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All remaining queries were answered and revised manuscript is acceptable now.
[# PeerJ Staff Note - this decision was reviewed and approved by Paula Soares, a PeerJ Section Editor covering this Section #]
Please address remaining concerns of the reviewer and amend manuscript accordingly.
Title:
The title of the manuscript is still not clear and my recommendation is:
Comparative transcriptome analysis of cucumber fruit tissues reveals novel regulatory genes in ascorbic acid biosynthesis.
Abstract:
Gene IDs must be italicized in the full main text and data.
This study has identified six putative candidate genes for regulating AsA biosynthesis in cucumber fruit. In the abstract, for three genes, their names (PMM, GME, and DHAR) are mentioned. While for the other three genes, their IDs [CsaV3_4G028360 (ERF), CsaV3_6G042110 (ERF), and CsaV3_6G032360 (MAPK)] have been mentioned. Write for all 6 genes their full functional names and IDs.
Introduction:
The introduction is clear.
Materials & Methods:
In the line 156, just mention PCA. Because the full term has already been mentioned in line 152. Please control all the full term and their stands in the full text. First time mention both full term and stand form and after that, just mention their stands form.
Results and Discussion:
The results and discussion are clear.
Reference:
The final references’ formats are not in the same format. Control all the references and get in the journal’s format.
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Please address concerns raised by both reviewers and amend your manuscript accordingly.
Title:
My recommendation for the title is:
Transcriptome analysis of different tissues of the pericarp from two cucumber cultivars reveals that compared to the mesocarp and endocarp, the exocarp has a higher nutritional level
Abstract:
1. Many terms have not mentioned their full names in the abstract. It is better to mention what their full stands for.
2. The abstract is a bit complex and hard to follow. It is better to rewrite and focus on your study's main outputs.
Introduction:
1. The introduction is too long and needs to be rewritten and connect some parts of the discussion.
2. In the whole text, is better to mention the specific terms' full name and what they stand for. After the first time, just mention what it stands for.
Materials & Methods:
1. Two cultivars (H28 and H105) of cucumber were used in the present study. It is better to add more information about their breeding background and history.
2. In the line 159 (|log2FoldChange| >1) equation needs to correct.
3. Total RNAs for transcriptome sequencing were extracted using TRIzol reagent (thermofisher, 1559601). For re-sequencing, usually, use a high-quality isolation kit. Please, clarify this issue in your study.
4. The units need to control and correct whole manuscripts.
5. Give more detail about the HISAT2 software and FPKM for standard expression.
6. Add an explanation about the statistical analysis, such as Pearson correlation, Heatmap, etc.
Results and Discussion:
1. Some details of results are better mentioned in material and methods and in results give more detail about your research output.
2. The present study is about transcriptome analysis and prediction of nutritional level in the cucumber pericarp. The main gap of the present study is that there is no biological study piece of evidence about how the output of the present study can link to the pathway of measured contents and their related genes.
Data (Figs & Tables):
1. It is necessary to do a grouping test for your results such as a t-test (for two groups) and ANOVA grouping for more than two groups.
2. In Figure 10 and part B, B11 needs to exchange B1. Please control and edit it.
The English language in the article needs to be improved throughout. There are many sentences that are not clear and it makes evaluating the science more challenging. I understand that the English might not be the authors first language, and so this comment should not be taken personally, but rather at face value.
Starting with the first sentence of the abstract "With the advancement of science and technology..." this is such a broad statement try to be more focused on the goal of the study.
Additionally, "To clarify the distribution and regulation of nutritional in cucumber fruit and explore whether cucumber exocarp can be consumed, the transcriptomes of three fruit tissues".
I think you mean "Nutritional value". But also this study doesn't examine or tell us anything about whether you should eat the cucumber fruit peels. especially not in the context of pesticide usage as was previously mentioned.
The literature review does not well cover the known literature and large chunks of it are self plagiarized from previously published works by the authors (eg https://www.sciencedirect.com/science/article/abs/pii/S0981942813003513). This is typically unacceptable and the entire section on AsA biosynthesis needs to be rewritten in new language.
Line71 -"Identify the relevant genes in these pathways and increase or decrease the content level of the substance by manipulating the relevant genes."
This is completely out of context and not clear at this point.
The figure legends are very sparse and not clear. They do not stand alone with out the rest of the manuscript and need to be improved.
I think the experimental design is fine and interesting! I would genuinely like to know how the different tissue types differ and there is plenty of evidence to suggest that cucumber peels produce important specialized metabolites.
Unfortunately, I don't think the correlation between transcriptome data and specialized metabolism data in this paper is very strong or rigorous.
Some of the methods are completely missing. ie how are GO and KEGG enrichments performed, what software is used. That is just not described at all.
Additionally, Some methods are described but the results are not presented ie PCA. I would like to see the PCA result.
The differential expression analysis description is not sufficient. I hope that the authors did not use FPKM values in the DESeq2 analysis, as that completely invalidates the statistics and is not permitted. Bottom line, it is just not clear. Results report analysis with edgeR and not just DESeq2, methods for edgeR are not reported at all.
The gene expression findings and vitamin measurements might be fine, but the correlation between them is quite weak. the authors make strong claims that need to be clarified or toned down.
If there are substantive differences in the Dry weight between the tissue types (ie line 326- Due to the main component of mesocarp is water) then perhaps the normalization of vitamin levels should be vs dry weight and not fresh weight. Perhaps freeze drying the tissue prior to weighing and performing analysis would yield better more comparable results. We therefore can not trust the data in Fig7 and again the correlation to the RNAseq analysis would make more sense. For example, it seems from figure 8 that genes in AsA synthesis are upregualted in exocarp tissue and less so in endocarp.
Once we're on the subject of these heat map figures, it's very hard to assess them a) the genes are not named, b) we don't know what Csa (ie genomic names) genes are used to represent these genes and c) how the expression is normalized.
This make the conclusions hard to track and limit the ability of the reader to asses if they are valid.
I appreciate the work in this manuscript. There is a very important question at hand - Which part of the cucumber fruit is most nutritional. Specifically, does removing the peel (as is done commonly by many consumers) remove valuable nutrients such as Vitamin C and E. I think the goal of the work was worth doing. Unfortunately, I think the manuscript suffers greatly from being disorganized, unclear and methodologically sparse. The authors thus make claims that are hard to substantiate based on the experiments performed. And so at this stage I can not recommend the manuscript for publication.
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