Profiling of the Salt Stress Responsive MicroRNA Landscape of C4 Genetic Model Species Setaria viridis (L.) Beauv

Round 1

Reviewer 1 Report

The manuscript by Pegler et al investigates the effect of salt stress on the root and shoot micro RNA profiles of two Setaria viridis accessions. The stress was applied as NaCl to 3 day old seedlings on MS plates for 7 days. High throughput sequencing was used, and the results were then validated by quantitative PCR (qPCR) for a subset of miRNAs. Two putative downstream target transcripts of miR160 and miR167 – belonging to the Auxin Response Factor (ARF8) gene family – were investigated by qPCR due to their high levels of complementarity. It is unclear whether any statistical tests have been performed to validate the significance of the findings. Proposed root phenotypic differences between the two accessions are not quantified. Comments below:

Major comments

The introduction is very long. I would suggest reducing the length of the introduction but about half.

p6, L277-284: The paper claims that after 10 days, A10 had more adventitious roots than ME034V, and the lateral roots of ME034V were longer than A10. I do not agree with these statements, as neither adventitious root number, nor lateral root length, are quantified in Figure 1. I suggest to add these data as graphs in Fig 1. If these data were not collected, then these statements should be moderated so as not to exaggerate the results.

P8 L313-316: The claim about lateral root elongation in ME034V (see comment above) is duplicated on p6 and p8. This should either be quantified, or not stated. It forms a theme throughout the MS as “the most pronounced phenotypic difference” between the two lines (L484-485). If this difference is central to the study, then it would be reasonable to quantify this difference.

Fig 4 C and E: Were the apparent differences (upregulation of miR395, downregulation of miR396) statistically significant? If not, then it should be stated, so as not to overstate the results/conclusions.

There is no statistical evidence that downregulation of miR160 leads to increased ARF1 expression in A10 shoots and roots (Fig 5C and D). A statistical test needs to be applied to the qPCR result, otherwise this conclusion is highly speculative. If there is no significant difference, then that should be reported.

The Figure legends should state i) what the error bars represent (SD or SEM), ii) how many replicates were analysed, iii) the meaning of the asterisks, iv) the statistical test(s) performed, v) p value.

Minor comments

Supplementary data are not provided, so these are impossible to assess.

How was the specificity of the primers determined? There is no mention of melt curve step following the qPCR.

L330, L331, L342: “Figure 2 heatmap” should simply be written as “figure 2”, because there is no other data shown except a heat map.

The resolution of the figures is so low that I can’t read the graph axes.

Do the two Setaria viridis strains used in the study (A10 and ME034V) greatly differ in their salt tolerance? It would be useful to mention.

Is there any difference between a crop species and a “cropping species”?

Author Response

First and foremost, on behalf of the authorship team, I would like to thank each of the three reviewers for taking the time and care to provide valuable comments and insightful feedback for the improvement of our originally submitted manuscript. Please find below our comment-by-comment responses in our attempt to adequately address each concern raised by the three reviewers post review of our originally submitted manuscript. Where appropriate, we have additionally identified where in the revised manuscript we have addressed each reviewer concern.
Kind regards,
Andrew Eamens

