Biochemical Characteristics and Elemental Composition Peculiarities of Rheum tataricum L. in Semi-Desert Conditions and of European Garden Rhubarb

Round 1

Reviewer 1 Report

This manuscript may be deserved for its publication in International Journal of Plant Biology but some points should be explained by the Authors.

1.     What chemical compound did the Authors have in mind when they used the expression 'beta-zetacarotene'?

2.     For the purpose of research, the Authors should indicate which parts of the tested plant were subjected to extraction?

3.     What device was used to dry to a constant weight the root peel and pulp? Please complete the information.

4.     Please complete the information regarding the type of samples that "were cut into small slices, dried at 25-30 ^oC and homogenised".

5.     Was it an AAS spectrophotometer Hitashi or Hitachi?

6.     Figure 3 requires an appropriate legend.

7.     How did the Authors determine the carotenoids in the samples tested? Please provide information in the Materials and Methods section.

8.     Captions for Tables 4, 5 and 6 should include not only the wording of roots but also leaves.

Author Response

Dear reviewer.

Thank you very much for your valuable comments. We have made all necessary changes according to your comments indicating them with red color

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Answers to the comments:

This manuscript may be deserved for its publication in International Journal of Plant Biology but some points should be explained by the Authors.

1. What chemical compound did the Authors have in mind when they used the expression 'beta-zetacarotene'?

Answer: we have added a chemical formula of the compound

2. For the purpose of research, the Authors should indicate which parts of the tested plant were subjected to extraction?

Answer: addressed

3. What device was used to dry to a constant weight the root peel and pulp? Please complete the information.

Answer addressed

4. Please complete the information regarding the type of samples that "were cut into small slices, dried at 25-30 ^oC and homogenised".

Answer addressed

5. Was it an AAS spectrophotometer Hitashi or Hitachi?

Answer the misprint has been corrected

6. Figure 3 requires an appropriate legend.

Answer addressed

7. How did the Authors determine the carotenoids in the samples tested? Please provide information in the Materials and Methods section.

Answer addressed

8. Captions for Tables 4, 5 and 6 should include not only the wording of roots but also leaves.

Answer corrected

 

Reviewer 2 Report

The manuscript is interesting in terms of knowledge of Rheum species. It is correctly written, methods are explained and results are supported and compared with published references.

My suggestion is Minor revision, and I am indication some changes in the manuscript: 

Abstract: beta-zetacarotene, beta-zatecarotene (as in Anstgract) or beta-zeacarotene (as in 3.3. Carotenoids) ???; you can write better using latin signs. please correct

Introduction: third line: stress, not stresses;  Nature, not capitalized. At the end of the sentence, you have to add references; after [3] reference, delete 2018.

(Fig. 1) is not correctly placed, because readers may understand that it represents garden rhubarb. Place it earlier in the sentence

Table 1: use the same number of decimals for every determination and standard deviations

Table 2: use decimals for the values

Table 4 and 5 may be joined, as there are so many tables in the manuscript

Table 5: use the same number of decimals in all the table and SD. Same for Table 6 and Table 7

Reference list must be corrected and wrote according to Instructions for authors

Author Response

Dear reviewer.

Thank you very much for your valuable comments. We have made all necessary changes according to your comments indicating them with red color

 

Answers to reviewer’s comments:

The manuscript is interesting in terms of knowledge of Rheum species. It is correctly written, methods are explained and results are supported and compared with published references.

My suggestion is Minor revision, and I am indication some changes in the manuscript: 

• Abstract: beta-zetacarotene, beta-zatecarotene (as in Anstgract) or beta-zeacarotene (as in 3.3. Carotenoids) ???; you can write better using latin signs. please correct

Answer addressed

• Introduction: third line: stress, not stresses;  Nature, not capitalized. At the end of the sentence, you have to add references; after [3] reference, delete 2018.

Answer addressed

• (Fig. 1) is not correctly placed, because readers may understand that it represents garden rhubarb. Place it earlier in the sentence

Answer addressed

• Table 1: use the same number of decimals for every determination and standard deviations

Answer addressed

• Table 2: use decimals for the values

Answer addressed

• Table 4 and 5 may be joined, as there are so many tables in the manuscript

Answer addressed

• Table 5: use the same number of decimals in all the table and SD. Same for Table 6 and Table 7

Answer addressed

• Reference list must be corrected and wrote according to Instructions for authors

Answer addressed

Reviewer 3 Report

The whole paper should be formatted following the Journal guidelines.

Editing, typing errors and the linguistic revision of the paper should be carried out.

Even if the subject is interisting, a deep revision, implementationand organization of paper should be carried out.

A more focused title should be added.

The abstract should be better summarized and organized in context, aim, material and methods, results, conclusion.

The aim and the novelty character of paper should be better marked.

Major details should be added in material and methods descriptions, particularly on samples and sampling. A graphical scheme also should be inserted.

The results are discussed in confused manner. The description and discussion of results should be greatly implemented and better organized. The results should be better correlated among them and correlated with data and literature.

All tables and figures should be checked and redisegned in a clearer manner, including legends, notes and statistical analysis.

The layout of paper should be checked.

The authors should be added considerations of results in conclusion.

Conclusion should be better reflect the results of paper and implemented with limits of research, advantages practical applications, future directions.

Author Response

Dear reviewer.

Thank you very much for your valuable comments. We have made all necessary changes according to your comments indicating them with red color

 

Answers to the comments

• The whole paper should be formatted following the Journal guidelines.

Answer addressed

• Editing, typing errors and the linguistic revision of the paper should be carried out.

Answer addressed

• if the subject is interisting, a deep revision, implementationand organization of paper should be carried out.

Answer we have done our best to improve the text according to the comments

• A more focused title should be added.

Answer addressed

• The abstract should be better summarized and organized in context, aim, material and methods, results, conclusion.

Answer addressed

• The aim and the novelty character of paper should be better marked.

Answer addressed

• Major details should be added in material and methods descriptions, particularly on samples and sampling. A graphical scheme also should be inserted.

Answer we have expanded Material and Methods section according to the comments. Though we have created a Table describing the main analytical results, we are not sure that it will improve the quality of Material Methods section as full description of the results are provided in the Discussion section

• The results are discussed in confused manner. The description and discussion of results should be greatly implemented and better organized. The results should be better correlated among them and correlated with data and literature.

Answer we have made appropriate changes in the discussion section

• All tables and figures should be checked and redisegned in a clearer manner, including legends, notes and statistical analysis.

Answer addressed

• The layout of paper should be checked.

Answer addressed

• authors should be added considerations of results in conclusion.

Answer we have made appropriate changes in the conclusion section

• Conclusion should be better reflect the results of paper and implemented with limits of research, advantages practical applications, future directions.

Answer we have made appropriate changes in the conclusion section

Reviewer 4 Report

The review of the manuscript „Biochemical characteristics and elemental composition of Rheum tataricum L.” by Golubkina et al. submitted to Int. J. Plant Biol.

The paper focus on minerals and biochemical comparison between two varieties of Rheum tataricum, the wild one and the cultivated one. However they are grown in different region and different soil. I supposed that also meteorological parameters were different (water supply, drought experience, temperature). All of them may influence on biochemical parameters such as proline, TP and AOA. It should be mention in the discussion.

The research was conducted in protected areas , information about the consent and its number should be placed at the end of the work. This also applies to the consent to extract legally protected species from the environment. I suspect that Russia has appropriate procedures in this regard.

Some minor remarks:

Table 1 - Please format the table better, as the units are now not very readable.

Figure 2 - poor quality - blurred text.

Figure 3 there is lack of specification of wave lengths. There is lack of full legend.

“Anthraquinone analogues are considered the most important constituents… showed a strong influence on the metabolites of rhubarb roots composition.” What is the link of this part with UV spectrum and rest of the analysis. You did not described methods to asses anthraquinone composition. However this analysis should be the most important one. I do not see link of this part to your results and whole paper. Please explain it and modified.

3.3. You have not presented the results for carotenoids.

Tables 4-6 there is no statistical analysis for leaves.

Literature no 46 is missing

Author Response

Dear reviewer.

Thank you very much for your valuable comments. We have made all necessary changes according to your comments indicating them with red color

 

 

Answers to the Comments

The review of the manuscript „Biochemical characteristics and elemental composition of Rheum tataricum L.” by Golubkina et al. submitted to Int. J. Plant Biol.

• The paper focus on minerals and biochemical comparison between two varieties of Rheum tataricum, the wild one and the cultivated one. However they are grown in different region and different soil. I supposed that also meteorological parameters were different (water supply, drought experience, temperature). All of them may influence on biochemical parameters such as proline, TP and AOA. It should be mention in the discussion.

Answer we have added the appropriate data to the discussion section

• The research was conducted in protected areas , information about the consent and its number should be placed at the end of the work. This also applies to the consent to extract legally protected species from the environment. I suspect that Russia has appropriate procedures in this regard.

Answer The work was achieved according to the agreement of scientific cooperation between Federal Scientific Center of Vegetable production, Voronezh State University of Forestry and Technologies and Bogdinsko-Baskunchak Nature Reserve,  22.02.2021. Such an agreement allows the participants to work at the territory of the Reserve and extract legally protected species. No other consent is necessary. The information has been added at the end of the work

• Some minor remarks:

1. i) Table 1 - Please format the table better, as the units are now not very readable.

