Functional Characterization of Glutathione Peroxidase Genes Reveals Their Contribution to the Rapid Range Expansion of Amaranthus Palmeri Under Stress Conditions

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this article, the author conducted a systematic analysis of the homologous genes of GPXs in various crops, comparing their sequence differences, gene structures, chromosome locations, and miRNA target structure networks. At the same time, through gene expression analysis, the relationship between GPXs and various stress conditions was explored. Furthermore, in Arabidopsis thaliana, gpx was found a regulatory effect on the oxidative stress response induced by glyphosate, verifying the aforementioned conjecture. The experimental design is reasonable, and the figures and tables in the article are exquisite, laying a solid theoretical foundation for the subsequent structural analysis and functional identification of GPX across species. However, there are still some minor issues as follows:

1. “ApGPX(protein)”, in line 286 and 293, are supposed to “ApGPX (proteins)”in accordance of it in line 297, please verify them in whole paper.
2. Line 104, the citation format for references in the text does not match the context, please review the entire document.
3. Line310, ApGPXare supposed ApGPX.
4. Line 251, the mentioned tissue of experimental materials are supposed a further specific information including time and spatial position.
5. Line 455, 4h instead of 4-h.
6. Line 445, the expression level of ApGPX4 during 12 to 48 h exhibited almost a totallyincreasing trend in figure 9, please carefully verify your description.By the way, there is a consideration whether demonstrate findings in form of a line chart which could reveal a more unambiguous trend of variation.
7. Line 508-509, the genus names written in Latin all need to be in italics such as “Oryza sativa”.
8. Line 498, there are spaces before and after “<”, please verify this problem in your manuscriptions.
9. Line 90-91, the form of protein “AtGPXL3”and “ABI2”is supposed to the regular script, please verify this character issue carefully in your manuscription.
10. Line 265, p-value are supposed to p-value.
11. Line 280, each biological duplication of the rosettes whether conduct the technical duplications?

Author Response

Reviewer 1

We sincerely thank the reviewers and the editors for your thorough review and positive assessment of our work. Their constructive comments have been invaluable in improving the quality of our manuscript. We have carefully addressed each point raised and highlighted them for easy tracking in the revised manuscript. Our point-by-point responses to all comments are provided below.

Comment 1: “ApGPX (protein)”, in line 286 and 293, are supposed to “ApGPX (proteins)” in accordance of it in line 297, please verify them in whole paper.

Response: Thank you for pointing this out. We have carefully verified and corrected the formatting to "ApGPX (proteins)" throughout the entire manuscript to ensure consistency.

Comment 2: Line 104, the citation format for references in the text does not match the context, please review the entire document.

Response: We apologize for this oversight. The citation style has been standardized throughout the manuscript to match the journal's requirements.

Comment 3: Line 310, ApGPX are supposed ApGPX.

Response: Thank you for your attention to detail. We have corrected this error in the revised manuscript.

Comment 4: Line 251, the mentioned tissue of experimental materials are supposed a further specific information including time and spatial position.

Response: Thank you for this insightful suggestion. We have revised Section 2.10 to include more specific information regarding the time of collection and the spatial position (fully expanded leaves) of the experimental materials.

Comment 5: Line 455, 4h instead of 4-h.

Response: This has been corrected to "4 h" according to your kind suggestion.

Comment 6: Line 445, the expression level of ApGPX4 during 12 to 48 h exhibited almost a totally increasing trend in figure 9, please carefully verify your description. By the way, there is a consideration whether demonstrate findings in form of a line chart which could reveal a more unambiguous trend of variation.

Response: Thank you for your kind review and careful observation. The description has been corrected in the revised manuscript to accurately reflect the data presented in Figure 9. Regarding the chart type, we agree that line charts can effectively display trends. However, for this specific dataset with multiple genes and treatments, the bar chart format was chosen to allow for clear visual comparison of expression levels at discrete time points. We believe this format remains effective for the presented analysis.

Comment 7: Line 508-509, the genus names written in Latin all need to be in italics such as “Oryza sativa”.

