Transcriptome Reveals the Specificity of Phyllostachys edulis ‘Pachyloen’ Shoots at Different Developmental Stages

Round 1

Reviewer 1 Report

The paper by Hu et al. reports results of monthly observations of expressed genes profile in two related bamboo clones differing in stem wall thickness. I am not expert in omics so I comment only general aspects of the paper. The omics part of the paper should be reviewed by other expert in this field.

I like the idea of the paper but consider the present paper not of a high quality leading me to suggest to be substantially reworked before eventual publication. The major weakness I consider poorly argued scientific necessity of the paper, rather simplistic description of methods, and, namely, the lack of relevant discussion of the obtained data and results. Some more detailed comments are indicated below.

 

General comments

• The paper just presents the results and lack fast any serious discussion, except of several “empty” statements that this study is important. I agree that the study may be important but this cannot be recognized otherwise than by confrontation with what has been already published in other literature. Here I think authors should discuss:
  • Why this study is needed? What particular scientific problem it should solve (in a better than using any other method)?
  • What is the knowledge of this topic in other plants. For example: is similar annual variation in number of expressed genes observed in other plants; how this is explained there; is it also related with production of thicker stems (wood)?
  • What is the situation in other grasses with thin and thick stem walls (or solid culms)?
  • In which way the present results may exactly be used for something real in bamboo industry
• The study compares expression profile in two bamboo clones. This has its advantages but also numerous disadvantages. Though the clones differ apparently in stem wall thickness, authors cannot be never 100% sure that the observed differences are just due to stem wall thickness and not by anything else. For example, could not be the difference due to different age of studied clones? Cannot similar difference be observed between other individual clones in other bamboo species irrespectively of their stem wall thickness? Is there any other than morphological evidence that cv. Pachyelon and standard moso bamboo are really related and that they do not represent different, more widely related species, where such differences in expression profile will be not much surprising? These critical aspects of the study design should be carefully discussed somewhere in the paper.
• The methods of sample collection and preparation (and seems me that also of the omics part) should be better explained. What exact part of the shoot was taken for the analysis – whole or only its apical part? How authors are sure they collected anatomically comparable parts of shoots. Were samples collected by the same person? Could sampling a bit different parts or parts of different size somehow influence the results? Environmental conditions are never the same – in which way they were the same in the experimental garden (I mean shoot shading, nutrient reserves in the soil, etc.)?
• Is there possible to estimate some uncertainty in quantification of gene number? Without that it is difficult to say if the observed differences are significant (e.g. in Fig. 2) and need to be further commented.
• I lack some more detailed information in the introduction on how stem wall is build in bamboo or other grasses and what genes are typically involved in this process. This prevents me (readers) to evaluate to what extent authors’ results are meaningful or not (as claimed for example in the abstract).
• Validation of gene expression using qPCR seems me bad form the statistical point of view. First, by comparing correspondence of estimates done by two methods, authors should test relationship qPCR=Seq (i.e. without intercept and with slope=1), not just any regression. Second, I see no reason why to logarithm the observed difference. This practice makes reported R2 values unreal and largely falsely positive.
• The English of the paper is principally not bad and it is understand what authors wanted to tell. However, I see necessary the language (and sometimes the grammar) of the paper to be revised by some English language service.

 

Minor comments

• l. 12: I would add “normal” or “standard” moso bamboo here.
• l. 21-22: too much “empty” statements.
• l. 28: I would expect some more general work to be cited here.
• l. 30: I would expect some more actual work (than from 2007) to be cited here. I also question whether the Norway spruce wood industry (or conifers wood in general to make a group comparable to “bamboo”) is not much economically important form the worldwide perspective.
• l. 32: some explanation of “moso bamboo” term should be added here.
• l. 33: some explanation of what is the “bamboo wall” should be added here and may be other term should be used instead (knowing nothing about previous works by the authors I would think this is some city wall made from bamboo stems or wood). Use the same term for this through the whole paper not to confuse readers about its meaning (I found several different names for this in the paper)
• l. 48: “unique expression genes” change to “uniquely expressed genes”.
• l. 64: something is perhaps lacking in this sentence.
• l. 83: explain meaning of “DEG” here. How DEGs were obtained, in which program? Are DEGs defined as differentially expressed genes between the two bamboo clones or among the sampling dates within one bamboo clone? This is very important for understanding the discussion.
• Fig 1. – I am not sure if this is some standard kind of a graph in omics studies but it looks very uninformative or difficult to understand (all looks very similar). Here I would prefer some other form how to present the information in some easy way to readers.
• l. 115: “difference in changes” sounds awkward.
• l. 118-121: the two sentences tells the same – delete one.
• l. 149: ? delete “terms of”.
• l. 152-155: difficult wording.
• l. 195-199 unnecessarily repeats the results and introduction.
• l. 208: delete “of culm wall” and add “culm” before “wall” at l. 209.
• l. 255: what means “active expression”? how this could be detected? Would not be better to speak here about “expressed genes” only?
• l. 226-230 is not much clear to me. This part should be written without usage of the “DEG” abbreviation.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Hu and colleagues describe in their manuscript the transcriptome of P. edulis ‘Pachyloen’ to find new insights into bamboo wall thickening. Shoots were monthly collected (SEP 2019 – APR 2020), so 8 samples, of P. edulis ‘Pachyloen’ and moso bamboo. RNA was isolated, deep sequenced and analyzed for differential expressed genes (DEG).