Reviewer 1
Major comments
The introduction is very long. I would suggest reducing the length of the introduction but about half.
• While the introduction is approximately 3 pages in length, background pertaining to (1) food security and alternative biofuel solutions, (2) prevalence and impact of soil salinization on plant growth (3) the use of Setaria as a model plant species to genetically characterise C4 biology, and (4) the biogenesis and action of microRNAs are all essential to appropriately introduce the aims of this study. In an attempt to address this concern of Reviewer 1, considerable portions of the Introduction have been removed in an attempt to reduce the length on the Introduction while maintaining the narrative of this section of the manuscript.
p6, L277-284: The paper claims that after 10 days, A10 had more adventitious roots than ME034V, and the lateral roots of ME034V were longer than A10. I do not agree with these statements, as neither adventitious root number, nor lateral root length, are quantified in Figure 1. I suggest to add these data as graphs in Fig 1. If these data were not collected, then these statements should be moderated so as not to exaggerate the results.
P8 L313-316: The claim about lateral root elongation in ME034V (see comment above) is duplicated on p6 and p8. This should either be quantified, or not stated. It forms a theme throughout the MS as “the most pronounced phenotypic difference” between the two lines (L484-485). If this difference is central to the study, then it would be reasonable to quantify this difference.
• While the authorship team is in complete agreeance with both of these related comments raised by Reviewer 1, this phenotypic measurement was not quantified in our study. To address this valid concern, the language throughout the manuscript has been moderated considerably in an attempt not to overstate the significance of the results presented here (i.e. Lines 160, 277,278, 283, 319, 490)
Fig 4 C and E: Were the apparent differences (upregulation of miR395, downregulation of miR396) statistically significant? If not, then it should be stated, so as not to overstate the results/conclusions.
• The absence of an asterisk in Fig 4C and E is indicative that the upregulation of miR395 and downregulation of miR396 respectively, were not deemed to be statistically significant. The Figure legend has been updated in the revised version of our manuscript to indicate the statistical analysis performed. Further, the language used in the text of the Results has been modified to only state the degree of accumulation change presented in the identified Figures (Lines 446, 451).
There is no statistical evidence that downregulation of miR160 leads to increased ARF1 expression in A10 shoots and roots (Fig 5C and D). A statistical test needs to be applied to the qPCR result, otherwise this conclusion is highly speculative. If there is no significant difference, then that should be reported.
• As stated above, the absence of an asterisk in Fig 5C and 5D is indicative that this result was determined to not be statistically significant. Again, the Figure 5 legend has been updated in the revised version of the manuscript to appropriately reflect the statistical analysis performed. Further, the text of the Results and Discussion sections states that the determined changes were ‘mild’.
The Figure legends should state i) what the error bars represent (SD or SEM), ii) how many replicates were analysed, iii) the meaning of the asterisks, iv) the statistical test(s) performed, v) p value.
• The authors kindly thank Reviewer 1 for the identification of this oversight in the original version of our manuscript. The appropriate Figure legends have been updated accordingly to state all statistical related analyses performed.
Minor comments
Supplementary data are not provided, so these are impossible to assess.
• The supplementary data files have been provided in the revised version of the manuscript. The authors apologise for failing to include the two Supp Tables in the originally submitted manuscript.
How was the specificity of the primers determined? There is no mention of melt curve step following the qPCR.
• All the primer pairs used for the reported RT-qPCR assessments were designed by the NCBI primer-BLAST online tools. Prior to use in the RT-qPCR analyses, all newly designed primer pairs were checked to determine that they only amplified a single amplicon of the correct size. Amplified PCR products were additionally sequenced to confirm that only the targeted product was being amplified by PCR. Finally, the specificity of each pair of primers was further confirmed via the formation of a single peak in each melt curve graph. The is standard practice in the Grof/Eamens laboratory and we do not feel that the inclusion of this preliminary data warrants inclusion in the study.
L330, L331, L342: “Figure 2 heatmap” should simply be written as “figure 2”, because there is no other data shown except a heat map.
• The word ‘heatmap’ has been deleted from L330, L333, 334, and L345. Now only Figure 2 is used as requested.
The resolution of the figures is so low that I can’t read the graph axes.
• AS required by Agronomy, all Figures are at a 300 dpi resolution and all images have been resized to ensure that all Figure included data is viewable by the reader.
Do the two Setaria viridis strains used in the study (A10 and ME034V) greatly differ in their salt tolerance? It would be useful to mention.
• There are several reports on the effects of salinity stress on Setaria viridis A10 and the domesticated Setaria italica species. However, presently, no data exists for the S. viridis ME034V accession as this accession has been more recently identified as readily amenable to Agrobacterium-mediated transformation.
Is there any difference between a crop species and a “cropping species”?
• The authors apologise for the use of these two interchangeable terms. We have removed the use of ‘cropping species’ in the revised version of the manuscript to avoid any additional reader confusion. We thank Reviewer 1 for identifying this issue.

Reviewer 2 Report

The manuscript presents an extremely current topic with the latest methodological approaches. It is a comprehensive and well written. I recommend that the manuscript be published after minor revision.

Title

Scientific name of the species in the title should be complete - Setaria viridis (L.) Beauv.

Abstract

Abstract should state hypotheses tested. It would be appropriate to mention specific results, similar to the ones in the last paragraph of Introduction.

The sentence in row 15 should begin “MicroRNAs…”

I propose to remove the keyword „gene expression regulation“, as the main object of the study is represented by microRNAs molecules and their regulatory potential under abiotic stress.

Introduction

On the other hand, the last paragraph of the Introduction should not contain a summary of the results, but the main hypotheses and intentions of the research. I recommend rewriting this paragraph.

The Latin name of the family Poaceae should be written in italics (rows 77, 78, 86, 88, 99 and 135) and similarly Latin name of S. viridis (row. 144). In the context of S. viridis genome annotation and transcriptomic datasets, the links to databases should be included.

Material and Methods

The methodology is written precisely and in detail.

I think readers would welcome a closer specification of the tested accessions, especially in terms of their genetic background.

Have the authors tested other concentrations of sodium chloride in their previous studies? What assumptions led them to apply this particular concentration?

Within the subchapter 2.2 is very often repeated information about the age of tested seedlings (10-days-old), which is redundant.

Result

The authors consistently interpret the individual results with emphasis on their application potential.

Unfortunately, individual figures, although very pedantic and comprehensive, but not enough sharp and legible.

Discussion

Is any reason to highlight the references 79, 80 and 83 (rows 753 and 754) in red?

I am sure that the authors take into account the complexity of regulatory mechanisms involving miRNAs molecules when interpreting their results.

Despite the abundance of specific miRNAs under induced stress conditions we should keep in mind that some miRNAs might be co-regulated by both environmental factors and developmental stimulus or different stress factors at the same time.

Conclusion

The conclusions are well written; include the main message of the manuscript and provide answers to the study objectives.

I would like to state that the authors carried out a scientifically consistent experiment with significant results for agronomic practice.

Author Response

First and foremost, on behalf of the authorship team, I would like to thank each of the three reviewers for taking the time and care to provide valuable comments and insightful feedback for the improvement of our originally submitted manuscript. Please find below our comment-by-comment responses in our attempt to adequately address each concern raised by the three reviewers post review of our originally submitted manuscript. Where appropriate, we have additionally identified where in the revised manuscript we have addressed each reviewer concern.
Kind regards,
Andrew Eamens

Reviewer 2
Title
Scientific name of the species in the title should be complete - Setaria viridis (L.) Beauv.
• We thank Reviewer 2 for identifying this oversight and have modified the revised manuscript’s title accordingly.
Abstract
Abstract should state hypotheses tested. It would be appropriate to mention specific results, similar to the ones in the last paragraph of Introduction.
• Both the aims of this study and the key validated microRNA abundance changes determined in this study changes have been stated in the Abstract of the revised manuscript.
The sentence in row 15 should begin “MicroRNAs…”
• The error has been fixed.
I propose to remove the keyword „gene expression regulation“, as the main object of the study is represented by microRNAs molecules and their regulatory potential under abiotic stress.
• The keyword ‘gene expression regulation’ has been removed. The authors thank Reviewer 2 for this helpful suggestion.
Introduction
On the other hand, the last paragraph of the Introduction should not contain a summary of the results, but the main hypotheses and intentions of the research. I recommend rewriting this paragraph.
• The authorship team respectively disagrees with this suggestion. It is standard practise in the journal Agronomy to provide a summary of the key results in the final paragraph of the Introduction. Further, the Introduction states the key aim of this study (Lines 137- 140), prior to highlighting the key results stemming from the assessed aims of the study.
The Latin name of the family Poaceae should be written in italics (rows 77, 78, 86, 88, 99 and 135) and similarly Latin name of S. viridis (row. 144). In the context of S. viridis genome annotation and transcriptomic datasets, the links to databases should be included.
• The plant family name can be written both in upright and/or italic text (see Spencer et al., 2007). The authors have therefore retained their preferred format.
• Error in row 144 has been addressed and the authors thank Reviewer 2 for identifying this oversight.
• A link has been included in the revised manuscript (line 574) to identify the BLAST search database used in this study. We thanks Reviewer 2 for identifying this oversight.
Spencer R., Cross R. (2007) Plant Names: A Guide to Botanical Nomenclature. CSIRO publishing. DOI: 10.1071/9780643097162.
Material and Methods
The methodology is written precisely and in detail.
I think readers would welcome a closer specification of the tested accessions, especially in terms of their genetic background.
• The name of the S.viridis accessions used in this study have been provided. The use of the two S. viridis accession in this study was purely based on both accessions demonstrated ability to be amenable to molecular modification via Agrobacterium-mediated transformation. Additional genetic information relating to the ME034V accession specifically is lacking, and the inclusion of such genetic background information would not add any further value to the manuscript.
Have the authors tested other concentrations of sodium chloride in their previous studies? What assumptions led them to apply this particular concentration?
• In the Grof/Eamens research laboratory, we have previously tested a range of NaCl concentrations for use in tissue culture based experimentation on a number of plant species. The use of 150 mM NaCl in this study was to provide a moderate and prolonged abiotic stress growth regime. Further, we have previously employed this saline growth regime in our previous study of Arabidopsis (https://www.mdpi.com/2223-7747/8/3/58/htm) for exactly the same reason as stated here for S. viridis, that is; to provide a moderate and prolonged salt stress growth regime. Therefore, we have selected the 150 mM NaCl stress regime for use across a range of plant species in an additional attempt to identify any conserved microRNA regulation trends in response to this identical salt stress treatment.
Within the subchapter 2.2 is very often repeated information about the age of tested seedlings (10-days-old), which is redundant.
• Thank you Reviewer 2 for identifying this issue. We have removed the use of seedling age in this section (and in other section where appropriate) of the revised manuscript in an attempt to address redundancy.
Result
The authors consistently interpret the individual results with emphasis on their application potential.
• The authors fail to see this issue. Further, any statement of potential application of the reported findings have been removed from the revised version of our manuscript in an attempt to address this concern.
Unfortunately, individual figures, although very pedantic and comprehensive, but not enough sharp and legible.
• The authors thank Reviewer 2 for identifying this issue. Updated versions of each Figure have been included in the revised manuscript in an attempt to address this concern of Reviewer 2.
Discussion
Is any reason to highlight the references 79, 80 and 83 (rows 753 and 754) in red?
• The authors thank Reviewer 2 for identifying this mistake. The errors have been amended in the revised version of the manuscript.
I am sure that the authors take into account the complexity of regulatory mechanisms involving miRNAs molecules when interpreting their results. Despite the abundance of specific miRNAs under induced stress conditions we should keep in mind that some miRNAs might be co-regulated by both environmental factors and developmental stimulus or different stress factors at the same time.
• The authorship team thanks Reviewer 2 for provide this highly informative insight. We have further acknowledged the high degree of cross-talk between the molecular pathways underpinning plant growth and abiotic stress response. Additional statements have been added to the revised manuscript to address this concern of Reviewer 2.
Conclusion
The conclusions are well written; include the main message of the manuscript and provide answers to the study objectives.
I would like to state that the authors carried out a scientifically consistent experiment with significant results for agronomic practice.
• The authors wish to thank Reviewer 2 for their positive review of our originally submitted manuscript, and for the many helpful and insightful suggestions for manuscript improvement made by Reviewer 2 in their assessment of the originally submitted manuscript: suggestions that have been included in the revised version of our manuscript.

