Genome-Wide Characterization of the HaALS Gene Family Reveals Its Potential Roles in Imazethapyr Tolerance in Sunflower (Helianthus annuus L.)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this study, the goal was to systematically identify and characterize the ALS gene family (HaALS) in sunflower (Helianthus annuus L.) across the genome; accordingly, members of the HaALS family were identified, the physicochemical properties of these genes were predicted, and their evolutionary relationships were examined through phylogenetic analyses. Additionally, conserved domains and gene structures were examined to reveal the structural diversity of HaALS paralogous genes. Furthermore, the expression profiles of HaALS genes and cis-regulatory elements in their promoter regions were analyzed to evaluate the transcriptional responses to imidazolinone-group herbicides, particularly imazethapyr application. In this regard, the study provides insights into the structural, evolutionary, and functional characteristics of the HaALS gene family in sunflower and aims to elucidate the potential roles of these genes in imazethapyr tolerance.

The introduction explains the topic and highlights the objectives and goals.

The Results and Discussion section is written in detail. Figures and tables are provided along with their captions. The text is clear and includes the necessary citations. In addition, the following corrections are recommended.

• For Figure 6, the colors should be updated as follows: red: sunflower; blue-green: lettuce; magenta: Arabidopsis
• Line 387-398: It can be seen that the HaALS4 and HaALS11 genes are significantly induced at 24 hours. However, the statement regarding HaALS11 that “amplification is greater at 48 hours” does not appear to fully align with the graph presented. According to the graph, HaALS11 expression in the resistant genotype reaches its peak at 24 hours and then decreases by 48 hours. Therefore, this statement needs to be reevaluated to ensure consistency with the graph.
• In the Materials and Methods section, statistical methods should be presented under a separate heading.

Author Response

Dear Reviewer,

 

Thanks a lot for having reviewed our manuscript (plants-4361047). We have revised the manuscript, and would like to submit it for your consideration. According to your comments and suggestions, we have made corresponding changes. The revisions have been highlighted in the revised manuscript.

 

I greatly appreciate both your help and that of the referees concerning improvement to this paper. Below you can find point-to-point responses to Reviewers’ comments. We hope that the revised version of the manuscript is now acceptable for publication in your journal.

 

I look forward to hearing from you soon.

 

We would like to express our sincere thanks again to you for the constructive and positive comments.

 

With best wishes,

 

Yours sincerely,

Pengyuan Xie

Response to Reviewer #1

Comment 1:

For Figure 6, the colors should be updated as follows: red: sunflower; blue-green: lettuce; magenta: Arabidopsis.

Response:

Thank you for your valuable suggestion. According to your comment, we have updated the color scheme in Figure 6 and unified the species-specific color coding throughout the figure and its legend. In the revised manuscript, the figure legend now explicitly states: “Red, sunflower (Helianthus annuus); blue-green, lettuce (Lactuca sativa); magenta, Arabidopsis (Arabidopsis thaliana).”

 

Comment 2:

Lines 387–398: It can be seen that the HaALS4 and HaALS11 genes are significantly induced at 24 hours. However, the statement regarding HaALS11 that “amplification is greater at 48 hours” does not appear to fully align with the graph presented. According to the graph, HaALS11 expression in the resistant genotype reaches its peak at 24 hours and then decreases by 48 hours. Therefore, this statement needs to be reevaluated to ensure consistency with the graph.

Response:

Thank you very much for your careful reading and for pointing out this inconsistency. We apologize for the inaccurate description of HaALS11 expression dynamics. According to your suggestion, we have carefully reevaluated Figure 11 and revised the related statements in the Results and Discussion sections to ensure full consistency with the qRT-PCR data. The related modifications are as follows:

Lines 386–388 (Results section 3.11): The original sentence “HaALS4 and HaALS11 displayed the most robust induction, with sharp increases already evident at 24 h and further amplification by 48 h” was revised to: “HaALS4 displayed the most robust and sustained induction, with sharp increases already evident at 24 h and further amplification to >10-fold by 48 h, whereas HaALS11 reached its peak induction (~6–7-fold) at 24 h and subsequently declined, yet remained above baseline at 48 h.”

Lines 367–371 (Discussion): The corresponding passage was revised to: “in the resistant background, HaALS4 showed a pronounced induction that rose to ~6-fold at 24 h and exceeded ~10-fold at 48 h relative to 0 h, while HaALS11 increased to ~6–7-fold at 24 h before partially declining yet remaining above baseline at 48 h.”

