Dear Dr Fouracre,

Thank you very much for submitting your Research Article entitled 'VAL genes regulate vegetative phase change via miR156-dependent and independent mechanisms' to PLOS Genetics.

The manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important topic but identified some concerns that we ask you address in a revised manuscript

We therefore ask you to modify the manuscript according to the review recommendations. Your revisions should address the specific points made by each reviewer.

In addition we ask that you:

1) Provide a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.

2) Upload a Striking Image with a corresponding caption to accompany your manuscript if one is available (either a new image or an existing one from within your manuscript). If this image is judged to be suitable, it may be featured on our website. Images should ideally be high resolution, eye-catching, single panel square images. For examples, please browse our archive. If your image is from someone other than yourself, please ensure that the artist has read and agreed to the terms and conditions of the Creative Commons Attribution License. Note: we cannot publish copyrighted images.

We hope to receive your revised manuscript within the next 30 days. If you anticipate any delay in its return, we would ask you to let us know the expected resubmission date by email to plosgenetics@plos.org.

If present, accompanying reviewer attachments should be 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.

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.

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.

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. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

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.

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, you will need to go to the link below and 'Revise Submission' in the 'Submissions Needing Revision' folder.

[LINK]

Please let us know if you have any questions while making these revisions.

Yours sincerely,

Claudia Köhler

Section Editor: Plant Genetics

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 paper provides a very detailed and comprehensive analysis of the role of the transcription factor VAL1 in regulating phase change in Arabidopsis. Overall I found it an impressive and high quality study, with elegant genetic analysis, so very suitable for PLoS Genetics.

The paper provides solid evidence that VAL1 and the related VAL2 gene promote phase change so that double mutants in particular have a prolonged juvenile phase. This is correlated with increases in miR156 levels, binding of VAL1 to RY elements in miR156 genes, and they also show that mutating the RY elements results in increased miR156 expression levels. However, val1 mutations continue to have an effect on phase change in quadruple mutants lacking most miR156 and miR157 gene activity, or in such mutants carrying miR156 transgenes lacking RY sites. Thus, although there is a good correlation of the effects of val1 mutations on phase change with increased miR156 expression, VAL1 can also act independently of miR156 and it is not very clear how much of the effects on phase change are due to the increases in miR156 activity. Consistent with this, they also show that VAL1 can regulate SPL9 activity independently of miR156/7, and that this is most likely indirect as VAL1 (a repressor) promotes SPL9 activity.

The paper also looks via ChIP at the effects of VAL1/2 on H3K27me3 and H2AK121ub levels at miR156 levels, with H3K27me3 levels increasing with time, and the rate of increase slowing in val1 mutants especially at miR156c. There seem to be little effect on val1 val2 double mutants on levels early in development (week one) although miR156c expression is clearly elevated at week one in the double mutants. Genetic analysis indicates that val1 mutation acts synergistically with clf or swn mutations affecting PRC2 (the H3K27me3 histone methyltransferase), this is interpreted as the genes acting in the same pathway which is plausible given the various redundancies involved but not straightforward, and is not well correlated with increases in miR156/7 levels, so may act via alternative pathways. There is also a decrease in H2AK119ub at miR156c in particular in val1 2 doubles, which may contribute to the elevated expression.

Another clear finding of the paper is that VAL1/2 affect overall expression levels of miR156 but not the dynamics, i.e. there is still a marked decrease in expression with time, suggesting there are other key regulators determining this drop. Reporter gene analysis suggests that VAL1 expression increases with time, perhaps contributing to increased H3K27me3 and repression with age. It might have been interesting to test this further, e.g. by VAL1 over/mis expression, but the paper already contains a very large amount of data.

In conclusion I think this is an important paper, well done and pretty honest in pointing out the things that don’t fit with a straightforward model. The picture that emerges is slightly cloudy, i.e VAL genes promote adult phase transition, in part by regulating epigenetic changes at miR156 and overall expression levels, but they are also acting independently via other less well defined pathways, and the relative significance of the two is not wholly clear. Nonetheless I find it a very thorough study and extremely suitable for PLoS Genetics.

Very minor comments – Materials and methods did not explain at well how miR156 and miR157 mature transcript levels were quantified. Probably this was by very standard methods, but they should be described in better detail. Lines 572 – 574 states that VAL1 VAL 2 repress SPL9 expression by miR156 independent means, however the results section showed the opposite e.g. expression of miR156 resistant SPL9 transgene is much higher in VAL1+ than in val1 mutants so VAL1 activity promotes SPL9 expression albeit likely indirectly.