Reviewer 3 Report

Manuscript ID Agronomy-805200 entitled "Profiling of the Salt Stress Responsive MicroRNA Landscape of C4 Genetic Model Species Setaria viridis”

Submitted to section: Crop Breeding and Genetics

Reviewer: 1

Ms. Ref. No.: Agronomy-805200

Authors: Joseph Pegler, Duc Quan Nguyen, Christopher Grof, Andrew Eamens*

Specific notes:

TITLE

Nothing to comment.

ABSTRACT

Line 18: Delete “we”

Line 24: "This analysis revealed accession-....", a script appears, is this correct?

KEYWORDS

Lines 30 and 31: The keywords do not seem to be keywords, since three of them also appear in the title, and another 3 are very extensive, please change/modify it ...

 INTRODUCTION

Lines 102 and 103: “…a short stature of 20 to 30 centimeters (20-30 cm) at maturity”. The statement seems to be duplicated, please clarify or correct

Line 140: Delete “we”

MATERIALS AND METHODS

Lines 169, 211, 225 and 226: Setaria viridis must be abbreviated

RESULTS

Lines 270, 299, 320, 352, 423, 433 and 458: Setaria viridis must be abbreviated

All the part of results are merely descriptive, because at no time do the values of the statistical analyzes appear. I understand that there is statistical analysis, because in some of the figures asterics appear above the bars (for example in figure 1), but the authors do not explain it in the legend either. Please, correct, add or explain everything related to the statistical analysis performed on the manuscript.

DISCUSSIÓN

Lines 753 and 754: The appointments numbers appear in red, please, change.

FIGURES

The 6 figures are incredible for the amount of information they can contribute to the article, but unfortunately it has been impossible for me to decipher their data, values of the axes,.... I would ask the authors to make a great effort to increase their resolution, size and clarity, otherwise they will not give any information to the possible readers.

