Unraveling the Epigenetic Landscape for Salt Tolerance in Plants

Round 1

Reviewer 1 Report

The submitted review manuscript entitled “Unraveling the epigenetic landscape for salt tolerance in plants” describes the Epigenetic modifications in plants under stress tolerance. This is an important study to understand their systems. This manuscript is recommended for publication in its current form.

Author Response

A note of thank you to the reviewer. Thank you for accepting it in its current form.

Reviewer 2 Report

In this review entitled “Unraveling the epigenetic landscape for salt tolerance in plants” is very well written and organized. It provides complete information about the current understanding gained on epigenetics changes affecting tolerance to salinity stress in plants. The story line begins with the method for determining changes in epigenome, the summary of salinity affecting epigenetic changes in plants. Subsequently, the authors described in details of how three modes, including DNA methylation, histone modifications, and RNA interference influence tolerance to salinity stress in plants. At last, the authors emphasized that an interplay between the main modes of epigenetic changes and the applications of epigenetic breeding in plants. Overall, this is an excellent review dealing with recent findings about epigenetics affecting salt tolerance in plants, and I approve of its publication.

 

However, I have some suggestions regarding the method part and the RNA interference part.

In the second part, when the authors describe the methods for detecting changes in epigenome, it’s better to include some terms and references of nuclease-based ChIP technologies, such as ultra-low-input micrococcal nuclease-based native ChIP (ULI-NChIP) (Amour et al., Nature Communications, (2015)), chromatin endogenous cleavage and high-throughput sequencing (ChEC-seq).

Several recent studies have implicated the non-coding RNAs that are longer than 200 bp are involved in gene expression under stress conditions, including salt stress response in Poplar species and Glycine max (Zhang et al., BMC Plant Biol, (2020); Ma et al., Front. Genet. (2019)). Such reports need to be cited in the present manuscript.

Several typos need to be corrected.

Such as “msh1 mutant” should be “msh1 mutant”, “hda710” should be “hda710”, “hdc1” should be “hdc1”, “MSh gene” should be “MSH gene”, “which are yet to b eovercome” should be “which are yet to be overcome”, “which undergo inbreeding as it” should be “which undergo inbreeding as ”, “which state that plants which are regenerated” should be “which state that plants are regenerated”. “. [93].” should be “[93].”. “N terminal lysine residues of H4, H3, 5 of HA2 and H2B” should be corrected.

 

 

 

Author Response

Thank you so much to the reviewer for sharing his words of wisdom. The ideas which were asked to addressed have been done in the revised draft. The answers are as follows.

In the second part, when the authors describe the methods for detecting changes in epigenome, it’s better to include some terms and references of nuclease-based ChIP technologies, such as ultra-low-input micrococcal nuclease-based native ChIP (ULI-NChIP) (Amour et al., Nature Communications, (2015)), chromatin endogenous cleavage and high-throughput sequencing (ChEC-seq).

Answer: Thank you reviewer for bringing this out. Though, these techniques are not friendly with plant system yet.

We have added this part with references.

Other refined ChIP based modes of egigenetic detection involves ultra-low-input native ChIP-seq which works efficiently with very sparse cell count [9] , though not worked out in plant system well. In this technique an ultra-low-input micrococcal nuclease-based native ChIP (ULI-NChIP) is used followed by sequencing method to generate genome-wide histone mark profiles [9]. Another helpful tool for studying epigenetic modifications is an enzymatic method which involves chromatin endogenous cleavage (ChEC), and with the aid of fusion protein comprising a chromatin protein of interest and MNase, which degrades unprotected DNA in presence of calcium [10]. This method has been heavily worked out in yeast but not in plants.

Several recent studies have implicated the non-coding RNAs that are longer than 200 bp are involved in gene expression under stress conditions, including salt stress response in Poplar species and Glycine max (Zhang et al., BMC Plant Biol, (2020); Ma et al., Front. Genet. (2019)). Such reports need to be cited in the present manuscript.

Answer: We have added a new section to it.

