Identification of SbWRKY Transcription Factors in Scutellaria baicalensis Georgi under Drought Stress and Their Relationship with Baicalin

Round 1

Reviewer 1 Report (Previous Reviewer 1)

Article Identification of SbWRKY transcription factors in Scutellaria baicalensis Georgi under drought stress and their relationship with baicalin by Lin Cheng, Jingjing Yu, Lichao Zhang, Yanying Yao, Zhuo Sun, Mei Han, Yonggang Zhang, Limin Yang examines the combined aspects of transcription and baicalin production.

The manuscript contains all the necessary sections, including the conclusion. The previously noted comments have been eliminated.

Improvement requires Figure 4 and 5 resolution

I have a question regarding the caption of Figure 11, since it is still difficult to understand what the lines without captions mean. The figures are a separate part of the manuscript and should be self-evident.

In general, the manuscript can be published.

Author Response

Q1.The manuscript contains all the necessary sections, including the conclusion. The previously noted comments have been eliminated.

Reply: We want to start by saying thanks for your correction recommendations for our paper. However, we apologise for not understanding what you meant by your recommendation and really hope you can clarify it for us.

Q2Improvement requires Figure 4 and 5 resolution

Reply:Thanks for your suggestion. Regarding Fig. 4 and Fig. 5, we have made every effort to increase its clarity. While the original image is clear, it becomes less so when it is inserted into the text and shrunk. For your convenience, we have attached the image as is.

Q3.I have a question regarding the caption of Figure 11, since it is still difficult to understand what the lines without captions mean. The figures are a separate part of the manuscript and should be self-evident.

Reply:We are extremely grateful to reviewer for pointing out this problem. For readers convenience, we have updated Fig. 11's legend to provide an explanation of the figures and colors in accordance with your recommendation.

Reviewer 2 Report (Previous Reviewer 2)

Authors have made significant improvements however there are still some minor corrections that need to be performed before publication like

Figure 12 - add error bar

need significant improvement in conclusions

in Abstract-A total of 60 ScWRKY transcription factorsSbWRKY TFs with highly conserved structural domains were identified with the protein lengths ranging from 138 to 752, and molecular weights  from 16.1 Da to 81.08 Da) based on the physicochemical analysis 

The results for molecular weight are consistent with amino acid length, however, isoelectric point size is not substantially correlated with 23 amino acid length.-remove this sentence from abstract.

Improve the abstract

Authors need to improve the MS significantly. Authors need to read MS line by line

Need to improve english

Author Response

Thank you for your suggestions, we have revised the manuscript according to your suggestions, and we hope you can give us valuable comments again!

Author Response File: Author Response.docx

Reviewer 3 Report (New Reviewer)

Thank you for inviting me to review the paper titled Identification of SbWRKY transcription factors in Scutellaria baicalensis Georgi under drought stress and their relationship with baicalin

The authors aim to clarify the connection between SbWRKY TFs involved in baicalin production and those involved in the response to drought stress.

Overall, the article is an nice transcript study. In the study, 14 DEGs were identified. But there are several drawbacks. Firstly, need to be reviewed in terms of writing

I also recommend to enrich introduction and discussion with the relevant papers. 

A clearer explanation of the relationship between expressed genes and metabolic pathways should be included in the results and discussion section.

How many RNA samples did you sequence? How many biological replicates in each stage? As you may know, The differential expression analysis without any biological replications is meaningless.

Needs editing by a native English speaker

Author Response

Thank you for your suggestions, we have revised the manuscript according to your suggestions, and we hope you can give us valuable comments again!

Author Response File: Author Response.docx

Reviewer 4 Report (New Reviewer)

