Identification and Expression Analysis of miR166 Gene Family in Response to Salt Stress in Chrysanthemum
and Yunwei Zhou *
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsmiRNAs are very important to understand the effects of abiotic stresses in plant research. This paper is well-designed, and the figures are well-prepared and informative. However, some changes are recommended to enhance the clarity and contextual relevance of the study: 1. Include the evaluation of salinity treatment's phenotypic effects by reporting growth parameters and providing visual evidence, such as pictures, to complement the molecular findings. 2. Clarify the selection of 12-hour time point for expression analysis. Additionally, justify the choice of roots and leaves as the only tissues analyzed for expression under salinity treatment. 3. Address the inconsistency between Figures 11 and 12. While Figure 12 provides a tissue-specific profiling of miR166 expression (including stems and flowers, which show higher expression levels than leaves and roots), these tissues are omitted from Figure 11, which examines the salinity effect. To improve coherence: -Present Figure 12 (tissue-specific expression) earlier in the manuscript, as it provides foundational context. -Expand Figure 11 to include all four tissues (roots, leaves, stems, and flowers) in the salinity analysis. 4. Consider incorporating additional time points to provide a more comprehensive understanding of miR166's role under salinity stress. This could also help to demonstrate its phenotypic effects on roots, leaves, stems, and flowers over time.
Author Response
Dear reviewer,
We are really appreciate for your excellent and professional revision of this manuscript. We have checked the manuscript according to the comments. After carefully studying, we have made corresponding changes on the manuscript and uploaded to the attachment. Hope these will make it more acceptable for publication.
If any other information or modification are needed, please let me know, thank you so much.
Yours sincerely,
Di Wang
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsGeneral comments
English language ans syntax need to be improved. Figures in the text should be named as (Fig. 1) or (Fig. 2) etc. Indicate in section M&M how miR166 were initially detected and add a reference. Discussion needs to be improved since after each statement based on the results of this work need to be compare with results obtained in other plants concerning miR116 isoforms.
Specific comments
Line 8, 200 mM
Line 10, naming them from cgr-miR166a to cgr-miR166j and their precursor could form
Line 13, which diverse stimuli? Name some of them
Line 15, HD-ZIPIII transcription factor
Line 21, salt stress; miR166; transcription factor
Line 27, nematodes (space) [2]
Line 29, miRNA (space) [5]
Polyadenylation (space) [4]
Strong resistance to what?
Line 61, 200 mM NaCl, and idem in 80; were watered with 45 mL of water (control) or with and aqueous solution containing 200 mM NaCl
Line 87, was determined (reference to previous work)
Line 1Line 161, roots (Fig. 1).
Line 171, decrease letter size in order to have the whole nucleotide sequence in one line, in mature miRNA
Line 182, (Fig. 2)
Line 194, (Fig. 3)
Line 226, ten precursors
Line 261, photoreactivity? Define the concept
Line 264, alkyl metabolism? Define the concept
Line 286, In addition to have
Line 306, why bars in Fig. 10 do not have statistical analyses?
Line 341-356, After each sentence with a statement it should be a comparison with what is known for miR166 in other plants. Do not place several statement without discussing each one with the literature.
Comments on the Quality of English Language
English language abd syntax need to be severely improved.
Author Response
Dear reviewer,
We are really appreciate for your excellent and professional revision of this manuscript. We have checked the manuscript according to the comments. After carefully studying, we have made corresponding changes on the manuscript and uploaded to the attachment. Hope these will make it more acceptable for publication.
If any other information or modification are needed, please let me know, thank you so much.
Yours sincerely,
Di Wang
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsDear authors, below is my comments to your work.
Abstract
"according to the small RNA library"— you should remove it from the abstract (it's redundant information here) and place the following information in the Materials and methods section;
"HD-ZipIII was validated to be the target gene"— not the target gene but the target transcription factor family?
I suggest five following keywords: Chrysanthemum; salt stress; transcription factors; target genes; microRNA, since "miR166" repeated from the article name
Introduction
33 line: miRNA/miRNA*, 37 miRNA* - What is the meaning of asterisks? You should remove it or make a proper description.
41-43 line: Please provide a reference paper to this sentence statement.
50 - 57 line: Provide the uniform spelling of plant species here and through the full text: A. thaliana or Arabidopsis thaliana, for instance.
60-61 line: "In the early stage of the research group"—please remove the word "group" since it creates a different meaning for the sentence; double-check the full text for such english formulations.
Materials and Methods
2.1. Which age/stage of plants was used in the experiment? Which species was used? Is it Chrysanthemum 'Niu 9717' (Chrysanthemum × grandiflora)? You should clearly indicate it.
