PONE-D-25-00360Desert-adapted tomato Solanum pennellii exhibit unique regulatory elements and stress-ready transcriptome patterns to drought.PLOS ONE

Dear Dr. Alvarez,

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.

The manuscript was reviewed by two independent experts in the field. Both the reviewers found the work interesting but raised several issues, which should be addressed for further consideration. The reviewers provide detailed comments in their reviews and point out the areas where the manuscript needs to be improved.

Please submit your revised manuscript by Apr 14 2025 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: https://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 Irfan, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1.Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for stating the following financial disclosure:

“This work was funded by grants FONDECYT-1210389 (JMA) FONDECYT-3220673 (to JSCR), FONDECYT-3220801 (to TM), ANID Millennium Nucleus in Data Science and Plant Resilience (PhytoLearning) NCN2024_047, and Instituto Milenio de Biología Integrativa iBio Chile ICN17_002 from “Agencia Nacional de Investigación y Desarrollo de Chile (ANID)”. JMA lab is also supported by National Science Foundation (NSF) Plant Genome Grant NSF-PGRP: IOS-1840761. JMJG is funded by Ministerio de Ciencia Inovación y Universidades of Spain (PID2023-151867OB-C33) and European Union's HORIZON-EIC-PATHFINDEROPEN no. 101098680 (project DARkWIN). This research was partially supported by the supercomputing infrastructure of the NLHPC (ECM-02).”

Please state what role the funders took in the study.  If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

If this statement is not correct you must amend it as needed.

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

3. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager.

4. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information .

5. We are unable to open your Supporting Information file supp. file 1 - network.cys and Supp. file2 - scripts.zip. Please kindly revise as necessary and re-upload.

[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?

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: Partly

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

**********

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

**********

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: Yes

Reviewer #2: Yes

**********

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: The manuscript by Contreras et al present a bioinformatics comparison of gene regulation in two close Solanum species using public data from a previous paper of their own. I like the style of the paper, straight to the point, but still I have comments for the authors:

L127 Please add FASTQC version

L129 Please add GCA assembly codes for both genomes

L129-131 Please rephraser and consider adding equation, in its current form it is not clear what's being done

L133 Doesn't it make more sense to fit instead of assign?

L135 Why were normalized expression values not computed by DEseq or kallisto, for instance in TPMs, why is the current approach used instead? Please justify. Also, are these the same values in Y-axis of Fig4B and Fig4C?

L138 Please provide source code/or a URL to code for the decision tree and explan also how FC is computed

L165 Please add GOATOOLS version

L173 Please add PLANTTFDB version and URL it possible

L174 Please explain the source of TSS or correct if you mean gene start; also explain whether raw or masked genomic sequences were used to scan motifs

L198 The authors should compare their obtained total orthologues to other estimates in the literature or databases to give the reader a sense of how accurate their simple approach is; in particular, as they reckon, the reciprocal blast way is highly sensitive to paralogues and multiple copies. Same problem would apply to assigning Araport gene ids, which I don't get why it was required (sorry if I missed it).

L211 Please help us understand these numbers: is this caused by the different number of genes in both species? Shouldn't both tests produce similar numbers of DEGs? Can this be explained by different variances across replicates?

L248 "minimal or no change" does not match FC > 0. Please see L138

L274 Did the authors compute enrichment for random gene groups (negative control). These should increase the confidence on this section and would help put in context L294.

L307 Why was it neccessary to verify it, how often the decision groups genes with different responsed? I apologize if I misunderstood this point.

L339 See L274, same idea here; how often random gene lists are enriched in motifs? Moreover, please let us know how many motifs from each family were scanned so that readers can gauge whether they were all equally likely to start with. Perhaps this could go in Fig5. Moreover, as motifs in PLANTFDB are mostly predictions, it is possible that some motifs are matched more that others due to differences in motif quality. This limitation should be discussed. I think this part of the work would greatly improve if the most significant motifs reported were confirmed by an ab initio approach, for instance based on the over-representation of DNA sequences, without prior knowledge of the motifs.

L378 It would help to see shared motifs as cis-elements mapped along orthologous promoters side to side, so readers can evaluate the conservation

L414 I find it hard to understand that the GRN are so different, is it because the number of shared cis-elements across orthologous is minimal? Perhaps this number should go into table 3 somehow. Please explain.

Reviewer #2: MAJOR ISSUES:

1. Why was Solanum chilense not considered for this study?

2. There are many instances where first person tone has been used for writing. Please consider using the third person, this is more formal and acceptable.

