Genome-Wide Identification, Characterization, and Expression Pattern of MYB Gene Family in Melastoma candidum

Round 1

Reviewer 1 Report

In the article entitled “Genome-wide identification, characterization, and expression pattern of MYB gene family in Melastoma candidum” the authors evaluate the MYB gene family through different strategies. Here I provided suggestions to improve the manuscript's final version.

The manuscript needs extensive revision for language and grammar.

Line 36: “and is characterized by approximately 50 to 53 amino acids at N-terminus.” It is characterized by a conserved MYB domain of ~52 amino acids located at the N-terminus.

Lines 45-46: since it is a requirement that transcription factors bind specific DNA sequences, please consider further explaining the idea in those lines. How specificity could be divided?

Lines 49-50: the authors expose that R2R3-MYB repeats are pivotal parts of the DNA binding process of both R2R3-MYB and R1R2R3-MYB genes. Please comment on the interaction of R1-MYB proteins in the DNA since they lack the R2R3-MYB repeat.

Lines 64-65: “R2R3-MYB is the largest because of their rapid expansion.” Dubos and colleagues suggest, considering Rosinski & Atchley's results, that the loss of the sequences encoding the R1 repeats from an R1R2R3-MYB gene ancestor may be responsible for the high number of R2R3- MYB proteins.

Lines 79-80: “In contrast to the R2R3-MYB genes, only a few MYB-related genes have been functionally studied.” R2R3-MYB genes are MYB-related genes. Please rewrite to clarify the sentence.

Lines 88-89: The genome was published in Jan 2023 (10.3389/fpls.2023.1126319). The data here analysis accessed in 2022. The genome had not been explored yet, but yes, there were, previously, genome resources to evaluate MYB genes.

Line 192: The authors used the α-tubulin gene as an internal control for normalization. It has been widely suggested that using two or more reference genes for RT-qPCR studies generates more reliable results. Please justify the use of one reference gene.

Line 198: The authors indicate the analysis of the distribution of MYB genes, but in line 203 they assume to present the conserved protein sequences. Was the analysis conducted with nucleotides and the amino acid sequences of MYB coding genes?

Lines 217-220: Please include evidence to support the direct association between structure similarity and biological function (without functional/in vivo experiments).

Lines 222-223: Please include evidence for the assumption that evolutionary relations affect biological functions.

Line 241: Please include evidence for the assumption that the protein sequence impact biological functions. Do the conserved structures differentially affect plants' phenotype, for example?

Lines 266-267: Rectangles with different colors represent different motifs, domains, UTRs, and exons, respectively. Respectively to which color? Please correlate the evaluated sequences and colors.

Lines 283-284: The authors analyzed the promoter region of MYB genes to identify their chief cis-acting elements. The identification of hormone metabolism-related motifs in MYBs promoter regions indicates, thus, that MYBs are being regulated by hormone metabolism-related genes, and not the contrary. This way, the resultant data do not allow us to determine if MYB genes play essential roles in the plant hormone metabolism… The authors must search for MYBs motif in the promoter regions of hormone metabolism-related genes to do so. Then would be possible to see if those TF are potentially regulating the hormone metabolism-associated genes.

Line 317: Please present the inconsistency criteria used to eliminate MYB sequences of the analysis.

There is much discussion in the Results section. And many results in the Discussion section. Please revise the text.

Line 361: The authors obtained 363 gene pairs in all identified MYBs. Figure 7 indicates 303 GO results. What about the remaining 60 genes? GO terms were not identified?

Lines 371-372: Did the results correlate MYB coding genes with other GO terms different from transcription regulatory activity? These findings must be discussed.

Line 458: It is not possible to make the correlation between gene ID in Table S7 and the evaluated genes in Figure 10. Were genes evaluated by qRT-PCR obtained from the results in Table S7? Please consider including the annotation of the DEGs in Table S7.

Lines 465-467: the discussion of RNA-seq results affirms that “saddlebrown module is crucial in flower development as it contains more genes related to flower development than other modules” and that enrichment of carotenoid biosynthesis genes indicates their potential roles in carotenoid metabolism. Nevertheless, the identification of differentially expressed genes may not directly correlate with protein production and alterations in phenotype. It is possible to see the upmodulation and downmodulation of genes in RNA-seq data, but we can not assume those traits are reflected in the M. candidum phenotype. Please adjust the text, restricting the analysis to the transcriptional pattern. Please indicate if these genes described as abundant are up or down-regulated.