Answer We have added the titled of the first two columns and the florets data for garden rhubarb

1. ii) Figure 2 - poor quality - blurred text.

Answer  addressed

iii) Figure 3 there is lack of specification of wave lengths. There is lack of full legend.

Answer  addressed

• “Anthraquinone analogues are considered the most important constituents… showed a strong influence on the metabolites of rhubarb roots composition.” What is the link of this part with UV spectrum and rest of the analysis. You did not described methods to asses anthraquinone composition. However this analysis should be the most important one. I do not see link of this part to your results and whole paper. Please explain it and modified.

Answer we have made appropriate changes in the discussion

• You have not presented the results for carotenoids.

Answer we have added the appropriate information

• Tables 4-6 there is no statistical analysis for leaves.

Answer we are extremely sorry. addressed

• no 46 is missing

Answer  addressed

Reviewer 5 Report

In the present manuscript, the mineral and chemical composition of Rheum tataricum in compariosn to the cultivated relative (garden rhubarb) was performed.

 

Although the topic is interesting aiminig to valorize wild unexpolited species, the concept has several methodological flaws.

First of all, harvesting stage is importnat for the chemical composition of plant matrices. It seems that the authors harvested wild plants at full blossom without indicating what is the most suitable phenological stage for the consumption of the wild species. Morever, the authors compare the chemical composition of the wild species with a cultivated relative which seems that it was cultivated before anthesis (hence in Table 1 there are no data for florets). Thereofre, such comparison is not valid due to differences in growth stages between the wild and cultivated plants.

Finally, the authors indicate that specific compounds (e.g beta-zatecarotene, proline, phenolics, malic acid) protect plants from oxidative stress without describing any stress factors applied. As soon as there is no control treatment, it is not possible to suggest that wild plants are subjected to environmental stress, especially when considering that plants are adapted to these conditions.

     

Author Response

Dear reviewer.

Thank you very much for your valuable comments. We have made all necessary changes according to your comments indicating them with red color

 

 

Answers to the Comments

In the present manuscript, the mineral and chemical composition of Rheum tataricum in compariosn to the cultivated relative (garden rhubarb) was performed.

 

Although the topic is interesting aiminig to valorize wild unexpolited species, the concept has several methodological flaws.

• First of all, harvesting stage is importnat for the chemical composition of plant matrices. It seems that the authors harvested wild plants at full blossom without indicating what is the most suitable phenological stage for the consumption of the wild species

Answer At present Rheum tataricum is not used for consumption as it is listed in the Red Book. But the species is interesting for selection process especially taking into account large mass of seeds produced. The beginning of May is the only time when it is possible to sample all plant tissues, including not only roots, but also leaves, stems and florets. By the middle of May  plant sheds seeds and the vegetation period ends. The appropriate data have been included in Material and Methods section

• Moreover, the authors compare the chemical composition of the wild species with a cultivated relative which seems that it was cultivated before anthesis (hence in Table 1 there are no data for florets). Thereofre, such comparison is not valid due to differences in growth stages between the wild and cultivated plants.

Answer Florets data for garden rhubarb have been added to Table 1

• Finally, the authors indicate that specific compounds (e.g beta-zatecarotene, proline, phenolics, malic acid) protect plants from oxidative stress without describing any stress factors applied. As soon as there is no control treatment, it is not possible to suggest that wild plants are subjected to environmental stress, especially when considering that plants are adapted to these conditions.

Answer The process of plants adaptation to environmental stresses is known to be intensively connected with accumulation of antioxidants, including carotenoids and polyphenols, organic acids and proline and that’s why we tried to compare the content of these compounds in R.tataricum grown in conditions of high salinity, water deficiency and high solar insolation with garden rhubarb grown in conditions of low environmental stress. In this respect we have added some characteristics of stress parameters to the appropriate data to the discussion section.

Round 2

Reviewer 3 Report

The authors have answered to suggestions and the paper is now improved. Only one suggestion should be better addressed; the authors should describe better the samples and samplings and should indicate the number of samples and samplings.

Author Response

Dear reviewer, thank you very much for your valuable help in improving the manuscript. We have made appropriate changes in the text and marked them by red color.

 

Answers to reviewer’s comments:

 

	(x)	( )	( )	( )

Comments and Suggestions for Authors

The authors have answered to suggestions and the paper is now improved. Only one suggestion should be better addressed; the authors should describe better the samples and samplings and should indicate the number of samples and samplings.

