Effects of Medium Supplements on Somatic Embryo Maturation and DNA Methylation in Pseudotsuga gaussenii Flous, a Species under Protection

Round 1

Reviewer 1 Report

The manuscript is technically sound and clearly written.
However it needs several revisions:

Line 54:  "it happens in which has never been" - what does in mean? I think he word 'locus' or 'site' is missing.
Lines 51-62: The description of methyltransferases family is slightly confusing. It is written that there are two types of DNA methylation,  maintenance methylation and de novo methylation (lines 51-52). But in the 61-62 lines it is written that DRM2 and CMT2 catalyze _the maintenance_ of asymmetric CHH methylation sites via _de novo_ methylation. What does it mean, if de novo and maintenance methylation are different types of methylation? 
Next, may be it will be better to describe maintenance methylation in all types of context at first, and then - de novo methylation by DRM2 in all contexts.
I also think you should specifically mention CMT3 methylase from CMT family (now it is just written that "the plant specific chromatin methylase (CMT) family is responsible for maintaining cytosine methylation in CHG nucleotide sequences"). 

Line 81: Were the full coding sequences of DNMTs cloned? Or these were the partial sequences?

Lines 88-101: please, describe more explicitly the type of each medium (solid or liquid). Were the explants  
in the suspension cultures cultivated on the shaker? If yes, what was the speed of shaker?

Line 155: Supplementary Table S3 (not Tables)

Line 169: I think it would be better to start the Results section from brief description of experiment (I mean, to write something like "To evaluate the effect of MGBG on SE, we performed a comparison between different variants of cultivation...")

Line 191-192: there were obviously visible SEs on all mature media (Figure 3b&e&h) - I cannot clearly see SE on Figure 3, h. Maybe, it will be better to add arrows pointing at the SEs on the Figure 3b&e&h.
 
Line 197: the size of MSE was smaller - statistical analysis confirming this conclusion should be presented.

Lines 197-199: I think it should be better to write that the density of embryos increased, or the number of MSE per 1 g of fresh tissue increased.

Lines 201-202: why elimination of maltose from mature media was better for SE maturation if the total number of MSE was not significantly improved? 

Line 218: "was lower" instead of just "lower"
Line 232: "As it can be seen from the Figure 7" instead of "As the Figure 7"
Lines 234-239: I cannot see that the expression level of DNA methyltransferase genes in SE tissue in maltose free medium decreased at the tenth week. For example, the level of  DRM-1 and 2 and MET1-2 was slightly higher in comparison with cultivation on the control medium at 10W. In addition the expression of CMT, MET1-1 and MET1-3 increased, not decreased, after one week of maturation on control medium, as well as the expression of MET1-2 and the expression of DRM1 and DRM2. Maybe, it would be better to make separate diagrams for each gene so that the differences could be seen more clearly.
Line 244. The branches of phylogenetic tree at the Figure 6 cannot be seen clearly. I think it should be better to use another kind of visualisation.
Lines 286-287: from the experiments of the study, one cannot assume that DNA methylation levels influenced SE morphogenesis. You can only affirm that PEG and maltose influence both on the methylation level and SE morphogenesis. The effect of methylation level on the SE, or vice versa, was not evaluated.

Also, what type of statistical test was used in all experiments?

 

 

 

 

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript addresses somatic embryogenesis (SE) in Pseudotsuga sinensis and the effect of maltose and PEG on the maturation of somatic embryos. Additionally, the authors performed DNA methylation quantification and gene expression of methyltransferase genes in different stages of the somatic embryogenesis process.

While this is the first report of somatic embryogenesis in this conifer species, the aims of the study are not clear since the establishment of the method is not the focus of the manuscript. Instead, the authors perform a series of experiments to study the effect of several media components on embryo maturation associated to DNA methylation analysis, but the relevance of these analyses are not explained.

I have several major concerns that should be addressed. Firstly, the objectives of the study need to be clarified, and the Material and Methods, Results and Discussion should be reorganized accordingly. Some sections of the Material and Methods are insufficiently described, for instance the design of the DNA methylation analysis is difficult to evaluate based on the provided detail of the plant material used. The discussion of the results is poor and does not highlight the significance of the study.   

Further comments on each section are detailed below.

Introduction

The authors start by describing the factors that can affect somatic embryogenesis without an introduction to somatic embryogenesis, and to the conditions commonly used for embryo development in conifers (and species closely related to Pseudotsuga sinensis). This is needed for a broader audience of researchers not familiar with this subject. The English and writing style need to be improved.

Lines 48-51: clarify the definition of DNA methylation as a way of modifying gene expression. Since the authors specifically focus on cytosine methylation the mention to purines is not required.

