Transfer Alert

This paper was transferred from another journal. As a result, its full editorial history (including decision letters, peer reviews and author responses) may not be present.

Dear Dr Wang,

Thank you very much for submitting your Research Article entitled 'Sugar inhibits brassinosteroid signaling by enhancing BIN2 phosphorylation of BZR1.' to PLOS Genetics. Your manuscript was fully evaluated at the editorial level and by two independent peer reviewers. The reviewers appreciated the attention to an important problem, but raised some substantial concerns about the current manuscript. Based on the reviews, we will not be able to accept this version of the manuscript, but we would be willing to review again a much-revised version. 

Should you decide to revise the manuscript for further consideration here, your revisions should address the specific points made by each reviewer. We will also require a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.

If you decide to revise the manuscript for further consideration at PLOS Genetics, please aim to resubmit within the next 60 days, unless it will take extra time to address the concerns of the reviewers, in which case we would appreciate an expected resubmission date by email to plosgenetics@plos.org.

If present, accompanying reviewer attachments are included with this email; please notify the journal office if any appear to be missing. They will also be available for download from the link below. You can use this link to log into the system when you are ready to submit a revised version, having first consulted our Submission Checklist.

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see our guidelines.

Please be aware that our data availability policy requires that all numerical data underlying graphs or summary statistics are included with the submission, and you will need to provide this upon resubmission if not already present. In addition, we do not permit the inclusion of phrases such as "data not shown" or "unpublished results" in manuscripts. All points should be backed up by data provided with the submission.

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool.  PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. 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 us at figures@plos.org.

PLOS has incorporated Similarity Check, powered by iThenticate, into its journal-wide submission system in order to screen submitted content for originality before publication. Each PLOS journal undertakes screening on a proportion of submitted articles. You will be contacted if needed following the screening process.

To resubmit, use the link below and 'Revise Submission' in the 'Submissions Needing Revision' folder.

[LINK]

We are sorry that we cannot be more positive about your manuscript at this stage. Please do not hesitate to contact us if you have any concerns or questions.

Yours sincerely,

Li-Jia Qu

Associate Editor

PLOS Genetics

Gregory P. Copenhaver

Editor-in-Chief

PLOS Genetics

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: The present manuscript describes that sugar inhibits brassinosteroid (BR) signaling pathway in the light, and BR induction of hypocotyl elongation in seedlings grown under light is inhibited by increasing concentration of sucrose. Through phenotypic analysis and immunoblot analysis, the authors demonstrate that sucrose inhibits the BR-induced hypocotyl elongation and dephosphorylation of BZR1 in light. Furthermore, the authors find that high sucrose inhibits BR-induced dephosphorylation of BZR1. Moreover, the authors present that sucrose inhibition of BR signaling in light dependents on the regulation of BIN2. This work illustrates an intricate three-way crosstalk whereby the combination of light and sugar signals modulate the BR signaling pathway to optimize growth according to both environmental and metabolic conditions, which is of novelty and interests to researchers in the fields of light, phytohormone and sugar signaling. However, the authors need to address the comments and concerns I raised below.

Major points:

1. The authors used BZR1-CFP and bzr1-1D-CFP to analyze the hypocotyl lengths (Figure 1), but did not include wild type or bzr1 /mutant or BZR1-RNAi plants. These data are not sufficient to support the authors’ conclusion that sucrose inhibits the BR-induced hypocotyl elongation and dephosphorylation of BZR1. The authors should perform these analyses again with at least wild type plants.

2. As shown in n Figures 2 A and C, as sucrose concentration increases, the protein levels of P-BZR1 and BZR1 increase accordingly, and P-BZR1 and BZR1 proteins are not at the similar levels at the very beginning. Therefore, it’s not appropriate to conclude that high sucrose inhibits BR-induced dephosphorylation of BZR1. Please clarify this complication.

3. The authors stated that high concentration of sucrose inhibits BR-induced dephosphorylation of BZR1. Unfortunately, the authors did not detect the protein levels of P-BZR1 and BZR1 in bin2-triple mutant, which is important to support their conclusion.

4. The authors conclude that sucrose inhibits BR signaling independent on 14-3-3 proteins, HXK1 and TOR. But the authors did not explain how high concentration of sucrose may inhibit BR signaling. Please discuss the possible molecular mechanisms. Moreover, the results showing that sucrose inhibition of BR signaling is independent of HXK1 and TOR should be included in the supplemental materials.

Minor points:

1. In Figures 1F and 3B, the loading control protein histone H3 is not at the same level.

2. In Figure S1B, “Mock” and “eBL” are not located at the central of the line. “Histone H3” does not show the band correctly. The similar problems are found in Figures 1, 2, 3, and 4.

3. The data shown in Figures 3C and 3D should be analyzed by student’s t test.

4. Given the authors’ conclusion that sucrose inhibits BR signaling independent of HXK1 and TOR, it appears that the trend of BZR1 phosphorylation in BZR1-CFP/Col-0 and the BZR1-CFP/gin2-1 seedlings shown in Figure 4C is not consistent with this conclusion. Please clarify.

