Evolutionary Relationships and Divergence of Filamin Gene Family Involved in Development and Stress in Cotton (Gossypium hirsutum L.)

Round 1

Reviewer 1 Report

The study “Evolutionary relationships and divergence of Filamin gene family involved in development and stress in cotton (Gossypium hirsutum L.)” comprehensively comprised with identification and systematic characterization of Filamin gene in cotton. Authors have tried all sorts of bioinformatics analysis and subsequently wet-lab validation of Filamin gene during developmental stages and multiple abiotic stress conditions in cotton which putatively prove the involvement of Filamin gene family in biological processes in cotton. The study is well executed with sound experimentations and representations. However, some modifications need to be rectified and incorporated by the authors to improve the manuscript for global readability and acceptance.  The comments observed during the review of the manuscripts are as follows:

General comments

Comment #1 – Authors must ensure to write uniformity in the names of the gene family in the whole document. They must write either “gene family members or genes instead of gene family or genes.

Comment #2 – Check the cis-acting at line number 21. Authors must write it as cis-acting in italicized font and must check it in the whole text of the manuscript to make it uniform in the revised version of the manuscript.

Comment #3 – It is just suggestion regarding abstract, if authors can add little more quantitative data.

Comment #4 – Authors have described the evolutionary pattern of the Filamin gene family members in various plant species but they have not included the phylogenetic analysis as a keyword. Please add the same.

Comment #5 – Line number 79, ‘23 species’ should be written as ‘Twenty-three species’

Comment #6 – Line number 90, the phrase ‘used to sequence align 23 species’ should be written as ‘used to align peptide sequences of Filamin proteins of 23 species’

Comment #7 – It seems to be repletions of methodology in the result section. Authors should mention only the findings in this section with minimum methodology description. 

Scientific comments

Comments #8 – In this study, fifty-four Filamin gene family members were identified and characterized in 23 species but authors have restricted to cotton for detailed description of this gene family. Why? In my opinion, authors must include this deficiency as a thematic area in the introduction as well as in the discussion part of the manuscript.

Comment #9- Why do polyethylene glycol was used at the rate of 22%? This was just a random concentration or purposefully it was taken to induce the drought stress. Please clarify. If possible, cite any reference, if followed any protocol.

Comment #10 – Why animal Filamin protein sequences were used as query search in blast analysis? Why not used the plant Filamin protein sequence to identify this gene family in cotton?

Comment #11 – Why authors have not performed functional annotations using gene ontology and blast2go analysis of the Flamin gene family members? If possible, it will be quite impressive to add the gene ontology analysis with Figure.

Comment #12 – Some figures are in blurred resolution so please improve the resolution at least minimum of 600dpi.

Comment #13 – Regarding the protein 3D structure prediction of Filamin proteins, why authors have not included Filamin protein sequences from other plants species?

Comment #14 – Authors have not mentioned about the house-keeping gene for normalization of qRT-PCR expression data? 

Comment #15 – Regarding the cis-acting elements and qRT-PCR analysis, authors have not discussed the findings with supportive references in the discussion section.

Comment #16 – The conclusion section must be comprehended more with quantitative data and final findings.

Author Response

1、Authors must ensure to write uniformity in the names of the gene family in the whole document. They must write either “gene family members or genes instead of gene family or genes.

Response: Thank you for your valuable comment. All “Filamin genes” in the manuscript have been replaced with “Filamin gene family members”. (Line18,20,63,84,97,114,122,123,147,149,154,158,193,196,202,205,206,207,212,228,229,230,236,237,238,239,240,245,246,248,263,264,273,275,277,279,280,288,293,429,630,652 and et.al)

2、Check the cis-acting at line number 21. Authors must write it as cis-acting in italicized font and must check it in the whole text of the manuscript to make it uniform in the revised version of the manuscript.

Response: Thank you for your comment. All cis-acting in the manuscript have been changed to italicized font. (Line22,80,147,150,152,428,433, 462,502,638,661,668,675,804 and 829)

3、It is just suggestion regarding abstract, if authors can add little more quantitative data.

Response: Thank you for your suggestion. Due to the limitation of the number of words in the Abstract. I have added some content related to fluorescence quantification in this part. (Line 20-26)

4、Authors have described the evolutionary pattern of the Filamin gene family members in various plant species but they have not included the phylogenetic analysis as a keyword. Please add the same.

Response: Thank you for your comments, I have added phylogenetic analysis as a keyword. (Line 35)

5、Line number 79, ‘23 species’ should be written as ‘Twenty-three species’

Response: Thank the reviewer for pointing out the problem. This problem has been corrected. (Line 96)

6、 Line number 90, the phrase ‘used to sequence align 23 species’ should be written as ‘used to align peptide sequences of Filamin proteins of 23 species’

Response: Thank the reviewer for pointing out the problem. Corrections have been completed. (Line 107 and 108)

7、 It seems to be repletions of methodology in the result section. Authors should mention only the findings in this section with minimum methodology description. 

Response: Thanks to the reviewers for suggestions about this part. The redundant methodology in the result section has been deleted. (Line 288-292,315,316,412,413,429-432 and 555-558)

Scientific comment

8、In this study, fifty-four Filamin gene family members were identified and characterized in 23 species but authors have restricted to cotton for detailed description of this gene family. Why? In my opinion, authors must include this deficiency as a thematic area in the introduction as well as in the discussion part of the manuscript.

