PONE-D-21-24817

Genome-wide identification and expression profiling of durian CYPome related to fruit ripening

PLOS ONE

Dear Dr. Sirikantaramas,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please address all points raised by the three reviewers. In addition, please also address the following:

• For expression analyses, be aware that fold-change values are not normally distributed and thus error bars should represent the upper and lower bounds of the confidence interval, rather than standard deviation/error, and statistical analyses should not assume normal distribution (or be performed on log fold change values which are normally distributed). If fold change is retained, explain clearly in the legend what comparison the fold change represents. 
• Include (or cite another paper that includes) the primers for the reference genes and explain how both were used (e.g. was the geometric mean of both housekeeping genes used, or were these used alone as individual reference genes in different experiments?).
• There is displacement of some letters in gene names in S5.

Please submit your revised manuscript by Oct 30 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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PLOS ONE

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"This research was funded by Chulalongkorn University (grant number GRU 6203023003-1) to S.S.

S.S. and H.S. acknowledge financial support from Franco-Thai Mobility Programme/PHC SIAM 2021- 2022 international exchange program N°46969PF. N.S. is supported by the Second Century Fund (C2F), Chulalongkorn University."

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: I Don't Know

Reviewer #3: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In the current work, Suntichaikamolkul et al. present a deep mining of the Durian genome and transcriptome libraries, leading to the identification of P450s that are potentially involved in Durian fruit ripening. They provide insight regarding the probable activity of the different P450 clans.

Major comments:

References for the bioinformatics tools used in this analysis (SATR, tblastn, MEME, htseq clustalW etc) should be included in the materials and methods secion.

Fig 1.: The legend lacks necessary information, including whether or not the tree is rooted, which external group was used, etc. Also, the leaves could be colored to be easier to read.

Fig 2.: More detail in the legend would help to explain the figure. Is it showing the degree of conservation of the Durian P450 for each group? How did you compute the degree of conservation? Furthermore, it would be interesting to redo the same figure with Arabidopsis P450 to be able to compare the motif conservation between Durian and Arabidopsis P450s.

The heat-map in Supplementary Fig. S4 should be included in the main body of work. However, it also needs more detail—how did you define these 5 clusters and which method did you use to produce them?

In regard to Line 237 “The expression profiles of nine randomly selected DzP450s were validated by RT-qPC:” Why did you not choose at least one or two genes within the clusters that you defined in Fig. S4? The C cluste is over-represented in your figure, and we do not know if the profiles for the other cluster are consistent?

Fig. S5: The legend needs to be clearer. You included the “ratio of RPKM-normalized read counts.” Is this an RPKM or a ratio of RPKM, and, if it is a ratio, what did you use as the reference?

Sup. Fig. S6: It is difficult to understand why the RPKM is between -1 and 1 because the legend is not clear.

Line 344 "CYP83 is involved in auxin oxidation:" This part only includes published information from other species and no Durian experiments, so you cannot make a claim regarding Durian CYP83 involvement in auxin oxidation. Furthermore, the authors stated that CYP83 has been lost in several plants, so you cannot truly know what the role of this gene is in Durian, as it could have a totally new activity.

Fig. 4C: Is this a fold change or an RPKM? The column title and the color scale legend are not in agreement. Also, the blue and red labels in Fig. 4A and 4B need more explanation, along with 4C.

In the section "Durian CYPome reveals phytohormone-crosstalk in the regulation of fruit ripening," there are no results from your analysis. Therefore, this should be included in the conclusion or another part.

Line 347 “This family has been reported to be lost from several plant genomes, such as rice, tomato, apple, grape, cucumber, strawberry, and watermelon:” if it has been reported you need to include the references.

Fig. S7: You need to include the method that was used. Can you add a table with the protein ID used in these different phylogeny tree? Is the tree rooted, and, if yes, with which species? How many branches are on the tree? How did you compute these bootstraps?

Reviewer #2: In this manuscript, the authors provide valuable data regarding the molecular mechanisms putatively regulating the ripening process of durian fruit ripening that may serve as a starting point for further studies involving functional characterization of the highlighted genes presented here. A better understanding of these mechanisms could lead to different biotechnological applications, positively impacting post-harvest shelf life and other organoleptic desirable traits.

