Genome-Wide Identification and Abiotic Stress Response Analysis of C2H2 Zinc Finger Protein Genes in Foxtail Millet (Setaria italica)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript by Qian Zhao et al. entitled ‘Genome-wide identification and abiotic stress response analysis of C2H2 zinc finger protein genes in foxtail millet (Setaria italica)’ is well written, clearly presented and does make a valuable contribution to the role of an important family of transcription factors in stress responses. It also considers applied aspects of the work, e.g. in agriculture. 
I have two points that the authors should consider.
1: The authors mention ploidy (and hence genome duplication) (line 247) and potential effects of this process on the enlargement of the C2H2-TF family. I think it would be most useful to refer to a landmark paper entitled ‘Genome duplication led to highly selective expansion of the Arabidopsis thaliana proteome’ (Trends Genet. (2004) 20, 461-464; doi: 10.1016/j.tig.2004.07.008). Essentially, the paper refers to important considerations of gene dosage and proposes that the expansion of the ‘regulatory capacity’ of organisms (e.g. through transcription regulatory mechanisms) can confer increased fitness. It also strengthens the arguments put forward by the authors and adds a mechanistic dimension.
2: I think referring to genes that can improve stress tolerance - particularly in an agricultural context - maybe a bit misleading not least because over-expressed genes often have negative effects on the gene balance and may incur significant costs. I think, ultimately it will be interesting to observe and describe natural millet variations and see if more drought resistant strains show different expression patters of C2H2 TFs.  

Minor consideration: I am not sure if the type of paper is really and “Article” rather than a e.g. “Perspective” or a “Review” since it does not, strictly speaking offer new data. 

Author Response

1. The authors mention ploidy (and hence genome duplication) (line 247) and potential effects of this process on the enlargement of the C2H2-TF family. I think it would be most useful to refer to a landmark paper entitled ‘Genome duplication led to highly selective expansion of the Arabidopsis thaliana proteome’ (Trends Genet. (2004) 20, 461-464; doi: 10.1016/j.tig.2004.07.008). Essentially, the paper refers to important considerations of gene dosage and proposes that the expansion of the ‘regulatory capacity’ of organisms (e.g. through transcription regulatory mechanisms) can confer increased fitness. It also strengthens the arguments put forward by the authors and adds a mechanistic dimension.

Response

-- We sincerely appreciate the reviewer for the valuable suggestion.

-- In response to your recommendation, we have cited the paper ‘Genome duplication led to highly selective expansion of the Arabidopsis thaliana proteome’ (Trends Genet.. (2004) 20, 461-464; doi: 10.1016/j.tig.2004.07.008) in the relevant content of line 247. This citation not only deepens the discussion on gene dosage effects and the mechanisms of regulatory capacity expansion in organisms but also further strengthens our argument regarding the impact of polyploidization on the expansion of the C2H2-TF family.

2. I think referring to genes that can improve stress tolerance - particularly in an agricultural context - maybe a bit misleading not least because over-expressed genes often have negative effects on the gene balance and may incur significant costs. I think, ultimately it will be interesting to observe and describe natural millet variations and see if more drought resistant strains show different expression patters of C2H2 TFs.  

Response

-- We sincerely appreciate the reviewer for the valuable suggestion.

-- “In response to your recommendation, we have revised the relevant content by changing the original phrase “improving stress tolerance” to “exploring stress tolerance regulatory mechanisms” to avoid overinterpretation of gene function applications and to focus on fundamental mechanism analysis.”

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors

Very interesting study. I consider after some minor revision that can see in the attached file, this submission might be accepted for publication.

Comments for author File: Comments.pdf

Author Response

thanks for review report.

Reviewer 3 Report

Comments and Suggestions for Authors

The Zhao et al. manuscript offers significant insights into the characterization of the C2H2-ZFP2 gene family and its role in enhancing abiotic stress resilience in Setaria italica. The work is more focused on in silico analysis and which also encloses a part of wet lab research. The manuscript is written according to journal’s aims and scope; applied experimentation is based on latest standards. The manuscript is title-focused and it covers all potential aspects of the gene family corresponding available information from plant model organism (A. thaliana) to various field crops resulting applied conclusions. A holistic approach for the functionality of the SiC2H2-ZFP2 gene family is provided in the manuscript. Findings are well discussed with other plant species homolog data.  The manuscript represents a good piece of research work; however, it does not follow the guidelines of the journal’s template [for example, introduction is NOT followed by materials and methods section]. Please check the template file of the journal https://www.mdpi.com/files/word-templates/agronomy-template.dot  for providing relative changes.

Author Response

1. The Zhao et al. manuscript offers significant insights into the characterization of the C2H2-ZFP2 gene family and its role in enhancing abiotic stress resilience in Setaria italica. The work is more focused on in silicoanalysis and which also encloses a part of wet lab research. The manuscript is written according to journal’s aims and scope; applied experimentation is based on latest standards. The manuscript is title-focused and it covers all potential aspects of the gene family corresponding available information from plant model organism (A. thaliana) to various field crops resulting applied conclusions. A holistic approach for the functionality of the SiC2H2-ZFP2 gene family is provided in the manuscript. Findings are well discussed with other plant species homolog data.  The manuscript represents a good piece of research work; however, it does not follow the guidelines of the journal’s template [for example, introduction is NOT followed by materials and methods section]. Please check the template file of the journal (https://www.mdpi.com/files/word-templates/agronomy-template.dot) for providing relative changes.

Response

-- Thank you for your valuable suggestions on our manuscript!

-- We have systematically adjusted the formatting of the entire manuscript in strict accordance with the Agronomy journal template (https://www.mdpi.com/files/word-templates/agronomy-template.dot). The specific revisions are as follows: The “Materials and Methods” section has been relocated to follow the “Introduction”, ensuring compliance with the journal's required standard structure: “Introduction → Materials and Methods → Results → Discussion → Conclusion”.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

Dear authors,

The work is very well-prepared, and the obtained results are convincing. I have some comments:

1. Fig. 2A should include the C2H2-ZFPs gene counts (67 genes) of foxtail millet (Setaria italica), as it is the subject used in this study.
2. The notation legend entry (ten motifs 1-10) of Figure 3B should be relocated next to its Figure.
3. Please provide an annotation for the legend entry of Fig.8, labeling as S24, D24. For example, S24: salt stress treatment (150 mmol/L NaCl) for 24h. Additionally, remove labels (A, B) that were listed in Fig. 8’s legend. Then rewrite the Fig.8’s legend:

“Figure 8. Expression levels of the SiC2H2 gene in foxtail millet (Yugu 56). A. Expression levels of the SiC2H2 gene under salt stress in foxtail millet (Yugu 56). B. Expression levels of the SiC2H2 gene under drought stress in foxtail millet (Yugu 56).”

For example, it could be rewritten as: “Figure 8. Expression levels of the SiC2H2 gene under salt and drought stress in foxtail millet (Yugu 56).”

4. Special attention needs to be paid to the genes and proteins mentioned in the study, using italics to differentiate genes from proteins. For gene, it is presented in italics. For instance, C2H2-ZFPs (Lines 130, 265, 307), C2H2 (Lines 136, 137), SiC2H2 (Line 169, 195, 280, 289, 436) denote for gene; therefore, they should be presented in italics. Please double-check the whole manuscript and revise it.
5. Requirements for paper formatting: Capitalize Each Word for the title and subsections.

- For title: “Genome-wide identification and abiotic stress response analysis of C2H2 zinc finger protein genes in foxtail millet (Setaria italica)”

→ Genome-wide Identification and Abiotic Stress Response Analysis of C2H2 Zinc Finger Protein Genes in Foxtail Millet (Setaria italica)”

- For headings:

For example, “2.1. Genomic identification, chromosomal localization, and physicochemical characterization of C2H2-ZFPs gene family members in foxtail millet”

→“2.1. Genomic Identification, Chromosomal Localization, and Physicochemical Characterization of C2H2-ZFPs Gene Family Members in Foxtail Millet”

- References section, authors have presented following the journal format [Author 1, A.B.; Author 2, C.D. Title of the article. Abbreviated Journal Name Year, Volume, page range.]. The volume should be italicized.

For example,

1. Liu, Y.; Khan, A.R.; Gan, Y. C2H2 zinc finger proteins response to abiotic stress in plants. Int. J. Mol. Sci. 2022, 23, e2730.

→ 1.  Liu, Y.; Khan, A.R.; Gan, Y. C2H2 zinc finger proteins response to abiotic stress in plants. Int. J. Mol. Sci. 2022, 23, e2730.  

6. Consider representing the manuscript as journal format: 1. Introduction → 2. Materials and Methods → 3. Results → 4. Discussion → 5. Conclusions
7. Revise: “responsively” (L155) → “respectively”

I have marked by highlighting the above comments on the manuscript. Please use it for easy tracking and revision.

