Molecular Mechanism of Exogenous Selenium Affecting the Nutritional Quality, Species and Content of Organic Selenium in Mustard

Round 1

Reviewer 1 Report

1. What is the meaning of "after treatment with the same Se source and different concentrations" in the note of Table 1?

2. The authors should recheck the data and statistical tests. I have some doubts regarding the statistics results of control treatment of MeSeCys and Se⁴⁺ in Table 1.

3. The control treatment in Fig. 2 through 6 should be just a single column, like Fig.1a.

4. It is recommended that authors indicate when the database was accessed, for example (KEGG, http://www.kegg.jp/kegg/pathway.html, accessed on 10 July 2022)

5. The quality of Figures 8 and 9 needs to be improved, and the relevant content in the figures is very unclear at present.

6. The author should add the dry weight and morphological characteristics of mustard sprouts under Na₂SeO₃ treatment, because many indicators in the manuscript are calculated by DW.

7. The recommended daily intake of Se for an adult is 55 micrograms. According to Fig. 3a, people would need just a few grams of dry mustard sprouts per day to exceed the recommended amount. How the authors intend to use these mustard sprouts?

8. Line 607, the result of photosynthetic pigment content is not seen in the manuscript.

Line 27, “and stored in plant leaves”. Is there any relevant data in the paper to support this conclusion?

9. The author should strengthen the analysis and discussion of Fig. 5. MDA, as an indicator of membrane lipid peroxidation, why its content was significantly lower than the control, in the case of sprouts whose growth was inhibited by high concentration Na₂SeO₃ treatment.

Minor editing of English language required.

Author Response

Thank you for your patience and review of our article, which was very helpful to us. We have made revisions to the entire text according to your suggestions,

1. What is the meaning of "after treatment with the same Se source and different concentrations" in the note of Table 1?

Thank you for your suggestion, we have modified the confusing statements.

2. The authors should recheck the data and statistical tests. I have some doubts regarding the statistics results of control treatment of MeSeCys and Se⁴⁺in Table 1.

Thank you for your suggestion. This may be due to the presence of trace amounts of selenium in experimental vessels and water, which belongs to the background content

3. The control treatment in Fig. 2 through 6 should be just a single column, like Fig.1a.

Thank you. We have made modifications to the image

4. It is recommended that authors indicate when the database was accessed, for example (KEGG, http://www.kegg.jp/kegg/pathway.html, accessed on 10 July 2022)

Thank you! We have added an analysis website and analysis time to the key database.

5. The quality of Figures 8 and 9 needs to be improved, and the relevant content in the figures is very unclear at present.

Okay, we have modified the image quality and resolution.

6. The author should add the dry weight and morphological characteristics of mustard sprouts under Na₂SeO₃treatment, because many indicators in the manuscript are calculated by DW.

We added different sodium selenite treatments to the supplementary materials to investigate the changes in water content of mustard seedlings.

7. The recommended daily intake of Se for an adult is 55 micrograms. According to Fig. 3a, people would need just a few grams of dry mustard sprouts per day to exceed the recommended amount. How the authors intend to use these mustard sprouts?

In this study, we recommend a selenium treatment concentration of 5-10mg/L, where the total selenium content in mustard is between 2-8mg/kg. If fresh vegetables are consumed, it should be around 0.2-0.8mg/kg, which is lower than the highest standard (400 µ g/day) based on daily consumption

8. Line 607, the result of photosynthetic pigment content is not seen in the manuscript.

Sorry, we missed the results of Figure 2 when organizing the article. We have added the data from Figure 2 in the results section.

Line 27, “and stored in plant leaves”. Is there any relevant data in the paper to support this conclusion?

Thank you for your suggestion. In other references, it is believed that these genes are generally expressed in plant leaves and are related to the synthesis of selenium amino acids

9. The author should strengthen the analysis and discussion of Fig. 5. MDA, as an indicator of membrane lipid peroxidation, why its content was significantly lower than the control, in the case of sprouts whose growth was inhibited by high concentration Na₂SeO₃treatment.

Thank you for your suggestion. Our preliminary results showed that when the concentration of sodium selenite was higher than 60mg/kg, it would cause damage to the plant, while when it was lower or equal to 40mg/kg, mustard was affected. However, its stress resistance increased, so this may be the reason why the MDA content was lower than the control.