7. Long noncoding RNA in plants affecting salinity stress

 Gene regulation by non-coding RNAs, has recently gained ground as another mode of epigenetic regulation. LncRNAs are a type of non-coding RNA that does not bears protein-coding ability while being able to regulate gene expressions at epigenetic, transcriptional, and post-transcriptional reach [83]. Plant lncRNAs are transcribed by RNA polymerases PolII, PolIV, and PolV. According to the positioning relative to the protein-coding genes in the genome, lncRNAs can be defined as natural antisense transcripts or lncNATs, intronic lncRNAs or intergenic lncRNAs lincRNAs, and sense lncRNAs [83]. lncRNA transcripts are shorter in size around 200bp and lack motifs, such as ORFs and Kozak consensus sequences [83]. lncRNAs generally have a 50 m7G cap and a 30 poly(A) tail and are processed as mRNA mimics. However, lncRNAs have shown low conservation in sequences among species and have low expression levels with tissue-specific expression patterns responding to various stresses in plants [84]. The existence of lnc and studies related to its role in stress is upcoming. Huanca-Mamani et al. in their study portrayed the role of lnRNA’s to be differentially expressed than the protein coding genes in maize, when they were exposed to a combination of boron and salinity stress [85]. Similar study have been carried out in Chenopodioum quinoa under salt stress conditions, highlighting the role of lncRNAs [86]. However, one of the earliest known lncRNA was DROUGHT-INDUCED LNCRNA (DRIR) identified in Arabidopsis, was noted to positively regulates drought and salinity stress responses via ABA mediated pathway [87]. Plants overexpressing DRIR display enhanced salt and drought tolerance through functioning at or upstream of the stage of gene transcription in the stress or ABA signaling. In cotton, lncRNA973 affects miR399 and its target gene PHO2 expression which are involved in response to salt stress. lncRNA973 can also modulate the expression of reactive-oxygen-species-scavenging (ROS) genes, transcription factors (TF’s) and stress-related processes especially under saline conditions [88]. On the otherhand, lncRNA354 works as a competing endogenous RNA of miR160b to regulate GhARF17/18 genes which effect plant growth and development, affecting auxin signaling under salt stress in cotton [89]. Since very less work have been carried out in terms of stress tolerance. This field of lcRNAs is an open avenue.

Table 4. lncRNAs mediated epigenetic changes under salinity stress. A summary of recently reported lncRNAs and their targeted genes are enlisted here.

Several typos need to be corrected.

Such as “msh1 mutant” should be “msh1 mutant”, “hda710” should be “hda710”, “hdc1” should be “hdc1”, “MSh gene” should be “MSH gene”, “which are yet to b eovercome” should be “which are yet to be overcome”, “which undergo inbreeding as it” should be “which undergo inbreeding as ”, “which state that plants which are regenerated” should be “which state that plants are regenerated”. “. [93].” should be “[93].”. “N terminal lysine residues of H4, H3, 5 of HA2 and H2B” should be corrected.

Answer

Addressed all the typos. Thank you for giving it a keen eye.

 

 

Reviewer 3 Report

The research presented by Suchismita et. al., introduces current advance in salt tolerance regrading epigenetic modification. However, current version of manuscript is not informative and scientific organized. Here are my comments:

1.The concept "epigenetic" is misused and overstated in this paper.

2. The title "Unraveling the epigenetic landscape for salt tolerance in plants “makes the readers confused, what exactly the author try to deliver to readers? About molecular mechanism, detecting methodology or breeding guidance? 

3. The manuscript is too wordy and unfocused. 9 sections with no sub-title and only 2 figures.

Author Response

A thank you note to the reviewer for going through the manuscript of Roy et al., though he did not find it informative and lacks scientific knowledge. 

The research presented by Suchismita et. al., introduces current advance in salt tolerance regrading epigenetic modification. However, current version of manuscript is not informative and scientific organized. Here are my comments:

1.The concept "epigenetic" is misused and overstated in this paper.

Answer:

We will be highly obliged if the reviewer could highlight a section in the Roy et al. draft, where the concept has been misused and overstated. We feel it's a review on epigenetics in plants focusing only on the aspect of salinity stress. Since, the topic we choose is epigenetics it should be overstated. 

2. The title "Unraveling the epigenetic landscape for salt tolerance in plants “makes the readers confused, what exactly the author tries to deliver to readers? About molecular mechanism, detecting methodology or breeding guidance?

Answer:

This review is an overall of what is known be it molecular mechanism which epigenetics is. Without molecular understanding we fail to understand the differences between epigenetics. Detection of epigenetics is very important, so we have tried to add a feather under the scope of this review. Breeding guidance is way out of, if at all the field of epigenetics is used to grow futuristic salt tolerant crop.

3. The manuscript is too wordy and unfocused. 9 sections with no sub-title and only 2 figures.

Answer

To address this, we already had 3 tables, we have added another table 4. This makes the MS crisp and can be read through just a glance if only looked at the table.

Figure 1 are a culmination of the molecular mechanisms which leads to epigenetic changes.

Figures 2 is an approach how these epigenetic changes can be used as a tool to raise future better crops.

We think 4 tables and 2 figures summarizes the paper very well.

Each bullet numbers are a section featuring different agenda. We thus have not subdivided it.

Round 2

Reviewer 3 Report

Although there are efforts were made by the author to improve the quality of the review, for current version manuscript of Roy et al.  However, I feel it still need major changes.

