PONE-D-21-13580

Exogenous putrescine attenuates the negative impact of drought stress by modulating physio-biochemical traits and gene expression in sugar beet (Beta vulgaris L.)

PLOS ONE

Dear Prof. Dr. Mohammad Anwar Hossain,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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We look forward to receiving your revised manuscript.

Kind regards,

Mohammad Golam Mostofa, PhD

Academic Editor

PLOS ONE

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This research received a grant from the project entitled ‘Strengthening Integrated Research Facilities of Bangladesh Sugarcane Research Institute’ of Bangladesh Sugarcrop Research Institute (Funding number: 224066000)

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Reviewers' comments:

Reviewer's Responses to Questions

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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4. 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 #1: No

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: Authors have very well showed that application of putrescine (Put) alleviates drought stress in two sugar beet genotypes. Authors have studied effect of Put application in sugar beets during drought stress in various biological processes such as physiological, biochemical, and transcriptional level. Moreover, Authors have demonstrated that Put application enhances antioxidant levels thereby reduces H2O2 and MDA level during drought stress in Sugar beet genotypes.

However, there are several formatting errors and in detail explanation needed in material method section and in every figure legend.

There are several grammatical errors in spacing, manuscript needs to be thoroughly checked for spacing errors.

Why it has been not considered to treat control plants also with Putrescine and normalize the data? Furthermore, why authors have not showed any images of plants for example how sugar beet plants looks after 10 days of drought stress and how Put treatment recovers severity of drought phenotype?

Introduction:

Line no. 67-68; reference error need to be checked.

Spacing errors.

Material and methods:

Line no. 115 to 117; explain in detail for how long (time) seeds were sterilized.

Line 121 to 125; This is the important information for readers, authors should mention whether Put was sprayed for every day/how many times per day?

Line 176: reference error, numbering not mentioned to reference.

Result and Discussion:

Result section is hard to follow. Authors should consider to rewrite it.

Line 226 to 236: written very poorly, Authors should consider rewriting this section carefully, What is Kaveri? The table 2 is very confusing, there are no units to Shoot dry wt, root dry wt, leaf area, LRWC. Again, new Kaveri genotype which is not mentioned in materials and method section!

Plant growth and parameters: At which day samples were collected/analyzed not mentioned.

At line 247 to 250, there is no flow to the sentence.

Line 302 to 306, there is no flow while reading, consider rewriting.

Antioxidant Enzyme Activity:

Line 405 to 406: Written wrong! Antioxidant enzyme levels were reduced after drought in both genotypes but its written increased; though its being increased in Put treatment compared to drought; but this has been not mentioned. The results written in Antioxidant enzyme activity need to rewrite carefully.

Relative gene expression:

If the relative gene expression is normalized to reference gene, then it should be mentioned and if it is compared to something then it is fold change or Log2fold change?

Authors need to consider checking the expression of Put biosynthesis genes namely, arginine decarboxylase (ADC) and Orn decarboxylase (ODC) in their sugar beet samples. Authors have checked expression of antioxidant genes after 10 days of drought, earlier time point is important. Moreover, whether does Put treatment to control sugar beet plants/genotypes alters the expression of these antioxidant genes has not been verified, short experiment would be to just treat sugar beet seedlings with Put for several timepoints and check expression of these antioxidant genes along with Put biosynthesis genes.

Figures:

Figure legends must be rewritten! In all figures, It is very confusing to determine the significant differences between the treatments, Authors should consider labeling significant differences by different letters and non-significance by same letter. This way it is easy for readers to follow. Moreover, Authors should explain in figure legend about the asterisk/ letters (if newly added) denotes significant differences between which treatment.

Reviewer #2: In the manuscript (ms) entitled “Exogenous putrescine attenuates the negative impact of drought stress by modulating physio-biochemical traits and gene expression in sugar beet (Beta vulgaris L.)” authors put quite a lot effort. Here they investigated the potential roles of exogenous putrescine (Put) in improving drought stress tolerance of two sugar beet genotypes by assessing morpho-physiological and biochemical traits, as well as gene expression.

