The Composition and the Content of ∆-5 Sterols, Fatty Acids, and the Activity of Acyl-Lipid Desaturases in the Shoots of Ephedra monosperma, Introduced in the Botanical Garden of the Cryolithozone of Yakutia

Round 1

Reviewer 1 Report

Title: The Composition and the Content of ∆-5 Sterols, Fatty Acids, and the Activity of Acyl-lipid Desaturases in the Shoots of Ephedra monosperma, Introduced in the Botanical Garden of the Cryolithozone of Yakutia.

The title and subject of the manuscript are very interesting, and coherent to the scope of the journal. This manuscript represents a good and original study with new results. The manuscript is well designed and written, and therefore could be acceptable after minor revision for possible publication in “horticulture”. However, some corrections should be considered as listed below:

The abstract is very clearly described and comprehensive.

Add latest references in the introduction. The introduction should be enriched based on new studies.

Line 33-34, please add the below reference at the end of sentence:

In regions with extreme climatic conditions, 33 there are several plant species that are excellent sources of phytochemicals (Eslamdoust 2022).

Eslamdoust, J. Bark content variations and the production of Populus deltoides as a source of bioenergy under temperate climate conditions. J. For. Res. 33, 1807–1815 (2022). https://doi.org/10.1007/s11676-022-01465-6.

Also, use the below reference in introduction:

Xie D, Ye J, Lu M, Wang S, You C and Li Y (2020) Comparsion of Activities of Fatty Acyl Desaturases and Elongases Among Six Teleosts With Different Feeding and Ecological Habits. Front. Mar. Sci. 7:117. doi: 10.3389/fmars.2020.00117.

Experimental design: it is poorly written. It is critical part of the research and worth’s to describe more details providing a good image of your experiments for readers.

The authors do not present the methodology used for the normality analysis in the M&M methodology

Discussion should provide a comprehensive review of the relevant literature, including recent studies and research findings in the field.

Conclusion is well written.

Remove all the errors including spacing and revise the entire manuscript by removing typographical errors.

Author Response

Response to Reviewer 1

Title: The Composition and the Content of ∆-5 Sterols, Fatty Acids, and the Activity of Acyl-lipid Desaturases in the Shoots of Ephedra monosperma, Introduced in the Botanical Garden of the Cryolithozone of Yakutia. 

The title and subject of the manuscript are very interesting, and coherent to the scope of the journal. This manuscript represents a good and original study with new results. The manuscript is well designed and written, and therefore could be acceptable after minor revision for possible publication in “horticulture”. However, some corrections should be considered as listed below: 

The abstract is very clearly described and comprehensive. 

Add latest references in the introduction. The introduction should be enriched based on new studies.

Response: Thank you, I added an analysis of new studies to the text of the manuscript:

Previously, it was shown that phosphatidic acid (PA) was the dominant lipid class of shoots E. monosperma [17]. In Ephedra alata lipids, PA was also the dominant class compared to phosphatidylcholine and phosphatidylethanolamine under control conditions [18]. A large proportion of PAs in the phospholipid profile is characteristic of some extremophile organisms [19]. The lipids of seeds of four ephedra species (E. nevadensis, E. viridis, E. przewalskii, and E. gerardiana) contain delta5-unsaturated polymethylene-interrupted fatty acids (∆5-UPIFA): ∆5,9- and ∆5,11-С18:2; ∆5,9,12-С18:3; ∆5,9,12,15-С18:4; ∆5,11-С20:2; ∆5,11,14-С20:3; and ∆5,11,14,17-С20:4 acids [20]. Our studies revealed that ∆5-UPIFA was detected in frost-tolerant coniferous [21] and in the aboveground parts of wintergreen species of horsetail [22].

Line 33-34, please add the below reference at the end of sentence:

In regions with extreme climatic conditions, 33 there are several plant species that are excellent sources of phytochemicals (Eslamdoust 2022).

Eslamdoust, J. Bark content variations and the production of Populus deltoides as a source of bioenergy under temperate climate conditions. J. For. Res. 33, 1807–1815 (2022). https://doi.org/10.1007/s11676-022-01465-6.

Response: Thanks for the comment, link added.

Also, use the below reference in introduction:

Xie D, Ye J, Lu M, Wang S, You C and Li Y (2020) Comparsion of Activities of Fatty Acyl Desaturases and Elongases Among Six Teleosts With Different Feeding and Ecological Habits. Front. Mar. Sci. 7:117. doi: 10.3389/fmars.2020.00117.

Response: I agree with the remark, the link has been added.

Experimental design: it is poorly written. It is critical part of the research and worth’s to describe more details providing a good image of your experiments for readers.

Response: Thanks for the note, the necessary information has been added to the experimental design.

The authors do not present the methodology used for the normality analysis in the M&M methodology

Response: Added a link to this protocol

Dudareva, L.V.; Semenova, N.V.; Nochsorov, V.V.; Rudikovskaya, E.G.; Petrov, К.А. The component composition of the phytosterols of the aerial part of the horsetail variegated Еquisétum variegatum Schleich. ex. Web. growing in North-East Yakutia.  Khimiya Rastitel'nogo Syr'ya 2020, 2, 133–139. https://doi.org/10.14258/jcprm.2020025555

GC/MS used in single quadrupole mode

Discussion should provide a comprehensive review of the relevant literature, including recent studies and research findings in the field.

Response: Thank you for your comment. We have expanded the literature review on the topic under studyю

Conclusion is well written.

Remove all the errors including spacing and revise the entire manuscript by removing typographical errors.

Response: Agree with the remark, the manuscript has been edited

The team of authors expresses their deep gratitude to the Reviewer for a deep professional analysis of our article and for valuable and useful comments. We hope that the changes made will help improve the manuscript.

Author Response File: Author Response.docx

Reviewer 2 Report

The subject matter of this article has a high research value and is a good reference for the study of the resistance function of metabolic substances, while the research method is detailed and clear. However, there are still some issues that need to be revised:

1. Line13-14, (-8.3 °C) and (-38.4 °C) are deleted, these two temperatures are the temperatures at which the experimental material was sampled, while this sentence in the abstract describes a knowledge and not the temperature set by the authors' experiment.

2. Line103, Figure 1 suggests that the four temperatures mentioned in the text or at least two times (july/sep, oct/dec) be removed so that the reader can see more visually the differences in plant phenotypes at the two important time points of low and non-low temperatures. As this study is part of a plant physiological work, the close relationship between physiology and phenotype allows the reader to better understand the authors' work.

3. Line 263-264 should be transferred to the 'Methods' section or to the figure notes in Figure 2.

4. In most of the figures, the horizontal coordinates or legends include the time of sampling and the temperature is shown in brackets. It is recommended that the authors explain this in the "Materials and Methods" section, or in the figure notes for the first appearance, and that the subsequent figures or tables be simply labelled. Otherwise it looks very messy.

5. Authors should upload the original data of all figures as attachments to the submission system.

Author Response

Response to Reviewer 2

The subject matter of this article has a high research value and is a good reference for the study of the resistance function of metabolic substances, while the research method is detailed and clear. However, there are still some issues that need to be revised:

1. Line13-14, (-8.3 °C) and (-38.4 °C) are deleted, these two temperatures are the temperatures at which the experimental material was sampled, while this sentence in the abstract describes a knowledge and not the temperature set by the authors' experiment.

Response: Thank you for your valuable advice, temperature has been removed from the annotation.

2. Line103, Figure 1 suggests that the four temperatures mentioned in the text or at least two times (july/sep, oct/dec) be removed so that the reader can see more visually the differences in plant phenotypes at the two important time points of low and non-low temperatures. As this study is part of a plant physiological work, the close relationship between physiology and phenotype allows the reader to better understand the authors' work.

Response: Thank you for your comment, four temperatures have been removed from all the figures.

3. Line 263-264 should be transferred to the 'Methods' section or to the figure notes in Figure 2.

Response: We agree with the remark, lines 263-264 were moved to the "Methods" section

4. In most of the figures, the horizontal coordinates or legends include the time of sampling and the temperature is shown in brackets. It is recommended that the authors explain this in the "Materials and Methods" section, or in the figure notes for the first appearance, and that the subsequent figures or tables be simply labelled. Otherwise it looks very messy.

Response: Thank you for the comment, corrections have been made to the drawings

5. Authors should upload the original data of all figures as attachments to the submission system.

Response: We agree with the remark, the source data is loaded

The team of authors expresses their deep gratitude to the Reviewer for a deep professional analysis of our article and for valuable and useful comments. We hope that the changes made will help improve the manuscript.

Author Response File: Author Response.docx

Reviewer 3 Report

In my opinion, the manuscript could be suitable for publication after some adjustments. The authors have performed the analysis of lipid composition in Ephedra monosperma shoots using chromatographic techniques such as HPTLC and GC-MS. In the introductory section the authors could include results of lipid analysis in other plant species to provide more context for the reader. In section 2.3 of materials and methods, they do not mention any reference to this protocol, therefore they should indicate if this method is developed by the authors or if it is part of the application notes developed by the companies that manufacture the equipment. The conclusions are very particular to the plant species analyzed in this research; however, an application of the potential of this research is not presented. The findings found in this study should be highlighted.

Ø  The name gas-liquid chromatography is no longer used, the correct name is gas chromatography - line 177.

Ø  According to the information reported in the manuscript, GC-MS with quadrupole analyzers were used for lipid analysis. I do not understand how they did tandem mass analysis if the analyzers were not triple quadrupole or two different analyzers in series type QTOF. Please clarify this information. 

Ø  This research does not specify how the tandem mass analyses were performed. If it is a triple quadrupole the analyzer used should include the conditions for selective reaction monitoring.

Ø  Correct the word electron impact by electron ionization in lines179 and 200. Since the early 2000s, the use of electron impact to refer to this hard ionization source has been discontinued.

Author Response

Response to Reviewer 3

In my opinion, the manuscript could be suitable for publication after some adjustments. The authors have performed the analysis of lipid composition in Ephedra monosperma shoots using chromatographic techniques such as HPTLC and GC-MS. In the introductory section the authors could include results of lipid analysis in other plant species to provide more context for the reader.

Response: Thank you for your valuable advice, in the introduction we added information about the composition of lipids of other plants.

In section 2.3 of materials and methods, they do not mention any reference to this protocol, therefore they should indicate if this method is developed by the authors or if it is part of the application notes developed by the companies that manufacture the equipment.

Response: Added a link to this protocol

Dudareva, L.V.; Semenova, N.V.; Nochsorov, V.V.; Rudikovskaya, E.G.; Petrov, К.А. The component composition of the phytosterols of the aerial part of the horsetail variegated Еquisétum variegatum Schleich. ex. Web. growing in North-East Yakutia.  Khimiya Rastitel'nogo Syr'ya 2020, 2, 133–139. https://doi.org/10.14258/jcprm.2020025555

The conclusions are very particular to the plant species analyzed in this research; however, an application of the potential of this research is not presented. The findings found in this study should be highlighted.

Response: Thanks for the helpful comment, added the following to the output:

Thus, the results obtained on the seasonal dynamics of lipids of ephedra shoots provide useful information at the physiological and biochemical level on the adaptation reactions of introduced valuable, rare and medicinal plants growing in the conditions of the Botanical Garden of permafrost ecosystems.

Ø  The name gas-liquid chromatography is no longer used, the correct name is gas chromatography - line 177. 

 Response: We agree with the remark, the name of the method has been corrected in the new edition, thank you.

Ø  According to the information reported in the manuscript, GC-MS with quadrupole analyzers were used for lipid analysis. I do not understand how they did tandem mass analysis if the analyzers were not triple quadrupole or two different analyzers in series type QTOF. Please clarify this information.  

 Response: We agree with the remark, the tandem device we have was used in this study in single quadrupole mode, not tandem (triple)

Ø  This research does not specify how the tandem mass analyses were performed. If it is a triple quadrupole the analyzer used should include the conditions for selective reaction monitoring.

 Response: We agree with the remark, the tandem device we have was used in this study in single quadrupole mode, not tandem (triple)

Ø  Correct the word electron impact by electron ionization in lines179 and 200. Since the early 2000s, the use of electron impact to refer to this hard ionization source has been discontinued.

Response: Thank you for your comment, the text has been updated accordingly.

The team of authors expresses their deep gratitude to the Reviewer for a deep professional analysis of our article and for valuable and useful comments. We hope that the changes made will help improve the manuscript.

Author Response File: Author Response.docx