Response: Thank you for this correction. All scientific genus and species names throughout the manuscript have been italicized.

Comment 8: Line 498, there are spaces before and after “<”, please verify this problem in your manuscript.

Response: Thank you for noting this formatting issue. We have checked the entire manuscript and corrected the formatting of statistical notations to ensure proper spacing.

Comment 9: Line 90-91, the form of protein “AtGPXL3” and “ABI2” is supposed to the regular script, please verify this character issue carefully in your manuscript.

Response: We appreciate the reviewer's time in reviewing our manuscript. All protein names are now presented in regular, non-italicized script throughout the revised text.

Comment 10: Line 265, p-value are supposed to p-value.

Response: This has been corrected to "p-value" in the revised manuscript.

Comment 11: Line 280, each biological duplication of the rosettes whether conduct the technical duplications?

Response: Thank you for seeking clarification. Yes, each biological replicate consisted of three technical replicates. This detail has been explicitly stated in the revised Materials and Methods section (Section 2.11) for full transparency.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

I carefully revised your manuscript entitled "Mechanistic investigation of glutathione peroxidase (GPX) genes mediated facilitation of rapid range expansion of Amaranthus palmeri".

Your analyses were well conducted and your goals were achieved and mostly clearly expressed throughout the manuscript.

Please, consider the following corrections and comments on your manuscript and incorporate them when appropriate.

 

Introduction

Lines 96-100: maize or rice OsGPX3? revise these sentences according to the reference of Paiva et al. 2019.

 

Materials and methods

Lines 145-148; 170-171: indicate tho which species these genes/protein sequences belong to (BnGPXs, TaGPXs, and CsGPXs; FUGPXs, SoGPXs).

Line 212: provide more detailed description on how putative miRNAs from A. palmeri were obtained from the AGRDB.

Line 251: "Total RNA was extracted from the leaves of A. palmeri…" Leaves used here are those obtained in 2.8 Plant material and treatment conditions? Please, clarify.

 

Results

For Supplementary file S3: provide units or some reference for numbers presented in this table. What do they refer to?

Lines 368-370: Revise: Supplementary file S3 or S4?

Lines 383-393: Please, from those CAREs listed at right side of figure 6, clarify which of them belong to the three major categories considered. In addition, in figure 6, CAREs located 2 kb upstream of APGPX genes do not seem to reflect the variability of CAREs presented in the right panel of the same figure.

 

Discussion

As the rapid range expansion of A. palmeri is mentioned in the title of your manuscript as possibly linked to the GPX genes funtion, please discuss this idea more cleary here. Try to explain how your results on the functions of these genes could be related to the invasiveness and aggresiveness of this plant.

Regarding Material and methods, lines 164-176: explain here why did you choose/considere/include the sequences of these species in the phylogenetic analysis of the GPX family.

Lines 502-503: "However, their functions in the stress responses of A. palmeri have not been extensively studied." Please, breafly  discuss here why it is importat to solve this issue.

 

Sincerely,

Reviewer.

Comments for author File: Comments.pdf

Author Response

Reviewer 2

We sincerely thank the reviewers and the editors for your positive assessment of our manuscript and your valuable suggestions, which have significantly improved our work. We have carefully addressed each comment in the revised manuscript, and our detailed responses are provided below.

Comment 1: (Introduction, Lines 96-100): maize or rice OsGPX3? Revise these sentences according to the reference of Paiva et al. 2019.

Response: Thank you for catching this error. The sentence has been revised to correctly refer to rice (Oryza sativa) OsGPX3 in accordance with the cited reference (Paiva et al., 2019).

Comment 2: (Materials and methods, Lines 145-148; 170-171): indicate to which species these genes/protein sequences belong to (BnGPXs, TaGPXs, and CsGPXs; FUGPXs, SoGPXs).

