Integrated Transcriptomics and Metabolomics Analysis Reveal Anthocyanin Biosynthesis for Petal Color Formation in Catharanthus roseus

Round 1

Reviewer 1 Report

The manuscript “Integrated transcriptomics and metabolomics analysis reveal anthocyanin biosynthesis for petals colour formation in Catharanthus roseus” comprehensively analyzed the metabolome and transcriptome of three distinct Catharanthus roseus varieties exhibiting different petal colours, and identified main anthocyanin components and candidate genes affecting floral colours. However, the following comments needs to be addressed before publishing.

1. Line 166, why RPS9 was chosen as a reference gene here? Are there any literatures for it?

 

2. To better understand how anthocyanins affected floral colours, I suggest that the writers use CIELab colour parameters to describe three floral colours, and correlation between colour parameters and anthocyanin components.

 

3. In Figure 1b, the contents of total anthocyanin in CR and CP were much higher than those in CW. Why it looks like the anthocyanins in CW were more abundant than those in CR in Figure 3b?

 

4. To better understand the pathway, I suggest that the writers added differential contents of main anthocyanin components in 3 samples in Figure 6.

 

5. In Figure 6, the expression patterns of FLS genes were opposite to total anthocyanin contents. Since flavonol pathway competes with anthocyanin pathway for substrates, how were the flavonol contents in three Catharanthus roseus varieties ?

 

6. The contents of anthocyanin contents were directly determined by enzymes encoding by structural genes in anthocyanin biosynthesis, while the expression levels of these structural genes were regulated by transcription factors. Therefore, I suggest the correlation between anthocyanin contents and structural genes, and correlation between structural genes and transcription factors are performed respectively in Figure 8.

Author Response

Dear Reviewer,

We would like to express our sincere appreciation to you for your valuable comments and suggestions on our manuscript “Integrated transcriptomics and metabolomics analysis reveal anthocyanin biosynthesis for petals colour formation in Catharanthus roseus”. We are grateful for the time and effort invested in reviewing our work.

We have carefully considered all the comments and have made revisions accordingly. In response to your comments, we would like to provide a point-by-point response:

1. Line 166, why RPS9 was chosen as a reference gene here? Are there any literatures for it?

Thank you very much for your reminding, The literature we refer to is “Selection and Validation of Reference Genes for Transcript Normalization in Gene Expression Studies in Catharanthus Roseus”.Specific information has been added to the references [28].

2. To better understand how anthocyanins affected floral colours, I suggest that the writers use CIELab colour parameters to describe three floral colours, and correlation between colour parameters and anthocyanin components.

Thank you very much for your suggestion. But it is a great pity that we did not find the relevant colorimeter and could not obtained CIElab color prameter, due to the limitation of conditions. However, we used the Royal Horticultural Society Color Chart(RHSCC) to determine type of color. You can check in the manuscript line 93-97 and line 177-178.

3. In Figure 1b, the contents of total anthocyanin in CR and CP were much higher than those in CW. Why it looks like the anthocyanins in CW were more abundant than those in CR in Figure 3b ?

Thanks for your question, we have reviewed the experimental data. Figure 3b aims to show the content of anthocyanins common to petals of different colors. In fact, the column shape of each group contains a common 6 anthocyanins. However, due to the large difference in content, the relative proportion of some anthocyanins is relatively small. The content of cyanidin 3-O-rutinoside is higher in CW, which may be the reason for the formation of a small amount of red color in the base of CW petals.

4. To better understand the pathway, I suggest that the writers added differential contents of main anthocyanin components in 3 samples in Figure 6.

Thanks for your suggestion, we made a heat map for the main anthocyanins shown in Figure 6, so as to represent the content of anthocyanins in different samples. You can check in the manuscript line 288.

5. In Figure 6, the expression patterns of FLS genes were opposite to total anthocyanin contents. Since flavonol pathway competes with anthocyanin pathway for substrates, how were the flavonol contents in three Catharanthus roseus varieties ?

Thank you for your question. Among the flavonols we detected, kaempferol and its derivatives had the highest content in CW, corresponding to compounds 28-32 in Figure 2b. Quercetin and its derivatives had the highest content in CR, corresponding to compounds 33-37 in Figure 2b. According to the transcriptional data, the pattern of high expression of FLS in CW is consistent with the large accumulation of kaempferol and its derivatives in CW, while the large accumulation of quercetin and its derivatives in CR may be related to the large expression of F3’H in CR, which may be the result of FLS competing with F3’H in dihydrokaempferol.

6. The contents of anthocyanin contents were directly determined by enzymes encoding by structural genes in anthocyanin biosynthesis, while the expression levels of these structural genes were regulated by transcription factors. Therefore, I suggest the correlation between anthocyanin contents and structural genes, and correlation between structural genes and transcription factors are performed respectively in Figure 8.

Thanks for your suggestion, we analyzed the correlation between anthocyanins and structural genes, structural genes and transcription factors respectively, and modified the content of section 3.7. We found that most anthocyanin content was significantly positively correlated with CHS(CRO_06G025530). FLS (CRO_06G000510) had a significant negative correlation with bHLH (CRO_07G004820) and F3’5’H (CRO_04G034940) had a significant correlation with WD40 (CRO_S000630).

We tried our best to improve the manuscript and made some changes to the manuscript. We would like to express our sincere thanks to you for your enthusiastic work and hope that the revision can be recognized. Thank you again for your comments and suggestions. Hope you have a nice day!

Sincerely,

Zhihua Liao

Author Response File: Author Response.docx

Reviewer 2 Report

1. For Figure 1a, there is no scale bar.

2. For Figure 1b, there is no statistical analysis in figure legend.

Author Response

Dear Reviewer,

We would like to express our sincere appreciation to you for your valuable comments and suggestions on our manuscript “Integrated transcriptomics and metabolomics analysis reveal anthocyanin biosynthesis for petals colour formation in Catharanthus roseus”. We are grateful for the time and effort invested in reviewing our work.

We have carefully considered all the comments and have made revisions accordingly. In response to your comments, we would like to provide a point-by-point response:

1.For Figure 1a, there is no scale bar.

Thank you very much for your careful check, we have added the scale in the lower right corner of Figure 1a.

2.For Figure 1b, there is no statistical analysis in figure legend.

Thank you for your careful inspection. We apologize for our carelessness. Both CP and CR are significantly different from CW at the 0.01 level, and we have added the statistical analysis in the legend instructions.

We tried our best to improve the manuscript and made some changes to the manuscript. We would like to express our sincere thanks to you for your enthusiastic work and hope that the revision can be recognized. Thank you again for your comments and suggestions. Hope you have a nice day!

Sincerely,

Zhihua Liao

Reviewer 3 Report

This authors comprehensively analyzed the metabolome and transcriptome of three distinct C. roseus varieties exhibiting different petal colors. Differential metabolites and genes for diverse colors were found. The manuscript is generally well written and read, However, I still have some minor questions and suggestions:

(1) The experimental materials were the petals of 3 varieties, containing big spot at the base of the petals? or not containing? Especially in white flower varieties, a small amount of anthocyanins may come from the spot,

(2) Line 229-230, the description in this manuscript is inconsistent with Table S4.

(3) There are two Table S1 in Supplementary files. Please check all table numbers in Supplementary files.

(4) Line 312-314, Integrated transcriptome and metabolome analysis found MYB (CRO_03G031380) had significant correlations with three anthocyanins. Please supplement the qRT-PCR verification results of this transcription factor.

(5) In Discussion section, the possible reason for the formation of white C. roseus petals.

(6) In Figure S2, it is recommended to add both the gene name and the gene number.

Quality of English Language is very well

Author Response

Dear Reviewer,

We would like to express our sincere appreciation to you for your valuable comments and suggestions on our manuscript “Integrated transcriptomics and metabolomics analysis reveal anthocyanin biosynthesis for petals colour formation in Catharanthus roseus”. We are grateful for the time and effort invested in reviewing our work.

We have carefully considered all the comments and have made revisions accordingly. In response to your comments, we would like to provide a point-by-point response:

1. The experimental materials were the petals of 3 varieties, containing big spot at the base of the petals? or not containing? Especially in white flower varieties, a small amount of anthocyanins may come from the spot.

Yes, we agree with you. We collected the whole flower containing big spot at the base of the petals. We hypothesize that the higher content of cyanidin 3-O-rutinoside in the CW is responsible for the formation of the red spots in the middle.

2. Line 229-230, the description in this manuscript is inconsistent with Table S4.

Thanks for your careful inspection, we have re-checked and verified the original data of the transcriptome to make it more accurate and concise. We also modified and corrected the corresponding lines 229-236。You can check it in the manuscript.

3. There are two Table S1 in Supplementary files. Please check all table numbers in Supplementary files.

Thank you for your careful checks. We apologize for our carelessness. We checked and renumbered the supplementary materials. There are now six tables and three pictures in the supplementary materials.

4. Line 312-314, Integrated transcriptome and metabolome analysis found MYB (CRO_03G031380) had significant correlations with three anthocyanins. Please supplement the qRT-PCR verification results of this transcription factor.

Thanks for your suggestion, we put the MYB (CRO_03G031380) qRT-PCR verification results in the supplementary material figure S3.

5. In Discussion section, the possible reason for the formation of white C. roseus petals.

Thank you very much for your suggestion. We add a discussion of the possible reason for the formation of white petals in lines 371-380 of the paper. We agreed that the high expression of FLS caused the reduse of substrate for anthocyanin pathway. The reduced anthocyanin content eventually led to the formation of white flowers. A small amount of DHK entered the anthocyanin synthesis pathway ,which was converted to cyanidin eventually, perhaps responsible for the formation of the red spot in the center of white petals.

6. In Figure S2, it is recommended to add both the gene name and the gene number.

Thank you for your suggestion. We have added both the gene name and gene number.

We tried our best to improve the manuscript and made some changes to the manuscript. We would like to express our sincere thanks to you for your enthusiastic work and hope that the revision can be recognized. Thank you again for your comments and suggestions. Hope you have a nice day!

Sincerely,

Zhihua Liao