Lines 67-68: very confusing sentence

Mat & Methods

Section 2.1. The authors mention SE has not been previously described, therefore the induction conditions should be detailed. In addition to the induction, also the other steps of the process are not detailed enough. For instance, for making the suspensions, how much tissue is used? In which proportion to the liquid medium? Not clear from the description if pre-maturation and maturation are performed in liquid or semi-solid medium.

Section 2.2. What is the meaning of “secondary week”? Is the cotyledon number the sole criterium to consider in the classification of abnormal embryos?

Section 2.3. Not clear which tissues were used for DNA methylation analysis. For instance, in the 10th week were only mature embryos used? What about abnormal embryos? Immature embryos?

Section 2.5. What were the PCR amplification conditions?

Section 2.6. The cited article referring to the reference gene used is from another conifer species (Scots pine) and the combined average expression of the three reference genes actin, glyceraldehyde-3-phosphate dehydrogenase, and ubiquitin was used for the normalization of gene expression data. Did the authors previously test this and other reference genes in P. sinensis to know if it is adequate to evaluate expression?

Results

It is very difficult to understand what the authors describe, the text/language is extremely confusing and, in some cases, not consistent with the results shown in Figures (e.g. line 217; 234-238). Legends of the figures also need rewriting. Additionally, the scale of the 3rd week graph in Figure 7 should be the same as the others for easier comparison.

A major issue here is the type of tissue used in the quantification of global DNA methylation, which is not described in detail, and in the legend of Figure 5 it is referred as “cultured tissue”, this is too vague.

Discussion

The text is extremely confusing, partially because the English language needs very extensive revision. Some of the literature cited is not relevant in this context (e.g. ref. 36, line 280). Moreover, the reference to human tumors in the discussion of DNA methyltransferases expression in P. sinensis is completely out of context, while relevant work in plants is not cited (lines 290-299).

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Introduction

Line 39 - Replace “sex cells” by “gametes”

Line 42 – be consistent in the use of SEis or SEs, in this case I guess it should be SEis

Lines 64-65 – please complete the information on which conifer species these observations have been made

Line 76 - DNA pyrimidine or cytosine? Not clear

Line 85 – the writing style is still poor as exemplified by this sentence starting with “And”.

Lines 93-94 – The sentence has not improved

Line 95 – I don´t understand the reasoning in this sentence. It seems that there are only two conifer species (spruce or Douglas fir) in which somatic embryogenesis has been established which is not true.

 

Material and Methods

Line 249 – this is still not clear. What is the meaning of “all SE cultures”? Specify the time at which the material was collected for DNA methylation analysis. Specify if isolated embryos were collected and include normal and abnormal embryos in the same sample.

Line 295-296 – I don´t understand what is meant with this sentence.

Line 312 – as stated in my previous report, reference 31 is not acceptable to justify the use of a single gene as reference. If the authors tested the 3 reference genes, they should show that glyceraldehyde-3-phosphate dehydrogenase gene was good enough to normalize gene expression

 

Results

Again, the language is not yet acceptable, I highlight only one of the several errors in the legend of Fig. 3 (1th week (a, d, g), 3ird week).

 

Discussion

The analysis of DNA methylation in tissues containing mostly mature embryos (complete maturation medium and maltose free) with tissues mostly containing proliferating or degraded embryo structures (PEG) is questionable. As the authors mention at the end of the Results, DNA methylation varies with the type of tissues and developmental stages and therefore this is reflected in the results obtained at the end of maturation. This issue is not discussed. Overall, the discussion did not improve when comparing to the previously submitted manuscript.

Author Response

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Author Response File: Author Response.pdf

Round 3

Reviewer 2 Report

Abstract needs English revision

Point 3 (previous report) was not addressed, which conifer species?

Point 4 (previous report) – no need to say DNA pyrimidine cytosine methylation, just DNA methylation

Point 5 (previous report) – not addressed, any sentence starting with “And” should be rephrased. I recommended an extensive English revision in my previous reports but it seems this recommendation was not considered by the authors

Point 6 (previous report) – the DNA methyltransferases (DNMTs) were not cloned, but only the partial sequences encoding them…

Line 166 – What is the meaning of “The cloning PCR were amplified as the KOD-Plus-Neo manufacturer’s protocol”? Please rephrase

Point 8 (previous report) – not completely addressed

Point 10 (previous report) – in reference 31 it is written: “The combined average expression of the three reference genes actin, glyceraldehyde-3-phosphate dehydrogenase, and ubiquitin was used for the normalization of gene expression data”. The first time I raised this issue was for you to justify why using only one reference gene and cite reference 31 since in that work in Pinus sylvestris 3 reference genes are used. So, you should only refer to citation 31 for the primers and not for the justifying the use of GADPH as control.

Point 11 (previous report) - legend of Fig. 3 is not yet correct

Line 322 - the work “remarkably” is used twice in the same sentence.

Conclusions – again, rephrase “supplement of MGBG of low concentrations”

Author Response

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Author Response File: Author Response.pdf