Identification and Expression Analysis of the bHLH Gene Family Members in Diospyros kaki
, Yujing Suo 1, Huawei Li 1, Songfeng Diao 1, Peng Sun 1, Lin Huang 1,* and Jianmin Fu 1,*Round 1
Reviewer 1 Report
In this work, the authors analyzed 59 bHLH family members from the “Xiaoguotianshi” persimmon transcriptome.
The concept of this work and the results presented are very interesting and could be helpful for researchers in further planning of experiments.
It is only necessary to add some information in the Introduction. Write why Diospyros kaki is an important plant for this kind of research. Also, add the importance of the biosynthesis of proanthocyanins in the plant. At the beginning of the discussion, this information was mentioned briefly, but I think that a little more should be written about it in the introduction.
Author Response
Dear Editor,
Thank you very much for offering us the opportunity to resubmit a revised version of our manuscript. Hereby, we submit the revised manuscript entitled “Identification and expression analysis of the bHLH gene family members related to proanthocyanin biosynthesis in Diospyros kaki.” (2245455) to Horticulturae. We appreciate the valuable comments and suggestions from you and the reviewers, which help us to improve and clarify the manuscript. We have discussed the comments carefully and tried our best to improve the manuscript accordingly.
Detailed responses to your and the reviewers’ comments are provided in the next sections. We hope these responses are satisfactory and that the revised version will be acceptable for publication.
Please do not hesitate to contact us with any questions and we are looking forward to your reply.
Thanks and Best wishes!
Yours sincerely,
Weijuan Han and Jianmin Fu
Response to Reviewer 1:
Q1:It is only necessary to add some information in the Introduction. Write why Diospyros kaki is an important plant for this kind of research. Also, add the importance of the biosynthesis of proanthocyanins in the plant. At the beginning of the discussion, this information was mentioned briefly, but I think that a little more should be written about it in the introduction.
A: Thanks for your positive comments. We have added these information in the introduction part (Line 26-31).
Persimmon (Diospyros kaki) is an important fruit tree. The high soluble proanthocyanin content leads to astringency taste of persimmon fruits, as the key characteristic of persimmon. Transcription factors (TFs) involved in proanthocyanin biosynthesis have been identified previously, for example, R2R3MYB, bHLH, and WD40 (MBW) complex regulates the structural genes of flavonoid/phenylpropanoid pathway.
Author Response File: 
Author Response.docx
Reviewer 2 Report
In their article authors identified and analyzed in more details the family of bHLH proteins in Diospyros kaki.
Analyses are very well performed, all methods and results are discussed appropriately. I do not have any major concerns.
However, I still have some minor comments:
1) Table 1: is quite long – would it be possible to somehow shorten it? The best to make each protein per 1 line (use “c.” instead of “cluster”, introduce an abbreviation for “Helix-loop-helix DNA-binding domain” or something similar)?
Explain in the legend what “instability index” and “GRAVY” exactly means.
2) Fig. 1: the resolution is quite small and not much information can be derived from it. Could it be possible to increase font size or to show only genes from D. Kaki (as definitely more than 59 proteins are present – potentially just leave colors according to groups from A. thaliana) to make the figure more clear?
Why only group “III f” is marked in the figure?
3) Table 2: please explain in more details what “Ka”, “Ks” and “Ka/Ks” mean and how were “seq_1” and “seq_2” sequences selected. Also alternative title might be used.
4) Fig. 4: similar to the Fig. 1, the resolution is small and not much information can be derived from the figure. Please, simplify it and explain in the legend what each color means (if anything).
5) Fig. 5: figure title is missing. Please explain what red color indicates – probably an average of all genes… Other color corresponds to particular genes analyzed? – please explain in the legend.
6) Line 34 (and line 130): its mentioned that A. thaliana bHLH genes cluster into 26 subfamilies – but only 12 families are subsequently used to classify D. Kaki genes – can you somehow explain in more details the difference?
7) Line 52: please indicate in the sentence where 59 bHLH transcription factors were identified.
8) Line 94: its mentioned that tertiary structure was predicted – where in the main text are these results presented?
9) Line 115: “removal of the redundant sequences” – please indicate in more details the nature of the redundancy a how these sequences were removed (how many of them)
10) Line 159: please change the title of the section as “Ka/Ks value” is not much suitable…
11) Lines 226-227: T3, T4, DAF and the number are not clear, please indicate in more details what do they mean (it would be better to be explained not only in the Methods section).
12) Line 231: what “T4VST3” means? (T4 vs T3)?
13) Typos: line 37 (“.” is missing at the end of the sentence), line 48 (a space missing before citations), line 131 (correct “()”), line 133 (correct “€”), line 134 (is the word “domains” missing at the end of the sentence?), lines 224 and 229 (check D. kaki font)
Author Response
Dear Editor,
Thank you very much for offering us the opportunity to resubmit a revised version of our manuscript. Hereby, we submit the revised manuscript entitled “Identification and expression analysis of the bHLH gene family members related to proanthocyanin biosynthesis in Diospyros kaki.” (2245455) to Horticulturae. We appreciate the valuable comments and suggestions from you and the reviewers, which help us to improve and clarify the manuscript. We have discussed the comments carefully and tried our best to improve the manuscript accordingly.
Detailed responses to your and the reviewers’ comments are provided in the next sections. We hope these responses are satisfactory and that the revised version will be acceptable for publication.
Please do not hesitate to contact us with any questions and we are looking forward to your reply.
Thanks and Best wishes!
Yours sincerely,
Weijuan Han and Jianmin Fu
Q1:(1)Table 1: is quite long – would it be possible to somehow shorten it? The best to make each protein per 1 line (use “c.” instead of “cluster”, introduce an abbreviation for “Helix-loop-helix DNA-binding domain” or something similar)?
- Explain in the legend what “instability index” and “GRAVY” exactly means.
A:Thank you for your suggestion.
- This is the id we obtained from the transcriptome raw data, ang it is inconvenient to change into abbreviated form. However, we have reformatted Table 1 so that the table does not look redundant and long
(2)Instability index is greater than 40, is unstable protein; instability index is less than 40, belongs to stable protein.
Grand aversion of hydrophilicity (GRAVY), larger negative values indicate better hydrophilicity and larger positive values indicate greater hydrophobia
Q2: Fig. 1: the resolution is quite small and not much information can be derived from it. Could it be possible to increase font size or to show only genes from D. Kaki (as definitely more than 59 proteins are present – potentially just leave colors according to groups from A. thaliana) to make the figure more clear?
Why only group “III f” is marked in the figure?
A:Thank you for your suggestion.
(1)The bHLH family members, particularly the III (f) subfamily is involved in PA and anthocyanin synthesis, and we focused on the function of involved in PA biosynthesis in this study, thus, we just marked III f in the figure 1
(2) Besides, only group “III f” marking in the figure does not affect the rigor of this manuscript and its reading by the audience. Beacuse of the family group information was shown in Table 1.
Q3: Table 2: please explain in more details what “Ka”, “Ks” and “Ka/Ks” mean and how were “seq_1” and “seq_2” sequences selected. Also alternative title might be used.
Q10: Line 159: please change the title of the section as “Ka/Ks value” is not much suitable…
A:Thank you for your suggestion.
“Ka”, “Ks” were explain in the materiala and methods part (Line 102-103). how were “seq_1” and “seq_2” sequences selected were shown in Line 103-106.
(1)Non-synonymous substitutions per non-synonymous site (Ka) and synonymous substitutions per synonymous site (Ks) were used to assess the selection pressure and divergence time of the bHLH gene family [32].
(2)The value of Ks and Ka of the orthologous bHLH gene pairs between A. thaliana and D. kaki and the paralogous DkbHLH gene pairs were choosed and calculated using the TBtools v1.0971 software [33].
(3) We have changed the title into “Selection pressure and differentiation time of the bHLH genes”
Q4: Fig. 4: similar to the Fig. 1, the resolution is small and not much information can be derived from the figure. Please, simplify it and explain in the legend what each color means (if anything).
A:Thank you for your suggestion.
We have added the high resolution figure in the manuscript. Colors are randomly generated at the time of prediction and have no particular significance.
Q5: Fig. 5: figure title is missing. Please explain what red color indicates – probably an average of all genes… Other color corresponds to particular genes analyzed? – please explain in the legend.
A:Thank you for your suggestion.
The title of Figure 5 was presented in Line 205-206.
Fig. 5. Gene expression analysis of bHLH genes in D. kaki fruit at five different stages. a) Heat map clustering of DkbHLH gene expression levels at five developmental stages, heatmaps depict the normalized gene expression values, which represent the mean value of three biological replicates.ï¼›b) Clustering analysis of DkbHLH genes, each line represents the normalized gene expression values for an individual transcript.
Q6: Line 34 (and line 130): its mentioned that A. thaliana bHLH genes cluster into 26 subfamilies – but only 12 families are subsequently used to classify D. Kaki genes – can you somehow explain in more details the difference?
A:Thank you for your suggestion.
However, this is normal, because the relationships between D.kaki and A. thaliana is not very closly.
Q7: Line 52: please indicate in the sentence where 59 bHLH transcription factors were identified.
A:Thank you for your suggestion.
We have changed this sentence into “In this study, 59 bHLH transcription factors were identified from the D. kaki transcriptome data. “
Q8: Line 94: its mentioned that tertiary structure was predicted – where in the main text are these results presented?
A:Thank you for your suggestion.
We have removed the secondary structure prediction part in the materials and methods.
Q9: Line 115: “removal of the redundant sequences” – please indicate in more details the nature of the redundancy a how these sequences were removed (how many of them)
A:Thank you for your suggestion.
We have changed this sentence into “Following the removal of the sequences without complete conserved domains”
Q11: Lines 226-227: T3, T4, DAF and the number are not clear, please indicate in more details what do they mean (it would be better to be explained not only in the Methods section).
Line 231: what “T4VST3” means? (T4 vs T3)?
A:Thank you for your suggestion.
- More detailswere presented in the materials and methods (Line67-68).
(2)We have changed this sentence into “Surprisingly, Cluster-15548.1 was differentially downregulated at T4 comparing to T3”
Q12: Typos: line 37 (“.” is missing at the end of the sentence), line 48 (a space missing before citations), line 131 (correct “()”), line 133 (correct “€”), line 134 (is the word “domains” missing at the end of the sentence?), lines 224 and 229 (check D. kaki font)
A: Thank you very much for your comments. We have changed these typos in the revised manuscript.
Author Response File: Author Response.docx
Reviewer 3 Report
Authors perform an in silico analysis of bHLH gene family in Diospyros kaki (persimmon) using previously published transcriptomic data. It is presented a list of bHlH genes identified, a phylogenetic analysis, a phylogenetic tree, expression data and a protein-protein interaction map among other results. All sections are poorly described and results are also poorly discussed. But the big issue is that authors previously published similar persimmon results in 2022 (bHLH genes, a phylogenetic analysis, a phylogenetic tree and a protein-protein interaction map; https://doi.org/10.1038/s41598-022-23742-4) and this antecedent is not mentioned in the introduction and in the discussion section in the submitted work. In the previous published work they report 13 bHLH genes and now are reporting 59, all with different names making impossible to compare both results. So it is not clear the methodological differences that could explain the different results and this makes difficult to determine the originality/novelty, significance of the content and the scientific soundness of the present work. Authors must rewrite the manuscript in order to clarify the differences and discuss the results with the previously published work.
Other major and minor points are included in the attached document.
Comments for author File: 
Comments.pdf
Author Response
Q1:Authors perform an in silico analysis of bHLH gene family in Diospyros kaki (persimmon) using previously published transcriptomic data. It is presented a list of bHlH genes identified, a phylogenetic analysis, a phylogenetic tree, expression data and a protein-protein interaction map among other results. All sections are poorly described and results are also poorly discussed. But the big issue is that authors previously published similar persimmon results in 2022 (bHLH genes, a phylogenetic analysis, a phylogenetic tree and a protein-protein interaction map; https://doi.org/10.1038/s41598-022-23742-4) and this antecedent is not mentioned in the introduction and in the discussion section in the submitted work. In the previous published work they report 13 bHLH genes and now are reporting 59, all with different names making impossible to compare both results. So it is not clear the methodological differences that could explain the different results and this makes difficult to determine the originality/novelty, significance of the content and the scientific soundness of the present work. Authors must rewrite the manuscript in order to clarify the differences and discuss the results with the previously published work.
Other major and minor points are included in the attached document.
A: Thank you very much for your comments. We previously published similar persimmon results in 2022 (bHLH genes, a phylogenetic analysis, a phylogenetic tree and a protein-protein interaction map; https://doi.org/10.1038/s41598-022-23742-4). This is true. However in the above studies, we only focused the bhlh proteins might involved the (R2R3MYB, bHLH, and WD40) MBW complex, so we just report 13 bHLH genes previously.
