Dear Dr. He,

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Naser A. Anjum, PhD

Academic Editor

PLOS ONE

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In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed.

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Additional Editor Comments:

REVIEWER #1

The manuscript entitled "Physiological and Bn-OASTL Gene Expression Responses to Cadmium Stresses in Tobacco Seedlings" presents a systematic study on the cloning and heterologous expression of the BnOASTL gene and its role in cadmium stress response. The research question is relevant, and the methodology is generally sound. Although the expected enhancement of Cd resistance and accumulation was not observed, the negative findings provide valuable insight for future studies on heavy metal stress mechanisms. The manuscript is generally well-organized and provides sufficient data to support its conclusions.

However, some issues in the abstract, introduction, methods, results discussion, and language presentation need to be addressed to further improve the manuscript's clarity, scientific rigor, and academic value. I recommend a minor revision before acceptance.

1. The title, abstract, and keywords are not fully consistent regarding the research subject, species, and gene information. For example, the title and abstract refer to Bn-OASTL and tobacco, while the keywords include “rapeseed (Brassica napus L.)” instead. Please ensure that these elements are precisely aligned and consistently reflect the main experimental system and research focus.

2. The abstract contains some ambiguous statements and lacks clear logical flow. In particular, the functional analysis of the gene did not show a significant effect in transgenic tobacco, but the discussion on the underlying mechanisms is insufficient. Furthermore, the scientific significance and future perspective of this study in the context of Cd-tolerant crop molecular design are not adequately highlighted. Please refine the abstract to clearly state the main findings (e.g., the lack of improvement in Cd tolerance/accumulation) and emphasize their scientific relevance to the field.

3. The introduction is overly lengthy and the literature review is somewhat scattered. I recommend condensing the introduction to make it more concise, with a clear focus on the scientific question and the innovative aspects of this study. Excessive background information should be removed to avoid redundancy. Please clearly articulate the rationale for selecting BnOASTL, the reason for using tobacco as the heterologous expression system, and the current knowledge gaps that this work aims to address. In addition, the introduction should include a more forward-looking discussion on the potential of the OASTL gene family in molecular breeding and heavy metal phytoremediation, highlighting future directions and scientific significance.

4. Provide more experimental details, such as specific reagent sources, PCR/qPCR conditions, and statistical methods to improve reproducibility. Ensure all primers, vectors, and key reagents are clearly described.

5. Enhance the interpretation of the results, especially the possible reasons why overexpression of BnOASTL did not improve Cd tolerance. Discuss the broader significance and possible directions for future research, such as the need for multi-gene manipulation or systems biology approaches.

6. Ensure that all figures and legends are sufficiently detailed, and clearly indicate statistical significance where applicable.

7. Please ensure that all references are properly formatted and include DOI numbers where available. Including DOIs for all cited literature is required by the journal and will improve the accessibility and traceability of your references.

REVIEWER #2

The manuscript entitled "Physiological and Bn-OASTL Gene Expression Responses to

Cadmium Stresses in Tobacco Seedlings" deal with an original subject within the plant soil interactions field. neertheless the ms at its present form is not suitable for publication:

-the title did not much the Ms. the is no physiological study it is just a monitoring of Cd and GSH contents

-in the ms authors present a work on tobacco plants however in conclusion they change to tomato???

-molecular analyses are goods however the discussion is not appropriated to the founded results. it is clear from results that theBn-OASTL Gene has no relatioship with the resistance beahavior within tobacco plant meaning thet tobacco did not use the g O-acetylserine(thiol)lyase pathway to overcome the Cd stress.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

Reviewer #1: Yes

Reviewer #2: Partly

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

Reviewer #1: The manuscript entitled "Physiological and Bn-OASTL Gene Expression Responses to Cadmium Stresses in Tobacco Seedlings" presents a systematic study on the cloning and heterologous expression of the BnOASTL gene and its role in cadmium stress response. The research question is relevant, and the methodology is generally sound. Although the expected enhancement of Cd resistance and accumulation was not observed, the negative findings provide valuable insight for future studies on heavy metal stress mechanisms. The manuscript is generally well-organized and provides sufficient data to support its conclusions.

