Genome-Wide Identification of R2R3-MYB Transcription Factor Family in Tartary Buckwheat (Fagopyrum tataricum) Identifies a Member Involved in Anthocyanin Biosynthesis

Round 1

Reviewer 1 Report

Deng et al., analyzed R2R3-MYB transcription factors comprehensively in the genome of Tartary buckwheat (Fagopyrum tataricum). The information is useful to characterize MYBs in F. tataricum and they identified FtMYB43 as a transcriptional activator of anthocyanin biosynthesis. The manuscript is well written, but several issues should be considered to improve it. 

Lines 53–64: There are a bunch of articles studying MYBs in various plant species. So, it’s not adequate to cite some specific studies here. The authors should explain the general aspects of MYBs referring to some review articles here.

Table 1 should be changed to supplemental material. It’s too large.

Lines 356–366: The relationship between the expression pattern and cis-elements should be explained more in detail. For example, are there common cis-elements in the promoters of FtMYB53, FtMYB90, and FtMYB135? 

Figure 8: Please provide the data from different independent lines in B and C as A. 

Minor points.

Line 17 and many others: “cis” should be in italics.

Line 46: I’m not sure if the phrase “which for encodes helices” is correct.

Line 87: What is the meaning of “published 2”?

Line 216–217: Please delete the comment in the brackets.

Line 301: Please spell out “MBSI” and explain the physiological role of the motif. 

I think some editing of English is required.

Author Response

Thank you for your valuable comments, we have revised the manuscript according to your suggestions as follows: 1.Lines 53–64: There are a bunch of articles studying MYBs in various plant species. So, it’s not adequate to cite some specific studies here. The authors should explain the general aspects of MYBs referring to some review articles here. Answer: Thank you for your suggestion, we have deleted the citation about specific MYBs here, and have adjusted this content according to some review articles about the general aspects of MYBs. 2.Table 1 should be changed to supplemental material. It’s too large. Answer: Thanks you for this suggestion, we have moved it to supplemental material (Table S2), and other supplemental materials have been adjusted accordingly. 3.Lines 356–366: The relationship between the expression pattern and cis-elements should be explained more in detail. For example, are there common cis-elements in the promoters of FtMYB53, FtMYB90, and FtMYB135?  Answer: Thank you for your suggestion, there are common cis-elements, such as TC-rich repeat、G-box、ARE、TGA-element and CGTCAC-motif in the promoters of FtMYB53, FtMYB90, and FtMYB135. We have added this information in the text. 4.Figure 8: Please provide the data from different independent lines in B and C as A.  Answer: Thank you for your suggestion, we have provided the data from different independent lines in B and C as A. Minor points. 5.Line 17 and many others: “cis” should be in italics.  Answer: Thanks, we have changed all the “cis” in this manuscript into “cis”. 6.Line 46: I’m not sure if the phrase “which for encodes helices” is correct. Answer: This phrase “which for encodes helices” is not correct, we have changed it to “which encodes for helices”. 7.Line 87: What is the meaning of “published 2”? Answer: This was an error which we have rectified and changed it to “The Tartary buckwheat genome has been sequenced and published [35]. 8.Line 216–217: Please delete the comment in the brackets. Answer: Thank you. We have deleted the comment in the brackets. 9.Line 301: Please spell out “MBSI” and explain the physiological role of the motif. Answer: MBSI is an MYB binding site involved in flavonoid biosynthetic genes regulation, and the sequence is TTTTTACGGTTA. We have provided this information in the text.

Reviewer 2 Report

To,

The Editor,

Agronomy, MDPI 

Manuscript ID: agronomy-2522204 

Subject: Submission of comments of the manuscript in “Agronomy" 

Dear Editor Agronomy, MDPI, 

Thank you very much for the invitation to consider a potential reviewer for the manuscript (ID: agronomy-2522204). My comments responses are furnished below as per each reviewer’s comments. 

