Comparative Proteomics Analysis of Primulina serrulata Leaves Reveals New Insight into the Formation of White Veins
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsDear Editors and Authors,
I read with interest the manuscript entitled “Comparative proteomics analysis of Primulina serrulata leaves reveals new insight into the formation of white veins”. In this study, we determined the Chl content of leaves and carried out a proteomic experiment to identify the differentially expressed proteins (DEPs) between WV plants and the control and characterize the role of DEPs in WV coloration. The subject of the article is important and has great relevance for the scientific environment of the study area. Therefore, the manuscript needs some adjustments so that it can then be forwarded to the publication process. The manuscript has the potential for publication in this journal Horticulturae and needs the following adjustments:
ABSTRACT
- The objective described in the Abstract must be similar to that described in the Introduction;
- Exclude repeated keywords in the title.
INTRODUCTION
- There is no need to start by mentioning that leaves are the organs of photosynthesis. This is obvious.
- This section needs to be expanded. There are only 4 short paragraphs.
MATERIAL AND METHODS
- Where was the study carried out? Cite location information.
- Mention the formulas used to quantify chlorophyll levels.
- Why weren't carotenoids measured?
RESULTS
- Don't you have the figures with Chla and Chlb?
- All results described must be followed by a Figure or Table citation at the end of the sentence. Review this throughout the text.
DISCUSSION
- This section needs to be reviewed carefully. There is a lot of generic information that needs to be related to the study. Review.
CONCLUSIONS
- Rewrite with more objective information.
- Some excerpts are already described in Resutlados and do not need to be repeated here.
Author Response
ABSTRACT
- The objective described in the Abstract must be similar to that described in the Introduction;
--- In the abstract, we incorporated a similar goal, stating: “our objective is to offer novel insights to assist in the breeding of WV plants”.
- Exclude repeated keywords in the title.
--- We omitted the repeated keyword 'comparative proteomics' and introduced additional keywords, including “differentially expressed proteins”, “functional enrichment analysis”, and “protein interaction network”.
INTRODUCTION
- There is no need to start by mentioning that leaves are the organs of photosynthesis. This is obvious.
--- This particular state has been removed from the document.
- This section needs to be expanded. There are only 4 short paragraphs.
--- The Introduction section has been extensively expanded, focusing primarily on aspects related to white coloration.
MATERIAL AND METHODS
- Where was the study carried out? Cite location information.
--- The chlorophyll determination was conducted at Zunyi Normal College, Guizhou Province, China, and the proteomic analysis was performed at BioNovoGene Company in Suzhou City, Jiangsu Province, China. These details have been incorporated into the manuscript.
- Mention the formulas used to quantify chlorophyll levels.
--- The formulas have been incorporated into the manuscript.
- Why weren't carotenoids measured?
--- We believed that carotenoids are directly responsible for the vivid yellow, orange, and red colors in plants. However, our focus was on coloration of white and green, which we hypothesized to be affected by chlorophyll biosynthesis. Consequently, we specifically measured the chlorophyll content.
RESULTS
- Don't you have the figures with Chla and Chlb?
--- We have included a new figure (Figure 2) that illustrates Chla and Chlb.
- All results described must be followed by a Figure or Table citation at the end of the sentence. Review this throughout the text.
--- Three supplemental tables have been included in our documentation. Due to the voluminous nature of some data sets, we have relegated these to the supplemental materials. Specifically, Table S1 lists 6,261 differentially expressed proteins (DEPs), Table S2 features 399 DEPs filtered by a log2 fold change (log2FC) of less than -0.58 or greater than 0.58, and Table S3 presents 69 DEPs screened by a fold change greater than 1.5.
DISCUSSION
- This section needs to be reviewed carefully. There is a lot of generic information that needs to be related to the study. Review.
---We have improved this section.
CONCLUSIONS
- Rewrite with more objective information.
