Genome-Wide Identification and Characterization of the ANS Gene Family in Pomegranate (Punica granatum L.)

Round 1

Reviewer 1 Report

The authors identify all the possible ANS genes in the pomegranate fruit and analyzed their encoded enzymes via different bioinformatics tools. Besides, transcriptomics changes of these genes were analyzed in three different variants of pomegranate. This study is very interesting but there remain a lot of grammatical issues to be solved and most importantly, the author should take note of the plagiarism issue of certain part in the manuscript.

Specific comments:
1) L20, "Pomegranate (Punica granatum L.) belongs to genus Punica in the family of Pomegranate and ..."

2) L40, start a new paragraph from "Anthocyanidin Synthase"

3) L40-41, "Anthocyanidin Synthase (ANS) involves in a series of metabolic reactions and it is a 2-ketoglutarate-dependent ..."

4) L50-52, " Overexpression of ANS can increase anthocyanin accumulation, while reducing the expression of ANS significantly decreases the anthocyanin level in plant, and this leads to the production of white flowers [23,24].

5) L69, "... local BLASTP against promegranate's proteins ...". L71, "... structural domain of ANS [25]."

6) L75, "... was conducted using the online tool ..."

7) move L73-76 to section 2.3 and change the section name accordingly.

8) Rewrite section 2.7 and 2.8 as the paragraphs are copy-paste directly from another article. Although the overall similarity of the manuscript is below the plagiarism threshold, the scientific community does not encourage plagiarism partially either. Please take note of some sentences in the introduction as well.

9) L117, delete ", with little variation in the number of amino acids."

10) L122, "Based on the instability factor, 28% of them are stable protein ..."

11) L137, "... by using the pomegranate ..."

12) L147, "... was carried out using Gene Structure ..."

13) Please improve the resolution of all figures.

14) There are a lot of grammatical issues in the manuscript, which I cannot list them down one by one. The authors should check the grammar or send the manuscript for English editing service.

Author Response

Dear Reviewer,

Thank you for your suggestions on the manuscript, we have revised the manuscript. The details are in Word.

Author Response File: Author Response.docx

Reviewer 2 Report

Dear, After reading the work, I congratulate you on your work and recommend doing or taking into account some small details suggested in the attached text that will allow you to improve your work.

Comments for author File: Comments.pdf

Author Response

Dear Reviewer,

Thank you for your suggestions on the manuscript, we have revised the manuscript. The details are in Word.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments to the Author

 

I find this report potentially useful in the field of horticulture crops improvement because of the information elucidating the evolutionary relationship of the 75 ANS gene family in pomegranate genes and genetic resources secondly Genome-wide identification and expression profile of the ANS gene family belongs to the ANS gene superfamily plays a critical role in regulating the accumulation of Anthocyanine thus, typically that the expression of the pomegranate ANS genes were species-specific and period-specific. The topic is of interest to horticulture and the manuscript has a potential for publishing after corrections, as mentioned below. Some other requirements and corrections are in the enclosed text.

 

1. There are many grammar and language issues that need to be fixed. I listed some of them.

Title: Genome-wide identification and Characterization of the ANS gene 1 family in pomegranate should add a full scientific name (Punica granatum L.) 

Introduction

Page 1, lines 25-26: Pomegranates are increasingly popular in the consumer market for their sweetness high economic value, nutritional value, medicinal value and health functions. (Please avoid repeated sentences. Please check throughout MS.

Page 1, line 28: change such as anti-oxidation to antioxidant

Page 1, line 29: The results of Wang et al. [7] Please follow the journal references format as listed.

Page 1, line 29: showed that pomegranate juice and pomegranate peel please avoid double words such as pomegranate.

Page 1, line 32: body omit it.

Page 1, line 35 and 42: State should be form, and oxygenase's should be oxygenases.

Page 2, line 46: The ANS gene was first isolated from a mutant of maize by transposon tagging. (Need proper citation). As similar in other plants….. Arabidopsis thaliana [17], Litchi chinensis  [18],  Mangifera indica  [19],  Malus pumila  [20]…..

