Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript “Unveiling CcR2R3-MYB: A Key Regulator of Leaf Pigmentation in Cymbidium Orchids” by Yan et al. investigates the role of the CcR2R3-MYB gene in leaf pigmentation of orchids. Using a combination of molecular biology, genetic, and biochemical techniques, the authors identified and cloned the CcR2R3-MYB gene from a variegated leaf mutant of Cymbidium. The study found that this gene is highly expressed in the mutant and encodes a nuclear-localized protein. Overexpression of CcR2R3-MYB in Dendrobium officinale, another orchid, resulted in reduced chlorophyll, carotenoid, and flavonoid content, leading to changes in leaf color. These findings suggest that CcR2R3-MYB plays a crucial role in regulating leaf pigmentation and could be a potential target for molecular breeding to develop new Cymbidium varieties with desirable leaf colors.

 

While the methodology presented holds promise, aspects of it would benefit from additional refinement to enhance their robustness and rigor. Specific areas for improvement are outlined below:

 

Introduction issues:

 

Lines 28-39. This part would be enhanced by providing a more comprehensive overview of leaf pigmentation in plants. This could include elaborating on the significance of various pigments, such as chlorophyll, carotenoids, and flavonoids, as well as their associated metabolic pathways.

 

Line 85: Arabidopsis. Please, put the genus in italics.

 

Lines 88-91. The inclusion of this RNAseq data in this article, particularly differential expression data of the gene under investigation, would strengthen the rationale for conducting functional studies. Additionally, identifying other differentially expressed genes associated with the CcR2R3-MYB gene could contribute to a more comprehensive understanding of the mechanisms regulating leaf coloration. The authors should consider including these results.

 

Lines 92-101. The phrasing of some sentences, such as those containing "this study" and "CcR2R3-MYB genes," is repetitive and could be restructured to enhance the reading experience.

 

Methodology issues:

 

Lines 124-125: “DNA was extracted from the leaves of Orchidaceae”. Please specify which species or cultivars were used in the article.

 

Lines 128-130: “The cDNA was obtained based on total RNA reverse transcribed using UEIris II RT-PCR System for First-Strand cDNA Synthesis Kit”. Please describe which conditions were used. Additionally, the authors should provide details on the cDNA quantification.

 

Lines 138-139: “Multiple sequence alignment analysis was performed using DNAMAN 8.0 software”. Please indicate which algorithm or heuristic was used, as well as whether nucleotide or amino acid sequences were aligned.

 

Lines 139-140: “Phylogenetic evolutionary trees were constructed using MEGA7 software”. Please specify the bootstrap number and the method used. Also, indicate if the evolutionary model had been previously determined.

 

Line 153: “Validation of positive clones by sequencing”. This sentence is out of context in the paragraph. Please restructure and indicate what type of sequencing was used.

 

Line 154: “Agrobacterium”. Please, put the genus in italics.

 

Lines 170-171: “Dendrobium protoplasts with consistent growth and growing period were selected and dark cultured in the pre-culture medium for 3 d”. The study's biggest limitation is that the authors didn't explain why they performed the functional validation in Dendrobium protoplasts rather than Cymbidium.  Additionally, they didn't address whether there are homologous genes to CcR2R3-MYB in Dendrobium that also play a role in pigmentation. Although the results show that CcR2R3-MYB is important in pigmentation,  applying these conclusions directly to Cymbidium may not be appropriate.

 

Results issues:

 

Most of the main figures in the article are of very low quality. Please correct this quality.

 

Discussion issues:

 

The article's discussion section lacks a thorough analysis of the molecular mechanisms underlying CcR2R3-MYB's regulation of pigmentation and the potential involvement of other genes. It offers limited comparison with other studies and insufficiently discusses the limitations of extrapolating results from Dendrobium officinale to Cymbidium. Additionally, the discussion lacks a dedicated section addressing the overall limitations of the study.

 

The manuscript contains some errors in the grammar and structure of the paragraphs that need to be corrected by a native language specialist.

Author Response

Reviewer1

Dear reviewers, thank you for your careful review and constructive suggestions regarding our manuscript. We have revised the manuscript in accordance with the comments and marked all the amends on our revised manuscript.

Introduction issues:

Comments 1: Lines 28-39. This part would be enhanced by providing a more comprehensive overview of leaf pigmentation in plants. This could include elaborating on the significance of various pigments, such as chlorophyll, carotenoids, and flavonoids, as well as their associated metabolic pathways.

Response 1: Thank you for kind suggestion, and we already Supplement the relevant content. The revised and supplementary contents have been marked red in the revised manuscript. We are willing to make further revise if necessary.

Comments 2: Line 85: Arabidopsis. Please, put the genus in italics.

Response 2: Thanks for the reviewer’s suggestion. We have revised its format (Red mark).

Comments 3: Lines 88-91. The inclusion of this RNAseq data in this article, particularly differential expression data of the gene under investigation, would strengthen the rationale for conducting functional studies. Additionally, identifying other differentially expressed genes associated with the CcR2R3-MYB gene could contribute to a more comprehensive understanding of the mechanisms regulating leaf coloration. The authors should consider including these results.

Response 3: Thanks for the reviewer’s suggestion. We have included this additional data in supplementary figures and tables (Figure S3, Table S4). However, these results are part of another study, and an article containing these data has been submitted to another journal and cannot be directly supplemented in the results. Our detailed reply to address the reviewers’ concerns is as follows:
A) The white regions of the leaves from variegated orchid mutants were used as the treatment group, while the green regions served as the control group, with the experiment conducted in triplicate (Figure 1). Using Illumina sequencing technology, we performed transcriptomic sequencing and analyzed differentially expressed genes, obtaining a high-quality dataset of 121,779,814 reads, which were assembled into 56,013 unigenes. Among these, 27,742 non-redundant unigenes were annotated (49.53%). KEGG analysis was conducted on 9,722 genes to identify metabolic pathways. GO analysis indicated that the majority of genes were associated with biochemical processes, cellular composition, and molecular functions. Additionally, gene expression analysis revealed 9,966 differentially expressed genes, of which 3,866 were upregulated and 6,100 were downregulated. Metabolic pathway analysis was performed on 2,265 highly expressed differentially expressed genes, yielding 115 pathway metabolic pathways. Notably, we identified differentially expressed structural genes involved in chlorophyll, carotenoid, and flavonoid metabolic pathways in the green and white regions: HEMG, CHLH, CHI, CHS, F3'H, F3H, FLS, C4H, F3'5'H, 3GT, DFR, ANR, LCY, and CYP, as well as regulatory genes: MYB, R2R3-MYB, bHLH and WD40. A total of 167 differentially expressed MYB genes were identified, with 55 upregulated and 112 downregulated. Among these, 15 were named R2R3-MYB (5 upregulated and 10 downregulated) (Supplementary Table S4). These data represent a valuable resource for studying the metabolic pathways and various biological mechanisms underlying the variegated traits in orchids.

 

   

Figure 1 Experimental material

1. B) Expression and Candidate Gene Screening of MYB Genes in Different Color Regions of Variegated Orchid Leaves. Using mutant and control (CK) plants as materials (Figure 1), we conducted transcriptome sequencing of the white, yellow, and green regions of the leaves, focusing on highly differentially expressed MYB genes and their EST sequences. Combining literature review and sequence alignment analysis, we used the 18S gene from Cymbidium (a genus of orchids) as an internal reference for RT-qPCR to detect the expression of 30 MYB unigenes across the three color regions. The results showed that all 30 MYB unigenes exhibited differential expression in the white and yellow regions of the leaf spots. Specifically, 5 MYB unigenes were upregulated in both regions; 7 MYB unigenes were downregulated in both regions; 6 MYB unigenes were downregulated in the yellow region but upregulated in the white region; and 12 MYB unigenes were upregulated in the yellow region but downregulated in the white region. Collectively, these findings indicate that MYB transcription factors differentially regulate the formation of variegated leaf spots in orchids. Furthermore, based on the differential expression of MYB genes, combined with literature and homologous sequence comparisons, we selected 8 R2R3-MYB transcription factors (Supplementary Figure S3), ultimately identifying Unigene0024556 (CcR2R3-MYB) as a candidate gene.

