Genome-Wide Classification and Evolutionary Analysis of the KNOX Gene Family in Plants

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Thank you for considering me to revise the manuscript entitled “Genome-wide classification and evolutionary analysis of the KNOX gene family in plants”. The manuscript provides fairly robust comparative information on KNOX genes in higher and lower plants. I suggest accepting the manuscript following minor revisions.

Suggestions

The manuscript needs English editing, rephrasing long sentences, resuming paragraphs that contain less important information, and combining related sentences.

Abstract

The abstract lacks the main purpose of this study and more details on the applied methodology of the assessed 118 species.

Introduction

The introduction is well written but still needs to be reorganized and improved. The importance of exploring the KNOX gene family in diverse plant species, rationale, and objectives need more clarification.

Materials and Methods

More information should be added for the assessed eleven lower plants as well as the other 47 higher plants out of 107. Lines 91-103: The scientific names of evaluated  60 horticultural plants (23 fruit trees, 24 vegetables, and 13 other plants) could be presented in a Table. More details are needed on the applied methodology for the collected genome of studied plants.

Results

The results are well written. Supplementary Figure 1 provided benefits information that could be added to the main text. The result section should present the obtained results and citations (as in lines 182, 218, 227, 258, 278, 303) should be delayed to be presented in the discussion section.

Discussion

The discussion needs more improvement with additional literature exploring KNOX genes in higher and lower plants. The contributions and benefits of the obtained results should be better clarified.

Line 457: A. thaliana should be in italics as well as all scientific names throughout the manuscript.

Conclusion

The conclusion should be improved and presented in a separate section

References

The reference list needs to be carefully revised, the style of MDPI should be applied, author names should be presented in the same style, the journal should be abbreviated, and scientific names should be in italics throughout the manuscript (as Prunus persica in line 566).  

Comments on the Quality of English Language

Moderate editing of English language required

Author Response

Dear editors and reviewers:

We sincerely thank the editors and reviewers who generously donated their valuable time to provide insightful suggestions for our manuscript (Manuscript ID: horticulturae-2648202). We have meticulously incorporated and considered each suggestion and comment, as indicated by the red highlights in the revised text. In the subsequent sections, we offer a comprehensive response to the comments of each reviewer and outline the corresponding revisions that have been made. In addition, this manuscript has been edited for MDPI language editing (English-72859).

Sincerely,

Zhi-Meng Gan, Xiao-Yan Ai, Chun-Gen Hu* and Jin-Zhi Zhang*

E-mail: jinzhizhang@mail.hzau.edu.cn (J.-Z Z) (corresponding author)

 

To reviewer #1:

Comment：The manuscript provides fairly robust comparative information on KNOX genes in higher and lower plants. I suggest accepting the manuscript following minor revisions.

Response: We sincerely appreciate your thorough examination of our paper and the positive comments. Each piece of advice was carefully considered, and we provided detailed responses accordingly. We hope that our responses have met your expectations and addressed any concerns.

Suggestion: The manuscript needs English editing, rephrasing long sentences, resuming paragraphs that contain less important information, and combining related sentences.

Response: Thank you for your valuable feedback. We acknowledge that the manuscript requires English editing to enhance its readability. We have carefully rephrased long sentences, condensed paragraphs that contain less important information, and consolidated related sentences to improve the overall clarity and coherence of the manuscript. For details, please refer to the section highlighted in red on page 1 line 41, 42; page2 line 66, and so on. We appreciate your suggestions and will ensure that the final version is revised accordingly. In addition, this manuscript has been edited for MDPI language editing (English-72859).

Q1: The abstract lacks the main purpose of this study and more details on the applied methodology of the assessed 118 species.

Response: Thank you for pointing out the problem. In the statement of the study's purpose, we realized that the explanation was not concise and clear. In response to this issue, we have revised and refined the purpose of the study. The Knotted1-like homeobox (KNOX) gene family plays a pivotal role in regulating meristem activity, organ differentiation, and cell meristematic identity. However, there has been a lack of large-scale, systematic, and comprehensive comparative analyses to explore their expression patterns and evolutionary mechanisms. In this study, we comprehensively identified KNOX gene family members across 118 plant species. Subsequently, we conducted a range of analyses, including comparative analysis, gene duplication and loss analysis, conserved motif analysis, collinearity analysis, replication type identification, expression pattern analysis, and pan-genome analysis on KNOX genes. Our aim was to provide a more comprehensive dataset on the KNOX gene family, enabling large-scale, systematic, and comprehensive comparative analyses to explore their expression patterns and evolutionary mechanisms. For details, please refer to the section highlighted in red on page 1 line 11, 30.

Q2: The introduction is well written but still needs to be reorganized and improved. The importance of exploring the KNOX gene family in diverse plant species, rationale, and objectives need more clarification.

Response: Thank you very much for your valuable suggestions. We acknowledge that the statement regarding the importance of exploring the KNOX gene family in diverse plant species was too concise. In response to this issue, we have reorganized and improved the introduction. The functions of KNOX genes mainly include the maintenance of apical meristems, leaf morphogenesis, stem elongation, and floral organ development. We have provided additional information about the functional mechanisms of class I KNOX family genes in different species. For more details, please refer to the section highlighted in red on page 2, lines 75-85.

Q3: More information should be added for the assessed eleven lower plants as well as the other 47 higher plants out of 107. Lines 91-103: The scientific names of evaluated 60 horticultural plants (23 fruit trees, 24 vegetables, and 13 other plants) could be presented in a Table. More details are needed on the applied methodology for the collected genome of studied plants.

Response: Thank you for your suggestions. To facilitate readers' clear understanding of the classification of these species, we have divided the species into four levels: Rhodophyta; Chlorophyta; Basal plants and Land plants based on their evolutionary relationships (https://www.plabipd.de/). And 107 land plants were divided into basal angiosperms, monocots and eudicots and non-angiosperms plants. Furthermore, we have sorted them based on their evolutionary relationships and used color coding to distinguish them, including indicating whether they are horticultural plants in Table S1. For details, please refer to the section highlighted in red on page 2 line 117, 214, Figure1, Figure2 and Table S1.

Q4: The results are well written. Supplementary Figure 1 provided benefits information that could be added to the main text. The result section should present the obtained results and citations (as in lines 182, 218, 227, 258, 278, 303) should be delayed to be presented in the discussion section.

Response: We greatly appreciate your thorough review and suggestions. During the manuscript preparation, we also contemplated the inclusion of Figure S1 in the main text. However, due to significant discrepancies in the dimensions of Figure 1 and Figure S1, which could affect the readability of these images, we opted to keep them separate. Regarding lines 182, 218, and 258, they expound upon the critical aspects of our study concerning gene families, serving as a prelude to the presentation of results. I believe that placing them within the results section is also feasible.

Q5: The discussion needs more improvement with additional literature exploring KNOX genes in higher and lower plants. The contributions and benefits of the obtained results should be better

Response: Thank you for your suggestions, we agree with your ideas that the discussion should be improved. For detailed information, please refer to the section 4 highlighted in red on page 13 line 543-552, 568-573.

Q6: Line 457: A. thaliana should be in italics as well as all scientific names throughout the manuscript.

Response: Thank you for pointing out the error. We have carefully checked all the scientific names throughout the manuscript and have applied italics formatting to A. thaliana and all other scientific names. For more details, please refer to the section highlighted in red on page 2, lines 75-85.

Q7: The conclusion should be improved and presented in a separate section

Response: Thank you for your suggestion. We agree with your ideas that the conclusion should be improved and presented in a separate section. For detailed information, please refer to the section 5 highlighted in red on page 14 line 599-616.

Q8: The reference list needs to be carefully revised, the style of MDPI should be applied, author names should be presented in the same style, the journal should be abbreviated, and scientific names should be in italics throughout the manuscript (as Prunus persica in line 566). 

Response: Thank you for your suggestion. We will carefully revise the reference list to adhere to the formatting requirements of MDPI. We will also ensure consistency in the presentation of author names. Additionally, we will abbreviate journal names and use italics for scientific names throughout the manuscript, such as "Prunus persica" "Brassica rapa (L.) and B. nigra (L.)" in line 566, 545 and so on.

Reviewer 2 Report

Comments and Suggestions for Authors

A very valuable publication which presents the analysis of the KNOX genes in 118 (11 lower and 107 higher) plants. Based on 118 genomes, the authors verified the occurrence of the KNOX gene and then, using bioinformatic tools performed phylogenetic analyses, identified gene duplication, loss and calculation of non-synonymous and synonymous substitution rates for 155 KNOX gene pairs. The results are reliable and valuable and expand knowledge on the KNOX gene family. With full responsibility I recommend the work for publication

Author Response

Dear editors and reviewers:

We sincerely thank the editors and reviewers who generously donated their valuable time to provide insightful suggestions for our manuscript (Manuscript ID: horticulturae-2648202). We have meticulously incorporated and considered each suggestion and comment, as indicated by the red highlights in the revised text. In the subsequent sections, we offer a comprehensive response to the comments of each reviewer and outline the corresponding revisions that have been made. In addition, this manuscript has been edited for MDPI language editing (English-72859).

Sincerely,

Zhi-Meng Gan, Xiao-Yan Ai, Chun-Gen Hu* and Jin-Zhi Zhang*

E-mail: jinzhizhang@mail.hzau.edu.cn (J.-Z Z) (corresponding author)

 

To reviewer #2:

Comment: A very valuable publication which presents the analysis of the KNOX genes in 118 (11 lower and 107 higher) plants. Based on 118 genomes, the authors verified the occurrence of the KNOX gene and then, using bioinformatic tools performed phylogenetic analyses, identified gene duplication, loss and calculation of non-synonymous and synonymous substitution rates for 155 KNOX gene pairs. The results are reliable and valuable and expand knowledge on the KNOX gene family. With full responsibility I recommend the work for publication.

Response: Thank you for your suggestions, and we are delighted to learn that you find our subject matter interesting.

Reviewer 3 Report

Comments and Suggestions for Authors

In this manuscript, the authors showed a total of 1425 KNOX genes from 118 plant species, which were divided into two classes (class I and II) after phylogenetic analysis. They observed that gene loss was more common than gene duplication in non-angiosperm plants. In addition, three motifs (M1, 19 M2, and M4) were present in nearly all KNOX genes, while four novel motifs (M7–M10) were lost in non-angiosperms plants. Gene expression pattern analysis in A. thaliana and O. sativa revealed that class I of KNOX genes exhibit conserved high expression in stems, indicating a constitutive function for these genes. In general, it is an interesting subject; however, the manuscript could be better presented.

 

- For me “higher plants” and “lower plants” make no sense in the evolutionary classification of plants. Apparently, the authors are naming algae as lower plants and this is not correct. Thus, I suggest that the authors divide the species into four levels:  Rhodophyta (other algae); Chlorophyta (Green algae); Basal plants (Klebsormidiophyceae, Chlorokybophyceae, Mesostigmatophyceae, Chaetosphaeridium, Charophyceae, Zygnematophyceae, Coleochaetophyceae) and Land plants (Embryophyta).

 

- In addition, I think that it would be more reliable to present the species from suppl table 1 (and along the manuscript) into these 4 main levels (Rhodophyta, Chlorophyta, Basal plants, and Land plants) instead of uniquely showing in alphabetic order. They can also divide land plants into angiosperms (basal angiosperms, monocots and eudicots) and non-angiosperms plants. These changes help the readers to understand the evolutionary classification.

 

- Lines 116 and 117, the authors state they used BLASTP to identify KNOX genes utilizing the KNOX protein sequences of A. thaliana and O. sativa as the query, however, BLASTP is a tool that searches proteins (not genes) using proteins as query.

 

 

- Line 273. The authors cite Figure S2a; however, this Figure is from expression analyses and it has no sense in the sentence.

 

- Lines 328 to 334; 357 to 361. These sentences are methodology and need to be removed from the results.

 

- With regard to the Pan-genome analysis, it is very important to confirm the genome sequencing coverage and assembly quality (finished or draft) from all species.

 

- The English needs to be checked carefully.  

Comments on the Quality of English Language

Moderate editing of English language required

Author Response

Dear editors and reviewers:

We sincerely thank the editors and reviewers who generously donated their valuable time to provide insightful suggestions for our manuscript (Manuscript ID: horticulturae-2648202). We have meticulously incorporated and considered each suggestion and comment, as indicated by the red highlights in the revised text. In the subsequent sections, we offer a comprehensive response to the comments of each reviewer and outline the corresponding revisions that have been made. In addition, this manuscript has been edited for MDPI language editing (English-72859).

Sincerely,

Zhi-Meng Gan, Xiao-Yan Ai, Chun-Gen Hu* and Jin-Zhi Zhang*

E-mail: jinzhizhang@mail.hzau.edu.cn (J.-Z Z) (corresponding author)

 

To reviewer #3:

Comment: In this manuscript, the authors showed a total of 1425 KNOX genes from 118 plant species, which were divided into two classes (class I and II) after phylogenetic analysis. They observed that gene loss was more common than gene duplication in non-angiosperm plants. In addition, three motifs (M1, 19 M2, and M4) were present in nearly all KNOX genes, while four novel motifs (M7–M10) were lost in non-angiosperms plants. Gene expression pattern analysis in A. thaliana and O. sativa revealed that class I of KNOX genes exhibit conserved high expression in stems, indicating a constitutive function for these genes. In general, it is an interesting subject; however, the manuscript could be better presented.