Page 2, line 53: and more omit it.

Page 2, line 56: characterized by bioinformatics tools.

Should be proteins they encoded. Omit the word “they”

Line 276: 01:91-93+59 +?

Line 280: pomegranate (Punica granatum L.) scientific name should be in italics. Please check throughout the manuscript. Please also check other plant's scientific names in the references part should be in italics. Such as Arabidopsis/ Mangifera/ Brassica napus and so on ….. I found a lot of references and the text size format is different please carefully check.

Line 142: et al. ?? please avoid such extra text.

 

2.      Does these genes register in NCBI? If so, please show the accession number for the convenience of information sharing.

3.      The results showed that the ANS gene family in pomegranate genes 75 ANS family members of pomegranate were localized in the cytoplasm. Is there genes? play a crucial role in plant growth and development and stress resistance? More pieces of evidence are needed. If not please discuss your results from other publications in the discussion part. The expression pattern of the ANS proteins should be analyzed through the GUS assay? There is no evidence to demonstrate that ANS participates in plant growth and development in the results section. Up/down-regulation or loss of function of some of the ANS and identifying the phenotypes, the authors can evaluate whether the ANS plays a crucial role in plant growth and development or stress resistance or not?

4.      It would be useful for the readers if the authors could also describe the ANS functional activity in the early stage of development (during embryogenesis)?

5.      It would be useful to describe the number of intron/exons.

6.      To compare the product function of twelve ANS genes in different tissues and organs of pomegranate, can the author? provide the Gene Ontology (GO) map.  

7.      The expression characteristics of pomegranate ANS genes were analyzed in three different varieties. Any validation of transcriptomic data of genes using qPCR? If yes please provide gene validation data which will authenticate your data for readers.

8.      Discussion needs to extend/polished.

9.       The paper is well organized, and the relevant published literature is completed cited and a minor revision is required.

 

Comments for author File: Comments.docx

Author Response

Dear Reviewer,

Thank you for your suggestions on the manuscript, we have revised the manuscript. The details are in Word.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

 

Specific comments:

1. L8, “to catalyze”, “to colored anthocyanidins”

2. L17, change to “variety-specific” since the compared pomegranates are same species.

3. L20, “Punica granatum L.”

4. L22, “… pomegranate was originated from the Middle East ...”

5. L24, “Currently, Shandong …”

6. L41, “Anthocyanidin Synthase (ANS) involves in a series of …”. ANS is protein or enzyme, not the pathway.

7. L44, “ANS is the key enzyme at the end of the anthocyanidin synthesis pathway and catalyzed the conversion of colorless leucoanthocyanidins to colored anthocyanidins.

8. L47, “Current studies have shown that ANS …”

9. L61-62, “Samples were taken from pomegranate seeds at …”

10. L66, “… was downloaded from NCBI …”

11. L68, “ … Arabidopsis thaliana … Anthocyanidin Synthase …” Italicized species name and non-italicized enzyme name.

12. L74, “… of the pomegranate ANS proteins were predicted …”. L75, remove “family”

13. L82, “… (NJ) method with the check …” remove “and”

14. L92, “… Calculator …”

15. L115, “The molecular weight of the pomegranate ANS family proteins ranged from …”

16. L119, “… most of the pomegranate ANS exhibited as acidic proteins.”

17. L121, remove “family”

18. L124, “… ANS gene family proteins.”

19. L126, “… pomegranate ANS proteins were composed …”. The structural predictions were done using the proteins not the genes, please double-check the rest of the writing.

20. L127, “The lowest proportion of these was β-turns …”

21. L128, “The majority of proteins had around …”. Again, what the authors refer is proteins, not genes.

22. L129, remove “ones”. L130 “ … the pomegranate ANS proteins, …” plural.

23. Table 2 first row, Irregular curling or Irregular coiling? Please standardize with the text written.

24. L134-135, “To investigate the phylogenetic relationships of the pomegranate ANS proteins, a phylogenetic tree was constructed.”

25. L137-143, “According to the analysis, the 75 pomegranate ANS proteins formed 29 paralogous proteins pairs. For example, PgANS7 and PgANS11, PgANS8 and PgANS12, PgANS23 and PgANS38, PgANS19 and PgANS29, PgANS13 and PgANS21, were paralogous proteins pairs. Of these, 21 pairs had 100% support from the 1000 bootstrapping test except for 8 paralogous proteins pairs, including PgANS8 and PgANS12, PgANS23 and PgANS39, PgANS17 140 and PgANS45, PgANS1 and PgANS4, PgANS34 and PgANS38, PgANS71 and PgANS72, PgANS40 and PgANS41, and 141 PgANS53 and PgANS54. This illustrates the robustness of the constructed phylogenetic tree, the similarity in protein sequences of the 21 pairs of pomegranate ANS proteins and their close affinity.”
    Since the phylogenetic tree was constructed based on the protein sequences (based on method and first sentence of section 3.3), please elaborate everything in the aspects of proteins instead of genes. The use of combined terminology “paralogous homologous genes” is also incorrect, the two terms “paralogous” and “homologous” should be mentioned separately.

26. L149-150, “Figure 1. (a) Phylogenetic tree of the pomegranate ANS proteins and (b) Genetic structure of the pomegranate ANS genes. UTR represents untranslated region, while CDS represent coding sequence.”

27. L153, “… and mapped by using the …”

28. L157, “… to colored anthocyanidins …”

29. L159-160, “Analysis of the conserved motifs of the pomegranate ANS proteins was conducted by using the online tool MEME, which yielded 10 potentially conserved motifs (Figure 2-b), …”

30. L161, “… were named as Motif 1-Motif 10.”

31. L163-172, please change all name PgANSXX to non-italicized format PgANSXX as here refer to the proteins, not the genes.

32. L174-175, change both to “… pomegranate ANS proteins”, remove the terms “gene”

33. L197, “… RNA-seq data …”. L198, “we acquired a total of 1,200,021,858 …”

34. Verification from the authors is required for section 3.7 (a new section added in the current manuscript version). Is the GO classification done using the 75 pomegranate ANS genes only or all the DEGs identified in the transcriptome data? If former, I do not understand why the ANS genes can be distributed across different gene functions. Besides, the sum of the total gene numbers in Figure 4 had exceeded 75. Lastly, in the method section, the authors mentioned that GO enrichment analysis was done. An enrichment test is different from basic GO classification, where the data shown in Figure 4 is unlikely an enrichment test for the transcriptome data.

By the way, I think that this GO classification section will not bring any extra value for the manuscript. In addition, the authors had clearly specified the functions of ANS genes in anthocyanins accumulation and responsible for the coloring of fruits and flowers, therefore, GO classification for these genes is unnecessary. I suggest to delete this newly added section.

35. L213, “Out of 75 aforementioned pomegranate ANS genes, we only detected the expression of 64 genes in the transcriptome datasets, while …”

36. L216, “A total of 21 genes (please list down all 21 genes) had similar …”. L218, “This suggested that most PgANS genes had ...”

37. L220-224, how about the expression of ANS genes at T1 and T2 when comparing the three varieties? Please elaborate before comparing the three varieties at T3 and variant-specific should be emphasized here.

38. L229, “Anthocyanidin Synthase (ANS), a key enzyme in the later stages of anthocyanidin synthesis pathway, has received widespread attention.”

39. L234, “ANS catalyzes …”

40. L238, “… pomegranate ANS genes were screened …”

41. L240, “… pomegranate ANS proteins contain …”

42. L245-247, please refer to comment #34.

43. L265, “… related to ANS in fruit of …”

44. L267, “… anthocyanidin synthesis pathway.”

45. Data Availability Statement is not available. Please upload the RNA-seq data into a suitable database such as NCBI SRA, and provide the accession number in Data Availability Statement (please refer to Horticulturae authors guideline and instructions).

Author Response

Dear Reviewers.
Thank you once again for your suggestions on our article, and we have revised the article according to your suggestions. The details are in Word.

Best wishes，

Huihui Ni

Author Response File: Author Response.docx