Line 497: “Detect the expression pattern of these 20 selected MYB genes.” Please inform which 20 genes.

Lines 534-537: caution is necessary when suggesting that the transcription process may be revealed by observing an elevated number of gene sequences in a genome. Those genes may or may not be expressed.

The discussion must be improved. The key point must not be restricted to comparing the number of MYBs identified among papers but to exploring what this number of genes may possibly implicate in the plant. In RNA-seq results, how many are up-modulated? How many are down-modulated? Those same genes (up- or down-modulated) remained modulated in the other samples evaluated by qRT-PCR? Was the phylogenetic distance among A. thaliana, M. candidum, and P. trichocarpa evident in the collinearity analyses? From lines 595 to 605, e.g., the discussion only explores literature. Please discuss more deeply the results integrating the obtained data.

Lines 611-612: “MYB family members participated in various developmental processes or stages of M. candidum.” Overall, the gene expression pattern appears to be not statistically significant. For example, for McMYB17a, the error bar makes it seem that there was no statistical difference among the samples. The authors consider that McMYB17a is more expressed in early-stage flowers. It is considered to be more expressed compared to which condition? Please revise the statistical significance in Figure 10.

Lines 622-623: Authors conclude that MYB genes possess huge potential for improving the application of M. candidum in the garden and pharmaceutical industry. Please explain how it will be applied. The gene family is highly diverse. The deletion of one of the genes may be supplanted by the activity of others. The effective biological function of each gene in M. candidum was not deciphered. This way,  in which context do the authors consider this gene family potential?

Please write the genus of A. thaliana, M. candidum, and P. trichocarpa in their first appearance in the main text.

Author Response

In the article entitled “Genome-wide identification, characterization, and expression pattern of MYB gene family in Melastoma candidum” the authors evaluate the MYB gene family through different strategies. Here I provided suggestions to improve the manuscript's final version.

Reply: Thank you very much for providing us with a lot of valuable and useful advice to improve the quality of our manuscript. Point-to-point responses were listed below.

Q1 The manuscript needs extensive revision for language and grammar.

Reply: We have thoroughly reviewed all the writing in this manuscript.

Q2 Line 36: “and is characterized by approximately 50 to 53 amino acids at N-terminus.” It is characterized by a conserved MYB domain of ~52 amino acids located at the N-terminus.

Reply: Corrected

Q3 Lines 45-46: since it is a requirement that transcription factors bind specific DNA sequences, please consider further explaining the idea in those lines. How specificity could be divided?

Reply: We have included additional reviews about DNA-binding specificities. (Lines 58-65).

Q4 Lines 49-50: the authors expose that R2R3-MYB repeats are pivotal parts of the DNA binding process of both R2R3-MYB and R1R2R3-MYB genes. Please comment on the interaction of R1-MYB proteins in the DNA since they lack the R2R3-MYB repeat.

Reply: We have added information about the role of R1 repeats in the MYB-DNA binding process. (Line 66-68)

Q5 Lines 64-65: “R2R3-MYB is the largest because of their rapid expansion.” Dubos and colleagues suggest, considering Rosinski & Atchley's results, that the loss of the sequences encoding the R1 repeats from an R1R2R3-MYB gene ancestor may be responsible for the high number of R2R3- MYB proteins.

Reply: After carefully reviewing the publications, we discovered that this is also a reason for the high numbers of R2R3 MYBs. Therefore, we have added this information to the text. (Lines 76-80)

Q6 Lines 79-80: “In contrast to the R2R3-MYB genes, only a few MYB-related genes have been functionally studied.” R2R3-MYB genes are MYB-related genes. Please rewrite to clarify the sentence.

Reply: MYB-related is a relative concept and different publications categorize it differently, causing confusion. Therefore, we removed this concept from the entire paper to avoid confusion.

Q7 Lines 88-89: The genome was published in Jan 2023 (10.3389/fpls.2023.1126319). The data here analysis accessed in 2022. The genome had not been explored yet, but yes, there were, previously, genome resources to evaluate MYB genes.

Reply: The genome was provided by Professor Zhou at Yat University, who uploaded it to the internet before its official publication.

Q8 Line 192: The authors used the α-tubulin gene as an internal control for normalization. It has been widely suggested that using two or more reference genes for RT-qPCR studies generates more reliable results. Please justify the use of one reference gene.

