Expression Analysis of Trihelix Transcription Factor Family in Strawberries and Functional Characterization of FvTrihelix6
, Syeda Anum Masood Bokhari 3, Qingxi Chen 1,* and Zhifeng Wen 1,*Round 1
Reviewer 1 Report
The paper of Fan et al. is well-presented and the English is clear and easy to read. The introduction is well structured and informative, the objectives are clear and well presented, the methods are well established and appropriated, the results are simple, well defined and easy to understand. The discussion is adequate. Nice work.
Some minor modifications need to be taken into account:
Title: It is better to delete Bioinformatics from the title (up to you !!!)
Line 79: Please add the chromosome number of strawberry and genome size.
Line 134: please verify in the subtitle 2.3 “d analyses “ ??
Line 167: you should describe briefly “The floral dip method” you mentioned, to be clear for readers
Line 172: you should use the hole word of “ORF” you mentioned for the 1st time. Open Reading frame ??
Line 182 and 183: you should add (GUS: β-glucuronidase, an enzyme common in bacteria) and its role
In the paragraph 2.8 (Lines 180-195): You may add pictures (or schematics) describing the different steps
Results Section :
Line 233: “Data S1’’ ? ? is that supplementary , because you did not uploaded this file with the manuscript
Line 234: put “bp” instead of “base pairs’’
Line 242: In the footnote of Table 1 you should put all the explanations of abbreviated words in the table (CDS and pI are missing from the footnote)
Line 278 and 279: delete “for example” (you have put all the genes in all the subfamilies)
Line 290: “Table S2” add it… you did not uploaded the supplementary data !!!
Figure 1, Page 8: You should enhance the quality of the images A, B and C (the legend is not clear)
Line 330: In the title of figure 4, avoid abreviations in the titles and use the hole word of TTF. You should also add the explanation of the colors (which represent up or down regulation) to be easy to read in the figure directly
Line 351: In the title of Figure 5 you should add the explanation of the SA and MeJA and detail how were applied. You may add this detail “For exogenous hormone treatment, methyl jasmonate or salicylic acid solution was sprayed on strawberry leaves, and distilled water was used as a control)
In all the document: Avoid that title of Figures or title of Tables are separated from the corresponding figure or table)
Line 382: in the title of Figure 6 you should add the WT is the wild type ?? non-transformed!!, used as control.
Line 421: “LB is left border and RB is right border” You should also add NOS and GUS. And you should add the legend (control, SA, GC) of figure 8C which represent control, SA treatment, and C. gloeosporioides inoculation.
Conclusion Section:
I suggest to add, If possible, a shematic presentation of the findings of this paper in conclusion or abstract section !!!
References Section:
You should very list of references, it is not uniform sometimes you put p (for the pagination) and sometimes not…etc ; verify instruction for authors
Author Response
Dear Editor,
Thank you for giving us the opportunity to submit a revised manuscript “Expression Analysis of Trihelix Transcription Factor Family in Strawberry, and Functional Characterization of FvTrihelix6” for publication in the Horticulturae Journal.
We appreciate the time and effort that you and the reviewers dedicated to providing feedback on our manuscript and are grateful for the insightful comments on and valuable improvements to our paper. We have incorporated the suggestions made by the reviewers. Those changes are markered within the manuscript. Please see below, for a point-by-point response to the reviewer’s comments and concerns. We have corrected all the grammatical and contextual mistakes. We would be happy to consider any further changes that you deem appropriate and look forward to working with the editorial staff through the final submission process.
Thank you again, and best regards,
Dr. Zhifeng Wen
College of Horticulture,
Fujian Agriculture and Forestry University
Response to Reviewer 1 Comments
Point 1: It is better to delete Bioinformatics from the title (up to you !!!)
Response: Thank you for your suggestion and we have replaced “Bioinformatics” with Genome-Wide Identification (Title Line 2). As we have identified Trihelix Transcription Factor genes and also performed their expression analysis.
Point 2: Line 79: Please add the chromosome number of strawberry and genome size.