SUPPLEMENTARY MATERIALS

Line 781: Setaria viridis must be abbreviated

Author Response

First and foremost, on behalf of the authorship team, I would like to thank each of the three reviewers for taking the time and care to provide valuable comments and insightful feedback for the improvement of our originally submitted manuscript. Please find below our comment-by-comment responses in our attempt to adequately address each concern raised by the three reviewers post review of our originally submitted manuscript. Where appropriate, we have additionally identified where in the revised manuscript we have addressed each reviewer concern.
Kind regards,
Andrew Eamens

Reviewer 3
TITLE
Nothing to comment.
ABSTRACT
Line 18: Delete “we”
• This sentence has been modified to remove ‘we’
Line 24: "This analysis revealed accession-....", a script appears, is this correct?
• Yes, the presence of ‘-‘ is intended for use here.
KEYWORDS
Lines 30 and 31: The keywords do not seem to be keywords, since three of them also appear in the title, and another 3 are very extensive, please change/modify it.
• The Keywords have been modified to address this concern. Those that remain are central to the main focus(s) of this study and should therefore be retained (keywords have been selected to assist in the identification this study in future literature searches).
INTRODUCTION
Lines 102 and 103: “…a short stature of 20 to 30 centimetres (20-30 cm) at maturity”. The statement seems to be duplicated, please clarify or correct
• Thank you to Reviewer 3 for identifying this error. The identified error has been fixed in the revised manuscript.
Line 140: Delete “we”
• The authors thank Reviewer 3 for identifying this error. The identified error has been fixed in the revised manuscript.
MATERIALS AND METHODS
Lines 169, 211, 225 and 226: Setaria viridis must be abbreviated
• The full name, Setaria viridis, has been kept in its full form on Lines 169, 211 and, 225 (Journal requirement for full names in subsection headings). On line 226, the abbreviation has been added.
RESULTS
Lines 270, 299, 320, 352, 423, 433 and 458: Setaria viridis must be abbreviated
• As stated above, following journal requirements, the full name for Setaria viridis remains in all subheadings. On lines 299, 320, 352, 423 and 458 the abbreviation has been used in the revised version of the manuscript.
All the part of results are merely descriptive, because at no time do the values of the statistical analyzes appear. I understand that there is statistical analysis, because in some of the figures asterics appear above the bars (for example in figure 1), but the authors do not explain it in the legend either. Please, correct, add or explain everything related to the statistical analysis performed on the manuscript.
• The authors apologise for this significant oversight, and thank Reviewer 3 for bringing this to the attention of the authorship team. We have now addressed this issue throughout the revised manuscript. In addition, the statistical analysis used is now detailed in each appropriate Figure legend.
DISCUSSIÓN
Lines 753 and 754: The appointments numbers appear in red, please, change.
• The authors thank Reviewer 3 for identifying this oversight. The error has been fixed in the revised version the manuscript.
FIGURES
The 6 figures are incredible for the amount of information they can contribute to the article, but unfortunately it has been impossible for me to decipher their data, values of the axes. I would ask the authors to make a great effort to increase their resolution, size and clarity, otherwise they will not give any information to the possible readers.
• All manuscript Figures have been enlarged and have been updated with higher resolution to address this reviewer criticism. The authors thank Reviewer 3 for identifying this issue of our originally submitted manuscript.
SUPPLEMENTARY MATERIALS
Line 781: Setaria viridis must be abbreviated
• The error has been fixed.

Round 2

Reviewer 1 Report

The authors have addressed my concerns and the manuscript is improved as a result.

Only a few minor, non-critical points remain:

In Table S2, there are no read counts for miR166g in the roots of either accession. Is this a typo?

I think a description of the melt curve step should be added to the qPCR cycle conditions that are described in the materials and methods section under heading 2.6. I agree that it is not standard practice to include melting curve data for a qPCR experiment in a scientific manuscript.

Author Response

Dear Reviewer,

Thank you for taking the time to again assess our revised manuscript. We additionally thank the reviewer for stating that the revised version of our originally submitted manuscript is 'improved' post addressing the reviewer's concerns.

As per your comments stemming from your 2nd round of review of our manuscript we have again revised the manuscript.

Namely, we thank the reviewer for identifying the typographical error in Table S2. A value of '0' should have been entered into each column of the table indicating our failure to detect the miR166g sRNA via sequencing.This has been amended in a revised Table S2.

Secondly, and as advised, we have included a further description of our lab's assessment of the suitability of each new pair of primers for use in our RT-qPCR quantifications of gene expression (please see lines 239 to 244 of the revised manuscript).

We hope that we have now made all corrections to our manuscript that address your concerns.

Kind regards,

Andrew Eamens (on behalf of the authorship team).