Metabolomic and Transcriptomic Analysis of Unique Floral Coloration in Osmanthus fragrans Cultivars
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors
The manuscript describes a valuable metabolic and transcriptional characterization of the floral pigments of Osmanthus fragrans at two floral stages (early flowering and peak flowering). The cultivar examined is 'Yanzhi Hong', which possesses a distinctive red-like color flower, as opposite to other cultivars that produce white, yellow or orange flowers.
The Authors analysed floral pigments composition and gene expression patterns, both as whole-transcriptome differential expression and by focusing on single genes of interest involved in the biosynthesis of floral pigments.
The study is well thought, the experiment appropriately conducted and the conclusion well supported by the results.
However, the description of the experimental procedures is lacking and the raw transcriptomic data are not publicly available.
The English need to be improved.
Here several more detailed comments:
Line 30: "during the early and full flowering stages". The genes were differentially expressed between these the two conditions? Please clarify.
Line 94-102: "were " instead of "should be" all throughout the paragraph.
Line 97: Please specify if BHT is butylated hydroxytoluene.
Line 98: For the sake of reproducibility, convert "r/min" to g's. Otherwise, specify the centrifuge model and the rotor radius.
Line 100: clarify the meaning/procedure for "concentration". Evaporation under vacuum?
Line 101: clarify what is MTBE.
Line 118-133: the verbal tense in this paragraph (imperative) is inappropriate. The Methods should descrive what has been done, whereas imperative suggest the Authors copy-pasted the protocol. Please correct. The paragraph 2.4 must be re-written.
Line 130-133: "Interventionary studies involving animals or humans, and other studies that require ethical approval, must list the authority that provided approval and the corresponding ethical approval code." This sentence should be at the end of the manuscript. It looks looks like the Authors DID NOT proofread this manuscript.
Line 140: Clarify what are the "offline data"
Line 140: "Liuyejingui" is a single word.
Line 141: Raw sequencing data (fastq files) must be made available through public repositories with an accession name/number.
Line 141: For the sake of reproducibility, more details are needed for the Differential expression analysis. A short description of the bioinformatic pipeline is necessary, with software names and versions. Which short reads aligner was used? How the aligned reads were filtered?
Which annotation was used to score raw counts? Which software was used for DEG analysis? the R suite? EdgeR? DESeq2?
Line 143: "Genes with a FoldChange absolute value ≥ 2 and an FDR value ≤ 0.05 were considered significantly expressed genes (DEGs)". Statistically significant genes need to be selected with a p-value (FDR) threshold, as generated by the software. LogFC can be optionally used for additional filtering but does not to define statistically significant genes. This, correctly, is clearly stated in Results at line 217-219 but shoould be indicated hgere in Methods.
Line 175-178: Please add letters A,B, and C to the three panels in Figure 1.
Line 176: "stage" instead of "period".
Line 195: Need more technical details on how this heatmap was generated, in particular about how the values were calculated.
Line 196-197: "Table 1 and Figure 2 illustrate that there is no difference in the carotenoid components in the petals of 'Yanzhi Hong' between the initial and peak flowering stages.". This would be better illustrated by two pie charts or two stacked bar charts.
Line 226: What does "GeneRatio" stand for? How was it calculated?
Line 255: Tabler 2 contains "Transcriptome sequencing data statistics". The authors mean Table S2 probably. How the functional annotation was conducted?
Line 269: The labels in Figure 6 are too small and not readable
Line 287-288: please indicate the meaning of the stars on Figure 7. Which p-values they indicate?
Line 292: please indicate the meaning of the labels 1 and 2 in Figure 8.
Line 292: please indicate the gene name. for example "LYG005369 (CHYE)" and not only "LYG005369". Also, indicating the biochemical pathway of each gene would help the interpretation of Figure 8.
Line 292: since hin Figure 8 the comparison is between gene expression in two floral stages and not between gene expression in different cultivars, I suggest to place the histograms pairwise, with expression level of each gene in the two stages side by side. This will also help make the trend between two stages more apparent.
Line 294: LYG009054, not LYG0090554.
Line 310: "the difference between 'Yanzhi Hong' and 'Liuye Jingui' during the peak flowering period is not significant". If the statistical significance has been tested (as it should), it is necessary to indicate p-values and to which comparison they are referred.
Comments on the Quality of English LanguageEnglish is adequate except minor issues that I highlighted in my comments.
Author Response
Thank you very much to the reviewer for taking the time to carefully review this manuscript and providing a lot of professional and constructive comments. We would like to respond to the comments of the reviewer one by one as follows:
Comments 1: [ Line 30: "during the early and full flowering stages". The genes were differentially expressed between these the two conditions? Please clarify.]
