Unexpected Low DNA Methylation in Transposable Elements at the 5′-CCGG Sites in Three Fruit Tree Cultivars
Round 1
Reviewer 1 Report
The manuscript “Unexpected low DNA methylation in transposable elements at the 5’-CCGG sites in three fruit tree species” aims to investigate DNA methylation among three fruit tree species (wild pear, Chinese plum and apple). The topic itself as well as the methods described in the manuscript are novel enough and the conclusion is very interesting. However, the manuscript is lacking in following aspects:
Methylation levels and patterns are affected by the developmental stage of the sampled plants, as well as the mode of its propagation. Therefore, a much more detailed overview of the investigated material needs to be given.
Although the authors do a good job of limiting the overall conclusions to 5’-CCGG sites, there is not enough attention in discussion is given to other studies based on other approaches of investigating methylation patterns in fruit crops.
The discussion regarding the effect of plant developmental stage on methylation is notable absent and the existing discussion is almost exclusively limited to apple.
In conclusion, the manuscript is well written but not enough attention is given to discussion related to previous studies on methylation in fruit crops and the overall implication of methylation as a process.
Author Response
The manuscript “Unexpected low DNA methylation in transposable elements at the 5’-CCGG sites in three fruit tree species” aims to investigate DNA methylation among three fruit tree species (wild pear, Chinese plum and apple). The topic itself as well as the methods described in the manuscript are novel enough and the conclusion is very interesting.
Reply: We appreciate the positive comments.
However, the manuscript is lacking in following aspects:
Methylation levels and patterns are affected by the developmental stage of the sampled plants, as well as the mode of its propagation. Therefore, a much more detailed overview of the investigated material needs to be given.
Reply: We appreciate the comments, and to which we fully agree. As said in the manuscript, all three fruit trees were asexually propagated. Indeed, we have been paying attention to sampling at the same developmental stage, i.e., “Genomic DNA was isolated from fully expanded young leaves of the three tree species”. We have further detailed the description now. Thanks!
Although the authors do a good job of limiting the overall conclusions to 5’-CCGG sites, there is not enough attention in discussion is given to other studies based on other approaches of investigating methylation patterns in fruit crops.
Reply: We appreciate the comments. As related to our replies to the following comments, other studies on DNA methylation in fruit trees are mainly about dynamics of DNA methylation during process of fruit development. Actually this aspect of study was first and best done in some vegetables, such as tomato. Our manuscript is focusing on a different aspect of DNA methylation. Still, we agree with the comments, and have added contents and cited relevant papers.
The discussion regarding the effect of plant developmental stage on methylation is notable absent and the existing discussion is almost exclusively limited to apple.
Reply: We appreciate the comments. We have added in the Discussion about effect of plant developmental stage on methylation. We have also added relevant studies on other aspects of DNA methylation in other fruits, as detailed below.
In conclusion, the manuscript is well written but not enough attention is given to discussion related to previous studies on methylation in fruit crops and the overall implication of methylation as a process.
Reply: We appreciate the comments. Indeed, there are studies focusing on aspects of dynamics of DNA methylation during fruit development and ripening. Originally, it did not occur to us that we should include this aspect of discussion because we thought this is not related with the major results of our manuscript, which are about our unexpected observation that the overall methylation level (especially of TEs) are marked lower in all the three fruit trees compared with other crops studied in our lab by the same method MSAP. In other words, our results are not emphasizing development. Per these comments, however, we realize that we should briefly refer to these studies, as they are also about fruit trees. Thus, we have added relevant studies of DNA methylation developmental dynamics on fruit trees. Also, we have added implications of our findings of unusual low methylation, especially in TEs, on biological features of asexually propagated plants including fruit tresses.
Reviewer 2 Report
Authors presented DNA methylation levels at 5'-CCGG sites (CpG context) in three fruit trees (wild pear, plum and apple) based on MSAP marker results.
Abstract:
Line 12: Please specify the exact other plant species which studied by the same method.
Introduction:
The introduction section well constructed. All of the cited references relevant to the manuscript.
Material and methods:
Line 61 - 64: Please add more information about the plant material which used for MSAP analysis. What was the scion cultivars? What was the developmental stage of plants when collected the leaves? The different developmental stage, different scion cultivars or different ages can cause differences between the DNA methylation levels.
Results:
The results are well represented. The figures and tables are informative.
Discussion and conclusion:
The discussion and conclusion are supported by results.
Author Response
Authors presented DNA methylation levels at 5'-CCGG sites (CpG context) in three fruit trees (wild pear, plum and apple) based on MSAP marker results.
Abstract:
Line 12: Please specify the exact other plant species which studied by the same method.
Reply: We appreciate the comments. We have added the missing information.
Introduction:
The introduction section is well constructed. All of the cited references relevant to the manuscript.
Reply: We appreciate the comments. Based on the other referee’s comments, we have added some more relevant references.
Material and methods:
Line 61 - 64: Please add more information about the plant material which used for MSAP analysis. What was the scion cultivars? What was the developmental stage of plants when collected the leaves? The different developmental stage, different scion cultivars or different ages can cause differences between the DNA methylation levels.
Reply: We appreciate the comments. We agree that these comments are highly relevant, and similar comments were also raised by the other referee. We have added information about the scion cultivars. We did not do sampling at different growth/developmental stages. We only sampled the leaf tissue at the same stage. Actually, we believe there should not be much difference across developmental stages of leaf, as shown in many plants and crops, including Arabidopsis, rice, maize etc. In contrast to mammalian animals in which different developmental stages and tissues/organ vary dramatically in DNA methylation, in plants, DNA methylation show dynamics only in certain specific terminal tissues, i.e., those that do not contribute hereditary information to the next generation, such as endosperm in monocots and fruits of dicots. Still, we have added relevant discussions about influence of development on DNA methylation, and developmental dynamics of DNA methylation in fruit development.
Results:
The results are well represented. The figures and tables are informative.
Reply: We appreciate the positive comments.
Discussion and conclusion:
The discussion and conclusion are supported by results.
Reply: We appreciate the positive comments.
Round 2
Reviewer 1 Report
Although several highly relevant studies have now been discussed in the amended version of the manuscript, most of my comments have not been adequately addressed, therefore I shall repeat them:
Methylation levels and patterns are affected by the developmental stage of the sampled plants, as well as the mode of its propagation. Therefore, a much more DETAILED OVERVIEW OF THE INVESTIGATED MATERIAL NEEDS TO BE GIVEN (information on the orchards, age of the trees, production system, rootstock etc.).
The discussion regarding the effect of plant developmental stage on methylation is notable absent and the existing discussion is almost exclusively limited to apple (pear and plum is underrepresented in the discussion).
Author Response
Reply to Comments by Referee 1 (2nd round)
Although several highly relevant studies have now been discussed in the amended version of the manuscript, most of my comments have not been adequately addressed, therefore I shall repeat them:
Methylation levels and patterns are affected by the developmental stage of the sampled plants, as well as the mode of its propagation. Therefore, a much more DETAILED OVERVIEW OF THE INVESTIGATED MATERIAL NEEDS TO BE GIVEN (information on the orchards, age of the trees, production system, rootstock etc.).
Reply: We apologize that it occurred to us that we did not fully understand these comments previously! We are grateful that we were given a second chance to address the comments and further revise the manuscript.
We agree that to an extent, DNA methylation levels and patterns are affected by developmental stage of the sampled plants, as well as the mode of its propagation. However, as the most stable epigenetic modification (relative to histone modifications, for example), there are certain properties that are not affected by external factors. For example, TEs in general are substantially more methylated than genes. Although different levels of DNA methylation found in different plant species should have been affected by differences in their growing environments and propagation models, the difference is mainly determined by genetic compositions, for example genome size and contents of TEs and their derivatives. As said in our manuscript, our study was not aimed to investigate DNA methylation dynamics in the contexts of developmental and environmental factors, because we only sampled one tissue at a single stage.
These explained, if we understand the comments correctly this time, the reviewer is concerned that our two main results, i.e., (1) DNA methylation at the 5’-CCGG sites in the leaf tissue of all three fruit trees are lower than expected, and especially (2) that TEs are not more methylated than genes at these sites in these fruit trees, can be due to unusual environmental and propagation models. In this respect, we agree that we should be more cautious in the conclusions, such as we only studied one tissue (leaf) and that the specific conditions under which the samples were taken should be described in more detail. We agree wot these and have now provided all these details (the orchards, age of the trees, production system, rootstock). All three fruit trees were grown under a common regular orchard in Northeastern China, and as commercial cuttings they were not unusually treated. Also, enlightened by these comments, we have changed “the three fruit species” to “three cultivars each of the three fruit tree species”, to more rigorous. Thanks!
The discussion regarding the effect of plant developmental stage on methylation is notable absent and the existing discussion is almost exclusively limited to apple (pear and plum is underrepresented in the discussion).
Reply: As also explained above, being involving only one tissue and one stage, this study could not address these issues. Accordingly, we thought we should limit discussions on these aspects. But, if we understand the comments correctly, we should be more conservative about our conclusions, as said above. We have done so. The reason why we mainly discuss in the context of apple was because only apple has the high-quality methylome and it was analyzed in aspect of relative methylation in TEs and genes, and therefor is highly relevant to out manuscript. Although there were reports on DNA methylation in the other two fruit trees or related species, they were mainly about fruit development, between-tissue differences and correlation of methylation with important traits. Based on this criticism, and also given that we are suggested to expand our discussion to include potential influences of conditions, we found some references in Pyrus that are relevant, and we have added these references now. All changes are tracked.
Finally, we realize that all comments should have intended to remind us of not overstating the results, which we admit indeed unconsciously occur. We have tried to avoid so. We agree that topological thinking is not appropriate in this type of empirical studies. Therefore, we have specified in all cases that we studied three fruit tree cultivars rather than three species. Many thanks!