Most of my comments focus on helping to make the text clearer and more consistent. However, the authors need to pay close attention to the statements made, particularly in the results and discussion section. In general, I suggest that authors change to a more moderate writing style, especially when speculating, which is not accompanied by additional data that clearly supports the speculations.

General comments:

-When the function of a CYP is cited, be careful to always mention in what species this function was studied.

-The expression “ripening stages” used in lines 251, 361, 428 and others is very ambiguous and seems to be used as synonym of ripe developmental stage and late developmental stages in some context, while in the legends is used to refer together to the five ripening stages analyzed.

-According to PLOS Data policy, all the single measurements used for Figure 3 must be available in a table.

Introduction:

- Line 44: Please change “Fruit qualities such as flavor, odor, and color” by “Fruit organoleptic properties such as flavor, aroma, and color.”

- Lines 64-66: Please avoid the repetitive use of “therefore.”

- Line 78:

- Lines 82-83: I would suggest replacing the expression “ripening repressor” and “ripening activator” with the relation of both compounds with ethylene since these expressions sound too broad.

- Lines 86-89: I suggest rewriting this sentence since it is unclear where or when this P450 burst appears and how it is related to phytohormone crosstalk.

Materials and methods:

- Line 92: Please mention which cultivars correspond to slow- and quick-postharvest in one sentence and not in different sections throughout the text.

- Line 95: immature1

- Line 96: harvest stage (mature?)

- Line 98: Please mention briefly the firmness values used for defining each developmental stage.

-It sounds a bit confusing that three biological replicates were collected in the “Plant material” section. However, in the following section, it is mentioned that five biological replicates were employed. I suggest clarifying that these three biological replicates were stored at -80 °C for qRT-PCR analysis.

- Regarding ethephon and 1-MCP treatments: it is unclear the experimental design. Five replicates were used for each treatment (15 fruits in total?). It is also not mentioned the developmental stage of treated fruits.

Results:

- Please indicate in the legend of figure 3, if the data presented correspond to the mean, and if the error bars correspond to standard deviation or standard error. Besides, indicate the number of biological replicates employed.

- I do not understand the idea of Figure 3C. Could you please clarify what you expect to show/demonstrate with this figure, or explain why the difference of expression level at the R stage between PM/CN and MT/KY is biologically relevant for elucidating the putative role CYPs during the ripening of durian fruit? I think that the pattern of expression of each CYP during the ripening process is more relevant than the total level of expression at the ripe stage. For example, “gene A” has a very high expression level at the ripe stage, but this level remained invariable during the whole ripening process. On the other hand, “gene B” has a lower level of expression compared to “gene A” at the ripe stage; however, the expression of “gene B” has gradually and significantly increased/decreased throughout the ripening process. Could you support that “gene A” is more relevant for the ripening process than a “gene B” based just on their expression level at the ripe developmental stage?

- Also, regarding figure 3, please homogenize “a” as the lowest value and “b” or “c” as the highest. Please, present the data in the order that it is mentioned in the text.

- I have a concern regarding the statistical analysis. Tukey HSD assumes a normal distribution. Was it tested?

-Figure 3B, section CYP88A: Is there no difference between IM1, IM2, M, and MR? or between IM1, IM2, and R in the CYP714E section?

-Lines 311-312: With the provided data, it is impossible to demonstrate that DzCYP88A is involved in GA biosynthesis and plays an essential role in accelerating the fruit ripening process in durian. For this, at least a functional characterization of DzCYP88A must be performed, including substrate tolerance assays and up or downregulation assays by agroinfiltration. Moreover, is there any evidence of the role of GA in durian fruit ripening??

-Line 337: It seems that only CYP714E is upregulated.

-I am not an expert in durian, but as a climacteric fruit, the ripening of durian must be characterized by a burst of ethylene. How would you explain that CYP714E is upregulated during the ripening process but is negatively regulated by the addition of ethephon?

-Line 343: Is there any evidence to support this sentence?

-Line 374: Is there any evidence to support that JA-Ile is a negative regulator of durian ripening?