Response 1: Thank you for pointing this out. We have changed “during the early and full flowering stages” to “at the peak flowering stage relative to the initial flowering stage”.[Line 29-30 in the revised manuscript]
Comments 2：[Line 94-102: "were " instead of "should be" all throughout the paragraph.,Line 97: Please specify if BHT is butylated hydroxytoluene.
Line 98: For the sake of reproducibility, convert "r/min" to g's. Otherwise, specify the centrifuge model and the rotor radius.
Line 100: clarify the meaning/procedure for "concentration". Evaporation under vacuum?
Line 101: clarify what is MTBE.]
Response 2: The main issues raised in paragraph 2.2 are multiple details. We have changed‘BHT’ to ‘butylated hydroxytoluene (BHT)’, ‘MTBE’ to ‘methyl tert-butyl ether (MTBE)’,‘12000 rpm’ to ‘13400 g’. The concentration of the extraction solution was indeed using evaporation under vacuum. Finally, The English grammars of this part have also been further revised. [Line 97-107 in the revised manuscript]
Comments 3：[Line 118-133: the verbal tense in this paragraph (imperative) is inappropriate. The Methods should descrive what has been done, whereas imperative suggest the Authors copy-pasted the protocol. Please correct. The paragraph 2.4 must be re-written.
Line 130-133: "Interventionary studies involving animals or humans, and other studies that require ethical approval, must list the authority that provided approval and the corresponding ethical approval code." This sentence should be at the end of the manuscript. It looks looks like the Authors DID NOT proofread this manuscript.]
Response 3: We are well Sorry, as for Line 130-133, it was indeed a result of our negligence in checking and retaining it when replacing the template. The paragraph 2.4 has been completely rewritten, polished, and checked. [Line 125-138 in the revised manuscript]
Comments 4：[Line 140: Clarify what are the "offline data"
Line 140: "Liuyejingui" is a single word.
Line 141: Raw sequencing data (fastq files) must be made available through public repositories with an accession name/number.
Line 141: For the sake of reproducibility, more details are needed for the Differential expression analysis. A short description of the bioinformatic pipeline is necessary, with software names and versions. Which short reads aligner was used? How the aligned reads were filtered?
Which annotation was used to score raw counts? Which software was used for DEG analysis? the R suite? EdgeR? DESeq2?
Line 143: "Genes with a FoldChange absolute value ≥ 2 and an FDR value ≤ 0.05 were considered significantly expressed genes (DEGs)". Statistically significant genes need to be selected with a p-value (FDR) threshold, as generated by the software. LogFC can be optionally used for additional filtering but does not to define statistically significant genes. This, correctly, is clearly stated in Results at line 217-219 but should be indicated here in Methods.]
Response 4: ‘Liuyejingui’ refers to ‘Liuye Jingui’ cultivar of Osmanthus fragrans, which had been obtained a high-quality reference genome and used for RT-qPCR analysis in this research to compare gene expression levels in different cultivars. The raw sequencing data for RNA-Seq had been uploaded to NCBI and the project number had been obtained (PRJNA1137553), but the accession number will be obtained a few days later. I will promptly notify the editor via email for updates. We had made detailed revisions to the comments from the reviewer regarding lines 140, 141, and 143. The paragraph 2.5 had been completely rewritten, polished, and checked. [Line 140-156 in the revised manuscript]。
Comments 5：[Line 175-178: Please add letters A, B, and C to the three panels in Figure 1.
Line 176: "stage" instead of "period".]
Response 5: The letters A, B and C had been added to the three panels in Figure 1. And we had changed ‘period’ to ‘stage’. [Line 175-185 in the revised manuscript].
Comments 6：[Line 195: Need more technical details on how this heatmap was generated, in particular about how the values were calculated.]
Response 6: The technical details for Figure 2 heat map were introduced in paragraph 2.4 of the revised manuscript. [Line 125-138 in the revised manuscript].
Comments 7：[Line 196-197: "Table 1 and Figure 2 illustrate that there is no difference in the carotenoid components in the petals of 'Yanzhi Hong' between the initial and peak flowering stages.". This would be better illustrated by two pie charts or two stacked bar charts.]
Response 7: Thank you very much for the professional suggestions from the reviewer. We had added ‘Figure 2 Proportion of carotenoid components in petals of ‘Yanzhi Hong’ at the initial and peak flowering stages’ so that the data could be presented more intuitively. With the intuitive presentation of Figure 2, we have slimmed down the proportion data in Table 1. [Line 205-212 in the revised manuscript].
Comments 8: [Line 226: What does "GeneRatio" stand for? How was it calculated?]
Response 8: In Gene Ontology (GO) enrichment analysis, the term "geneRatio" refers to the ratio of genes associated with a particular GO term to the total number of genes that were input for the enrichment analysis. Identify the number of genes associated with a specific GO term: This is the count of genes from input list that are annotated with the particular GO term. Count the total number of input genes: This is the total number of genes that were submitted for the GO enrichment analysis. Calculate the ratio: The geneRatio is then calculated as the number of genes associated with the GO term divided by the total number of input genes.
