Integrated Metabolome and Transcriptome Analyses Provide New Insights into the Leaf Color Changes in Osmanthus fragrans cv. ‘Wucaigui’

Round 1

Reviewer 1 Report

I have completed the review of manuscript #forests-2948991. Author shave investigated transcriptome and metabolome of ornamental plant Osmanthus fragrans ‘Wucaigui’ to examine the role of different genes in different patterns of leaf coloration. 

I have few concerns regarding the presented work.

1. What was the rationale/objective of undertaking this work? Was it only academic? Or the work has some applied aspects!

2. how could authors determine the various types of leaves based on leaf color. Was there a criterion? Or it was just visualization1 If so, what about the validation? A robust response is required.

3. What standards were used for identification of the metabolites? Details should have been there? 

4. What is ‘Wucaigui’?

5. What is meant by 'hot topic'?

6. Which metabolites are responsible for imparting different coloration to plant leaves? A listing is not of values as regards the theme of the paper!

Where are the tables and figures?

A moderate editing is required.

Author Response

Comments and Suggestions for Authors

I have completed the review of manuscript #forests-2948991. Author shave investigated transcriptome and metabolome of ornamental plant Osmanthus fragrans ‘Wucaigui’ to examine the role of different genes in different patterns of leaf coloration.

Response: Thanks for your careful reading and fair evaluation of our work. We respect your comments and have dealt with each point seriously in the following responses.

I have few concerns regarding the presented work.

1. What was the rationale/objective of undertaking this work? Was it only academic? Or the work has some applied aspects!

Response: We appreciate your review and thanks for your inquiry! The rationale for conducting this research on Osmanthus fragrans from its long history of traditional use as a well-known ornamental plant for its specialties, including plant shape, flower fragrance, and leaf coloration. Despite widespread use of this plant in China, there is a lack of comprehensive scientific addressing genetical mechanisms of its leaf coloration. Therefore, this study aims to systematically investigate the transcriptome and metabolome of Osmanthus fragrans variety ‘Wucaigui’, to provide scientific understanding for the role of different genes and their functions in leaf coloration, this work seeks to contribute to the development of artificial regulate and control technology in breeding and production of Osmanthus fragrans and other ornamental plants.

2. how could authors determine the various types of leaves based on leaf color. Was there a criterion? Or it was just visualization1 If so, what about the validation? A robust response is required.

Response: Thank you for your academical comments! We first of all performed phenological observation on Osmanthus fragrans ‘Wucaigui’ by the period of spring and autumn, we found that the color of its new leaves transformed from red to yellow-green, and furthermore to completely green. In order to validation, the Royal Horticultural Society (RHS) Color Chart was used for comparing colored leaves in six stages, namely S1 (gray-brown RHS 166A), S2 (medium-brown RHS 165A), S3 (dark green RHS 152C), S4 (strong yellow-green RHS 144B), S5 (yellow-green RHS 143B), and S6 (medium-olive green RHS 137B). The sampling time were determined according to those above leaf colors. Above revisions were added at the section Materials and Methods in our manuscript from line 112-115.

3. What standards were used for identification of the metabolites? Details should have been there?

Response: Thanks for your concern on standards! This study was supported by Metware which is a biological company who purchased standards of the metabolites. We have already sent a request asking for detailed information of standards; however, it may take some time for Metware to reply. More information of their anthocyanin assay and carotenoid assay can be seen from their website.

https://www.metwarebio.com/anthocyanin-targeted-metabolomics-service/

https://www.metwarebio.com/metabolomics-services/carotenoid-targeted-metabolomics/

4. What is ‘Wucaigui’?

Response: Thanks for your inquiry! The sentence “The plant material O. fragrans ‘Wucaigui’ is a mutant variety from O.fragrans var. thunbergii, among all O.fragrans we observed, it has a unique characteristic which is long period of leaf color changes.” has been added in line 106-108.

5. What is meant by 'hot topic'?

Response: Thank you for mentioning that! We think 'hot topic' can be confusing in this case, therefore, we reorganized this sentence as ‘study of leaf color changes on ornamental plants is one of the mostly concentrated topics at horticulture research field.’ (in line 54-55)

6. Which metabolites are responsible for imparting different coloration to plant leaves? A listing is not of values as regards the theme of the paper!

Response: Thank you for your constructive suggestion! We presented in sentences as ‘In the early stage of leaf color change, the anthocyanin content was high, and the leaves of O. fragrans ‘Wucaigui’ appeared red. With development, the anthocyanin content in the leaves gradually decreased, and the carotenoid and chlorophyll contents gradually increased, until finally the leaves appear green.” has been replaced by “In the early stage of leaf color change, the anthocyanin (Rutin and Quercetin-3-O-glucoside) content was high, and the leaves of O. fragrans ‘Wucaigui’ appeared red. With development, the anthocyanin content in the leaves gradually decreased, and the carotenoid (lutein and zeaxanthin) and chlorophyll (chlorophyll a and chlorophyll b) contents gradually increased, until finally the leaves appear green.’ (line 368-373)

Moreover, three tables (Table 1, Table 2, and Table 3) of values for metabolites has been attached to the manuscript.

7. Where are the tables and figures?

Response: Thank you for pointing that out! Tables and figures were uploaded to submission system, and we managed to fill them in our manuscript.

8. Comments on the Quality of English Language

A moderate editing is required.

Response: Thank you for careful review! We have made some improvements on English Language of our manuscript for avoiding misunderstandings. A edited version of our manuscript was uploaded to the submission system..

Reviewer 2 Report

Using integrated metabolome and transcriptome analyses, the manuscript investigates the mechanisms driving leaf color changes in Osmanthus fragrans 'Wucaigui'. It successfully identifies key metabolites and genes associated with different developmental stages, offering insights into regulating leaf color variation. The authors identified key metabolites and genes associated with different developmental stages, shedding light on the metabolic and transcriptional regulation of leaf color variation. The introduction provides a comprehensive background on plant leaf color variation, highlighting its significance in both natural and breeding contexts. It effectively sets the stage for the study by discussing the role of pigments and the importance of molecular approaches in understanding leaf color changes. However, the introduction could benefit from more specific references to previous research on Osmanthus fragrans and a clearer delineation of the research gap addressed by the current study. The materials and methods section is well-structured and provides detailed protocols for chlorophyll, carotenoid, and anthocyanin extraction and determination, as well as transcriptome sequencing and analysis. T

The results section presents a comprehensive metabolome and transcriptome data analysis, supported by statistical analyses. However, the presentation would benefit from integrating figures or tables for improved visualization.

Major Comments:

RNAseq sequencing quality check with PCA or MDS should be included in the supplementary.

WCGNA hub gene selection is not elaborated correctly, the criteria for GS and MM should clearaly mansion.

Tables are very hard to fallow for reader try to make it easy to read

Minor Comments:

The abstract should be more concise. Consider condensing the description of the leaf color changes and focusing on the key findings.

The identification of key metabolites and genes is pivotal. Could you elaborate on the criteria used to select these key components?

Please clarify the statistical methods used for metabolite identification and analysis.

Line 102: Clarify whether the metabolite and transcriptome analyses were conducted on the same leaf samples.

Line 177: Please provide more details on the criteria used for determining significantly differentially expressed genes.

Line 233+: Ensure consistency in reporting the number of novel genes across the manuscript.

Author Response

Comments and Suggestions for Authors

Using integrated metabolome and transcriptome analyses, the manuscript investigates the mechanisms driving leaf color changes in Osmanthus fragrans 'Wucaigui'. It successfully identifies key metabolites and genes associated with different developmental stages, offering insights into regulating leaf color variation. The authors identified key metabolites and genes associated with different developmental stages, shedding light on the metabolic and transcriptional regulation of leaf color variation. The introduction provides a comprehensive background on plant leaf color variation, highlighting its significance in both natural and breeding contexts. It effectively sets the stage for the study by discussing the role of pigments and the importance of molecular approaches in understanding leaf color changes. However, the introduction could benefit from more specific references to previous research on Osmanthus fragrans and a clearer delineation of the research gap addressed by the current study. The materials and methods section is well-structured and provides detailed protocols for chlorophyll, carotenoid, and anthocyanin extraction and determination, as well as transcriptome sequencing and analysis.

The results section presents a comprehensive metabolome and transcriptome data analysis, supported by statistical analyses. However, the presentation would benefit from integrating figures or tables for improved visualization.

Response: Thanks for your careful reading and fair evaluation of our work! From previous studies on leaf color changes of Osmanthus fragrans, untargeted metabolomics analyses were performed such as in article “Changes of coloration and pigment composition during leaf development of Osmanthus fragrans color group cultivar”, in our research, targeted metabolomics approach with accurate identification and quantification of anthocyanins and carotenoids were carried for more precise integrated metabolome and transcriptome analyses.

Major Comments:

1. RNAseq sequencing quality check with PCA or MDS should be included in the supplementary.

Response: Thank you for reminding us on this issue! A figure of RNAseq sequencing quality check with PCA has been added in supplementary in Figure S1.

Figure S1. PCA of RNAseq sequencing quality check.

2. WCGNA hub gene selection is not elaborated correctly, the criteria for GS and MM should clearly mansion.

Response: Thanks for your comments on this point! The sentences of “We determined the feature modules of interest and calculated the feature module values (MM > 0.9) and gene significance value (GS > 0.5) to identify hub genes.” has been added in line 211-213.

3. Tables are very hard to fallow for reader try to make it easy to read

Response: Thank you for pointing out this issue! We agreed that the tables for metabolites can be difficult for our readers, some alternatives were tried, however, based on integrality of data, we believed the complete original tables are necessary.

Minor Comments:

4. The abstract should be more concise. Consider condensing the description of the leaf color changes and focusing on the key findings.

Response: Thanks for your kindly advise!

The sentence “In Osmanthus fragrans ‘Wucaigui’, which has a high ornamental value, the leaf color change is very rich.” has been replaced by “ Osmanthus fragrans is listed as one of the most important traditional ornamental plant species in China. A new cultivar O. fragrans ‘Wucaigui’ has a very diversified form in terms of leaf colors, which leaf color changes from red to yellow-green and finally to dark green.” in line 13-15.

The sentences “The positive regulation of OfUGE3 led to an increase in the lutein content, which resulted in the leaves changing from grayish brown to moderate brown; during the change from moderate brown to dark greenish yellow, the positive regulation of three genes (OfHOU4L, OfATL1B, and OfUGE5) increased the content of pelargonidin-3-O-(6-O-p-coumaroyl)-glucoside, and the red color of leaves gradually faded to dark greenish yellow and then to strong yellow green; the positive regulation of OfEIF1AX increased the content of neoxanthin; the stages in which the color changed from strong yellow green to yellow green and then to moderate olive green were positively regulated by OfUGE3, which resulted in higher α-carotene content.” have been added in line 28-36. 

The sentence “As the leaves changed from red to yellow-green and finally to dark green, we identified six stages with different leaf colors: grayish brown, moderate brown, dark greenish yellow, strong yellow green, yellow green, and moderate olive green.” has been deleted.

The sentence “This work seeks to contribute to the development of artificial regulate and control technology in breeding and production of O. fragrans and other ornamental plants.” has been added in line 38-40.

5. The identification of key metabolites and genesis pivotal. Could you elaborate on the criteria used to select these key components?

Response: Thanks for your inquiry! For criteria of metabolites, we performed OPLS-DA analysis by using SIMCA software, according to their VIP value from the result, a list of metabolites with high value were selected as key components. The gene expression levels were measured using the exon model per kilobase fragment per million mapping fragments to eliminate the influence of different gene lengths and sequencing differences on gene expression calculation. Transcripts with a fold change > 2 and false discovery rate (FDR) < 0.05 were considered to be differentially expressed during plant development.

6. Please clarify the statistical methods used for metabolite identification and analysis.

Response: The statistical methods for analyzing the metabolome data of Osmanthus fragrans ‘Wucaigui’ leaves at different developmental stages were analyzed using an orthogonal partial least squares-discriminant analysis (OPLS-DA) with SIMCA 14.1 software. (line 224-227)

7. Line 102:Clarify whether the metabolite and transcriptome analyses were conducted on the same leaf samples.

Response: Thanks for your inquiry! Our metabolite and transcriptome analyses were conducted based on same leaf samples from same part of plant materials.

8. Line 177:Please provide more details on the criteria used for determining significantly differentially expressed genes.

Response: Thanks for your contributed suggestion! The sentence “Transcripts with -2 and Q value ≤ 0.05, were considered to be significantly differentially expressed during plant development” has been replaced by “Transcripts with a fold change > 2 and false discovery rate (FDR) < 0.05 were considered to be differentially expressed during plant development.” in line 199-201.

9. Line 233+:Ensure consistency in reporting the number of novel genes across the manuscript.

Response: Thank you for reminding us on this issue! We have checked carefully through entire manuscript and the number of novel genes is consistent.

Author Response File: Author Response.pdf

Reviewer 3 Report

This study confirmed the combined metabolome and transcriptome analysis of the leaf color change process in O. fragrans. The authors then determined which trait modules had the highest correlation with key metabolites and selected relevant candidate genes. Overall, the article is well written and the data is nicely presented. I have a few small comments.

- It would be nice to add a bit more detail about the plant material. The author said that the leaves change from red to green. Please describes the time or date of sampling (S1~S6).

- What are the full names of the genes in result 3.5?

Author Response

Reviewer 3

This study confirmed the combined metabolome and transcriptome analysis of the leaf color change process in O. fragrans. The authors then determined which trait modules had the highest correlation with key metabolites and selected relevant candidate genes. Overall, the article is well written and the data is nicely presented. I have a few small comments.

Response: Thanks for your careful reading and fair evaluation of our work! We respect your comments and have dealt with each point seriously in the following responses.

1. It would be nice to add a bit more detail about the plant material. The author said that the leaves change from red to green. Please describes the time or date of sampling (S1~S6).

Response: Thank you for reminding us on this section! Our plant material Osmanthus fragrans ‘Wucaigui’is a mutant variety from Osmanthus fragrans var. thunbergii, among all Osmanthus fragrans we observed, ‘Wucaigui’has a unique characteristic which is long period of leaf color changes. The sentence “The sampling dates were 25th (S1) on March, 05th (S2), 15th (S3), 25th (S4) on April, and 05th (S5), 15th (S6) on May 2022.” has been added in line 115-116.

2. What are the full names of the genes in result 3.5

Response: Thanks for your inquiry! Full names of the genes in result 3.5 have been added to the supplementary Table S3.

 Table S3. Full names of hub genes.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Authors have addressed to the queries, concerns and suggestions. The manuscript is much improved now.

I have a couple of suggestions that can be taken care at the proofing stage.

1. Authors should incorporate the following change in the title

Osmanthus fragrans ‘Wucaigui’ be changed to Osmanthus fragrans cv. ‘Wucaigui’

2. Pl include the authority and family name in the first line of the abstract.

By and large it is fine.