Genome-Wide Identification and Expression Analysis of the VILLIN Gene Family in Soybean

Round 1

Reviewer 1 Report

The paper titled with ‘Genome-wide Identification …. Family in Soybean’ by Zhou et. al. has focused on characterization of the VLN gene family in soybean. They have also explored expression pattern of GmVLNs in various tissues of soybean. Although the authors attempted to focus on relevant areas of research, the manuscript requires further improvement before acceptance. 

Following are the few comments for the authors to further improve this manuscript. 

1. Authors should explain the aspects of linkage between Profilin and Villin in introduction section as Profilin has been reported in several study for its functional similarities with Villin in relation to Actin protein. Please check few studies mentioned below. 

Marleen Van Troys, Joël Vandekerckhove, Christophe Ampe, Structural modules in actin-binding proteins: towards a new classification, Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, Volume 1448, Issue 3, 1999, Pages 323-348, ISSN 0167-4889, https://doi.org/10.1016/S0167-4889(98)00152-9 

Pandey, D.K., Chaudhary, B. Synchronous Transcription of Cytoskeleton-Associated Genes is Critical to Cotton Fiber Elongation. J Plant Growth Regul 38, 1037–1061 (2019). https://doi.org/10.1007/s00344-019-09913-0 

Yoo M-J, Wendel JF (2014) Comparative Evolutionary and Developmental Dynamics of the Cotton (Gossypium hirsutum) Fiber Transcriptome. PLoS Genet 10(1): e1004073. https://doi.org/10.1371/journal.pgen.1004073 

2. Page No. 3, Paragraph 2, Authors should focus more on functional part in this study if claiming about analysis of functional aspects of GmVLN in statement. 

3. Figure 5a: authors are suggested to provide better quality image. Also mention confidence score of String based result of protein- protein interaction. 

4. Figure 5b, What is the accuracy level of prediction, mention either in material method or in legend of figure. 

5. Please provide better quality image for Figure 6. 

6. Figure 7, What is the statistical parameter used to analyze expression data and making of graph? mention significance difference level in each graph. 

7. Specify, details of tissue selected for RNA extraction for gene expression analysis in material and method section. 

8. Botanical name should be italicized. Please check entire manuscript for the same. 

9. Material and Methods, Section 4.1. Authors are suggested to reframe the statements for better understanding to the readers. 

10. Material and Methods, Section 4.1. Section 4.3, Section 4.4, and section 4.5.  These sections need to be re-written for better understanding to the readers. 

11. Section 4.6, Rather than using ‘day/night’, suggested to use ‘light/dark’. 

 

12. Section 4.7, Mention quality of RNA measured through Nanodrop. Which reference gene was used for normalization of gene Expression? Mention it. Also provide Ct value of each gene authors obtained though qRT-PCR in a separate table as supplementary data.

Author Response

Reviewer #1:

The paper titled with ‘Genome-wide Identification …. Family in Soybean’ by Zhou et. al. has focused on characterization of the VLN gene family in soybean. They have also explored expression pattern of GmVLNs in various tissues of soybean. Although the authors attempted to focus on relevant areas of research, the manuscript requires further improvement before acceptance.

Following are the few comments for the authors to further improve this manuscript.

1. Authors should explain the aspects of linkage between Profilin and Villin in introduction section as Profilin has been reported in several study for its functional similarities with Villin in relation to Actin protein. Please check few studies mentioned below.

 

Marleen Van Troys, Joël Vandekerckhove, Christophe Ampe, Structural modules in actin-binding proteins: towards a new classification, Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, Volume 1448, Issue 3, 1999, Pages 323-348, ISSN 0167-4889, https://doi.org/10.1016/S0167-4889(98)00152-9

 

Pandey, D.K., Chaudhary, B. Synchronous Transcription of Cytoskeleton-Associated Genes is Critical to Cotton Fiber Elongation. J Plant Growth Regul 38, 1037–1061 (2019). https://doi.org/10.1007/s00344-019-09913-0

 

Yoo M-J, Wendel JF (2014) Comparative Evolutionary and Developmental Dynamics of the Cotton (Gossypium hirsutum) Fiber Transcriptome. PLoS Genet 10(1): e1004073. https://doi.org/10.1371/journal.pgen.1004073

Response: Yes, it is. In the new revised manuscript, a brief description was made of their participation. Profilin and Villin have similar functions, and both belong to ABP proteins. At present, the function of the Profilin protein is recognized as binding to the actin monomer, participating in the reorganization of the actin filament, and then regulating cytoskeleton dynamics. Current research has shown that Villin not only has the function of action binding but also has the functions of severing, capping, clearing, or bundling action fillings. In our understanding, both Profilin and Villin are involved in the regulation of cytoskeleton dynamics, but it is unknown whether Villin and Profilin or other ABPs jointly regulate cytoskeleton dynamics. In future research, relevant molecular mechanisms need to be explained to explain the functional relationship between Villin and Profilin. We will continue to pay attention to research in this area and try to explain the relationship between them.

 

2. Page No. 3, Paragraph 2, Authors should focus more on functional part in this study if claiming about analysis of functional aspects of GmVLN in statement.

