Integrated Transcriptome and Metabolome Analysis Revealed Mechanism Underlying Anthocyanin Biosynthesis During Flower Color Formation in Lagerstroemia indica
Round 1
Reviewer 1 Report (Previous Reviewer 1)
Comments and Suggestions for AuthorsTitle: Integrated transcriptome and metabolome analysis revealed mechanism underlying anthocyanin biosynthesis during flower color formation in Lagerstroemia indica
The manuscript has been revised in a previous version, where 7 points had been suggested.
In this resubmitted manuscript, the authors have incorporated all seven suggestions, which have improved the presentation of the paper.
Please include the Conclusions as other section
5. Conclusions.
Move here Lines 408-423, page 15
After this minor suggestion, the paper should be accepted for publication.
Author Response
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Author Response File: 
Author Response.pdf
Reviewer 2 Report (Previous Reviewer 3)
Comments and Suggestions for AuthorsThe manuscript is a revised version, the authors considered the suggestions.
Author Response
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Author Response File: Author Response.pdf
Reviewer 3 Report (Previous Reviewer 4)
Comments and Suggestions for AuthorsThe authors did significant work to understand the genetic regulation of the pigment production of Lagerstroemia indica, in the anthocyanin pathway. The authors successfully found differences for samples with contrasting colors. Nevertheless, there is an important missing link in the work: the authors did not explain the importance of anthocyanin to the work. Their results indicate that anthocyanins are just the 6th most prevalent group of flavonoids in the cells. They should explain with references or reasons why they decided to study the biosynthesis pathway of these compounds. Is the color of anthocyanin more striking than flavonols? Is the biosynthesis of flavones, tannins, and flavanones already understood, and then the next science problem is the anthocyanins? The authors must explain their reasoning or the work looks like they picked a random class from the metabolite analysis and worked on it. I have other suggestions that I hope will be helpful.
**Title: OK
**Abstract: OK
**Keywords: OK
- Introduction:
The current flow is:
Biochemistry of the flower color and nutraceutical properties of plant chromophores > Secondary metabolites as plant chromophores, especially flavonoids and derivatives molecules > Biosynthesis of flavonoids and examples of genes related to it > The impact of plant hormones on the flavonoid biosynthesis > Specific effect of ethylene, a plant hormone > Description and uses of Lagerstroemia indica > Used germplasm and objective of this study
The flow could improve. If the authors will study anthocyanin biosynthesis, the authors should not discuss much about flavonoids and chalcones, focusing the introduction only on anthocyanins. Additionally, the 4th and 5th paragraphs could be merged because they deal with the same point: the impact of plant hormones (auxin and ethylene) on the biosynthesis of chromophores. Finally, the authors should explain the effect of plant hormones on the biosynthesis of anthocyanins, not flavonoids, because the focus of the study is on anthocyanins, not flavonoids. Therefore, I recommend the following flow:
Biochemistry of the flower color and nutraceutical properties of plant chromophores > Secondary metabolites as plant chromophores, especially anthocyanins and related molecules > Biosynthesis of anthocyanins and examples of genes related to it > The impact of plant hormones, especially auxin, and ethylene, on the anthocyanin biosynthesis > Description and uses of Lagerstroemia indica > Used germplasm and objective of this study
- Materials and Methods
2.1. Plant Materials: OK
2.2. Detection of total flavonoids in Lagerstroemia indica petals for metabolomic analysis: OK
2.3. Transcriptome Analysis Method for Lagerstroemia indica: OK
2.4. Quantitative Real-Time PCR (qRT-PCR): OK
2.5. Transcription factors analysis: OK
2.6. Statistical Analysis: OK
3. Results
3.1. Principal Component Analysis of Metabolites: OK
3.2. Analysis of differential metabolites:
-Please explain the reasoning for considering anthocyanins as the main pigment of the plant. Viewing Figure 1D, the main pigments are flavonols. If the anthocyanins are the pigments in the highest amount by mass, please make a figure showing the mass distribution per flavonoid group. If this is not the reason for studying anthocyanins, explain the reasoning.
