natural ripening fruit should be high, but its expression is low against the expectation. Provide the reason(s) for its low expression level. Provide the data if the existence of the auxin responsive motifs in the promoter region of DzERF6 and DzERF9, if any. In the current Fig. 8, the role of DzERF6 was not well shown. DzERF6 acts as a repressor in the unripe fruit. Modify the schematic flow showing the DzERF6 function. 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. 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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. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ [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: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes 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: Yes 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 the present study, the ERF gene family was investigated in durian fruit. Two ERF genes (DzEFR6 and DzERF9), which regulated durian fruit ripening, were identified and their regulation on ethylene biosynthesis were discussed. The manuscript was well organized, and the results presented in this study were clear and informative. Thus, I suggested it to be published after some revisions. 1. In the material and method, how the authors determined the maturation of fruit? Why 1 day, 3 days, and 5 days were selected in this study. I think that the maturity indication should be described in the material and method, and the photos of unripe, midripe, and ripe samples should be added. 2. It is suggested to describe the concentrations of ethephon and 1-MCP used in this study. In addition, in Line 137, how many days were the fruit stored until the ethephon-induced samples ripened. 3. In this study, the authors found that DzEFR6 was a transcriptional repressor, while DzERF9 was a transcriptional activator of fruit ripening. What was the sequence difference between the two genes? Were functions of DzEFR6 and DzERF9 investigated in other plants previously? 4. The expression of DzEFR6 and DzERF9 in response to auxin treatment was investigated in leaves. This purpose of this study was to characterize the ERF genes in fruit during the ripening. Thus, it was difficult to understand why leaves were used instead of fruit in the auxin treatment experiment. Reviewer #2: This work tried to clarify the role of the ERF gene family as transcriptional activators and repressors of fruit ripening in durian. The results and conclusion are reasonable at least for this reviewer. Please consider the following items to enhance the value of your work. L134-136 As far as the reviewer reads how to apply ethephon and 1-MCP from your previous study, the related description is not so long. Therefore, how about adding such a description in the current manuscript? L141-142 It is unclear how to apply auxin. L235-239, and Figs. 6 & 7 This reviewer is not sure whether we can carry out parametric methods for the data of fold change. In any case, nowadays we should not use Duncan’s multiple range test. Please confirm and reconsider your statistical analysis. If part of the results is changed, you should revise the descriptions related to them. ****** 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/. 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. 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Revision 1
21 Jul 2021 Author Response Dear Editor, We do appreciate your valuable comments. We did try to do our best and revised our manuscript accordingly. In Fig. 2, explanation for motifs 3-10 should be added in the legend. Response: Many thanks for your valuable comment. The explanation for motifs 3-10 has been added to the Figure 2 legend (Line 284-285) and Discussion (line 406-413). 2. Resolution for some Figures are quite low, provide good ones. Response: Following your comment, all source files have been uploaded with higher resolution (at least 350 dpi). 3. In Fig.7D, the expression of DzERF9 in the natural ripening fruit should be high, but its expression is low against the expectation. Provide the reason(s) for its low expression level. Response: Dear Editor, According to your comment, we think you mean Fig. 6D. In Fig. 6D, the relative expression levels of DzERF9 under three different ripening conditions, natural (control), ethylene-induced, and 1-MCP-delayed ripening were quantified by using the 2−ΔΔCt method, and levels were normalized by the geometric mean of reference genes and the natural ripening condition which was set to 1 (as the control) (Line 365-366). Considering the Ct value of natural ripening, it was significantly higher than 1-MCP and dramatically lower than ethephon, consistent with our expectation. 4. Provide the data if the existence of the auxin responsive motifs in the promoter region of DzERF6 and DzERF9, if any. Response: Many thanks for raising this interesting point. Our in silico analyses of the 2-kb promoter regions located upstream of the translation start site of DzERF6 and DzERF9 confirmed the existence of auxin response elements (AuxREs; TGTCTC) which are the binding sites for auxin response factor (ARF) transcription factor family (Line 484-486). The promoter regions have been added to the manuscript as Supplementary Fig. S5. 