In the reviewed manuscript, the authors carried out domains feature, chromosomal location, motif prediction, gene structure, cis-acting elements as well as the expression pattern of R2R3-MYB transcription factors were analyzed comprehensively in Tartary buckwheat using a bioinformatic approach. Additionally, one R2R3-MYB gene was verified by transgenic Arabidopsis. Results indicated that a total of 152 R2R3-MYB genes were identified with special R2R3 domains and were distributed on 8 chromosomes of Tartary buckwheat, and were further classified into 25 sub-categories via phylogenetic analysis in terms of the R2R3-MYB genes family classification principles of Arabidopsis thaliana. This classification was further supported by analysis of exon-intron structure, motif, and cis-elements. Tandem and segmental duplication existed among the R2R3-MYB gene family of Tartary buckwheat, and there were 5, 8, 27, and 36 FtR2R3-MYB homologous genes respectively, comparing with Zea mays, Oryza sativa, Arabidopsis thaliana, and Solanum melongena by synteny analysis. The expression pattern of FtR2R3-MYB genes in different tissue and under salt stress and different light condition showed that members had tissue-specific expression levels and that these members may play diverse functions in plant growth and adaptation to varying environments. In addition, one of the FtR2R3-MYB gene family, FtMYB43, a homologue of AtTT2 was verified to improve the production of anthocyanin in transgenic Arabidopsis by overexpression, implying that it may regulate the expression levels of most structural genes of anthocyanin biosynthesis pathway in Arabidopsis seedling. These results provide more insights into the structure and function of the R2R3-MYB gene family and may accelerate the breeding of ornamental buckwheat cultivars. There are some minor and major comments. The author should revise as per my comments and suggestions. 

1. I have read the entire manuscript and my initial comment is that manuscript is poorly written. I have significant concerns about the grammar and vocabulary of the manuscript; therefore, I recommend the authors to use an English proofreading service.
2. The abstract does not reflect the whole story, revise it
3. The key words must be in alphabetical order.
4. The writing style of the paper is very poor. There are many grammatical mistakes. Long sentences with noticeable grammatical mistakes are frequently present throughout the manuscript. There are many typos mistakes in this whole manuscript. The author should check the whole manuscript.
5. The introduction part is not impressive and systematic. In the introduction part, the authors should elaborate on the scientific issues in plant research. The Content of the introduction is effective in essence but very poorly presented, significant improvements are needed in presenting the proper background of the work undertaken
6. The figures are quite low resolution and difficult to make out. Higher-resolution versions will be needed for publication. Further, text in figure is not readable, for example, in Figures 1, 3, 4A, 5 and 6.
7. qRT-PCR methodology provided is also very vague and confusing. Please provide more details like what was the calibrator used in the study. I assume the authors have used the control as the calibrator. If so, the authors should not include the control within the bar graph as it represents the fold change between the treated vs control and a fold change of “1” for the ‘control’ doesn’t make any sense.  Also, would be good to provide details on what reagents (details of probes used, if any, if SYBR was used then details for that, etc.) and real time PCR machine were used in the current study.
8. The problem is not with the research, but with the form of the description, which is too poor in my opinion. The literature review and discussion should be improved. The discussion should be interpreted with the results as well as discussed in relation to the present literature and authors must cite recently published research article in introduction and discussion on genome-wide identification in wheat, for instance, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8880425/

a.    https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8303626/

b.    https://www.frontiersin.org/articles/10.3389/fpls.2021.663118/full

c.    https://pubmed.ncbi.nlm.nih.gov/33673010/

d.    https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8395832/

e.    https://bmcplantbiol.biomedcentral.com/articles/10.1186/s12870-020-02576-0

9. The conclusion section is very poor, hence, the author should emphasize this in a better way.

To,

The Editor,

Agronomy, MDPI 

Manuscript ID: agronomy-2522204 

Subject: Submission of comments of the manuscript in “Agronomy" 

Dear Editor Agronomy, MDPI, 

Thank you very much for the invitation to consider a potential reviewer for the manuscript (ID: agronomy-2522204). My comments responses are furnished below as per each reviewer’s comments. 

In the reviewed manuscript, the authors carried out domains feature, chromosomal location, motif prediction, gene structure, cis-acting elements as well as the expression pattern of R2R3-MYB transcription factors were analyzed comprehensively in Tartary buckwheat using a bioinformatic approach. Additionally, one R2R3-MYB gene was verified by transgenic Arabidopsis. Results indicated that a total of 152 R2R3-MYB genes were identified with special R2R3 domains and were distributed on 8 chromosomes of Tartary buckwheat, and were further classified into 25 sub-categories via phylogenetic analysis in terms of the R2R3-MYB genes family classification principles of Arabidopsis thaliana. This classification was further supported by analysis of exon-intron structure, motif, and cis-elements. Tandem and segmental duplication existed among the R2R3-MYB gene family of Tartary buckwheat, and there were 5, 8, 27, and 36 FtR2R3-MYB homologous genes respectively, comparing with Zea mays, Oryza sativa, Arabidopsis thaliana, and Solanum melongena by synteny analysis. The expression pattern of FtR2R3-MYB genes in different tissue and under salt stress and different light condition showed that members had tissue-specific expression levels and that these members may play diverse functions in plant growth and adaptation to varying environments. In addition, one of the FtR2R3-MYB gene family, FtMYB43, a homologue of AtTT2 was verified to improve the production of anthocyanin in transgenic Arabidopsis by overexpression, implying that it may regulate the expression levels of most structural genes of anthocyanin biosynthesis pathway in Arabidopsis seedling. These results provide more insights into the structure and function of the R2R3-MYB gene family and may accelerate the breeding of ornamental buckwheat cultivars. There are some minor and major comments. The author should revise as per my comments and suggestions. 

