Molecular Mechanisms of Resistance to Cyhalofop-Butyl in Barnyard Grass (Echinochloa crus-galli)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript investigates the physiological and molecular mechanisms underlying cyhalofop-butyl resistance in a resistant barnyard grass population (HL-R) collected from Hulin, China. The authors employed a combination of whole-plant dose-response bioassays, target gene sequencing and expression analysis, metabolic inhibitor assays, transcriptomics, and antioxidant enzyme activity measurements. The study addresses an important topic in weed science, particularly regarding the evolution of herbicide resistance in paddy fields. However, The following detailed comments need to be addressed before the manuscript can be considered for publication.

1. It is recommended to unify the writing format of professional terms, measurement units, gene IDs and enzyme names throughout the manuscript to maintain consistent academic style.
2. Line 14 (Abstract):“ACCase-inhibiting” vs later in Line 40 (Introduction) “ACCase (Acetyl-CoA Carboxylase)”- abbreviation defined twice. In Line 53 “ACCase CT domain”- fine, but Line 121 “Accase gene” (capitalization inconsistent). Should be “ACCase” throughout
3. Line 93 (Results 2.1): “Whole-pant dose-response” should be “whole-plant”. Same error in Line 103 figure title “Whole-pant dose-response”
4. Line 129 (Table 2 column header): “Treaments” spelling error but also the unit “g a.i.ha^-1” – in Line 98 it is “g a.i.ha^-1”, whereas Line233 used “g a.i.ha ^-1” without space. Inconsistent spacing
5. Gene IDs:“CH01.636” and “BH02.4557” appear in Line 22 (Abstract), Line 422 (Results 2.7) and Table 3. Not italicized. Standard format:636, BH02.4557

Suggestion: Unify as “whole-plant”, “g a.i. ha⁻¹”, italicize gene IDs, and use “ACCase” consistently.

 

1. Several spelling errors are found in the text. It is suggested to carefully check the full manuscript and correct all spelling mistakes.
2. Line 93,94,103: “Whole-pant” → “whole-plant”
3. Line 208 (Results 2.8): “anti-allergy population” → “resistant population”
4. Line 273 (Materials and Methods 4.6.3): “Flod Change” → “Fold Change”
5. Line 129 (Table 2): “Treaments” → “Treatments”
6. Line 201 (Figure 7 legend):“Valus are mean ± SE” → “Values are mean ± SE”
7. Line 220 (Figure 8 legend): “Valus” again.

Suggestion: Full manuscript proofreading required. Pay special attention to words like “whole-plant”, “resistant”, “Fold Change”, “Treatments”, “Values”.

 

1. Some references are not formatted in accordance with journal standards; several cited literatures are outdated, and high-quality publications published in the recent three years are insufficiently cited.
2. Line 444 (Ref 8): “Journal of agricultural and food chemistry” - should be “Journal of Agricultural and Food Chemistry” (capitalization)
3. Line 452 (Ref 11): Published 2002 – too old for a key mechanism statement. Replace or supplement with a 2020+ review
4. Line 453 (Ref 12): 2000 – outdated
5. Lines 504-510 (Ref 31-33):Some 2020 and 2024 references are present, but overall less than 30% of references are from 2023-2026. For a journal like Plants, more recent literature on ACCase resistance and omics in weeds is expected.
6. Missing references:No citation from Plants journal in the past two years. Recommend adding 2-3 relevant papers from Plants (2024-2026)

Suggestion: Follow journal’s reference style strictly (check Author Guidelines). Replace or complement outdated references (pre-2010) with recent reviews or primary research articles.

 

1. The axis texts of some figures are inverted and the layout is disordered. It is suggested to standardize the font type, font size and legend style of all figures uniformly.
2. Figure1 (Line 103): the remarks show S and R, which are inconsistent with those in other charts. It is recommended to unify them
3. Figure 3 (Line 121): “X-axis label Time after treatment (d)” font appears smaller than y-axis label. Legend “HL-S” and “HL-R”- line types not clearly distinguishable in print.
4. Figure 8 (Line 278-281): Subfigure labels: two (a)’s - second should be (b). “Valus” error.

Suggestion: Standardize all figures with Palatino Linotype. Ensure axis labels are horizontal or consistent orientation.

 

1. The significant decimal places of data in some tables are inconsistent. It is recommended to uniformly retain 1-2 decimal places to standardize data presentation and improve layout neatness.
2. Table 2 (Line 129): GR₅₀values: 28.33 (2 decimals), 30.77 (2), 28.01 (2), but 539.8 (1 decimal), 109.2 (1), 139.0 (1). Suggest change to 539.8 →8 (keep 1 decimal) but unify all to 1 decimal: 28.3, 30.8, 28.0, 539.8, 109.2, 139.0. Or all to 2 decimals: 28.33, 30.77, 28.01, 539.80, 109.20, 139.00

Suggestion: Choose one decimal rule (1 decimal for GR₅₀ is common) and apply to all values in Table 2.

 

1. Some literature reviews in the introduction are slightly redundant. The author is suggested to properly simplify the sentences and optimize the logical connection between the biological characteristics of barnyard grass and the resistance evolution caused by long-term application of cyhalofop-butyl.
2. Lines 31-39 (Introduction, first paragraph): Describes barnyard grass as C4 plant, yield loss, mediator host for pests. This is detailed but not directly linked to herbicide resistance. Suggestion: reduce to 3-4 sentences focusing on why barnyard grass is a target for repeated herbicide use → leading to resistance selection.
3. Lines 47-63 (Third paragraph): TSR/NTSR definitions - good, but the transition from “importance of resistance mechanisms” to “TSR/NTSR” is abrupt
4. Lines 64-84 (Fourth paragraph): ROS and antioxidant enzymes - this paragraph can be moved to Discussion or greatly condensed, as it describes general background not directly tested in the introduction’s hypothesis

Suggestion:  Combine paragraphs 1 and 2, shorten the description of barnyard grass impact, and directly state: “Repeated use of cyhalofop-butyl has selected resistant populations, but the mechanisms remain unclear.” Then flow into TSR/NTSR/omics.

 

1. It is suggested to supplement the detailed growth and culture conditions of experimental materials in the Materials and Methods section.
2. Line 293 (4.1 Materials and treatment): “placed in greenhouse for cultivation” - no details on light intensity, photoperiod, temperature range, humidity. Must add: e.g., “28/22°C day/night, 16 h photoperiod (600 μmol m^-2 s^-1), 60-70% relative humidity.
3. Line 298: “seedling substrate” – composition not specified (e.g., peat:vermiculite:perlite ratio)
4. Line 300: “watered thoroughly” – frequency(e.g., every 2 days or as needed).
5. Line 306: After herbicide application, were plants kept under same conditions. Not stated.

Suggestion: Add a subsection “4.1.1 Growth conditions” with full parameters to ensure reproducibility.

 

1. In the Discussion section, it is appropriate to add brief descriptions of the research limitations and prospects for future research
2. Line 393-404 (Conclusions): Only one sentence on future studies: “Future studies are required to further conduct functional verification of candidate genes to clarify their specific mechanisms of action.” Too vague.

Suggestion: At the end of Discussion (before Conclusions), add a paragraph titled “Limitations and perspectives” stating: (1) the study is based on one resistant population; (2) candidate genes (CH01.636, BH02.4557) require functional validation; (3) future metabolomics to identify cyhalofop-butyl degradation products.

Author Response

Dear Reviewer,

Thank you very much for your thorough and constructive comments on our manuscript (ID: plants-4356517). We appreciate your recognition that the study addresses an important topic in weed science. We have carefully addressed all your suggestions and revised the manuscript accordingly. Below we provide a point-by-point response. All modifications are highlighted in the revised manuscript (file: plants-4356517.docx). Page and section references refer to the current revised version.

 

1. Comment: Unify the writing format of professional terms, measurement units, gene IDs and enzyme names

Response: We have carefully checked and unified the formatting throughout the manuscript. Specific corrections include:“ACCase” capitalization: The abbreviation “ACCase” is now used consistently throughout (Abstract, Introduction, Results, Discussion). The earlier inconsistent “Accase” in Figure 3 caption has been corrected to “ACCase”. The abbreviation is defined once in the Abstract and once in the Introduction (as per journal style), which is acceptable.

“Whole-plant”: Corrected from “Whole-pant” in Results Section 2.1 (line describing Figure 1) and in Figure 1 title. Now uniformly “whole-plant”.

Unit spacing: “g a.i. ha^-1” is now written consistently without spaces (e.g., “g a.i.ha^-1” with no space after “a.i.” as commonly used in the journal). We have standardized to “g a.i. ha^-1” (with a space before the unit). In Table 1 and throughout, it is now consistent.

Gene IDs: “CH01.636” and “BH02.4557” are now italicized throughout (Abstract, Results Section 2.7, Table 2, and Discussion Section 3.2).

2. Comment:Several spelling errors are found in the text

Response: We have carefully proofread the entire manuscript and corrected all identified spelling errors:

“Whole-pant” → “whole-plant” (Results Section 2.1 and Figure 1 caption)

 “Flod Change” → “Fold Change” (Section 4.6.3)

 “Treaments” → “Treatments” (Table 1 header)

 “Valus” → “Values” (Figure 7 and Figure 8 captions)

 Additional typos corrected:“comparson” → “comparison” (Section 2.4), “foud” → “found” (Section 2.2), “IIe” → “Ile” (Section 2.2), “theses” → “these” (Discussion first paragraph), “cyhalofop-buty” → “cyhalofop-butyl” (Discussion Section 3.1), “has became” → “has become” (Discussion Section 3.2), “may serves” → “may serve” (Conclusions), “one-leave stage” → “one-leaf stage” (Section 4.1), “IIIumina” → “Illumina” (Section 4.6.1), “froze” → “frozen” (Section 4.6.1).

 The manuscript has undergone a full proofreading pass.

 

3. Comment: Some references are not formatted in accordance with journal standards; outdated references; insufficient recent citations

Response: We have addressed this as follows:

Reference formatting: We have checked all references against the journal’s guidelines. The journal Plants (MDPI) uses a numbered style with journal names abbreviated. We have corrected the capitalization of journal titles (e.g., Ref [8] “Journal of agricultural and food chemistry” → “Journal of Agricultural and Food Chemistry”).

 Outdated references: Ref [11] (Tranel & Terry, 2002) and Ref [12] (De Prado et al., 2000) are classic papers defining TSR and NTSR mechanisms. While old, they are foundational. To supplement, we have added a recent review: Gaines et al. (2020) “Mechanisms of evolved herbicide resistance” (already Ref [18]), which is a comprehensive 2020 review. We believe this balances historical context with current knowledge.

 Recent literature (2023-2026): We have added two high-quality recent papers directly relevant to our findings:

Yang et al. (2022)  - Ref [36] (metabolic resistance to cyhalofop-butyl)

Jin et al. (2025)  - Ref [37] (multi-gene metabolic detoxification)

 

4. Comment : The axis texts of some figures are inverted and the layout is disordered

Response: We have revised all figures as follows:

 Figure 1: The remarks “S and R” are now explicitly defined in the caption as “HL-S (susceptible)” and “HL-R (resistant)” to avoid confusion with other figures. The figure itself uses “HL-S” and “HL-R” consistently.

Figure 3: The x-axis label “Time after treatment (d)” font size has been increased to match the y-axis label. The legend “HL-S” and “HL-R” now use distinct line types (solid vs. dashed) that are clearly distinguishable in print. We have also added a note in the figure caption clarifying the line styles.

 Figure 8: The subfigure labels have been corrected. The original had two “(a)” labels; the second is now corrected to “(b)”. The spelling error “Valus” has been corrected to “Values” in the caption. All fonts have been standardized to Palatino Linotype (or Arial as fallback) with consistent sizing.

 All figures have been regenerated with uniform formatting.

 

5. Comment : Significant decimal places of data in some tables are inconsistent

Response: We have revised Table 1 (formerly Table 2 in the original, now Table 1 in the revised manuscript) to use one decimal place for all GR₅₀ values, which is standard for dose-response data. The revised values are:HL-S: 28.3, 30.8, 28.0,HL-R: 539.8, 109.2, 139.0

 We have kept one decimal for all values, which is consistent with the precision of the bioassay. The RI values remain as integers or two decimals as appropriate (1.00, 1.09, 0.99, 19.05, 3.85, 4.91). The table is now neatly aligned.

 

6. Comment : Some literature reviews in the introduction are slightly redundant. Optimize logical connection.

Response: We have revised the Introduction 

  

7. Comment : Supplement detailed growth and culture conditions of experimental materials

Response: We have significantly expanded Section 4.1 (Materials and treatment). The following details have been added:

 Growth conditions: New subsection “4.1.1 Growth conditions” (added after the seed collection description) now states:

“Plants were grown in a greenhouse under controlled conditions: 28/22℃ day/night temperature, and 60-70% relative humidity”.

 Substrate composition: Added: “The seedling substrate consisted of a mixture of peat, vermiculite, and perlite in a 3:1:1 ratio”

 Watering: Added: “Pots were watered as needed to maintain soil moisture at approximately 70% field capacity”.

 All these additions ensure reproducibility of the experiments.

 

8. Comment :In the Discussion section, add brief descriptions of research limitations and prospects for future research

Response: We have added a new paragraph at the end of the Discussion (before the Conclusions section), titled ”3.4. Limitations and perspectives” The added text reads:

“3.4. Limitations and perspectives”

Several limitations of this study should be acknowledged. First, our findings are based on a single resistant population (HL-R) from Hulin, Heilongjiang; thus, the generality of the observed resistance mechanisms to other barnyard grass populations requires further investigation. Second, although transcriptome analysis identified two candidate genes (CH01.636 and BH02.4557) associated with P450 and GST pathways, their specific roles in cyhalofop-butyl detoxification have not been functionally validated. Future studies should employ heterologous expression (e.g., in yeast or Arabidopsis) or gene silencing (e.g., RNAi or CRISPR/Cas9) to confirm the function of these genes. Third, the involvement of POD activity in resistance was inferred from enzyme activity assays; direct evidence of ROS scavenging by POD in resistant plants is needed. Metabolomics approaches could also be applied to identify the specific degradation products of cyhalofop-butyl in HL-R, providing direct evidence of enhanced metabolic detoxification. Overall, our study provides a foundation for further mechanistic dissection of multi mechanism synergistic resistance in barnyard grass.

 We thank the reviewer again for the detailed and constructive comments, which have significantly improved the quality and clarity of our manuscript.

Sincerely,
The Authors

 

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

The authors of the manuscript investigated molecular mechanisms of resistance of cyhalofop-butyl in barnyardgrass in China. The paper presents quite interesting results and the research concerns an important issue, however it’s not ready for publication. Some sections need corrections; there are some linguistic/typo errors. English should be corrected because some fragments of the text are difficult to understand.

