Mammalian Sex Hormones as Steroid-Structured Compounds in Wheat Seedling: Template of the Cytosine Methylation Alteration and Retrotransposon Polymorphisms with iPBS and CRED-iBPS Techniques

Round 1

Reviewer 1 Report

The authors studied the effect of mammalian sex hormones as steroid-like compounds on wheat seedlings: Cytosine methylation template and retrotransposon polymorphisms using iPBS and CRED-iBPS techniques

Is a well researched and documented study, however I would have a few comments to make:

specify the manufacturer of the reagents used

also specify the model and manufacturer of the equipment used

explain what Tm and CG stand from table 1

as you have detailed what iPBS analysis is, explain for CRED-iPBS as well

Figures 1 and 3 are unclear. Please increase clarity

Author Response

Responses to Comments of Reviewer 1

General Response:

First of all, we thank the potential reviewer for her/his valuable time and also raised helpful comments and suggestions. In this step of revision, we have tried to respond to all comments and addressed all questions. We hope the revised version of manuscript gets positive feedback from you and will be acceptable for publication in the
Applied Sciences journal. All revised parts have been highlighted in blue.

 

Sincerely,

Dr. Aras Turkoglu

Comments

 

Comment 1# Specify the manufacturer of the reagents used

Response to Comment 1#

Line129-133, Line153: Added the manufacturer of the reagents used in line with the reviewer's suggestion.

 

Comment 2# also specify the model and manufacturer of the equipment used

Response to Comment 2#

Line145: Added the model and manufacturer of the equipment used.

 

Comment 3# explain what Tm and CG stand from table 1

Response to Comment 3#

Line171: Tm and CG explained and added for Table 1

 

Comment 4# as you have detailed what iPBS analysis is, explain for CRED-iPBS as well

Response to Comment 4#

CRED-iPBS is described in the main text of manuscript; Line105-107 “After DNA restriction digestion with methylation–sensitive enzymes such as HpaII and MspI, the CRED–iPBS method is used to identify methylation changes that occur in different tissues or between different development stages [22-24]”Line161-169 “To perform CRED–iPBS analysis, a quantity of 1,000 ng of DNA sample from each treatment was individually digestion at 37°C for 2 hours, using 1 µl of HpaII and 1 µl of MspI, according to the instructions provided by the manufacturer (Thermo Scien-tific, Waltham, USA). The digested DNA, which corresponds to each endonuclease, was used in the PCR mix as a surrogate for undigested gDNA. The amplification pro-cedure was carried out using the primers shown in Table 1. The PCR procedural steps were analogous to those used in the previously mentioned iPBS study. The differentia-tion of iPBS and CRED–iPBS PCR products on the basis of their base size was achieved by using the electrophoresis technique[11,13,19,22–25].”Line214-216 “The results of the CRED–iPBS assay are indicated as the percentage of polymorphism in CRED–iPBS assays that were digested by MspI and HpaII”

 

Comment 5# Figures 1 and 3 are unclear. Please increase clarity

Response to Comment 5# We made increase the clarity of this figures.

 

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Dear authors,

The manuscript entitled “Mammalian Sex Hormones as Steroid-Structured Compounds in Wheat Seedling: Template of the Cytosine Methylation Alteration and Retrotransposon Polymorphisms with iPBS and CRED-iBPS Techniques” report an investigation on the effects of MSHs on DNA damage and DNA methylation of wheat (Triticum aestivum L.) during the seedling growth stage, using the CRED–iPBS assay and iPBS analysis to determine DNA methylation status. Some authors have published articles related to the theme of the manuscript found in databases (Sciencedirect, Pubmed, MDPI, Web of science, etc). The language (English) are satisfactory (I suggest the final revision)! However, you need to change some details/information in the abstract, Introduction, Methods, results, discussion and conclusions.

1. Abstract: Adequate, but I suggest rewrite and add information:

- There are many acronyms! I suggest informing them the meaning of CRED-iPBS and iPBS.

- The abstract is well written, with details of the methods used. However, at the end, I suggest highlighting the advantages/limitations of the method and analysis applied.

2. Introduction section: It is well written, but I suggest:

- I suggest highlighting the "innovative" proposal of the study, as well as the advantages / disadvantages, at the end of the introduction.

