Untargeted Metabolite Profiling of Camellia tetracocca’s Response to an Empoasca onukii Attack Using GC-MS and LC-MS

Round 1

Reviewer 1 Report

The authors have performed GC/MS and LC/MS analysis in order to study metabolite profiles of Empoasca onukii-attacked Camellia tetracocca

Major comments:

1. It was not possible for me to open Supplementary files.

2. Did authors use any of standards for GC-MS or LC-MS analysis? If positive, please add this to the Materials and methods.

Minor comments:

1. few double spaces, please check throughout the manuscript

2. “Figure 2. Total ion current (TIC) chromatogram of damaged leaves attacked by E. onukii of C. tetracocca and healthy leaves”

·        E. onukii should be italic

·        Missing the explanation for E1,2,3 and N1,2,3

3. line 314-15 : L-phenylalanine, L-tyrosine, citrulline, trans-4-hydroxy-L-proline, aspartate, and L-alanine

·        correct the size of letters for the relative configuration (correct throughout the manuscript)

·        trans should be italic (correct throughout the manuscript)

4. line 338: missing the reference Wan and Xia

5. line 364 “we utilized ChemDraw”: this is part for materials and methods

6. line 370: “Arabidopsis thaliana” should be italic

Author Response

Dear reviewer,

 

  Thank you for returning our manuscript entitled “Untargeted metabolite profiling of Camellia tetracocca's response to an Empoasca onukii attack using GC-MS and LC-MS” (Manuscript ID: agronomy-2201368) with valuable comments and suggestions from you. We have revised the manuscript comprehensively based on these comments and suggestions, and here would like to submit the revised manuscript for publication in Agronomy.

   We have improved the manuscript and made some changes in the manuscript. We also responded to all comments point-by-point. The file “manuscript_20230227.docx” is a clean revised manuscript with the track changes. Point-by-point responses to the reviewer are listed below this letter. We hope that our revised paper is suitable for publication in Agronomy.

 

 

    Yours faithfully,

    First author, PhD Ni Zhang

    Corresponding author, Prof. Yuehua Song

Major comments:

1. It was not possible for me to open Supplementary files.

Response：Thank you very much for your suggestion. We have put the figures in the supplementary materials into the manuscript. However, the table of all differential metabolites is too large to be put into the manuscript. Therefore, we can use it as an attached materials.

2. Did authors use any of standards for GC-MS or LC-MS analysis? If positive, please add this to the Materials and methods.

Response：We have add the data processing standards for GC-MS analysis to the Materials and methods.

Minor comments:

1. few double spaces, please check throughout the manuscript.

Response：We are sorry for our carelessness, we have checked the manuscript about double spaces.

2. “Figure 2. Total ion current (TIC) chromatogram of damaged leaves attacked by E. onukiiof C. tetracocca and healthy leaves”

•  E. onukii should be italic.
• Missing the explanation for E1,2,3 and N1,2,3.

Response：  E. onukii has been changed to italic.

           We have added this explanation for E1,2,3 and N1,2,3. E1, E2 and E3 represents the groups of the leafhopper feeding, N1, N2 and N3 represents the groups of leafhopper not feeding.

3. line 314-15: L-phenylalanine, L-tyrosine, citrulline, trans-4-hydroxy-L-proline, aspartate, and L-alanine

• correct the size of letters for the relative configuration (correct throughout the manuscript)
• trans should be italic (correct throughout the manuscript)

Response：Thanks for your suggestion, we have corrected the size of letters for the relative configuration in the manuscript. For instance, l-phenylalanine, l-tyrosine, citrulline, trans-4-hydroxy-l-proline, aspartate, and l-alanine. In addition, we have changed “trans” changed to italic “trans” in the manuscript.

3. line 338: missing the reference Wan and Xia.

Response：We have supplemented the reference and revised it according to the format required by MDPI.

5. line 364 “we utilized ChemDraw”: this is part for materials and methods.

Response：“we utilized ChemDraw”. This sentence has been moved to materials and methods.

6. line 370: “Arabidopsis thaliana”should be italic.

Response：The latin name “Arabidopsis thaliana” has been changed to italic “Arabidopsis thaliana”.