These are methods of previous study
“Identification of MYB-bHLH-WD40 complex members in C-PCNA persimmon
To identify MYB-bHLH-WD40 Complex Members in the persimmon genome, local tBLASTp (https://blast.ncbi.nlm.nih.gov/) (E-value ≤ 1e−5) was performed using the sequences of homologous MBW complex proteins in Arabidopsis thaliana16,31, Malus × domestica32, Vitis vinifera33,34,35, and Fragaria × ananassa17 (Table S2). The online website Pfam (https://pfam.xfam.org/) was used to remove sequences without conserved MYB_DNA-binding (PF00249.30, PF13921.5), bHLH-MYC_N (PF14215.5), or WD40 (PF00400.31) domains (Table S2). Multiple sequence alignments and phylogenetic analysis were implemented using Clustal X36 and MEGA 537 software, respectively.”
Therefore, in this study, all bhlh transcriptor factors of D. kaki were identified, the number of total bhlh TFs is 59 > MBW complex bhlh member 13, it is no doubt. And I think this study will provide theoretical reference for further exploring the molecular characteristics and biological functions of the D. kaki bhlh gene.
Author Response File: Author Response.docx
Round 2
Reviewer 3 Report
Despite authors clarify the point about the previously work regarding bHLH genes in the reviewers answer, they did not include any information about this study in the introduction and discussion sections. Since my point of view, this is an inappropriate self-citations procedure because they do not mention important and relevant information. Just an example, they say in introduction: "The bHLH gene family has been recently identified in different plants including Arabidopsis [4], potato [5], tomato [6], apple [7], and rice [8]." What about their published similar persimmon results published in 2022. The previously published data is not relevant in the present paper? I also think that the present paper will provide theoretical reference for further exploring the molecular characteristics and biological functions of the D. kaki bHLH gene, but this information should be discussed, compared, and contrasted with the actual literature, including your previously published work.
On the other hand, in the revised version, authors did not correct or clarify important mistakes/omissions indicated in the revised attached file. Next I just mention some of them but i respectfully encourage them to deeply revise the previously attached file and take into account the comments and suggestions.
Note: All line numbers correspond to the revised version (not clean version)
Major and Minor Points:
1.- Line 95: It is indicated a Table S4, which was not included and there are not Table S1, S2 and S3.
2.- Figure legends are not self explanatory. In figures 1, 2, 3 and 4, the legend is just a title.
3.- Line 146 says: "Motifs 1 and 2 were present in all DkbHLH proteins." But, This is not true since motif 1 is not present in cluster 17857.0 and in cluster 6987.43498. Motif 2 is not present in cluster 6987.10953.
4.- Figure 4: What do colors mean? All bHLH proteins were considered? Which MYB proteins were considered? The nine reported in reference 20?
5.- Figure 5: What does red line represent?
6.- Line 240-241: Considering that Cluster 15548.1 is the protein involved in PA biosynthesis, why it was not compared in Table 2 and highlighted in Figure 4.
7.- In lines 248-250 is declared: "Previous studies have shown that 120 (T3) to 140 (T4) DAF may be the critical phase for the “dilution effect”, and 140 (T4) to 160 (T5) DAF may be the critical phase for crucial phase for the “coagulation effect” (unpublished)." But in the published work it is declared: ""These results indicated that 120–140 DAF could be the critical phase for the “dilution effect." So those unpublished data are already published.
8.- Line 253: I see the main downregulation in T2 vs T3 in figure 5.
9.- Genes are named as proteins and vicecersa. Species scientific names are not in italics.
Author Response
Dear Editor,
Thank you very much for offering us the opportunity to resubmit a revised version of our manuscript. Hereby, we submit the revised manuscript entitled “Identification and expression analysis of the bHLH gene family members related to proanthocyanin biosynthesis in Diospyros kaki.” (2245455) to Horticulturae. We appreciate the valuable comments and suggestions from you and the reviewers, which help us to improve and clarify the manuscript. We have discussed the comments carefully and tried our best to improve the manuscript accordingly.
Detailed responses to your and the reviewers’ comments are provided in the next sections. We hope these responses are satisfactory and that the revised version will be acceptable for publication.
Please do not hesitate to contact us with any questions and we are looking forward to your reply.
Thanks and Best wishes!
Yours sincerely,
Weijuan Han and Jianmin Fu
Despite authors clarify the point about the previously work regarding bHLH genes in the reviewers answer, they did not include any information about this study in the introduction and discussion sections. Since my point of view, this is an inappropriate self-citations procedure because they do not mention important and relevant information. Just an example, they say in introduction: "The bHLH gene family has been recently identified in different plants including Arabidopsis [4], potato [5], tomato [6], apple [7], and rice [8]." What about their published similar persimmon results published in 2022. The previously published data is not relevant in the present paper? I also think that the present paper will provide theoretical reference for further exploring the molecular characteristics and biological functions of the D. kaki bHLH gene, but this information should be discussed, compared, and contrasted with the actual literature, including your previously published work.
Thank you very much for your comments. We previously published similar persimmon results in 2022 (bHLH genes, a phylogenetic analysis, a phylogenetic tree and a protein-protein interaction map; https://doi.org/10.1038/s41598-022-23742-4). This is true. However in the above studies, we only focused the bhlh proteins might involved the (R2R3MYB, bHLH, and WD40) MBW complex, so we just report 13 bHLH genes previously. We have only analyzed some of them and it is not appropriate to compare them to the total number of other species. Thank you!
On the other hand, in the revised version, authors did not correct or clarify important mistakes/omissions indicated in the revised attached file. Next I just mention some of them but i respectfully encourage them to deeply revise the previously attached file and take into account the comments and suggestions.
Note: All line numbers correspond to the revised version (not clean version)
Major and Minor Points:
1.- Line 95: It is indicated a Table S4, which was not included and there are not Table S1, S2 and S3.
We have removed table S4
2.- Figure legends are not self explanatory. In figures 1, 2, 3 and 4, the legend is just a title.
We have added the explaination to the figure legend.
Fig. 1. Phylogenetic analysis of the D. kaki bHLH proteins compared to A. thaliana. Different colors represent different family groups, and the detail groups were shown in Table 1.
Fig.2. Phylogenetic tree (left) and the conserved motifs (right) of the D. kaki bHLH proteins. Different color represent different motif.
Fig. 3. GO annotation of 59 bHLH transcription factors. The dot size represent gene number; and different color represent the value of qValue.
Fig. 4. The protein-protein interaction network between bHLH and MYB proteins. Different colors have no additional meaning
3.- Line 146 says: "Motifs 1 and 2 were present in all DkbHLH proteins." But, This is not true since motif 1 is not present in cluster 17857.0 and in cluster 6987.43498. Motif 2 is not present in cluster 6987.10953.
We have changed into “Motifs 1 and 2 were present in nearly all DkbHLH proteins.”
4.- Figure 4: What do colors mean? All bHLH proteins were considered? Which MYB proteins were considered? The nine reported in reference 20?
Sorry, color have no meanings, we have added the explaination to the figure legend. We used all bhlh proteins, and we added in the material approach how the MYB gene was obtained.
5.- Figure 5: What does red line represent?
The red line is the fitted gene expression trend for this cluster, which we have added to the figure notes
6.- Line 240-241: Considering that Cluster 15548.1 is the protein involved in PA biosynthesis, why it was not compared in Table 2 and highlighted in Figure 4.
Table 2 only contains gene pairs with homologous relationships, because kaks analysis is to determine the degree of divergence of homologous genes and further determine the evolutionary relationship, Cluster 15548.1 did not predict homologous genes with high similarity, so this analysis was not performed
7.- In lines 248-250 is declared: "Previous studies have shown that 120 (T3) to 140 (T4) DAF may be the critical phase for the “dilution effect”, and 140 (T4) to 160 (T5) DAF may be the critical phase for crucial phase for the “coagulation effect” (unpublished)." But in the published work it is declared: ""These results indicated that 120–140 DAF could be the critical phase for the “dilution effect." So those unpublished data are already published.
We have removed the unpublished article and cited the reference.
8.- Line 253: I see the main downregulation in T2 vs T3 in figure 5.
The function of the same family gene is diverse, and it is normal to have down- or up-regulation in different cluster. We analyzed this part based on the pre-publication results, and although there is down-regulation in T2 vs T3, it is not within our scope of concern.
- - Genes are named as proteins and vicecersa. Species scientific names are not in italics.
We have checked and revised in the manuscript.
Author Response File: Author Response.docx