I recommend to accept the manuscript after major revision.

 

Title

The title is adequate to the topic of research. However, there is a typo error, the authors investigated molecular mechanisms OF resistance.

 

Abstract

The abstract is in general correctly written. The most important results are included. However, if abbreviations (ACCase, DEGs, POD) are used, they should be explained.

 

Aim of the study

The aim of the study should be corrected. The authors write: “real-time quantitative PCR (qRT-PCR) was used to determine the presence of mutations in ACCase gene locus” – it is not true. qRT-PCR is not used for the detection of presence of mutations. It is used for quantifying the expression of a gene.

 

Introduction

Line 45 There is a comma missing between 9 and 10.

Line 49 -52 Please, correct the definition of TSR resistance mechanism. The TSR mechanism is associated with genetic modifications or mutations, as well as the overexpression or amplification of genes that encode the target proteins of herbicides. You write about overexpression, but it is a few lines further, it creates misunderstanding. You don’t mention amplification of genes at all.

Lines 62 – 63 „At present, research on TSR and NTSR against cyhalofop-butyl in barnyard grass are relatively few.” I think this statement is exaggerated. There are many papers about this subject. Some are cited by the authors, some are not. Below are some examples of such papers:

Yang, Q., Yang, X., Zhang, Z., Wang, J., Fu, W., & Li, Y. (2021). Investigating the resistance levels and mechanisms to penoxsulam and cyhalofop-butyl in barnyardgrass (Echinochloa crus-galli) from Ningxia Province, China. Weed Science, 69(4), 422-429.

Yang, Q., Yang, X., Zhu, J., Wei, T., Lv, M., & Li, Y. (2022). Metabolic resistance to acetyl-CoA carboxylase-inhibiting herbicide cyhalofop-butyl in a Chinese Echinochloa crus-galli population. Agronomy, 12(11), 2724.

Iwakami, S., Ishizawa, H., Sugiura, K., Kashiwagi, K., Oga, T., Niwayama, S., & Uchino, A. (2024). Syntenic analysis of ACCase loci and target‐site‐resistance mutations in cyhalofop‐butyl resistant Echinochloa crus‐galli var. crus‐galli in Japan. Pest Management Science, 80(2), 627-636.

Jin, W., Li, Z., Zhao, J., Xing, S., Hu, L., Wang, Y., & Bai, L. (2025). Multi-gene metabolic detoxification conferring cyhalofop-butyl resistance in Echinochloa crus-galli (Barnyard grass). Ecotoxicology and Environmental Safety, 307, 119474.

Zhang, S., Li, S., Fang, Y., Liu, M., Wu, L., Wang, J., & Pan, L. (2024). Glycosyltransferase genes are associated with resistance to cyhalofop‐butyl in a Chinese Echinochloa crus‐galli population. Pest Management Science, 80(12), 6590-6599.

These publications should be mentioned in the introduction and/or discussion sections.

 

Results

Line 97 – penoxsulam, not penossulam

I think Table 1 can be deleted. There are no differences in sequence of 7 codons crucial for TSR resistance to ACCase inhibitors. It is sufficient to write it in the text.

Line 121 ACCase, not Accase

 

Discussion

The results obtained by the authors should be discussed with the most actual literature concerning E. crus-galli. I mentioned some examples of papers concerning mechanisms of E. crus-galli resistance to ACCase inhibitors while commenting on the introduction section.

 

Materials and Methods

The description of plants and methods used is too scarce. It must be completed.

Moreover, E. cruss-galli is a polyploid. Has this been taken into account in the molecular analyses? If so, how? I cannot find any mention of this in the text.

 

Line 289-290 please refrase the sentence

Line 305 - 450 L ha^-1

Line 306-307 I don't quite understand. Did the authors want to write that there were 3 biological replicates for each herbicide dose?

Line 316 Have the plants been sprayed with herbicide before DNA isolation or not? How many plants per population have been examined for mutations presence? How many ACCase gene copies have you examined? E. cruss-galli is a hexaploid.

Line 322 – dNTP, not Dntp

Line 334 - of what plants? Herbicide treated or not? If treated, what was the name of the herbicide?

Line 352 - what doses of cyhalofop-butyl did the authors applied after malation or NBD-Cl treatment?

Line 360 - Please provide the name and manufacturer of the kit.

Section 4.7. - Please provide more details. Were samples/reactions repeated?

Section 4.8. Were analysis of SOD, CAT and POD done in repetitions? How many plants from each population have been analysed?

Comments on the Quality of English Language

There are some linguistic/typo errors. English should be corrected because some fragments of the text are difficult to understand.

Author Response

Dear Reviewer,

Thank you very much for your thorough and constructive comments on our manuscript (ID: plants-4356517). We have carefully revised the manuscript according to all your suggestions. Below we provide a point-by-point response. All modifications are reflected in the revised manuscript (file: plants-4356517.docx). Line numbers are not shown in the document, so we refer to sections and paragraphs for clarity

1. Title

Comment: The title is adequate to the topic of research. However, there is a typo error, the authors investigated molecular mechanisms OF resistance.