3. Material and Methods section: The methodological proposal is appropriate to the manuscript, but I suggest:

- Page 3, in “2.1. Plant material, seed germination and treatments” section: The authors wrote: “The wheat seeds were subjected to preliminary washing process using tap water to eliminate any extraneous contaminants”. I suggest indicating the possible contaminants! I suggest informing storage/packaging data of the plant/samples until the time of the tests performed. I suggest indicating the references used for the procedures adopted.

- Page 3, line 129: The authors wrote: “Seeds were treatment with MSH at various concentrations, including 0, 0.05, 0.5 and 5 μM.” How were these concentrations defined? Any previous protocol/study? If yes, inform!

4. Results section (or “Results and discussion”)?

Wouldn't it be more interesting to combine the "results” with the "discussion" to better describe the findings and compare them with other works published in the literature?? I suggest expanding the discussions!

- Page 4, in “3.1. iPBS assays” section: Were the experiments done in triplicate or more? Was any statistical analysis performed to interpret the data? There are authors from the Department of Mathematical and Statistical Methods!!!?? I Suggest statistical treatment! Enter the deviations from the data in Figure 2!!! Same for Figure 4!

5. Discussion section (or “Results and discussion”)?

Wouldn't it be more interesting to combine the "results” with the "discussion" to better describe the findings and compare them with other works published in the literature?? I suggest expanding the discussions!

- I suggest, at the end of the section, to write a paragraph summarizing the findings and their impacts on the research proposal.

- Conclusion section: I suggest highlighting  the "innovative" proposal of the study, as well as the advantages/ disadvantages/limitations.

* Figures: Adequate! Enter the deviations from the data in Figure 2!!! Same for Figure 4!

* References: Please, check if the references are in accordance with the journal's rules.

Dear editor,

The manuscript entitled “Mammalian Sex Hormones as Steroid-Structured Compounds in Wheat Seedling: Template of the Cytosine Methylation Alteration and Retrotransposon Polymorphisms with iPBS and CRED-iBPS Techniques” report an investigation on the effects of MSHs on DNA damage and DNA methylation of wheat (Triticum aestivum L.) during the seedling growth stage, using the CRED–iPBS assay and iPBS analysis to determine DNA methylation status. Some authors have published articles related to the theme of the manuscript found in databases (Sciencedirect, Pubmed, MDPI, Web of science, etc). The language (English) are satisfactory (I suggest the final revision)! However, you need to change some details/information in the abstract, Introduction, Methods, results, discussion and conclusions.

1. Abstract: Adequate, but I suggest rewrite and add information:

- There are many acronyms! I suggest informing them the meaning of CRED-iPBS and iPBS.

- The abstract is well written, with details of the methods used. However, at the end, I suggest highlighting the advantages/limitations of the method and analysis applied.

2. Introduction section: It is well written, but I suggest:

- I suggest highlighting the "innovative" proposal of the study, as well as the advantages / disadvantages, at the end of the introduction.

3. Material and Methods section: The methodological proposal is appropriate to the manuscript, but I suggest:

- Page 3, in “2.1. Plant material, seed germination and treatments” section: The authors wrote: “The wheat seeds were subjected to preliminary washing process using tap water to eliminate any extraneous contaminants”. I suggest indicating the possible contaminants! I suggest informing storage/packaging data of the plant/samples until the time of the tests performed. I suggest indicating the references used for the procedures adopted.

- Page 3, line 129: The authors wrote: “Seeds were treatment with MSH at various concentrations, including 0, 0.05, 0.5 and 5 μM.” How were these concentrations defined? Any previous protocol/study? If yes, inform!

4. Results section (or “Results and discussion”)?

Wouldn't it be more interesting to combine the "results” with the "discussion" to better describe the findings and compare them with other works published in the literature?? I suggest expanding the discussions!

- Page 4, in “3.1. iPBS assays” section: Were the experiments done in triplicate or more? Was any statistical analysis performed to interpret the data? There are authors from the Department of Mathematical and Statistical Methods!!!?? I Suggest statistical treatment! Enter the deviations from the data in Figure 2!!! Same for Figure 4!

5. Discussion section (or “Results and discussion”)?

Wouldn't it be more interesting to combine the "results” with the "discussion" to better describe the findings and compare them with other works published in the literature?? I suggest expanding the discussions!

- I suggest, at the end of the section, to write a paragraph summarizing the findings and their impacts on the research proposal.