Response: We thank the reviewer for this suggestion to enhance clarity. The manuscript has been revised to explicitly state the species names:

Section 2.1. Second, protein sequences of GPX from A. Arabidopsis (AtGPXs) [11], Brassica napus (BnGPXs) [31], wheat (TaGPXs) [32], and cucumber (CsGPXs) [33] were collected from the NCBI (https://www.ncbi.nlm.nih.gov/) and Ensembl Plants (https://plants.ensembl.org/) databases.

Section 2.3. The GPX protein sequences for Amaranthus hypochondriacus (AhGPXs), Amaranthus tuberculatus (AtuGPXs), Amaranthus hybridus (AhyGPXs), Amaranthus cruentus (AcGPXs), A. thaliana (AtGPXs), Panicum virgatum (PvGPXs), Portulaca amilis (FUGPXs), and Solanum lycopersicum (SlGPXs) were obtained from the Phytozome v13 and Amaranth Genomic Resource (AGRDB) [34] databases using a BLAST search with an e-value of 1e-5. A phylogenetic analysis was performed on the protein sequences of 8 ApGPXs, 7 AhGPXs, 7 AtuGPXs, 4 AhyGPXs, 5 AcGPXs, 8 AtGPXs, 6 FUGPXs, 7 SlGPXs, and 10 PvGPXs utilizing MEGA 11 software.

Comment 3: (Materials and methods, Line 212): provide more detailed description on how putative miRNAs from A. palmeri were obtained from the AGRDB.

Response: Thank you for this suggestion. We have revised the text to provide the specific link: “Putative miRNAs of A. palmeri were obtained from the online AGRDB (nbpgr.ernet.in:8080/AmaranthGRD/downloads.aspx) database.”

Comment 4: (Materials and methods, Line 251): "Total RNA was extracted from the leaves of A. palmeri…" Leaves used here are those obtained in 2.8 Plant material and treatment conditions? Please, clarify.

Response: Yes, that is correct. We have revised Section 2.10 for clarity: "Total RNA was extracted from the fully expanded treatment leaves collected at 0 h, 12 h, 24 h, 36 h, 48 h, and 72 h as described in Section 2.8 using TRIzol reagent (Invitrogen, Gaithersburg, MD, USA), following the manufacturer's guidelines."

Comment 5: (Results, Supplementary file S3): provide units or some reference for numbers presented in this table. What do they refer to?

Response: We thank the reviewer for pointing out this lack of clarity. The numbers in Supplementary file S3 represent the percentage composition of each secondary structure element (alpha helix, extended strand, etc.) within the ApGPX proteins. This has been clearly indicated in the revised supplementary file S3.

Comment 6: (Results, Lines 368-370): Revise: Supplementary file S3 or S4?

Response: Thank you for identifying this inconsistency. The correct reference is Supplementary file S4, and this has been corrected in the revised manuscript.

Comment 7: (Results, Lines 383-393): Please, from those CAREs listed at right side of figure 6, clarify which of them belong to the three major categories considered. In addition, in figure 6, CAREs located 2 kb upstream of APGPX genes do not seem to reflect the variability of CAREs presented in the right panel of the same figure.

 Response: We thank the reviewer for the suggestion. The manuscript has been revised to clarify the categorization of the cis-acting regulatory elements (CAREs) in the figure legend and results text. Furthermore, Figure 6 has been updated to more accurately depict the diversity of CAREs present in the ApGPX promoter regions.

Comment 8: (Discussion): As the rapid range expansion of A. palmeri is mentioned in the title of your manuscript as possibly linked to the GPX genes funtion, please discuss this idea more cleary here. Try to explain how your results on the functions of these genes could be related to the invasiveness and aggresiveness of this plant

Response: We thank the reviewer for reviewing our manuscript. We have revised the Discussion section (Lines 703-714) based on your kind comment:

This study presents the first genomic characterization of the GPX gene family in A. palmeri. The expression profiles and structural features of the ApGPX genes provide strong insights into their potential role in A. palmeri’s remarkable invasiveness. The observed robust upregulation of ApGPXs under glufosinate ammonium, high temperature, and osmotic stress; their potential for rapid miRNA-mediated regulation due to the lack of UTRs; and their strategic localization across key cellular compartments collectively suggest a highly efficient system for managing oxidative stress. We hypothesize that this enhanced antioxidant capacity constitutes a significant adaptive trait, enabling A. palmeri to thrive under the diverse abiotic stresses during range expansion. This molecular adaptability likely influences its survival and competitive dominance and facilitate its rapid spread. Further studies are required to validate the proposed functional link between ApGPX activity and A. palmeri ecological success.