Response: Thank you for your comment. We have added the chromosome number (2 n = 8 x = 56) and genome size (∼ 240 Mb) (Line 83-84).
Point 3: Line 134: please verify in the subtitle 2.3 “d analyses”??
Response: Thanks for your suggestion and we have deleted “d analyses” (Line 139).
Point 4: Line 167: you should describe briefly “The floral dip method” you mentioned, to be clear for readers.
Response: Thanks for your suggestion. We have added the required information. The inflorescences of all the Arabidopsis plants were dipped for a few seconds into the 5% sucrose solution containing 0.05% (vol/vol) Silwet L-77 and resuspended Agrobacterium cells carrying 35S::construct FvTrihelix6 (Line 176-178).
Point 5: Line 172: you should use the hole word of “ORF” you mentioned for the 1st time. Open Reading frame ??
Response: Thanks for your suggestion we have used the whole word of ORF as “Open Reading frame” (Line 181).
Point 6: Line 182 and 183: you should add (GUS: β-glucuronidase, an enzyme common in bacteria) and its role.
Response: Thank you for your suggestion. The bacterial β-glucuronidase (GUS) gene is often introduced into plants as a reporter gene fused to a promoter because of its several advantages over other reporter genes. We have added the description of GUS to the manuscript (Line 189 and 190).
Point 7: In the paragraph 2.8 (Lines 180-195): You may add pictures (or schematics) describing the different steps.
Response: Thank you for your suggestion. We have described the steps of “Cloning of FvTrihelix6 promoter, GUS protein staining, and GUS activity assay” clearly in the manuscript. We think that a schematics description is not required (or schematics) in the materials and methods part.
Point 8: Line 233: “Data S1”?? is that supplementary, because you did not uploaded this file with the manuscript.
Response: Thanks for your suggestion and we apologize for this mistake. We have uploaded the Data S1.
Point 9: Line 234: put “bp” instead of “base pairs”
Response: Thank you for your comment. Done as suggested. (Line 245).
Point 10: Line 242: In the footnote of Table 1 you should put all the explanations of abbreviated words in the table (CDS and pI are missing from the footnote)
Response: Thank you for your suggestion. We have added the explanations of CDS and pI (Line 253-254).
Point 11: Line 278 and 279: delete “for example” (you have put all the genes in all the subfamilies)
Response: Thank you for your suggestion. We have deleted “for example” (Line 282-283).
Point 12: Line 290: “Table S2” add it… you did not uploaded the supplementary data !!!
Response: Thanks for your suggestion and we apologize for this mistake. We have uploaded Table S2.
Point 13: Figure 1, Page 8: You should enhance the quality of the images A, B, and C (the legend is not clear)
Response: Thanks for your suggestion. We have enhanced the quality of Figure 1 (Figure 1 Line 270) and modified the legend.
Point 14: Line 330: In the title of figure 4, avoid abreviations in the titles and use the hole word of TTF. You should also add the explanation of the colors (which represent up or down regulation) to be easy to read in the figure directly
Response: Thank you for your suggestion. We have added the whole word of TTF and the explanation of the colors (Line 342-344).
Point 15: Line 351: In the title of Figure 5 you should add the explanation of the SA and MeJA and detail how were applied. You may add this detail “For exogenous hormone treatment, methyl jasmonate or salicylic acid solution was sprayed on strawberry leaves, and distilled water was used as a control)
Response: Thank you for your suggestion. We have added an explanation of the SA and MeJA, as well as details on how they were applied to the document——Strawberry leaves were sprayed with methyl jasmonate or salicylic acid solution for exogenous hormone treatment, and distilled water served as a control (Line 364-365).
Point 16: In all the document: Avoid that title of Figures or title of Tables are separated from the corresponding figure or table)
Response: Thank you for your suggestion.
Point 17: Line 382: in the title of Figure 6 you should add the WT is the wild type ?? non-transformed!!, used as control.
Response: Thank you for your suggestion. We have used “the wild type” instead of “WT” (Line 391-396).
Point 18: I suggest to add. If possible, a shematic presentation of the findings of this paper in conclusion or abstract section!!!