Reviewer 2 Report

1. introduction needs to be updated. most of the information useless and needs updated work.  the last paragraph of the introduction should explained the rational of the research work, and should focus the research gap, what question was answered. comparision to the previously publsihed work must be mentioned. 

2. old citation should be updated with new citation from 2018 to 2022.

3. some methods are not explained , so detailed steps are need to be mentioned. 

4.  the result are presented well, but need a proper english check especaily in the transcriptomics analysis section. 

5. discussion needs to be improve with proper and valid citation from standard jornals,. also aviod selfcitation.

6. conclusion should give a home take message and must have recomendation, also must clear question for future research. what was acchieved and what need to be done.

 

1. introduction needs to be updated. most of the information useless and needs updated work.  the last paragraph of the introduction should explained the rational of the research work, and should focus the research gap, what question was answered. comparision to the previously publsihed work must be mentioned. 

2. old citation should be updated with new citation from 2018 to 2022.

3. some methods are not explained , so detailed steps are need to be mentioned. 

4.  the result are presented well, but need a proper english check especaily in the transcriptomics analysis section. 

5. discussion needs to be improve with proper and valid citation from standard jornals,. also aviod selfcitation.

6. conclusion should give a home take message and must have recomendation, also must clear question for future research. what was acchieved and what need to be done.

 

Author Response

Thank you for your patience and review of our article, which was very helpful to us. We have made revisions to the entire text according to your suggestions,

1. introduction needs to be updated. most of the information useless and needs updated work.  the last paragraph of the introduction should explained the rational of the research work, and should focus the research gap, what question was answered. comparision to the previously publsihed work must be mentioned. 

Thank you, we have made modifications in the introduction section.

2. old citation should be updated with new citation from 2018 to 2022.

Thank you for your suggestion. We have added some new references

3. some methods are not explained, so detailed steps are need to be mentioned. 

Thank you! We have added a description of physiological indicator measurement in the materials and methods

4. the result are presented well, but need a proper english check especaily in the transcriptomics analysis section. 

Thank you for your suggestion. We have rewritten the description of transcriptome data.

5. discussion needs to be improve with proper and valid citation from standard jornals,. also aviod selfcitation.

Thank you for your suggestion. We have made modifications to the discussion section

6. conclusion should give a home take message and must have recomendation, also must clear question for future research. what was acchieved and what need to be done.

Thank you for your suggestion. We have made modifications to the summary section and added the significance and outlook of the results.

Reviewer 3 Report

The manuscript number agronomy-2366949 entitled “Molecular Mechanism of Exogenous Selenium Affecting the Nutritional Quality, Species and Content of Organic Selenium in Mustard” aims to decipher the role of sodium selenite on leaf mustard. In general, the manuscript is well written but requires some corrections/clarifications.

 

The abstract is well written however I was confused with the treatment used. Was it used sodium selenite (Na₂SeO₃) or sodium sulfite (Na₂SO₃)? Please verify it throughout the manuscript.

 

Also in the abstract, the authors mention the content of Vc. What is Vc? Is it vitamin C or another compound? Please clarify.

 

Line 64, “plants have abiotic stress” plants do not have abiotic stress they suffer from abiotic stress. Please correct it.

 

“For each treatment, select mustard with the same growth trend and wash it with ultra-pure water for three times.” I suppose that I should read: ‘For each treatment, select mustard with the same growth trend was washed with ultra-pure water for three times.’ Am I right? (Lines 98 and 99).

 

Please make sure to use the same tense throughout the Materials and Methods section. As sometimes it is used in the present and in others in the past. Please verify lines: 99-101.

 

How many plants were used for each treatment? Was used 5 plants per treatment? Please clarify.

 

Please verify lines 104-106. It seems that two different sentences were put together and forgotten to be reviewed.

 

“Chlorophyll and carotenoids were extracted by 95% ethanol and measured by UV-visible spectrophotometer at 665, 649, and 470 nm separately” Please provide a reference and more details of the methodology used as well as the formulae used for the calculation of each pigment. Lines 107 and 108. Please, provide the number of measurements for each parameter.