The author introduces about the molecular analysis techniques of epigenome analysis, genes involved in epigenetic regulation of salinity tolerance and respond, proposed an idea for epigenetic breeding for raising salinity tolerant crop plants. The concept of epigenetic Here are my comments:

1.     In section 2, authors introduced analytic techniques for epigenetic analysis, some of them are using for whole genome level analysis for methylation site (Like High-throughput sequencing based techniques and ChIP based techniques), some of them are using for single gene analysis (Like HRM and PCR based techniques), and the analysis purpose and output data for gene function study are varied. The purpose for utilizing these techniques in epigenomic detection and its application should be made clear to the reader. In this part, an informative illustration will help to improve readability.

2.     In section 3,4,5,6 and 7, the authors listed recent advances in understanding epigenetic regulators in salinity tolerance and respond. In most cases, epigenetic pathway such as miRNA, methylation, histone modification, lncRNA participated in regulation of stress tolerance, which final resulted is to change the expression of target gene. But for crop breeding, why not breeding scientist directly editing the target salinity tolerance genes? The advantages/side effects of change upstream epigenetic regulators (which are normally target multiple genes) were not discussed in the context.

3.     In section 9, the authors proposal an idea for epigenetic breeding for raising salinity tolerant crop, which sounds new and interesting.  Screening target epi-alleles for utilization in crop breeding. Overall, screening api-alleles are technically feasible，but which kinds of techniques could be using for creating epigenetic edited salinity tolerant crops?  To achieve "GMO-free raising of crop plants"? For indentifing new epigenetic mechanism, this story is ok. But for breeding, it is far away from it. This still requires a lot of technological breakthroughs and commercial efforts. I suggest author add more detailed about possible breeding route for epigenetic improvement.

 

 

Author Response

Although there are efforts were made by the author to improve the quality of the review, for current version manuscript of Roy et al.  However, I feel it still need major changes.

Response: Thank you so much for the valuable inputs. We have further worked to bring in some changes.

The author introduces about the molecular analysis techniques of epigenome analysis, genes involved in epigenetic regulation of salinity tolerance and respond, proposed an idea for epigenetic breeding for raising salinity tolerant crop plants. The concept of epigenetic Here are my comments:

1. In section 2, authors introduced analytic techniques for epigenetic analysis, some of them are using for whole genome level analysis for methylation site (Like High-throughput sequencing based techniques and ChIP based techniques), some of them are using for single gene analysis (Like HRM and PCR based techniques), and the analysis purpose and output data for gene function study are varied. The purpose for utilizing these techniques in epigenomic detection and its application should be made clear to the reader. In this part, an informative illustration will help to improve readability.Response: We have introduced figure 2 describing the schematics of the widely used methodologies to identify epigenetic changes.
2. In section 3,4,5,6 and 7, the authors listed recent advances in understanding epigenetic regulators in salinity tolerance and respond. In most cases, epigenetic pathway such as miRNA, methylation, histone modification, lncRNA participated in regulation of stress tolerance, which final resulted is to change the expression of target gene. But for crop breeding, why not breeding scientist directly editing the target salinity tolerance genes? The advantages/side effects of change upstream epigenetic regulators (which are normally target multiple genes) were not discussed in the context.

Thank you for pointing this. "Breeding scientist directly editing the target salinity tolerance genes" via editing falls under genomic modifications of CRISPR, TALENS etc. Under this review we did not focus on that aspect. We tried to highlight how epigenetic changes which might occur naturally or by chemical interventions causing them to have epigenetic effect as i.e methylation, acetylation can be used to generate saline stress tolerant plants.

1. In section 9, the authors proposal an idea for epigenetic breeding for raising salinity tolerant crop, which sounds new and interesting.  Screening target epi-alleles for utilization in crop breeding. Overall, screening api-alleles are technically feasible，but which kinds of techniques could be using for creating epigenetic edited salinity tolerant crops?  To achieve "GMO-free raising of crop plants"? For indentifing new epigenetic mechanism, this story is ok. But for breeding, it is far away from it. This still requires a lot of technological breakthroughs and commercial efforts. I suggest author add more detailed about possible breeding route for epigenetic improvement.

Response: In this review we give a brief attempt of the idea that epigenetic breeding does exist. To explain its aspect, we have added a paragraph. To describe the entire the process we think it is beyond the scope of this current review.

The added paragraph as follows:

Classical crop breeding is a powerful method to obtain crops with valued agronomical traits, but its potential is gradually being compromised by the limitation of genetic variation. Resorting to the epigenome as a source of variation could serve as a promising alternative. Dealing with abiotic stress with the help of epigenetic breeding still at its infancy. The usage of these epigenetic tools for trait improvement of crops will also be beneficial for GMO-free raising of crop plants. A future strategy for ways of generating salinity tolerant plants using epigenetic breeding is given in Fig 3. Epigenetic breeding will provide some advantages over classical breeding in terms of selection of elite varieties, providing a broad spectrum of resistance to abiotic stress and also a balance between important agronomic traits [108]. This kind of breeding with be time and cost effective and most importantly will have more acceptance than GMO’s to be grown worldwide [108].

 

Round 3

Reviewer 3 Report

Accept in present form