PONE-D-19-24237

Transcriptome analysis of Curcumawenyujin from Haikou and Wenzhou and a comparison of the main substances and related genes of Rhizoma Curcumae

PLOS ONE

Dear Dr. Lilan,

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.

Both reviewers pointed out deficiencies in description of methodological details, conditions and sample details and conditions. According to Publication Criterion #3 You are required to describe your methods and samples in sufficient detail to ensure replicability. This extends also to purchased products that may not be sufficiently described or characterized, or whose purchase locations were not provided.

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Christian Schönbach, Dr.rer.nat.

Academic Editor

PLOS ONE

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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: Yes

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

Reviewer #1: Yes

Reviewer #2: 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: Yes

Reviewer #2: Yes

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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: Yes

Reviewer #2: 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: Curcumae Rhizoma, known as Ezhu (Chinese), and Curcumae Radix, known as Yujin (Chinese), are different plant parts coming from three same species of Curcuma according to China Pharmacopoeia. The Chinese Pharmacopoeia recorded that Curcumae Radix should be the dry radix of Curcuma wenyujin Y. H. Chen and C. Ling, C. longa L., C. kwangsiensis S. G. Lee, and C. phaeocaulis Valeton. And Curcumae Rhizoma should be the dry rhizomes derived from the above-mentioned species except C. longa L. They are similar in source but different in medicinal parts. Curcumae Rhizoma and Curcumae Radix are confused in variety and source, even in clinical trials by some nonprofessional workers. So, it is important for us to make them clear. Also, genotype and environment interaction is high for secondary metabolites in Curcuma sp. The main objective of the present study was to analyze the constituents of essential oils such as terpene, curcumin, polysaccharide, starch, and other important substances from C. Wenyujin rhizomes from the traditional (Wenzhou) and introduced (Haikou) production areas. In this study authors also attempted comparative RNAseq profiles of C. Wenyujin rhizomes produced in traditional (Wenzhou) and introduced (Haikou) areas. The study has some interesting findings in the poorly understood crop species of Curcuma.

However, authors need to clarify following points.

The introduction part seems to be too lengthy – cab be shortened.

L.71: The terminology 'Species variety' is not botanically correct. Authors may use the term ‘genotype’ instead.

L.86: There is nothing called “lime soil”. Is it alkaline soil?

L.120-122: Give a suitable reference for this statement.

L.146: What do you mean by treatment conditions?

L.146-153: Authors silent on experimental conditions. Similar sampling and growing conditions are important for studying secondary metabolites and comparative transcriptomics.

The environmental and weather conditions of the two provinces need to be given as supplementary table to understand the differences in secondary metabolites and related genes.

L.154: Protocol for RNA isolation is not mentioned. Also specify the tissue from which RNA was isolated.

In case of RNASeq, number of biological replicates sequences is not clear. Also, replicates need to be compared at sequence level and prove that they are not significantly different.

L.209: Sufficient experimental details viz., temperature, pressure and slice thickness need to be given on boiling and drying of samples?

L.234: What is the sample? rhizome or leaf, or at what stage?

L. 248: Is it a curcumin or curcuminoids

L.268: Polysaccharide and starch content methodology are too elaborate and need to shorten with suitable references.

L 361………..

In many literatures, the C. weeyujin is referred as synonymous to C. aromatica. In that case can authors compare the present transcritomes with already available C. aromatica transcriptome to draw the meaningful conclusions.

Hope above suggestions help authors to improve the manuscript.

Reviewer #2: Language correction is essential before acceptance. Method used for RNA isolation is not mentioned (what mentioned as RNAse free DNAse, Takara; is just an enzyme and is not the method used for RNA extraction).

Minor Corrections Suggested

1) Line no. 38, 39: better replace ‘was’ with were.

2) Species name should always be in small letters, throughout the manuscript its written as CurcumaWenyujin , few line numbers are mentioned ( Line no.119, 134, 148, 342, 350, 347, 355, 506, 509, 522, 528, 538, 541, 544, 546, 549, 558, 562, 563, 574, 582, 583, 594, 609, 611, 625, 627, 644, 650).

3) Make sure that Curcuma wenyujin is in italics (not followed).

4) Line no. 265: Rephrase the sentence in a better conveyable form.

5) Line no. 297: spelling correction; ‘using’ is written as uing.

6) Line no. 329: better replace ‘was’ with were.

7) Line no. 561 is contradictory to Line no. 564 (about polysaccharide).

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Reviewer #1: No

Reviewer #2: No

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Attachments

Attachment

Submitted filename: PONE-D-19-24237_reviewer.pdf

PONE-D-19-24237R1

Transcriptome analysis of Curcuma wenyujin from Haikou and Wenzhou and a comparison of the main constituents and related genes of Rhizoma Curcumae

PLOS ONE

Dear Dr. Lu Lilan,

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.