Reply: Using two or more internal reference genes is a rigorous approach. However, our research species, M. candidum, is relatively understudied, there are limited reference options available to us. Prior to conducting RT-qPCR, we synthesized the Actin7, EF1A, 18S, GAPDH, βTUBB4, and UBQ10 of M. candidum, and conducted a semi-quantitative test to determine the most suitable internal reference gene. Through this process, we found that α-tubulin provided the best results. Additionally, many studies currently only use one internal reference gene, and not every species has multiple internal reference genes available, so we believe it is reasonable to use α-tubulin as the sole reference gene in this research.

Q9 Line 198: The authors indicate the analysis of the distribution of MYB genes, but in line 203 they assume to present the conserved protein sequences. Was the analysis conducted with nucleotides and the amino acid sequences of MYB coding genes?

Reply: Distribution of MYB genes was used to show the distribution of MYB genes on the chromosomes, it was produced by the gtf file of the genome, not referring to the sequences. Figure 1 showed the evolutionary relations among different MYB members, here we used the coding protein sequences in. However, we found that some MYBs were not clustered well when we used the full-length coding protein sequence, thus, we used the conserved protein sequences in Figure S1 to perform evolutionary relations again (New Line 223).

Q10 Lines 217-220: Please include evidence to support the direct association between structure similarity and biological function (without functional/in vivo experiments).

Reply: Yes. Because this research didn’t involve any direct functional experiments, and instead, we inferred our results based on previous references as cited before this sentence. (Line 234-235).

Q11 Lines 222-223: Please include evidence for the assumption that evolutionary relations affect biological functions.

Reply: Same to Q9, both two sentences were inferred from the publication cited in Lines 234-235.

Q12 Line 241: Please include evidence for the assumption that the protein sequence impact biological functions. Do the conserved structures differentially affect plants' phenotype, for example?

Reply: Here, our objective is to illustrate the characteristics of the R2R3 domain without delving into its structure or biological functions.

Q13 Lines 266-267: Rectangles with different colors represent different motifs, domains, UTRs, and exons, respectively. Respectively to which color? Please correlate the evaluated sequences and colors.

Reply: Actually, there are also some legends in the corresponding position motif analysis, domains analysis, and gene structure, because there are so many colors, so we deleted this sentence. (Lines 284-285)

Q14 Lines 283-284: The authors analyzed the promoter region of MYB genes to identify their chief cis-acting elements. The identification of hormone metabolism-related motifs in MYBs promoter regions indicates, thus, that MYBs are being regulated by hormone metabolism-related genes, and not the contrary. This way, the resultant data do not allow us to determine if MYB genes play essential roles in the plant hormone metabolism… The authors must search for MYBs motif in the promoter regions of hormone metabolism-related genes to do so. Then would be possible to see if those TF are potentially regulating the hormone metabolism-associated genes.

Reply: That's a great question. As you mentioned, the cis-acting elements are in the promoter region of MYBs. Therefore, it can be inferred that MYBs are the regulated objects, and are likely regulated by upstream hormone or hormone metabolism-related genes. We have rewritten this sentence to improve clarity. (Lines 302-304)

Q15 Line 317: Please present the inconsistency criteria used to eliminate MYB sequences of the analysis.

Reply: The criteria used were stated in Lines 260-263, after domain analysis, we found each type of MYBs has its own characters, though we could find the homologous genes in Arabidopsis by blast, however, the domains are not consistent with the common character of corresponding types. So, we think these genes should be deleted.

Q16 There is much discussion in the Results section. And many results in the Discussion section. Please revise the text.

Reply: We have made some changes to the results and discussion part.

Q17 Line 361: The authors obtained 363 gene pairs in all identified MYBs. Figure 7 indicates 303 GO results. What about the remaining 60 genes? GO terms were not identified?

Reply: We have checked the primary data, here we conducted collinearity analysis for all the identified 421 MYBs, among them, we found 363 gene pairs. The GO result was conducted for all 421 MYBs. We have modified it in the text. New Line 380

Q18 Lines 371-372: Did the results correlate MYB coding genes with other GO terms different from transcription regulatory activity? These findings must be discussed.

Reply: We have conducted GO analysis for these 23 MYBs again, their enriched GO terms were listed in Table S8, and further discussions were performed in Lines 630-637.

Q19 Line 458: It is not possible to make the correlation between gene ID in Table S7 and the evaluated genes in Figure 10. Were genes evaluated by qRT-PCR obtained from the results in Table S7? Please consider including the annotation of the DEGs in Table S7.