Lines 15–16 (Abstract): The abstract was also updated accordingly to reflect the distinct temporal patterns of HaALS4 and HaALS11.

 

Comment 3:

In the Materials and Methods section, statistical methods should be presented under a separate heading.

Response:

Thank you for your valuable advice. According to your suggestion, we have extracted the statistical analysis procedures from the qRT-PCR subsection and added a new independent subsection entitled “2.8 Statistical Analysis” in the Materials and Methods section. The revised text reads as follows:

Lines 36–37: “Relative expression levels were calculated using the 2^(−ΔΔCt) method, with three technical replicates per biological replicate. Gene expression was normalized to an internal reference gene (ACT2).”

Lines 38–40 (new Section 4.8): “Statistical Analysis. Differential expression between treatment and control groups, as well as between susceptible (S) and resistant (R) genotypes at each time point, was assessed using one-way analysis of variance (ANOVA) followed by Tukey’s honestly significant difference (HSD) test. Upregulation was considered significant when relative expression was ≥ 2-fold, and downregulation was considered significant when relative expression was ≤ 0.5-fold. Statistical significance was defined at P < 0.05. All statistical analyses were performed using SPSS or R software, and data are presented as mean ± standard error (SE) of three biological replicates.”

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript entitled "Genome-wide characterization of the HaALS gene family reveals its potential roles in imazethapyr tolerance in sunflower (Helianthus annuus L.)" by Xie et al. aims to identify and characterize acetolactate synthase (ALS)-related genes in the sunflower genome.

First, the authors performed a comprehensive bioinformatics analysis and identified 11 putative members of the HaALS gene family, which were subsequently classified into three distinct groups (Groups a–c). Second, the authors characterized the physicochemical properties of these genes and proteins and predicted the transcriptional regulatory networks in which they may be involved. Finally, the authors examined the herbicide-induced expression patterns of the HaALS gene family and investigated their potential roles in phenotypic and photosynthetic responses to imazethapyr stress.

Although the authors present a substantial amount of data and extensive analyses, relevant literature citations are often missing or not appropriately incorporated throughout the manuscript. In addition, several issues require further clarification, as detailed below.

Specific Comments

1. Abstract: The authors have described the experimental results and significance in detail. 

For lines 16–23, it is recommended to select only the most critical results, as the detailed content can be elaborated on within the specific results sections. 

For lines 25–37, it is suggested that the authors focus on key phenotypic descriptions. The total word count for the abstract should be kept concise, ideally within 200–250 words.

1. Line 156 (Table 1): It is recommended to add a "Chromosome ID" column and a "Gene ID" column for better clarity.
2. Line 159: The sentence could be revised for better flow. For example: "The 11 HaALS genes are unevenly localized across 10 of the sunflower chromosomes (Figure 1), with no ALS members detected on chromosomes 3, 8, 11, 12, 13, 14, and 17."
3. Missing References: The authors need to thoroughly proofread the manuscript, update the references, and add appropriate citations to support their results, hypotheses, and analyses throughout the text. Specifically, please add references to the following locations:
• Line 183: Add reference to support the analysis.
• Line 193: Update and check references.
• Lines 198–199: Add a reference to support this hypothesis.
• Line 225: Add a reference.
• Line 241: Add a reference.
• Line 246: Add a reference to support this hypothesis.
• Lines 260 and 264: Add references.
4. Figure Numbering: It appears that Figure 5 is missing from the manuscript. Please double-check and correct the sequential numbering of the figures.
5. Line 271 (Figure 6): It is highly recommended to maintain consistent nomenclature between the figure legend and the figure subtitle. For example, the subtitle reads, "...ALS gene family in sunflower, lettuce, and Arabidopsis," whereas the figure legend uses the scientific names: "Helianthus annuus", "Lactuca sativa", and "Arabidopsis thaliana". Please unify these terms.
6. Lines 272–273: Please clearly specify the color coding in the text or legend (e.g., "Red represents sunflower; blue-green represents lettuce; magenta represents Arabidopsis").
7. Line 306 (Figure 8): Similar to the comment on Figure 6, please ensure the nomenclature is consistent between the figure legend and the figure subtitle.
8. Lines 311–312: The authors state, "For the vast majority of gene pairs, Ka/Ks < 1, with values mostly ranging from 0 to approximately 0.85, indicating that the HaALS family has evolved primarily under purifying selection." Please add an appropriate reference to support the conclusion regarding purifying selection thresholds.
9. Line 340 (Figure 10A): The authors present a word cloud of transcription factors (TFs). Do the varying word sizes represent quantitative differences among the TFs? If not, Panel A may be unnecessary and misleading. It is highly recommended to replace or supplement this with a comprehensive table detailing all regulatory networks between the TFs and the gene family in the supplementary materials.
10. Line 386: The phrase "...the untreated control by several-fold" is vague. Does this indicate a 5-fold difference, a 50-fold difference, or something else? Please clarify the specific fold change or provide a precise numerical range.