 

Comments 4: Lines 92-101. The phrasing of some sentences, such as those containing "this study" and "CcR2R3-MYB genes," is repetitive and could be restructured to enhance the reading experience.

Response 4: We have re-worked the first paragraph as suggested (Red mark).

Methodology issues:

Comments 5: Lines 124-125: “DNA was extracted from the leaves of Orchidaceae”. Please specify which species or cultivars were used in the article.

Response 5: Thanks for this question. We have done the revision in the manuscript (Red mark).

Comments 6: Lines 128-130: “The cDNA was obtained based on total RNA reverse transcribed using UEIris II RT-PCR System for First-Strand cDNA Synthesis Kit”. Please describe which conditions were used. Additionally, the authors should provide details on the cDNA quantification.

Response 6: Thanks for this question. We have done the revision in the manuscript (Red mark).

Comments 7: Lines 138-139: “Multiple sequence alignment analysis was performed using DNAMAN 8.0 software”. Please indicate which algorithm or heuristic was used, as well as whether nucleotide or amino acid sequences were aligned.

Response 7: Thanks for this question. We have done the revision in the manuscript (Red mark).

Comments 8: Lines 139-140: “Phylogenetic evolutionary trees were constructed using MEGA7 software”. Please specify the bootstrap number and the method used. Also, indicate if the evolutionary model had been previously determined.

Response 8: Thanks for this question. We have done the revision in the manuscript (Red mark)

Comments 9: Line 153: “Validation of positive clones by sequencing”. This sentence is out of context in the paragraph. Please restructure and indicate what type of sequencing was used.

Response 9: Thank you for bringing this issue to the fore. We have moved this to the methods 2.4 section.

Comments 10: Line 154: “Agrobacterium”. Please, put the genus in italics.

Response 10: Thanks for this question. We have done the revision in the manuscript (Red mark).

Comments 11: Lines 170-171: “Dendrobium protoplasts with consistent growth and growing period were selected and dark cultured in the pre-culture medium for 3 d”. The study's biggest limitation is that the authors didn't explain why they performed the functional validation in Dendrobium protoplasts rather than Cymbidium. Additionally, they didn't address whether there are homologous genes to CcR2R3-MYB in Dendrobium that also play a role in pigmentation. Although the results show that CcR2R3-MYB is important in pigmentation,  applying these conclusions directly to Cymbidium may not be appropriate.

Response 11: Thanks for this question. Our detailed reply to address the reviewers’ concerns is as follows: Orchids (family Orchidaceae) are monocotyledonous plants, generally challenging for inducing callus tissue or somatic embryos from leaves. Current reports on genetic transformation in orchids primarily focus on species such as Phalaenopsis, Dendrobium, and Oncidium Gower Ramsey. Genetic transformation in the genus Cymbidium is rarely reported, with only a few instances for Cymbidium goeringii (Rchb.f.) Rchb.F × Cymbidium hybrid (Xu, 2011) and Cymbidium sinense (Xie et al., 2015), but with extremely low transformation efficiencies and no subsequent reports on transgenic plants. Our research team has established a genetic transformation system for Dendrobium officinale and has obtained multiple transgenic plants. Therefore, this study focuses on the functional analysis of the CcR2R3-MYB gene using Dendrobium officinale as the recipient plant.

