Coumarin Inhibits Primary Root Growth by Modulating Auxin Signaling via Neddylation
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors in this manuscript try to elucidate the mechanism of action of coumarin for root inhibition in Arabidopsis thaliana. They screened mutants and identified auxin signaling mutants that are insensitive to coumarin root inhibition. They propose that coumarin functions in the neddylation pathway, altering auxin signaling and thus affecting root growth. The manuscript is clear written and easy to follow.
Some comments
line 93 what is DAB, authors should provide full name from all abbreviations
line 104 hoe sure are the authors that ACTIN2 can be used as a reference gene, is its expression stable after coumarin threatment, at least a second reference gene should be tested and yield similar results
line 126 the authors could show that data on different concentrations of coumarin, at least as supplementary, they could prove useful to other researchers
line 128-129 cytological analysis alone is not enough to claim that the roots of treated plants have reduced cell divisions, a mitotic index could be used, though understandably could be beyond the scope of the paper
fig1 D The authors should link H2O2 levels with cell division, the levels of H2O2 are somewhat increased but its not statistically significant. Also, the experiment was performed with 2 hours treatment but the cyclinB1 and root measurement were performed after 1 day of treatment.
line 149 what other mutants were screened? this information should be provided because it could be useful to other researchers.
Fig 2 The photo of the plants under microscope should be provided not only the graph.
Fig 4 Both coumarin and MLN4924 at the concentrations used cause an extreme root inhibition to 15-17 % of the control. That may mask some effect, the manuscript would benefit if they used concetrations that reduce root length at about 50%. then some synergistic actions could be revealed.
line 173 why the authors overexpressed RUB1 and not AXR1 or ECR1?please explain
Finally the authors try to show that coumarin regulates auxin signaling through neddylation. Since its auxin responses that are altered the manuscript would benefit greatly if auxin signaling was visualized through a reporter line like DR5:GFP or DII VENUS, before and after treatment with coumarin and MLN4924.
Author Response
Reviewer 1
Comment 1：line 93 what is DAB, authors should provide full name from all abbreviations
Response 1：We are grateful for the reviewers’ suggestions and have made adjustments accordingly.
Comment 2：line 104 hoe sure are the authors that ACTIN2 can be used as a reference gene, is its expression stable after coumarin threatment, at least a second reference gene should be tested and yield similar results.
Response 2：We are grateful for the reviewers’ suggestions. As a typical housekeeping gene, ACTIN2 is commonly used as a reference gene for relative gene expression. Its expression is exceptionally stable and is largely unaffected by environmental conditions. In our qPCR experiments, we also used another reference gene, GAPC (GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE C SUBUNIT), and the results obtained were consistent with those when using ACTIN2 as the reference.
Comment 3：line 126 the authors could show that data on different concentrations of coumarin, at least as supplementary, they could prove useful to other researchers.
Response 3：We are grateful for the reviewers’ suggestions. In Supplementary Figure S1, we have included a dose-response graph showing the inhibition of primary root growth by coumarin.
Comment 4：line 128-129 cytological analysis alone is not enough to claim that the roots of treated plants have reduced cell divisions, a mitotic index could be used, though understandably could be beyond the scope of the paper
Response 4：We thank the reviewer for the suggestion. The proCycB1;1-GUS marker line is specifically used to analyze the cell division index. In Figure 1C, the GUS staining intensity in this line was significantly decreased under coumarin treatment, indicating that coumarin inhibits cell division activity in the root tip.
Comment 5：fig1 D The authors should link H2O2 levels with cell division, the levels of H2O2 are somewhat increased but its not statistically significant. Also, the experiment was performed with 2 hours treatment but the cyclinB1 and root measurement were performed after 1 day of treatment.
Response 5：We thank the reviewer for the comments. The production and accumulation of reactive oxygen species (ROS) in plants under stress is a very rapid process, with significant differences observable within approximately 2 hours. In contrast, the effect on cell division is more delayed, requiring 24 hours for any differences to be detected. Therefore, we employed different treatment durations.
Comment 6：line 149 what other mutants were screened? this information should be provided because it could be useful to other researchers.
Response 6：We thank the reviewer for the suggestion. Our laboratory has collected over a thousand Arabidopsis mutants involved in the biosynthesis and signal transduction of various plant hormones. We utilized these mutants to screen for mutants with altered sensitivity to coumarin. We have now included the details of the mutant screening procedure in the manuscript.
Comment 7：Fig 2 The photo of the plants under microscope should be provided not only the graph.
Response 7：We are grateful for the reviewers’ suggestions. We have added images of root tip cells from the axr1-3 mutant and wild-type Col under coumarin treatment to Figure 2.
Comment 8：Fig 4 Both coumarin and MLN4924 at the concentrations used cause an extreme root inhibition to 15-17 % of the control. That may mask some effect, the manuscript would benefit if they used concetrations that reduce root length at about 50%. then some synergistic actions could be revealed.
Response 8：We are grateful for the reviewers’ suggestions. We reduced the coumarin concentration by half and lowered the concentration of MLN4924 to 2µM. This greatly alleviated the inhibition of root growth, allowing for a clearer observation of the relationship between coumarin and MLN4924.