With regards,

Comments for author File: Comments.zip

Author Response

1. Fig. 2A should include the C2H2-ZFPs gene counts (67 genes) of foxtail millet (Setaria italica), as it is the subject used in this study.

Response

-- Thank you for your valuable suggestions.

-- As requested, the C2H2-ZFPs gene count (67 genes) of foxtail millet (Setaria italica) has been added to Fig. 2A. This revision clarifies the core subject of the study, ensuring consistency between the figure and the main text. The updated legend now includes the annotation, facilitating readers' understanding of the research scope. Thank you again for your guidance in enhancing the manuscript's rigor.

2. The notation legend entry (ten motifs 1-10) of Figure 3B should be relocated next to its Figure.

Response

-- Thank you for your valuable suggestions.

-- The notation legend entry (ten motifs 1-10) of Figure 3B has been relocated adjacent to the figure, ensuring that the legend corresponds to the graphical content. The revised legend is formatted to enhance readability, allowing readers to easily interpret the distribution of each motif.

3. Please provide an annotation for the legend entry of Fig.8, labeling as S24, D24. For example, S24: salt stress treatment (150 mmol/L NaCl) for 24h. Additionally, remove labels (A, B) that were listed in Fig. 8’s legend. Then rewrite the Fig.8’s legend: “Figure 8. Expression levels of the SiC2H2 gene in foxtail millet (Yugu 56). A. Expression levels of theSiC2H2 gene under salt stress in foxtail millet (Yugu 56). B. Expression levels of the SiC2H2 gene under drought stress in foxtail millet (Yugu 56).” For example, it could be rewritten as: “Figure 8. Expression levels of the SiC2H2 gene under salt and drought stress in foxtail millet (Yugu 56).”

Response

-- Thank you for your valuable suggestions.

-- The legend of Fig. 8 has been revised as requested, with annotations added for S24 and D24 (“S24: Salt stress treatment (150 mmol/L NaCl) for 24 h; D24: Drought stress treatment (20% PEG-6000) for 24 h”) and the original labels (A, B) removed. The title is now revised to “Figure 8. Expression levels of the SiC2H2 gene under salt and drought stress in foxtail millet (Yugu 56)”, integrating stress treatment conditions with gene expression data to enhance the figure's comprehensiveness and readability.

4. Special attention needs to be paid to the genes and proteins mentioned in the study, using italics to differentiate genes from proteins. For gene, it is presented in italics. For instance, C2H2-ZFPs (Lines 130, 265, 307), C2H2 (Lines 136, 137), SiC2H2 (Line 169, 195, 280, 289, 436) denote for gene; therefore, they should be presented in italics. Please double-check the whole manuscript and revise it.

Response

-- Thank you for your valuable suggestions.

-- We have added italics to the gene names you specified, including C2H2-ZFPs (Lines 130, 265, 307), C2H2 (Lines 136, 137), and SiC2H2 (Lines 169, 195, 280, 289, 436), and used a text search function to uniformly italicize all gene symbols (such as SiC2H2-1, C2H2-TF, etc.) throughout the manuscript while keeping protein names in roman type. Additionally, a new annotation "Gene names are italicized (e.g., SiC2H2), while protein names are in roman type (e.g., SiC2H2 protein)" has been added to the "Materials and Methods" section to ensure formatting consistency. These revisions have significantly enhanced the academic rigor of the paper, and we thank you again for your meticulous guidance!

5. Requirements for paper formatting: Capitalize Each Word for the title and subsections.

- For title: “Genome-wide identification and abiotic stress response analysis of C2H2 zinc finger protein genes in foxtail millet (Setaria italica)”

→ Genome-wide Identification and Abiotic Stress Response Analysis of C2H2 Zinc Finger Protein Genes in Foxtail Millet (Setaria italica)”

- For headings:

For example, “2.1. Genomic identification, chromosomal localization, and physicochemical characterization of C2H2-ZFPs gene family members in foxtail millet”

→“2.1. Genomic Identification, Chromosomal Localization, and Physicochemical Characterization of C2H2-ZFPs Gene Family Members in Foxtail Millet”

- References section, authors have presented following the journal format [Author 1, A.B.; Author 2, C.D. Title of the article. Abbreviated Journal Name Year, Volume, page range.]. The volume should be italicized.

For example,

1. Liu, Y.; Khan, A.R.; Gan, Y. C2H2 zinc finger proteins response to abiotic stress in plants. Int. J. Mol. Sci. 2022, 23, e2730.