Author Response

Dear Reviewer,

 

Thanks a lot for having reviewed our manuscript (plants-4361047). We have revised the manuscript, and would like to submit it for your consideration. According to your comments and suggestions, we have made corresponding changes. The revisions have been highlighted in the revised manuscript.

 

I greatly appreciate both your help and that of the referees concerning improvement to this paper. Below you can find point-to-point responses to Reviewers’ comments. We hope that the revised version of the manuscript is now acceptable for publication in your journal.

 

I look forward to hearing from you soon.

 

We would like to express our sincere thanks again to you for the constructive and positive comments.

 

With best wishes,

 

Yours sincerely,

Pengyuan Xie

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

I go through the manuscript and found it a very interesting however lacks scientific novelty and significance as well as written in a very non scientific language. The manuscript need a major revision by reanalyzing the genome wide section. Putting some more data and analyzing some new sights. I suggest the authors to read some similar work high quality papers such as 10.1016/j.scienta.2022.111341 and make correction to the current manuscript. Exactly follow the given article and make corrections accordingly. I hope the authors will make significant and serious revision. Secondly, the abstract is too long and written in a very non-scientific way. The introduction is too long and contains too much irrelevant information. Other parts such as materials and methods, results, discussion also require a significant revision. Thirdly, the figures needs to be redrawn using some professional software such as PRISM or others. Add one graphical abstract/schematic illustration figure to the manuscript to encapsulate the whole story in one picture. 

After making a significant and serious revision I accept the manuscript for publication. Good luck.

Author Response

Dear Reviewer,

 

Thanks a lot for having reviewed our manuscript (plants-4361047). We have revised the manuscript, and would like to submit it for your consideration. According to your comments and suggestions, we have made corresponding changes. The revisions have been highlighted in the revised manuscript.

 

I greatly appreciate both your help and that of the referees concerning improvement to this paper. Below you can find point-to-point responses to Reviewers’ comments. We hope that the revised version of the manuscript is now acceptable for publication in your journal.

 

I look forward to hearing from you soon.

 

We would like to express our sincere thanks again to you for the constructive and positive comments.

 

With best wishes,

 

Yours sincerely,

Pengyuan Xie

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have addressed all of comments and there are no further comments. 

Author Response

Response to Reviewer #2
We sincerely thank the reviewer for the thorough evaluation and positive recognition of our revised manuscript. We greatly appreciate the constructive comments and valuable suggestions provided during the previous round of review, which have substantially improved the scientific quality, logical coherence, and clarity of our work. All comments have been carefully considered, and corresponding revisions have been fully incorporated into the manuscript as requested. We are grateful for the time and effort the reviewer has devoted to assessing our work, which has been instrumental in refining this manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

Please compress and shorten the introduction upto three scientific and logical paragraphs with the last paragraph must include the key problem, hypothesis, aims, objectives and implications of the study. Also make corrections to the minor typos (Line 576 etc) such as the first letter in some words of headings are capital. I think keep the first letter of first word capital while following small letter. Also,  interlink all the citations to the corresponding references (Clickable) and the mention of table and figure in main text to the corresponding table and figure (Clickable). Make correction to the schematic illustration figure Imidazolinone Tolerance as Imidazolinone tolerance. The figure legends needs to be scientific and without the use of abbreviations. Figure legends should be independent of abbreviations. Make corrections. Some text in most of the figures are invisible. Almost invisible such as 7, 10, 11 etc. Please make corrections. Please produce high quality figures and enlarge the figures in the file to be more clear and high visibility. Line 16 the scientific names and genes should be kept italic throughout the manuscript. protein names should be kept straight. 

Author Response

Please see the attachment.

Author Response File: Author Response.docx