Response: Thank you for your suggestion. We have added a schematic presentation of the findings of this paper in the abstract section (Abstract).
Point 19: You should very list of references, it is not uniform sometimes you put p (for the pagination) and sometimes not…etc; verify instruction for authors
Response: Thank you for your suggestion and we apologize for this mistake. We have uniformed the references (References Line 568-690).
Author Response File: 
Author Response.docx
Reviewer 2 Report
This study performed a comprehensive analysis of the trihelix family in strawberries, resulting in identification of thirty trihelix family members. It uses a series of methods to find answers to important questions. It makes use of a variety of precise methods that are suitable for achieving the objectives set. A large amount of data has been generated. These data are processed separately and in context. Expression levels were measured in different tissues (root, stolon, leaf, flower, and fruit) after infection with C. gloeosporioides or salicylic acid and jasmonic acid spraying. GFP, GUS and qPCR methods were used to map the mechanisms of Trihelix genes. Can GFP of GUS sequences modify the localization of Trihelixes? Moreover, FvTrihelix6 enhances resistance against pathogens through the SA and JA signaling pathways in transgenic A. thaliana plants. Does the indigenous AtTrihelix genes have similar effect? Why did You used tobacco leaves for transient expression analysis instead of Arabidopsis? Graphs are sufficiently detailed and easy to expound. The text contains occasional typos, but it is nevertheless easy to read and uses scientific language appropriately. The literature used is fairly extensive. Names of genes and gene families should be in Italics. Tolerance against abiotic stresses exists, the term ‘resistance’ is an exaggeration. I cannot find the Latin name of tobacco. At first appearance, please provide the descriptor of F. vesca and A. thaliana.
The text contains occasional typos.
Author Response
Dear Editor,
Thank you for giving us the opportunity to submit a revised manuscript “Expression Analysis of Trihelix Transcription Factor Family in Strawberry, and Functional Characterization of FvTrihelix6” for publication in the Horticulturae Journal.
We appreciate the time and effort that you and the reviewers dedicated to providing feedback on our manuscript and are grateful for the insightful comments on and valuable improvements to our paper. We have incorporated the suggestions made by the reviewers. Those changes are markered within the manuscript. Please see below, for a point-by-point response to the reviewer’s comments and concerns. We have corrected all the grammatical and contextual mistakes. We would be happy to consider any further changes that you deem appropriate and look forward to working with the editorial staff through the final submission process.
Thank you again, and best regards,
Dr. Zhifeng Wen
College of Horticulture,
Fujian Agriculture and Forestry University
Response to Reviewer 2 Comments
This study performed a comprehensive analysis of the trihelix family in strawberries, resulting in identification of thirty trihelix family members. It uses a series of methods to find answers to important questions. It makes use of a variety of precise methods that are suitable for achieving the objectives set. A large amount of data has been generated. These data are processed separately and in context. Expression levels were measured in different tissues (root, stolon, leaf, flower, and fruit) after infection with C. gloeosporioides or salicylic acid and jasmonic acid spraying. GFP, GUS and qPCR methods were used to map the mechanisms of Trihelix genes. Can GFP of GUS sequences modify the localization of Trihelixes? Moreover, FvTrihelix6 enhances resistance against pathogens through the SA and JA signaling pathways in transgenic A. thaliana plants. Does the indigenous AtTrihelix genes have similar effect? Why did You used tobacco leaves for transient expression analysis instead of Arabidopsis? Graphs are sufficiently detailed and easy to expound. The text contains occasional typos, but it is nevertheless easy to read and uses scientific language appropriately. The literature used is fairly extensive. Names of genes and gene families should be in Italics. Tolerance against abiotic stresses exists, the term ‘resistance’ is an exaggeration. I cannot find the Latin name of tobacco. At first appearance, please provide the descriptor of F. vesca and A. thaliana.
Point 1: Can GFP of GUS sequences modify the localization of Trihelixes?
Response: Thank you for your comment. GFP of GUS sequences cannot modify the localization of Trihelixes.
