MicroRNA Identification and Integrated Network Analyses for Age-Dependent Flavonoid Biosynthesis in Ginkgo biloba

Round 1

Reviewer 1 Report (Previous Reviewer 1)

Dear Authors,

thank you for considering the comments and your answers and clarifications to the questions. I wish you success in your further research work.

Author Response

Many thanks for your carefully reviewing our manuscript.

Reviewer 2 Report (Previous Reviewer 2)

Manuscript review: "MicroRNA expression and integrated network analyses of age-dependent flavonoid biosynthesis regulation in Ginkgo biloba"

Authors investigated the miRNA profiles of G. biloba seedlings at three different ages through the construction of sRNA libraries and examined the relationships between miRNA expression patterns and flavonoid contents in leaves sampled from ginkgo plants at different ages through comprehensive metabolome, sRNA, and coexpression network analyses.

The description of the methodology (except 2.7) is good. Unfortunately, the subsection "Statistical analysis" is written very briefly. All the statistical methods used in the manuscript are not listed here. This should be supplemented. In addition, there is no information on meeting the assumptions required for ANOVA.

The quality of Figure 1 is very poor. It should be improved. In addition, the LSD or HSD values (the method is not mentioned in the methodology) are missing next to the individual Figures.

The quality of Figure 2 is very poor. It should be improved.

Line 182: "correlation > 0.85" provide the exact values of the correlation coefficients and the level of significance.

The quality of Figure 3 is very poor. It should be improved.

The quality of Figure 4 is very poor. It should be improved.

The quality of Figure 5 is very poor. It should be improved. In addition, the LSD or HSD values (the method is not mentioned in the methodology) are missing next to the individual Figures.

The quality of Figure 6 is very poor. It should be improved.

Figure S6: No information on the significance of coefficients in regression models.

Papier needs major revision.

Author Response

Point 1: The description of the methodology (except 2.7) is good. Unfortunately, the subsection "Statistical analysis" is written very briefly. All the statistical methods used in the manuscript are not listed here. This should be supplemented. In addition, there is no information on meeting the assumptions required for ANOVA.

Response 1: Many thanks for your carefully reviewing our manuscript and putting forward many valuable comments and suggestions. According to your suggestion, we have revised the description of the "Statistical analysis" in the materials and methods (please see line 131-134). Furthermore, we have added more detailed information for ANOVA (please see lines 151-153 and 272-274).

Point 2: The quality of Figure 1 is very poor. It should be improved. In addition, the LSD or HSD values (the method is not mentioned in the methodology) are missing next to the individual Figures.

Response 2: Thank you for pointing out this issue. We carefully checked the figures in our manuscript, and we re-uploaded them in the revised manuscript. Furthermore, we have added more detailed information for statistical analysis (please see line 151-153).

Point 3: The quality of Figure 2 is very poor. It should be improved.

Response 3: We have re-uploaded the Figure 3 in the revised manuscript.

Point 4: Line 182: "correlation > 0.85" provide the exact values of the correlation coefficients and the level of significance.

Response 4: Sorry for this inappropriate description. We have revised it and the exact values of the correlation coefficients is 0.859 (line 180-182). Furthermore, we have added the level of significance (line 183).

Point 5: The quality of Figure 3 is very poor. It should be improved.

Response 5: We have re-uploaded the Figure 3 in the revised manuscript.

Point 6: The quality of Figure 4 is very poor. It should be improved.

Response 6: We have re-uploaded the Figure 4 in the revised manuscript.

Point 7: The quality of Figure 5 is very poor. It should be improved. In addition, the LSD or HSD values (the method is not mentioned in the methodology) are missing next to the individual Figures.

Response 7: We have re-uploaded the Figure 5 in the revised manuscript. Furthermore, we have added more detailed information for statistical analysis (please see line 272-274).

Point 8: The quality of Figure 6 is very poor. It should be improved.

Response 8: We have We have re-uploaded the Figure 6 in the revised manuscript.

Point 9: Figure S6: No information on the significance of coefficients in regression models.

Response 9: According to your suggestion, we have added significance analysis of coefficients in regression models.

Reviewer 3 Report (New Reviewer)

To,

The Chief Editor,

Forests, MDPI,

Manuscript ID: forests-2551392

Subject: Submission of comments on the manuscript in “Forests"

 

Dear Chief Editor Forests, MDPI,

Thank you very much for the invitation to consider a potential reviewer for the manuscript (ID: forests-2551392). My comments responses are furnished below as per each reviewer’s comments. 

 Dear Chief Editor,

The reviewed manuscript authors profiled miRNAs in G. biloba through high-throughput sequencing of leaf samples from 1-, 4-, and 7-year-old ginkgo plants and identified 28 miRNAs related to the aging pathway, according to their miRNA expression patterns. Correlation analysis of age-related miRNAs and major flavonoid compounds screened 17 vital miRNAs, including miR535a, miR166a, miR171a, and miR396. Interactive miRNA–transcription factor network analysis suggested that a pivotal miR535a-SPL module may be involved in the flavonoid biosynthesis and aging pathways in G. biloba through post-transcriptional regulation. Our findings provide insights into the age-dependent regulatory roles of miRNAs in flavonoid biosynthesis. Instead, authors have to improve their manuscripts with many non-clear meanings, inaccuracies, and the authors need to address the following issues.

1. I have read the entire manuscript and my initial comment is that manuscript is poorly written. I have significant concerns about the grammar and vocabulary of the manuscript; therefore, I recommend the authors to used an English proofreading service.

1. The abstract does not reflect the whole story, revise it.

2. Introduction grammatical issues appear to be most prevalent in the introduction, making for very confusing reading. Further, the introduction is short but has no clear thread.
3. Figures are quite low resolution and text in figure is not readable, for example, 3, 4, 5, and 6.
4. The discussion should be interpreted with the results as well as discussed in relation to the present literature.
5. In Material and Methods:- indicate how many replicates assayed in each analysis/parameter. The number of samples or biological and technical replicates should be mentioned for each parameter in the methods.
6. The conclusion section is very poorly written. It should be extensively improved.
7. References: shall have to correct the whole References according to the ”Instructions for the Authors”, e.g. the Journal name must be abbreviated, journal name in italics, the year must be bold and you shall have to use the abbreviated number of the Journals cited. Further, some references the title to paper in title case some are in small letter case, hence please follow the journal instruction. Moreover, the scientific name must be italics. Please check the all refernce carefully.

Thank you and Best wishes

 

 

 

 

 

To,

The Chief Editor,

Forests, MDPI,

Manuscript ID: forests-2551392

Subject: Submission of comments on the manuscript in “Forests"

 

Dear Chief Editor Forests, MDPI,

Thank you very much for the invitation to consider a potential reviewer for the manuscript (ID: forests-2551392). My comments responses are furnished below as per each reviewer’s comments. 

 Dear Chief Editor,

The reviewed manuscript authors profiled miRNAs in G. biloba through high-throughput sequencing of leaf samples from 1-, 4-, and 7-year-old ginkgo plants and identified 28 miRNAs related to the aging pathway, according to their miRNA expression patterns. Correlation analysis of age-related miRNAs and major flavonoid compounds screened 17 vital miRNAs, including miR535a, miR166a, miR171a, and miR396. Interactive miRNA–transcription factor network analysis suggested that a pivotal miR535a-SPL module may be involved in the flavonoid biosynthesis and aging pathways in G. biloba through post-transcriptional regulation. Our findings provide insights into the age-dependent regulatory roles of miRNAs in flavonoid biosynthesis. Instead, authors have to improve their manuscripts with many non-clear meanings, inaccuracies, and the authors need to address the following issues.

1. I have read the entire manuscript and my initial comment is that manuscript is poorly written. I have significant concerns about the grammar and vocabulary of the manuscript; therefore, I recommend the authors to used an English proofreading service.

1. The abstract does not reflect the whole story, revise it.

2. Introduction grammatical issues appear to be most prevalent in the introduction, making for very confusing reading. Further, the introduction is short but has no clear thread.
3. Figures are quite low resolution and text in figure is not readable, for example, 3, 4, 5, and 6.
4. The discussion should be interpreted with the results as well as discussed in relation to the present literature.
5. In Material and Methods:- indicate how many replicates assayed in each analysis/parameter. The number of samples or biological and technical replicates should be mentioned for each parameter in the methods.
6. The conclusion section is very poorly written. It should be extensively improved.
7. References: shall have to correct the whole References according to the ”Instructions for the Authors”, e.g. the Journal name must be abbreviated, journal name in italics, the year must be bold and you shall have to use the abbreviated number of the Journals cited. Further, some references the title to paper in title case some are in small letter case, hence please follow the journal instruction. Moreover, the scientific name must be italics. Please check the all refernce carefully.

Thank you and Best wishes

 

 

 

 

 

Author Response

Point 1: The abstract does not reflect the whole story, revise it.

Response 1: Many thanks for your carefully reviewing our manuscript and putting forward many valuable comments and suggestions. According to your suggestion, we have revised abstract section to better summarize the content of our whole study. Additionally, we also made some modifications to correct grammar errors in the abstract section (lines 9 and 16).

Point 2: Introduction grammatical issues appear to be most prevalent in the introduction, making for very confusing reading. Further, the introduction is short but has no clear thread.

Response 2: Thanks for your suggestion. We have reworded the introduction section and highlighted the logicality of the introduction. Furthermore, we have modified the language of the whole manuscript. We also carefully checked for grammatical problems and corrected minor mistakes throughout the manuscript. Additionally, we have used the language editing service from Textcheck to improve the English language of our manuscript (for a certificate, please see http://www.textcheck.com/certificate/index/PBEoyy).

Point 3: Figures are quite low resolution and text in figure is not readable, for example, 3, 4, 5, and 6.

Response 3: Thank you for pointing out this issue. We have re-uploaded the all figures in the revised manuscript.

Point 4: The discussion should be interpreted with the results as well as discussed in relation to the present literature.

Response 4: According to your suggestion, we have added the discussion of previous studies and our results. Specifically, we deleted some content from the discussion. For example, in the first paragraph, we removed not relevant content and added comparison of previous studies and our results (line 341-346). In the third paragraph, we eliminated some unrelated discussions that did not pertain to our results. Additionally, we added a paragraph discussing plant hormones, as hormones are a significant part of our findings (line 396-404). Finally, we made some modifications to correct grammar errors in the discussion section (lines 339, 347-350, 354, 359 and so on).

Point 5: In Material and Methods: - indicate how many replicates assayed in each analysis/parameter. The number of samples or biological and technical replicates should be mentioned for each parameter in the methods.

Response 5: Thank you for pointing out this issue. We have added more information on the experiment design in the materials and methods. Please see line 131-134 in the revised manuscript. “The expression levels of miRNAs are presented as means ± standard deviation (SD) of at three times detection” were corrected as “Statistical data were presented as means ± standard deviation (SD) of three biological replicates.” In addition, we have also added relevant information in the figure legends (Figure 1 and Figure 5).

Point 6: The conclusion section is very poorly written. It should be extensively improved.

Response 6: As suggested, we have revised the conclusion section in the revised manuscript. Please see line 424-428.

Point 7: References: shall have to correct the whole References according to the” Instructions for the Authors”, e.g., the Journal name must be abbreviated, journal name in italics, the year must be bold and you shall have to use the abbreviated number of the Journals cited. Further, some references the title to paper in title case some are in small letter case, hence please follow the journal instruction. Moreover, the scientific name must be italics. Please check the all reference carefully.

Response 7: Thanks for your reminding. We have carefully checked all the references and corrected all the mistakes, such as references 8, 22, 25, 41, 44, and 46.

Author Response File: Author Response.docx

Round 2

Reviewer 3 Report (New Reviewer)

Dear Chief Editor,

Thank you for providing the opportunity to review the revised manuscript. The authors have addressed all comments and incorporated changes suggested by reviewers during the first round of revisions. The revised version of the manuscript is improved as expected. Based on these revisions, now this study is a suitable contribution to the Forests. I recommend the manuscript for publication.

Thank you

With best regards

Dear Chief Editor,

Thank you for providing the opportunity to review the revised manuscript. The authors have addressed all comments and incorporated changes suggested by reviewers during the first round of revisions. The revised version of the manuscript is improved as expected. Based on these revisions, now this study is a suitable contribution to the Forests. I recommend the manuscript for publication.

Thank you

With best regards

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

Dear Authors,

your research is well established and written, however, the methodology and discussion require further elaboration. 

Throughout the manuscript, the names of species of trees and plants are given incompletely. The authorship of the species is missing, e.g. Ginkgo biloba L.

Rows 83-87: sRNA nor SRNA. Please check through the whole manuscript.

Please describe in more detail the construction of sRNA libraries (type of kit etc.).

Row 93: miRBase is the database not software. There is missing link on this database. The same for KEGG database (row 106).

Row 127: Missing information for G. biloba genome.

Fig. 1: there is missing "nt" for sRNA size on the graph

Rows: 150-154: not clear meaning (Fig. 2). What exactly does this tell us, as no specific miRNAs are identified on the graphs? I leave it up to the authors, but I would classify this image as supplementary.

Rows: 174-175: If the authors have such information, it is necessary to cite it, because their research did not focus on both types (gymnosperms and angiosperms).

The paragraph 3.4: Are these results from a previous study? This paragraph seems confusing, as it intertwines the results obtained in the given study with the previous results of the authors. Paragraph 3.5 is of a similar nature, which I recommend reworking, as the current study was not focused on the regulatory processes of photosynthesis.

I consider the discussion to be the weakest part of the manuscript. The authors confront their results with only slightly more than 20 other publications, while these are mainly publications that report results mainly for crops and not trees. Not all areas of the presented research are addressed in the discussion. There are many more publications in scientific databases that address the issue of miRNA reregulation in ginkgo and are not listed in the discussion.

Author Response

Please see the attachment

Author Response File: Author Response.docx

Reviewer 2 Report

Review of manuscript "MicroRNA expression and integrated network analyses of age-dependent flavonoid biosynthesis regulation in Ginkgo biloba"

General comments:
Manuscript provided insights into the regulatory roles of miRNAs in flavonoid biosynthesis in response to plant age.
The leaves of 1-, 4- and 7-year-old ginkgo trees were analyzed.

Detailed comments:
Line 113; "R" is not a Pearson’s correlation coefficient!!!
Lines 142, 161: "correlation coefficient R2" - "R2" is not correlation coefficient. This is the determination factor. The authors should contact a statistician.
Quality of Figures is very poor.
Figure 3 needs statistical analysis.
Line 225: "cor"?
Line 243: "Tukey’s test" - No information in the M&M section about the use of this method.
Line 417: "Author 1"?!

My suggestion:
Figure 5B: What method was used for grouping?
Linr 272: "Figure 6." not "Figure6."
Figure S1: Specify critical values.
Figure S2: "log10" - zapis "10" nie jest potrzebne w logarytmie dziesiętnym
Figure S3: What method was used for grouping?
Figure S4: What method was used for grouping?
Figure S5 contains regression coefficients rather than correlation coefficients. This should be corrected.
Figure S6: What method was used for grouping?

Paper needs major revision.

Author Response

Please see the attachment

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Dear Authors,

thank you for your effort. I wish you every success in your future research.