Reviewer #2: In addition to the known mechanism of juvenile-to-adult phase transition that relies on the VAL-mediated PRC recruitment and repression of miR156 and miR157, the authors describe a novel finding that VAL TFs act in a mode that involves transcriptional regulation of the SPL genes but is independent of miR156. VAL TFs are shown to moderate the transcription level of the miR156 miR157 genes but their temporal decline during development is shown to be VAL-independent. Based on a VAL1-HA coIP and MS experiment, the authors identify the AtBMI1A as a robust VAL1 interactor, confirming previous studies, and suggest that the spectrum of VAL1 interactors is relatively stable during the first 3 weeks of shoot development when the juvenile-to-adult phase transition takes place.

The manuscript is clearly written, methods are well-described, the experiments are carefully conducted and interpreted, and the data presented supports the conclusions. Even though the exact mechanism of how the VAL TFs contribute to the miR156-independent mode of action is not uncovered, the manuscript opens and starts filling a gap in our understanding of the VAL-mediated juvenile-to-adult transition and the role of the VAL transcription factors in general.

I have several comments:

1. Line 157 – val2-4 is a newly described val2 allele – has expression of the locus bee tested to determine the effect of the T-DNA insertion?

2. Fig 1E (line 163), Fig.4D – The blade base angle results are not intuitive. For WT, the expectation would be an increase of blade base angle in adult leaves. In contrast, the angle decreases in leaf 5 compared to leaf 3. Can the authors indicate how the blade angle is defined in this particular quantification?

3. Fig1C – the color-coding should be changed as the shades of blue and green are difficult to be differentiated in pdf or print

4. Lines 164-166: A comparison is made between val1-5(sd); val 2-3 and val1-2;val2-3, where the latter is not shown (analysed) and a reference to previous works (in which the mutants are grown in vitro on MS medium) is made. I would argue that the difference may be caused by different growth conditions and if such comparison should be made, the mutants should be tested in parallel. I therefore think that the association between the val1-5(sd) semi-dominance and VAL2 interaction is not sufficiently justified.

5. Lines 203-204 (Fig 2A,B, S2 Fig) While I agree with the increase of miR156 in val mutants, I don´t think evidence supports the general conclusion that miR157 is increased upon loss of VAL activity. Increase is robustly visible in val1-2; val2-1 (Fig S2) but not in the other mutant/allelic combinations. In fact, there is significantly less miR157 in val1-5(sd); val 2-3 in youngest leaf primordia.

6. Line 229: formulation: “…we examined PRC2 activity in the val1-5(sd); val2-3…” PRC2 activity was not examined (and most likely it would be comparable if “only” recruitment is impaired). Please reformulate.

7. ChIP-qPCR – the rationale behind the difference in quantification is unclear – Fig3B – H3K27me3 related to H3 and normalized to STM (H3K27me3 target) and Fig. 3F – H2Aub related to input and normalized to ACT7 (H3K27me3/H2Aub non-target). The stability of STM and ACT7 in terms of the modification occupancy is not shown.

Line764, line 510 – 512: VAL1-HA coIP experiment- the age of plants at harvest should be indicated in methods as well as in the main text/legend.

**********

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 Fouracre,

We are pleased to inform you that your manuscript entitled "VAL genes regulate vegetative phase change via miR156-dependent and independent mechanisms" 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,

Claudia Köhler

Section Editor: Plant Genetics

PLOS Genetics

Claudia Köhler

Section Editor: Plant Genetics

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: authors have addressed my comments.

Reviewer #2: The revised version of the manuscript addresses all the comments raised by the reviewers and led in mostly to minor modifications of the manuscript.

One exception to this is changing the quantification strategy in an anti-H2Aub ChIP experiment, which changed the conclusions of that experiment (Fig 3F, results lines 254 - 258, discussion lines 517-523 of changes-tracked manuscript), newly suggesting limited effect of the VAL TFs on the temporal deposition of H2Aub at MIR156A and MIR156C genes. This demonstrates the fragile nature of ChIP quantification strategies, especially in cases of limited effect/trend. The conclusions on H3K27me3 deposition however seem to be robust, having been observed repeatedly and with a clear trend. Although the particular conclusion of the one experiment has been altered, this does not change the main findings and conclusions of the manuscript.

All the other reviewer comments have in my opinion been sufficiently and carefully addressed.

**********

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

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

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-21-00390R1

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.