Point 2: Moreover, FvTrihelix6 enhances resistance against pathogens through the SA and JA signaling pathways in transgenic A. thaliana plants. Does the indigenous AtTrihelix genes have similar effect?
Response: Thank you for your comment. In this study, FvTrihelix6 was transferred into Arabidopsis thaliana and it was found that the resistance to C. higginsianum was enhanced. Resistance to C. higginsianum may exist in AtTrihelix genes but we have not investigated this aspect.
Point 3: Why did You used tobacco leaves for transient expression analysis instead of Arabidopsis?
Response: Thank you for your comment. Tobacco leaves were selected as the plant material for transient expression analysis because they gave excellent transformation efficiency and allowed multiple transient expression assays on a single large leaf (Y. Yang et al. 2001).
Point 4: Graphs are sufficiently detailed and easy to expound. The text contains occasional typos, but it is nevertheless easy to read and uses scientific language appropriately. The literature used is fairly extensive. Names of genes and gene families should be in Italics.
Response: Thanks for your suggestion and we apologize for this mistake. Gene names are already italicized.
Point 5: I cannot find the Latin name of tobacco. At first appearance, please provide the descriptor of F. vesca and A. thaliana.
Response: We have added the Latin name of tobacco and the descriptor of F. vesca and A. thaliana. (Line 197)
Author Response File: Author Response.docx
Reviewer 3 Report
The manuscript reports important discoveries about the trihelix transcription factor (TTF) in strawberries. However, there are a few things that need clarification and improvement.
Abstract
The abstract must be rewritten with more information.
- Describe the results of the in-silico characterization of the trihelix tf briefly.
- What is C. gleosporioides/ higginsuanum? An abstract is the first thing that we read, so it has to informative. Please write in full when mentioning something for the first time.
Introduction
Line 78 - The authors might want to check the statement and make sure that the claim is valid. There is one study on TTF in pineapple from a simple Google search. Most importantly, the authors missed one publication on the characterization of GT-2 factors which are the members of TTF in strawberries. Please cite and include the papers in the introduction and identify the knowledge gap that must be addressed to justify the current study.
This paper also characterizes TTF in F. vesca but mentions F.ananassa in the introduction. Can the authors explain why the study was conducted in F. vesca instead of F. ananassa?
Materials and method
2.1
Which part of the strawberries was sprayed with the spore suspension?
Line 116 - Do not use 'we' to describe the method section
2.3
Line 134 - The term 'strawberry TFF genes' refers to which species of strawberry? Please re-write the sentence for clarity
2.5
Line 157 - Actin gene from which species of strawberry?
Results
3.1, Line 236 - What does 'the majority....supposed to' mean in the sentence? What about the rest of the genes? Aren't they supposed to reside in the nucleus as well, since they are transcription factors?
3.2, The title of this section does not reflect the content since the section also covers the evolutionary relationship of the TTF. It is better to move the evolutionary relationship of the TTF to 3.3.
3.9, Line 387 - a different microscope was mentioned here to monitor the subcellular localization. Which one? After all, the name of the instrument should not be mentioned in the result section.
3.10, The authors repeat the methodologies here. Please re-write the section and also describe the results.
3.11, What is the purpose of this experiment? I don't find it relevant for this study. The results obtained do not answer any research question. I suggest removing this section.
Discussion
The first paragraph of the introduction section is a repetition of the introduction section. Please remove it. Focus on discussing the results and minimizing redundancy. Include a discussion on the results from the bioinformatic analysis.
Conclusion
Line 527 - What does 'Part of genes..' means?
Line 532,533 - Following the suggestion above, please remove the conclusion on the recombinant protein expression.
The English language of the manuscript must be improved. There are errors in sentence structure and choice of words that make the sentences ambiguous. The authors use simple and short sentences in several places. The sentences are better combined to form complex sentences. Please standardize the use of short form. For example, Trihelix TF, trihelix genes, and TTF have been used interchangeably. TTF, among other short forms, was not written in full when it was first mentioned. Please check the whole manuscript. In other cases, the short form was written in full again.