3.3. Transcriptome sequencing and analysis
-Table S6 is missing in the supplementary material. Please add it for review.
3.4. Identification and Enrichment of DEGs: OK
3.5. DEGs were involved in the anthocyanin synthesis pathway
-Please explain the reasoning for studying genes related to anthocyanins. Anthocyanins are the 6th largest group in the Figure 1. Did they have the largest difference in gene expression? Were their metabolites particularly impacting the color? There is no problem with focusing on anthocyanins, but the authors must explain their reasoning and discuss why focus on them.
-Please explain the reasoning for commenting on DEGs related only to auxins. Was the majority of the hormone-related DEGs linked to auxins, for example? There is no problem to limit the discussion to one plant hormone, but they must present and discuss their reasoning for this filtering.
3.6. Models of anthocyanin biosynthetic pathways and qRT-PCR verification
-Line 271: Do the authors really mean “Wisteria?” Lagerstroemia indica is not from the Wisteria genus.
3.7. Correlation analysis of anthocyanin synthesis pathways
-Line 288: Do the authors really mean “Crassulacea plants?” Lagerstroemia indica is not from the Crassulaceae family.
4. Discussion
-The discussion focuses on anthocyanin, but there is no explanation of what is different with anthocyanin compared to the other flavonoids. The authors keep repeating that anthocyanins are the most important flavonoid, but they did not present any data or reference indicating that they have a stronger colors or are present in higher amounts. Please provide this explanation with reasonable values.
4.1. Structural gene analysis of anthocyanin synthesis pathway: OK
4.2. Correlation of transcription factors involved in anthocyanin synthesis in L. indica: OK
4.3. Auxins may be involved in the regulation of anthocyanin synthesis in L. indica
-I suggest splitting the last paragraph from this subsection and creating the section “5. Conclusions.”
** Supplementary Materials: OK
**Author Contributions: OK
** Funding: OK
** Data Availability Statement: OK
** Conflicts of Interest: OK
**References:
-Most references capitalize all the nouns of the articles’ titles. Therefore, please fix the references that are not in this format. Therefore, for example, the authors should write “A Novel Integrated Method for Large-Scale Detection, Identification, and Quantification of Widely Targeted Metabolites: Application in the Study of Rice Metabolomics” instead of “A novel integrated method for large-scale detection, identification, and quantification of widely targeted metabolites: application in the study of rice metabolomics.”
Author Response
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Author Response File: Author Response.pdf
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
Comments and Suggestions for Authorshorticulturae-3199830
Integrated transcriptome and metabolome analysis revealed mechanism underlying anthocyanin biosynthesis during flower color formation in Lagerstroemia indica
This paper report valuable insights into the regulatory mechanisms underlying anthocyanin accumulation and color formation in L. indica petals, and identifies several potential genes, laying the groundwork for further research on regulatory mechanisms and genetic improvement of L. indica.
Below are some suggestions for authors, which could be incorporated into the manuscript.
a- Include some bibliography that supports this paragraph and expands the economic relevance of the species, related to its commercialization, export and other data of importance for people who consult the Journal
b- 2.1. Plant Materials
This section should be improved, including some additional information such as: access to herbaria numbers, if they exist, where are they deposited? What botanist or institution classifies the species?
When collecting samples, is there a specific weight for the samples?
2.2. Detection of total flavonoids in Lagerstroemia indica petals for metabolomic analysis
Please state the analytical quality of the solvents and the company of origin
Please be more specific about the equipment used in your analysis, including the country of origin of the equipment.
c- 3.7. Correlation analysis of anthocyanin synthesis pathways The analysis revealed that most of these genes related to anthocyanin synthesis are positively correlated with the accumulation of the 22 anthocyanin metabolites (Figure 7), which may be associated with 274 color changes.
Mention or include the correlation values ​​or ranges obtained.
d- 4. Discussion
Paragraph page 13, 325-335
Values ​​could be included when high levels of flavonoids are mentioned, compared to which species?
e- It includes a conclusions section highlighting the relevant results of the research, future perspectives and potential contribution to the market of these species.
The paper could be considered for publication, after the suggestions have been answered.
Comments on the Quality of English LanguageMinor language revisions requested
Author Response
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Author Response File: Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors used inntegrated transcriptome and metabolome analysis to find mechanisms underlying anthocyanin biosynthesis during flower color formation in Lagerstroemia indica. Multi-omics studies like these are taking a very good approach to understanding the underlying biology.
The work is interesting, but there are several points that could be improved.
The main issue can be found in the data availability statement:
"Data Availability Statement: All the data presented in this study are available in the manuscript." This is ridiculous, where are the reads, TPM values, analyses, script, etc. The authors present no supplementary data to support their conclusions. It is for this reason that I recommend rejection of the paper. To withhold the underlying data is unacceptable in this era of scientific publishing. I would expect to see many GB of supporting data, instead, the authors claim the data is in the paper. It is not! They only show graphics derived from the data... Such a blatant misunderstanding does not bode well for the underlying, presented, science.
The manuscript is filled with vague and uninformative statements. For example, the authors state " auxin may play a role in regulating anthocyanin biosynthesis in L. indica". Well, common sense and multitudes of previous, published, research studies have already shown this to be true. It doesn't seem like a novel finding at all.
The introduction is way too long. Many of these facts should be contrasted with current findings in the Results section.
Such statements as "RNA-seq analysis annotated a total of 35,505 genes" are woefully vague and uninformative. What does this even mean? The authors should clarify ambiguous statements, telling clearly what was done and how. I see that later in the manuscript, the authors state: "By assembling new transcripts with StringTie and annotating them using databases such as Pfam, GO, and KEGG, a total of 35,505 genes were annotated." Now this is a little better, but actually what the authors need to do is give metrics for how many PFAMs, GO terms, and KEGG accessions were found. Be specific, because vagueness really hurts this paper. Furthermore, the authors state that "Among these, Pfam, GO (Gene Ontology), and KEGG anotated 2,388, 17,204, and 6,760 genes, respectively." This is less than 35,505, so the authors should clearly state where the other annotations are coming from. Also, there are probably many overlaps, making the total annotations from those mentioned analyses less.
The figures generally look good, but the font should be fixed in all figures (inconsistent font/sizes). The figures need cleaning; the text is of variable size and often difficult to read. For example, in Figure 1, the text is of many sizes and fonts which looks unprofessional.
Comments on the Quality of English Languagemany vague statements, need to be specific
Author Response
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Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsI reviewed the manuscript “Integrated transcriptome and metabolome analysis revealed mechanism underlying anthocyanin biosynthesis during flower color formation in Lagerstroemia indica” (horticulturae-3199830).
The manuscript is clearly described, and the results obtained are supported with experimental evidence.
Some points:
1. It is suggested to the authors to check letter size in Figures, I understand there is much information, but some axes sizes are too small and it is not easy to read them.
2. Please include the composition of “internal standard extraction solution” mentioned in line 115.
Author Response
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Author Response File: Author Response.pdf
Reviewer 4 Report
Comments and Suggestions for Authors The authors performed a functional genomics analysis of half-sibling ornamental trees with contrasting colors in their petals to discover the biochemical pathways related to different flower pigment. Extracting and analyzing mRNA from woody plants is complicated, and the authors succeed in this struggle. I found small writing details that the authors must fix. I hope my comments are helpful.
**Title: OK
**Abstract: OK
**Keywords: OK
1. Introduction
-The current flow is:
Biochemistry of the flower color > Biochemical groups of pigments and some biochemical synthetic pathways > Transcription factors related to the pigment synthetic pathways > Plant hormones as activators of pigment synthesis > The importance of “Lagerstroemia indica” > Lack of plants with specific colors and objective of this work.
The flow is fair, and I do not see the need to change it. Good job.
-Line 38: It is strange mentioning “beet pigments” for a plant that is not beet. Please use a biochemical synonym for this chemical.
-Line 83: Please write the full scientific name the first time it appears in the main text. Therefore, please write “Lagerstroemia indica” at the beginning of the line and then write “L. indica.”
2. Materials and Methods
2.1. Plant Materials: OK
2.2. Detection of total flavonoids in Lagerstroemia indica petals for metabolomic analysis:
-Line 114: Please explain if the extraction solution is 70% volume/volume (v/v), weight/weight (w/w), or weight/volume (w/v).
-Line 117: Please provide the maker and model of the centrifuge and provide the g-force, not the rpm. Different centrifuges at the same rpm may have yield different g-forces.
-Lines 124, 125: Please explain if the formic acid concentration is 0.1% volume/volume (v/v), weight/weight (w/w), or weight/volume (w/v).
2.3. Transcriptome Analysis Method for Lagerstroemia indica: OK
2.4. Quantitative Real-Time PCR (qRT-PCR): OK
2.5. Transcription factors analysis: OK
2.6. Statistical Analysis:
-PCA is a type of statistical analysis. Please explain here how the authors performed this analysis.
3. Results
3.1. Principal Component Analysis of Metabolites
-I suggest adding the scree plot to let the readers understand how many PCs are needed to reach 90% or 95% of the total variability.
3.2. Analysis of differential metabolites: OK
3.3. Transcriptome sequencing and analysis: OK
3.4. Identification and Enrichment of DEGs: OK
3.5. DEGs were involved in the anthocyanin synthesis pathway: OK
3.6. Models of anthocyanin biosynthetic pathways and qRT-PCR verification: OK
3.7. Correlation analysis of anthocyanin synthesis pathways: OK
4. Discussion
4.1. Structural gene analysis of anthocyanin synthesis pathway: OK
4.2. Correlation of transcription factors involved in anthocyanin synthesis in L. indica:
-The authors must write the scientific name in regular text because the title is italicized, and scientific names must always be written in a different format than the text around them.
4.3. Auxins may be involved in the regulation of anthocyanin synthesis in L. indica
-The authors must write the scientific name in regular text because the title is italicized, and scientific names must always be written in a different format than the text around them.
-Line 377: Please italicize “Malus domestica.”
5. Conclusions
-There is no conclusion in the document. At the end of the abstract, the authors claimed that “This study provides valuable insights into the regulatory mechanisms underlying anthocyanin accumulation and color formation in L. indica petals, and identifies several potential genes, laying the groundwork for further research on regulatory mechanisms and genetic improvement of L. indica.” However, there is nothing in the text elaborating this thought. The authors must explain how the results of this document will lay the ground for future works.
**Supplementary material: OK
**Author Contributions: OK
**Funding: OK
**Data Availability Statement:
-The raw data is absent in the manuscript and supplementary material. I recommend the Data Availability Statement: “The data that support the findings of this study are available from the corresponding author, [author initials], upon reasonable request.”
**Conflicts of Interest: OK
**References: OK
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors did not address my points. Instead, they now state:
"The data presented in this study are available on request from the corresponding author"
Which is totally unacceptable and does nothing to make the underlying data available. I need to be able to access the data, and so do the readers. Not 'on request'. Put them in a public archive. You can share the links for exclusive access during review, and then you need to make them public after publications.
English is OK could use a copy edit
Author Response
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Author Response File: Author Response.pdf
Round 3
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors attempted to address my previous comments (unsuccessfully). I had remarked that they need to make their data available. The data is still not available to me, the reviewer, during the time of review. It should be privately shared with reviewers during the review phase, then later published. Just saying 'submitted to NCBI' doesn't mean much.
Furthermore, almost nothing in the paper is properly cited. The authors mention a great deal of software and databases they used while failing to provide proper bibliographic information. Companies should have their locations listed, and, if applicable, websites.
Still a long way to go, I'm afraid, before this paper is ready for publication.
Comments on the Quality of English LanguageEnglish needs careful consideration.
Author Response
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Author Response File: Author Response.pdf