1. I have read the entire manuscript and my initial comment is that manuscript is poorly written. I have significant concerns about the grammar and vocabulary of the manuscript; therefore, I recommend the authors to use an English proofreading service.
2. The abstract does not reflect the whole story, revise it
3. The key words must be in alphabetical order.
4. The writing style of the paper is very poor. There are many grammatical mistakes. Long sentences with noticeable grammatical mistakes are frequently present throughout the manuscript. There are many typos mistakes in this whole manuscript. The author should check the whole manuscript.
5. The introduction part is not impressive and systematic. In the introduction part, the authors should elaborate on the scientific issues in plant research. The Content of the introduction is effective in essence but very poorly presented, significant improvements are needed in presenting the proper background of the work undertaken
6. The figures are quite low resolution and difficult to make out. Higher-resolution versions will be needed for publication. Further, text in figure is not readable, for example, in Figures 1, 3, 4A, 5 and 6.
7. qRT-PCR methodology provided is also very vague and confusing. Please provide more details like what was the calibrator used in the study. I assume the authors have used the control as the calibrator. If so, the authors should not include the control within the bar graph as it represents the fold change between the treated vs control and a fold change of “1” for the ‘control’ doesn’t make any sense.  Also, would be good to provide details on what reagents (details of probes used, if any, if SYBR was used then details for that, etc.) and real time PCR machine were used in the current study.
8. The problem is not with the research, but with the form of the description, which is too poor in my opinion. The literature review and discussion should be improved. The discussion should be interpreted with the results as well as discussed in relation to the present literature and authors must cite recently published research article in introduction and discussion on genome-wide identification in wheat, for instance, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8880425/