--- We have rewritten this section to include more objective information.
- Some excerpts are already described in Resutlados and do not need to be repeated here.
--- The repetitive description has been removed.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe manuscript horticulturae-2758817 describes a TMT-labeled proteomics-based analysis to examine the protein-level biogenesis of white-veined (WV) leaves of Primulina serrulata. The manuscript is well-written, interesting, and has relevant results for readers. However, some issues should be addressed before further consideration.
1. Lines 39-40: Revise this passage since it seems to be confusing.
2. Line 69: Details of the collection site to obtain P. serrulata leaves must be provided. In addition, the status of the parent plants (e.g., age, phytosanitary, strata, etc) and if the plants were wild or cultivated must also be provided.
3. Line 72: Particular experimental details of Chl quantification following the reported method should be informed. For instance, plant material amount.
4. Line 73: The number of biological replicates must be informed.
5. Line 75: The sample amount must be informed. Additionally, the number of technical replicates must be informed.
6. Line 109: The brand, model and type of MS instrument should be informed.
7. In general, full experimental details must be provided in the M&M section to ensure reproducibility.
8. Line 141: The statistical processing to obtain the DEPS and other statistical analyses must be informed. For instance, the details of building the volcano plot (Figure 3) are missing.
9. Line 160: A deeper description of the results in Figure 3 can be added.
1 10. The discussion section can be improved since it summarizes results and seems to be a list of ideas without connectivity. I recommend building a better-organized discussion.
Comments on the Quality of English LanguageDetailed scrutiny to revise some grammar and stylistic issues should be performed throughout the manuscript.
Author Response
- Lines 39-40: Revise this passage since it seems to be confusing.
--- The first paragraph of the Introduction section has been rewritten.
- Line 69: Details of the collection site to obtain P. serrulata leaves must be provided. In addition, the status of the parent plants (e.g., age, phytosanitary, strata, etc) and if the plants were wild or cultivated must also be provided.
--- Details about the collection site for P. serrulata leaves are included in the Materials and Methods section. Information regarding the status of the parent plants is also provided in the same paragraph.
- Line 72: Particular experimental details of Chl quantification following the reported method should be informed. For instance, plant material amount.
--- We have included the formulas used for quantifying chlorophyll levels, along with the fresh leaf weight, which is 0.5 grams.
- Line 73: The number of biological replicates must be informed.
--- There were three biological replicates for each group, and the related description has been revised.
- Line 75: The sample amount must be informed. Additionally, the number of technical replicates must be informed.
--- The quantity of the sample used (100 μg) is detailed in the manuscript as follows: “Next, 100 μg of protein solution was added to DTT to a final concentration of 10 mM”.
--- Due to the expensive cost of proteomics testing, we did not perform technical replicates.
- Line 109: The brand, model and type of MS instrument should be informed.
--- Mass spectrometry (MS) analysis was performed using an Easy-nLC 1200 Liquid Chromatography System and an Orbitrap Fusion Lumos Mass Spectrometer (Thermo Fisher Scientific, USA). This description has been incorporated into the manuscript.
- In general, full experimental details must be provided in the M&M section to ensure reproducibility.
--- The detailed experimental process has been thoroughly revised.
- Line 141: The statistical processing to obtain the DEPS and other statistical analyses must be informed. For instance, the details of building the volcano plot (Figure 3) are missing.
--- A new paragraph detailing the statistical processing used to identify the DEPs and generate the volcano plot has been added to the manuscript.
- Line 160: A deeper description of the results in Figure 3 can be added.
--- A deeper description of the results in Figure 3 (now Figure 4) has been added.
- The discussion section can be improved since it summarizes results and seems to be a list of ideas without connectivity. I recommend building a better-organized discussion.
--- We have improved the discussion section.