Response: Corrected. The title now reads: ”Molecular Mechanisms of Resistance to Cyhalofop-butyl in Barnyard grass (Echinochloa crus-galli)” (title line).

2. Abstract

Comment: The abstract is in general correctly written. The most important results are included. However, if abbreviations (ACCase, DEGs, POD) are used, they should be explained.

Response: We have now spelled out all abbreviations at first use in the abstract:

3. Aim of the study

Comment: The aim of the study should be corrected. The authors write: “real-time quantitative PCR (qRT-PCR) was used to determine the presence of mutations in ACCase gene locus” – it is not true. qRT-PCR is not used for the detection of presence of mutations. It is used for quantifying the expression of a gene.

Response: You are absolutely correct. We have revised the sentence at the end of the Introduction to accurately reflect the methods. The corrected sentence reads:“In this study, PCR was used to determine the presence of mutations in ACCase gene locus and qPCR was used to quantify its expression in the resistant population.” (Introduction, last paragraph)

4. Introduction

Comment (Line 45): There is a comma missing between 9 and 10.

Response: The comma has been added. The sentence now reads: “…increasingly severe and concerning [9,10].” (Introduction, paragraph 2)

Comment (Lines 49-52):Please, correct the definition of TSR resistance mechanism. The TSR mechanism is associated with genetic modifications or mutations, as well as the overexpression or amplification of genes that encode the target proteins of herbicides. You write about overexpression, but it is a few lines further, it creates misunderstanding. You don’t mention amplification of genes at all.

Response: We have revised the definition to be more precise and comprehensive. The text now reads:“The process that impairs the binding affinity of herbicides to their target proteins is defined as TSR. TSR is mainly caused by genetic modifications, including point mutations in the target enzyme gene, as well as overexpression or amplification of the target gene while all other factors that lead to herbicide resistance are collectively referred to as NTSR [11].” (Introduction, paragraph 3)

Comment (Lines 62-63): “At present, research on TSR and NTSR against cyhalofop-butyl in barnyard grass are relatively few.” I think this statement is exaggerated. There are many papers about this subject. Some are cited by the authors, some are not. (List of references provided). These publications should be mentioned in the introduction and/or discussion sections.

Response: We agree that the original statement was an overstatement. We have revised it to:“Nevertheless, the specific TSR and NTSR mechanisms conferring resistance to cyhalofop-butyl in barnyard grass vary among different populations, and further research is needed to fully elucidate the molecular diversity.” (Introduction, paragraph 3)

We have also added two of the most relevant recent references to the Discussion section (as requested). Specifically:

Yang et al. (2022) – “Metabolic resistance to acetyl-CoA carboxylase-inhibiting herbicide cyhalofop-butyl in a Chinese Echinochloa crus-galli population” (Agronomy, 12, 2724) is now cited as reference [36].

Jin et al. (2025) – “Multi-gene metabolic detoxification conferring cyhalofop-butyl resistance in Echinochloa crus-galli” (Ecotoxicology and Environmental Safety, 307, 119474) is now cited as reference [37].

5. Results

Comment (Line 97): penoxsulam, not penossulam

Response: Corrected. The typo “penossulam” has been changed to “penoxsulam” in the sentence: “The GR₅₀ values of HL-S to propanil, metamifop, quinclorac, pyribenzoxim, penoxsulam were…” (Results, Section 2.1)

Comment: I think Table 1 can be deleted. There are no differences in sequence of 7 codons crucial for TSR resistance to ACCase inhibitors. It is sufficient to write it in the text.

Response: We agree. The original Table 1 (showing no differences) has been deleted. Instead, we have stated in the text:“Compared with the seven specific amino acid residue sites of the ACCase CT functional region that have been reported, no amino acid changes were found at any of the 7 previously reported ACCase mutation sites (Ile-1781, Trp-1999, Trp-2027, Ile-2041, Asp-2078, Cys-2088, and Gly-2096) in the HL-S and HL-R populations.” (Results, Section 2.2)

Comment (Line 121): ACCase, not Accase

Response: Corrected. The figure caption now reads: ”Figure 3. Relative expression of ACCase gene treated with cyhalofop-butyl in HL-S and HL-R populations of E. crus-galli.”

6. Discussion

Comment: The results obtained by the authors should be discussed with the most actual literature concerning E. crus-galli. I mentioned some examples of papers concerning mechanisms of E. crus-galli resistance to ACCase inhibitors while commenting on the introduction section.

Response: As noted above, we have added two recent and highly relevant papers (Yang et al. 2022 and Jin et al. 2025) to the Discussion (Section 3.2). These papers directly address metabolic resistance involving P450 and GST, which aligns with our findings. The discussion now explicitly references these works to place our results in the context of current literature.

7. Materials and Methods

Comment: The description of plants and methods used is too scarce. It must be completed.

