Comprehensive Histological, Endogenous Hormone, and Transcriptome Analysis to Reveal the Mechanism of Hormone Regulation Mediating Pepper (Capsicum annum L.) Fruit Size
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsTang et al performed a study about the molecular and hormone mechanism behind the pepper fruit size. The study was well conducted and the manuscript is well written. I really enjoyed reading the paper. Congratulations. I have only minor comments:
1.- Please add the scientific name of the species. The name "pepper]" is frequently used for plant species from the genus Capsicum and Piper. I guess you are working with Capsicum annum, but please confirm this in the manuscript.
2.- It was difficult to understand the supplementary figures because the figure legends were not included.
3.- Line 273: Use uppercase for the number 2 in mm2
4.- For the heatmaps, indicate the color scale units. Is is z-score?
5.- I suggest to include a model in order to integrate the results obtained in the present study with the literature.
Author Response
The first reviewer’s comments: Tang et al performed a study about the molecular and hormone mechanism behind the pepper fruit size. The study was well conducted and the manuscript is well written. I really enjoyed reading the paper. Congratulations. I have only minor comments: 1. Please add the scientific name of the species. The name "pepper" is frequently used for plant species from the genus Capsicum and Piper. I guess you are working with Capsicum annum, but please confirm this in the manuscript. Response: Thanks for pointing this out. The scientific name of pepper (Capsicum annum L.) has been added to the manuscript. (Lines 4, 40) 2. It was difficult to understand the supplementary figures because the figure legends were not included. Response: Thank you for your comment. The figure legend of the supplementary figures is located in the 'Supplementary Materials ' chapter of the manuscript. (Lines 714-729) 3. Line 273: Use uppercase for the number 2 in mm2 Response: Thanks for pointing this out. The problem was corrected in the revised manuscript. (Lines 250) 4. For the heatmaps, indicate the color scale units. Is is z-score? Response: Sorry for the confusion. The data in the heat map of manuscript were normalized (row z-score), and the color scale units are z-score. Color scale units were added to the revised figures. 5. I suggest to include a model in order to integrate the results obtained in the present study with the literature. Response: Thank you for your valuable advice. In the conclusion chapter, we add a model to show the results of the study. (Lines 707)
Author Response File: Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsGeneral remark to AUTHORS:
- Authors used 58 different literature sources in this Manuscript, which is quite a large quantity.
- From 58 different literature sources used, 18 references or 31% belong to the period from 2021-2025 years. 12 references or 21% belong to the period from 2016-2020 years. And even 28 references or 48% belong to earlier periods of 2015 year.
- Almost 50% of the literature is older than 2015, or older than ten years.
- It is quite contradictory to write the latest research and use older literature.
- Only in the 11 literature references "Pepper" is the main subject of investigation.
- Also in the 11 literature references "Tomato" is the main subject of investigation.
- If Authors investigation is addressed to pepper that must been visible also from literature references where main subject of investigation is also pepper.
- Due to the importance of the topic and this research, it would be desirable for the Authors to find minimum 4 literature references on the pepper with the same research topic (as is this investigation) from the period 2021-2025. Two references write in the Chapter 1.Introduction and two references write in the Chapter 4.Discussion.
Rows 40 to 107 (in:Introduction)
Authors wrote: Content of Introduction.
Reviewer: find 2 new literature references on the pepper with the same research topic (as is this investigation) from the period 2021-2025 and write it in the Introduction.
Reviewer: Please, correct this remark.
Row 157 (in: 2.3 Hormone quantification)
Authors wrote: ....previously described by Liu et al. [32].
Reviewer: Delete: "by Liu et al".
Reviewer: write: ...described [32].
Reviewer: Please, correct this remark.
Row 179 (in: 2.5 Integrative phytohormone and transcriptome analysis)
Authors wrote: .... by referring to the method of Liu et al. [1]....
Reviewer: Delete: "referring to the method of Liu et al.".
Reviewer: write: ... by the cited method [1].
Reviewer: Please, correct this remark.
Row 203 (in: 2.7 RT-qPCR analysis)
Authors wrote: .... referenced by Liu et al. [32]....
Reviewer: Delete: " Liu et al.".
Reviewer: write: ... by the cited method [32].
Reviewer: Please, correct this remark.
Rows 678 to 839 (in:Discussion)
Authors wrote: Content of Discussion.
Reviewer: find 2 new literature references on the pepper with the same research topic (as is this investigation) from the period 2021-2025 and write it in the Introduction.
Reviewer: Please, correct this remark.
Row 708 (in: Discussion)
Authors wrote: .... Recently, Han et al. [42] found....
Reviewer: Delete: " Han et al. [42]"
Reviewer: write: ... Recently was found...
Reviewer: Please, correct this remark.
Row 710 (in: Discussion)
Authors wrote: .... during fruit softening.
Reviewer: write: " during fruit softening [42]."
Reviewer: Please, correct this remark.
Row 835 (in: Discussion)
Authors wrote: Borovsky et al. [58] found....