→ 1.  Liu, Y.; Khan, A.R.; Gan, Y. C2H2 zinc finger proteins response to abiotic stress in plants. Int. J. Mol. Sci. 2022, 23, e2730. 

6. Consider representing the manuscript as journal format: 1. Introduction → 2. Materials and Methods → 3. Results → 4. Discussion → 5. Conclusions
7. Revise: “responsively” (L155) → “respectively”

Response

-- Thank you for your valuable suggestions.

-- The following revisions have been made:

1. Title and subsection headers:Adjusted to "Capitalize Each Word" format, e.g., the original title “Genome-wide identification...” revised to “Genome-wide Identification...”, with all subheadings (e.g., “2.1 Genomic identification...”) similarly updated to “2.1 Genomic Identification...” to ensure capitalization of major terms;
2. Reference formatting: Volume numbers italicized uniformly, e.g., "Int. J. Mol. Sci. 2022, 23, e2730" corrected to "Int. J. Mol. Sci. 2022, 23, e2730", in strict compliance with the journal's style;
3. Manuscript structure: Reorganized as "1. Introduction → 2. Materials and Methods → 3. Results → 4. Discussion → 5. Conclusions", aligning with the journal template;
4. Word correction: "Responsively" in Line 155 revised to "respectively" for lexical accuracy.

Author Response File: Author Response.pdf

Reviewer 5 Report

Comments and Suggestions for Authors

The manuscript contains quite interesting research results on the C2H2-32 ZFP family in foxtail millet (Setaria italica) and indicates potential genetic markers that can be used to improve plant resistance to abiotic stress.

I would like to ask the authors what is preventing their research results from being quickly used in the breeding of new varieties? What problems still need to be solved?

Comments

Results

The figures and tables are necessary and legible.

Table 1. I suggest removing the information about Subcellular location from the table and placing it below the table, which will further improve the readability of the table.

Similarly, in Table 2 with the Multiplicity information.

Discussion

It is appropriate.

Materials and Methods

When was the research conducted? Where?

Please provide a brief description of the millet variety studied.

Who was the producer of the statistical software used for data analysis?

References

Quite a large number of publications (58). I suggest focusing on the most recent publications (from the last 10 years) and removing the oldest ones.

Author Response

1. Table 1. I suggest removing the information about Subcellular location from the table and placing it below the table, which will further improve the readability of the table.Similarly, in Table 2 with the Multiplicity information.

Response

-- Thank you for your valuable suggestions.

-- We have strictly followed your comments to make the following revisions: In Table 1, the subcellular localization column was removed, and relevant information such as “SiC2H2-11 and SiC2H2-66 are localized in chloroplasts, while the rest are localized in the nuclear” was added as a footnote below the table. Meanwhile, in Table 2, the columns for “Inhibition” and “Multiplicity” were removed. These modifications have optimized the table layout through information stratification.

2. When was the research conducted? Where?

Response

-- This study was conducted from February to May 2025 at Jilin Agricultural University. The experimental phases are outlined as follows:

1. February–March 2025: Planting foxtail millet, followed by salt/drought stress treatment and sampling of five-leaf-stage seedlings;
2. March–April 2025: Completion of genomic data collection, bioinformatics analysis, and gene expression validation in foxtail millet;
3. April–May 2025: Data consolidation, manuscript drafting, and related work.

3. Please provide a brief description of the millet variety studied.

Response

-- Thank you for your comment. The foxtail millet variety used in this study is ‘Yugu 56’, which was bred by the Anyang Academy of Agricultural Sciences, Henan Province. This variety demonstrates good adaptability in the North China summer millet region, the northwest early-maturing region, and the northeast region. Field natural identification has shown that it possesses strong drought resistance, making it suitable for exploring drought-tolerance mechanisms in millet.

4. Who was the producer of the statistical software used for data analysis?

References

Response

-- Thank you for your inquiry. The producers of the statistical software used for data analysis have been supplemented as follows:

5. Quite a large number of publications (58). I suggest focusing on the most recent publications (from the last 10 years) and removing the oldest ones.

Response

-- Thank you for your suggestions regarding the references

-- We have made the following adjustments as per your advice: First, four older references were removed to enhance the timeliness of the bibliography. Some pre-2015 references, however, were retained because they describe core bioinformatics methods, which are essential methodological citations. Removing these would compromise the logical integrity of the study. The references now focus on research from the past decade while preserving key methodological foundations to ensure the rigor of the study background. Thank you again for your professional guidance!

Author Response File: Author Response.pdf