Response: Thank you for your question. First of all, there are few studies of Filamin gene family in plants, mainly on individual Filamin gene family members in Arabidopsis, maize and chlamydomonas. In plants, systematic studies on the whole family are still lacking. Cotton is an important cash crop, it provides important natural fiber and oil seed, so the research of cotton reproductive and development is of great significance for cotton breeding. Our laboratory mainly focuses on the study of cotton, so we have made a detailed analysis of the role of cotton in this family. We have filled this gap in the introduction and discussion. (Line 62-65,68-76 and 605-609)

9、Why do polyethylene glycol was used at the rate of 22%? This was just a random concentration or purposefully it was taken to induce the drought stress. Please clarify. If possible, cite any reference, if followed any protocol.

Response: Thanks to the reviewers for suggestions on this part，I used 20% PEG, which has been corrected and references have been added. (Line 168 and 817-819)

10、Why animal Filamin protein sequences were used as query search in blast analysis? Why not used the plant Filamin protein sequence to identify this gene family in cotton?

Response: Thanks for the suggestions on this part. Because there is currently no systematic analysis of this family in plants, but there is systematic analysis in animals. So it is used animal Filamin protein sequences were used as query search in blast analysis.

11、Why authors have not performed functional annotations using gene ontology and blast2go analysis of the Filamin gene family members? If possible, it will be quite impressive to add the gene ontology analysis with Figure.

Response: Thanks to the reviewers for suggestions on this part. I used the gene id of cotton Filamin gene family members to conduct a simple GO analysis on them, and sho w the results in Figure S5. (Line 136,137,651-655)

12、Some figures are in blurred resolution so please improve the resolution at least minimum of 600dpi.

Response: Thank the reviewer for pointing out the problem, I've reset all the pictures to 600dpi.

13、Regarding the protein 3D structure prediction of Filamin proteins, why authors have not included Filamin protein sequences from other plants species?

Response: Thank the reviewer for pointing out the problem. Our research mainly focus on cotton, so only the tertiary structures of cotton protein are predicted. Due to the low reliability of direct modeling, we downloaded the PDB file of Arabidopsis in AlphaFold website (https://www.alphafold.ebi.ac.uk/), and then constructed the model according to the two cotton protein sequences closest to Arabidopsis.

14、Authors have not mentioned about the house-keeping gene for normalization of qRT-PCR expression data? 

Response: Thanks to the reviewer for suggestions. I have added the house-keeping gene of fluorescence quantification to the article. (Line 175, Table S5)

15、Regarding the cis-acting elements and qRT-PCR analysis, authors have not discussed the findings with supportive references in the discussion section.

Response: Thank the reviewer for pointing out the problem, I have added the supportive references about cis-acting elements and qRT-PCR analysis in the discussion section to the manuscript. (Line 643,647,653,855 and 862)

16、 The conclusion section must be comprehended more with quantitative data and final findings.

Response: Thanks to the reviewer for suggestions on this part，I have added fluorescence quantitative results and final findings to the conclusion. (Line 660-669)

Reviewer 2 Report

This MS (genes-2056137, Evolutionary relationships and divergence of Filamin gene family involved in development and stress in cotton (Gossypium hirsutum L.) is interesting as a research topic because it uses a database to identify Filamin genes involved in reproductive development and abiotic stress in cotton to elucidate their functional networks by inferring candidate genes and protein interactions.

Fluorescence quantitative analysis has not been represented in materials and methods section.

To determine the role of GH_A02G0519 and GH_D02G0539 in plants, it’s needed to knockout these genes which inactivate or remove the genes. Silencing of these genes will determine their function in plants based on the reduction of tolerance to stress and their silencing will decrease reproductive development. Further gene silencing experiments are required to determine their function in plants.

How could you conclude that GH_A02G0216 and GH_D02G0235 are involved in abiotc stresses. You just measured their expression values in response to some stresses and explained that the expression levels of GH_A02G0216 and GH_D02G0235 decreased significantly after low temperature and PEG treatment, but the expression levels of GH_A02G0216 and GH_D02G0235 increased after 1h NaCl treatment and then decreased again, while the expression levels increased after high temperature stress. However, their expression values have shown irregular trend at specific times.

There is no discussion of the possible mechanisms of genes and proteins involved in abiotic stress and how they could regulate plant tolerance to these stresses.

I suggest if you can identify the biological pathways or process in the nodes interaction you can make circles or any shape to highlight it with caption.

Author Response

Response: Thank the reviewer for pointing out the problem, the specific procedures and steps of fluorescence quantification have been supplemented in materials and methods. (Line 176-181)

Because these two genes (GH_A02G0519 and GH_D02G0539) are mainly involved in the double fertilization process of cotton, and the double fertilization process of cotton after flowering is hard to observed and verified temporarily, this experiment may not be completed temporarily. If conditions permit, gene silencing will be performed later.

The role of these two genes (GH_A02G0216 and GH_D02G0235) in abiotic stress is only explored based on the fluorescence quantification under stress. Because we mainly focus on the role of this family in reproductive development, we speculate that these two genes may have obvious down-regulation under the action of low temperature and PEG based on the fluorescence quantification under stress. It follows that they may be related to abiotic stress.

Regarding to the last question, I have performed a simple GO analysis, found the enrichment pathways of these two genes and related literature, and further elaborated their roles in abiotic stress. (Line 651-656)