Reviewer 3 Report
Comments and Suggestions for AuthorsThe manuscript “Comparative proteomics analysis of Primulina serrulata leaves reveals new insight into the formation of white veins” by Dou et al sent for publication to Horticulturae deals with interesting topic such TMT-labeled proteomics analysis and white vein formation in P. serrulata. The manuscript will be of interest to the scientific community working on the topic of ornamental plants and breeding. The present work deserve to be published after addressing some comments listed below:
Minor remarks:
L99: I’m not sure the word “desaulted “ is correct.
L129: “RAN-seq” should be replaced by “RNA-seq”
The english language should be verified by native speaking person.
Introduction:
The introduction is well written and point the main problems related to the topic.
Materials and methods:
L75: The authors should give the composition of the lysis buffer.
Results:
This part is somehow well written. But I have a question. The authors identified 6261 proteins and inside them only 69 DEPs (25 up- and 44 down regulated). I am surprised about the low numbet of the identified DEPs. Any comments on that?
A table with the 69 DEPs should be added.
In addition, a supplementary table with the all proteins and data should be added. In addition, I am wondering if the authors deposited the proteomics data in a public database (PRIDE etc…) which is the common way of sharing info inside scientific community.
Discussion:
This section is well written and supported by results.
Overall, the manuscript deserved to be published after addressing the comments and improve the manuscript.
Comments on the Quality of English Language
Author Response
L99: I’m not sure the word “desaulted “ is correct.
--- It should be “desalted”. Thank you for your thorough revision.
L129: “RAN-seq” should be replaced by “RNA-seq”
--- We apologize for our oversight, and the issue has been corrected.
The english language should be verified by native speaking person.
--- The manuscript has been polished by a native English speaker.
Introduction:
The introduction is well written and point the main problems related to the topic.
--- Thank you for your positive feedback! However, in response to another reviewer's comment about the simplicity of this section, we have expanded the Introduction.
Materials and methods:
L75: The authors should give the composition of the lysis buffer.
--- Lysis Buffer Composition: 7M Urea, 4% SDS, 30mM HEPES, 1mM PMSF, 2mM EDTA, 10mM DTT, 1X Protease Inhibitor. This lysis buffer composition has been included in the manuscript.
Results:
This part is somehow well written. But I have a question. The authors identified 6261 proteins and inside them only 69 DEPs (25 up- and 44 down regulated). I am surprised about the low numbet of the identified DEPs. Any comments on that?
--- The DEPs with statistical significance were numbered as 399 at first but we pursued a more realistic and accurate approach by applying the BH FDR method to adjust the P-values. In calculating the DEPs, we referenced these adjusted P-values. As a result, many proteins with large fold changes but with P-values greater than 0.05 were not included in the list of differentially expressed proteins.
--- Three supplemental tables have been included in our documentation. Due to the voluminous nature of some data sets, we have relegated these to the supplemental materials. Specifically, Table S1 lists 6,261 DEPs, Table S2 features 399 DEPs filtered by a log2 fold change (log2FC) of less than -0.58 or greater than 0.58, and Table S3 presents only 69 DEPs screened by a fold change greater than 1.5.
--- We have incorporated the relevant contents into the manuscript.
In addition, a supplementary table with the all proteins and data should be added. In addition, I am wondering if the authors deposited the proteomics data in a public database (PRIDE etc…) which is the common way of sharing info inside scientific community.
--- Three supplementary tables have been added.
--- We attempted to deposit the proteomics data using the PRIDE Submission Tool (version 2.7.3), but encountered a failure. The Java program displayed a message indicating a potential issue with the internet connection: “Are you connected to the Internet via a proxy? You can manually update the proxy server details in the config/config.props file present in the px-submission-tool folder”.
Discussion:
This section is well written and supported by results.
--- Thank you for your positive feedback! However, in response to another reviewer's suggestion that this part needed improvement, we have made substantial revisions to this section.
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsDear,
The authors made most of the suggestions proposed in the previous version. Therefore, the manuscript has potential for publication.