- Conclusion section: I suggest highlighting  the "innovative" proposal of the study, as well as the advantages/ disadvantages/limitations.

* Figures: Adequate! Enter the deviations from the data in Figure 2!!! Same for Figure 4!

* References: Please, check if the references are in accordance with the journal's rules.

Author Response

Responses to Comments of Reviewer 2

General Response:

First of all, we thank the potential reviewer for her/his valuable time and also raised helpful comments and suggestions. In this step of revision, we have tried to respond to all comments and addressed all questions. We hope the revised version of manuscript gets positive feedback from you and will be acceptable for publication in the Applied Sciences journal. All revised parts have been highlighted in red.

 

Sincerely,

Dr. Aras Turkoglu

Comments

 

Abstract: Adequate, but I suggest rewrite and add information:

 

Comment 1# There are many acronyms! I suggest informing them the meaning of CRED-iPBS and iPBS.

Response to Comment 1#

Line31: In abstract, added for CRED-iPBS “Coupled restriction enzyme digestion- inter primer binding site”

Abbreviations are explained where they were first used.

Line59; available for MSH

Line96; also added for PCR

Line100-101; available for CRED-iPBS and iPBS

Line195; available for GTS

 

Comment 2# The abstract is well written, with details of the methods used. However, at the end, I suggest highlighting the advantages/limitations of the method and analysis applied.

Response to Comment 2#

We Added in abstract this sentences “Upon comprehensive examination of the results, it was seen that the employed methodology successfully detected alterations in cytosine methylation of genomic DNA (gDNA) as well as changes in the pattern of genomic instability.”

 

2. Introduction section: It is well written, but I suggest:

 

Comment 3# I suggest highlighting the "innovative" proposal of the study, as well as the advantages / disadvantages, at the end of the introduction.

Response to Comment 3#

This sentence was highlighting in the main text.; The study of epigenetic changes can be approached from many different angles. To detect changes in DNA, one of these methods involves the use of PCR (polymerase chain reaction) in combination with cytosine methylation–sensitive enzymes [19]. This method is used to identify methylation changes. The use of cytosine methylation–sensitive enzymes enable high–throughput studies of this epigenetic regulatory mechanism. In addition, the technique is simple and cost–efficient [20,21]. Restriction enzyme coupled digestion and inter-primer binding site (iPBS) methodology, also known as CRED–iPBS, is a method that is both efficient and cost–effective for studying the methylation status of plant DNA [13,22-24].

 

3. Material and Methods section: The methodological proposal is appropriate to the manuscript, but I suggest:

 

Comment 4# Page 3, in “2.1. Plant material, seed germination and treatments” section: The authors wrote: “The wheat seeds were subjected to preliminary washing process using tap water to eliminate any extraneous contaminants”. I suggest indicating the possible contaminants! I suggest informing storage/packaging data of the plant/samples until the time of the tests performed. I suggest indicating the references used for the procedures adopted.

 

Response to Comment 4#

Line126-127: added “such as dust, soil, chemical residue.”

Line144: Added reference for section “2.1. Plant material, seed germination and treatments”

 

Comment 5# Page 3, line 129: The authors wrote: “Seeds were treatment with MSH at various concentrations, including 0, 0.05, 0.5 and 5 μM.” How were these concentrations defined? Any previous protocol/study? If yes, inform!

Response to Comment 5#

No. To the best of our knowledge, these concentrations were planned for the first time in wheat by this study.

 

4. Results section (or “Results and discussion”)?

 

Comment 6# Wouldn't it be more interesting to combine the "results” with the "discussion" to better describe the findings and compare them with other works published in the literature?? I suggest expanding the discussions!

Response to Comment 6#

Thank you very much for the suggestion. However, we think that writing the conclusion and discussion separately will provide a clearer understanding of the manuscript.

 

Comment 7# Page 4, in “3.1. iPBS assays” section: Were the experiments done in triplicate or more? Was any statistical analysis performed to interpret the data? There are authors from the Department of Mathematical and Statistical Methods!!!?? I Suggest statistical treatment! Enter the deviations from the data in Figure 2!!! Same for Figure 4!

Response to Comment 7#

Dear reviewer, in this study analyzed data made with TOTAL LAB software, in this study data is like molecular weight and it is not numeric data, therefore, analyze method was different.