Author Response File: Author Response.pdf

Reviewer 2 Report

The paper presents an untargeted assessment, using GC-MS and LC-MS methods, of the impact of pest damage of Camellia tetracocca by the tea green leafhopper E.onukki. Details of the method of tea leaf cultivation and collection is given. Detail of the untargeted metabolomics methods should be more clearly described. A leading section to explain that samples were divided into two workflows of GC and LC analysis and the differences would help. A figure for this would be good. The sample prep being one section including derviatisation, the separation and methodology on the MS then described. Ideally GC-MS and then LC-MS. Some detail of the chemicals is missing. THe internal standard is not in the chemicals and the amount added unclear in one method. It wasn't clear which method had been used to generate the TIC results shown in Figure 2. This figure doesn't show much other than there are multiple peaks. As there is nothing to draw from it, suggest not needed or amended with peaks identified or Mass Spectra that identifies certain metabolites. 

THere is a lot of focus on fact that you have a GC-MS method and an LC-MS/MS but once you have data you should just be bringing that together and assessing as a full dataset? Does it matter how the data was obtained by this point? 

At the moment some of your figures are total ion chromatograms that don't show any peaks identified but just show a difference. These aren't particularly useful for connecting to the text. They show what the GC-MS method chromatogram looks like, but doesn't show what the actual results are. 

Some of the figures used are Total Ion chromatograms which just show unidentified peaks. Figures that show the data, evidence of particular metabolites in the damaged leaves vs healthy leaves would be more useful. 

Is the figure of structures the metabolites you have identified of interest or is that from the literature. It's not clear to me in the way it has been worded. 

Author Response

Dear reviewer,

 

    Thank you for returning our manuscript entitled “Untargeted metabolite profiling of Camellia tetracocca's response to an Empoasca onukii attack using GC-MS and LC-MS” (Manuscript ID: agronomy-2201368) with valuable comments and suggestions from you. We have revised the manuscript comprehensively based on these comments and suggestions, and here would like to submit the revised manuscript for publication in Agronomy.

    We have improved the manuscript and made some changes in the manuscript. We also responded to all comments point-by-point. The file “manuscript_20230227.docx” is a clean revised manuscript with the track changes. Point-by-point responses to the reviewer are listed below this letter. We hope that our revised paper is suitable for publication in Agronomy.

 

 

    Yours faithfully,

    First author, PhD Ni Zhang

    Corresponding author, Prof. Yuehua Song

The paper presents an untargeted assessment, using GC-MS and LC-MS methods, of the impact of pest damage of Camellia tetracocca by the tea green leafhopper E.onukki.

1. Details of the method of tea leaf cultivation and collection is given.

Response：We have supplemented the method of tea leaf cultivation and collection.

2. Detail of the untargeted metabolomics methods should be more clearly described. The sample prep being one section including derviatisation, the separation and methodology on the MS then described.Ideally GC-MS and then LC-MS. Some detail of the chemicals is missing. The internal standard is not in the chemicals and the amount added unclear in one method.

Response: Thank you very much for your suggestion. We have added the internal standard and their amount in the manuscript.

3. It wasn't clear which method had been used to generate the TIC results shown in Figure 2. This figure doesn't show much other than there are multiple peaks. As there is nothing to draw from it, suggest not needed or amended with peaks identified or Mass Spectra that identifies certain metabolites.

Response: The peak in Fig.2 is generated by the GC-MS method. We have supplemented this method. In addition, we have supplemented GC-MS primary mass spectrometry of l-phenylalanine and LC-MS secondary mass spectrometry of compound epigallocatechin -7-O-gallate.

4. There is a lot of focus on fact that you have a GC-MS method and an LC-MS/MS but once you have data you should just be bringing that together and assessing as a full dataset? Does it matter how the data was obtained by this point? 

Response: We have combined all the differential metabolites to form a data set for clustering and enrichment pathway analysis based on the results of GC-MS and LC-MS. Additionally, Figures 7, 8 and 10 were added in the revised manuscript.

5. At the moment some of your figures are total ion chromatograms that don't show any peaks identified but just show a difference. These aren't particularly useful for connecting to the text. They show what the GC-MS method chromatogram looks like, but doesn't show what the actual results are. Some of the figures used are Total Ion chromatograms which just show unidentified peaks. Figures that show the data, evidence of particular metabolites in the damaged leaves vs healthy leaves would be more useful. 

Response: Thank you very much for your comments. We have reflected the difference between the two groups of ion peaks taking GC-MS as an example in the manuscript. Due to the wide variety of metabolites, the spectra of GC primary mass spectrometry and LC-MS secondary mass spectrometry need to be searched separately for each compound, and not all of them can be put together in the manuscript. Therefore, we have selected the mass spectra of one compound from GC-MS and LC-MS to supplement in the revised text. These two compounds were up-regulated by the E.onukki after C. tetracocca.

Response:

6. Is the figure of structures the metabolites you have identified of interest or is that from the literature. It's not clear to me in the way it has been worded. 

Response: These metabolites are the up-regulating compounds derived from the E. onukii after sucking the C. tetracocca. On this basis, we have reviewed other literature further. After the combination of two factors, we summarized these compounds. Therefore, we re-modify the figure note.

Author Response File: Author Response.pdf

Reviewer 3 Report

Please find comments in the attached file.

Comments for author File: Comments.pdf

Author Response

Dear reviewer,

 

    Thank you for returning our manuscript entitled “Untargeted metabolite profiling of Camellia tetracocca's response to an Empoasca onukii attack using GC-MS and LC-MS” (Manuscript ID: agronomy-2201368) with valuable comments and suggestions from you. We have revised the manuscript comprehensively based on these comments and suggestions, and here would like to submit the revised manuscript for publication in Agronomy.

    We have improved the manuscript and made some changes in the manuscript. We also responded to all comments point-by-point. The file “manuscript_20230227.docx” is a clean revised manuscript with the track changes. Point-by-point responses to the reviewer are listed below this letter. We hope that our revised paper is suitable for publication in Agronomy.

 

 

    Yours faithfully,

    First author, PhD Ni Zhang

    Corresponding author, Prof. Yuehua Song

1. Unrelevant for the topic."Seed Fossils of C. tetracoca" from the Neogene period to the Quater- 32 nary Period have been revealed in Sunjiaqing, Yuntou Dashan at the junction of Pu'an 33 and Qinglong, Guizhou Prov., China. The discovery suggests that world's tea history has 34 lasted for more than 1 million years [3].

Response：Thank you very much for your suggestion. We have deleted this sentence.

2. “at metabolome levels”

Response：“at metabolome levels” has deleted.

3. phenloics

Response：phenloics has been changed to phenols.

4. Line 121-122 . It is suggested to change methanol-water (...) to methanol-water (...) solution.

Response：The sentence has been modified. methanol-water (V:V=1:1, containing 4 μg/mL of L-2- chlorophenylalanine) solution.

5. Please add the instrument used to ground the samples.

Response：We have add the instrument used to ground the samples. Full-automatic sample fast grinding instrument (Wonbio-E, Shanghai Wonbio Biotechnology Co., Ltd.

6. The supernatant (150μL) in a glass vial was dried completely in a freeze concentration centifugal dryer.

Response：The sentence has been modified. The supernatant was transferred in a glass vial and dried completely in a freeze concentration centifugal dryer.

7. Please specify which internal standards were used.

Response：We have added internal standards.

8. “Isolate”should be changed to “seperate”.

Response：“Isolate” has been changed to “seperate”.

9. “sucked up”should be changed to “collected”.

Response：“sucked up” has been changed to “collected”.

10. These two numbers“ 1%” should be changed to “0.1%”.

Response：“o.1%” has been changed to “0.1%”.

11. “comducted”should be changed to “conducted”.

Response：“comducted” has been changed to “conducted”.

12. “M/Z”should be changed to “m/z”.

Response：“M/Z” has been changed to “m/z”.

13. Unnecessary in this section.

“GC-MS has mostly been adopted in the analysis of the volatile flavor and semi-volatile compounds from plant [35]. LC-MS is more suitable for phenolics,  terpenes, and N-containing and S-containing compounds [36]. According to our summary of the literatures, there has not been any untargeted metabolomics studies of C. tetracocca that combined GC-MS and LC-MS, especially for leaves of C. tetracocca attacked by E. onukii.”

Response：This section has been deleted.

14. Please add this information “MetaboAnalyst 4.0”also in methods section.

Response：We have add this information also in methods section.

15. “creates”should be changed to “created”.

Response：“creates” has been changed to “created”.

16. Use the complete name(Gln, Glu, Asn, Asp).

Response：We have used the complete name (glutamine, glutamic acid, asparagine, aspartic).

17. Format the reference according to MDPI format.

Response：We have supplemented the reference and revised it according to the format required by MDPI.

18. Line 351. Please add reference.

Response：We have added reference.

 

 

 

 

 

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

All the comments are addressed and the manuscript can now be accepted.

Reviewer 3 Report

The reviewer thanks the authors for the reply. All the comments are well addressed. The manuscript is accepted in the current form.