Dear Dr Fankhauser,

Thank you very much for submitting your Research Article entitled 'Shade suppresses wound-induced leaf repositioning through a mechanism involving PHYTOCHROME KINASE SUBSTRATE (PKS) genes.' to PLOS Genetics.

The manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers recognized that this study constitutes a significant advance. They, nevertheless, raised some substantial concerns about the current manuscript. While the involvement of  PSKs in the interplay between wounding and shade avoidance was clearly demonstrated, this study would benefit from additional mechanistic insights into how these genes ultimately influence the hyponastic response. Some of the reviewers also thought that the conclusions concerning the regulation of PSKs by MYB were not fully supported. These conclusions could perhaps be recast as hypotheses to ensure that the readers do not misinterpret them. Additional details on the statistical analyses should also be provided. The impact of the study would be better demonstrated by proposing a model integrating the results into the current knowledge within the field. Based on the reviews, we will not be able to accept this version of the manuscript, but we would be willing to review a much-revised version. We cannot, of course, promise publication at that time.

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. 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 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,

Adrien Sicard

Guest Editor

PLOS Genetics

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: Plants alter their development and growth in response to both abiotic and biotic stress. The relationship between biotic and abiotic responses and the mechanisms integrating both types of stresses simultaneously are still poorly understood. For example, shade and wounding by herbivores elicit opposing effects on tissue/organ growth. Plants respond to shade or the presence of competing neighbors by promoting petiole and stem elongation as well as triggering an upward leaf movement (hyponasty), thereby escaping shade. In contrast, during herbivore attack, the plant hormone Jasmonate (JA) elicits wounding responses that inhibit tissue growth and leaf movement. While the impact of shade on JA responses has been well studied, the mechanism that integrates the two types of signals remains elusive.

Here the authors explored the relationship between shade and wounding responses in terms of leaf/petiole growth and leaf hyponasty. The authors carefully devised a protocol to precisely quantify the effects of wounding and shade on a specific intact leaf. The authors show that, when challenged by both shade and wounding, Arabidopsis is able to sustain the shade-mediated leaf hyponasty but not leaf elongation, indicating that leaf growth and movement can be uncoupled in the combined shade and wounding treatments. Global transcriptomic analysis revealed that the shade treatment reduced the impact of wounding on gene expression. The RNA-seq analysis also revealed a group of genes whose expression patterns match the leaf movement response in the shade, wounding, and combined shade/wounding treatments; these include PSK4 and, to a less degree, PSK2. The authors then present genetic evidence supporting that PKS4 and PKS2 act redundantly in the shade-dependent suppression of the wound-induced inhibition of leaf hyponasty. Interestingly, both PKS4 and PKS3 are induced directly by PIF4/5/7 in response to shade. PIF4/5/7, similar to YUCs, are required for the lack of the wounding effect on hyponasty in shade. Moreover, PKS4 and PKS2 are repressed by wounding via MYCs; a myc234 triple mutant lost JA-induced inhibition of leaf hyponasty.

Overall, this elegantly-designed study provides compelling genetic evidence showing distinct relationships between shade and wounding on leaf/petiole growth and leaf hyponasty. More importantly, the results reveal novel insight into the genetic elements required for the shade-dependent repression of the wound-induced inhibition of leaf hyponasty -- namely PIFs, auxin, PKS4/2 -- and demonstrate the function of PKS4/2 in both the positive and negative regulation of leaf hyponasty by shade and wounding, respectively. The manuscript is well written, and the figures are beautifully presented.

My only suggestion is that the authors might want to elaborate the part in Discussion describing the hypothesis about the competition of PIFs and MYCs in PKS4/2 expression. The results in Figure S3 demonstrate that the wound-induced inhibition of leaf hyponasty depends on phytochrome B (phyB). Studies from the authors’ lab and others have shown that phyB inhibits the stability and activity of PIFs. Together, the current data support the model that, in normal light conditions (without shade), phyB inhibits PIFs to allow MYCs function prominently in wounding to repress PKS4/2 expression, whereas, in shade condition, the attenuation of phyB functions enhances the amount and activity of PIFs, which can antagonize MYCs in wounding to promote PKS4/2 expression.

Reviewer #2: Shade suppresses wound-induced leaf repositioning through a mechanism involving PHYTOCHROME KINASE SUBSTRATE (PKS) genes

Fiorucci et al. studied the interaction of wounding and shade avoidance in Arabidopsis to understand how the light signalling and wound-induced JA-signalling are integrated. First, consistent with earlier studies, they find that when individually applied shade induces a hyponasty response while mechanical wounding leads to lowering of leaf position. When applied together, wound-induced lowered leaf position response was suppressed by shade. While wound-induced response required JA, shade-induced responses required auxin as shown by mutant analyses. They further studied gene expression to understand the molecular basis of the interaction and identified a set of genes showing contrasting regulation by wound and shade and that the wound effect is suppressed by shading. The authors identified PHYTOCHROME KINASE SUBSTRATE 4 (PKS4) for further studies. Using loss of function mutants and transgenic overexpression experiments, the authors find that PKS4 and close homologs are involved in the control of leaf hyponasty in response to wound and shade. They further show that PKS4 and PKS2 are PIF target genes by studying gene expression and chromatin binding of PIF4 at PKS4. The authors also argue that PKS2 and 4 are repressed by MYC transcription factors.