Comment 9: (Discussion, Regarding Material and methods, lines 164-176): explain here why did you choose/consider/include the sequences of these species in the phylogenetic analysis of the GPX family.

Response: This is an excellent suggestion. We have revised the Discussion (Lines 606-616) to include a sentence explaining the phylogenetic strategy:

To gain meaningful evolutionary insights, a phylogenetic analysis was conducted between ApGPX proteins and those from AhGPXs, AtuGPXs, AhyGPXs, AcGPXs, AtGPXs, FUGPXs, SlGPXs, and PvGPXs. This approach ensured the inclusion of lineage-specific conservation, ecological diversity, and well-annotated GPX proteins. The analysis revealed that ApGPX genes clustered into distinct clades, indicating conserved functional divergence. Most ApGPXs grouped closely with other Amaranthus homologs, indicating strong evolutionary conservation within the genus. Notably, ApGPX7 and ApGPX8 aligned with more distantly related species, suggesting potential neofunctionalization or adaptation to unique stress conditions. These findings imply that while certain ApGPX genes maintain ancestral roles, others may have acquired novel functions to potentially enhance A. palmeri resistance to environmental conditions.

Comment 10: (Discussion, Lines 502-503): "However, their functions in the stress responses of A. palmeri have not been extensively studied." Please, briefly discuss here why it is important to solve this issue.

Response: We agree that stating the importance of this research gap strengthens the manuscript. We have revised the sentence (Lines 570-573) to read: “However, their functions in the stress responses of A. palmeri have not been extensively studied. Understanding ApGPX functions is essential to explore the molecular mechanisms of A. palmeri’s stress tolerance, which may explain its invasiveness and herbicide resistance, and guide the development of targeted weed management and stress-resilient crop strategies.”

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript addresses an important topic on Amaranthus palmeri stress tolerance and GPX gene family characterization. The work combines bioinformatics, expression profiling, and comparative analysis. However, several issues must be addressed before the manuscript is suitable for publication, including formatting of scientific names and gene names, clarity of methods, statistical consistency, and the appropriateness of the title.

Major comments

Comment 1:

The current title does not fully reflect the study. The work is primarily a genome-wide identification and characterization of the GPX gene family in A. palmeri, not a mechanistic investigation of range expansion. Please revise the title.

Comment 2:

All scientific names must be italicized (e.g., Amaranthus palmeri, Arabidopsis thaliana, Oryza sativa). Please correct consistently throughout the manuscript. At first mention of each species, provide the full scientific name with author citation where appropriate.

Comment 3:

Gene symbols (ApGPX, AtGPX, OsGPX, etc.) should be italicized. Proteins should remain in regular font. Ensure that abbreviations like ApGPXs do not italicize the 's' for plural form.

Comment 4:

The number of biological and technical replicates is unclear. For each treatment, clearly state the replicates used in qPCR, oxidative burst assays, and other experiments.

Comment 5:

When listing GPX proteins from other species, specify the plant species they belong to (e.g., AhGPXs from Amaranthus hypochondriacus, AtGPXs from Arabidopsis thaliana).

Comment 6:

Figure 9 and 10; the qPCR results appear to be presented as fold-change relative to 0 h. In this case, use the term 'expression change' rather than 'expression level'.

Comment 7:

The lettering system indicating significant differences is inconsistent. For example, ApGPX2 in Figure 9C at 4 h should have different lettering. Similar issues occur in Figures 9D and 10B. Please recheck all statistical analyses and correct figure notations.

