PONE-D-20-05724

Integrated analysis of differential expression and alternative splicing in microalgae grown under heterotrophic condition

PLOS ONE

Dear Dr Panahi,

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.

It is a simple bioinformatic study to compare the data of transcriptional and post-transcriptional RNA. Study will be important if it could have been supported lab experimentation.  Authors stated, “ The results also indicated that prevalence of Intron retention is associated with down-regulation of the genes involved in carotenoid biosynthesis”. This needs to be supported with the practical experimentation to validate your statement and make study useful to wide readership of Plos-One. Results and Discussion parts are not clearly written. I would appreciate if you could sperate them and write more lucidly. Please state, how your current study differed from “Systems biology approach identifies functional modules and regulatory hubs related to secondary metabolites accumulation after transition from autotrophic to heterotrophic growth condition in microalgae” that published recently.

We would appreciate receiving your revised manuscript by within six months. When you are 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Shashi Kumar, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments:

It is a simple bioinformatic study to compare the data of transcriptional and post-transcriptional RNA. Study will be important if it could have been supported lab experimentation. Authors stated, “ The results also indicated that prevalence of Intron retention is associated with down-regulation of the genes involved in carotenoid biosynthesis”. This needs to be supported with the practical experimentation to validate your statement and make study useful to wide readership of Plos-One. Results and Discussion parts are not clearly written. I would appreciate if you could sperate them and write more lucidly. Please state, how your current study differed from “Systems biology approach identifies functional modules and regulatory hubs related to secondary metabolites accumulation after transition from autotrophic to heterotrophic growth condition in microalgae” that published recently.

Journal requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for stating the following in the Acknowledgments Section of your manuscript:

'The authors would like to thank Agricultural Biotechnology Research Institute of Iran for

financial support.'

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

'No'

1. Please clarify the sources of funding (financial or material support) for your study. List the grants or organizations that supported your study, including funding received from your institution.
2. State what role the funders took in the study. If the funders had no role in your study, please state: “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”
3. If any authors received a salary from any of your funders, please state which authors and which funders.
4. If you did not receive any funding for this study, please state: “The authors received no specific funding for this work.”
5. Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

3. Please upload a copy of Figure 6, to which you refer in your text. If the figure is no longer to be included as part of the submission please remove all reference to it within the text.

4. Please include a separate caption for each figure in your manuscript.

5. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information

[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

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy 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: 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: The article is a well-written, needed, and helpful to understand the relationship between growth conditions and differential gene expression pattern. The interesting thing that I liked is how alternative splicing events can change the mode of metabolism in response to growth condition change. I thought this article and previous article about identification of functional modules and regulatory hubs may help us to study the principal factors that can change specific metabolism to get specific product for commercial production.

1. Kindly correct the word “evenets”. ( Abstract-line No.5)

Reviewer #2: Summary:

This paper is an analysis of the transcriptional and post transcriptional responses of algae grown under heterotrophic conditions for the production of high value metabolites. The ability of algae to reduce the nutrients in the environment while still extracting a usable product is an important factor in moving towards energy sustainability and effective nutrient management in an algal cultivation. In order to efficiently proceed with the growth of algae for industrial productions under controlled conditions, understanding the influence of different nutrient quality parameters and nutrients is important. This paper brings a light forward knowledge supporting this understanding.

Overall Impression:

This work appears to bring new knowledge on the transcriptional and post-transcriptional responses of alga towards the growth conditions influenced by nutrient inputs. However, the details of nutrient parameters in two conditions are not stated by the authors and also how they are influencing above process. It should require more clarifications for claiming their suitability to novelty.

General Editing comments:

The paper requires a detailed round of editing. There are some examples of missing legends and references in the text. The user should be able to read the caption and have all the information required to understand the figure or table.

Specific Comments:

a) Add a brief description of the autotrophic and heterotrophic culture conditions and nutrient parameters used in this study. Because this work emphasizes the growth conditions and it will help in understanding the context.

b)Mention the correct test and control sample size and their replications; ie. In the material and methods section Page No. 9 first paragraphs (The sample size of autotrophic growth conditions and their test timings).

c)Page number 11, figure 2B: The data presented here are quite interesting. However, authors need to make a connection between these data with the possible importance/influence of the algal metabolite production.

d)In the result section, functional impact of DEG and DAS genes, in third paragraph. The authors state that “Transcription regulator activities of DEGs are significant …” Are these statistical differences observed after the analytical experiment? If so, where are the stats to prove the statements in this paragraph? If a statistical analysis was completed, please add the test statistic and p values either embedded in the text or in a graph figure legend.

e)Figure legend ‘C’ is present in the text, but absent in all mentioned figures.

f)Page No. 8 reference ‘Huang & Xu, 2015’ is present in the text, but absent in the reference list

g)Page No. 15 line number 13th journal description is present in the reference section but author detail is absent in text as well as in reference.

h)Page No. 15 Kong et al. Journal page number missing

i)Page No. 15 Panahi et al., typographic error in year.

j)Page No. 16 Ravichandran et al. and Xiao et al. References journal page number missing.

k)Page No. 16 Panahi et al., typographic error in journal page number

l)Page No. 16 Schurch & Schofield (2016) Journal name not mentioned

m)Reference journal name consistency; either follow full name/abbreviation/italic pattern/non italic.

**********

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 to be viewed.]

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 us at figures@plos.org. Please note that Supporting Information files do not need this step.

Attachments

Attachment

Submitted filename: article_comments.pdf

Integrative analysis of gene expression and alternative splicing in microalgae grown under heterotrophic condition

PONE-D-20-05724R1

Dear Dr. Panahi,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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.

With kind regards,

Shashi Kumar, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Thanks for making all corrections as suggested by reviewers.

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: Yes

**********

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: 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

**********

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)

**********

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