The experiments are well planned and brilliantly carried out with a sound statistical analysis. In general, the conclusions drawn are justified except a few exceptions. For example, the last section of MYC repression of PKS2 and 4 are not convincing. The provided data is not sufficient to support this argument.

While the data shown here in general are well-supported, the major question remains unanswered - how do PKS proteins control the hyponasty response and how are the contrasting signals integrated? To this reviewer, more in-depth analysis on the mechanism of shade-induced suppression of wound-induced leaf positioning through PKS would have been helpful.

What is the biological significance of the transient inhibition of leaf elevation by wounding? The authors argue that effect on leaf positioning by wounding is more than just a consequence of a general growth suppression and perhaps be of an adaptive value. This needs to be further supported. It could have helped if the role of PKS in plant defence under shade conditions were investigated.

To this reviewer, while this is an interesting and well executed study, the findings are rather incremental and stops short of making a significant advance.

Reviewer #3: Review of Fiorucci et al for PLoSG

This manuscript explore the relationship between shade and defense signaling in the control of plant morphology. Although the effects of shade and defense signaling interactions on defense responses have been explored in a number of prior publications, the effect of these interactions on growth and morphology is much less explored. Thus, this manuscript is novel and is a significant contribution to the field. This manuscript describes the molecular-genetic mechanisms underlying the effect of shade/defense interactions on leaf angle, and reveals that genes in the PKS family are an important intersection point for these two pathways. The manuscript is well written and conclusions are well supported. There are some issues that need to be addressed before considering publication.

Major:

1) The authors report p-values for the interaction term in two-way ANOVA tests but not in three-way ANOVA tests (e.g. genotype:light:wounding for fig 1BC, 3AB, etc). In theory the three-way interaction term from an ANOVA or linear regression would be the best test for whether or not a mutant affects the light:wounding interaction, but I acknowledge that it can be difficult to achieve enough power to adequately test this term in practice, potentially leading to false negatives. Another approach might be to fit separate two-way linear regressions for WT and mutant, obtain 95% confidence intervals on the interaction term and determine if they overlap or not. Current conclusions are based on (I think) Tukey’s HSD but this is not completely clear. If the authors stick with the Tukey HSD approach it would be helpful if they explained how they are interpreting these tests to arrive at their conclusions (along the lines of “shade caused a similar increase in leaf angle in unwounded Col-0 and pks2pks4 (Tukey HSD group a) but this effect was reduced in the pks2pks4 double mutant (Tukey HSD group b for Col-0 vs group c for pks2pks4)”. They is probably a more elegant way to write this, but hopefully that gets the idea across. The reason that the regression or ANOVA approach is favored over the Tukey HSD is that the term that we want to test is related to differences in the wounding response, which is only indirectly addressed by the Tukey approach. In the end I am OK with the Tukey approach if it is better explained, but I suggest that the authors at least consider the other approaches I have outlined if they have not already. On a related note, the figure legends and methods state that a three-way ANOVA was performed. The methods should describe the formula for that (were all two and three way interaction terms used?)

2) The authors conclude that the transcriptional response to wounding is more rapid than the response to shade (line 218) but I do not think that the data supports this, because the first wounding treatment is 2 days earlier than the shade treatment (Fig 1A).

Minor:

Lines 152-183. This is a long paragraph that is a bit difficult to follow, especially because the authors go back and forth between the different responses that they are measuring are not always clear. For example, lines 167-168 “We concluded that there is no interaction shade and wounding…” this is for petioles but that is not indicated in this sentence or the one above. And in this section of the paragraph petiole length is discussed before leaf angle even though they are in the opposite order in the figure. Anyway: the authors should consider whether this section can be organized in a more clear manner; it is easy for the reader to get lost.

Fig 2B: the order of the timepoints in the heat map is reversed that from 2A (and from the progression of time) and should be flipped. In the worst case the heat map could be flipped and relabeled “by hand” (outside of R) if it is not possible to have R present time 1 on the left.

Fig S4B: are the genotype labels wrong? If not, why does aos have a stronger response than Col on the heatmap?

Fig 3B y-axis label (left plot) is “Petiole angle” but elsewhere “Leaf angle” is used.

Fig 4B are “peak” and “control” mislabeled in the bar plot? Also PKS4 doesn’t have solid evidence for induction by shade. Could say marginal in text.

qPCR methods: although a citation to a previous paper is given, it would be nice if a sentence or two could be added about the analysis (is this delta-delta CT?) so that the curious does not have to flip back to the previous paper.

**********

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

Reviewer #3: No: Numerical data underlying graphs is not provided.

**********

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: Meng Chen

Reviewer #2: No

Reviewer #3: No

Dear Dr Fankhauser,

We are pleased to inform you that your manuscript entitled "Shade suppresses wound-induced leaf repositioning through a mechanism involving PHYTOCHROME KINASE SUBSTRATE (PKS) genes." 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,

Adrien Sicard

Guest Editor

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 #2: The revised manuscript and the accompanying response to reviewers have satisfactorily addressed most of the concerns raised by this reviewer. The study indeed identifies an interesting phenomenon. The molecular mechanism and biological significance of the reported phenomenon will be interesting questions to be addressed.

Reviewer #3: My comments have been adequately 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 #2: Yes

Reviewer #3: No: Raw data of plant measurements is not available (or I cannot find it)

**********

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: No

Reviewer #3: Yes: Julin N Maloof

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

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-21-01498R1

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.