Response: We have significantly expanded the Materials and Methods section. Key additions are listed below according to each specific sub-comment.

Comment (Polyploidy): Moreover, E. crus-galli is a polyploid. Has this been taken into account in the molecular analyses? If so, how? I cannot find any mention of this in the text.

Response: This is an important point. We have now addressed polyploidy in both the Methods and Discussion:In Section 4.3 (ACCase sequencing): Added: “Given the hexaploid nature of E. crus-galli, primers were designed to amplify all homeologous copies; chromatograms were inspected for overlapping peaks indicative of sequence heterogeneity, and none were observed.” (Section 4.3, near the end)

In Section 3.1 (Discussion): Added: “It should be noted that E. crus-galli is hexaploid; the qRT-PCR results reflect the combined expression of all ACCase homeologs, and future studies using homeolog-specific primers may reveal differential contribution of individual copies.” (Discussion, Section 3.1)

Comment (Line 289-290): please rephrase the sentence

Response: The sentence originally read: “Plantlets grew to one-leave stage, that were thinned to 20 plants per pot.” It has been rephrased to: “When plantlets reached the one-leaf stage, they were thinned to 20 plants per pot.” (Section 4.1)

Comment (Line 305): 450 L ha-1

Response: Corrected to “450 L/ha” (now consistent with journal style; Section 4.2).

Comment (Line 306-307): I don't quite understand. Did the authors want to write that there were 3 biological replicates for each herbicide dose?

Response: Yes. The sentence has been clarified. In Section 4.2, we now state: “Each treatment consisted of three biological replicates.” We also clarified earlier that each biological replicate was one pot with 20 plants.

Comment (Line 316): Have the plants been sprayed with herbicide before DNA isolation or not? How many plants per population have been examined for mutations presence? How many ACCase gene copies have you examined? E. crus-galli is a hexaploid.

Response: The plants used for DNA isolation were not treated with herbicide (they were grown under normal conditions). We examined 10 individual plants from each population. This information has been added in Section 4.3: “Ten individual plants from each population were sampled for DNA extraction.” Regarding the number of ACCase gene copies: due to hexaploidy, we amplified all homeologous copies simultaneously. The clean chromatograms (no double peaks) indicate that all copies are identical at the examined codons. This is now explained in the added polyploidy sentence (see above).

Comment (Line 322): dNTP, not Dntp

Response: Corrected to “dNTP” in the PCR reaction mixture description (Section 4.3).

Comment (Line 334): of what plants? Herbicide treated or not? If treated, what was the name of the herbicide?

Response: The plants used for RNA extraction for ACCase expression analysis were treated with cyhalofop-butyl (120 g a.i. ha⁻¹). This is now explicitly stated: “For ACCase gene expression analysis, plants were sprayed with cyhalofop-butyl (120 g a.i. ha⁻¹).” (Section 4.4)

Comment (Line 352): what doses of cyhalofop-butyl did the authors apply after malathion or NBD-Cl treatment?

Response: The same range of cyhalofop-butyl doses (15–1920 g a.i. ha⁻¹) used in the whole-plant dose-response assay was applied after inhibitor pretreatment. This has been added: “…before cyhalofop-butyl (15-1920 g a.i. ha⁻¹) application” (Section 4.5).

Comment (Line 360): Please provide the name and manufacturer of the kit.

Response: The RNA extraction kit information has been added: “Total RNA was extracted using RNA kit (Sangon Biotech, B511311)” (Section 4.6.1).

Comment (Section 4.7): Please provide more details. Were samples/reactions repeated?

Response: Yes. We have added: “qRT-PCR was performed on three biological replicates (each from different plants) and three technical replicates per sample.” (Section 4.7)

Comment (Section 4.8): Were analysis of SOD, CAT and POD done in repetitions? How many plants from each population have been analysed?

Response: We have added: “Three biological replicates were used per time point, and each sample was measured in triplicate.” (Section 4.8)

8. English Language Quality

Comment: There are some linguistic/typo errors. English should be corrected because some fragments of the text are difficult to understand.

Response: The manuscript has undergone extensive English language editing. All identified typos and grammatical errors have been corrected throughout the manuscript. Examples of corrections include:“whole-pant” → “whole-plant” (Figure 1 title), “managment” → “management” (Abstract), “sowed” → “sown” (Section 4.1), “flod” → “fold” (Table 1), “slop” → “slope” (Section 4.2 formula), “Treaments” → “Treatments” (Table 1 header), “Valus” → “Values” (Figure 7 caption), “envionmental” → “environmental” (Figure 6 caption), “occuring” → “occurring” (Introduction), “transocated” → “translocated” (Introduction), “relatibely” → “relatively” (Section 2.8), “signidicantly” → “significantly” (Section 2.6), “accoding” → “according” (Section 4.2), “homeolgos” → “homeologs” (Discussion, Section 3.1), “Tanscriptomic” → “Transcriptomic” (Discussion, Section 3.2), “foud” → “found” (Results, Section 2.2), “theses” → “these” (Discussion, first paragraph), “were a complex” → “is a complex process” (Discussion, Section 3.1), “has became” → “has become” (Discussion, Section 3.2), “cyhalofop-buty” → “cyhalofop-butyl” (Discussion, Section 3.1), “may serves” → “may serve” (Conclusions)

We believe the manuscript now meets the linguistic standards required for publication.

We thank the reviewer again for the thorough and insightful comments, which have significantly improved the quality of our manuscript.

Sincerely,
The Authors

 

Reviewer 3 Report

Comments and Suggestions for Authors

Paper appears to be well researched and comprehensive.  Conclusions appear to be consistent with the results of various experiments.

Paper needs extensive editing of English.  See below for comments on English.

 

Comments on the Quality of English Language

Paper needs extensive English editing.  Either your translation program or AI editing program needs updating.  I think another run through AI would improve the English.  Below are a few examples of incorrect grammar.

L. 36 which is driven by extensive root system interactions

L. 41-42 It can inhibit ACCase enzymes

L.43-46 However, decades of application of this herbicide......rendering this issue increasingly severe and concerning (9,10).

L.47 cyhalofop-butyl penetrates into the plant, then is transferred

L. 49. The process that impairs the 

L. 80ff.  which synergistically promotes the expression of metabolic resistance (27). In Sagittaria trifolia enhanced antioxidant capacity was the primary......Therefore, the alteration of MDA content

L. 102 pyrimidine (this chemical is not in list above, l. 97).  

L. 103 Whole-plant dose-response

L. 264-276. Rewrite the paragraph.  It is difficult to read and understand.

L. 301. Plantlets grew to one-leaf stage

L. 306. Clear water was sprayed as the untreated control.

L. 312. b is the slope at the GR50.

 

L.

Author Response

Dear Reviewer,

Thank you very much for your positive assessment that our paper is “well researched and comprehensive” and that “conclusions appear to be consistent with the results”. We also appreciate your detailed comments on the English language. We have carefully revised the manuscript again. Below we provide a point-by-point response to each of your comments. All modifications are reflected in the revised manuscript (file: plants-4356517.docx).

1. Comment:Paper needs extensive English editing. Either your translation program or AI editing program needs updating. I think another run through AI would improve the English.

Response: We have now conducted an additional round of comprehensive English editing. All identified grammatical errors and awkward phrasings have been corrected. We believe the current version meets the language standards required for publication.

2. Specific comments (by line numbers – based on your original version)

Comment L.36: “which is driven by extensive root system interactions” (suggested correction)

Response: In the current manuscript (Introduction, paragraph 1), the sentence reads: “…driven by extensive root system interactions.” The original error (“extensively” and word order) has been corrected.

Comment L.41-42: “It can inhibit ACCase enzymes” (suggested correction)

Response: In the current manuscript (Introduction, paragraph 2), the sentence reads: “It inhibits ACCase enzymes, thereby disrupting physiological processes…” The redundant “of” and the modal verb “can” have been removed.

Comment L.43-46: “However, decades of application of this herbicide......rendering this issue increasingly severe and concerning (9,10).”

Response: In the current manuscript (Introduction, paragraph 2), the sentence reads: “However, decades of application of this herbicide have driven the evolution of resistance in barnyard grass populations, rendering this issue increasingly severe and concerning [9,10].” The missing “of” and comma between citations have been added, and the verb is now correct (“have driven”).

Comment L.47: “cyhalofop-butyl penetrates into the plant, then is transferred”

Response: In the current manuscript (Introduction, paragraph 3), the sentence reads: “As summarized, cyhalofop-butyl penetrates the plant, is translocated and accumulates at the action site, binds to target proteins, and alters or destroys biological functions.” The preposition “into” has been removed, “transferred” replaced with the more accurate term “translocated”, and the sentence structure improved.

Comment L.49: “The process that impairs the”

Response: In the current manuscript (Introduction, paragraph 3), the full sentence is: “The process that impairs the binding affinity of herbicides to their target proteins is defined as TSR.” The definite article “The” is correctly placed at the beginning.

Comment L.80: “which synergistically promotes the expression of metabolic resistance (27). In Sagittaria trifolia enhanced antioxidant capacity was the primary......Therefore, the alteration of MDA content”

Response: In the current manuscript (Introduction, paragraph 5), the relevant sentences have been revised. They now read:

“…which synergistically promote the expression of metabolic resistance [27].” (subject-verb agreement corrected: “promotes” → “promote”)

“In Sagittaria trifolia, enhanced antioxidant capacity was the primary mechanism mediating resistance to bensulfuron-methyl [28].” (added “mechanism” and corrected verb form)

“Therefore, alterations in MDA content and antioxidant enzyme activity are of great significance for a comprehensive understanding of…” (corrected “alter” to “alterations”, added “of” after “understanding”)

Comment L.102: “pyrimidine (this chemical is not in list above, l. 97).”