 

The same procedure for the determination of soluble sugars and protein content should be made. (Reference and more details about the procedure as well as the number of measurements, n).

 

“The content of MDA was determined using the kit (A003-3-5) of Nanjing Jiancheng Bioengineering Co., Ltd. (Nanjing, China). The activities of glutathione peroxidase GSH, peroxidase POD, superoxide dismutase and SOD catalase CAT were determined using the kits (AKPR008M, AKAO005C, AKAO001C, AKAO003-1M) of Beijing Box Biotechnology Co., Ltd. (Beijing, China).” Lines 115-119. 

I would write as: ‘The content of MDA was determined using the kit (A003-3-5) of Nanjing Jiancheng Bioengineering Co., Ltd. (Nanjing, China). The activities of glutathione peroxidase (GSH, EC XXXX), peroxidase (POD, EC XXXX), superoxide dismutase (SOD, EC XXXX), and catalase (CAT, EC XXXX) were determined using the kits (AKPR008M - XXX, AKAO005C - XXX, AKAO001C - XXX, AKAO003-1M - XXX) of Beijing Box Biotechnology Co., Ltd. (Beijing, China).’

 

Please provide information on which kit is for what in the ‘Determination of growth indicators’ and ‘Determination of mustard antioxidant system’.

 

Is the 0.2 g dry sample from the previously digested one of 0.5 g? Please clarify.

 

In the ‘Results’ section, the authors start to show data related to biomass and germination rate, however, those parameters do not make it in the section of materials and methods. Please rectify.

 

What is “CK” in figures 1b and c, 3a, 4, 5, and 6? Please clarify it in the figure caption.

 

In lines 199 and 209, “However” should start with a lower letter. 

 

Why did the authors present the data in some parameters per dry weight and others per fresh weight? To uniformize, is it possible to present the data for all the parameters as per dry weight? 

 

Where is the Figure 2? Please correct it.

 

Could the authors provide a better quality and larger image of Figure 8? It is very difficult to read. Same for Figure 9.

 

“SeMet, SeCys, MeSeCys“, please provide the full name the first time each one is mentioned in the text.

In the discussion, please make sure to always use the same nomenclature. As the authors sometimes use Vc or Vitamin C, please uniformize. Also after a “;” use lower lettering, please see it throughout the whole manuscript.

I would like to know what influenced the authors to choose the concentrations of Se. Also, do you consider the chosen concentrations appropriate? Why? I would like to see the context of it in the manuscript. Thank you.

 

Author Response

Thank you for your patience and review of our article, which was very helpful to us. We have made revisions to the entire text according to your suggestions,

The manuscript number agronomy-2366949 entitled “Molecular Mechanism of Exogenous Selenium Affecting the Nutritional Quality, Species and Content of Organic Selenium in Mustard” aims to decipher the role of sodium selenite on leaf mustard. In general, the manuscript is well written but requires some corrections/clarifications.

1. The abstract is well written however I was confused with the treatment used. Was it used sodium selenite (Na2SeO3) or sodium sulfite (Na2SO3)? Please verify it throughout the manuscript.
2. Also in the abstract, the authors mention the content of Vc. What is Vc? Is it vitamin C or another compound? Please clarify.
3. Line 64, “plants have abiotic stress” plants do not have abiotic stress they suffer from abiotic stress. Please correct it.
4. “For each treatment, select mustard with the same growth trend and wash it with ultra-pure water for three times.” I suppose that I should read: ‘For each treatment, select mustard with the same growth trend was washed with ultra-pure water for three times.’ Am I right? (Lines 98 and 99).
5. Please make sure to use the same tense throughout the Materials and Methods section. As sometimes it is used in the present and in others in the past. Please verify lines: 99-101.
6. How many plants were used for each treatment? Was used 5 plants per treatment? Please clarify.
7. Please verify lines 104-106. It seems that two different sentences were put together and forgotten to be reviewed.
8. “Chlorophyll and carotenoids were extracted by 95% ethanol and measured by UV-visible spectrophotometer at 665, 649, and 470 nm separately” Please provide a reference and more details of the methodology used as well as the formulae used for the calculation of each pigment. Lines 107 and 108. Please, provide the number of measurements for each parameter.

Thank you for your suggestion. We have added detailed steps for determining photosynthetic pigments in the material method

9. The same procedure for the determination of soluble sugars and protein content should be made. (Reference and more details about the procedure as well as the number of measurements, n).

Thank you for your suggestion. We have completed this part of the assay using the reagent kit. Readers can refer to the operating instructions of the reagent kits.

10. “The content of MDA was determined using the kit (A003-3-5) of Nanjing Jiancheng Bioengineering Co., Ltd. (Nanjing, China). The activities of glutathione peroxidase GSH, peroxidase POD, superoxide dismutase and SOD catalase CAT were determined using the kits (AKPR008M, AKAO005C, AKAO001C, AKAO003-1M) of Beijing Box Biotechnology Co., Ltd. (Beijing, China).” Lines 115-119. 

I would write as: ‘The content of MDA was determined using the kit (A003-3-5) of Nanjing Jiancheng Bioengineering Co., Ltd. (Nanjing, China). The activities of glutathione peroxidase (GSH, EC XXXX), peroxidase (POD, EC XXXX), superoxide dismutase (SOD, EC XXXX), and catalase (CAT, EC XXXX) were determined using the kits (AKPR008M - XXX, AKAO005C - XXX, AKAO001C - XXX, AKAO003-1M - XXX) of Beijing Box Biotechnology Co., Ltd. (Beijing, China).’

12. Please provide information on which kit is for what in the ‘Determination of growth indicators’ and ‘Determination of mustard antioxidant system’.
13. Is the 0.2 g dry sample from the previously digested one of 0.5 g? Please clarify.

Here, 0.5g of the sample was used to determine the total selenium content, and 0.2g of the sample was used to determine the selenium species content. Due to the use of different equipment, the weight requirements for the sample were different, and the same processed sample was divided into two parts for use.

14. In the ‘Results’ section, the authors start to show data related to biomass and germination rate, however, those parameters do not make it in the section of materials and methods. Please rectify.
15. What is “CK” in figures 1b and c, 3a, 4, 5, and 6? Please clarify it in the figure caption.
16. In lines 199 and 209, “However” should start with a lower letter. 
17. Why did the authors present the data in some parameters per dry weight and others per fresh weight? To uniformize, is it possible to present the data for all the parameters as per dry weight? 

A: Thank you for your suggestion. Because some parameters, such as enzyme activity, MDA content, soluble sugar, soluble protein, chlorophyll content, etc., can only be measured using fresh samples, while content of flavonoids and total phenolic requires high performance liquid chromatography, selenium species and total selenium content need to be measured using HG-AFS, and can only be measured using dry samples, the measurement parameters have different performance results.

18. Where is the Figure 2? Please correct it.

Thank you, we have added the picture in the article

19. Could the authors provide a better quality and larger image of Figure 8? It is very difficult to read. Same for Figure 9.

Thank you. We have adjusted the picture definition and size in the article.

20. “SeMet, SeCys, MeSeCys“, please provide the full name the first time each one is mentioned in the text.

Thank you. We have added the detailed name of the abbreviation in the article

21. In the discussion, please make sure to always use the same nomenclature. As the authors sometimes use Vc or Vitamin C, please uniformize. Also after a “;” use lower lettering, please see it throughout the whole manuscript.

Thank you. We have standardized the abbreviation format in the text.

22. I would like to know what influenced the authors to choose the concentrations of Se. Also, do you consider the chosen concentrations appropriate? Why? I would like to see the context of it in the manuscript. Thank you.

We have used a wide range of treatment concentrations (0-100mg/L) in leguminous plants and other plants in the early stage, and have also conducted preliminary experiments on mustard. We found that treatment concentrations higher than 40mg/L resulted in low germination and survival rates of seedlings, making it meaningless for the experiment. Therefore, we chose a treatment concentration of 0-40mg/L. Please refer to the following (Cheng, H., Li, L., Dong, J., Wang, S., Wu, S., Rao, S., Li, L., Cheng, S., & Li, L. (2023). Transcriptome and physiological determination reveal the effects of selenite on the growth and selenium metabolism in mung bean sprouts. Food Research International, 112880.)