5. Discussion section (or “Results and discussion”)?

 

Comment 8# Wouldn’t it be more interesting to combine the "results” with the "discussion" to better describe the findings and compare them with other works published in the literature?? I suggest expanding the discussions!

Response to Comment 8#

We think that writing the conclusion and discussion separately will provide a clearer understanding of the manuscript.

In the discussion section, it was written extensively, supported by current studies.

 

Comment 9# I suggest, at the end of the section, to write a paragraph summarizing the findings and their impacts on the research proposal.

Response to Comment 9#

The findings are summarized in detail in the results section.

Line196-205: for iPBS assays

Line234-238 and Line244-246: for CRED-iPBS assays

 

 

6. Conclusion section: 

 

Comment 10# I suggest highlighting the "innovative" proposal of the study, as well as the advantages/ disadvantages/limitations.

Response to Comment 10# ‘In the present study, for the first time we investigated the effects of four different MSHs at four different concentrations on the genetic and epigenetic stability of wheat plants at the seedling growth stage using CRED-iPBS and iPBS analyses.’ and ‘Nevertheless, further studies are recommended to better understand the function that MSHs play in the molecular mechanisms affecting wheat plant growth. In conclusion, our work shows that MSHs induce genetic and epigenetic changes and play an active role in maintaining genomic stability.’ was added.

 

 

7. Figures:

 

Comment 11# Adequate! Enter the deviations from the data in Figure 2!!! Same for Figure 4!

Response to Comment 11# Dear reviewer, in this study analyzed data made with TOTAL LAB software, in this study data is like molecular weight and it is not numeric data, therefore, analyze method was different.

 

8. References:

 

Comment 12# Please, check if the references are in accordance with the journal's rules.

Response to Comment 12#

All references were checked again.

Author Response File: Author Response.pdf

Reviewer 3 Report

The article titled "Mammalian Sex Hormones as Steroid-Structured Compounds in Wheat Seedling: Template of the Cytosine Methylation Alteration and Retrotransposon Polymorphisms with iPBS and CRED-iBPS Techniques", discuss a good point and may be a kind of warning of using the growth hormones in agriculture. The idea of the manuscript is very good and also the work plan is good but I have some comments.

1-Title: should be shortening because it is too long and why it contains reterotransposon and nothing mentioned in the main text of the manuscript

Abstract: it is ok

Introduction:

1-Introduction is very short and did not contain the importance of the studied crop (wheat).

2-Aime of the study is not quite clear; authors should concentrate on the aim of the work.

Materials and methods:

Plant material collection and identification should be included in the materials part.

In page 4 line no.152, there is no final extension cycle which is 7-10 minutes, I think it is missed.

-the quantity and the concentration of the undigested DNA before digestion not indicated.

- Table 2 and 3, can be substituted by dendogram (Phylogeny) based on the band presence absence using the STATICA 5 PROGRAM.

Discussion

Is written well but it is very short and needs more interpretation-more citation and recent references.

Conclusion:

Is too long and should accurate and more concentrated on what the valuable results was obtained and also convey resulted in this study should be listed as well.

 

Author Response

Responses to Comments of Reviewer 3

General Response:

First of all, we thank the potential reviewer for her/his valuable time and also raised helpful comments and suggestions. In this step of revision, we have tried to respond to all comments and addressed all questions. We hope the revised version of manuscript gets positive feedback from you and will be acceptable for publication in the Applied Sciences journal. All revised parts have been highlighted in yellow.

 

Sincerely,

Dr. Aras Turkoglu

Comments

 

1-Title

 

Comment 1# should be shortening because it is too long and why it contains retrotransposon and nothing mentioned in the main text of the manuscript.

Response to Comment 1#

We added the new sentence in the main text. Also, the markers used were designed according to the retrotransposon regions. Mentioned on Line 106, Line21, Line22, Line 23, Line 25, Line26, Line30, Line41, Line44, line69 and Line 87.

 

2- Abstract:  it is ok

 

3. Introduction:

 

Comment 2# Plant material collection and identification should be included in the materials part.

Response to Comment 2#

Line 123-125: Added where plant material is sourced from

 

Comment 3# Aime of the study is not quite clear; authors should concentrate on the aim of the work.

