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  • Aamir Ali Shaikh 1,†,
  • Sadaruddin Chachar 2,*,† and
  • Pingxian Zhang 1,*
  • et al.

Reviewer 1: Anonymous Reviewer 2: Slawomir Sowa Reviewer 3: Anonymous

Round 1

Reviewer 1 Report

This is a very comprehensive review on plant epigenetics with thorough background on general epigenetics. A review for wide range of audience with different level of understanding on DNA methylation, Shaikh et al. discussed what are the known features of DNA methylation in plants, described the molecular mechanisms, roles, and interplays of methylases and demethylases, explained different types of methylation (5mC, 6mA), the effect of methylation or methylation-regulation, and discussed existing and potential applications by DNA methylation. The paper is unambiguous without any concerns.

Author Response

Reviewer #1: This is a very comprehensive review on plant epigenetics with thorough background on general epigenetics. A review for wide range of audience with different level of understanding on DNA methylation, Shaikh et al. discussed what are the known features of DNA methylation in plants, described the molecular mechanisms, roles, and interplays of methylases and demethylases, explained different types of methylation (5mC, 6mA), the effect of methylation or methylation-regulation, and discussed existing and potential applications by DNA methylation. The paper is unambiguous without any concerns.

Response: We sincerely thank the reviewer for the supportive and encouraging comments.

Reviewer 2 Report

This is an interesting paper on important for plant breeding topic. Apart form some style and spell check I would recommend to:

improve the discussion by adding more about disadvantages of epigenome editing in plant breeding (eg https://doi.org/10.1371/journal.pgen.1009034)

describing the methodology of RdDM that can be applied in breeding programs

at least mention the new genomic techniques and the potential problem of classification as GMOs

spelling of Latin names must be harmonised eg Arabidopsis thaliana – italic

line 81 -Cytosine methylation plays a significant role. And TE needs explanation here.

eg. Transposable Elements silencing of genes.

Line 124- sequence context

Line 183 Chlamydomonas reinhardtii  (italic) the same with all Latin names

Line 248 “writers, eraser, readers - this terms are not used in the text. please explain the term in the text.

Line 551 (Table) ????

Line 801 5mA  -this is not described in the text. The methylation of adenine. Should it be 6mA?

Author Response

Reviewer #2: This is an interesting paper on important for plant breeding topic. Apart form some style and spell check I would recommend to:

  1. improve the discussion by adding more about disadvantages of epigenome editing in plant breeding (eg https://doi.org/10.1371/journal.pgen.1009034)

Response: We do sincerely thank for this comment. We and agree with your suggestion, and we have revised the manuscript, accordingly, added the following text for disadvantages of epigenome editing in plant breeding.

“The extensive use of induced DNA methylation in plant breeding is hindered by a number of limitations. On a biological level, the selection of genes to target, the durable maintenance of induced epialleles, and the occurrence of off-target effects. On the other hand, improper legislation involving new breeding techniques has been introduced, which has restricted research and the adoption of sophisticated breeding approaches [195]”.

  1. describing the methodology of RdDM that can be applied in breeding programs

Response: Thanks for this valuable comment. We understand and agree with your suggestion, and we have revised our manuscript, added the new heading for methodology of RdDM that can be applied in breeding programs.

“Potential Applications of RdDM in Crop Improvement

RdDM is a biological mechanism in which non-coding RNA molecules direct the addition of DNA methylation to specific sequence [270]. The mechanism underlying the sequence-specificity of RdDM is well understood. RdDM can be 'tricked' into targeting and silencing endogenous genes in a highly specific, which offers a variety of potential biotechnological and breeding applications. Numerous methods can be employed to induce RdDM based DNA methylation and silencing of specific genes. One approach, known as virus-induced gene silencing (VIGS), involves introducing a portion of the target gene's promoter region into a virus [271]. As the virus replicates its own RNA, it will generate the chunk of promoter sequence as part of its own RNA, which is foreign to the plant. The viral RNA being foreign will be targeted for PTGS and processed into sRNAs, some of which will be complementary to the promoter of the original target gene. Certain sRNAs add DNA methylation to the target gene by recruiting RdDM machinery. Various studies have used this approach such as fruit ripening gene in tomato and a gene affecting flower pigmentation in Petunia have been silenced using Cucumber Mosaic Virus to recruit RdDM to silence the gene [272]. Arabidopsis FWA locus has also been silenced using VIGS, which resulted in delayed flowering [271]. RdDM-induced changes can occasionally be retained and inherited through multiple generations without outside intervention or manipulation, implying that RdDM can be a useful tool for targeted epigenome editing”.