The deep sequencing of the bamboos at different developmental stages is of scientific interest, but a thorough revision of the manuscript is necessary – especially the writing must be improved.

Figure 2 describes the obtained expressed gene numbers per sequencing at different time points. The authors state that P. edulis ‘Pachyloen’ expressed more unique genes than moso bamboo, except in March. One cannot make this statement, because there is no statistic. Just one sample was sequenced / month. One would need to sequence ten samples of P. edulis ‘Pachyloen’ and moso bamboo / month to evaluate if P. edulis ‘Pachyloen’ expressed more unique genes than moso bamboo. Thus I strongly suggest to delete this figure and analysis. Data shown in Table 1 are sufficient.

I would like to urge the authors to re-think the usefulness to present all figures and tables.

The method of qrt-RT-PCR must be described in more detail. Annotate the genes which have been tested in the qrt-RT-PCR analysis and why have these genes been chosen.

Figure legend S2 is missing.

 

Minor comments:

1. 30, delete „, which is recognized….“ – repetition of first sentence
2. 34 delete “and so on”, instead declare the listing as “for example”
3. 35 what is the “law of bamboo shoot formation”? wrong term?!
4. 41 check for additional space before “mechanisms”
5. 60 & 61, it’s samples not simples, I assume
6. 64 rephrase sentence
7. 67 one cannot detect or extract quality, one can measure quality. Rephrase sentence
8. 78 remove brackets. Make it own sentence “The data source website is listed in S1.”
9. 106 rephrase sentence, wording and syntax error
10. 116-119, rephrase sentence. Scientific writing demanded, for example: “Shoots were dormant in January and February, while bamboo shoots germinated beginning in March.”
11. 127 typo, space missing
12. 142 typo, space missing
13. 149 lab jargon, unigenes
14. 156 Table 2, Percentage with 2 decimal places is not useful
15. 168 intersected?
16. 174 Table 3, Does #1 make sense, if it is only one gene?
17. 189 results cannot be stable, delete
18. 195-197 delete sentences, not scientific
19. 202 delete et al.
20. 205, syntax error, in addition, this sentence implies that the presented data are not trustworthy, because a high quality reference genome is lacking. I suggest to weaken this statement, if this study should be published
21. 210-212 wording and syntax error
22. 217 delete “thas is, the shoots began to grow”
23. 218, 219 repetition of information within one sentence
24. 240 wording, solid foundation

 

 

 

 

 

 

 

 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Only minority of my general comments have been handled in the new version (I expected authors will reply them point by point as is the standard in other IF journals) so I cannot be satisfied with authors’ revision. The paper has improved a bit in some aspects but the overall quality of the paper, namely regarding the quality of the discussion is still poor for standard scientific paper. I am still also not sure from what is written whether the reported differences cannot be due to some kind methodical bias and that they are really related to the process of culm wall thickening.

Regarding my doubts about the existence of some methodical biases during sampling: authors now indicate they sampled material 1 cm from the top of the shoot. Why not including the apex with the apical meristem? Why this is the right part where the culm thinckness is determined? Sampling 1 cm from the top (how large part is not indicated in the paper) I can imagine in larger shoots but was this really also the case of underground shoot buds? Would not such sampling (I expect they sampled similar weight of material) cover different portion of nodes and internodes in the buds and in the tall culms? Could not affect this the reported results (gene numbers and spectrum), i.e. if anatomically different parts are compared (practically the whole culm in the bud versus only the very top part in several meters high mature culm)? 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf