Variation in the Calyx Color in Two Styrax japonicus Varieties Is Attributed to Varied Anthocyanin Levels as Revealed by Integrated Metabolomic and Transcriptomic Analyses

Round 1

Reviewer 1 Report

I read the whole manuscript and came to the conclusion that the manuscript is very well written and has the ability to create interest to the readers. So, I have no objection for this manuscript except some minor grammar and spelling errors. 

 

Author Response

Dear reviewer:

Thank you very much for your comments concerning our manuscript (forests-2459833) submitted to Forests.

Those comments have been very helpful for revising and improving our paper. To modify the remaining grammatical mistakes, the manuscript has been procured language editing services again.  I apologize the spelling errors in the manuscript, and these errors had been corrected.

Thank you again for your advices.

Reviewer 2 Report

Comments for author File: Comments.docx

Author Response

Manuscript ID: forests-2459833

Title: Variation in the calyx color in two Styrax japonicus varieties is attributed to varied anthocyanin levels as revealed by integrated metabolomic and transcriptomic analyses

Dear reviewer:

Thank you very much for your comments concerning our manuscript (forests-2459833) submitted to Forests.

Those comments have been very helpful for revising and improving our paper, and have provided important guidance for our research. We have studied the comments carefully and have made corrections accordingly.

Our itemized responses to your questions, comments and suggestions are as follows:

Comment 1: The introduction part is very long in addition the authors have developed the results a lot, normally the introduction is generally having the main functions of presenting the subject and announcing the plan of a written document. Also, I will have well developed the abbreviation R2R3-MYB TFs their usefulness.

Response: Thanks for the kind comment. We have removed the part of the introduction that were involved in the results. Lines 40-45, lines 65-66 and lines 70-75 that related to the results in the introduction have been deleted.

Comment 2: since you have dosed the flavonoids the anthocyanins, you must also dose the other phenolic acids, in short the total polyhonols.

Response: Thanks for the kind comment. In the current research, we assessed the contents of flavonoids and anthocyanins, but did not measure the phenolic acids content. There are two main considerations.

• Many studies on the mechanism of pigmentation in plants have shown that flavonoids, especially anthocyanins are the main determinants of coloration in floral organs. In this study, 202 differentially accumulated metabolites (DAMs) were identified in the calyxes of GA and RA. Among the 202 DAMs, two classes of components with the highest proportion were the flavonoids and anthocyanins, while the phenolic acids accounted for only 5.94%. In addition, the top 10 up-regulated metabolites were mainly anthocyanins, while no phenolic acids were detected. Combined with the transcriptome analysis, we mainly focused on the regulatory mechanism of anthocyanin accumulation.
• In recent years, medical values of different parts of japonicus have been investigated. The extraction analysis of different parts (including the calyx) of S. japonicus, including phenolic acids which have exhibited important antioxidant, anti-inflammatory, antiproliferative and neuroprotective properties, is also one of the ongoing research projects of our research group. We would like to present the results in the future.

Comment 3: For figure 3 I prefer that each figure must be mentioned in the part that suits it.

Response: Thanks for the kind comment. We have broken up the different parts of Figure 3 and inserted them where suit in the results. (Page 6, line 204, Figure3 and line 219, Figure 4)

Comment 4: Lack of references for comparison of results in the part of the discussion, the first two paragraphs.

Response: Thanks for the kind suggestion. Three references have been added to compare with our results. (Page 12, line 402 and Page 13, line 414)

Comment 5: Also, it is possible to add physico-chemical analyzes to characterize the plant, Ash, heavy metals, oraganic matter, humidity, ......

Response: Thanks for the kind comment. We would like to add these physico-chemical analysis in the next article relating to extraction analysis of different parts (including the calyx) of S. japonicus. In the current study, we want to clarify the secondary metabolism pathways, their regulators, and the changes in the transcription and metabolite levels in the calyx of S. japonicus regulating sepal color.

Comment 6: For the conclusion it is preferable to add a perspective of this work, explain the sentence (These mechanisms should be further studied)

Response: Thanks for the kind comment. A perspective of this work has been added in the conclusion. (Page 15, lines 502-504)

We explain the sentence “These mechanisms should be further studied” as follows:

Integrative analysis of metabolomics and transcriptomics data of GA and RA revealed the DAMs and DEGs related to calyx pigmentation. Besides the anthocyanin biosynthesis pathways, the DEGs also enriched in other pathways, such as “Photosynthesis - antenna proteins pathway”, “Ras signaling pathway” and “Plant-pathogen interaction pathway”, which suggested that the differences in anthocyanin accumulation may also be related to other metabolic pathways. These pathways might have certain correlation with anthocyanin biosynthesis, which should be further studied.

Thank you again for your advices.

Reviewer 3 Report

Title: Variation in the calyx color in two Styrax japonicus varieties is attributed to varied anthocyanin levels as revealed by integrated metabolomic and transcriptomic analyse.

In this paper, the authors investigate the metabolism of anthocyanins in the cups of S. Japonicus. Based on the obtained metabolome and transcriptome data, it was shown that the level of anthocyanins in the cups of RA was higher than in the cup of GA. Metabolism is closely related to the five late biosynthesis genes of the anthocyanin biosynthesis pathway. MYB1, MYB82, and MYB113 were the three likely transcription factors responsible for anthocyanin accumulation.

The work was done at a good methodological level. The presented results are beyond doubt. However, I had a few questions and comments, which I give below.

Line 36. Example with lily and magnolia add a references.

Line 97. Why do you use the abbreviation GA and RA, but not GL and RL, or GC and RC? Just wondering.

Line 114. You claim to have collected 25 calyxes for analysis, but then (line 118) you are talking about 30 calyxes. How many calyxes were there and how much is it per mass.

Line 127. On which device were the data received?

Line 135. Rutin standard, specify the manufacturer.

Line 171. Total RNA was isolated using a set of the specified manufacturer?

Line 185. Why was only one housekeeping gene (GAPDH) used? It is necessary to use at least 2 genes to normalize the data.

Line 209. The first sentence in the paragraph must be deleted, repeats the next sentence.

Figure 2. Indicate in the figure caption what A and B are.

Figure 3. The letters A,B,C... must be in parentheses. (A), (B), (C)…

Figure 5,6. The captions in the figure are poorly distinguishable. Improve the quality of the captions.

Fig.S5. Low information content of this figure due to poor image quality

Figure S4. Make captions on a horizontal scale in English.

Figure 6. Zoom in on the image

Author Response

Dear reviewer:

Thank you very much for your comments concerning our manuscript (forests-2459833) submitted to Forests.

Those comments have been very helpful for revising and improving our paper, and have provided important guidance for our research. We have studied the comments carefully and have made corrections accordingly.

Our itemized responses to your questions, comments and suggestions are as follows:

Comment 1: Line 36. Example with lily and magnolia add a references.

Response: Thanks for the kind comment. We have added the corresponding references (Page 1, line 37, references [2, 3]).

Comment 2: Line 97. Why do you use the abbreviation GA and RA, but not GL and RL, or GC and RC? Just wondering.

Response: Thanks for the kind comment. We have previously graded the flowering capacity of S. japonicus, numbering A-D. The "A" in GA and RA represents the flowering capacity of Red Linglong and Green Linglong. Therefore, we simply used the abbreviations "GA" and" RA" for metabolomic and transcriptomic analyses.

Comment 3: Line 114. You claim to have collected 25 calyxes for analysis, but then (line 118) you are talking about 30 calyxes. How many calyxes were there and how much is it per mass.

Response: Thanks for the kind reminder. The 25 calyxes of each biological replication were collected and immediately frozen in liquid nitrogen for subsequent physiological and molecular tests, including the assessment of total flavonoid and anthocyanin contents, the measurement of cell sap pH, extraction of secondary metabolites and total RNA. While approximately 30 fully pigmented calyxes were randomly sampled for determining the values of L^∗, a^∗, and b^∗, since liquid nitrogen frozen samples were not suitable for the determination of this index. We have corrected the description to make it clearer. (Page 3, lines 105-106)

Comment 4: Line 127. On which device were the data received?

Response: Thanks for the kind comment. The data was received on an Agilent Cary 60 UV-visible spectrophotometer (Agilent Technology Co., Ltd., China). We have added the device in the manuscript. (Page 3, lines 114-115)

Comment 5: Line 135. Rutin standard, specify the manufacturer.

Response: Thanks for the kind reminder. We have added the manufacturer of rutin standard in the manuscript. (Page 4, lines 122-123)

Comment 6: Line 171. Total RNA was isolated using a set of the specified manufacturer?

Response: Total RNA was extracted from the calyxes of GA and RA according to the manufacturer of RNA extraction kit (DP441, TIANGEN bio-chemical Technology Co., Ltd., Beijing, China).

Comment 7: Line 185. Why was only one housekeeping gene (GAPDH) used? It is necessary to use at least 2 genes to normalize the data.

Response: Thanks for the kind comments. We have added another housekeeping gene (PP2A) to validate the transcriptome results, which made sure that 2 genes were used to normalize the data.

Comment 8: Line 209. The first sentence in the paragraph must be deleted, repeats the next sentence.

Response: Thanks for the kind reminder. The first sentence in the paragraph has been deleted. (Page 5, line 198)

Comment 9: Figure 2. Indicate in the figure caption what A and B are.

Response: Thanks for the kind suggestion. We have added the information of A and B in Figure 2.

Comment 10: Figure 3. The letters A,B,C... must be in parentheses. (A), (B), (C)…

Response: Thanks for the kind comment. We found that the instructions for authors of Forests showed that label “A, B…” or “a, b…” is recommended to use for describing each part in figures with more than one part. Therefore, we used “A, B…”. (https://www.mdpi.com/authors/layout#_bookmark39)

Comment 11: Figure 5,6. The captions in the figure are poorly distinguishable. Improve the quality of the captions.

Response: Thanks for the kind suggestion. The size of captions in the figure have been enlarged to make them clear.

Comment 12: Fig.S5. Low information content of this figure due to poor image quality

Response: Thanks for the kind reminder. The image quality has been improved to make the information content clear.

Comment 13: Figure S4. Make captions on a horizontal scale in English.

Response: Thanks for the kind suggestion. We apologize for the mistake. The captions have been corrected on a horizontal scale in English.

Comment 14: Figure 6. Zoom in on the image

Response: Thanks for the kind comment. We have enlarged the image to make it clearer.

Thank you again for your advices.