Author Response
Dear Editor,
Thank you for giving us the opportunity to submit a revised manuscript “Expression Analysis of Trihelix Transcription Factor Family in Strawberry, and Functional Characterization of FvTrihelix6” for publication in the Horticulturae Journal.
We appreciate the time and effort that you and the reviewers dedicated to providing feedback on our manuscript and are grateful for the insightful comments on and valuable improvements to our paper. We have incorporated the suggestions made by the reviewers. Those changes are markered within the manuscript. Please see below, for a point-by-point response to the reviewer’s comments and concerns. We have corrected all the grammatical and contextual mistakes. We would be happy to consider any further changes that you deem appropriate and look forward to working with the editorial staff through the final submission process.
Thank you again, and best regards,
Dr. Zhifeng Wen
College of Horticulture,
Fujian Agriculture and Forestry University
Response to Reviewer 3 Comments
Point 1: Describe the results of the in-silico characterization of the trihelix tf briefly. Response: Thank you for your suggestion and we have added the results of in-silico characterization of the Trihelix TF (Line 15-18).
Point 2: What is C. gleosporioides / C. higginsuanum? An abstract is the first thing that we read, so it has to informative. Please write in full when mentioning something for the first time.
Response: Thank you for your comment. We have added the full names of C. gleosporioides and C. higginsuanum (Line 19 and Line 24).
Point 3: Line 78 - The authors might want to check the statement and make sure that the claim is valid. There is one study on TTF in pineapple from a simple Google search. Most importantly, the authors missed one publication on the characterization of GT-2 factors which are the members of TTF in strawberries. Please cite and include the papers in the introduction and identify the knowledge gap that must be addressed to justify the current study.
Response: Thanks for your suggestion. We have cited more studies to justify the current study (Line 45, 51-53).
Point 4: This paper also characterizes TTF in F. vesca but mentions F.ananassa in the introduction. Can the authors explain why the study was conducted in F. vesca instead of F. ananassa?
Response: Thank you for the suggestion. F. vesca is diploid and an excellent model material for studying strawberry disease resistance in the lab. Therefore, we selected this for our study.
Point 5: Which part of the strawberries was sprayed with the spore suspension?
Response: Thank you for your suggestion. We sprayed leaves with spore suspension in this study.
Point 6: Line 116 - Do not use 'we' to describe the method section.
Response: Thank you for your suggestion. We have deleted 'we' and modified the expression (Line 121-123).
Point 7: Line 134 - The term 'strawberry TFF genes' refers to which species of strawberry? Please re-write the sentence for clarity.
Response: Thank you for your suggestion. The strawberry TFF genes refers to F. vesca. We have modified the information (Line 140).
Point 8: Line 157 - Actin gene from which species of strawberry?
Response: Thank you for your comment. The Actin gene is from F.vesca specie. We have supplemented the information (Line 163).
Point 9: 3.1, Line 236 - What does 'the majority...supposed to' mean in the sentence? What about the rest of the genes? Aren't they supposed to reside in the nucleus as well, since they are transcription factors?
Response: Thank you for your suggestion. We have added that ‘Twenty-nine FvTrihelix genes were supposed to reside in the nucleus’ (Line 247).
Point 10: 3.2, The title of this section does not reflect the content since the section also covers the evolutionary relationship of the TTF. It is better to move the evolutionary relationship of the TTF to 3.3.
Response: Thank you for your suggestion. We have modified the title (3.2 Line 255).
Point 11: 3.9, Line 387 - a different microscope was mentioned here to monitor the subcellular localization. Which one? After all, the name of the instrument should not be mentioned in the result section.
Response: Thank you for your suggestion. We have deleted the name of the microscope (Line 402).
Point 12: 3.10, The authors repeat the methodologies here. Please re-write the section and also describe the results.
Response: Thank you for your suggestion. We have rewritten the section and have deleted methodologies in the 3.10 (Line 416-419).
Point 13: 3.11, What is the purpose of this experiment? I don't find it relevant for this study. The results obtained do not answer any research question. I suggest removing this section.