Response to Comment 3#

Line116-120: available “This study was designed to investigate the effects of MSH (17 β–estradiol, estrogen, testosterone, and progesterone) on DNA damage and DNA methylation of wheat plants during the seedling growth stage. In addition, the CRED–iPBS method was used in combination with iPBS markers to determine the DNA methylation status of wheat plants.”

 

3. Material and Methods:

 

Comment 4# Plant material collection and identification should be included in the materials part.

Response to Comment 4#

Line 123-125: Added where plant material is sourced from

 

Comment 5# In page 4-line no.152, there is no final extension cycle which is 7-10 minutes, I think it is missed.

Response to Comment 5#

Sentence “and final extension for ten minutes at 72°C.” added in line with reviewer's suggestion

 

Comment 6# the quantity and the concentration of the undigested DNA before digestion not indicated.

Response to Comment 6#

Line165-167: Available “To perform CRED–iPBS analysis, a quantity of 1,000 ng of DNA sample from each treatment was individually digestion at 37°C for 2 hours, using 1 µl of HpaII and 1 µl of MspI, according to the instructions provided by the manufacturer”

 

Comment 7# Table 2 and 3, can be substituted by dendrogram (Phylogeny) based on the band presence absence using the STATICA 5 PROGRAM.

Response to Comment 7#

The study only determined methylation and genomic instability as there was no phylogenetic relationship.

 

4. Discussion:

 

Comment 8# Is written well but it is very short and needs more interpretation-more citation and recent references.

Response to Comment 8#

Dear reviewer, you know better, the MSH study is limited in plant especially in plant genetic such as the epigenetic, thus we discussed all the work in this field.

 

5. Conclusion section: 

 

Comment 9# Is too long and should accurate and more concentrated on what the valuable results was obtained and also convey resulted in this study should be listed as well.

Response to Comment 9#

Line90-95: sentences added (In the context of progesterone therapy, an inverse relationship was found between the concentration of progesterone and the levels of GTS, indicating a reduction in GTS as the concentration of progesterone increased. Based on the mean proportion of poly-morphisms detected in HpaII and MspI digestion, it may be deduced that elevated amounts of MSHs (17 β–estradiol, progesterone, and estrogen) would provide a safe-guarding impact against hypermethylation.)

Author Response File: Author Response.pdf

Reviewer 4 Report

As far as I understood the authors are trying to investigate the biological effects of mammalian sex hormones on on DNA damage and DNA methylation of wheat (Triticum aestivum L.)

1- The first question I would ask why you selected wheat plants as a model organism for this study, we know that plants do not have sex chromosomes

2- Have you tested other steroids (not related to sex hormones) like dexamethason as a control

3- Please unify the format of all your graphs and include statistical analysis

 

Moderate editing of English language required

Author Response

Responses to Comments of Reviewer 4

General Response:

First of all, we thank the potential reviewer for her/his valuable time and also raised helpful comments and suggestions. In this step of revision, we have tried to respond to all comments and addressed all questions. We hope the revised version of manuscript gets positive feedback from you and will be acceptable for publication in the Applied Sciences journal. All revised parts have been highlighted in green.

 

Sincerely,

Dr. Aras Turkoglu

Comments

 

Comment 1# The first question I would ask why you selected wheat plants as a model organism for this study, we know that plants do not have sex chromosomes.

Response to Comment 1#

Relevant literature information is explained in the introduction;

Line67-77: “These hormones were once thought to be found only in animals, but at the turn of the 20th century, their presence in plants was discovered [6]. As a result of advances in technology, a significant number of researchers have published results indicating that MSHs are naturally present in plants and that their concentrations vary depending on the plant species, tissue, and stage of development [7,8].”

“The number of studies on the existence of MSH in plants has increased, prompting the need to investigate the role these hormones play in plant life [9]. Recent studies have focused on evaluating the effects of exogenous application of these compounds on plant growth and development, as well as their tolerance to various abiotic and bio-tic stimuli and their metabolic mechanism of action [10,11]”

 

Comment 2# Have you tested other steroids (not related to sex hormones) like dexamethasone as a control

Response to Comment 2#

We did not test.

 

Comment 3# Please unify the format of all your graphs and include statistical analysis.

Response to Comment 3# all graphs  and table was rearranged.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

I suggest accepting the manuscript, after the proposed corrections!

Reviewer 4 Report

I am pleased to accept the manuscript for publication in its present form

Minor editing of English language required