Response: Thank you for pointing out this point. This study mainly focuses on the characteristics analysis of soybean VLN, and has changed its “function” to “characteristics”. We hope that this study can provide a basis for future VLN gene function research. In future research, we will consider conducting functional research based on your suggestions.

 

3. Figure 5a: authors are suggested to provide better quality image. Also mention confidence score of String based result of protein- protein interaction.

Response: Based on your valuable suggestions, data 5A has been modified, and the predicted interaction protein scores have been added to Supplementary Table 6.

4. Figure 5b, What is the accuracy level of prediction, mention either in material method or in legend of figure.

Response: Thank you very much for your valuable suggestion. Due to the update of the website, we apologize for not being able to recover the original data. Based on reliable protein 3D structure prediction parameters, we have reconstructed the 3D structure of the soybean VLN protein. The screening parameters and usage model have been added to Supplementary Table 8. By the way, the selection criteria for each parameter have been modified in Section 4.2.

5. Please provide better quality image for Figure 6.

Response: Thank you for pointing out this point. It has been revised in the new manuscript.

6. Figure 7, What is the statistical parameter used to analyze expression data and making of graph? mention significance difference level in each graph.

Response: Sincerely thank you for pointing out this point. We have corrected it in the new manuscript. By the way, we have added statistical indicators of these figures.

7. Specify, details of tissue selected for RNA extraction for gene expression analysis in material and method section.

Response: Thank you for your suggestion. The details of RNA extraction have been described in the new manuscript.

 

8. Botanical name should be italicized. Please check entire manuscript for the same.

Response: We have carefully checked and made modifications.

 

9. Material and Methods, Section 4.1. Authors are suggested to reframe the statements for better understanding to the readers.

Response: In the method, modifications have been made based on your suggestions.

 

10. Material and Methods, Section 4.1. Section 4.3, Section 4.4, and section 4.5.  These sections need to be re-written for better understanding to the readers.

Response: In the method, modifications have been made based on your suggestions.

 

11. Section 4.6, Rather than using ‘day/night’, suggested to use ‘light/dark’.

Response: We have made modifications based on your suggestion.

 

12. Section 4.7, Mention quality of RNA measured through Nanodrop. Which reference gene was used for normalization of gene Expression? Mention it. Also provide Ct value of each gene authors obtained though qRT-PCR in a separate table as supplementary data.

Response: Thank you for pointing out this point. In the qRT experiment, the reference gene ACT11(Glyma.18g290800) was used, it has been added to the method, and the Ct value is in supplementary Table 10.

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript is very important to provide some basic information about the VLN genes in soybean and optimize the Family in Soybean for further breeding program in this culture a wide growing area. Modern methods of research and analysis of the obtained results were used.  

The structure of the manuscript is excellent. 

The title is appropriate for the very idea of the manuscript. 

The summary is presented clearly and precisely in a concise form. 

Research results are well presented and comprehensively explained.  

In the discussion part, the authors have tried to give and at the same time comment on the results of other authors' research. 

The reference is sufficient and written in accordance with the guidelines of the author of this journal.  

It might be good if the authors wrote a few sentences about the practical application of the new information about soybeans.

I think this manuscript is scientifically correct and can be accepted for publication in this prestigious journal.  

Author Response

Thank you very much for your suggestion. We have revised some sentences in the manuscript and sorted out some writing ideas. At the same time, adjustments have been made to the relevant figures, and important experimental data has been added to the supplementary table.

Reviewer 3 Report

These are the major issues in the manuscript

1) There is a substantial lack of methods description.

It is not clear whether you analyze proteins or gene sequences which is not adequately mentioned. Ex: pag 13 "we downloaded soybean genome data from the Phytozome website" - are these proteins actually? How did you analyze the genome data with PFAM domains? This is typically done on the aa sequences. Same for section 4.4 - what data did you use for each analysis? genes, proteins, or genome? Specify better

Methods pieces are sprinkled in some of the results. Section 2.1 "A significant e-value of 1e-5 was..." this needs to go into the methods and needs to be enriched with a better description.

You mention the TBtools platform in the results section which is a user interface for the main methods. This does not provide a sufficient description of the Bioinformatics analysis. The methods section lacks information about the underlying algorithms, tools, and filtering strategies. What are the tools underlying TBtools that you used?

Another example is 2.2 ". To acquire candidate VLN protein sequences, the query sequences were used to blast the genome database of every species with TBtools." - how exactly is this done? This is not sufficient

2) Results incoherence

", the protein sequences corresponding to PF00626 and PF02209 are used as query sequences" - these are Pfam profiles, not sequences. I am not sure how did you use them to find your hits. This is ambiguous

"The encoding genes for these GmVLN proteins were given the names GmVLN1a, GmVLN1b, GmVLN2a, GmVLN2b, GmVLN3a, GmVLN3b, GmVLN4a, GmVLN4b, GmVLN5a, and GmVLN5b respectively" - how did you name these? Is there any strategy behind it?

Figure 2 - what is the reference sequence of the MSA? Gelsolin 5 and 6 are full of gaps which makes me think that there is something lacking in your search or comparative analysis. Gelsolin 5 and 6 don't look like to be in your proteins, can you check if they are complete?

pag 7 "The results of three-dimensional structure prediction indicate that all GmVLN proteins are highly similar in spatial structure, which may reflect their functional similarity (Figure 5B)." This statement is not supported by quantification, do you have any structure alignment analysis that supports the fact that they are similar?

Figure 5A -  What type of interaction do you use in STRING? There are several scores that can be used

section 2.6 "the parallel genes" - paralogous genes?

Figure 7 - Can you please group better the images? C-K can be presented according to paralogous genes 

Figure 8A - How did you do the clustering of the groups (both y and x axes)

Figure 8C - Y-axis - what are the units?

3) English language

In several cases, the use of adverbs is not clear.

Ex: 4.6 ". Besides, seedlings grown in the same environment are also used for tissue specific expression analysis, but the photoperiod was 14 h day/10 h night." in this case but means a contrast with the previous part of the sentence. Can you re-write it for clarity?

Please read the text carefully, especially for the results and methods. Some sections are unclear. 

Ex: pag 13 - 4.1 "These selected sequences conformed again through the SMART website (SMART: Main page (embl-heidelberg.de)) [54], and the deletion contained not the gelsolin and headpiece domain sequences, the other left were regard as soybean VLN sequences." This is not correct English

Ex: 4.4 - "Soybean VLN family protein, gene sequence was downloaded from the Phytozome websites (Phytozome info: G. max Wm82. a4. v1 (doe. Gov)) [51], then based on online website NCBI batch CD search prediction conserved domain (Welcome to NCBI Batch CD-search (nih.Gov)) and MEME (MEME-Submission form (meme-suite.org)) [58] prediction conserved motif, finally all these results were visualized by TBtools." same as the previous comment.

Please check the species' scientific names that need to be italicized. I noticed that there are several multiple spaces between the words and web links are not properly formatted. Please curate the text for these issues.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Good to see that authors have made required change to improve this manuscript. 

 

Author Response

Thank you again for your feedback on the modifications

Reviewer 3 Report

I appreciate your effort to address my concerns. Thank you for adding information to the methods and enhancing the manuscript results/methods. 

For future work in Bioinformatics, and strongly recommend using standard terminal-based tools, not GUI-based tools. This will help you to understand better the methods that you are using and will result in more clarity to the Bioinformatics community.

I still don't see the following points (see comments in italic):

pag 12: To identify all the possible VLN in soybean, we downloaded soybean protein data from the Phytozome website (Phytozome info:G.max Wm82.a4.v1 (doe.gov)) [51], according to the conserved Pfam profiles of PF00626 and PF02209 to identify all members 400 of the soybean VLN in several sequences to a big file of TBtools (Pfam is now hosted by InterPro (xfam.org)) [52], the number of threads was set to 2, the E-value was set to 1e-5, the number of hits was set to 500, and the number of alignments was set to 250. A total of 12 transcript IDs were extracted. Then protein sequences of 12 transcript IDs were extracted with Fasta Extract of TBtools. 

* Please clarify if you downloaded the proteins and performed a PFAM domain search. Why did you extract Transcript IDs from these proteins and then extract the proteins again with TBtools? This approach is not clear. Did you start with transcripts instead? Please address the methodological approach better

* What method did you use for the PFAM search? Did you use InterProScan or another profile search? Please specify

pag 7 - The results of three-dimensional structure prediction indicate that GmVLN proteins in the same subgroup are highly similar in spatial structure, which may reflect their functional similarity (Figure 5B); the parameters and usage models for predicting the three-dimensional structure of proteins are presented in Supplementary Table 8.  

* Please remove the statement of similarity. As you mentioned in your response, this is a speculation. All the results in the manuscript need to be supported by solid data.

New Figure 7 - thank you for improving the Figure. Can you please make the "*", "**", "***" bigger? The asterisk color is not visible 

pag 12 - When predicting the three-dimensional structure of proteins, global model quality evaluation (GMQE), coverage, and sequence identity (Seq Identity) are important screening parameters. The GMQE is between 0 and 1, and the closer it is to 1, the better the quality of the model used. The coverage rate represents the degree to which the target protein sequence is covered by the template protein sequence; the seq identity represents the alignment and matching degree of amino acids, and the higher the identity, the more reliable the model and the more accurate the predicted structure.

* Sure, but what are GMQE scores for your proteins? Are they mostly > 0.75 or similar? What is the quality of the structures you predicted? Are they good? Please specify if you want to include this analysis in the paper

Figure 7 B-K, Figure 8C - Please provide the units only on the y-axis. You are mixing with the title of the barplot; the y-axis contains units. In the case of 8C it should be "Relative expression". Units are missing in Figure 7 B-K, please specify

 

Author Response

Please see the attachment

Author Response File: Author Response.docx

Round 3

Reviewer 3 Report

Thank you for providing the improved methods, it now looks more clear and the Bioinformatics methods are described in details.