PONE-D-21-17629

The framework of plant regeneration in duckweed (Lemna turonifera) comprises genetic transcript regulation and cyclohexane release

PLOS ONE

Dear Dr. Sun,

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Academic Editor

PLOS ONE

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(No. 32071620 ), Tianjin Science and technology project (19ZYPTSN00030)."

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Initials of the authors who received each award: L.Yang. , JG. Sun

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The full name of each funder: Present research has been supported by National Natural Science Foundation of China (No. 32071620), Tianjin Science and technology project (19ZYPTSN00030)

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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: No

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: N/A

Reviewer #3: Yes

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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: No

Reviewer #2: No

Reviewer #3: No

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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

Reviewer #3: No

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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: This study provides the new insights related to duckweed regeneration. Unfortunately, the results have not been well presented and have not been interpreted correctly. Also, manuscript needs to be edited by a native English editor.

Other my comments:

- Please revise abstract to highlight hypothesis/objective of work

- I recommend revising Abstract. Content is not connected.

- Lines 77-78: please revise it (e.g. double "with")

- Line 83: Transcriptome analysis identifies Genes and Genomes (KEGG): KEGG is database for pathway analysis, not abbreviation of " Transcriptome analysis identifies Genes and Genomes".

- Lines 89-91: this sentence should be rewritten. It is unclear.

- Line 94: "Pathways were" instead of "pathway was".

- Line 190: "AUX/IAA and GH3 has " it should be "have"

- Authors used a lot of "have/has been up/down-regulated" I do not recommend expressing results in this way.

- Line 202: double "in"

- In discussion, the results repeated. Please interpret the key results.

- Line 260: "has" should be changed to "have".

- Line 274: it needs to edit. Why "were"

- Lines 292-295: Unclear, please re-write them.

- Materials and methods are not perfect and more details have to add.

- How to analyze pathways, and selecting DEGs.

- Line 312: How were RNA samples extracted? Which method or kit?

Reviewer #2: In this study, the authors duckweed regeneration molecular mechanism by regenerating callus. The authors identified that mainly auxin, cytokinin and VOCs related genes and compounds play a key role in callus regeneration. Overall, there is no major issue. However, the current version is not acceptable in its present form and demands improvement. Some of the suggestions are as follows:

� The unit is not consistent throughout the text. Please follow the standard unit style, i.e., mg L-1.

� Have you submitted the raw RNA-seq data to any repository?

� Line 85-105, this section is all about KEGG and GO results. The authors did not explain the results of the number of DEGs and related information. Please carefully check and add DEG-related information.

� In fig 3 and 4, please define the gene name abbreviations in the captions.

� Line 142-153, please properly cite Fig 5a/b/c in the text at the suitable place.

� There are several spacing errors in the text.

� Table 2 and 3 are not cited anywhere in the text.

� The English language needs improvement.

� It would have been a nice addition if the authors can add a mechanistic flow diagram showing the molecular mechanism controlling regeneration.

� The revised can be considered for possible publication after minor revision.

Reviewer #3: 1. The motivation of the study is interesting, however, the hypothesis is not clearly described. In Line 263, the hypothesis is not an accurate hypothesis.

2. In the manuscript, ‘genetic transcript regulation’ of the title is not proper, because there is no temporal comparison of the transcript level or upstream/downstream relationship of gene expression to explain the transcriptional regulation.

3. There is no clear description in the Materials & Methods.

� Please define the plant materials of regenerating callus and callus.

� In “VOCs Collection and analysis”, the material quantity used for VOC collection and analysis and the analysis methods for quantitative data.

� There is no description about RNA isolation.

� Before “Sequencing data filtering and transcript assembly”, there is no description about RNA sequencing. Please describe the method clearly, including the plant materials and the gene expression comparison between RG and CL or Cyclohexane and CL.

� There is no description about the application of VOC on callus, including cyclohexane, 4-methyl-2-pentanol and 1, 3-dimethyl benzene. The dosage? The quantity of callus in the treatment?...

4. The callus induction medium (2 mg/L BA) is different from the formula (1 mg/L BA) in the cited reference 35 and 36, please explain.

5. Title of Table 2, 3, 4 and Fig. 3, 4, 6 needs to be revised. For example, Table 2. Gene expression in plant regeneration of Auxin. What is “plant regeneration of auxin”? Please revised them.

6. Line 150-152, please check the exact fold change of cyclohexane.

7. Line 218, Genome?. There is no study of genome sequencing in this study.

8. There is no scale bar shown in Fig. 1a and Fig. 6.

9. Line 255 and Line 261, “the regeneration of callus” and “the root formation”. Exact morphological change of callus treated with cyclohexane is the root differentiation or induction from callus in this study. The term “the regeneration of callus” may be ambiguous with “the plant regeneration from callus”. Please define and specify the different terms in the study.

10. I sincerely suggested that the authors use a professional editing service to avoid mistakes in English scientific writing and typing error.

� Line 27, lower case of G in Genetic

� Line 56-58, the sentences are not complete. vitro-> in vitro; novo-> de novo; incubate->incubated; callus-> callus induction….

� Line 95, wrong spelling of repaire

� Line 285, formated?

� Line 245, and?

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Attachments

Attachment

Submitted filename: Comments to PONE-D-21-17629.pdf

PONE-D-21-17629R1Regeneration of duckweed (Lemna turonifera) involves genetic molecular regulation and cyclohexane releasePLOS ONE

Dear Dr. Sun,

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 21 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.

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,

Jen-Tsung Chen, Ph.D.

Academic Editor

PLOS ONE

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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.

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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: (No Response)

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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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: No

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: No

**********

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: No

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

**********

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: No

Reviewer #2: No

Reviewer #3: No

**********

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 satisfactorily addressed all the comments, and I have no more comments. Thus, I endorsed the submission for acceptance in its current form.

Congratulations!

Reviewer #3: The fulltext has been carefully revised according to the suggestions and comments of the reviewers and improved the quality of the manuscript. In my opinion, the manuscript can be acceptable for publication but after the minor revision as suggested.

1. For the description of fold change in RNA sequencing data, the 4nd decimal place seems redundant. Please consider to round off to second or third decimal place seems redundant.

2. Line 142, please substitute symbol “x” to * in the fulltext.

3. Please improve the format of Table 2, 3 and 4.

[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.

Regeneration of duckweed (Lemna turonifera) involves genetic molecular regulation and cyclohexane release

PONE-D-21-17629R2

Dear Dr. Sun,

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.

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Kind regards,

Jen-Tsung Chen, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: