Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Comments for Authors:
General comments:

The manuscript horticulturae-3314513 has been checked for its suitability to Horticulturae Journal.  Originality of this manuscript is not clear. Introduction needs revision. Methods and results are easy to understand but discussion must be improved. This manuscript could be accepted with some corrections. Please response to the following specific comments:

Specific comments
L9, 28: TFs or TF? Please confirm

L29—32: please add on TFs more in details with more ref.

L44: “In recent years, an increasing number of trihelix genes have been cloned and characterized”-need ref. to support this info.

L46, 53, 61: check the format, it may be Maréchal et al. [12] and Shibata et al. [14]…….

L71-85:

(a)   This paragraph lacks any supporting references.

(b)   add more on watermelon importance in world perspective as well as China (where research conducted)

(c)   minimize the research gap- How do TFs associated to biotic and abiotic stress function in watermelon?

Methodology: Well described.

Results and Discussion: results well described but discussion should be improved.

Q1: Motif 1 was the most conserved and appeared in all trihelix genes, why

Q2: some genes showed significant differences in ex pression among different tissues. -why?

Q3: Why did Cla97C08G19147040 and Cla97C10G197980 genes only involve in the response to high temperature?

Author Response

Reponses to reviewer 1:

The manuscript horticulturae-3314513 has been checked for its suitability to Horticulturae Journal.  Originality of this manuscript is not clear. Introduction needs revision. Methods and results are easy to understand but discussion must be improved. This manuscript could be accepted with some corrections. Please response to the following specific comments:

Specific comments

Comment 1: L9, 28: TFs or TF? Please confirm

Response 1: Thank you for pointing this out. This sentence has been revised with transcription factors (TFs), L9, 28.

 

Comment 2: L29—32: please add on TFs more in details with more ref.

Response 2: We agree with this comment. More references have been listed in the main text and discussion.

 

Comment 3: L44: “In recent years, an increasing number of trihelix genes have been cloned and characterized”-need ref. to support this info.

Response 3: Thank you for the comments. The following sections list multiple examples of trihelix gene functions; please refer to lines 46-70 of the revised version for details.

 

Comment 4: L46, 53, 61: check the format, it may be Maréchal et al. [12] and Shibata et al. [14]…….

Response 4: Thank you for the suggestion. These sentences have been revised in the revised manuscript.

 

Comment 5: L71-85: (a)   This paragraph lacks any supporting references. (b)   add more on watermelon importance in world perspective as well as China (where research conducted). (c)   minimize the research gap- How do TFs associated to biotic and abiotic stress function in watermelon?

Response 5: Thank you for pointing this out. We have made the corresponding revisions, which can be seen in line 71-90.

 

Comment 6: Q1: Motif 1 was the most conserved and appeared in all trihelix genes, why

Response 6: Thank you for the comments. Motif 1 is highly functionally important and plays a central role in the trihelix gene family. This conservation could be due to its involvement in essential processes that are critical for the basic function of trihelix transcription factors across all members. The reason for its presence in all trihelix genes likely relates to the key biological functions these genes serve, such as regulating stress responses, growth, or development, which require conserved structural motifs to maintain their activity across diverse conditions. The conservation of Motif 1 may reflect its essential role in maintaining the structural integrity and functional activity of trihelix TFs, regardless of specific lineage or environmental adaptation.

 

Comment 7: Q2: some genes showed significant differences in expression among different tissues. -why?

Response 7: Thank you for the comments. In different tissue cells, gene expression patterns vary to a certain extent. Various cell types differ in their functions and properties due to the expression patterns of different genes that play an important role in regulating cell function and characteristics.

 

Comment 8: Q3: Why did Cla97C08G19147040 and Cla97C10G197980 genes only involve in the response to high temperature?

Response 8: Thank you for the comments. In plants, tissue-specific expression of transcription factor family genes is typically due to tissue-specific promoters, regulatory factors, and epigenetic modifications that influence gene expression patterns. In this article, these two genes exhibit tissue specificity, indicating their unique functions in specific tissues.

Reviewer 2 Report

Comments and Suggestions for Authors

Summary

This manuscript presents a genome-wide identification and analysis of trihelix genes in watermelon (Citrullus lanatus). This study identifies 29 trihelix genes in the watermelon genome, analyzes their physicochemical properties, chromosomal distribution, gene structure, and phylogenetic relationships. The authors also investigate the expression patterns of these genes in different tissues and under various biotic and abiotic stresses using transcriptome data. The research aims to provide a foundation for further functional studies of trihelix transcription factors in watermelon and contribute to molecular breeding efforts.

 

Major comments

While the study provides a comprehensive analysis of trihelix genes in watermelon, it lacks a clear explanation of how this research advances our understanding beyond what is already known about trihelix genes in other plant species. The authors should emphasize the unique aspects of their findings in watermelon and their potential implications for crop improvement.

The methods section lacks detail in some areas, particularly regarding the criteria used for identifying trihelix genes. The authors should provide more information on the specific parameters and thresholds used in their bioinformatic analyses to ensure reproducibility.

The manuscript presents a large amount of data, but the interpretation and discussion of the results could be more in-depth. The authors should provide more context for their findings, explaining how they compare to similar studies in other plant species and what new insights they offer about trihelix gene function in watermelon.

The study relies heavily on in silico analyses and expression data. While this provides valuable information, the lack of functional validation experiments limits the strength of the conclusions. The authors should consider including some preliminary functional studies or clearly state the need for such studies in future research.

 

Minor comments

Some figures, particularly the phylogenetic trees and heatmaps, may be difficult to read due to their complexity. The authors should consider simplifying these figures or providing higher resolution versions.

The naming convention for the identified trihelix genes is not clearly explained. A consistent and logical naming system should be adopted and explained in the manuscript.

The statistical methods used for the expression analysis should be more clearly described, including any normalization procedures and significance thresholds.

While generally well-written, there are some instances of awkward phrasing and grammatical errors throughout the manuscript. A thorough proofreading would improve the overall readability.

Consider moving some of the detailed data tables to supplementary materials to improve the flow of the main text.

 

Author Response

Reponses to reviewer 2:

This manuscript presents a genome-wide identification and analysis of trihelix genes in watermelon (Citrullus lanatus). This study identifies 29 trihelix genes in the watermelon genome, analyzes their physicochemical properties, chromosomal distribution, gene structure, and phylogenetic relationships. The authors also investigate the expression patterns of these genes in different tissues and under various biotic and abiotic stresses using transcriptome data. The research aims to provide a foundation for further functional studies of trihelix transcription factors in watermelon and contribute to molecular breeding efforts.

 

Major comments:

Comment 1: While the study provides a comprehensive analysis of trihelix genes in watermelon, it lacks a clear explanation of how this research advances our understanding beyond what is already known about trihelix genes in other plant species. The authors should emphasize the unique aspects of their findings in watermelon and their potential implications for crop improvement.

Response 1: Thank you for the suggestion. In the conclusion section, we discuss the specific roles of the trihelix family genes in watermelon, compare them to studies of trihelix genes in other species, and emphasize the uniqueness of the discovery of trihelix family genes in watermelon and their potential implications for crop improvement. For detailed information, please refer to lines 419-440 of the revised manuscript.

 

Comment 2: The methods section lacks detail in some areas, particularly regarding the criteria used for identifying trihelix genes. The authors should provide more information on the specific parameters and thresholds used in their bioinformatic analyses to ensure reproducibility.

Response 2: Thank you for the suggestion. We have provided information on the specific parameters and thresholds used in their bioinformatic analyses. We used the HMMER 3.0 software to search for the trihelix gene family using the Hidden Markov Model (HMM) file (PF02704), with the threshold set to E-value < 1 × 10^-5. For details, please refer to line 96 of the revised manuscript.

 

Comment 3: The manuscript presents a large amount of data, but the interpretation and discussion of the results could be more in-depth. The authors should provide more context for their findings, explaining how they compare to similar studies in other plant species and what new insights they offer about trihelix gene function in watermelon.

Response 3: Thank you for the suggestion. We have already made revisions in the conclusion section.

 

Comment 4: The study relies heavily on in silico analyses and expression data. While this provides valuable information, the lack of functional validation experiments limits the strength of the conclusions. The authors should consider including some preliminary functional studies or clearly state the need for such studies in future research.

Response 4: Thank you for the comments. Functional validation is our next key task, and we have added an explanation in the conclusion section.

 

Minor comments

Comment 5: Some figures, particularly the phylogenetic trees and heatmaps, may be difficult to read due to their complexity. The authors should consider simplifying these figures or providing higher resolution versions.

Response 5: Thank you for the comments. However, we believed that the figures in the manuscript are readable with the high quality. The reviewer could read it in the manuscript with word version (the figures are not compressed), but not PDF version (the figures are compressed). The figures in the manuscript (Word version) are clear.

 

Comment 6: The naming convention for the identified trihelix genes is not clearly explained. A consistent and logical naming system should be adopted and explained in the manuscript.

Response 6: Thank you for the suggestion. Those 29 trihelix genes were named ClGT1 to ClGT29 according to their order on the chromosomes. And we have revised at the results section and Table 2 in the manuscript.

 

Comment 7: The statistical methods used for the expression analysis should be more clearly described, including any normalization procedures and significance thresholds.

Response 7: Thank you for the comments. In the Materials and Methods section under "RNA-seq Re-analysis of Transcriptome Sequencing Data," we utilized StringTie for normalization and DESeq2 for differential expression gene analysis. For further details, please refer to lines 121-129 of the revised manuscript.

 

Comment 8: While generally well-written, there are some instances of awkward phrasing and grammatical errors throughout the manuscript. A thorough proofreading would improve the overall readability.

Response 8: Thank you for the suggestion. We have already polished the entire manuscript.

 

Comment 9: Consider moving some of the detailed data tables to supplementary materials to improve the flow of the main text.

Response 9: Thank you for your valuable comments on our manuscript. Regarding the suggestion to move some of the detailed data tables to the supplementary materials, after careful consideration, we believe that retaining these tables in the main text is crucial for presenting the research results in a complete manner. Moving the tables to the supplementary materials might affect the direct access and understanding of the data by the readers. Therefore, we have decided to keep these tables in the main text to ensure the clear communication of information and transparency of the research findings. However, we are open to optimizing the layout based on your further suggestions to improve the flow of the manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

 

Dear authors,

Hope you are doing well.

I find your manuscript interesting, but I think it needs some major changes to be understood by readers. Below I indicate my suggested changes focused on improving the manuscript.

Major concerns

·       Names of Trihelix genes. Because this gene family has not been previously reported in watermelon, I think it is a good opportunity for you to assign names to each of the genes. For example: ClGT-1 is Cla97C01G001230, ClGT-2 is Cla97C01G010480 and so on. This will make it easier for other researchers to review the information. Moreover, the manuscript can be read and interpreted more easily.

·       Introduction. Lines 44-47. I think this paragraph, as written, gives too much background on functional studies. From my point of view it gives a lot of detail about this. However, this is not a functionality study. Therefore, I think it is more correct to focus on the background on genomic information, protein domains, evolutionary relationships, gene subfamilies, expression under different types of stress, and so on.

·       Statistical information must be included for gene expression.

·       Protein motifs "The results of the motif prediction analysis indicated that with a limit of 195 10 motifs, none of the trihelix genes contained all motifs.” If there is no common motif/domain present in all proteins (TFs), how are they sure that they are trihelix TFs? Where is the helix-loop-helix-loop-helix domain? You should show a multiple alignment where the domain present in all proteins can be observed, since this should be highly conserved. This must be discussed.

·       Description of material and methods. Authors should be more specific with the description of materials and methods. For example, they do not give information about the type of plant used for stress treatments: in vitro plant? greenhouse plant? field? It is important to take this information into account to be able to compare your RT-qPCR results with previously published transcriptome analysis results.

·       Description of the results. Sometimes the results are poorly described. For example, in Figure 9, I think it is necessary to describe the figure in more detail, not just mention that they are correlated. That, of course, is important, but it is also important to highlight how the expression of each of the genes varies.

·       The discussion must be improved. All the elements are there but they seem to be disconnected. From my point of view, I think a common thread is needed. There is little discussion about the genomic part (number of genes, synteny, motifs and protein domains, etc.) and everything seems to be disconnected. Without a doubt this section should be improved.

·       Conclusion. I think this section replicates the end of the discussion, therefore, it should be improved.

Minor concerns

·       Line 41. Please, include this reference.

·       Material and methods. Web pages should be include the last date of access.

·       Material and methods. GSDS. Please, indicate what version of this software was used.

·       Line 140-145. I think this paragraph should be a separate subsection of RNA extraction and RT-qPCR analysis.

·       Please, change qRT-PCR by RT-qPCR.

·       Please indicate how many ug of total RNA were used for cDNA synthesis and indicate the amplicon size for each primers (Table 1).

·       Figure 1. What is the meaning of Cla97? All information within the figures, including abbreviations, should be explained in the legends to facilitate reading and understanding.

·       Line 179-189. You mention that there are several subfamilies but that does not appear in the introduction. It is important to include it.

·       Figure 2 legend. Pease, change cucumber by C. sativus.

·       Lines 234-235. It is not clear whether they refer to RNAseq or qPCR analyses. Please indicate it.

·       Figure 6. Please, explain what the color scale means, the numbers inside the rectangles, and so on. The same for other figures with color scale.

Best regards,

 

 

Author Response

Reponses to reviewer 3:

I find your manuscript interesting, but I think it needs some major changes to be understood by readers. Below I indicate my suggested changes focused on improving the manuscript.

Major concerns

Comment 1: Names of Trihelix genes. Because this gene family has not been previously reported in watermelon, I think it is a good opportunity for you to assign names to each of the genes. For example: ClGT-1 is Cla97C01G001230, ClGT-2 is Cla97C01G010480 and so on. This will make it easier for other researchers to review the information. Moreover, the manuscript can be read and interpreted more easily.

Response 1: Thank you for the suggestion. Those 29 trihelix genes were named ClGT1 to ClGT29 according to their order on the chromosomes. And we have revised at the results section and Table 2 in the manuscript.

 

Comment 2: Introduction. Lines 44-47. I think this paragraph, as written, gives too much background on functional studies. From my point of view it gives a lot of detail about this. However, this is not a functionality study. Therefore, I think it is more correct to focus on the background on genomic information, protein domains, evolutionary relationships, gene subfamilies, expression under different types of stress, and so on.

Response 2: Thank you for your suggestion. The background on genomic information, protein domains, evolutionary relationships, and gene subfamilies has already been discussed in the first two paragraphs. The core of the article focuses on the functional study of trihelix genes under stress, so this section emphasizes the background on functional research.

 

Comment 3: Statistical information must be included for gene expression.

Response 3: Thank you for the comments. The article focuses on the expression analysis of trihelix genes under stress and selects representative genes for RT-qPCR validation.

 

Comment 4: Protein motifs "The results of the motif prediction analysis indicated that with a limit of 195 10 motifs, none of the trihelix genes contained all motifs.” If there is no common motif/domain present in all proteins (TFs), how are they sure that they are trihelix TFs? Where is the helix-loop-helix-loop-helix domain? You should show a multiple alignment where the domain present in all proteins can be observed, since this should be highly conserved. This must be discussed.

Response 4: Thank you for the comments. Motif 1 was the most conserved and appeared in all trihelix genes. The analysis and explanation have already been provided in the article. L203-204.

 

Comment 5: Description of material and methods. Authors should be more specific with the description of materials and methods. For example, they do not give information about the type of plant used for stress treatments: in vitro plant? greenhouse plant? field? It is important to take this information into account to be able to compare your RT-qPCR results with previously published transcriptome analysis results.

Response 5: Thank you for your suggestion. We have already made revisions in the material and methods. To ensure consistency in the results, we conducted stress treatments on watermelon materials based on the methods used in previous transcriptomic studies. All treatments were carried out in a controlled climate chamber, as described in the article. L150-152.

 

Comment 6: Description of the results. Sometimes the results are poorly described. For example, in Figure 9, I think it is necessary to describe the figure in more detail, not just mention that they are correlated. That, of course, is important, but it is also important to highlight how the expression of each of the genes varies.

Response 6: Thank you for your suggestion. The description of Figure 9 is intended to validate the consistency of the results with the transcriptomic data. The expression values have already been detailed in the transcriptomic analysis, so further description here would be somewhat repetitive.

 

Comment 7: The discussion must be improved. All the elements are there but they seem to be disconnected. From my point of view, I think a common thread is needed. There is little discussion about the genomic part (number of genes, synteny, motifs and protein domains, etc.) and everything seems to be disconnected. Without a doubt this section should be improved.

Response 7: Thank you for your suggestion. We have rephrased the content of this section.

 

Comment 8: Conclusion. I think this section replicates the end of the discussion, therefore, it should be improved.

Response 8: Based on your suggestion, we have revised the conclusion section, as detailed in the manuscript.

 

Minor concerns

Comment 9: Line 41. Please, include this reference.

Response 9: Thank you for your suggestion. Relevant literature is already provided in line 41 of the manuscript.

 

Comment 10: Material and methods. Web pages should be include the last date of access.

Response 10: Thank you for your suggestion. We have updated the latest URL.

 

Comment 11: Material and methods. GSDS. Please, indicate what version of this software was used.

Response 11: Thank you for the comments. GSDS 2.0 was used. We have updated the latest URL.

 

Comment 12: Line 140-145. I think this paragraph should be a separate subsection of RNA extraction and RT-qPCR analysis.