  1. at least mention the new genomic techniques and the potential problem of classification as GMOs

Response: Thanks. We have added discussion as you suggested.

“Numerous GMO regulations are based on out-of-date definitions that do not take into account new plant breeding techniques and are incompatible with modern genome editing technology. The European Court of Justice ruled in July 2018 that plants resulting from genome editing methods, including ones that do not employ recombinant DNA, are subject to current GMO regulations [196]. Some nations, such as Canada, define the safety of novel crops based on the end product's attributes (product-based) rather than the technology used to create it (process-based). The United States has implemented a hybrid system in which only plants featuring novel traits are subject to stringent regulations [197]. The Australian government deregulated "DNA free" gene editing methods in 2019. It is critical to amend regulations governing novel plant breeding techniques, since it has both scientific and commercial implications [198]”.

  1. spelling of Latin names must be harmonised eg Arabidopsis thaliana– italic

Response: We sincerely thank the reviewer for this comment. We have carefully checked the whole manuscript. Now, all the Latin names are harmonized and written as italic.

  1. line 81 -Cytosine methylation plays a significant role. And TE needs explanation here. eg. Transposable Elements silencing of genes.

Response: We sincerely thank the reviewer for this constructive comment, the following text has been added to manuscript.

The proliferation and accumulation of TE sequences are also influenced by epigenetic mechanisms. TEs are targeted for DNA methylation by small RNA (sRNA)–mediated mechanisms in several eukaryotic lineages [34]. DICER-LIKE RNase enzymes in plants generate 24-bp sRNA that directs ARGONAUTE and other downstream proteins to complementary DNA sequences, promoting and sustaining DNA and histone methylation [35,36]. In Arabidopsis TE methylation has been found to silence transposition, as evidenced by drastically increased levels of TE transcription in met1 methylation mutants [37,38]. Silencing TEs near genes may also inhibit the generation of aberrant transcripts via read-through transcription beyond TE termini [39], in addition to preventing the proliferation of new TE sequences. However, methylated sequences can influence the expression of nearby genes, typically reducing expression [40]. Expression of flowering time gene FWA has been found to be correlated with the methylation status of nearby SINE-like TE. Hence, changes in gene expression caused by methylation of neighboring TEs may have a deleterious impact on gene and genome function.

  1. Line 124-sequence context

Response: Thanks, we have revised it accordingly.

  1. Line 183 Chlamydomonas reinhardtii (italic) the same with all Latin names

Response: Done.

  1. Line 248 “writers, eraser, readers - this terms are not used in the text. please explain the term in the text.

Response: We sincerely thank the reviewer for this helpful comment, the following explanation has been added to the text.

“These methylation modifications are enzymatically regulated by writers: that introduce various chemical modifications on DNA and histones, readers: the specialized domain comprising proteins that recognize and interpret such alterations and erasers: the unique set of enzymes capable of erasing these chemical tags (Table. 1)”.

  1. Line 551 (Table) ????

Response: We are sorry for the mistake. It should be Table 2. Now we have added this table in the revised manuscript. In Table 2, we have summarized recent advance on epigenetic mechanisms involved in different abiotic stresses in plants.

  1. Line 801 5mA-this is not described in the text. The methylation of adenine. Should it be 6mA?

Response: We are sorry for the mistake. 5mA has been changed to 6mA.

We would like to take this opportunity to offer our gratitude to you for the helpful input about how to improve the scientific rigor and utility of our manuscript. Many thanks!