Response: Thank you for your suggestion. We will continue to perform more recombinant protein experiment study functions of FvTrihelix6, so we kept this section (3.11 Line 444-462).
Point 14: The first paragraph of the introduction section is a repetition of the introduction section. Please remove it. Focus on discussing the results and minimizing redundancy. Include a discussion on the results from the bioinformatic analysis.
Response: We are grateful for your suggestion. Indeed, this paragraph is a continuation of the introduction therefore the first paragraph of the discussion has been omitted (Line 463-482).
Point 15: Line 527 - What does 'Part of genes' means?
Response: Thank you for your suggestion. We have deleted 'Part of genes' and have added 'Ten genes' (Line 544).
Point 16: Line 532,533 - Following the suggestion above, please remove the conclusion on the recombinant protein expression.
Response: Thank you for your suggestion. We will continue to perform more recombinant protein experiment study functions of FvTrihelix6, so we kept this section (Line 549-550).
Point 17: The English language of the manuscript must be improved. There are errors in sentence structure and choice of words that make the sentences ambiguous. The authors use simple and short sentences in several places. The sentences are better combined to form complex sentences. Please standardize the use of short form. For example, Trihelix TF, trihelix genes, and TTF have been used interchangeably. TTF, among other short forms, was not written in full when it was first mentioned. Please check the whole manuscript. In other cases, the short form was written in full again.
Response: Thank you for your comment. We have improved the language of the manuscript.
Author Response File: Author Response.docx
Round 2
Reviewer 3 Report
I just have one follow-up comment on the recombinant protein expression. I understand that the authors have carried out the experiment and feel like they need to retain it. But, the authors did not clearly justify it in their response file. Referring to the first sentence, 'To further study the function of FvTrihelix6, we expressed it in the prokaryotic expression system'. Can you explain how the successful expression of the recombinant protein in E.coli inform the protein function? The protein under study is a eukaryotic transcription factor. One must express it in plants and observe the binding with the target sequence or alternatively carry out a yeast one-hybrid experiment to confirm the role of a transcription factor. In my professional opinion, since it is an incomplete experiment and as I commented before, did not answer any research question or achieve the intended objective (to study the function), there is no justification to include it in the current manuscript. The authors can complete the experiment and include it in another manuscript.
The English language is satisfactory.
Author Response
Dear Editor,
Thank you for giving us the opportunity to re-submit a revised manuscript “Expression Analysis of Trihelix Transcription Factor Family in Strawberry, and Functional Characterization of FvTrihelix6” for publication in the Horticulturae Journal.
We appreciate the time and effort that you and the reviewers dedicated to providing feedback on our manuscript and are grateful for the insightful comments on and valuable improvements to our paper. We have incorporated the suggestions made by the reviewers. Those changes are markered within the manuscript. Please see below, for a point-by-point response to the reviewer’s comments and concerns. We have corrected all the grammatical and contextual mistakes. We would be happy to consider any further changes that you deem appropriate and look forward to working with the editorial staff through the final submission process.
Thank you again, and best regards,
Dr. Zhifeng Wen
College of Horticulture,
Fujian Agriculture and Forestry University
Response to Reviewer 3 Comments
Point 1: I just have one follow-up comment on the recombinant protein expression. I understand that the authors have carried out the experiment and feel like they need to retain it. But, the authors did not clearly justify it in their response file. Referring to the first sentence, 'To further study the function of FvTrihelix6, we expressed it in the prokaryotic expression system'. Can you explain how the successful expression of the recombinant protein in E.coli inform the protein function? The protein under study is a eukaryotic transcription factor. One must express it in plants and observe the binding with the target sequence or alternatively carry out a yeast one-hybrid experiment to confirm the role of a transcription factor. In my professional opinion, since it is an incomplete experiment and as I commented before, did not answer any research question or achieve the intended objective (to study the function), there is no justification to include it in the current manuscript. The authors can complete the experiment and include it in another manuscript.
Response: Thank you for your suggestion and we have deleted the part of the recombinant prokaryotic protein experiment.