In my opinion, the findings of this study would be pretty interesting and important for the readers from related field. However, the ms in the current state lacks cohesiveness in scientific writing and prepared carelessly in terms of writing norm. Based on the following observations the article in its current status is not suitable to publish in this journal.

1. In the abstract, the authors mainly discussed the methods and listed the overall results instead of presenting the main findings. Authors are suggested to synthesize the main findings here highlighting special techniques/methods used.

2. The main drawback of this ms is poor presentation and explanation of results. In presenting comparative results, authors in most of the cases just have used the terms ‘significant’, ‘prominent increase’, ‘increased/decreased’. In this case, authors should focus on quantifying (where possible) the recovery of drought-induced losses in sugar beet in response to Put treatments. Additionally, authors should include a comparative phenotype figure on Put-induced drought tolerance in sugar beet to strengthen their claim.

3. The subsequent discussion supporting the results has also been very superficial. For instance, in Line (L)-270, authors mentioned “these results have been confirmed in other studies”. How their results confirmed by others? Authors should clearly express what they meant here?

4. The ms requires several rounds of English polishing. Specifically, there are lots of typos, long sentences and scientifically incorrect sentences in the ms, which might confuse the readers to get clear insights. Inconsistency in writing style (abbreviation, citation etc.) should also address very seriously.

Some specific concerns, but by no means an exhaustive list, mentioned below should be addressed to improve the current status of the ms:

-L67: Citation style is found inconsistent

-while discussing the results in L225: surprisingly, in table 2, no ‘'BSRI Sugar beet 2’ was found!! Here they mentioned the genotype name 'Kaveri' instead of 'BSRI Sugar beet 2'. It seems authors carelessly mentioned ‘Kaveri’ here!! Or, something else, please clarify.

-L231-232: “In the case of SBT-010, although SDW and LA recovered

most under 0.6 mM and 0.9 mM Put, respectively, but no positive effect was observed for RDW”. As said earlier, from such types of ‘English’ readers might lose their interest. Please improve the English standard to have readers’ interest.

L258: chlorophyll "a" and "b" should write in italic!

L280: Results explained on 'fluorescence parameters' is not understandable at all!

L281: What is meant by OJIP? Author should elaborately mention when any abbreviation appears first.

L294-296: Unnecessary discussion.

L405-411: Very hard to understand the activity of enzymes. This explanation is not even consistent with Fig. 4!

L469: Authors mentioned ‘it was revealed that cluster-A was mostly characterized by…….’ They should avoid such kind of vague and non-scientific wording to present the data.

L485-491: Authors should discuss the PCA results more elaborately to clearly focus the main traits contributed greatly to Put-mediated drought tolerance in sugar beet.

L519-520: In the last line of conclusion “Importantly, Put at a medium dose (0.6 mM) was found to be most effective in modulating the morpho-physiological and biochemical processes in sugar beet plants under drought stress. I wonder how authors reached on a conclusion like this! Besides, the term ‘medium dose’ is a vague and non-scientific word!

-the figure and table legends are not complete. Authors should give more care on writing it completely.

-in table 4: in presenting the Pro data of SBT-010, it seems authors mistakenly put the same letter ‘g’ for both ‘4.81 ± 0.48’ and ‘15.05 ± 0.07’. There must have significant difference between them! Authors should seriously address such kind of issue.

Further recommendation

It would be pretty interesting and worth reading for the related readers if authors could produce a flow/process diagram on Put-mediated drought tolerance mechanism in sugar beet based on the findings of the present study.

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Reviewer #1: No

Reviewer #2: Yes: Gopal Saha

[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.

PONE-D-21-13580R1

Exogenous putrescine attenuates the negative impact of drought stress by modulating physio-biochemical traits and gene expression in sugar beet (Beta vulgaris L.)

PLOS ONE

Dear Dr. Hossain,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Oct 15 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Mohammad Golam Mostofa, PhD

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments (if provided):

Authors should present the figures by placing the data of two varieties side by side in one figure for better comparison. Authors are also suggested to use either SEs or SDs in the tables and figures. There are still many English issues throughout the manuscript.