1) Major flaws: Statistical tests should be mentioned and incorporated. I do not see any high quality statistics-based parameters in the Methods and result figures. Furthermore, throughout the text, the authors mention secondary metabolites and some omic approaches (such as transcriptomics and genomics). I recommend providing data and insights regarding metabolome and metabolomic analyses. Metabolomics is not mentioned anywhere in the text, and I consider this a significant flaw, given the manuscript's focus and introduction on metabolites. For instance, in the introduction, secondary metabolites are mentioned, raising readers' expectations of seeing metabolomic results. Additionally, gene expression analyses require validation through metabolite and protein-based analyses. PCR or transcriptomics alone are not sufficient to confirm metabolite synthesis. Moreover, for the analysis and validation of results related to transcription factors, analyses focused on protein quantification should be conducted. Without these validation analyses and other phenotype parameters, I believe the current manuscript appears to be more of a preliminary study than a full research article;   I recommend a significant improvement in the Conclusion: the sentence "this experiment was to study the..." is not grammatically correct, nor is "after analyzing." Moreover, it is better to write shorter sentences. It is crucial to provide more details about the new insights provided in this study. There is no need of mentioning the research objective in the conclusion. The current version of the conclusion does not highlight any interesting insights from the conducted research. In other words, the authors should include more result details in the conclusion.    Considering these major issues, I do not recommend manuscript acceptance.      Other comments of mine include:     2) Throughout the manuscript: Gene names should be italicized to distinguish them from proteins. Additionally, when referring to proteins, please avoid using the term "expression." Like other proteins, transcription factors are "accumulated" but not "expressed." Only genes are expressed. In this context, the phrase "expression of key enzymes" is also incorrect.     2.1) In several parts of the manuscript, the authors mention "expression" for transcription factor genes whose names are not italicized.     3) In the Abstract: The authors might briefly introduce the importance of analyzing baicalin; I recommend using "TFs are responsive" instead of "TFs can respond," and 'drought stress conditions' instead of 'drought stress' situations (abstract and discussion).     4) In the Introduction: When the authors introduce the plant species, they mention Chinese medicine. I recommend providing information and cultivation potential regarding this plant species in other countries as well.     5) Plant species names should be italicized (for example, Zea mays and Arabidopsis thaliana), and the full plant species name should be provided for other mentioned other crops (e.g., wheat and rice).    6) I recommend writing “drought stress conditions” instead of “drought stresses” (e.g, title of the topic 2.5)

Please see my comments to the authors and then revise the other manuscript topics accordingly.

Author Response

Thank you for your suggestions, we have revised the manuscript according to your suggestions, and we hope you can give us valuable comments again!

Round 2

Reviewer 4 Report (New Reviewer)

In my first reviewer report, I mentioned metabolomics, protein quantification analyses for validation, and the need of including statistical information into result figures and tables. The authors did not incorporate any data about this omic approach and protein-base analyses, and did not update tables and figures captions or legends regarding statistical information, and other aspects. Thus, I do not see improvements regarding my major recommendations. Actually I think the authors may consider spend much more time to incorporate these changes. I submitted my first report just a few days ago. 

Author Response

Q. In my first reviewer report, I mentioned metabolomics, protein quantification analyses for validation, and the need of including statistical information into result figures and tables. The authors did not incorporate any data about this omic approach and protein-base analyses, and did not update tables and figures captions or legends regarding statistical information, and other aspects. Thus, I do not see improvements regarding my major recommendations. Actually I think the authors may consider spend much more time to incorporate these changes. I submitted my first report just a few days ago.

Reply: We are extremely grateful to reviewer for pointing out this problem. The reason why we did not provide data on metabolome and protein quantification analysis was explained in the last cover letter, and metabolome and protein quantification will be the focus of our future research. Questions about the titles or legends of the graphs for statistical information and other aspects were revised, and the method of significance analysis was added in 2.8, and the legends of Fig 10 and 11 have been modified for clarity.

Round 3

Reviewer 4 Report (New Reviewer)

I have no more comments.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Article Identification of ScWRKY transcription factors in Scutellaria baicalensis Georgi under drought stress and their relationship with baicalin by Yonggang Zhang et al., discusses the regulation of baikolin synthesis by inducing transcription factor binding. This aspect of epigenetic regulation suggests that moderate drought is favorable for baikolin biogenesis, in contrast to strong drought. Although this statement is self-evident, but the authors proposed a variant of the molecular aspect in the understanding of this issue.

The manuscript is designed in accordance with the rules and contains the necessary sections.

There are some minor design flaws.

In materials and methods, it is necessary to indicate the volumes of pots, substrate, lighting (mode and intensity), daily fluctuations in temperature and humidity, in which room or grow box the plants were grown. How many repetitions were there and how the degree of reliability of the obtained data was assessed.

So, table 1 should be designed so that the primer fits on line 1, especially since other columns contain a maximum of 2 digits. This looks messy.

The quality of the images should be improved, as it is difficult to make out the inscriptions.

I think these corrections will not take much time and the manuscript will be prepared.

Author Response

  We appreciate your wise counsel and have made the necessary modifications. Still, we might need your guidance in the future as some of our knowledge may not be complete.

Reviewer 1’ s Comments：

Q1.The manuscript is designed in accordance with the rules and contains the necessary sections.

Reply:We appreciate the reviewer’s positive evaluation of our work.

 

Q2.In materials and methods, it is necessary to indicate the volumes of pots, substrate, lighting (mode and intensity), daily fluctuations in temperature and humidity, in which room or grow box the plants were grown. How many repetitions were there and how the degree of reliability of the obtained data was assessed.

Reply:Thanks for your suggestion. Based on your suggestions we have added the suggested missing parts to 2.1 Plant material

Q3.So, table 1 should be designed so that the primer fits on line 1, especially since other columns contain a maximum of 2 digits. This looks messy.