The homology of CcR2R3-MYB is as follows: DhMYB1 from Dendrobium hybrid (84.66%, JX846911.1), Dsp.MYB1 from Dendrobium sp (83.84%, AF485892.1), Pemyb-related from Phalaenopsis amabilis (81.74%), PeMYB16 from Phalaenopsis equestris (70.74%, XM_020726637.1, KF769481.1), Cemyb-related from Cymbidium ensifolium (72.75%, MK282456.1), DcMYBML1 from Dendrobium crumenatum (82.74%, GQ981643.1), AtMYB106 from Arabidopsis thaliana (52.89%, AT3G01140.1), and TaMYB16 (62.46%) and TaMYB77 (54.46%) from Triticum aestivum (JF951918.1, KU674900.1). The highest similarity is with DhMYB1 from Dendrobium hybrid. Among closely related species, the recipient plant used in this study, Dendrobium officinale, is not included. This implies that if R2R3 subfamily genes are present in Dendrobium officinale, they are distantly related to CcR2R3-MYB. Moreover, the leaf color of transgenic Dendrobium officinale plants is significantly different from that of wild-type Dendrobium officinale plants. This study focuses on the functional analysis of the CcR2R3-MYB gene. Additionally, there are currently no reports on the function of R2R3-MYB genes in wild-type Dendrobium officinale.

 

Results issues:

Comments 12: Most of the main figures in the article are of very low quality. Please correct this quality.

Response 12: Thank you for bringing this question up. We have remade the figure as suggested.

 

Discussion issues:

Comments 13: The article's discussion section lacks a thorough analysis of the molecular mechanisms underlying CcR2R3-MYB's regulation of pigmentation and the potential involvement of other genes. It offers limited comparison with other studies and insufficiently discusses the limitations of extrapolating results from Dendrobium officinale to Cymbidium. Additionally, the discussion lacks a dedicated section addressing the overall limitations of the study.
Response 13: We thank the reviewer for the helpful suggestion. The Discussion section has been modified in the revised manuscript (Red mark).
Comments 14: The manuscript contains some errors in the grammar and structure of the paragraphs that need to be corrected by a native language specialist.

Response 14: We have asked native English speakers to help us with the revision. If it still fails to meet the reviewer's requirements, please do not hesitate to let us know that we are willing to continue to revise our manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript contributes significantly to understanding the role of CcR2R3-MYB in leaf variegation in Cymbidium orchids, a critical trait in ornamental plant breeding. The findings are valuable for advancing molecular breeding strategies in ornamental horticulture.

However, it could benefit from the following enhancements:

 

1/ Line 17-18: Phylogenetic analysis is described well, but the significance of its relationship with AtMYB106 and DhMYB1 could be expanded. For example, briefly explain why this connection is biologically or functionally relevant.

 

2/ Line 131-133: While the sequence of primers is provided, the rationale for selecting specific primer regions is missing.

 

3/ Line 135-136: Using the ExPASy server for molecular weight and pI predictions is a standard approach. However, consider mentioning why these parameters were essential for the study.

 

4/ Line 244-250: The phylogenetic analysis in Figure 4A provides compelling results. However, the bootstrap values' confidence could be discussed further to address the robustness of evolutionary relationships.

 

5/ Line 281-290: Subcellular localization is well-explained. A comparative analysis of localization probabilities (nucleus vs. mitochondria) with other MYB proteins could provide deeper insights.

 

6/ Line 370-374: The statement about MYB transcription factors not dealing with leaf coloration is inaccurate, as some studies may have touched on this indirectly. Please revise: "Despite extensive studies on MYB transcription factors, their specific role in leaf coloration remains underexplored."

 

7/ Line 381-385: The hypothesis regarding the regulation of cell morphogenesis and leaf color is interesting but speculative. Please strengthen this hypothesis by referencing similar findings from other studies or providing additional data from this study to support it.

 

 

8 / Please address grammatical inconsistencies and improve phrasing where clarity is lacking.

Author Response

Reviewer2
Dear reviewers, thank you for your careful review and constructive suggestions regarding our manuscript. We have revised the manuscript in accordance with the comments and marked all the amends on our revised manuscript.

Comments 1: 1/ Line 17-18: Phylogenetic analysis is described well, but the significance of its relationship with AtMYB106 and DhMYB1 could be expanded. For example, briefly explain why this connection is biologically or functionally relevant.

Response 1: Thank you very much for your suggestions and recommendations. We already revised it in the revised manuscript (Red mark).

 

Comments 2: 2/ Line 131-133: While the sequence of primers is provided, the rationale for selecting specific primer regions is missing.

Response 2: Thank you for your valuable comments on our manuscript. We appreciate your suggestion regarding the rationale for selecting specific primer regions. We chose primer regions based on highly conserved regions within the target gene sequences. This approach ensures that the primers will specifically bind to the intended target, minimizing the risk of non-specific amplification. For instance, in the case of the CcR2R3-MYB gene, we identified conserved domains through multiple sequence alignments with closely related genes such as AtMYB106 and DhMYB1. This alignment process helped us to select primer regions that are both conserved and specific to the CcR2R3-MYB gene, thereby enhancing the reliability of our PCR results

 

Comments 3: 3/ Line 135-136: Using the ExPASy server for molecular weight and pI predictions is a standard approach. However, consider mentioning why these parameters were essential for the study.

Response 3: Thank you for your suggestion and advice. In this paper, we supplemented and improved the above part of surgery (Red mark).

 

Comments 4: 4/ Line 244-250: The phylogenetic analysis in Figure 4A provides compelling results. However, the bootstrap values' confidence could be discussed further to address the robustness of evolutionary relationships.

Response 4: We appreciate the reviewer's suggestion for further discussion on the confidence of bootstrap values and the robustness of evolutionary relationships. In our study, we have already employed the standard Bootstrap method to assess the support for the branches of the phylogenetic tree. However, we understand the reviewer's concerns. We generated 1000 bootstrap replicate trees using the NJ, Bayesian and ML methods and calculated the bootstrap support values for each branch. The results showed that both the NJ, Bayesian and ML trees exhibited high topological consistency.

 

Comments 5: 5/ Line 281-290: Subcellular localization is well-explained. A comparative analysis of localization probabilities (nucleus vs. mitochondria) with other MYB proteins could provide deeper insights.

Response 5: We appreciate the reviewer's affirmation of the subcellular localization section and agree with the suggestion that a comparative analysis of localization probabilities with other MYB proteins can provide deeper insights. Our study shows that CcR2R3-MYB is primarily localized in the nucleus, which is consistent with its function in regulating gene expression. The nucleus is the main site for gene expression regulation, and the localization of CcR2R3-MYB in the nucleus suggests that it may be involved in transcriptional regulation. CcR2R3-MYB shares similar subcellular localization with other MYB proteins (such as DhMYB1, AmMYBML1, and AtMYB106), mainly in the nucleus. This indicates that these MYB proteins influence plant morphogenesis and metabolic processes by regulating gene expression in the nucleus. Although some MYB proteins may function in other subcellular organelles, our study did not find significant localization of CcR2R3-MYB in these organelles. By using various online prediction tools, we further verified the subcellular localization of these MYB proteins, providing deeper insights into their functions. We had also commented and supplemented this part in the discussion section (Red mark).

 

Comments 6: 6/ Line 370-374: The statement about MYB transcription factors not dealing with leaf coloration is inaccurate, as some studies may have touched on this indirectly. Please revise: "Despite extensive studies on MYB transcription factors, their specific role in leaf coloration remains underexplored."

Response 6: We thank reviewer for the suggestion and we agree with the suggested changes (Red mark).

 

Comments 7: 7/ Line 381-385: The hypothesis regarding the regulation of cell morphogenesis and leaf color is interesting but speculative. Please strengthen this hypothesis by referencing similar findings from other studies or providing additional data from this study to support it.

Response 7: We thank the reviewer and agree, the sentence has been changed as suggested (Red mark).

 

Comments 8: 8 / Please address grammatical inconsistencies and improve phrasing where clarity is lacking.

Response 8: We have asked native English speakers to help us with the revision (Red mark).

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

I have carefully read the article. I believe that the data and results presented in this article hold significance in breeding strategies of ornamental plants. The authors successfully isolated and characterized a R2R3-MYB transcription factor, from a mutant variant of Cymbidium and their findings highlight the association between the CcR2R3-MYB gene and leaf color variation in D. officinale. In my opinion, the manuscript solves an interesting issue, but in the current version there are several problematic things that need to be revised.

- R2R3-MYB is a gene family. There is usually more than one copy of the MYB gene in a plant. It is also known that two historical duplication events in the orchid genome. Did the authors test for copies of the MYB gene in Cymbidium Orchids?

- Since the authors used non-directed mutagenesis to obtain mutant plants, it is likely that the resulting mutants were not only for the CcR2R3-MYB gene, but also in other genes associated with chlorophyll and flavonoid biosynthesis. Transcriptome analysis should be performed to reveal the full picture. These considerations can be added to the discussion chapter.

Comments for authors:

-44: form helix-helix-helix (HLH) - correct to helix-loop-helix (HLH)

- Introduction

It would be good to add information about the genome of the object being studied.

 

2. Materials and Methods. 2.2. Cloning and Sequence Analysis of CcR2R3-MYB

- The question remains: is this the only copy of the gene?

 

Figure 3.

- Three-stage structure -  better: predicted three-stage structure

Figure 4.

PeMYB16: Phalaenopsis equestris; Pemyb-related: Phalaenopsis equestris; Are these different objects/ gene? Why were these plants, including monocots, chosen for the dendrogram?

 

Discussion

- 406 «Our study results demonstrated a significant decrease in flavonoid content in CcR2R3-MYB transgenic plants compared to wild-type plants. This finding suggests an active involvement of CcR2R3-MYB in the regulation of gene expression related to flavonoid metabolism». This fact is well known, here it is better to say here that the study confirms the involvement of the MYB gene in regulating the expression of genes associated with flavonoid biosynthesis. Add references to publications on this issue.

- 410 «Specifically, the subgroup 9 gene CcR2R3-MYB acts as a negative regulator of the flavonoid pathway» What could this be related to?

- 421 «Furthermore, the analysis of total carotenoid content revealed a significant decrease in carotenoid levels in transgenic plants compared to wild-type plants, suggesting the potential involvement of this gene in carotenoid metabolism». Perhaps this is caused by dysfunction of other genes in mutants?

Author Response

Reviewer 3
Dear reviewers, thank you for your careful review and constructive suggestions regarding our manuscript. We have revised the manuscript in accordance with the comments and marked all the amends on our revised manuscript.

Comments 1: R2R3-MYB is a gene family. There is usually more than one copy of the MYB gene in a plant. It is also known that two historical duplication events in the orchid genome. Did the authors test for copies of the MYB gene in Cymbidium Orchids?

Response 1: Thank you very much for your attention to our research and the valuable questions you raised. Your reminder about the R2R3-MYB gene family usually having multiple copies in plants, as well as the two historical duplication events in the orchid genome, is of great significance for us to further improve our research. We screened T3 transgenic plants through a 3:1 segregation ratio, which usually indicates that the transgene is likely to be a single-copy insertion. We will select plants from the T3 generation as materials for subsequent experiments to ensure the stable inheritance and expression of the transgene in the offspring.

Comments 2: Since the authors used non-directed mutagenesis to obtain mutant plants, it is likely that the resulting mutants were not only for the CcR2R3-MYB gene, but also in other genes associated with chlorophyll and flavonoid biosynthesis. Transcriptome analysis should be performed to reveal the full picture. These considerations can be added to the discussion chapter.

Response 2: Thank you very much for your careful review and valuable suggestions. You pointed out that since we used non-directed mutagenesis to obtain mutant plants, it is likely that the resulting mutants were not only for the CcR2R3-MYB gene, but also in other genes associated with chlorophyll and flavonoid biosynthesis. This is a very insightful comment. We fully agree with you and believe that transcriptome analysis is crucial for revealing the full picture of the mutants.

In our current study, although we have not yet performed transcriptome analysis on these mutants, there are related studies showing that the MYB gene family plays an important role in regulating chlorophyll and flavonoid biosynthesis. For example, in tomatoes, SlMYB72 has been found to regulate the metabolism of chlorophylls, carotenoids, and flavonoids. Additionally, in tea plants, GOLDEN 2-LIKE transcription factors CsGLK1 and CsGLK2 can coordinate UV-B-induced chlorophyll biosynthesis and flavonoid accumulation. These research findings indicate that members of the MYB gene family may affect plant pigment metabolism through complex regulatory networks.