2.2. line 86: "significantly differentially expressed" should be rephrased as "differentially expressed". Also, please provide which thresholds of P-values and fold change thresholds were used to consider a gene as differentially expressed. Which parameters of BLASTN were used during analysis?
2.3. Title "Prediction of the secondary structure" - structure of what? miRNA? Please add to the title.
2.4. Which parameters of DNAMAN were applied?
2.5. Which databases were used to download miR166 precursor sequences of A. thaliana, O. sativa, Z. mays, S. bicolor, Medicago truncatula, G. max, and Vitis vinifera?
MEGA: Which parameters of NJ were used? What number of bootstraps?
2.6. Line 113: (version 2.096)was—need to add space. Please check the full text for such misspellings.
2.7. Which algorithm and parameters were used to reconstruct the phylogenetic tree?
Results
All the legends on figures should be self-explanatory. You need to add some important information for them:
Figure 1 - Add species name;
Figure 3 - Not conservative analysis but identified conserved motif?
Figure 4 - Which algorithm was used for MSA?
Figure 5 - Meaning of red-labeled nodes? Meaning of violet, yellow, and green background? Add cgr abbreviation meaning — what species it is.
Figure 6, Figure 9 - Meaning of numbers? Which algorithm is used for reconstruction?
Figure 7. There is not a good color legend, since it overlaps heavily between different elements, and it is almost impossible to understand. Please, for each element, set a unique color and remove the gradient and use a solid fill instead.
Figure 8. - Very small fonts — please make them at least 1.5x bigger.
Figure 2 — Could you get the image of better quality? It is difficult to see nucleotides on this figure.
Title "3.4. Analysis of phylogenetic evolution" should be renamed to Phylogenetic analysis of identified MIR166 or similarly. "Phylogenetic evolution" is not correct.
Discussion
If possible, for better reader experience, draw the figure with the overall scheme of probable mechanisms of miRNA166 action of Chrysanthemum that you found during your study. You could indicate possible pathways, regulators, downstream genes, and regulation of miRNA166 with HD-ZIPIII genes, etc.
Data Availability Statement
Please write this section. Here you should indicate which data you provide in Supplementary Materials for this article and any other relevant data you shared.
Comments on the Quality of English LanguageYou should improve the overall quality of English language before resubmitting the new version of manuscript.
60-61 line: "In the early stage of the research group"—please remove the word "group" since it creates a different meaning for the sentence; double-check the full text for such english formulations.
Author Response
Dear reviewer,
We are really appreciate for your excellent and professional revision of this manuscript. We have checked the manuscript according to the comments. After carefully studying, we have made corresponding changes on the manuscript and uploaded to the attachment. Hope these will make it more acceptable for publication.
If any other information or modification are needed, please let me know, thank you so much.
Yours sincerely,
Di Wang
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript doesn't provide enough information about the relationship between miR166 and salinity stress and doesn't show any phenotypic effects. The information requested in the previous revision is needed to be accepted for publication.
Author Response
Dear reviewer,
We are really appreciate for your excellent and professional revision of this manuscript. We have checked the manuscript according to the comments. After carefully studying, we have made corresponding changes on the manuscript and uploaded to the attachment. Hope these will make it more acceptable for publication.
If any other information or modification are needed, please let me know, thank you so much.
Yours sincerely,
Di Wang
Author Response File: Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe language has been improved but there are still some aspects that can be improved.
Line 8, Chrysanthemum nankinense
Line 9, Chrysanthemum in italics
Line 13, like the phytohormones ABA and IAA
Line 16, which one of the miRNA166? or all of them?
Line 30, on miRNA in terrestrial plants
Lien 33, and are subjected to polyadenylation
Line 44, and there are
Line 173, cgr-miRNA166a to cgr-miNA166j
Line 315, Table 2, reduce the size of letter in column 1 to put the text in one line and define the target gene n 5 or it is ab hypothetiical protein?
Line 358, which miRNA166?
Line 364, In Chrysanthemum, there are seeral miRNA166 showing the conserved sequence
Comments on the Quality of English Language
English still can be improved
Author Response
Dear reviewer,
We are really appreciate for your excellent and professional revision of this manuscript. We have checked the manuscript according to the comments. After carefully studying, we have made corresponding changes on the manuscript and uploaded to the attachment. Hope these will make it more acceptable for publication.
If any other information or modification are needed, please let me know, thank you so much.
Yours sincerely,
Di Wang
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsDear authors, I think you did a good job in revising your initial manuscript. Most of my comments were taken into account. Good luck with your research!
Author Response
Reviewer #3：Dear authors, I think you did a good job in revising your initial manuscript. Most of my comments were taken into account. Good luck with your research!
Response: We greatly appreciate your excellent and professional comments on this manuscript. If you need any further information, please feel free to contact me.