3. In many instances, there is no consistency in the use of terminology.

4. In many instances, some sentences are long and complex, which might hinder readability. Consider breaking down complex sentences into simpler ones for better clarity.

5. The discussion of ERF transcription factors and their roles in stress responses is good. However, it might benefit from a more detailed explanation of how these factors mechanistically contribute to stress resilience.

6. There is should a section for conclusion and future directions. Which is missing.

MINOR ISSUES:

1.Line 114: "BLASTp" should be defined for readers unfamiliar with it, perhaps as "Basic Local Alignment Search Tool for proteins (BLASTp)."

2.Line 198-201: The numbers of unique genes are inconsistent. If 21,104 is the number of orthologous genes, the unique genes should differ. Double-check these figures for accuracy. Consider rephrasing for clarity: "Additionally, we identified 13,325 unique genes in S. lycopersicum and 21,104 in S. pennellii."

3.Line 202-203: The explanation for non-orthologous genes is clear but consider adding more detail on how paralogs might affect the analysis.

4. Line 204-205: Specify the conditions of the "control" and "drought" treatments for clarity.

5. Line 206: Moreira et al. (2022) is not properly cited.

6. Line 207-209: The sentence is complex; consider breaking it into two for clarity. For example, "PCA revealed distinct expression patterns in orthologs. Species differences influenced these patterns, consistent with clade distances, and by environmental conditions such as drought."

7. Line 210-214: The comparison of differentially expressed genes is clear but ensure that the methodology for determining differential expression is described elsewhere in the document. Consider adding a brief discussion on the biological significance of the findings, especially regarding the implications of differential gene expression in response to drought.

8. Line 231-233: Consider briefly explaining why the decision-tree algorithm method was chosen and how it categorizes orthologs.

9. Lines 266-273: The statement "S. pennellii appears to be preadapted to drought" is a strong conclusion. Consider discussing potential evolutionary mechanisms or historical environmental conditions that may have contributed to this preadaptation.

10. Lines 279-284: Consider rephrasing for clarity: "Orthologs with increased expression under drought in both species were enriched for genes involved in seed dormancy, water deprivation, response to abscisic acid (ABA), and oxidative stress."

11.Lines 333-339: Consider rephrasing for clarity: "Using the FIMO tool, we scanned transcription factor binding motifs (TFBMs) in the promoters of genes associated with each expression model in both species.

12.Line 432: "pathways pathway" is a repetition. It should be corrected to "pathways."

13. Line 454: The sentence structure is awkward: "Notably, nearly one-third of drought-responsive orthologs were associated with a stress-ready state, representing 20% of all orthologs analyzed." Consider rephrasing for clarity: "Notably, nearly one-third of drought-responsive orthologs, representing 20% of all orthologs analyzed, were associated with a stress-ready state."

14. Line 460: The phrase "providing weaker evidence for pre-adaptation in that context r (6)" contains a typographical error with the letter "r." It should be removed.

15. Line 471: The phrase "but S. pennellii appears to adopt a more energy-efficient strategy during stress" could be more concise: "suggesting that S. pennellii adopts a more energy-efficient strategy during stress."

16. Line 481: The sentence is split awkwardly: "ERF proteins are multifunctional regulators known to enhance resilience to various stressors, including drought, salinity, extreme temperatures, and heavy metals, and are involved in processes such as root hair development, leaf senescence, and proline synthesis for osmoprotection (42)." Consider breaking it into two sentences for clarity.

7. PLOS authors have the option to publish the peer review history of their article (https://journals.plos.org/plosone/s/editorial-and-peer-review-process#loc-peer-review-history). If published, this will include your full peer review and any attached files. Do you want your identity to be public for this peer review? YES

7a. If you answered "Yes" above, that you would like your identity to be public. Vishnutej Ellur

**********

6. 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:  Vishnutej Ellur

**********

[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.

Desert-adapted tomato Solanum pennellii exhibit unique regulatory elements and stress-ready transcriptome patterns to drought.

PONE-D-25-00360R1

Dear Dr. Alvarez,

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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager®  and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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 Irfan, Ph.D.

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 #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: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

**********

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: The authors have significantly improved the paper by clarifying the points raised by both referees, congrats

Reviewer #2: I am happy with the changes made. I will recommend accepting the manuscript. My suugestions were considered and answered.

**********

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: Yes:  Bruno Contreras Moreira

Reviewer #2: No

**********