Response 12: Thank you for your suggestion. We merged the content of these two sections into a separate subsection.

 

Comment 13: Please, change qRT-PCR by RT-qPCR.

Response 13: Thank you for your suggestion. We have made the changes.

 

Comment 14: Please indicate how many ug of total RNA were used for cDNA synthesis and indicate the amplicon size for each primers (Table 1).

Response 14: Thank you for the comments. 1 µg of total RNA was used for the reverse transcription reaction.

 

Comment 15: Figure 1. What is the meaning of Cla97? All information within the figures, including abbreviations, should be explained in the legends to facilitate reading and understanding.

Response 15: Thank you for the comments. Cla97 is the prefix for the watermelon gene ID, where "Cla" stands for the abbreviation of the watermelon's Latin name, Citrullus lanatus, and "97" represents the designation of the watermelon material 97103 used for genome sequencing.

 

Comment 16:

Line 179-189. You mention that there are several subfamilies but that does not appear in the introduction. It is important to include it.

Response 16: Thank you for your suggestion. We have highlighted the information about the subfamilies in the first paragraph of the discussion section.

 

Comment 17: Figure 2 legend. Pease, change cucumber by C. sativus.

Response 17: Thank you for your suggestion. We have made the replacement.

 

Comment 18: Lines 234-235. It is not clear whether they refer to RNAseq or qPCR analyses. Please indicate it.

Response 18: Thank you for your suggestion. The results here are based on RNA-seq, which we have explained in the text.

 

Comment 19: Figure 6. Please, explain what the color scale means, the numbers inside the rectangles, and so on. The same for other figures with color scale.

Response 19: Thank you for your suggestion. We have provided explanations for the relevant Figure in the text. In each figure,the color scale represents the level of expression, with red indicating high expression, and the deeper the color, the higher the expression. Blue represents low expression, and the deeper the color, the lower the expression. the number in the table represent the log2(FPKM+1) values.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Authors have successfully revised their manuscript according to reviewers' comments. 

I recommend the manuscript for publication as it is. 

Congratulations on the excellent work!"

Author Response

Comment 1: Authors have successfully revised their manuscript according to reviewers' comments.

I recommend the manuscript for publication as it is.

Congratulations on the excellent work!"

Response 1: Thank you very much for your positive feedback and for recommending our manuscript for publication. We are delighted to hear that you found our revisions satisfactory and that you consider our work to be of high quality. Your support and encouragement mean a great deal to us, and we are grateful for the time and effort you have invested in reviewing our manuscript.

We look forward to contributing to the field with our research and are excited about the prospect of sharing our findings with the broader scientific community.

Once again, thank you for your kind words and for your valuable input throughout the review process.

Reviewer 3 Report

Comments and Suggestions for Authors

Dear authors,

Hope you are doing well.

Thank you for making the suggested changes.

First of all, I would like to make a clarification about the study, as it has been designed and carried out. From my point of view, this is not a functional study (although this can always be discussed), rather it is a characterization study of the Trihelix gene family in watermelon. When functional studies are carried out, that is, the role of one of the genes in a certain process is demonstrated (for example, abiotic stress), additional experiments are carried out. Among these experiments are cloning and overexpression studies in vivo, silencing of genes, and so on.

I would like to insist that you include an image of an alignment that shows the domino, where the conserved amino acid residues of the domain are observed. This is important, since the conservation of the amino acid sequence in transcription factors will determine their structure and, therefore, their function. If you prefer, you can include it as a supplementary figure.

Regarding this previous comment “Comment 10: Material and methods. Web pages should be include the last date of access.”, I can't see the changes. I don’t see the date of last access.

Best regards,

Author Response

Comment 1: I would like to insist that you include an image of an alignment that shows the domino, where the conserved amino acid residues of the domain are observed. This is important, since the conservation of the amino acid sequence in transcription factors will determine their structure and, therefore, their function. If you prefer, you can include it as a supplementary figure.

Response 1: Thank you for your valuable suggestion. We have included the sequence alignment diagram of the conserved domains as supplementary material and provided a detailed description of the results in Lines 166-168 of the revised manuscript.

 

Comment 2: Regarding this previous comment “Comment 10: Material and methods. Web pages should be include the last date of access.”, I can't see the changes. I don’t see the date of last access.

Response 2: We sincerely apologize for the oversight in our initial submission. In the revised manuscript, we have now added access dates for all web-based resources cited throughout the text.