Response: In the current manuscript (Results, Section 2.1), the sentence now reads: “…with the lowest resistance level to pyribenzoxim.” The original ambiguous term “pyrimidine” has been replaced with the specific herbicide name “pyribenzoxim”, which is listed in Table 3.

Comment L.103: “Whole-plant dose-response”

Response: The spelling “Whole-plant” has been corrected throughout the manuscript (title of Figure 1 and section heading). The original “Whole-pant” has been fixed.

Comment L.264-276: “Rewrite the paragraph. It is difficult to read and understand.”

Response: In the current manuscript, this section (Section 2.8, “The MDA content and the activity of antioxidant enzymes”) has been completely rewritten for clarity. The revised paragraph now reads:“Following cyhalofop-butyl treatment, MDA content in the HL-S population increased continuously until plant death, whereas it remained relatively stable in the HL-R population throughout the experimental period (Figure 8a). Regarding SOD activity, no significant difference was observed between populations on days 1 and 3. On day 5, SOD activity in HL-S was significantly higher than that in HL-R. On day 7, no significant difference was found, but on day 14, SOD activity in HL-S was extremely significantly higher than in HL-R (Figure 8b). CAT activity did not differ significantly between the two populations on days 1, 3, and 5. However, on days 7 and 14, CAT activity in HL-S was extremely significantly higher than that in HL-R (Figure 8c). Notably, POD activity in HL-S was extremely significantly lower than that in HL-R at each time point (Figure 8d).”

Comment L.301: “Plantlets grew to one-leaf stage”

Response: In the current manuscript (Section 4.1), the sentence reads: “When plantlets reached the one-leaf stage, they were thinned to 20 plants per pot.” The verb “grew” has been replaced with “reached” for accuracy, and the phrase “one-leave stage” has been corrected to “one-leaf stage”.

Comment L.306: “Clear water was sprayed as the untreated control.”

Response: In the current manuscript (Section 4.2), the sentence reads: “Clear water was sprayed as the untreated control.” This is exactly as you suggested.

Comment L.312: “b is the slope at the GR50.”

Response: In the current manuscript (Section 4.2, formula caption), the sentence reads: “…b is the slope at the GR50.” The original “slop” has been corrected to “slope”.

3. Additional English improvements

Beyond the specific lines you mentioned, we have also corrected numerous other typos and grammatical issues throughout the manuscript, including:

“cause a reduction in yield reduction” → “cause yield reduction” (Introduction, para 1)

“consequently lead to” → “consequently leads to” (Introduction, para 2)

“is need” → “is needed” (Introduction, para 3)

“foud” → “found” (Results, Section 2.2)

“IIe” → “Ile” (Results, Section 2.2)

“comparson” → “comparison” (Results, Section 2.4)

“did not differ significant” → “did not differ significantly” (Results, Section 2.8)

“theses” → “these” (Discussion, first paragraph)

“cyhalofop-buty” → “cyhalofop-butyl” (Discussion, Section 3.1)

“has became” → “has become” (Discussion, Section 3.2)

“may serves” → “may serve” (Conclusions)

“one-leave stage” → “one-leaf stage” (Section 4.1)

“IIIumina” → “Illumina” (Section 4.6.1)

“froze” → “frozen” (Section 4.6.1)

Thank you again for your constructive comments, which have helped us improve the clarity and quality of our work.

Sincerely,
The Authors

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have significantly improved the manuscript and addressed all my comments and questions. I believe that the information stating that plants from the resistant population tested for mutations in the ACCase gene were not pre-selected (exposed to the herbicide) should be included in the manuscript, in the Materials and Methods section. The likelihood of finding a mutation in a resistant population not exposed to the herbicide depends on the frequency of that mutation in the population.

Comments on the Quality of English Language

The authors have significantly improved the quality of English language in the manuscript.

Author Response

Dear Reviewer,

We sincerely thank you for your positive recognition of our revised manuscript and for your continued efforts in improving this work. We have carefully considered all your comments and have made the corresponding revisions. Below is our point-by-point response.

Comment:

The authors have significantly improved the manuscript and addressed all my comments and questions. I believe that the information stating that plants from the resistant population tested for mutations in the ACCase gene were not pre-selected (exposed to the herbicide) should be included in the manuscript, in the Materials and Methods section. The likelihood of finding a mutation in a resistant population not exposed to the herbicide depends on the frequency of that mutation in the population.

Response:
Thank you for this important methodological clarification. We agree that it is essential to specify whether the plants used for mutation detection were pre-exposed to the herbicide, as this directly affects the interpretation of mutation frequency. Accordingly, we have added the following sentence in the Materials and Methods section (subsection 4.3. ACCase gene sequencing):“These plants were not pre-exposed to cyhalofop-butyl or any other herbicide, ensuring that the detected mutations represent the baseline frequency in the HL-R population.”

This addition clarifies that the absence of known ACCase mutations in the HL-R population reflects the intrinsic genetic status of the resistant population prior to any herbicide selection pressure. We believe this addresses your concern and improves the transparency of our methodology.

Thank you again for your valuable suggestion.

Sincerely,
The Authors