Reviewer: Delete: " Borovsky et al. [58] "
Reviewer: write: "Earlier was found..."
Reviewer: Please, correct this remark.
Row 836 (in: Discussion)
Authors wrote: .... of pepper fruit.
Reviewer: write: " of pepper fruit [58]."
Reviewer: Please, correct this remark.
Row 838 (in: Discussion)
Authors wrote: ...those of Borovsky et al. [58]....
Reviewer: Delete: " those of Borovsky et al. [58]"
Reviewer: write: ..."with mentioned above"...
Reviewer: Please, correct this remark.
Row 839 (in: Discussion)
Authors wrote: .... in this study.
Reviewer: write: " in this study [58]."
Reviewer: Please, correct this remark.
Comments for author File: Comments.pdf
Author Response
The second reviewer’s comments:
Authors used 58 different literature sources in this Manuscript, which is quite a large quantity. From 58 different literature sources used, 18 references or 31% belong to the period from 2021-2025 years. 12 references or 21% belong to the period from 2016-2020 years. And even 28 references or 48% belong to earlier periods of 2015 year. Almost 50% of the literature is older than 2015, or older than ten years. It is quite contradictory to write the latest research and use older literature. Only in the 11 literature references "Pepper" is the main subject of investigation. Also in the 11 literature references "Tomato" is the main subject of investigation. If Authors investigation is addressed to pepper that must been visible also from literature references where main subject of investigation is also pepper. Due to the importance of the topic and this research, it would be desirable for the Authors to find minimum 4 literature references on the pepper with the same research topic (as is this investigation) from the period 2021-2025. Two references write in the Chapter 1. Introduction and two references write in the Chapter 4. Discussion.
Response: Thank you for your valuable advice. We added two references (period 2021-2025) related to pepper fruit size in the introduction and discussion chapters, respectively. (Line 100-105, Line 561-563, Line 611-612)
- find 2 new literature references on the pepper with the same research topic (as is this investigation) from the period 2021-2025 and write it in the Introduction.
Response: Thank you for your valuable advice. We added two references (period 2021-2025) related to pepper fruit size in the introduction chapters. (Line 100-105)
- Authors wrote: ....previously described by Liu et al. [32].
Response: Thank you for your comment. It has been corrected in the revised manuscript. (Line 164)
- Authors wrote: .... by referring to the method of Liu et al. [1]....,Reviewer: Delete: "referring to the method of Liu et al.".
Response: Thank you for your comment. It has been corrected in the revised manuscript. (Line 186)
- Authors wrote: .... referenced by Liu et al. [32]...., Reviewer: Delete: " Liu et al.".
Response: Thank you for your comment. It has been corrected in the revised manuscript. (Line 209)
- Reviewer: find 2 new literature references on the pepper with the same research topic (as is this investigation) from the period 2021-2025 and write it in the discussion.
Response: Thank you for your valuable advice. We added two references (period 2021-2025) related to pepper fruit size in the discussion chapters. (Line 561-563, Line 611-612)
6.Authors wrote: .... Recently, Han et al. [42] found....,Reviewer: Delete: " Han et al. [42]"
Response: Thank you for your comment. It has been corrected in the revised manuscript. (Line 543)
- Authors wrote: .... during fruit softeningï¼›Reviewer: write: " during fruit softening [42]."
Response: Thank you for your comment. It has been corrected in the revised manuscript. (Line 545)
- Authors wrote: Borovsky et al. [58] found....ï¼›Reviewer: Delete: " Borovsky et al. [58] "ï¼›Reviewer: write: "Earlier was found..."
Response: Thank you for your comment. It has been corrected in the revised manuscript. (Line 673)
- Authors wrote: .... of pepper fruit; Reviewer: write: " of pepper fruit [58]."
Response: Thank you for your comment. It has been corrected in the revised manuscript. (Line 673-674)
- Authors wrote: ...those of Borovsky et al. [58]....; Reviewer: Delete: " those of Borovsky et al. [58]"; Reviewer: write: ..."with mentioned above"...
Response: Thank you for your comment. It has been corrected in the revised manuscript. (Line 676)
- Authors wrote: .... in this study; Reviewer: write: " in this study [58]."
Response: Thank you for your comment. It has been corrected in the revised manuscript. (Line 677)
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThe manuscript by Sijie Tang et al. aims to analyze the molecular mechanism underlying pepper fruit size differences from the perspectives of histology, physiology, and transcriptomics.
This study is well designed and conducted, and the results obtained have the potential to be used in the future breeding of pepper. However, there are several issues that need to be addressed to improve the readability and presentation of the results.
- please explain how this two recombinant pepper lines were obtained and why exactly these two lines were selected for this analysis (despite the size of fruit) ?
- in paragraph 2.2 „Histological analysis of fruit pericarp tissue”, the microscope brand and magnification should be indicated and also in the Figure 2 a.
- line 142 – the type of microtome should be mentioned
- how many slides were observed under the microscope per line and per 10, 20, and 30 DAA respectively?