Answer: addressed.

Reviewer 4 Report

The authors have modyfied the manuscript and I trust their responses. However some minor issues should be adressed.

Table 1 - the units are divided between two lines

Figures 2, 5 and 6 there are lack of SD on the figures.

 

Author Response

Dear reviewer, thank you very much for your valuable help in improving the manuscript. We have made appropriate changes in the text and marked them by red color.

Answers to reviewer 4 comments

The authors have modyfied the manuscript and I trust their responses. However some minor issues should be adressed.

Table 1 - the units are divided between two lines

Answer:  we have deleted the second line.

Figures 2, 5 and 6 there are lack of SD on the figures.

Answer: addressed.

Reviewer 5 Report

AFter the explanations of the authors, I suggest they remove all the results related to the cultivated species and focus only on the characterization of the wild one (R. tataricum), as the title indicates.

Author Response

Dear reviewer, thank you very much for your valuable help in improving the manuscript. We have made appropriate changes in the text and marked them by red color.

Answers to reviewer 5 comments:

	Yes	Can be improved	Must be improved	Not applicable
Does the introduction provide sufficient background and include all relevant references?	( )	( )	(x)	( )
Are all the cited references relevant to the research?	(x)	( )	( )	( )
Is the research design appropriate?	( )	( )	(x)	( )
Are the methods adequately described?	(x)	( )	( )	( )
Are the results clearly presented?	( )	( )	(x)	( )
Are the conclusions supported by the results?	( )	( )	(x)	( )

Comments and Suggestions for Authors

AFter the explanations of the authors, I suggest they remove all the results related to the cultivated species and focus only on the characterization of the wild one (R. tataricum), as the title indicates.

Answers:

i) we have modified the title of the manuscript and the end of Introduction section, indicating the importance of tataricum and garden rhubarb comparison. Two more references have been included.

ii) the same approach has been used in Results and Discussion section.

and

iii) we have fully revised the Conclusions.

Round 3

Reviewer 5 Report

Although the authors insist on the comparison of wild and cultivated species, the new additions in the text regarding sampling revealed another methodological flaw. In particular, sampling of only five plants is not appropriate, especially in the case of wild plants where a great variation should be expected or in the case of cuultivated ones where one leaf per plant was used. There are specific protocols regarding the sampling method of wild plants that should be followed. As it is, the authors captured only a portion of variation in plants and soil of wild plants. 

Therefore, based on the new evidence I think that methodological flaws do not allow the publication of the manuscript and no further consideration is needed. 

 

Author Response

Answers to the reviewer’s comments.

Although the authors insist on the comparison of wild and cultivated species, the new additions in the text regarding sampling revealed another methodological flaw. In particular, sampling of only five plants is not appropriate, especially in the case of wild plants where a great variation should be expected or in the case of cuultivated ones where one leaf per plant was used. There are specific protocols regarding the sampling method of wild plants that should be followed. As it is, the authors captured only a portion of variation in plants and soil of wild plants. 

Therefore, based on the new evidence I think that methodological flaws do not allow the publication of the manuscript and no further consideration is needed. 

Answers:

Dear reviewer. In response to your comments we should like to indicate the following:

1)  R. tataricum is listed in the Red Book and five plants with three replicates each year of investigation are representative in our opinion. Please, take also in account that we have worked in a Nature Reserve with serious restrictions of plant sampling and only the Agreement of Scientific Cooperation allowed us to collect the needed material. Moreover, the diameter of plant’s leaves is about 0.5 m and, therefore, the mixture of 5 leaves from five plants makes representative samples, in our opinion. Besides, to check soil characteristics we used a broad soil sampling of the territory, which showed relatively low variability of mineral composition and salinity in the rhubarb area. We used a systematic uniform random sampling at fixed intervals along equally spaced 3 parallel transects (Wulfsohn, D. Sampling techniques for plants and soil. Landbauforsch, Volkenrode, 2010, 340, 3-30). This sampling approach offers an appropriate balance between the estimation precision and the time spent to obtain representative samples. Finally, we collected 5 plant samples of R. tataricum with three replicates each year by sampling the site according to its natural distribution within the research area in Bogdinsko-Baskunchak Nature Reserve. The needed modifications and a further reference were added to the text.

• As for garden rhubarb, we are sorry that our description was inaccurate, i.e.: all leaves from each of the 5 sampled plants were collected, with three replicates each year, dried and homogenized to obtain a mixed sample for the analysis.
• Furthermore, we included the garden rhubarb data because the simple description of new biochemical parameters of tataricum without any comparison would not have allowed to evaluate plant peculiarities and utilization prospects.

Author Response File: Author Response.doc