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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 #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

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 #1: Yes

Reviewer #2: Partly

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

Reviewer #1: Yes

Reviewer #2: No

**********

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 #1: Yes

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 #1: Yes

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 #1: Dear Authors,

If possible enlarge the newly added plant photographs for better visualization.

Please check the manuscript once again for grammatical mistakes, I see at some places there are spacing errors.

Thank you.

Reviewer #2: I thank Islam et al. for their effort in revising and updating the status of the manuscript (ms). In the revised ms authors remarkably improved the results writing and English standard.

However, I have a major concern on the current data presentation style both in tables and figures. I am surprised why authors presented data (table 2-4) in separate panels for two different varieties. Authors must justify this issue first. To me, it would have been better to present data in one panel after running a two-way ANOVA. This will provide detailed information on main and interactive effects. Therefore, the reader will be able to assess the treatments across different concentrations of Putrescine (Put) and variety. I suggest the authors to consult with a professional statistician to get better insight about the analysis. Likewise, for better visibility of the treatment effects in figures, authors are recommended to present the data of two varieties combinedly as single comparative group bar graph (for each parameter) instead of two separate figures.

Besides, there are some other concerns listed below and authors should address these before publication of the ms:

L126-127(Materials & Methods)-Authors should tell about the commercial formulation of Put, e. g., form, company name etc. they have been used for treatment.

L454-478 (Results & discussion)- The Relative gene expression section has not been well written as per data presented in the fig. 5. To me, in most of the cases the relative expression of antioxidant genes contrasts between the two varieties in response to Put treatments!! Authors should discuss the results accordingly and focus this issue with possible facts.

L563-564 (Conclusion)-the authors concluded “However considering most of the traits under study, 0.3 mM TU was of effective for the variety BSRI Sugar beet 2 whereas 0.6 mM TU was found was found effective for the variety SBT-010” – what actually authors meant by ‘TU’? Importantly, how authors recommended two different doses of Put are suitable for drought tolerance of two sugar beet varieties, since authors analyzed and presented the interaction data separately?? In that case authors may specifically mention the traits/parameters of two varieties improved for drought tolerance in response to Put application.

Finally, the figures of phenotypic differences of treatment effects were supposed to be presented in a separate figure, authors provided it along with the schematic diagram in a less convincing way! However, for better understanding for the readers the authors should give the discussion on this Put-induced drought tolerance mechanism (fig. 7) separately as individual section other than depicting it only in the figure legend! And, authors are advised to present this figure in a more reader friendly way explaining all the items (e.g., arrow, color code etc.) of the figure in the figure legend.

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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 #1: No

Reviewer #2: Yes: Gopal Saha

[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.

Exogenous putrescine attenuates the negative impact of drought stress by modulating physio-biochemical traits and gene expression in sugar beet (Beta vulgaris L.)

PONE-D-21-13580R2

Dear Dr. Hossain,

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.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Mohammad Golam Mostofa, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Please address the minor issues raised by Reviewer 2 during proof reading.

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: All comments have been addressed

**********

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: I thank Islam et al. for their effort in revising and updating the status of the manuscript (ms). In the revised ms authors remarkably addressed the major concern on statistical issues and data presentation style, especially the tables and figures.

Before publication authors should address the following issues for better readability of the article:

For all figures (1-6): the lettering on the top of the bar should be positioned nicely comparing with other font size in fig.

Fig 3: Why the comparative phenotypic effect of D+0.6 mM Put & D+0.9 mM Put have not been presented? Please add these if they are available!

Fig 5: Please use contrasting color code for bars instead of current format for better understanding. The scaling font in the X & Y axis looks unusually large!

Fig 6: In the PCA plot (both 1&2) the variables/treatments names are overlapped due to space problem. Authors may consider customizing the writing/presenting style either using symbol, or omitting them and re-writing in distant understandable format & font.

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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: Gopal Saha