- why the clean reads were mapped to the pepper (‘Zunla’) reference genome within RNA sequencing?
- please add some explanations regarding the significant difference between the expression patterns of the transcription factors in the modules between the two analzed pepper varieties. Why Authors assumed that these transcription factors may be candidate gene targets for regulating pepper fruit size.
- at the end of Discussion chapter should be add at least few sentences about the directions of further exploring the molecular regulation mechanism of pepper fruit growth and size.
Author Response
The third reviewer’s comments:
The manuscript by Sijie Tang et al. aims to analyze the molecular mechanism underlying pepper fruit size differences from the perspectives of histology, physiology, and transcriptomics.This study is well designed and conducted, and the results obtained have the potential to be used in the future breeding of pepper. However, there are several issues that need to be addressed to improve the readability and presentation of the results.
- please explain how this two recombinant pepper lines were obtained and why exactly these two lines were selected for this analysis (despite the size of fruit)?
Response:Two pepper varieties were derived from the germplasm resource bank of Hunan Vegetable Research Institute (Line 128). During the previous field experiment, the fruits of QB6 and CXJ82 varieties completed enlargement on the 30 th day after flowering. Furthermore, On the 50 th day after flowering, the fruits of the two varieties completed the color conversion, and the peel showed red. The QB6 and CXJ82 varieties showed consistent maturity, which helped to eliminate the effect of growth duration on fruit size. Therefore, QB6 and CXJ82 were identified as the research materials for this study.
- in paragraph 2.2 “Histological analysis of fruit pericarp tissue”, the microscope brand and magnification should be indicated and also in the Figure 2 a.
Response:Thanks for pointing this out. The microscope brand (NIKON Eclipse E100, Japan) and magnification (20 ⅹ) have been added in the revised manuscript and Figure 2 a. (Line 149-150)
- line 142, the type of microtome should be mentioned.
Response:Thank you for your advice. The type of microtome (Leica, Wetzlar, Germany) have been added in the revised manuscript. (Line 147)
- how many slides were observed under the microscope per line and per 10, 20, and 30 DAA respectively?
Response: Three biological replicates were performed on two pepper varieties at each fruit development stage. Six cells were randomly selected from each cell layer (First, Sixth, Ninth layer) for area and length measurement. (Line 150-152)
- why the clean reads were mapped to the pepper (‘Zunla’) reference genome within RNA sequencing?
Response: Thank you for your comment. Because ‘Zunla’ is one of the earliest sequenced pepper varieties, its assembled genome quality is excellent in continuity, integrity and accuracy. Furthermore, it belongs to Capsicum annum cultivated species with QB6 and CXJ82.
- please add some explanations regarding the significant difference between the expression patterns of the transcription factors in the modules between the two analzed pepper varieties.
Response:Weighted gene co-expression network analysis is mainly used to identify co-expression modules related to phenotypes, and genes in the same module exhibit the same expression pattern. In the 3.8 WGCNA chapter, we describe the expression patterns of genes in the blue, brown, green, and black modules (Line 467-470). The genes in the blue and brown modules were highly expressed in QB6 fruit, while the genes in the green and black modules were highly expressed in CXJ82 fruit. The expression pattern of the module can represent the expression pattern of all genes in the whole module, including transcription factors. Therefore, in the 3.8 WGCNA section, we did not further emphasize the expression pattern of transcription factors in each module, which may lead to redundancy.
- Why Authors assumed that these transcription factors may be candidate gene targets for regulating pepper fruit size.
Response:(1) The blue and brown modules were positively correlated with QB6 fruit samples (large fruit type), and the genes in these two modules were highly expressed in QB6 fruit (Figure 8 and Figure S8). The green and black modules were positively correlated with CXJ82 fruit samples (small fruit type), and the genes in these two modules were highly expressed in CXJ82 fruit (Figure 9 and Figure S8).
(2) We only constructed a regulatory network of highly expressed (FPKM > 40) transcription factors in four modules. The higher transcriptional abundance of transcription factors in QB6 or CXJ82 fruits also suggests that they play a more important role in regulating fruit size.
(3) In the regulatory network, these transcription factors showed a high correlation with multiple cell division, cell wall polysaccharide metabolism, and nucleotide metabolism-related genes.
Therefore, in the above four modules, transcription factors with high expression and high correlation with multiple cell division, cell wall polysaccharide metabolism, and nucleotide metabolism-related genes were identified as candidate genes for regulating pepper fruit size.
- at the end of Discussion chapter should be add at least few sentences about the directions of further exploring the molecular regulation mechanism of pepper fruit growth and size.
Response:At the end of the discussion chapter, the research direction of further exploring the regulation mechanism of pepper fruit size has been added (Line 677-680). “In the next step, functional validation of transcription factors and hormone signal transduction factors in the regulatory network will be conducted, which will include gene silencing and complementation analysis, to determine their roles in regulating fruit growth and fruit size.”
Author Response File: Author Response.pdf