However, some issues in the abstract, introduction, methods, results discussion, and language presentation need to be addressed to further improve the manuscript's clarity, scientific rigor, and academic value. I recommend a minor revision before acceptance.

1. The title, abstract, and keywords are not fully consistent regarding the research subject, species, and gene information. For example, the title and abstract refer to Bn-OASTL and tobacco, while the keywords include “rapeseed (Brassica napus L.)” instead. Please ensure that these elements are precisely aligned and consistently reflect the main experimental system and research focus.

2. The abstract contains some ambiguous statements and lacks clear logical flow. In particular, the functional analysis of the gene did not show a significant effect in transgenic tobacco, but the discussion on the underlying mechanisms is insufficient. Furthermore, the scientific significance and future perspective of this study in the context of Cd-tolerant crop molecular design are not adequately highlighted. Please refine the abstract to clearly state the main findings (e.g., the lack of improvement in Cd tolerance/accumulation) and emphasize their scientific relevance to the field.

3. The introduction is overly lengthy and the literature review is somewhat scattered. I recommend condensing the introduction to make it more concise, with a clear focus on the scientific question and the innovative aspects of this study. Excessive background information should be removed to avoid redundancy. Please clearly articulate the rationale for selecting BnOASTL, the reason for using tobacco as the heterologous expression system, and the current knowledge gaps that this work aims to address. In addition, the introduction should include a more forward-looking discussion on the potential of the OASTL gene family in molecular breeding and heavy metal phytoremediation, highlighting future directions and scientific significance.

4. Provide more experimental details, such as specific reagent sources, PCR/qPCR conditions, and statistical methods to improve reproducibility. Ensure all primers, vectors, and key reagents are clearly described.

5. Enhance the interpretation of the results, especially the possible reasons why overexpression of BnOASTL did not improve Cd tolerance. Discuss the broader significance and possible directions for future research, such as the need for multi-gene manipulation or systems biology approaches.

6. Ensure that all figures and legends are sufficiently detailed, and clearly indicate statistical significance where applicable.

7. Please ensure that all references are properly formatted and include DOI numbers where available. Including DOIs for all cited literature is required by the journal and will improve the accessibility and traceability of your references.

In summary, I recommend minor revision. The authors should carefully address the points above to enhance the scientific rigor, clarity, and academic value of the manuscript. After revision, the manuscript will be suitable for publication.

Reviewer #2: The manuscript entitled "Physiological and Bn-OASTL Gene Expression Responses to

Cadmium Stresses in Tobacco Seedlings" deal with an original subject within the plant soil interactions field. neertheless the ms at its present form is not suitable for publication:

-the title did not much the Ms. the is no physiological study it is just a monitoring of Cd and GSH contents

-in the ms authors present a work on tobacco plants however in conclusion they change to tomato???

-molecular analyses are goods however the discussion is not appropriated to the founded results. it is clear from results that theBn-OASTL Gene has no relatioship with the resistance beahavior within tobacco plant meaning thet tobacco did not use the g O-acetylserine(thiol)lyase pathway to overcome the cd stress

**********

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 #1: No

Reviewer #2: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

Monitoring of Cd and GSH Contents and Bn-OASTL Expression in Transgenic Tobacco Seedlings in Response to Cd Stress

PONE-D-25-39819R1

Dear Dr. He,

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.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Debasis Chakrabarty

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: (No Response)

Reviewer #3: All comments have been addressed

Reviewer #4: (No Response)

**********

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

Reviewer #1: Yes

Reviewer #3: Yes

Reviewer #4: (No Response)

**********

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

Reviewer #1: Yes

Reviewer #3: Yes

Reviewer #4: (No Response)

**********

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

The PLOS Data policy

Reviewer #1: Yes

Reviewer #3: Yes

Reviewer #4: (No Response)

**********

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

Reviewer #1: Yes

Reviewer #3: Yes

Reviewer #4: (No Response)

**********

Reviewer #1: (No Response)

Reviewer #3: The MS titled "Monitoring of Cd and GSH Contents and Bn-OASTL Expression in Transgenic Tobacco Seedlings in Response to Cd Stress" has been well revised. And I have no additional comment.

Reviewer #4: (No Response)

**********

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 #1: Yes:  Hucheng XING

Reviewer #3: No

Reviewer #4: No

**********