5. In the current Fig. 8, the role of DzERF6 was not well shown. DzERF6 acts as a repressor in the unripe fruit. Modify the schematic flow showing the DzERF6 function. Response: Many thanks for your valuable comment. As a transcriptional repressor of fruit ripening, we propose that DzERF6 acts via negatively regulating the transcription of ethylene biosynthetic genes. Accordingly, this proposed function has been added to our schematic flow. The figure legend has been revised accordingly (Line 504-506). Dear Reviewer 1, We do appreciate your valuable comments. We did try to do our best and revised our manuscript accordingly. 1. In the material and method, how the authors determined the maturation of fruit? Why 1 day, 3 days, and 5 days were selected in this study. I think that the maturity indication should be described in the material and method, and the photos of unripe, midripe, and ripe samples should be added. Response: Many thanks for your valuable comment. Since durian is a climacteric fruit, the fruit is commonly harvested at a commercially mature stage (105 days after anthesis for Monthong cultivar as mentioned in the manuscript, Line 122). At this stage, the fruit is considered to be at the unripe stage. After leaving the fruit at room temperature for a few days, durian fruit undergoes a post-harvest ripening process during which climacteric ethylene biosynthesis and respiration rate increase dramatically which then accelerate the post-harvest ripening process (Ketsa and Daengkanit, 1998, link: https://www.tandfonline.com/doi/abs/10.1080/14620316.1998.11511017). Therefore, we do not use “day after anthesis: DAA” during post-harvest ripening process which includes midripe and ripe stages. Preferably, to determine the ripeness of the fruit, we count the days during post-harvest ripening process as “days after harvest” and measure fruit firmness as an indication of fruit ripening. Accordingly, fruits which were harvested at the commercially mature stage were kept at room temperature (30 °C) for post-harvest ripening until reaching a firmness of 3.4 ± 0.81 N (∼3 days after harvest) (for midripe stage) and 1.55 ± 0.45 N (∼5 days after harvest) (for ripe stage) (Khaksar et al., 2019). We have revised this section accordingly and added the information of fruit firmness (Line 123-127). Following your comment, the photos of unripe, midripe, and ripe durian samples have been added as Supplementary Fig. S1 (Line 130-131). 2. It is suggested to describe the concentrations of ethephon and 1-MCP used in this study. In addition, in Line 137, how many days were the fruit stored until the ethephon-induced samples ripened. Response: Following your comment, the concentrations of ethephon and 1-MCP were added (Line 137-141). In addition, the control and treated samples were kept at room temperature (30 °C) (for ∼3 days) until the ethephon-induced samples ripened. This point was added to the manuscript (Line 142). 3. In this study, the authors found that DzEFR6 was a transcriptional repressor, while DzERF9 was a transcriptional activator of fruit ripening. What was the sequence difference between the two genes? Were functions of DzEFR6 and DzERF9 investigated in other plants previously? Response: Many thanks for your valuable comment. Following your suggestion, we analyzed and compared the sequences of DzERF6 and DzERF9. Notably, the amino acid sequence analysis of DzERF9 showed regions of acidic amino acid-rich, including Gln-rich and/or Ser/Thr-rich amino acid sequences which are often designated as transcriptional activation domains [Liu et al., 1999]. However, our sequence analysis of DzERF6 revealed the existence of regions rich in DLN(L/F)xP, which are often associated with transcriptional repression [Ohta et al., 2001]. This point has been added to our discussion (Line 432-436). The functions of both DzERF6 and DzERF9 have been previously investigated. Functional characterization of ERF6 from tomato (SlERF6) suggested its role as a transcriptional repressor via negatively regulating transcription of ethylene biosynthetic genes (Line 421-423). Functional characterization of ERF9 from banana (MaERF9) suggested its role as a transcriptional activator of fruit ripening via trans-activating the ethylene biosynthetic genes (Line 425-427). 