PONE-D-24-43694Transcriptomic analysis reveals cloquintocet-mexyl-inducible genes in hexaploid wheat (Triticum aestivum L.)PLOS ONE

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PLOS ONE

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Dear authors

The authors have done interesting research entitled “Transcriptomic analysis reveals cloquintocet-mexyl-inducible genes in hexaploid wheat (Triticum aestivum L.)”. They have performed transcriptome sequencing of the wheat to find the inducible DEGs involved in some herbicide metabolisms. They have identified candidate genes responsible for halauxifen acid-detoxification and comprehensively discussed the related molecular mechanisms. They have shown that a novel member of the CYP81A subfamily of cytochrome P450s (CYPs) (CYP81A-5A) is capable of catalyzing synthetic auxin detoxification. This finding can be used in the genetic engineering of new herbicide-resistant wheat lines.

I believe that this manuscript is worthy to be accepted.

Regards,

Reviewer #2: The authors conducted a study to identify halauxifen acid (HA) detoxification genes using RNA-Seq. They compared untreated leaf tissue with leaf tissue treated with Cloquintocet-mexyl (CM). Through this analysis, they discovered the specific expression of CYP81A-5A and its homologues (CYP81A-5B and CYP81A-5D) in untreated leaf tissue, as well as in leaf tissue treated with CM and HM over time. While the findings of the study are innovative, further research is needed to fully support the claim that CYP81A-5A plays a role in herbicide detoxification. The results section should provide more detailed information on the number of differentially expressed genes (DEGs) and the gene ontology (GO) terms associated with transferase and oxidoreductase activity.

In summary, this study sheds light on potential HA detoxification genes, but additional studies are necessary to confirm the role of CYP81A-5A in this process.

Abstract:

Authors should provide a more detailed abstract regarding the number of reads that mapped to genes, as well as gene ontology analysis of differentially expressed genes.

Material and methods:

1. Please ensure that the names of all chemicals, the company name, and the percentage purity for each chemical are included in the Materials and Methods section of the manuscript.

2. The wording of line 98 should be formalize.

3. During the seedling stage, how was the plant irrigation performed before the treatment was administered?

4. When an abbreviation is used for the first time, it is not necessary to use the full name for subsequent mentions.

5. In line 120, why only one biological replicate per treatment per timepoint from each experimental replication was used for TaqMan RT-qPCR. It is advisable to include 3-5 biological replicates for each experimental condition in order to accurately measure experimental variation and establish the statistical significance of results. Additionally, it is recommended to conduct at least 3 technical replicates for each cDNA sample to assess assay variability and reduce the likelihood of pipetting errors.

Results:

1. Line 192, add the number of genes found through gene annotation that belong to the following categories: annotations, herbicide/xenobiotic metabolism, stress/defense response, amino acid metabolism, heavy metal binding, and encoded transcription factors. Specifically, indicate which genes were upregulated and which were downregulated in the category of herbicide/xenobiotic metabolism.

2. Line 195, The names of repressed genes need to be included.

3. In line 196, the author stated, "CM represses a few stress-related genes and likely triggers some level of oxidative stress, possibly to fine-tune safener-mediated transcriptional regulation." Author should be providing clarification on the number of genes that were suppressed.

4. The end of sentence of line 220 should be removed and relocated to the Materials and Methods.

5. In line 221, the author should specify the number of Gene Ontology (GO) terms associated with transferase activity, including both glutathione S-transferases (GST) and UDP-glucuronosyltransferases (UGTs), as well as oxidoreductase activity.

6. Line 236 should be shifted to M&M.

7. Which plant is involved with stress responses in UGT85As at line 247? Please include the name of the plant at the end of the sentence.

8. In line 257, the author stated that, "With the exception of TraesCS5A02G472300, these genes are located in tandem gene clusters encoding similar enzymes (Table S5)." Provide clarification on which specific genes are being referred to in this statement?

9. Line 271 should be relocated to the Methodology section of the report, as it is relevant to the methodology employed in the study, rather than the results obtained.

Discussion:

The discussion is too lengthy. It would be beneficial to eliminate redundant and overly informative sentences. For example, the sentence "Results of previous LC-MS [35] and phenotypic experiments [34] indicate that CYP81A-5D alone..." is repeated multiple times throughout the manuscript in the results, discussion, and conclusion sections. It is recommended to streamline the content and avoid unnecessary repetition for better clarity and conciseness.

Conclusion:

It is not customary to include references in the conclusion. Please remove them and instead, highlight the key findings of the study in the conclusion. Lines 415-420 should be shortened, and it is recommended that authors bold their key results for emphasis.

Figures:

- The red boxes in Supplementary Figures S1 and S2 were not sharpened.

- The supplementary figure S2 was not sharpened.

- In Supplementary Figure S4, there are errors in the significant letters for the 12- hours after treatment. Make the necessary corrections.

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Reviewer #1: Yes:  Ali Moghadam

Reviewer #2: Yes:  Maryam Salami

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Submitted filename: PONE-D-24-43694.docx

Transcriptomic analysis reveals cloquintocet-mexyl-inducible genes in hexaploid wheat (Triticum aestivum L.)

PONE-D-24-43694R1

Dear Dr. Landau,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Bahram Heidari

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: (No Response)

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #2: Yes:  Maryam Salami

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