Reviewer 3 Report

Dear authors

In order to improve this manuscript I would like to make some suggestions: 

I do not agree with the statement "This will eventually help breeders" (line 117) considering the title you have chosen for this manuscript. The Introduction part of the manuscript should be written more concisely and without going back to some scientific information already discussed in the other parts of the manuscript. For a clearer structure of the manuscript, I propose that the objectives of the manuscript be in close connection with the way of presenting the scientific documentation on the subchapters. The objectives of the manuscript are: "In this review, we explain the DNA methylation machinery, diversity, and dynamics in plants as well as the agronomic traits associated with DNA methylation. We outlined recent technological developments in epigenome editing tools, as well as the biological and legal constraints that must yet be surmounted. We also highlight the knowledge gaps that need to be filled in order to fully comprehend the role of DNAmethylation in plant breeding. This will eventually help breeders. "

From my point of view, the restructuring of the manuscript and its writing in a more concise way will also determine a decrease in the number of pages. The conclusions (if considered necessary to be highlighted) must be concise and highlight very clearly the importance of this bibliographic study for unconventional plant breeding. The novelty of the manuscript must be more clearly and concisely highlighted.

Author Response

Reviewer #3: Dear authors

In order to improve this manuscript, I would like to make some suggestions:

I do not agree with the statement "This will eventually help breeders" (line 117) considering the title you have chosen for this manuscript. The Introduction part of the manuscript should be written more concisely and without going back to some scientific information already discussed in the other parts of the manuscript. For a clearer structure of the manuscript, I propose that the objectives of the manuscript be in close connection with the way of presenting the scientific documentation on the subchapters. The objectives of the manuscript are: "In this review, we explain the DNA methylation machinery, diversity, and dynamics in plants as well as the agronomic traits associated with DNA methylation. We outlined recent technological developments in epigenome editing tools, as well as the biological and legal constraints that must yet be surmounted. We also highlight the knowledge gaps that need to be filled in order to fully comprehend the role of DNA methylation in plant breeding. This will eventually help breeders. "

From my point of view, the restructuring of the manuscript and its writing in a more concise way will also determine a decrease in the number of pages. The conclusions (if considered necessary to be highlighted) must be concise and highlight very clearly the importance of this bibliographic study for unconventional plant breeding. The novelty of the manuscript must be more clearly and concisely highlighted.

Response: We do sincerely thank the reviewer for this constructive comment. We agree with this reviewer for the comments to reconstruct the objectives of this review manuscript. Now we have clearly and concisely rewritten our manuscript. We summarized key points listed as below.

1) We have re-constructed our chapters and subchapters. To make it concise and clear, this manuscript has been numbered with each chapter and subchapter. Now this revision contains six chapters:

  1. Introduction
  2. DNA 5mC in plants

2.1 Distribution of 5mC in plant genome

2.2 Derivatives of 5mC

2.3 Writers, erasers and readers of DNA methylation

  1. DNA 6mA in plants
  2. Molecular and biological function of DNA methylation

4.1 Molecular functions of DNA methylation

4.2 Dynamic DNA methylation in plant development

4.3 DNA Methylation Heritability in Plants

4.4 DNA Methylation in response to stresses

  1. Epigenetics Prospective for Plant Breeding

5.1 Applications of Methylation modifications in breeding

5.2 Potential Applications of RdDM in Crop Improvement

5.3 DNA methylation and Heterosis

5.5 Challenges and opportunities for DNA methylation in breeding applications

  1. Conclusion and future perspectives

2) Guided by your suggestions, we have carefully revised the objectives to make them close connection with the way of our topic presented in this review. It was:

“In this review, we explain the DNA 5mC methylation machinery, diversity and dynamics, molecular and biological functions in plants. We also discuss the newly identified N6-methyladenosine (6mA) dynamic distribution and responses to developmental signals in model plants such as Arabidopsis thaliana and rice (Oryza sativa). Especially for DNA methyltransferases and demethylases that catalyzed specific DNA modifications (5mC, 6mA), recent progresses and understanding in DNA methylation and demethylation in genetic regulation and functions with emphasis on DNA methylation in plants. We also highlight the knowledge gaps, and discuss challenges and opportunities for exploitation of DNA methylation in breeding applications.”

3) We have highlighted the conclusion and addressed future perspectives.

5) Together with Reviewer #2’s comments, we have carefully modified our manuscript accordingly.

5) We have modified our title “Recent Advances in DNA Methylation and Their Potential Breeding Applications in Plants”, and added a new Table 2 in this revised version.

We would like to take this opportunity to offer our gratitude to you for the helpful input about how to improve the scientific rigor and utility of our manuscript. Many thanks!

Round 2

Reviewer 3 Report

Dear authors 

after the major revision of this manuscript, my decision is Accept in present form.