PONE-D-22-11553

System network analysis of Rosmarinus officinalis transcriptome ─ key genes in biosynthesis of secondary metabolites

PLOS ONE

Dear Dr. Moghadam,

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PLOS ONE

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Reviewers' comments:

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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: Partly

Reviewer #3: Yes

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

Reviewer #1: I Don't Know

Reviewer #2: No

Reviewer #3: Yes

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

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Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

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

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Reviewer #1: Yes

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: The manuscript (research article) explores the relationship between crucial secondary metabolites and their biosynthetic genes in the rosemary. The article’s topic fits within the scope of PLOS ONE, and the argument is of interest to a broad audience. However, I have got some comments:

1. In the manuscript, the order of references is chaotic. Please check reference outputs.

2. It seems that the author is unclear about the form of the gene and protein (the name of the gene should be in italics).

3. The author used RT-PCR to confirm the function of hub genes, but only

chose three genes. It is necessary to add more genes to improve the reliability of conclusions.

4. Figures 3, 4, and 5, please, combine them.

5. Line 107: The author pointed out that ‘Many plant species treated with methyl jasmonate are able to produce compounds such as alkaloids, terpenoids, coumarins, and phenolic compounds [2]. On the other hand, the accumulation of rosmarinic acid in cell culture of methyl jasmonate-treated Menta balsamea has been proven [27].’. To our knowledge, the articles about the effect of methyl Jasmonate in Rosemary have been published. Please add relevant literature.

Yao D, Chen Y, Xu X, et al. Exploring the Effect of Methyl Jasmonate on the Expression of microRNAs Involved in Biosynthesis of Active Compounds of Rosemary Cell Suspension Cultures through RNA-Sequencing[J]. International journal of molecular sciences, 2022, 23(7): 3704.

Yao D, Zhang Z, Chen Y, et al. Transcriptome Analysis Reveals Differentially Expressed Genes That Regulate Biosynthesis of the Active Compounds with Methyl Jasmonate in Rosemary Suspension Cells[J]. Genes, 2021, 13(1): 67.

Reviewer #2: The paper needs revision, and the following questions/points should be further clarified prior to its acceptance for publication.

1. The author disregards the manuscript writing format. All citations in the text are cited in the wrong way. The citation should begin with [1]. The authors are doing the exact opposite. This reflects authors lack of expertise in scientific manuscript writing. Please revised your manuscript.

2. The references also writing in the wrong way and format. Please refer PLOS ONE format.

3. The introduction provides background information to establish the context of the study, but you should add more details about Rosmarinus spp. Benefits, uses in industry and others in other family representatives. Add more citations to show the species' significance in this study.

4. The materials and methods section needs revision and lacking many important details that make this study impossible to replicate. The arrangement of material and methods need to be improve and started with Plant material. Here are the following questions that need to be answered:

• Line 113: explain why the samples treated for 12 and 24 hours? Add the references.

• Line 113: How many leaves (g) were collected for each treatment? How many replications for each treatment? Are those biological replications?

• What data the statistical analysis used for? Please mention in the text. Please add the company and country for SAS 9.4 software (company, country). Also, for the rest software and equipments in the text.

5. The results provide interesting information but are described in a boring way. Some figures have low quality and they do not show what they should. In some cases, it is difficult to read hub gene. Figure 1 is not necessary.

• What does the colour indicate in Figure 5? Please specify in the text how the colours red, green, and white are represented in the scale and in the image.

• I suggest splitting the Figure 7. Current arrangement makes the hub genes are difficult to read.

• Figure 1 is not necessary.

6. The discussion is quite interesting, but difficult to follow due to incorrect citation structure.

7. They many grammatical errors throughout the manuscript that need to be fixed.

8. I recommend English editing by native specialists. It was difficult to read some sentences of the main text.

Reviewer #3: The manuscript by Eisa Foroozan et al. aim to reveal the key genes involved in the biosynthesis of secondary metabolites in Rosmarinus officinalis. The topic is valuable and is well within the scope of journal. But its main disadvantage is related to the unsuitable method of plant materials collection. Sometimes the text is confusing and some concepts are not clear or correct (see specific comments below). Thus, it must be substantially improved.

My concerns are the following:

1) keywords: It should be listed in alphabetical order.

2) Introduction: I don’t think the opening paragraph (line 43-47) placed here is necessary, which is not closely related to the main point of this paper. Instead, authors should directly introduce the special value of Rosmarinus officinalis in the beginning.

3) Line 49: Rosmarinic acid, carnosic acid, carnosol, and essential oils has which important medicinal value should be described more detailedly.

4) Line 54-56: Authors should simplify the statements to improve the readability and scientificity of the paper. My suggestion: Although the secondary metabolites have diverse functions in plant, including regulating growth (citing literature), interacting with the environment (citing literature), and ameliorating abiotic and biotic stress (citing literature), the structure and metabolic pathways of secondary metabolites are still unknown.

5) Plant selection and treatment: The selection of plant materials in this paper is not convincible only based on the previous literatures. Because the results drawn by other researchers cannot be same as yours due to different climate, growth conditions, and treatment methods. Therefore, I suggest authors need supplement the relevant data of the content of rosmarinic acid in Menta balsamea under methyl jasmonate treatments.

6) The figures and tables cited in the end of each sentence should be enclosed in brackets (see Line 137, 140, 141, 143 and other places).

7) Line 143: change ‘the major modules’ to ‘the three major modules’.

8) Line 148: Our results revealed that secondary metabolites have significant correlation with the co-expression modules.

9) Line 152-154: ‘While rosmarinic acid… brown4 module.’ I can’t understand what the authors mean. This sentence should be rewritten.

10) Line 157: (Table 2 & 3)

11) Line 166: replace ‘effect’ to ‘affect’.

12) Line 173: Which important family of proteins was identified in all modules except for Yellow?

13) Line 182: It should be Table S1 rather than S1 Table.

14) Line 187: This sentence should be revised as ‘The main systems are listed as follows: Channels/Pores, Electrochemical Potential-driven Transporters, Primary Active Transporters, Group Translocators, Transmembrane Electron Carriers, Accessory Factors Involved in Transport, and Incompletely Characterized Transport Systems.’

15) Authors are responsible for preparing their papers in correct English language. Some sentences are difficult to understand (e.g., Line 193-196), and the manuscript requires substantial grammatical revisions in the present form.

17) Fig. 7: The image resolution is bad. Please export the picture with the suitable format, such as jpg., tiff.

16) Discussion: In general, the discussion is too long and it should be shorten. If authors detect the content of rosmarinic acid in Menta balsamea, the discussion can be connected to the results better. Line 209-234, this part is mainly the replication of the results. Line 211: change ‘a disease’ to ‘disease’. The GO enrichment analysis and KEGG enrichment analysis need to add the relevant figures as supplementary materials.

17) Table 4: Don’t make the table on the different pages.

18) Line 547: Miss the title ‘Figure caption’.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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System network analysis of Rosmarinus officinalis transcriptome and metabolome ─ key genes in biosynthesis of secondary metabolites

PONE-D-22-11553R1

Dear Dr. Moghadam,

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.

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Wujun Ma

Academic Editor

PLOS ONE

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