To better understand the possible mutations in other genes in our mutants and how these mutations affect chlorophyll and flavonoid biosynthesis, we plan to conduct transcriptome analysis in our future research. This will help us reveal other genes that may have been affected besides the CcR2R3-MYB gene, thereby gaining a more comprehensive understanding of the physiological and molecular mechanisms of the mutants.

We will add these considerations to the discussion section to emphasize the potential multi-gene effects of non-directed mutagenesis and to illustrate the necessity of conducting transcriptome analysis (Red mark). Thank you again for your suggestions, which will greatly promote the in-depth development and improvement of our research.

 

Comments for authors:

Comments 3: -44: form helix-helix-helix (HLH) - correct to helix-loop-helix (HLH)

Response 3: Thank you very much for your suggestions and recommendations. We already revised it in the revised manuscript (Red mark).

 

- Introduction

Comments 4: It would be good to add information about the genome of the object being studied.

Response 4: We are extremely grateful for your review of our study and the valuable suggestion to include information about the genome of the subject under study. We fully agree with your perspective and have made the corresponding additions in the manuscript (Red mark).

 

Comments 5: Materials and Methods. 2.2. Cloning and Sequence Analysis of CcR2R3-MYB

- The question remains: is this the only copy of the gene?

Response 5: We will select plants from the T3 generation as materials for subsequent experiments to ensure the stable inheritance and expression of the transgene in the offspring.

 

Comments 6: Figure 3.

- Three-stage structure -  better: predicted three-stage structure

Response 6: We thank the reviewer to pointing it out and have made the amendments as suggested (Red mark).

 

Comments 7: Figure 4.

PeMYB16: Phalaenopsis equestris; Pemyb-related: Phalaenopsis equestris; Are these different objects/ gene? Why were these plants, including monocots, chosen for the dendrogram?

Response 7: Thank you for your valuable comments. PeMYB16 and Pemyb-related are both genes found in Phalaenopsis equestris. PeMYB16 is a well-known R2R3-MYB transcription factor involved in anthocyanin biosynthesis, while Pemyb-related refers to homologous genes that are functionally or structurally similar to PeMYB16. We chose to include monocots and other plants in the phylogenetic tree analysis to compare the CcR2R3-MYB gene from Cymbidium with known homologs in monocots, revealing the conservation and diversity of these genes in evolution, as well as their functional similarities in processes such as pigment synthesis, cell morphogenesis, and stress responses. This helps to infer the potential functions of the CcR2R3-MYB gene in Cymbidium, providing a theoretical basis for subsequent experimental validation. We have supplemented the manuscript with detailed information on gene function annotations and phylogenetic tree analysis to enhance the scientific rigor and completeness of our study.

 

Discussion

Comments 8: - 406 «Our study results demonstrated a significant decrease in flavonoid content in CcR2R3-MYB transgenic plants compared to wild-type plants. This finding suggests an active involvement of CcR2R3-MYB in the regulation of gene expression related to flavonoid metabolism». This fact is well known, here it is better to say here that the study confirms the involvement of the MYB gene in regulating the expression of genes associated with flavonoid biosynthesis. Add references to publications on this issue.

Response 8: We thank the reviewer for their suggestion, and have amended the manuscript as such (Red mark).

 

Comments 9: - 410 «Specifically, the subgroup 9 gene CcR2R3-MYB acts as a negative regulator of the flavonoid pathway» What could this be related to?

Response 9: Thank the reviewer for pointing it out. The subgroup 9 R2R3-MYB genes are conserved across different plant species, suggesting that they have evolved to play similar roles in regulating secondary metabolism. The negative regulation of the flavonoid pathway by CcR2R3-MYB in Cymbidium is consistent with the functions of homologous genes in other plants, such as AtMYB106 in Arabidopsis, which is involved in the negative control of flowering time and may also regulate secondary metabolism.