Considering that the article mainly describes the number of DEGs, we replaced the bubble diagram with the bar chart of DEGs GO enrichment. [Line 261-263 in the revised manuscript].
Comments 9: [Line 255: Tabler 2 contains "Transcriptome sequencing data statistics". The authors mean Table S2 probably. How the functional annotation was conducted?]
Response 9: Sorry for the annotation error here. Table S2, annotated results of DEGs related to carotenoid and flavonoid metabolism, had been added in the revised manuscript. So Table 2 here had been corrected to Table S3. [Line 293 in the revised manuscript].
The method of gene function annotation had been introduced in paragraph 2.5, based on the KEGG annotation results and KEGG PATHWAY map00906. [Line 153-156 in the revised manuscript].
Comments 10: [Line 269: The labels in Figure 6 are too small and not readable]
Response 10: Due to the fact that the main factor causing the color change of ‘Yanzhi Hong’ is the carotene components, β-carotene, lycopene, γ-carotene, α-carotene and (E/Z)-phytoene. A new readable Figure 6 had been obtained by re-analyzing between these 5 components and carotenoid metabolism pathway genes. [Line 304-307 in the revised manuscript].
Comments 11: [Line 287-288: please indicate the meaning of the stars on Figure 7. Which p-values they indicate?]
Response 11: Asterisks indicate the significant difference between the initial and peak flowering stages revealed by t-test: *p < 0.05, **p < 0.01. [Line 323-3027 in the revised manuscript].
Comments 12: [Line 292: please indicate the meaning of the labels 1 and 2 in Figure 8.
Line 292: please indicate the gene name. for example "LYG005369 (CHYE)" and not only "LYG005369". Also, indicating the biochemical pathway of each gene would help the interpretation of Figure 8.
Line 292: since hin Figure 8 the comparison is between gene expression in two floral stages and not between gene expression in different cultivars, I suggest to place the histograms pairwise, with expression level of each gene in the two stages side by side. This will also help make the trend between two stages more apparent.
Line 294: LYG009054, not LYG0090554.
Line 310: "the difference between 'Yanzhi Hong' and 'Liuye Jingui' during the peak flowering period is not significant". If the statistical significance has been tested (as it should), it is necessary to indicate p-values and to which comparison they are referred.]
Response 12: The labels 1 and 2 respectively stand for the initial and peak flowering stage. In order to demonstrate the relationship between genes and metabolites more clearly, we have added biochemical pathways in this figure. Due to the fact that this figure mainly compares the gene expression between different cultivars at the same flowering stage, we believe that it is reasonable to place the expression levels of each gene in different cultivars at the same stage side by side. And other details had also been corrected. [Line 333-370 in the revised manuscript].
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe manuscript "Metabolomic and Transcriptomic Analysis of Unique Floral 2 Coloration in Osmanthus fragrans Cultivars" describes studies on red coulored flower cultivar of an important Chinese ornamental crop. The main concern with the design is that metabolomic and transcriptomic experiment are generally achieved comparing the treatment (in this case, two flower stages of a cultivar) in reference to a check, which is not included in this analysis. Hence, there are certain limitations with the obtained results, especially because this lack of testers is not mentioned (at least in Discussion, there must be an argument for this situation). Then, an RT-qPCR assay to validate some gene expression is done with two additional genotypes, bit they are not described in Material and Methods (though there are a mention in the Abstracts). Also, tittles of tables (especially Table2) must be self-explanatory. I have made some additional comments in the attached file.
Comments for author File: Comments.pdf
Author Response
Thanks to the reviewer for timely review and professional suggestions. Our response to the reviewer's comments is as follows:
Comments 1: [ Family is Oleaceae.]
Response 1: Thank you for pointing this out. We have changed “the Osmanthus family and genus” to “to the Oleaceae family and Osmanthus genus”.[Line 48 in the revised manuscript]
Comments 2: [ Why a tester cultivar presenting a different color was not assayed? Results in metabolomic and transcriptomic studies must have a reference.]
Response 2: An introduction about the comparing cultivars of ‘Liuye Jingui’ and ‘Gecheng Dangui’ had been added in 2.1 plant materials. “Two other cultivars, 'Liuye Jingui' and 'Gecheng Dangui' previous studied [15], used for RT-qPCR analysis of genes for lycopene cyclase and carotene hydroxylase, were obtained from the nursery at Huazhong Agricultural University campus (114°21'3"E, 30°28'43"N).” [Line 90-93 in the revised manuscript]
Comments 3: [Please give more information about data on Table 2.]
Response 3: The detailed information about data on Table 2 had been introduced in paragraph 2.5 of the revised manuscript. [Line 139-156 in the revised manuscript]
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsThe present version of the manuscript is acceptable for publication. All changes were done according my previous suggestions.