-Lines 414-46: I am confused by these sentences. First, you present ABA as a positive regulator of ripening in tomato and banana. However, in these two sentences, you say that CYP707A (involved in ABA inactivation) might play a role in accelerating the ripening process.

-Line 425: Is there metabolomic data available to support the affirmation that GA content decreases during durian ripening?

-Line 426: positively regulates.

-Lines 425-429: These assumptions contradict those stated previously in lines 304-312. First, you presented CYP88A as a key gene for GA biosynthesis, induced by ethephon and highly expressed at the ripe developmental stage (stage characterized by high levels of ethylene), speculating with a role of GA in the ripening process. Now, your crosstalk GA-ABA model is based on a decrease of GA content during the ripening.

Reviewer #3: Authors have identified 355 genes related to the cytochrome P450 gene family in Durio zibethinus and were further distributed in 10 groups consisting of 56 families. Additionally, they have also characterized the motifs and phylogenetic analysis. They have further performed the expression analysis and have validated some selected genes using the real-time PCR. The findings of current study represent an important step towards comprehending the molecular regulation of CYPs related to durian fruit ripening. However, the main problem was in the introduction and result and discussion parts. I wrote some my suggestions about this sections;

1. Introduction part; Authors should explain the significance of this study at the end of Introduction part.

2. Materials and methods part; Authors should explain Statistical Analysis in the Materials and methods part.

3. Lines 169-170, 174,…… When providing location details for a vendor, both city and country are mentioned (city, country). In case of US-based vendors, city, state suffice.

4. For the gene family expansion and evolution of novel functions, gene duplication and divergence are essential steps in the plant genome. Authors should add these researches related to Gene Duplication and Syntenic Analysis in revised manuscript.

5. Authors should carefully recheck the manuscript for scientific styles. There are some grammar mistakes; a detailed revision for English is necessary.

6. Authors should consider adding one short paragraph with a conclusion.

7. In the result and discussion section, the authors should add or discuss the following research papers:DOI：10.1007/s13258-013-0170-9 (Genome-wide identification, annotation and characterization of novel thermostable cytochrome P450 monooxygenases from the thermophilic biomass-degrading fungi Thielavia terrestris and Myceliophthora thermophila); DOI: 10.3389/fgene.2020.00044 (The Cytochrome P450 Monooxygenase Inventory of Grapevine (Vitis vinifera L.): Genome-Wide Identification, Evolutionary Characterization and Expression Analysis); DOI：10.1186/s12864-017-4425-8 (Global identification, structural analysis and expression characterization of cytochrome P450 monooxygenase superfamily in rice)

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Reviewer #1: Yes: Axel Poulet

Reviewer #2: No

Reviewer #3: No

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PONE-D-21-24817R1Genome-wide identification and expression profiling of durian CYPome related to fruit ripeningPLOS ONE

Dear Dr. Sirikantaramas,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not yet fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please address the following points prior to resubmission:

• Table S7a, S7b, S7c should be separate supp tables (S7, S8, S9).
• Fig 1 legend (L223 track changes version) – please reword to clarify what you mean by “bars in the trees”
• Fig 4
  • Bar charts are not logical with these x axes (esp. as y intercept not at 0) – please change to an appropriate alternative (e.g. scatter charts, box plots, violin plots)
  • Please make the x axis labels consistent (can also remove unnecessary repetition) – e.g. “Relative Expression (log FC)” together with an explanation of calculation in the methods would be sufficient.

Please submit your revised manuscript by Dec 25 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Frances Sussmilch

Academic Editor

PLOS ONE

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Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In the current work, Suntichaikamolkul et al present a deep mining of the Durian genome and transcriptome libraries leading to the identification of all P450s that are potentially involved in Durian fruit ripening. They provide insight of the probable activity of the different P450s clan. In this current version all the comments made during the first round of review has been addressed.

Reviewer #2: (No Response)

Reviewer #3: (No Response)

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Axel Poulet

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Genome-wide identification and expression profiling of durian CYPome related to fruit ripening

PONE-D-21-24817R2

Dear Dr. Sirikantaramas,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Frances Sussmilch

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: