An HPLC-automated Derivatization for Glutathione and Related Thiols Analysis in Brassica rapa L.

Round 1

Reviewer 1 Report

In the manuscript entitled "Analytical methods for determination of glutathione and glutathione disulfide in Brassica rapa L." the authors improved the detection method of thiols (glutathione, cysteine, ϒ-GC) with HPLC. The research work is well-planed and performed, the methods were described in details, and the presentation of the results are clear. In general, the manuscript is written in good manner and order. However, few corrections are required:

Line #69: The abbreviation of 5,5 γ-dithiobis-(2-nitrobenzoic) acid is DTNB

Line #71: OPA is ortho-phthalaldehyde, please check the correct name

Line #73: SBD-F is 7-fluorobenzo-2-oxa-1,3-diazol-4-sulfonate, please check the correct name

Further questions and comments:

In Figure 7. the authors presented the oxidized and reduced form of Cys, γ-GC and GSH. The results are contradictory with the already published results. Using different methods, even with HPLC, the measured amount of reduced GSH is always higher than the oxidised GSSG form in control conditions in different plant species. For example in broccoli Mills et al. (1997) measured with HPLC the GSH and GSSG contents (https://doi.org/10.1006/jfca.1997.0526), but many other research proved that reduced GSH form presents in a higher proportion in whole shoot or root extracts. Can you please explain this contradiction?

In conclusion, I think this manuscript provides interesting results and the integration of this method can improve thiol detection with HPLC.

Author Response

Discussion Sheet for Reviewer 1

 

In the manuscript entitled "Analytical methods for determination of glutathione and glutathione disulfide in Brassica rapa L." the authors improved the detection method of thiols (glutathione, cysteine, ϒ-GC) with HPLC. The research work is well-planed and performed, the methods were described in details, and the presentation of the results are clear. In general, the manuscript is written in good manner and order. However, few corrections are required:

Thank you for your revisions and considerations. We have revised our manuscript taking into account all your comments. Please find below the specific answers to comments listed below.  All the revisions are marked in red and indicated with reference to the actual specific page/line.

 

Point 1: Line #69: The abbreviation of 5,5 γ-dithiobis-(2-nitrobenzoic) acid is DTNB

Response 1: Corrected as indicated (line #80)

 

Point 2: Line #71: OPA is ortho-phthalaldehyde, please check the correct name

Response 2: Corrected as indicated  (line #82)

 

Point 3: Line #73: SBD-F is 7-fluorobenzo-2-oxa-1,3-diazol-4-sulfonate, please check the correct name

Response 3: Corrected as indicated  (line #83)

 

Q4: Further questions and comments:

In Figure 7. the authors presented the oxidized and reduced form of Cys, γ-GC and GSH. The results are contradictory with the already published results. Using different methods, even with HPLC, the measured amount of reduced GSH is always higher than the oxidised GSSG form in control conditions in different plant species. For example in broccoli Mills et al. (1997) measured with HPLC the GSH and GSSG contents (https://doi.org/10.1006/jfca.1997.0526), but many other research proved that reduced GSH form presents in a higher proportion in whole shoot or root extracts. Can you please explain this conadiction?

Response 4: We have reviewed Figure 7.  Indeed, checking the raw data we noticed that data of reduced glutathione had been reversed with those of oxidized glutathione. The figure has been reworked and replaced.

 

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript titled “Analytical methods for determination of glutathione and glu-tathione disulfide in Brassica rapa L.” Manuscript ID: agronomy-1201961, by Nacca et al. is a very interesting piece of research on the determination of glutathione and other sulphur derivatives in plant tissues. In this study, the authors proposed an improved automated method for the determination of glutathione, cysteine, and γ-glutamylcysteine in plant tissue. Although the idea is interesting, the study is poorly planned, and the data were not analyzed adequately. Moreover, the figures are poorly presented.

Major concerns

1. The title is very descriptive and misleading. The term “glutathione and glu-tathione” in the title is very confusing. I would recommend rephrasing the title and remove one “glutathione” to make it straightforward and to ensure clarity.
2. Although the abstract is well-written, however, it is very descriptive and does not present the main findings of the work. About two-thirds of the abstract just for describing the methodology. I could recommend re-writing the abstract to articulate more clearly the novelty of the results and the main findings. It needs to be more informative rather than describing the methodology. According to the “Instructions for Authors of Agronomy”, the abstract should be an objective representation of the article: it must not contain results that are not presented and substantiated in the main text and should not exaggerate the main conclusions. (please see https://www.mdpi.com/journal/agronomy/instructions).
3. The materials and methods are poorly described, particularly the method validation. Although the authors briefly described some of the method validation parameters within the “results” section, they did not mention them within the “materials and methods” section. The methods should be described in detail. Moreover, they did not present these data in an appropriate I could recommend that:-

• Adding a new section within the “materials and methods” for the method validation to include (1) Extraction recovery %, (2) Limit of detection (LOD) and limit of quantification (LOQ), and (3) Reproducibility for relative retention times and relative peak areas for each compound.
• Adding a new table/graph to present the method validation parameters properly.

4. As I mentioned above although the idea is interesting, the study is poorly planned, particularly in terms of application. The authors used their method to determine oxidized and reduced content of Cys, γ-GC, and GSH in florets, leaf blades, and stems of
   Brassica rapa. However, I believe that this is not enough to evaluate the method. I could recommend that the method should be evaluated by comparing the endogenous content of Cys, γ-GC, and GSH within the same tissue, for example, leaves, under stressful conditions (salinity, drought, ..etc) or under enriched sulphur nutrition. For instance, the authors mentioned that “the young plants were separated into 4 groups with different sulphur nutrition (K2SO4: 0.01, 0.05, 0.5 and 2 mM) in modified Hoagland medium”, however, they did not present any related data to this experiment. I believe this experiment might be good to evaluate the method. Please, analyze the endogenous content of Cys, γ-GC, and GSH under this enriched sulphur nutrition.
5. The figure presentation is also very poor and should be enhanced before publishing. Most of the figures are scanned graphs with low resolution and inappropriate Kindly, reconstruct all figures to enhance their presentation and ensure clarity.
6. Finally, Although the language used in the manuscript is easy to follow and understand, however, the manuscript should be carefully and deeply revised for grammar and English use, since some other mistakes were found throughout the whole paper.

Comments for author File: Comments.pdf

Author Response

Thank you for your revisions and considerations. We have revised our manuscript taking into account all your comments. The specific answers to comments are listed below.  All the revisions are marked in red and indicated with reference to the actual specific page/line.

 

Author Response File: Author Response.pdf

Reviewer 3 Report

The paper entitled: ” Analytical methods for determination of glutathione and glu3 tathione disulfide in Brassica rapa L.”  is well structured and documented. The analysis methods are rigorously presented and the results are meticulously interpreted statistically providing numerous correlations.

Even if the paper is well structured, 2 fundamental aspects are missing for this paper to be published:

1. The purpose of this abstract work is missing
2. The conclusion of this manuscript is missing

Author Response

Thank you for your revisions and considerations. We have revised our manuscript taking into account your comments.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Thanks for addressing my comments and suggestions. The revised manuscript looks much better.

Reviewer 3 Report

The authors responded satisfactorily to all my comments.