Reply:We are extremely grateful to reviewer for pointing out this problem.We have simplified Table 1 and changed the header to make it more concise and understandable。

Q4.The quality of the images should be improved, as it is difficult to make out the inscriptions.

Reply:We are grateful for the suggestion. Regarding the problem of unclear pictures in the manuscript we have the original picture clarity is still possible, but put into the manuscript to reduce the size of its become unclear, we can provide the original picture.

Reviewer 2 Report

The manuscript titled "Identification of ScWRKY transcription factors in Scutellaria baicalensis Georgi under drought stress and their relationship with baicalin" by Cheng et al. will be more comprehensible, informative, and impactful for the readership of Agronomy Journal if the authors consider the comments provided below to improve the MS.

In the abstract section, the statement "we analyzed the physical, chemical, evolutionary relations, conservation structure, and expression of the WRKY gene family in Scutellaria baicalensis" lacks clarity regarding the intended meaning of "physical" and "chemical." Clarification on the specific aspects of the physical and chemical analyses would be beneficial to readers.

The concluding statement in the abstract, "These results indicate that drought stress affects the expression of ScWRKY transcription factors, key enzyme genes, and baicalin content. They provide a theoretical basis for further functional studies of ScWRKY transcription factors," could be refined for a more succinct and conclusive tone. Moreover, acknowledging the well-established role of WRKY transcription factors in drought stress response might add context to the study's novelty.

The inclusion of Figure 1, illustrating the Baicalin synthesis pathway, in the Introduction section requires clarification. Providing a brief explanation for its relevance to the topic at hand will help readers understand its purpose.

Under the materials and methods section, the subheading "Screening and identification of WRKY transcription factors in Scutellaria baicalensis" should be revised for greater clarity and precision. A more detailed and informative subheading that reflects the specific methodology employed would be beneficial.

The provided table headings are currently inadequate in terms of information and clarity. For instance, "Table 1. RqT-PCR primers" could be enhanced to offer more descriptive information about the content of the table. A revised table heading that concisely conveys the content and purpose of the table would be preferable.

No

Author Response

  We appreciate your wise counsel and have made the necessary modifications. Still, we might need your guidance in the future as some of our knowledge may not be complete.

Reviewer 2’ s Comments：

Q1. In the abstract section, the statement "we analyzed the physical, chemical, evolutionary relations, conservation structure, and expression of the WRKY gene family in Scutellaria baicalensis" lacks clarity regarding the intended meaning of "physical" and "chemical." Clarification on the specific aspects of the physical and chemical analyses would be beneficial to readers.

Reply:We are extremely grateful to reviewer for pointing out this problem.Physical and chemical properties are not well distinguished, so we have changed physicochemical properties to physicochemical properties.

 

Q2. The concluding statement in the abstract, "These results indicate that drought stress affects the expression of ScWRKY transcription factors, key enzyme genes, and baicalin content. They provide a theoretical basis for further functional studies of ScWRKY transcription factors," could be refined for a more succinct and conclusive tone. Moreover, acknowledging the well-established role of WRKY transcription factors in drought stress response might add context to the study's novelty.

Reply:We are grateful for the suggestion. We have rewritten the conclusion section of the summary

 

Q3. The inclusion of Figure 1, illustrating the Baicalin synthesis pathway, in the Introduction section requires clarification. Providing a brief explanation for its relevance to the topic at hand will help readers understand its purpose.

Reply:Thank you for pointing out this problem. We have explained Figure 1 in more detail and improved it somewhat in the manuscript.

Q4.Under the materials and methods section, the subheading "Screening and identification of WRKY transcription factors in Scutellaria baicalensis" should be revised for greater clarity and precision. A more detailed and informative subheading that reflects the specific methodology employed would be beneficial.

Reply:We are grateful for the suggestion. We've changed “Screening and identification of WRKY transcription factors in Scutellaria baicalensis” to “Screening of WRKY transcription factors in Scutellaria baicalensis and prediction of their properties”.

 

Q5. The provided table headings are currently inadequate in terms of information and clarity. For instance, "Table 1. RqT-PCR primers" could be enhanced to offer more descriptive information about the content of the table. A revised table heading that concisely conveys the content and purpose of the table would be preferable.

Reply:Thank you for your pertinent suggestions. We simplified Table 1 by describing it in more detail in "2.6 Extraction of Scutellaria baicalensis RNA and real-time fluorescence quantitative PCR" and changed the header to make it more specific.

Thank you again for your positive comments and valuable suggestions to improve the quality of our manuscript. If there are any other modifications we could make, we would like very much to modify them and wo really appreciate your help. Thank you very much for your help.