Comment 8:

There is inconsistency between Actin7 and Actin8 (P18L631) being referred to as the most stable reference gene. Please clarify which was used for normalization in final analyses.

Author Response

Reviewer 3

We are grateful to Reviewers and the editors for their time to review our manuscript and constructive feedback, which has significantly improved the quality and clarity of our manuscript. We have carefully addressed each of the major comments, and our detailed responses and the corresponding revisions are outlined below.

Comment 1: The current title does not fully reflect the study. The work is primarily a genome-wide identification and characterization of the GPX gene family in A. palmeri, not a mechanistic investigation of range expansion. Please revise the title.

Response: We agree with this assessment. The title has been revised to more accurately reflect the core content of our study. The revised title is: "Functional characterization of glutathione peroxidase genes reveals their contribution to the rapid range expansion of Amaranthus palmeri under stress conditions”.                                                               

Comment 2: All scientific names must be italicized (e.g., Amaranthus palmeri, Arabidopsis thaliana, Oryza sativa). Please correct consistently throughout the manuscript. At first mention of each species, provide the full scientific name with author citation where appropriate.

Response: Thank you for your insightful review. We have italicized and revised all scientific names in the revised manuscript according to your valuable suggestion.

Comment 3: Gene symbols (ApGPX, AtGPX, OsGPX, etc.) should be italicized. Proteins should remain in regular font. Ensure that abbreviations like ApGPXs do not italicize the 's' for plural form.

Response: We thank the reviewer for pointing out this important formatting detail. The manuscript has been thoroughly revised to ensure that all gene symbols are consistently italicized, while protein names are in regular font. We have also corrected the plural forms to ensure the 's' is not italicized.

Comment 4: The number of biological and technical replicates is unclear. For each treatment, clearly state the replicates used in qPCR, oxidative burst assays, and other experiments.

Response: We apologize for any lack of clarity. The Materials and Methods sections have been revised to explicitly state the replicate structure for each experiment:

Section 2.8 (Plant material and treatment): Clarified that each treatment consisted of three biological replicates.

Section 2.10 (qPCR analysis): Specified that each biological replicate was analyzed with three technical replicates.

Section 2.11 (Oxidative burst assay): Explicitly stated that the assay was performed with three biological replicates, and subsequent qPCR analysis used three technical replicates per biological sample.

Comment 5: When listing GPX proteins from other species, specify the plant species they belong to (e.g., AhGPXs from Amaranthus hypochondriacus, AtGPXs from Arabidopsis thaliana).

Response Thank you for this suggestion to improve clarity. We have revised Section 2.3 to explicitly state the full species name for each set of GPX proteins immediately upon their introduction:

2.3. Phylogenetic analysis of the GPX gene family

The GPX protein sequences for Amaranthus hypochondriacus (AhGPXs), Amaranthus tuberculatus (AtuGPXs), Amaranthus hybridus (AhyGPXs), Amaranthus cruentus (AcGPXs), A. thaliana (AtGPXs), Panicum virgatum (PvGPXs), Portulaca amilis (FUGPXs), and Solanum lycopersicum (SlGPXs) were obtained from the Phytozome v13 and Amaranth Genomic Resource (AGRDB) [34] databases using a BLAST search with an e-value of 1e-5. A phylogenetic analysis was performed on the protein sequences of 8 ApGPXs, 7 AhGPXs, 7 AtuGPXs, 4 AhyGPXs, 5 AcGPXs, 8 AtGPXs, 6 FUGPXs, 7 SlGPXs, and 10 PvGPXs utilizing MEGA 11 software.

Comment 6: Figure 9 and 10; the qPCR results appear to be presented as fold-change relative to 0 h. In this case, use the term 'expression change' rather than 'expression level'.

Response: Thank you for this precise correction. We have revised the figure 9 and 10 to ensure terminological accuracy according to your kind suggestion.

Comment 7: The lettering system indicating significant differences is inconsistent. For example, ApGPX2 in Figure 9C at 4 h should have different lettering. Similar issues occur in Figures 9D and 10B. Please recheck all statistical analyses and correct figure notations.