a.    https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8303626/

b.    https://www.frontiersin.org/articles/10.3389/fpls.2021.663118/full

c.    https://pubmed.ncbi.nlm.nih.gov/33673010/

d.    https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8395832/

e.    https://bmcplantbiol.biomedcentral.com/articles/10.1186/s12870-020-02576-0

9. The conclusion section is very poor, hence, the author should emphasize this in a better way.

Author Response

Thanks for the in depth review of our manuscript, and we have revised according the minor and major comments mentioned as follows: 1.I have read the entire manuscript and my initial comment is that manuscript is poorly written. I have significant concerns about the grammar and vocabulary of the manuscript; therefore, I recommend the authors to use an English proofreading service. Answer: Thank you for your suggestion, we asked a native English speaker in the field to revise this manuscript, and in addition to those sections, including abstract, introduction, discussion and conclusion, were revised respectively, other revised details are shown in the revised version of the manuscript and listed point-by-point as follows: (1)Line 42-45, “The MYB structural domain, which contains four types of MYB proteins (1R-, R2R3-, 3R-, and 4R-MYB proteins) based on the number of N-terminal MYB domain with imperfect repeats, is where the MYB TFs family gets its name [1-2].” has been changed to “The MYB structural domain, which contains four types of MYB proteins, namely, 1R-, R2R3-, 3R-, and 4R-MYB proteins. The TFs family name is based on these number of N-terminal MYB domain with imperfect repeats, is where the MYB TFs family gets its name [1-2].” (2)Line 45-46, “ Each R domain is comprised of approximately 50 amino residues including conservative Trp (W), which for encodes helices.” has been changed to “Each R domain is comprised of approximately 50 amino residues, including conserved Trp (W), which encodes for helices. ” (3)Line 74-76, “...... and a large number of studies suggested flavonoids are beneficial for human health, including sterilization, anti-infection, oxidation resistance and arteriosclerosis of prevention [18, 22-26].” has been changed to “.......and a large number of studies suggested that flavonoids are beneficial for human health, where they function in sterilization, anti-infection, oxidation resistance and prevention of arteriosclerosis [18, 22-26].” (4)Line 84-86, “However, systematic and comprehensive bioinformatics analysis of R2R3-MYB TFs of Tartary buckwheat remains elusive. ” has been changed to “However, systematic and comprehensive bioinformatics analysis of R2R3-MYB TFs of Tartary buckwheat remains elusive, though which, more MYBs TFs involved in regulation of flavonoid biosynthesis may be identified.” (5)Line 100-101, “Seedlings of seven days were collected and snap frozen in liquid N2 , then stored at -80 oC for further use. ” has been changed to “Seven-days-old seedlings were collected and snap-frozen in liquid N2, then stored at -80 oC until use.” (6)Line 111-114, “Furthermore, based on the AtR2R3-MYBs proteins query using BLASTP programs to retrieve local Tartary buckwheat protein data, which obtained the preliminary homologous sequences of Tartary buckwheat which was followed by removal of the repeat sequences. ” has been changed to “ Furthermore, based on the AtR2R3-MYBs proteins query using BLASTP programs to retrieve local Tartary buckwheat protein data was performed, which obtained the preliminary homologous sequences of Tartary buckwheat. This was followed by the removal of the repeat sequences.” (7)Line 115-117, “Additionally, the candidates were submitted to Expasy and SMART software to test the conserved domain of R2R3-MYB proteins and deleted protein sequences without these typical characteristics.” has been changed to “The candidates were then submitted to Expasy and SMART software for conserved domain of R2R3-MYB proteins analysis, where proteins sequences without these typical characteristics were deleted.” (8)Line 120-121, “Physicochemical properties and subcellular localization and secondary structure of identified FtR2R3-MYB proteins above were analyzed...... ” has been changed to “Physicochemical properties, subcellular localization, and secondary structure of identified FtR2R3-MYB proteins above were analyzed......”. (9)Line 129, “The multiple sequences alignment of these FtR2R3-MYB proteins was......” has been changed to “The multiple sequence alignment of these FtR2R3-MYB proteins was......” (10)Line 173-174, “The CDS of FtMYB43 was amplified cloned into the pC1300S-GFP vector by homologous recombination method, and the specific primer sequences were listed in Table S5.” has been changed to “The CDS of FtMYB43 was amplified and cloned into the pC1300S-GFP vector by homologous recombination method, and the specific primer sequences are listed in Table S5.” (11)Line 175, “The vector Ghd7:RFP was used as reference which is located in the nuclear.” has been changed to “The vector Ghd7:RFP was used as a reference which is located in the nucleus.” (12)Line 178-179, “Fluorescence signals of GFP or RFP were observed using confocal laser scanning microcopy (STELLARIS 8). ” has been changed to “Fluorescence signals of GFP or RFP were observed using confocal laser scanning microscopy (STELLARIS 8). ” (13)Line 199-200, “ The extracting solutions were measured at absorptions......” has been changed to “The extracted solutions were measured at absorptions......” (14)Line 56, 61, 295, 496, 504, 507, “low-temperature” has been changed to “low temperature ”. (15)Line 330, “Investigation on the tissue-specific expression of these FtR2R3-MYBs,” has been changed to “Investigation of the tissue-specific expression of these FtR2R3-MYBs,” (16)Line 407, “ therefore, FtMYB43 might well has the similar function. ” has been changed to “ therefore, FtMYB43 might as well has the similar function. ” (17)Line 412-413, “ These