Genome-Wide Identification and Analysis of the Nuclear Factor Y Gene Family in the Woodland Strawberry Fragaria vesca
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript describes a detailed bioinformatic analysis of the NF-Y gene 2 family in woodland strawberry Fragaria vesca. The almost exclusive in silico approach must be included in the title, such as "In silico genome-wide identification and analysis of the NF-Y gene 2 family in woodland strawberry Fragaria vesca". Analytical bioinformatic tools are appropriatte and deeply used. However I found some problems with the wet lab assay. For instance: "We amplified and cloned the coding sequences of FveNF-YA3, FveNF-YA4, and 154 FveNF-YC5 into the GFP::pCAMBIA 1302 vector", which was the source plant material fro amplifying and cloning? Then, "...the Agrobacterium culture was resuspended in an infiltration 158 medium and then introduced into the leaves of Nicotiana benthamiana." is stated in Material and Methods but "...we further explored this phenomenon in sweet 351 potato by transiently expressing FvNF-YA3, FvNF-YA4, and FvNF-YC5 proteins as GFP 352 fusion constructs" is mentioned in Results (though tobacco is informed again in Figure 1.
Comments on the Quality of English LanguageRevision by native English is reuqired.
Author Response
Comments 1: The manuscript describes a detailed bioinformatic analysis of the NF-Y gene 2 family in woodland strawberry Fragaria vesca. The almost exclusive in silico approach must be included in the title, such as "In silico genome-wide identification and analysis of the NF-Y gene 2 family in woodland strawberry Fragaria vesca". Analytical bioinformatic tools are appropriatte and deeply used. However I found some problems with the wet lab assay. For instance: "We amplified and cloned the coding sequences of FveNF-YA3, FveNF-YA4, and 154 FveNF-YC5 into the GFP::pCAMBIA 1302 vector", which was the source plant material fro amplifying and cloning? Then, "...the Agrobacterium culture was resuspended in an infiltration 158 medium and then introduced into the leaves of Nicotiana benthamiana." is stated in Material and Methods but "...we further explored this phenomenon in sweet 351 potato by transiently expressing FvNF-YA3, FvNF-YA4, and FvNF-YC5 proteins as GFP 352 fusion constructs" is mentioned in Results (though tobacco is informed again in Figure 1.
Response 1: Thank you for your insightful comments and suggestions. The strawberry served as the source plant material for amplification and cloning. We regret the error in our manuscript where we mistakenly referred to strawberries as sweet potatoes. This oversight has been corrected throughout the manuscript (Sections 2.9 and 3.9) to ensure accuracy. We appreciate your attention to detail in pointing this out.
Reviewer 2 Report
Comments and Suggestions for AuthorsReview report: Horticulturae
Yang Zhou, Feiyan Gao, Wenjie Zhao, Tianjia Liu*, and Mengzhao Wang*
Genome-wide identification and analysis of the NF-Y gene family in woodland strawberry Fragaria vesca
The manuscript provides a comprehensive genomic and functional analysis of the Nuclear Factor Y (NF-Y) transcription factors in woodland strawberry, Fragaria vesca. This study is significant as it expands our understanding of the NF-Y family, which plays crucial roles in plant development and stress responses. The authors have successfully identified and characterized multiple NF-Y genes, presenting detailed phylogenetic relationships, gene structures, chromosomal locations, and expression profiles.
- The manuscript is well-structured and generally clear. However, the discussion could be expanded to compare these findings with other species more thoroughly to highlight the unique or common features of NF-Y genes in F. vesca. Thus, is suggest incorporating a comparative analysis with NF-Y genes in other well-studied species, such as Arabidopsis thaliana and rice, to provide deeper evolutionary insights. This could be supported by references such as Swain et al., 2017, which discusses NF-Y genes in Arabidopsis.
Suggested reference: Swain, S., Myers, Z. A., Siriwardana, C. L., & Holt III, B. F. (2017). The multifaceted roles of NUCLEAR FACTOR-Y in Arabidopsis thaliana development and stress responses. Biochimica et Biophysica Acta (BBA)-Gene Regulatory Mechanisms, 1860(5), 636-644. https://doi.org/10.1016/j.bbagrm.2016.10.012
- The figures are informative and enhance the understanding of the text. Nonetheless, some figures, particularly those representing phylogenetic trees and gene structures, are dense and may be challenging to visualize. I suggest adding these figures in their original quality as supplementary file.
- The manuscript does not clearly state the statistical methods (e.g., cutoff values for motif significance) used to analyze expression data. Please add more details on the statistical tests adopted in this study.
- The methods used for the identification and analysis of NF-Y genes are rigorous and appropriate. However, the manuscript could benefit from additional functional experiments, such as gene knockdown or overexpression, to validate the roles of these genes in stress responses or development. The authors must add future perspectives that should include functional validation using CRISPR/Cas9 or RNAi to strengthen the conclusions about the roles of NF-Y genes in stress response and development.
This manuscript is a valuable addition to the field of plant molecular genetics and offers significant insights into the NF-Y gene family in woodland strawberry. With the suggested revisions, particularly those related to expanding the discussion and including functional validation, this paper will be a robust contribution to the literature.
Comments on the Quality of English LanguageMinor editing of English language required
Author Response
Please see the attachment.
Author Response File: 
Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsDear authors,
Hope you are doing well.
After reviewing your manuscript, I believe it is of high interest to learn more about the evolution of the NF-Y gene family in woodland strawberry. However, I believe that the manuscript needs a thorough revision and the correction of some points that I indicate below.
Major concerns
1. From my point of view, I think that some parts of the manuscript should be written in the passive voice. The use of personal pronouns such as "we" should be avoided.
2. Supplementary information is mentioned but is not accessible. (lines 155, 182, 293,…).
3. “To determine the relationship between FveNF-Y proteins and AtNF-Y proteins, we performed a phylogenetic analysis using NF-Y genes from both strawberry and Arabidopsis thaliana.” I think that the study of phylogenetic relationships between proteins using gene sequences is not correct. Is it possible that the sentence was incorrectly written? Lines 185-187, this part is well explained. Please, review this part through the manuscript.
4. 3.3 Prediction of miRNA target site of FveNF-Y genes. I suggest including a supplementary table with miRNA names or expand the Table 1 with this information for each gene.
5. Sometimes the authors refer to other works in the results section. For instance, lines 327-329, 178-179, 348-350… I think this needs to be corrected, as that should be part of the discussion. In the results, you should only focus on describing your observations. Reference should only be made to other works on very specific occasions.
6. There is a lack of information on the methodology used to perform gene expression analyses.
7. The description of the expression analysis should be improved. For example, NF-YB genes appear to be more expressed in all tissues. That is, they seem to have ubiquitous expression, while others such as NF-YB10 and NF-YB11 are less expressed. Authors should identify particular patterns of expression to later discuss them in the discussion section.
8. Discussion should be improved. Authors did not include discussion about cis elements, miRNAs, domains and motifs, … This is one of the most important parts of the manuscript.
9. This previous research should be considered for inclusion in the manuscript, either in the introduction or in the discussion: https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2022.1009747/full
10. Conclusions. Lines 435-438. This sentence is very general. I think I should go a little deeper. Perhaps, divide it into: 1. Conservation and evaluation of gene and protein sequences (including gene structure, synteny, phylogeny, cis elements,...); 2. miRNA targets; 3. Gene expression. It is not necessary to expand, but to make clear the main findings and biological implications.
Minor concerns
11. Line 16. “FveNF-Ys in Fragaria vesca” I suggest change it by “NF-Ys in Fragaria vesca”.
12. Line 17. My recommendation is that you use “woodland strawberry” or “F. vesca” to avoid confusion with the commercial strawberry.