Response: We sincerely appreciate the reviewer's careful attention to detail in identifying the inconsistencies in the statistical lettering. As requested, we have completely rechecked all statistical analyses for the qPCR data. This involved re-running the two-way ANOVA and post-hoc tests. The figures now present a consistent and correct representation of significant differences between time points for each gene.

Comment 8: There is inconsistency between Actin7 and Actin8 being referred to as the most stable reference gene. Please clarify which was used for normalization in final analyses.

Response: We thank the reviewer for seeking this clarification. The stability analysis using five different algorithms identified Actin8 as the most stable reference gene overall. Therefore, Actin8 was selected and used for the normalization of all qPCR data presented in the study. We have clarified this point in both the Results (Section 3.7) and Methods (Section 2.10) sections.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

1. Include additional physiological measurements such as MDA content, antioxidant enzyme activities, or phenotypic assays under herbicide or stress treatments to strengthen the functional validation.
2. The qPCR experiments analyzed only six of the eight ApGPX genes. And others？
3. Actin8 is a suitable reference gene for stress conditions?
4. Figures 9 and 10 indicate significance using letters, but the statistical methods and exact p-values are not clearly described.
5. Some figures (e.g., Figures 4 and 5) have fonts that are too small and difficult to read.
6. Figure 3 lacks clear labels for the control versus experimental groups.
7. Results and discussion overlap. The discussion section repeats many descriptive results rather than focusing on interpretation and comparison with previous studies.
8. The MS contains numerous grammatical errors and awkward expressions.
9. The abstract is overly long and does not clearly highlight the key findings and novelty.
10. Several references do not fully comply with Agronomy style, particularly in terms of format for volume, issue, and page numbers.
Please carefully check and revise all references according to journal guidelines.
11.  Clarify the number of biological and technical replicates for all experiments.
12. Provide supplementary tables for raw qPCR data and Ka/Ks calculations.
13. Please discuss the potential roles of ApGPXs in regulating ROS homeostasis and herbicide detoxification.

Author Response

We sincerely thank the reviewers and editors for their time and effort in evaluating our manuscript and providing thoughtful, constructive feedback. We have carefully considered each comment and revised the manuscript accordingly. All revisions have been highlighted in green in the revised manuscript for ease of reference, and our detailed point-by-point responses to each comment are provided below.

 

1. Include additional physiological measurements such as MDA content, antioxidant enzyme activities, or phenotypic assays under herbicide or stress treatments to strengthen the functional validation.

Response: Thank you very much for your insightful and constructive suggestion. We fully agree with your recommendation, which has greatly contributed to refining the direction of our research. In this study, our primary objective was to identify and isolate candidate genes potentially involved in herbicide resistance. As you rightly pointed out, further functional validation—such as physiological index measurements and phenotypic assays under herbicide or stress conditions—is essential to investigate the underlying mechanisms. We recognize the importance of these follow-up experiments and are committed to conducting them in future research phases. Due to the time and resources required for these comprehensive analyses, they were beyond the scope of the current study. Nevertheless, we have incorporated your suggestion into our long-term research plan and will pursue these investigations to strengthen and expand upon our initial findings.

 

2. The qPCR experiments analyzed only six of the eight ApGPX genes. And others?

Response: Thank you very much for your insightful comment. We selected six ApGPX genes for qPCR analysis based on their consistent amplification and robust primer performance under stress conditions. The remaining two genes exhibited low amplification efficiency and inconsistent results, which could compromise data reliability. To ensure the accuracy and reproducibility of our findings, we excluded these two genes from the qPCR experiments.

 

3. Actin8 is a suitable reference gene for stress conditions?

Response: Thank you for your valuable comment and for highlighting the importance of reference gene selection. We chose Actin8 due to its consistently stable expression across all stress conditions, as verified through multiple validation tools, including NormFinder, GeNorm, and RefFinder. This ensured accurate normalization of our qPCR data and enhanced the reliability of our gene expression analysis.

 

4. Figures 9 and 10 indicate significance using letters, but the statistical methods and exact p-values are not clearly described.

Response: Thank you for your time to review our manuscript and valuable comment. We have revised the manuscript to clearly describe the statistical methods used for the qPCR analysis in Section 2.11. Specifically, significance was determined using two-way ANOVA followed by Tukey’s HSD test, with a threshold of P < 0.05. We have also updated the figure legends to include the exact P-value criteria and clarify how the lettering system reflects statistical differences among treatments. We appreciate your suggestion, which has helped improve the transparency and rigor of our data presentation.

 

5. Some figures (e.g., Figures 4 and 5) have fonts that are too small and difficult to read.

Response: Thank you for your time and helpful observation. We have revised Figures 4 and 5 to improve readability by increasing the font size and enhancing overall clarity. We appreciate your attention to presentation quality and believe these adjustments will make the figures easier to interpret.

 

6. Figure 3 lacks clear labels for the control versus experimental groups.

Response: Thank you for your valuable comment. We have revised Figure 3 to include clear labels distinguishing the control and experimental groups. The updated figure and legend now provide improved clarity to help readers accurately interpret the data. We appreciate your attention to detail.

 

7. Results and discussion overlap. The discussion section repeats many descriptive results rather than focusing on interpretation and comparison with previous studies.

Response: Thank you for your valuable comment. We have revised the discussion section to reduce repetition of descriptive results and focus more on interpretation, biological relevance, and comparison with previous studies. We appreciate your suggestion, which helped improve the clarity and depth of our manuscript.

 

8. The MS contains numerous grammatical errors and awkward expressions.

Response: Thank you for your valuable comment. We have carefully reviewed the manuscript and corrected grammatical errors and awkward phrasing throughout. The revised version has been thoroughly edited to improve clarity, readability, and overall language quality. We appreciate your feedback, which helped enhance the presentation of our work.

 

9. The abstract is overly long and does not clearly highlight the key findings and novelty.

Response: Thank you for your valuable feedback. We have revised the abstract to make it more concise and focused. The revised manuscript now clearly emphasizes the key findings and highlights the novel aspects of our study. We appreciate your comment, which helped improve the clarity and impact of the abstract.

 

10. Several references do not fully comply with Agronomy style, particularly in terms of format for volume, issue, and page numbers. Please carefully check and revise all references according to journal guidelines.

Response: Thank you for your careful review and helpful comment. We have thoroughly checked all references and revised them to ensure full compliance with the Agronomy journal’s formatting guidelines.

11. Clarify the number of biological and technical replicates for all experiments.

Response: Thank you for your thoughtful comment. We have clarified the number of biological and technical replicates in the revised manuscript. All experiments were conducted with three biological replicates and three technical replicates to ensure the reliability and reproducibility of the results. We appreciate your valuable suggestion.

 

12. Provide supplementary tables for raw qPCR data and Ka/Ks calculations.

Response: Thank you for your thoughtful suggestion. To support transparency during the review process, we have provided supplementary tables containing the raw qPCR data and Ka/Ks calculations. However, these files are intended solely for peer review and will not be included in the final published version of the article. The data will remain available upon reasonable request from the corresponding author.

 

13. Please discuss the potential roles of ApGPXs in regulating ROS homeostasis and herbicide detoxification.

Response: Thank you for this important suggestion. We have revised the manuscript to address the potential roles of ApGPX genes in regulating ROS homeostasis and herbicide detoxification (Line 601-607).

 

We would like to express our sincere gratitude to the reviewers and editors once again for dedicating their valuable time to evaluate our manuscript and provide insightful comments. Your thoughtful feedback has greatly contributed to improving the quality and clarity of our work. We hope that the revised version now meets the expectations of both the reviewers and the journal's requirements.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have addressed all my comments and concerns. I have no further comments.

Author Response

We are grateful to reviewers and the editors for their time to review our manuscript and constructive feedback, which has significantly improved the quality and clarity of our manuscript.

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript now meets the publication standards of Agronomy