results suggest that FtMYB43 is a transcript factor that plays a positive regulation role. ” has been changed to “ These results suggest that FtMYB43 is a transcription factor that plays a positive regulatory role. ” (18)Line 421-422, “ while no obvious pigment accumulation were observed in the wild-type (WT) seedling (Figure 8A). ” has been changed to “ while no obvious pigment accumulation was observed in the wild-type (WT) seedlings (Figure 8A). ” (19)Line 422-425, “After extraction, the extracted solution exhibited red color, being indicative of the high anthocyanin content in the transgenic Arabidopsis seedlings, while the extracted solution of wild type exhibited green color with lower anthocyanin content (Figure 8B). ” has been changed to “After extraction, the extracted solution exhibited red color, indicative of the high anthocyanin content in the transgenic Arabidopsis seedlings, while the extracted solution of the wild type exhibited green color with lower anthocyanin content (Figure 8B).” (20)Line 427-428, “The results showed that except UFGT, the other structural genes in anthocyanin biosynthesis pathway......” has been changed to “The results showed that except for UFGT, the other structural genes in the anthocyanin biosynthesis pathway......” (21)Line 430-432, “ These results suggested that FtMYB43 played an important role as a positive regulator in the anthocyanin biosynthesis. ” has been changed to “These results suggested that FtMYB43 played an important role as a positive regulator in anthocyanin biosynthesis.” 2.The abstract does not reflect the whole story, revise it Answer: Thanks a lot, and we have revised the abstract as follows: Abstract: Tartary buckwheat (Fagopyrum tataricum Gaertn.) belongs to the family of Polygonaceae and is used as a multi-functional plant. R2R3-MYB transcription factors play a crucial part in plant growth and many biological processes, where they regulate their internal environment. To date, there is no documented systematic research on the R2R3-MYB gene family in Tartary buckwheat. Here, domains feature, chromosomal location, motif prediction, gene structure, cis-acting elements as well as the expression pattern of R2R3-MYB transcription factors were analyzed comprehensively in Tartary buckwheat using a bioinformatic approach. Additionally, the function of one member was verified by transgenic Arabidopsis. Results indicated that a total of 152 R2R3-MYB genes were identified with special R2R3 domains and were distributed on 8 chromosomes of Tartary buckwheat. They were further classified into 25 sub-categories via phylogenetic analysis in terms of the R2R3-MYB genes family classification principles of Arabidopsis thaliana. This classification was further supported by analysis of exon-intron structure, motif, and cis-elements. Tandem and segmental duplication existed among the R2R3-MYB gene family of Tartary buckwheat, and there were 5, 8, 27, and 36 FtR2R3-MYB homologous genes respectively, when comparing with Zea mays, Oryza sativa, Arabidopsis thaliana, and Solanum melongena by synteny analysis. The expression pattern of FtR2R3-MYB genes in different tissue and under salt stress and different light condition showed that members had tissue-specific expression levels and that these members may play diverse functions in plant growth and adaptation to varying environments. In addition, one of the FtR2R3-MYB genes, FtMYB43, a homologue of AtTT2, clustered with R2R3-MYB from other plant species, which have been reported to be involved in the regulation of anthocyanin or proanthocyanidin biosynthesis. This gene was located in the nucleus, and had transcriptional activation activity, indicating that FtMYB43 may be a positive transcript factor of anthocyanin or proanthocyanidin biosynthesis. Moreover, the function of FtMYB43 was further verified to improve the production of anthocyanin in transgenic Arabidopsis by overexpression, and qRT-PCR assay implied that FtMYB43 may regulate the expression levels of most structural genes of anthocyanin biosynthesis pathway in Arabidopsis seedling. These results provide more insights into the structure and function of the R2R3-MYB gene family and may accelerate the breeding of ornamental buckwheat cultivars. 3.The key words must be in alphabetical order. Answer: We have arranged the key words in an alphabetical order: Keywords: anthocyanin biosynthesis; bioinformation analysis; R2R3-MYB gene family; Tartary buckwheat 4.The writing style of the paper is very poor. There are many grammatical mistakes. Long sentences with noticeable grammatical mistakes are frequently present throughout the manuscript. There are many typos mistakes in this whole manuscript. The author should check the whole manuscript. Answer: We have checked the whole manuscript, and we have revised the grammatical mistakes, long sentences and typos mistakes, the details are listed as follows: (1)Line 42-45, “The MYB structural domain, which contains four types of MYB proteins (1R-, R2R3-, 3R-, and 4R-MYB proteins) based on the number of N-terminal MYB domain with imperfect repeats, is where the MYB TFs family gets its name [1-2].” has been changed to “The MYB structural domain, which contains four types of MYB proteins, namely, 1R-, R2R3-, 3R-, and 4R-MYB proteins. The TFs family name is based on these number of N-terminal MYB domain with imperfect repeats, is where the MYB TFs family gets its name [1-2].” (2)Line 45-46, “ Each R domain is comprised of approximately 50 amino residues including conservative Trp (W), which for encodes helices.” has been changed to “Each R domain is comprised of approximately 50 amino residues, including conserved Trp (W), which encodes for helices. ” (3)Line 111-114, “Furthermore, based on the AtR2R3-MYBs proteins query using BLASTP programs to retrieve local Tartary buckwheat protein data, which obtained the preliminary homologous sequences of Tartary buckwheat which was followed by removal of the repeat sequences. ” has been changed to “ Furthermore, based on the AtR2R3-MYBs proteins query using BLASTP programs to retrieve local Tartary buckwheat protein data was performed, which obtained the preliminary homologous sequences of Tartary buckwheat. This was followed by the removal of the repeat sequences.” (4)Line 120-121, “Physicochemical properties and subcellular localization and secondary structure of identified FtR2R3-MYB proteins above were analyzed...... ” has been changed to “Physicochemical properties, subcellular localization, and secondary structure of identified FtR2R3-MYB proteins above were analyzed......”. (5)Line 129, “The multiple sequences alignment of these FtR2R3-MYB proteins was......” has been changed to “The multiple sequence alignment of these FtR2R3-MYB proteins was......” (6)Line 173-174, “The CDS of FtMYB43 was amplified cloned into the pC1300S-GFP vector by homologous recombination method, and the specific primer sequences were listed in Table S5.” has been changed to “The CDS of FtMYB43 was amplified and cloned into the pC1300S-GFP vector by homologous recombination method, and the specific primer sequences are listed in Table S5.” (7)Line 175, “The vector Ghd7:RFP was used as reference which is located in the nuclear.” has been changed to “The vector Ghd7:RFP was used as a reference which is located in the nucleus.” (8)Line 178-179, “Fluorescence signals of GFP or RFP were observed using confocal laser scanning microcopy (STELLARIS 8). ” has been changed to “Fluorescence signals of GFP or RFP were observed using confocal laser scanning microscopy (STELLARIS 8). ” (9)Line 199-200, “ The extracting solutions were measured at absorptions......” has been changed to “The extracted solutions were measured at absorptions......” (10)Line 56, 61, 295, 496, 504, 507, “low-temperature” has been changed to “low temperature ”. (11)Line 330, “Investigation on the tissue-specific expression of these FtR2R3-MYBs,” has been changed to “Investigation of the tissue-specific expression of these FtR2R3-MYBs,” (12)Line 407, “ therefore, FtMYB43 might well has the similar function. ” has been changed to “ therefore, FtMYB43 might as well has the similar function. ” (13)Line 412-413, “ These results suggest that FtMYB43 is a transcript factor that plays a positive regulation role. ” has been changed to “ These results suggest that FtMYB43 is a transcription factor that plays a positive regulatory role. ” (14)Line 421-422, “ while no obvious pigment accumulation were observed in the wild-type (WT) seedling (Figure 8A). ” has been changed to “ while no obvious pigment accumulation was observed in the wild-type (WT) seedlings (Figure 8A). ” (15)Line 422-425, “After extraction, the extracted solution exhibited red color, being indicative of the high anthocyanin content in the transgenic Arabidopsis seedlings, while the extracted solution of wild type exhibited green color with lower anthocyanin content (Figure 8B). ” has been changed to “After extraction, the extracted solution exhibited red color, indicative of the high anthocyanin content in the transgenic Arabidopsis seedlings, while the extracted solution of the wild type exhibited green color with lower anthocyanin content (Figure 8B).” (16)Line 430-432, “ These results suggested that FtMYB43 played an important role as a positive regulator in the anthocyanin biosynthesis. ” has been changed to “These results suggested that FtMYB43 played an important role as a positive regulator in anthocyanin biosynthesis.” 5.The introduction part is not impressive and systematic. In the introduction part, the authors should elaborate on the scientific issues in plant research. The Content of the introduction is effective in essence but very poorly presented, significant improvements are needed in presenting the proper background of the work undertaken. Answer: Thank you for your suggestion, we have revised the introduction section: 1. Introduction In most eukaryotes, transcription factors (TFs) are a class of proteins that exhibit polytypism, multifunction, universality, and multidomain structure. They also have distinctive structures called structural domains [1-2]. The MYB structural domain contains four types of MYB proteins namely, 1R-, R2R3-, 3R-, and 4R-MYB. The MYB TFs family name is based on these number of N-terminal MYB domain with imperfect repeats. [1-2]. Each R domain is comprised of approximately 50 amino residues including conserved Trp (W), which encodes for helices. This conserved Trp cluster and helix-turn-helix (HTH) combines with DNA resulting in execution of protein functions [2]. MYB TFs family is one of the largest families in plants and the first MYB gene was isolated from Zea mays [2]. Since then, an increasing number of MYB genes have been discovered in other species (http://planttfdb.gao-lab.org/). The largest subfamily of MYB TFs, R2R3-MYB TFs, are involved in regulating a variety of processes [3-4], including plant morphology [5-7], secondary metabolism (such as flavonoid [8], especially anthocyanin biosynthesis [9]; carotenoid biosynthesis [10]; lignin biosynthesis [11]), biotic and abiotic response [12], and other life processes. They either individually or in combination with other TFs [11-12]. Therefore, research of R2R3-MYB TFs has attracted attention from both domestic and foreign researchers [1]. At present, R2R3-MYB gene families have been identified in many plants, including Arabidopsis thaliana (126) [2], Oryza sativa (130) [13], Hypericum perforatum (109) [14], Camellia sinensis (122) [15] among others [16-17]. Tartary buckwheat (Fagopyrum tataricum Gaertn.) is a kind of important pseudocereal and domesticated crop that belongs to the genus Fagopyrum within the family Polygonaceae. Tartary buckwheat is native to the mountains of southwest China and widely grown around the world, including in Asia, America, and Europe. Tartary buckwheat has been characterized as a naturally functionality plant, which is a combination of medicine, nutrient, and