13. Line 60-61. Please, change “seed morphology, seed germination.” by “seed morphology and germination.”
14. Paragraph 45-65 is focused on the role of NF-Ys in growth and development, while paragraph 66-85 is focused on the role of this transcription factors in stress responses. I suggest that this be reflected at the beginning of the paragraphs. For example, for the first paragraph: “Plant NF-Y complexes, as well as individual members of each NF-Y subunit family, play important roles in regulating various growth and development processes. For instance, in Arabidopsis, an NF-Y complex…” For the second paragraph: “NF-Y proteins have been also identified as crucial regulators of stress tolerance. For example..”.
15. Line 91. “stages” by “developmental stages”.
16. Line 93. “strawberry” by “F. vesca” or “woodland strawberry”.
17. Line 97. I suggest change this sentence “In order to identify NF-Y genes in the F. vesca genome, we employed a combination of three methods.” by “In order to identify NF-Y genes in the F. vesca genome, three methods were applied sequentially.
18. Line 127. “gene” by “genes”.
19. miRNA target site prediction. I think it should be mentioned briefly in the abstract.
20. Please, include access date for each web.
21. FveNF-YA1 is not showed in Table 1.
22. Lines 178-180. This information is part of the discussion. Please, to remove from this section.
23. Figure 1. Bootstrap values can not be displayed.
24. Line 219-220. I suggest being very careful with assertions. Functional studies are needed to verify this.
25. Figure 4. I recommend expanding the synteny graph. The same for Figure 5.
26. Lines 336-338. This is part of the discussion.
27. Line 343. “An expression profile…” by “Expression profile…”.
28. Lines 403-410. I think that some references are lacking.
29. Line 434. 21 or 23 NF-Y transcription factors?
Best regards,
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Reviewer 4 Report
Comments and Suggestions for AuthorsThe article is suitable for publication in the journal in question. Meets the journal's standards regarding formatting, language and scientific rigor. With some revisions, it will be an excellent contribution to the field of plant genomics and molecular biology.
- The title is clear and informative, giving a good indication of the study's focus on the NF-Y gene family in woodland strawberry Fragaria vesca.
- The abstract provides a comprehensive summary of the study, including objectives, methods, key findings, and implications. It effectively encapsulates the study's scope and significance.
Introduction
- The introduction is thorough and well-structured. It provides a detailed background on the NF-Y gene family, explaining their roles in plant development and stress responses. The introduction sets the context by highlighting the gap in research regarding the NF-Y gene family in strawberries.
- The introduction effectively establishes the relevance of the study, linking the importance of NF-Y genes to both fundamental plant biology and potential agricultural applications.
- The references are comprehensive, covering a wide range of relevant studies. They include seminal works and recent research, providing a solid foundation for the study.
- The references are formatted according to the journal’s guidelines. They are consistently presented, with all necessary details included.
- The references are well-integrated into the text, supporting the statements made in the introduction and discussion.
- Figures and tables are clear and appropriately labeled. They complement the text well and are referenced correctly within the text.
- The manuscript is well-written, with few grammatical errors. Sentences are clear and concise, effectively conveying the study’s findings and their significance.
- The manuscript flows well, with each section transitioning smoothly to the next. The argument is coherent, and the findings are presented in a logical order.
- The conclusion succinctly summarizes the key findings of the study and their implications. It reiterates the importance of the research and suggests potential future directions.
- The conclusion effectively ties the study’s findings back to the broader context outlined in the introduction.
Recommendations for Improvement
- While the manuscript is well-written, a few minor language edits could improve clarity. For example, simplifying complex sentences and ensuring consistent use of tense.
- Consider enhancing the clarity of figures and tables. High-quality visuals can significantly improve the reader’s understanding of the data.
Provide more detailed descriptions of certain methodological steps:
- The methodology mentions the use of NF-Y gene protein sequences from Arabidopsis as query sequences and the use of the BLASTP program with a cutoff value of e < 1e-5. However, it would be beneficial to specify the detailed criteria for selecting the query sequences. For instance: How were the 30 NF-Y gene sequences from Arabidopsis selected? Were all sequences considered, or only those with known higher functional relevance? What other parameters besides the cutoff value were used? Were additional filters applied, such as minimum alignment coverage or minimum sequence identity?
- Although the methodology mentions the use of MUSCLE software for protein alignment and MEGA11 for phylogenetic tree construction, specific details about the alignment parameters used in MUSCLE are not provided. For example: What specific parameters were used in MUSCLE for the alignment? Were default values used, or were specific adjustments made to optimize the alignment? In addition to the already mentioned parameters (Maximum Likelihood method, JTT model, Gamma distribution, partial deletion, and 1000 bootstrap replications), it would be useful to detail any other adjustments made in MEGA11 for phylogenetic tree construction.
- The methodology mentions the use of MEME-Suite to identify conserved motifs, but specific details about the parameters used in the analysis are lacking. For example: What parameters were set for the maximum number of motifs to be identified? Was a specific value used for motif length? What significance criteria were applied to accept a motif as conserved?
- Although MCScanX was used to analyze gene duplication events, details on the specific parameters used are missing. For example: What configuration parameters were used in MCScanX? Were there any changes to the default values to better fit the results to the strawberry genome specifics? How were syntenic regions between NF-Y genes of strawberry, Arabidopsis, and rice defined?
- The methodology mentions the use of the psRNATarget website for miRNA target site prediction but does not specify the parameters used. For instance: What parameters were set for the target site prediction? Were default values used, or were specific adjustments made? What criteria were used to consider a target site as valid? Was any additional filtering applied to eliminate false-positive predictions?
- The methodology mentions the use of PlantCARE for predicting cis-regulatory elements, but specific details are missing. For example: What parameters were set for the analysis? Was the entire 2 kb upstream region considered, or were additional criteria applied? How were the identified cis-regulatory elements categorized and interpreted? Was any filtering applied to select elements with higher functional relevance?
- Although the methodology mentions the use of ColabFold and GOR IV for protein structure prediction, specific details about the parameters used are lacking. For instance: What specific configurations were used in ColabFold for three-dimensional structure prediction? Were default parameters used, or were specific adjustments made? What parameters were set in the GOR IV algorithm for secondary structure prediction?
The discussion could be expanded to include a more in-depth comparison with similar studies in other plant species. This would help contextualize the findings further.
- The discussion could be enriched with a more detailed comparison of the results found in strawberries with similar studies in other plant species, such as *Arabidopsis thaliana*, rice, canola, and soybean. This would include an analysis of similarities and differences in the numbers of identified NF-Y genes, gene structures, conserved motifs, and expression patterns. It would be interesting to discuss how the conserved functions of NF-Y genes in different species (e.g., in seed development, stress tolerance) manifest in strawberries. Additionally, discussing any potentially new or specific function of strawberries identified in the study would be valuable.
- The discussion could delve deeper into the functional implications of the specific conserved motifs identified in each NF-Y gene subfamily. This includes discussing how these motifs may influence protein-protein interactions, DNA binding, and gene regulation. Comparing the conserved motifs found in strawberries with those in other species could provide insights into the functional evolution of NF-Y genes.
- A more detailed discussion of how post-transcriptional regulation by miRNAs can affect the expression and function of NF-Y genes in strawberries is warranted. For instance, how the presence of multiple miRNA target sites in certain genes might influence their regulation under different stress conditions or developmental phases. Comparing miRNA regulation of NF-Y genes in strawberries with other plants could provide insights into the conservation or divergence of this regulatory mechanism.
- Discuss how the cis-regulatory elements identified in the promoters of NF-Y genes might modulate gene expression in response to different environmental and hormonal signals. Include specific examples of elements and their known functions in other species. Addressing how knowledge of cis-regulatory elements and NF-Y gene regulation can be applied in breeding programs to develop strawberry varieties with improved stress resistance.
- Discuss in more detail the specific functions of NF-Y genes that show tissue-specific expression. For example, the elevated expression of FveNF-YB2 in mature pollen suggests a crucial role in pollen development. Relate the expression patterns of NF-Y genes to plant developmental processes such as embryogenesis, seed development, and organogenesis, with examples from other species to contextualize the findings.
- Discuss how the predicted secondary and three-dimensional structures of NF-Y proteins might influence their functions and interactions. This can include a discussion on the importance of α-helices, extended chains, and random coil regions in protein function. Comparing the predicted structures of NF-Y proteins in strawberries with known NF-Y protein structures in other species could provide insights into structural and functional conservation.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsAuthors have accepted most of my suggestions and the scientific quality of the manuscript is now acceptable.
Author Response
Comments 1: Authors have accepted most of my suggestions and the scientific quality of the manuscript is now acceptable.
Response 1: Thank you for your detailed review and constructive feedback. We are pleased to hear that you find the scientific quality of our manuscript acceptable following the revisions. Your suggestions have been invaluable in enhancing the overall quality of our work.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors responded to all of the comments on the previous version, so it can now be accepted for publication.
Comments on the Quality of English LanguageMinor editing of English language required
Author Response
Comments 1: The authors responded to all of the comments on the previous version, so it can now be accepted for publication.
Response 1: Thank you for your positive feedback and for accepting our manuscript for publication. We greatly appreciate your thorough review and valuable suggestions, which have significantly improved the quality of our work.
Reviewer 3 Report
Comments and Suggestions for AuthorsDear authors,
Thank you for taking my previous comments into account. I think the manuscript is much better and more informative. I am only going to ask you for a few changes that I indicate below.
1. Supplementary tables must be named and numbered in the same order as they appear in the text. For example, Table S4 (line 191) should be Table S1, Table S5 should be Table S2 (line 196), ...
2. Previous “Major concern 5”.
“5. Sometimes the authors refer to other works in the results section. For instance, lines 327-329, 178-179, 348-350… I think this needs to be corrected, as that should be part of the discussion. In the results, you should only focus on describing your observations. Referenceshould only be made to other works on very specific occasions.
Response: Thank you for the suggestion. References to other works (line 283, line 452…) in the resultssection have been moved to the discussion section, focusing the results solely on our observations.”
New comment: some changes are not reflected in the new version. Please, see [34] (line 282), [39] (line 292), [40] (line 302), [41] (line 303), [42] (line 306), … and review through the results section.
Best regards,
Author Response
Comments 1: Supplementary tables must be named and numbered in the same order as they appear in the text. For example, Table S4 (line 191) should be Table S1, Table S5 should be Table S2 (line 196), ...
Response 1: Thank you for suggestion. We have renamed and renumbered the supplementary tables to ensure they appear in the same order as referenced in the text. For example, Table S4 is now Table S1, and Table S5 is now Table S2.
Comments 2: Previous “Major concern 5”.
“5. Sometimes the authors refer to other works in the results section. For instance, lines 327-329, 178-179, 348-350… I think this needs to be corrected, as that should be part of the discussion. In the results, you should only focus on describing your observations. Referenceshould only be made to other works on very specific occasions.
Response: Thank you for the suggestion. References to other works (line 283, line 452…) in the resultssection have been moved to the discussion section, focusing the results solely on our observations.”
New comment: some changes are not reflected in the new version. Please, see [34] (line 282), [39] (line 292), [40] (line 302), [41] (line 303), [42] (line 306), … and review through the results section.
Response 2: We appreciate your guidance on this matter. We have moved references to other works from the results section to the discussion section to focus solely on our observations in the results.
Specifically, we have addressed citations such as [34] (line 282), [39] (line 292), [40] (line 302), [41] (line 303), [42] (line 306), among others, ensuring they are now appropriately placed in the discussion section. We have thoroughly reviewed the entire results section to ensure no references to other works remain.