4. The expression of DzEFR6 and DzERF9 in response to auxin treatment was investigated in leaves. This purpose of this study was to characterize the ERF genes in fruit during the ripening. Thus, it was difficult to understand why leaves were used instead of fruit in the auxin treatment experiment. Response: Many thanks for raising this point. Unfortunately, during conducting this experiment, we did not have durian fruits to be used in our experiment because it was not the fruit season in Thailand. Therefore, we only had access to the leaves of durian trees. We hope you accept our explanation. Although we performed our experiment in durian leaves, the results provided a strong evidence that auxin affects the expression of both DzERF6 and DzERF9. More details regarding the auxin treatment have been added to the Materials and methods (Line 145-148). Dear Reviewer 2, We do appreciate your valuable comments. We did try to do our best and revised our manuscript accordingly. L134-136 As far as the reviewer reads how to apply ethephon and 1-MCP from your previous study, the related description is not so long. Therefore, how about adding such a description in the current manuscript? Response: Many thanks for your valuable comment. Following your suggestion, detailed description has been added to the manuscript (Line 137-144). L141-142 It is unclear how to apply auxin. Response: Many thanks for your valuable comment. Following your suggestion, detailed information regarding the auxin treatment has been added to the manuscript (Line 145-148). L235-239, and Figs. 6 & 7 This reviewer is not sure whether we can carry out parametric methods for the data of fold change. In any case, nowadays we should not use Duncan’s multiple range test. Please confirm and reconsider your statistical analysis. If part of the results is changed, you should revise the descriptions related to them. Response: We do appreciate your valuable suggestion. Following your comment, we reconsidered our statistical analysis. We carried out our statistical analysis of RT-qPCR data using the method described in Steibel et al. (2009) which consists of the analysis of cycles to threshold values (Ct) for the target and reference genes according to a linear mixed model (Model I of the report of Steibel et al. (2009)). This approach is more accurate and powerful than existing alternatives (parametric methods) for RT-qPCR data analysis and has been extensively exploited in many studies, such as: Pathi KM, Rink P, Budhagatapalli N, Betz R, Saado I, Hiekel S, Becker M, Djamei A and Kumlehn J (2020) Engineering Smut Resistance in Maize by Site-Directed Mutagenesis of LIPOXYGENASE 3. Front. Plant Sci. 11:543895. doi: 10.3389/fpls.2020.543895 Becker, M., Ngo, N.S. & Schenk, M.K.A. Silicon reduces the iron uptake in rice and induces iron homeostasis related genes. Sci Rep 10, 5079 (2020). https://doi.org/10.1038/s41598-020-61718-4 Angrimani DSR, Francischini MCP, Brito MM, Vannucchi CI. Prostatic hyperplasia: Vascularization, hemodynamic and hormonal analysis of dogs treated with finasteride or orchiectomy. PLoS One. 2020 Jun 25;15(6):e0234714. doi: 10.1371/journal.pone.0234714 Khaksar G and Sirikantaramas S (2020) Auxin Response Factor 2A Is Part of the Regulatory Network Mediating Fruit Ripening Through Auxin-Ethylene Crosstalk in Durian. Front. Plant Sci. 11:543747. doi: 10.3389/fpls.2020.543747 Dimopoulou, A., Theologidis, I., Liebmann, B. et al. Bacillus amyloliquefaciens MBI600 differentially induces tomato defense signaling pathways depending on plant part and dose of application. Sci Rep 9, 19120 (2019). https://doi.org/10.1038/s41598-019-55645-2 Ayuso M, Fernández A, Núñez Y, Benítez R, Isabel B, Barragán C, et al. (2015) Comparative Analysis of Muscle Transcriptome between Pig Genotypes Identifies Genes and Regulatory Mechanisms Associated to Growth, Fatness and Metabolism. PLoS ONE 10(12): e0145162. doi:10.1371/journal.pone.0145162 Pe ́rez-Montarelo D, Ferna ́ndez A, Barraga ́n C, Noguera JL, Folch JM, et al. (2013) Transcriptional Characterization of Porcine Leptin and Leptin Receptor Genes. PLoS ONE 8(6): e66398. doi:10.1371/journal.pone.0066398 Accordingly, we revised the manuscript text as follows: Materials and methods (Line 241-247), Fig. 6 legend (Line 362 & 367), and Fig. 7 legend (Line 381-382). Attachments Attachment Submitted filename: Response to Editor and Reviewers.docx https://doi.org/10.1371/journal.pone.0252367.r002
26 Jul 2021 Decision Letter - Takaya Moriguchi, Editor Transcriptome-wide identification and expression profiling of the ERF gene family suggest roles as transcriptional activators and repressors of fruit ripening in durian PONE-D-21-15671R1 Dear Dr. Sirikantaramas, 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, Takaya Moriguchi Academic Editor PLOS ONE Additional Editor Comments (optional): Dear Dr. Supaart Sirikantaramas, Chulalongkorn University I have satisfied your revision. Reviewers' comments: https://doi.org/10.1371/journal.pone.0252367.r003
Formally Accepted
29 Jul 2021 Acceptance Letter - Takaya Moriguchi, Editor PONE-D-21-15671R1 Transcriptome-wide identification and expression profiling of the ERF gene family suggest roles as transcriptional activators and repressors of fruit ripening in durian Dear Dr. Sirikantaramas: 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. Takaya Moriguchi Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0252367.r004

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