 

Comments 10: - 421 «Furthermore, the analysis of total carotenoid content revealed a significant decrease in carotenoid levels in transgenic plants compared to wild-type plants, suggesting the potential involvement of this gene in carotenoid metabolism». Perhaps this is caused by dysfunction of other genes in mutants?

Response 10: Thank you for your valuable comments. Our study found a significant decrease in carotenoid levels in transgenic plants overexpressing CcR2R3-MYB, suggesting its potential involvement in carotenoid metabolism. While our focus has been on CcR2R3-MYB, we acknowledge that non-targeted mutagenesis could lead to dysfunctions in other genes, affecting carotenoid levels. To further investigate, we plan to conduct transcriptome analysis and qRT-PCR validation to identify and verify the expression changes of genes related to carotenoid metabolism, and to study their potential interactions with CcR2R3-MYB. This will help provide a more comprehensive understanding of the regulatory mechanisms underlying carotenoid metabolism.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I appreciate the authors' response. Upon thorough examination of the manuscript, it is evident that certain aspects have improved in order to meet the criteria for acceptance.

The authors should justify their use of Dendrobium protoplasts in the methodology or discussion section before the manuscript can be accepted for publication.

Author Response

Reviewer 1
Dear reviewers, thank you for your careful review and constructive suggestions regarding our manuscript. We have revised the manuscript in accordance with the comments and marked all the amends on our revised manuscript.

Comments 1: The authors should justify their use of Dendrobium protoplasts in the methodology or discussion section before the manuscript can be accepted for publication.

Response 1: Thank you for your insightful comments regarding the use of Dendrobium protoplasts in our study. We appreciate your concerns and would like to provide a detailed justification for our methodological choice. Rationale for Using Dendrobium Protoplasts: Our study aims to elucidate the role of the CcR2R3-MYB transcription factor in leaf pigmentation. High Transformation Efficiency: Protoplasts, lacking rigid cell walls, offer a highly efficient platform for genetic transformation. This feature allows for rapid and direct evaluation of gene function, which is crucial for our investigation of CcR2R3-MYB (Wu et al., 2020), Cell-Level Analysis: Protoplasts enable the study of gene function at the cellular level, minimizing the complexity associated with tissue- and organ-level analyses. This approach is particularly suitable for investigating the direct effects of CcR2R3-MYB on chlorophyll and pigment metabolism (Chen et al., 2021). Precedents in the Literature: The use of protoplasts for gene transformation and functional analysis is well-established in plant research. For example, Wu et al. (2020) successfully employed Dendrobium protoplasts to study the role of key genes in flower pigmentation. Similarly, Chen et al. (2021) utilized protoplasts to investigate chlorophyll metabolism, highlighting the robustness and reliability of this system for functional genomics. Effectiveness of Our Approach: Our results demonstrate that overexpression of CcR2R3-MYB in Dendrobium protoplasts led to significant reductions in chlorophyll content and altered leaf coloration (Figure 7). These findings are consistent with our observations in transgenic Dendrobium plants, thereby validating the use of protoplasts as a reliable model system. The observed phenotypic changes provide compelling evidence of CcR2R3-MYB's regulatory role in leaf pigmentation. In light of these points, we believe that the use of Dendrobium protoplasts was both rational and essential for our study.

We will incorporate a detailed explanation of our rationale into the "Methods" sections of our manuscript to provide further clarity (Red mark).

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Based on the authors' detailed responses and thorough revisions, it is evident that they have addressed nearly all the comments. The manuscript has been significantly improved, demonstrating the authors' substantial effort in refining their work, integrating the necessary corrections, and aligning their study with the suggestions.

 

 

Given the comprehensive nature of these revisions and the significant enhancements made, I consider the manuscript well-prepared and suitable for publication in its current form.

Author Response

Reviewer2
Dear reviewers,

Thank you very much for your positive feedback and suggestions. We are delighted to hear that our manuscript has been accepted for publication.

Thank you again for your time and support.

Author Response File: Author Response.pdf