Reply: Table S7 displays the 6104 DEGs identified in the RNA-seq analysis. WGCNA analysis is also based on these DEGs. Because this paper mainly focused on MYBs, the genes in qRT-PCR were obtained from the result of Figure 7b. Here also included some 95 differentially expressed MYBs in Table S7, we have added genes symbol names in Table S7.

Q20 Lines 465-467: the discussion of RNA-seq results affirms that “saddlebrown module is crucial in flower development as it contains more genes related to flower development than other modules” and that enrichment of carotenoid biosynthesis genes indicates their potential roles in carotenoid metabolism. Nevertheless, the identification of differentially expressed genes may not directly correlate with protein production and alterations in phenotype. It is possible to see the upmodulation and downmodulation of genes in RNA-seq data, but we can not assume those traits are reflected in the M. candidum phenotype. Please adjust the text, restricting the analysis to the transcriptional pattern. Please indicate if these genes described as abundant are up or down-regulated.

Reply: As you mentioned, phenotypes can be influenced by many factors, including gene expression levels, post-transcriptional modifications, translation, post-translational modifications, and even the environment. As this research is an initial study on M. candidum, we conducted a WGCNA to build relations between MYBs and other genes in this section. Due to the lack of verification system in this species, we inferred the function of genes in each module through GO and KEGG analysis. Our findings revealed that many genes related to pigment biosynthesis, flower development, and flavonoid biosynthesis were enriched in the saddlebrown module (Figure 8e and f), leading us to infer that this module plays a key role in flower development. Our goal for further work was to build a co-expression network between MYBs and other genes here. So we think the description of upmodulation and downmodulation of genes also should focus on MBYs. Descriptions of up-regulated and down-regulated genes have been added in Lines 476-478.

Q21 Line 497: “Detect the expression pattern of these 20 selected MYB genes.” Please inform which 20 genes.

Reply: Because there are a lot of MYBs in this study, in previous analyses we have obtained 23 MYBs which is close to the flowering of M. candidum by GO analysis, here we selected 20 of 23 MYBs to detect their expression pattern in different tissues or developmental stages. We have modified this sentence (Lines 509-510).

Q22 Lines 534-537: caution is necessary when suggesting that the transcription process may be revealed by observing an elevated number of gene sequences in a genome. Those genes may or may not be expressed.

Reply: That's a great question. In our identification process, we only focused on the gene members based on the genome of M. candidum at the DNA level. However, gene expression occurs at the transcriptional level and is influenced by various factors, such as tissues, treatments, and surrounding conditions. Therefore, we cannot currently confirm whether these genes are expressed. Upon further review, we also realized we cannot conclude from the former sentence. We have reorganized the discussion part.

Q23 The discussion must be improved. The key point must not be restricted to comparing the number of MYBs identified among papers but to exploring what this number of genes may possibly implicate in the plant. In RNA-seq results, how many are up-modulated? How many are down-modulated? Those same genes (up- or down-modulated) remained modulated in the other samples evaluated by qRT-PCR? Was the phylogenetic distance among A. thaliana, M. candidum, and P. trichocarpa evident in the collinearity analyses? From lines 595 to 605, e.g., the discussion only explores literature. Please discuss more deeply the results integrating the obtained data.

Reply: The discussion of MYB number has been improved, and some possible reasons were discussed.

In Figure 10, we conducted qRT-PCR for some 20 selected MYBs. Amongst, McGLK1c, MYB12b, MYB11, MYB21a-c, MYB113h, MYB17b, MYB32 are also the differentially expressed MYBs in RNA-seq, we have compared them in both RNA-seq and qRT-PCR results (Lines 511-517).

The phylogenetic distance surely has close relations with collinearity analysis, we have added these in the discussion part. (Lines 609-618)

We have made some changes in the Discussion part by integrating our results.

Q24 Lines 611-612: “MYB family members participated in various developmental processes or stages of M. candidum.” Overall, the gene expression pattern appears to be not statistically significant. For example, for McMYB17a, the error bar makes it seem that there was no statistical difference among the samples. The authors consider that McMYB17a is more expressed in early-stage flowers. It is considered to be more expressed compared to which condition? Please revise the statistical significance in Figure 10.

Reply: Because some biological repeats of qRT-PCR are not well enough, so the big error bars lead to imperfect One-way ANOVA analysis results. We have carried out qRT-PCR again for these genes using the corresponding tissues and modified the qRT-PCR result in Figure 10.

This investigation entailed conducting qRT-PCR across numerous tissues and developmental stages, with the One-way ANOVA conducted via SPSS software. As such, our comparisons were made between different tissues or developmental stages, without referencing any specific conditions.

Q25 Lines 622-623: Authors conclude that MYB genes possess huge potential for improving the application of M. candidum in the garden and pharmaceutical industry. Please explain how it will be applied. The gene family is highly diverse. The deletion of one of the genes may be supplanted by the activity of others. The effective biological function of each gene in M. candidum was not deciphered. This way, in which context do the authors consider this gene family potential?

Reply: The reviewer is very meticulous and rigorous. Yes, this conclusion in the text cannot be drawn from the current study, so we have adjusted the final conclusion here. (Lines 677-679)

Q26 Please write the genus of A. thaliana, M. candidum, and P. trichocarpa in their first appearance in the main text.

Reply: We have added the full Latin name in the first appearance.

Reviewer 2 Report

The manuscript by Li et al., “Genome-wide identification, characterization, and expression 2 pattern of MYB gene family in Melastoma candidum” reports on the first identification and classification of the MYB gene family in the M. candidum ornamental species.

The manuscript brings an important amount of information of potentially big importance for the little studied species, with overall properly conducted analysis. However, there are some fundamental issues that have to be addressed to improve this paper. My main concern is with the overall writing style, particularly in the results and discussion section. The impression is that the data should be better and more proficiently described. In multiple occasions, details are missing, terminology used is rather essential and not properly selected (also from the basic English language point of view). I encourage the authors to consult the vast amount of similar research available for different species and to accordingly adjust their manuscript. The information is there it just needs a good scientific edit. This would not take very long for an experienced scientific author, but could take quite a while by someone with less experience in English scientific writing. This should significantly improve the validity and significance of the present study. Although I made several suggestions in the attached file, my review cannot be considered exhaustive as the manuscript would need a sort of edit first. Some of the general comments are as follows:

-          Introduction – sufficient with suitable references, objectives clearly stated

-          M&M section need to be improved by adding details on the tools used and methodology, having in mind that not all of the readers are familiar with the applied methods and techniques (see details in the attached file); technical details for data presentation are often lacking (e.g. explanation of the elements/symbols present in figures)

-          Although citations can be integrated in the Results section, it should be only for clarification purpose and not to advance any hypothesis or explain why a given result was observed – the authors should modify accordingly such text sections in the Results (see some details in the attached file) and move similar comments in the Discussion section

-          Revise carefully all the captions to make them standalone and adopt the commonly used terminology when describing symbol colours, shape etc. (please consult similar literature), and where indicated provide the description of figure panels

-          English should be revised (in particular the article and preposition use) and the terminology checked for suitability

Other specific comments are present in the attached file.

Comments for author File: Comments.pdf

I am not a native speaker but the overal impression is that an editing is needed, from the grammatical and style point of view.

Author Response

Comments and Suggestions for Authors

The manuscript by Li et al., “Genome-wide identification, characterization, and expression 2 pattern of MYB gene family in Melastoma candidum” reports on the first identification and classification of the MYB gene family in the M. candidum ornamental species.

The manuscript brings an important amount of information of potentially big importance for the little studied species, with overall properly conducted analysis. However, there are some fundamental issues that have to be addressed to improve this paper. My main concern is with the overall writing style, particularly in the results and discussion section. The impression is that the data should be better and more proficiently described. In multiple occasions, details are missing, terminology used is rather essential and not properly selected (also from the basic English language point of view). I encourage the authors to consult the vast amount of similar research available for different species and to accordingly adjust their manuscript. The information is there it just needs a good scientific edit. This would not take very long for an experienced scientific author, but could take quite a while by someone with less experience in English scientific writing. This should significantly improve the validity and significance of the present study. Although I made several suggestions in the attached file, my review cannot be considered exhaustive as the manuscript would need a sort of edit first. Some of the general comments are as follows:

Reply: We thank the reviewer for the review and the valuable comments. We have revised the manuscript based on your comments. Specific changes are listed below.

Q1 Introduction – sufficient with suitable references, objectives clearly stated

Reply: We have made revisions to the introduction section of the manuscript.

Q2 M&M section need to be improved by adding details on the tools used and methodology, having in mind that not all of the readers are familiar with the applied methods and techniques (see details in the attached file); technical details for data presentation are often lacking (e.g. explanation of the elements/symbols present in figures)

Reply: TBtool is an integrated software that boasts a relatively simple operation and has been introduced in its published literature. As we have already cited it in this manuscript, we think that the current introduction is sufficient for publication.

Q3 Although citations can be integrated in the Results section, it should be only for clarification purpose and not to advance any hypothesis or explain why a given result was observed – the authors should modify accordingly such text sections in the Results (see some details in the attached file) and move similar comments in the Discussion section

Reply: Yes, we have made changes for the results and discussion section

Q4 Revise carefully all the captions to make them standalone and adopt the commonly used terminology when describing symbol colours, shape etc. (please consult similar literature), and where indicated provide the description of figure panels

Reply: We revised the manuscript based on feedback and suggestions.

Q5 English should be revised (in particular the article and preposition use) and the terminology checked for suitability

Reply: We have thoroughly reviewed all the writing in this manuscript.

Q6 Other specific comments are present in the attached file.

Reply: We have modified the paper according to the attached file.

Round 2

Reviewer 1 Report

Dear authors,

Thank you for your efforts in revising the manuscript. After reading through your replies and revision, I have the following comments to be considered before the final publication of your manuscript.

When the authors affirm that “due to the lack of a genome for Melastoma candidum the MYB gene family had not been previously identified in this species” it gives the impression that this manuscript is publishing the genome. To avoid this misunderstanding, the authors could consider rewriting this sentence as “due to the recent availability of Melastoma candidum genome, this is the fists time that MYB gene family was identified in this species” - or in a similar way.

Line 36: “and is characterized by approximately 50 to 53 amino acids at N-terminus.” This sentence was highlighted not because of the number of amino acids. To correctly inform the reader is necessary to specify that this family of genes is characterized by a conserved MYB domain located at the N-terminus portion. Please correct the sentence in the manuscript.

Line 238: The authors can only affirm that the similarity in sequences reflects similar functions by evaluating the biological function experimentally, as through gene knockout or silencing. The authors can suggest that similar protein sequences may reflect similarities in their functions based on literature evidence. But, biologically, only after in vivo experiments can functions be determined. 

Lines 238-240: Although McMYB124a, 238 McMYB124b, and McMYB88 have no close evolutionary relations with other members of R2R3, their biological function may only be attested if the authors perform in vivo analysis, e.g., through gene knockout, or other genetic strategies. The authors can only suggest that these dissimilarities may be reflected in biological functions compared to other R2R3 members.

Author Response

Thank you for your careful review of our article. Your insightful comments are instrumental in improving completeness and rigour of the manuscript, and we have made the modifications in the corresponding positions according to your requirements. In addition we checked the writing of whole text again.

1. When the authors affirm that “due to the lack of a genome for Melastoma candidum the MYB gene family had not been previously identified in this species” it gives the impression that this manuscript is publishing the genome. To avoid this misunderstanding, the authors could consider rewriting this sentence as “due to the recent availability of Melastoma candidum genome, this is the fists time that MYB gene family was identified in this species” - or in a similar way.

Reply: we have corrected here according your recommendation (Lines 15-16)

2. Line 36: “and is characterized by approximately 50 to 53 amino acids at N-terminus.” This sentence was highlighted not because of the number of amino acids. To correctly inform the reader is necessary to specify that this family of genes is characterized by a conserved MYB domain located at the N-terminus portion. Please correct the sentence in the manuscript.

Reply: we have corrected here to “is characterized by a conserved MYB DNA binding domain located” (Line 35)

3. Line 238: The authors can only affirm that the similarity in sequences reflects similar functions by evaluating the biological function experimentally, as through gene knockout or silencing. The authors can suggest that similar protein sequences may reflect similarities in their functions based on literature evidence. But, biologically, only after in vivo experiments can functions be determined. 

Reply: Refer to the reply in Q4

4. Lines 238-240: Although McMYB124a, McMYB124b, and McMYB88 have no close evolutionary relations with other members of R2R3, their biological function may only be attested if the authors perform in vivo analysis, e.g., through gene knockout, or other genetic strategies. The authors can only suggest that these dissimilarities may be reflected in biological functions compared to other R2R3 members.

Reply: We have reorganized these two sentences, and added one transitional sentence to ensure the rigour of the description (Lines 232-237)