Reviewer #2: Brassinosteroids (BRs) are a family of plant steroid hormones that play crucial roles throughout plant growth and development. Through BRI receptor, BR signal inhibits BIN2 kinase and results in dephosphorylation and activation of the master transcription factors BZR1 and BES1 to regulate thousands of BR target genes. In dark, BR promotes hypocotyl growth, while under light, BR inhibit hypocotyl elongation. It was shown that sugar-TOR signaling promote hypocotyl growth by stabilizing BZR1 protein when light grown seedlings shift to darkness (ref1). However, the mechanism of how BR inhibit seedling hypocotyl elongation is still unclear. The authors reported an interesting finding that under light high sucrose inhibits BR signaling by stabilizing BIN2 kinase and therefore increasing BZR1 phosphorylation and degradation/inactivation. This paper connects BR signaling with nutrient status and provides interesting observation that BIN2 and BZR1 protein levels correlate with sugar inhibition

Specific concerns

1. As hypocotyl length is one measurement of BR/BZR1/BES1 activity, it would be nice to have seedling pics of those various sugar & BL treatments, at least for Fig 1, 2,3. Reference 1 is a good example for combing phenotypes and quantitative measurements.

2. do authors also have other evidence for BZR1 activity, such as target gene expression change by Q-RT-PCR and binding of BZR1 to its target genes by ChIP-PCR? Reference 2 provides RT and ChIP-PCR target genes. The reason is that WB exposure varies from gel to gel, which makes it hard to compare between samples. For example, in fig 4 D and F, the measurements of hypocotyl length are not consistent with the BZR1 band intensity between samples treated w/ and w/o Estradiol.

3. BIN2 has been reported to be a direct target of TOR-S6K signaling (ref 3). When the authors claimed that the sugar inhibition is independent of TOR, the possibility was not ruled out that high sugar might reduce TOR activity, which releases BIN2 inhibition and results in decreased BZR1 activity.

Minor issues:

1. Fig 3E, the label needs to correspond to samples.

2. In some figures, such as Fig1D and E, western exposure time varies a lot that makes the similar samples look so different (for example, Suc 0, eBL Vs Mannitol 0, eBL)

Reference

1. Zhang Z, Zhu J Y, Roh J, et al. TOR signaling promotes accumulation of BZR1 to balance growth with carbon availability in Arabidopsis[J]. Current Biology, 2016, 26(14): 1854-1860.

2. Oh E, Zhu J Y, Wang Z Y. Interaction between BZR1 and PIF4 integrates brassinosteroid and environmental responses[J]. Nature cell biology, 2012, 14(8): 802-809.

3. Xiong F, Zhang R, Meng Z, et al. Brassinosteriod Insensitive 2 (BIN2) acts as a downstream effector of the Target of Rapamycin (TOR) signaling pathway to regulate photoautotrophic growth in Arabidopsis[J]. New Phytologist, 2017, 213(1): 233-249.

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

**********

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: No

Dear Dr Wang,

We are pleased to inform you that your manuscript entitled "Sugar inhibits brassinosteroid signaling by enhancing BIN2 phosphorylation of BZR1." has been editorially accepted for publication in PLOS Genetics. Congratulations!

Before your submission can be formally accepted and sent to production you will need to complete our formatting changes, which you will receive in a follow up email. Please be aware that it may take several days for you to receive this email; during this time no action is required by you. Please note: the accept date on your published article will reflect the date of this provisional acceptance, but your manuscript will not be scheduled for publication until the required changes have been made.

Once your paper is formally accepted, an uncorrected proof of your manuscript will be published online ahead of the final version, unless you’ve already opted out via the online submission form. If, for any reason, you do not want an earlier version of your manuscript published online or are unsure if you have already indicated as such, please let the journal staff know immediately at plosgenetics@plos.org.

In the meantime, please log into Editorial Manager at https://www.editorialmanager.com/pgenetics/, click the "Update My Information" link at the top of the page, and update your user information to ensure an efficient production and billing process. Note that PLOS requires an ORCID iD for all corresponding authors. Therefore, 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.

If you have a press-related query, or would like to know about making your underlying data available (as you will be aware, this is required for publication), please see the end of this email. If your institution or institutions have a press office, please notify them about your upcoming article at this point, to enable them to help maximise its impact. Inform journal staff as soon as possible if you are preparing a press release for your article and need a publication date.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Genetics!

Yours sincerely,

Li-Jia Qu

Section Editor: Plant Genetics

PLOS Genetics

Gregory Copenhaver

Editor-in-Chief

PLOS Genetics

www.plosgenetics.org

Twitter: @PLOSGenetics

----------------------------------------------------

Comments from the reviewers (if applicable):

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: The authors have addressed almost all of my comments and concerns, and I am satisfied with the revised manuscript.

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

**********

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

----------------------------------------------------

Data Deposition

If you have submitted a Research Article or Front Matter that has associated data that are not suitable for deposition in a subject-specific public repository (such as GenBank or ArrayExpress), one way to make that data available is to deposit it in the Dryad Digital Repository. As you may recall, we ask all authors to agree to make data available; this is one way to achieve that. A full list of recommended repositories can be found on our website.

The following link will take you to the Dryad record for your article, so you won't have to re‐enter its bibliographic information, and can upload your files directly: 

http://datadryad.org/submit?journalID=pgenetics&manu=PGENETICS-D-20-01404R1

More information about depositing data in Dryad is available at http://www.datadryad.org/depositing. If you experience any difficulties in submitting your data, please contact help@datadryad.org for support.

Additionally, please be aware that our data availability policy requires that all numerical data underlying display items are included with the submission, and you will need to provide this before we can formally accept your manuscript, if not already present.

----------------------------------------------------

Press Queries

If you or your institution will be preparing press materials for this manuscript, or if you need to know your paper's publication date for media purposes, please inform the journal staff as soon as possible so that your submission can be scheduled accordingly. Your manuscript will remain under a strict press embargo until the publication date and time. This means an early version of your manuscript will not be published ahead of your final version. PLOS Genetics may also choose to issue a press release for your article. If there's anything the journal should know or you'd like more information, please get in touch via plosgenetics@plos.org.