 

Response: Thank you for your suggestions, and we are delighted to learn that you find our subject matter interesting. We have carefully reviewed your suggestions for improving the presentation of our manuscript and have taken them into consideration in our revisions.

Q1: For me “higher plants” and “lower plants” make no sense in the evolutionary classification of plants. Apparently, the authors are naming algae as lower plants and this is not correct. Thus, I suggest that the authors divide the species into four levels:  Rhodophyta (other algae); Chlorophyta (Green algae); Basal plants (Klebsormidiophyceae, Chlorokybophyceae, Mesostigmatophyceae, Chaetosphaeridium, Charophyceae, Zygnematophyceae, Coleochaetophyceae) and Land plants (Embryophyta).

Response: Thank you for your thoughtful suggestions and comments regarding the evolutionary classification of plants in our study. We agree with your ideas that divide the species into four levels: Rhodophyta; Chlorophyta; Basal plants and Land plants (https://www.plabipd.de/). We hope it will help the readers to understand the evolutionary classification and the innovative aspects of our work. For detailed information, please refer to the section highlighted in red on line 97, 170, Figure1, Figure2 and Table S1.

Q2: In addition, I think that it would be more reliable to present the species from suppl table 1 (and along the manuscript) into these 4 main levels (Rhodophyta, Chlorophyta, Basal plants, and Land plants) instead of uniquely showing in alphabetic order. They can also divide land plants into angiosperms (basal angiosperms, monocots and eudicots) and non-angiosperms plants. These changes help the readers to understand the evolutionary classification.

Response: Thank you for your suggestions, all the species we selected were divided into four levels: Rhodophyta; Chlorophyta; Basal plants and Land plants. And 107 land plants were divided into basal angiosperms, monocots and eudicots and non-angiosperms plants. For details, please refer to the section highlighted in red on line 90, 170, Figure1, Figure2. To facilitate readers' clear understanding of the classification of these species, we have sorted them based on their evolutionary relationships and used color coding to distinguish them, including indicating whether they are horticultural plants in Table S1.

 

Q3: Lines 116 and 117, the authors state they used BLASTP to identify KNOX genes utilizing the KNOX protein sequences of A. thaliana and O. sativa as the query, however, BLASTP is a tool that searches proteins (not genes) using proteins as query.

Response: Thank you for your suggestion. We agree with your ideas that BLASTP is a tool which can searches proteins using proteins as query. But in this study, we utilized the identified KNOX protein sequences of A. thaliana and O. sativa as the query, while translating the representative transcripts of different species into proteins as the database (using representative transcripts to represent the genes) for homologous BLASTP. This method is used for identifying members of a gene family and is complementary to the HMM model identification method. Finally, we integrated the results of both methods for greater accuracy.

Q4: Line 273. The authors cite Figure S2a; however, this Figure is from expression analyses and it has no sense in the sentence.

Response: Thank you for pointing out the error. and we sincerely apologize for the oversight in the manuscript. The correct reference should be to Table S1, which allows readers to find the number of KNOX gene family members present in different species by querying it.

Q5: Lines 328 to 334; 357 to 361. These sentences are methodology and need to be removed from the results.

Response: Thank you for your suggestions. These sentences describe the specific details of the dataset that I collected, which indeed belong to the methodology section. Hence, we will relocate these details to Section 2.1 of the Materials and Methods, as found on page 3, lines 122-130.

Q6: With regard to the Pan-genome analysis, it is very important to confirm the genome sequencing coverage and assembly quality (finished or draft) from all species.

Response: Thank you very much for your suggestions. We agree with your ideas that the quality of genome assembly is crucial for pan-genome analysis. Among the 17 species genomes used in this study, 8 were assembled into chromosomes, 3 scaffolds, and 6 contigs. For further statistical information, please refer to section 2.1 Data Collection of Materials and Methods, line 111, which mentions the CPBD database (http://citrus.hzau.edu.cn/statistics.php#genomeInfo).

Q7: The English needs to be checked carefully.

Response: This manuscript has been edited for MDPI language editing (English-72859).

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The authors accepted the large majority of my suggestions and improved the manuscript satisfactorily. Thus, I agree with the acceptance of this manuscript for publication.