health [18-20]. Edible seeds and sprouts of Tartary buckwheat not only contain rich nutrients, including dietary fiber, protein, starch, numerous amino acids, etc., but also contain various bioactive compounds, especially abundant flavonoids, including rutin, anthocyanins, proanthocyanidins [20-21]. A large number of studies suggested flavonoids are beneficial for human health, where they function in sterilization, anti-infection, oxidation resistance and prevention of arteriosclerosis [18, 22-26]. Therefore, Tartary buckwheat has become a popular daily food and has found favor in the consumer’s eyes [27]. Flavonoid biosynthesis requires not only synthetase enzymes but also transcription factors for modulation [28-29]. Numerous works of literatures reported that R2R3-MYB TFs are involved in flavonoids biosynthesis processes in many species, such as Zea mays, Arabidopsis thaliana, Solanum lycopersicum, Malus domestica, and Medicago truncatula [17, 30-32]. Recent studies suggested that significant accumulations of flavonoid have been detected through overexpression of FtPinG0002593000.01, FtPinG0009153900.01, and FtPinG0004608100.01 (GenBank ID: MT333222) of R2R3-MYB members in Tartary buckwheat [3, 33-34]. However, systematic and comprehensive bioinformatics analysis of R2R3-MYB TFs of Tartary buckwheat remains elusive, though which, more MYBs TFs involved in regulation of flavonoid biosynthesis may be identified. The Tartary buckwheat genome has been sequenced and published [35], which is useful for researching its gene families and gene functions. In this study, 152 R2R3-MYB genes were identified from the Tartary buckwheat genome and bioinformatics analysis including structure, chromosomal distribution, conserved motifs, phylogeny, cis-acting elements, synteny, and expression patterns were conducted. Additionally, one FtR2R3-MYB gene was verified to regulate anthocyanin accumulation. These results provide a research basis and scientific reference for subsequent functional studies of Tartary buckwheat transcription factors. 6.The figures are quite low resolution and difficult to make out. Higher-resolution versions will be needed for publication. Further, text in figure is not readable, for example, in Figures 1, 3, 4A, 5 and 6. Answer: Thank you for your suggestion, we have improved the resolution of all figures to over 300 dpi, and the text in the original figure 1, 3, 4A, 5 and 6, submitted in the system, can be readable clearly when they are enlarged. We have reduced the size of these figures in the text for typography. 7.qRT-PCR methodology provided is also very vague and confusing. Please provide more details like what was the calibrator used in the study. I assume the authors have used the control as the calibrator. If so, the authors should not include the control within the bar graph as it represents the fold change between the treated vs control and a fold change of “1” for the ‘control’ doesn’t make any sense.  Also, would be good to provide details on what reagents (details of probes used, if any, if SYBR was used then details for that, etc.) and real time PCR machine were used in the current study. Answer: Thank you for your suggestion. We used AtActin [44] as internal control, and WT as the control when comparing the expression level of genes from WT and transgenic Arabidopsis. The regents we used for qRT-PCR was SYBR Green Super mix (Bio-Rad, USA), and real time PCR machine is the C1000TM thermal cycler coupled with a CFX96TM detection module (Bio-Rad, USA). We have added this information in the revised manuscript. 8.The problem is not with the research, but with the form of the description, which is too poor in my opinion. The literature review and discussion should be improved. The discussion should be interpreted with the results as well as discussed in relation to the present literature and authors must cite recently published research article in introduction and discussion on genome-wide identification in wheat, for instance, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8880425/ a.    https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8303626/ b.    https://www.frontiersin.org/articles/10.3389/fpls.2021.663118/full c.    https://pubmed.ncbi.nlm.nih.gov/33673010/ d.    https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8395832/ e.    https://bmcplantbiol.biomedcentral.com/articles/10.1186/s12870-020-02576-0 Answer: Thank you for your suggestion. We have improved the literature review and discussion, please see the revised version, and we have cited recently published research article in discussion on genome-wide identification in wheat [61-64]. 9. The conclusion section is very poor, hence, the author should emphasize this in a better way. Answer: Thank you for your suggestion. We have revised the conclusion section： In conclusion, a genome-wide search identified a total of 152 R2R3-MYB TF genes in the Tartary buckwheat genome, and their physicochemical property, phylogeny, gene structure, synteny, cis-acting elements, conserved domain, and motifs as well as gene expression profiles were systematically analyzed by bioinformatics methods. The functions of FtR2R3-MYB gene family members were relatively conserved and numerous genes selected purification pressure during evolution. Most FtR2R3-MYB genes may participate in plant growth and respond to salt and light treatments or other abiotic stresses according to their expression patterns and cis-elements prediction. In addition, one member of FtR2R3-MYB gene family FtMYB43, congenerous to AtMYB123 (AtTT2) and belonging to the SG5 subgroup, was verified to be a positive TF by subcellular localization and transcriptional activity assay. FtMYB43 was further identified to positively regulate anthocyanin biosynthesis in transgenic Arabidopsis. This study might offer important information for research on the function of other gene families and provide excellent genetic resources for breeding of Tartary buckwheat.

Reviewer 3 Report

The document presents relevant information, the comments are really minimal since it is very well presented from the beginning to the end. Up-to-date references and a well-managed technical language

Please revise all “et al” to write correctly

Author Response

Thank you for your comments, and we have revised according to your suggestions as follows: 1. Please revise all “et al” to write correctly . Answer: Thank you, and we have revised all “et al” to wrote them correctly as “et al.” throughout the manuscript. 2. Line 101, change for further use by UNTIL USE. Answer: Thanks you, we have changed “for further use” to “until use”. 3. Line 165, et al (please check) Answer: We have changed “et al” here to “et al.” 4. Line 216-217, Please check ERROR! REFERENCE SOURCE NOT FOUND. Answer: In the word file, this sentence is “A total of 152 R2R3-MYB genes were identified from the Tartary buckwheat genome based on the number of MYB DNA-binding domains with the name of FtMYB1~ FtMYB152 according to their chromosome physical position (Table 1 and Figure 1).”, We don’t know why the citation “(Table 1 and Figure 1)” changed to “(ERROR! REFERENCE SOURCE NOT FOUND.)”, maybe due to the citation format. Since Table 1 is suggested to be changed to a supplemental material by another reviewer, so the citation is changed to “(Table S2 and Figure 1)”.

Round 2

Reviewer 1 Report

I could not find the involvement of data from independent lines in Fig. 8B and 8C as Fig. 8A. Please provide those as #1, #2, and #3 in Fig. 8A)

Author Response

Question: I could not find the involvement of data from independent lines in Fig. 8B and 8C as Fig. 8A. Please provide those as #1, #2, and #3 in Fig. 8A). Answer: Because each line of transgenic Arabidopsis seedlings was carried out as one biological repeat for total anthocyanin content measurement and expression level analysis , therefore, the data was the mean of three biological repeats ±SD.

Reviewer 2 Report

Dear Chief Editor,

Thank you for providing the opportunity to review the revised manuscript. The authors have addressed all comments and incorporated changes suggested by reviewers during the first round of revisions. The revised version of the manuscript is improved as expected. Based on these revisions, now this study is a suitable contribution to the Agronomy. I recommend the manuscript for publication.

Thank you

With best regards

Dear Chief Editor,

Thank you for providing the opportunity to review the revised manuscript. The authors have addressed all comments and incorporated changes suggested by reviewers during the first round of revisions. The revised version of the manuscript is improved as expected. Based on these revisions, now this study is a suitable contribution to the Agronomy. I recommend the manuscript for publication.

Thank you

With best regards

Author Response

Reviewer：Thank you for providing the opportunity to review the revised manuscript. The authors have addressed all comments and incorporated changes suggested by reviewers during the first round of revisions. The revised version of the manuscript is improved as expected. Based on these revisions, now this study is a suitable contribution to the Agronomy. I recommend the manuscript for publication. Answer: Thank you for this feedback.