While some of the major issuses raised by reviewers in the previous round were addressed more work is required improve descriptions of methods and language to meet publicaiton criteria #3 and #5. Especially, add parameters and thresholds to methods applied, explain rational for chosing a particular statistical test (here univariate ANOVA), and most importantly revisit and revise the DEG analysis part to ensure enrichment results are statically correctly supported. In additon your are required to improve lablelling and quality of figures.

We would appreciate receiving your revised manuscript by May 23 2020 11:59PM. 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,

Christian Schönbach, Dr.rer.nat.

Academic Editor

PLOS ONE

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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 #2: All comments have been addressed

Reviewer #3: (No Response)

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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 #2: Yes

Reviewer #3: Partly

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

Reviewer #2: Yes

Reviewer #3: No

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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

Reviewer #3: Yes

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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

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: Authors incorporated the suggestions into their manuscript and as a result the MS quality has been improved. The revised MS can be accepted as it is.

Reviewer #3: The authors analyzed C. wenyujin rhizomes in two different locations and showed that the difference in metabolome and transcriptome were consistent.

The conclusion sounds OK but there are so many points to be improved in figures, statistical analysis and sentences.

The comparison along the metabolic pathway, and their conclusion would be OK. However, the distribution of transcriptome should be evaluated and normalized before detail statistical analysis such as DEG. Also, the detail about clustering analysis and enrichment analysis are not clear to evaluate them.

All of the figures were looks very low in resolution (converted from compressed JPEG?) and most of the labels are difficult to read, even I downloaded each tiff file.

Use vector format images or, at least, more high resolution and low compression images.

It is strongly recommended to submit the whole manuscript to an English editorial service.

There are detail comments below.

l85. ~ indicating that the oil content of Curcuma rhizomes can

85 be affected by factors such as soil quality and climate.

> Add some reference if there are some preceding related studies.

l. 192

> It would be specify the name of the test and parameters since "DESeq" is a name of a function in the R package rather than a name of a method.

l. 282 Univariate analysis of variance was applied to determine the significance of the results among different treatments.

> The target of the analysis unclear. Does it means "different species", and the variables are compound components? Since they have only two groups, are there any rational reason to apply univariate ANOVA instead of t-test or U-test?

Through three replications, the average value of each parameter was calculated, and the standard error (SE) of the mean value was obtained. Univariate analysis of variance was applied to determine the significance of the results among different treatments. Multidimensional tests were considered significant at a P value < 0.05. SPSS V.13 was used for statistical analysis.

l 327 41.74 Gb

> If it means giga "byte", show the amount in giga "base pair" instead.

l. 328 Q30 percentages

> It should be more concretely and use general term.

For example, "Number of samples which Phred quality score is higher than 30."

l. 340 1.00e-05

> It would be better to use scientific format (superscripts) instead of computational format.

l 340

> In the DEG analysis the number of up regulated genes were much larger than that of down regulated. The authors should concern about experimental bias between samples or consider to apply some compensation such as quantile normalization.

Fig 4 shows the ratio of DEG unigenes in each ontology is highly correlated

with that of all annotated unigenes.

> It seems that DEG are not correlate with their functions but almost randomly sampled from the all unigenes, so that the significance of enrichment analysis is rather doughtful.

Fig. 7

> Make clear the number of genes shown in this figure, is it 3720? If it is so make clear how and why these genes were chosen. It is not clear what means "up/down regulated" in this figure, I mean compared with what values.

> And the labels in the columns should be replaced by their real labels (T01 = HK1, T02 = HK2, T03 = HK3, T04 = WZ1, T05 = 393 WZ2, T06 = WZ3).

l. 409 A comparison of DEGs in 26 COG classifications between HK and WZ is shown in Fig 8.

> It is not clear that the numbers of DEG in this figure means up regulated or down regulated. And for enrichment analysis, it is also important to show the number of annotated genes in each category and the ratio of DEG to the annotated genes.

l. 454

> It seems meaningless to show the sequence id such like c118842.grraph-co, show gene name or protein name instead. It would be much better to show them in the pathway map also.

Fig. 9

> Arrange the plots for FPKM and rt-PCR side by side when the comparison about the same gene is important.

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Reviewer #2: No

Reviewer #3: Yes: Naoaki ONO

[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.

Transcriptome analysis of Curcuma wenyujin from Haikou and Wenzhou and a comparison of the main constituents and related genes of Rhizoma Curcumae

PONE-D-19-24237R2

Dear Dr. Lilan,

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,

Christian Schönbach, Dr.rer.nat.

Section 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

Reviewer #4: 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: Yes

Reviewer #4: Yes

**********

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

Reviewer #1: Yes

Reviewer #4: N/A

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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

Reviewer #4: 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 #4: 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: All the comments have been addressed. Manuscript Transcriptome analysis of Curcuma wenyujin from Haikou and Wenzhou and a comparison of the main constituents and related genes of Rhizoma Curcumae is now acceptable.

Reviewer #4: the revised manuscript has complied all the query of the reviewers and meets all the mandate of the journal.

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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 #4: Yes: Enketeswara subudhi