Comment 9：line 173 why the authors overexpressed RUB1 and not AXR1 or ECR1?please explain
Response 9：We are grateful for the reviewers’ suggestions. AXR1 and ECR1 constitute the NEDD8-activating enzyme (NAE), which acts on the NEDD8 protein encoded by *RUB1*. To achieve a more robust enhancement of neddylation, we therefore overexpressed the RUB1 gene.
Comment 10：Finally the authors try to show that coumarin regulates auxin signaling through neddylation. Since its auxin responses that are altered the manuscript would benefit greatly if auxin signaling was visualized through a reporter line like DR5:GFP or DII VENUS, before and after treatment with coumarin and MLN4924.
Response 10：We are grateful for the reviewers’ suggestions. We are very grateful for the reviewer’s insightful suggestion, which we fully endorse. Unfortunately, the DR5:GFP and DII-VENUS marker lines are not currently available in our lab. We are actively taking steps to acquire these lines from other institutions. We concur that the interaction between coumarin and auxin warrants further investigation, and we plan to address this in our subsequent work, aiming to present more definitive findings in a future manuscript. We appreciate your guidance once again.
Reviewer 2 Report
Comments and Suggestions for Authors- Line 149, minor grammar edit to "an Arabidopsis mutants"
- Section 3.2, please clarify a bit more how you designed the screening. Is the screening done against a general mutational library or a specific list of mutational lines? If specific, how did you pick the lines?
- I would recommend the authors to add a schematic graph to the paper to illutrate the signaling pathway of coumarin-NEDDlyation-Auxin pathway better.
- Is the root growth inhibition upon coumarin treatment dose dependent on Col-0/axr1-3/ecr1-1 background? For experiments in section 3.4, given that the root upon the treatment of only coumarin or MLN4924 for the dose tested is already pretty short, I would think any additive effect is not obvious. I would suggest test a lower dose of both compound and the combo effect.
- It seems to me that both axr2 and ecr1 mutants are only partially insensitive to coumarin while axr1 mutant seems almost totally insensivie, please provide some hypothesis (probably in discussion) around it.
Author Response
Comment 1：Line 149, minor grammar edit to "an Arabidopsis mutants"
Response 1：We thank the reviewer for pointing out this error. We have corrected it in the manuscript.
Comment 2：Section 3.2, please clarify a bit more how you designed the screening. Is the screening done against a general mutational library or a specific list of mutational lines? If specific, how did you pick the lines?
Response 2：We thank the reviewer for the suggestion. Our laboratory has collected over a thousand Arabidopsis mutants involved in the biosynthesis and signal transduction of various plant hormones. We utilized these mutants to screen for mutants with altered sensitivity to coumarin. We have now included the details of the mutant screening procedure in the manuscript.
Comment 3：I would recommend the authors to add a schematic graph to the paper to illutrate the signaling pathway of coumarin-NEDDlyation-Auxin pathway better.
Response 3：We thank the reviewer for the suggestion. However, the process of neddylation is extremely complex and involves numerous proteins. This is further complicated by the auxin-mediated regulation of AUX/IAA proteins. In this study, we have only investigated a small fraction of this intricate network. Therefore, we believe it would be premature to use a schematic diagram to illustrate the coumarin-Neddylation-auxin relationship at this stage. We will continue this research to fully elucidate the complex interplay between coumarin, auxin, and neddylation. At that point, a schematic model would be more appropriate.
Comment 4：Is the root growth inhibition upon coumarin treatment dose dependent on Col-0/axr1-3/ecr1-1 background? For experiments in section 3.4, given that the root upon the treatment of only coumarin or MLN4924 for the dose tested is already pretty short, I would think any additive effect is not obvious. I would suggest test a lower dose of both compound and the combo effect.
Response 4：We are grateful for the reviewers’ suggestions. We reduced the coumarin concentration by half and lowered the concentration of MLN4924 to 2µM. This greatly alleviated the inhibition of root growth, allowing for a clearer observation of the relationship between coumarin and MLN4924.
Comment 5：It seems to me that both axr2 and ecr1 mutants are only partially insensitive to coumarin while axr1 mutant seems almost totally insensivie, please provide some hypothesis (probably in discussion) around it.
Response 5：Thank you for the suggestion. The axr1-3 mutant is completely insensitive to the coumarin-induced inhibition of root growth, while the axr2-1 mutant is only partially insensitive. This suggests that the function of coumarin in inhibiting root growth depends on neddylation, which affects the degradation of AUX/IAA proteins. Since AXR2 is just one of many AUX/IAA proteins targeted for neddylation-dependent degradation, it is possible that coumarin also affects the degradation of other AUX/IAA proteins. As suggested by the reviewer, we have added that to the Discussion section to address this point.
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsIn this revised manuscript the authors successfully incorporated the reviewers' comments, greatly improving the work presented. This work demonstrates interesting interactions between coumarin and auxin signaling; of great interest to both auxin and allelopathy scientific communities.
