The Role of Reactive Oxygen Species in the In Vitro Germination and Growth of the Petunia (Petunia hybrida E. Vilm.) Male Gametophyte
and Marat R. Khaliluev 1,2,*
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsAttached file
Comments for author File: 
Comments.pdf
Author Response
Dear Reviewer,
On behalf of ourselves and the co-authors, we thank you for your appreciation of our manuscript and valuable comments. We thank the reviewer for his high assessment of our manuscript. We are confident that your comments and corrections will make our manuscript better.
Remark 1: Keywords: Petunia hybrida E. Vilm male gametophyte. These keywords already appear in the title, put others.
Response 1: Thank you for your valuable comment. We agree with the reviewer's remark. We have excluded these keywords and added pollen grain (PG) germination (L. 27).
Remark 2: Although the object of study is pollen grains and pollen tube growth, it is necessary to clearly describe the growing conditions and provide bibliography with information on the soil, irrigation, from planting cuttings to flowering. What were the climatic conditions of the greenhouse? (L. 88-92)
Response 2: We thank the reviewer for valuable comment. We took into account the reviewer’s comments and made appropriate additions to the text of the manuscript (L. 142–167).
Remark 3: The following variants were used for pollen cultivation: H2O2 at a concentration of 1, 5, or 10 μM and DPI, at a concentration of 0.5 μM, 1 μM, 0.1 mM, or 1 mM Why they used these concentrations is necessary to physiologically substantiate?
Response 3: Thank you for your valuable comment. We agree with the reviewer's remark. In our work, we used hydrogen peroxide because it is the most stable ROS and, presumably, the most important in terms of physiological activity [19]. We have made the necessary corrections in the text (L. 175-176).
Remark 4: A low germination rate has been characteristic of the SI clone PGs in all our longterm experiments [23]. The self-citations are interesting; However, it is best not to personalize past scientific products. Why? is what you expect to read in a piece of writing. Write the reason for the low germination rate of pollen grains in plants with an incompatible system.
Response 4: Thank you for your valuable comment. Unfortunately, we cannot unambiguously answer the question why SI petunia clones are characterized by a low germination rate. We can only state it as a fact. Perhaps this is due to differences between genotypes of SC and SI petunia clones.
Remark 5: According to our data, two main phytohormones that stimulate PG germination and PT growth are IAA and ABA. Specify what are the conditions that stimulate the activity of these hormones, in addition to those indicated; such as environmental, genetic background of the crop, compatibility, among others that could be described.
Response 5: Thank you for your valuable comment and interesting question. This proposal talks about stimulating of PG germination and PT growth by these phytohormones both in vitro and in vivo. The purpose of this work was to demonstrate the participation, necessity and importance of ROS for germination and growth of PTs in an in vitro model system. We have been studying hormonal balance in the progamic phase of fertilization for many years. The first our article on this topic was published in 2003 and we refer to it (14). We recently published a review article in the Horticulturae journal on the role of phytohormones in the progamic phase of fertilization ([17] Zakharova, E.V.; Khaliluev, M.R.; Kovaleva, L.V. Hormonal Signaling in the Progamic Phase of Fertilization in Plants. Horticulturae 2022, 8, 365, doi:10.3390/horticulturae8050365). In this review article, we tried to systematize the available literature data.
Remark 6: Figure 2 shows the ROS staining in the male gametophyte of petunia. The petunia PGs secrete ROS as early as the beginning of activation (Figure 2A–C) Those that the white arrows point to, related to the secretion of ROS is almost imperceptible.
Response 6: Quite right. Most likely, these are non-viable PGs that exhibit a bright fluorescence associated with the ROS generation. This confirms our assumption that ROS generation is necessary to activate the germination of viable PGs.
Remark 7: In line with the modern view on the exudate as the medium of pollen–stigma interaction, the authors postulate that one of the ROS functions on the stigma is the support and/or stimulation of pollen germination. Makes a biochemical explanation related to metabolism
Response 7: We thank the reviewer for the interesting question. This aspect is well described in the work of our colleagues [8-10]. The purpose of this work was to demonstrate the participation, necessity and importance of ROS for germination and growth of PTs in an in vitro model system. We continue to study the influence of ROS on the germination and growth of PTs in vivo, as well as their role in the process of arresting the growth of PTs as a result of the gametophytic self-incompatibility. We look forward to publishing these results soon.
Remark 8: It remains to be widely discussed and concluded, why use compatible and incompatible systems.
Response 8: We use compatible and self-incompatible systems in order to show how the phenomena we study behave on different genotypes. Аdditionally, the main question for us is the ROS participation in the process of self-incompatibility, arresting the PT growth in pistil tissues. These in vivo experiments are already being completed and, we hope, will be published soon. We have written a Conclusion section (L 408-417).
Remark 9: Figure 1 is difficult to interpret
Response 9: Thank you for your valuable comment. We agree with the reviewer's remark. We have improved the quality of Figure 1 for better understanding.
Once again, we are so grateful for your review and valuable comments. In addition, we also send a resubmitted Word document with your and other reviewers' comments. We hope that the resubmitted version of the article has become better and more understandable to readers.
Additionally, we used the MDPI editing service to improve the English language.
Best regards,
Marat Khaliluev and co-authors.
Reviewer 2 Report
Comments and Suggestions for Authors1. Abstract. Line 15-17. What is the meaning of ‘appearing as acceleration and inhibition of these processes, respectively, depending on the concentration and the duration of cultivation’. It is confusing. Is the process accelerated by high concentration or by low concentration? What role does cultivation time play? The abstract still needs to describe the results clearly.
2. The Introduction is too long and lacks logic, and it talks about the research progress in general, which fails to grasp the research focus of this paper, and makes it impossible for readers to quickly find the research purpose, main research content and innovation of the manuscript.
3. Line 101, Why choose to record the results every hour during 3-h pollen cultivation? Is there any reference?
4. There are too few results in the manuscript to support the research content of the manuscript. What about hormones and Ca2+?
5. The manuscript lacks conclusions, does not lead to accurate data, and cannot find specific results from the study.
Comments on the Quality of English LanguageThe writing of English needs to be improved.
Author Response
Dear Reviewer,
On behalf of ourselves and the co-authors, we thank you for your appreciation of our manuscript and valuable comments. We thank the reviewer for his high assessment of our manuscript. We are confident that your comments and corrections will make our manuscript better.
Remark 1: Abstract. Line 15-17. What is the meaning of ‘appearing as acceleration and inhibition of these processes, respectively, depending on the concentration and the duration of cultivation’. It is confusing. Is the process accelerated by high concentration or by low concentration? What role does cultivation time play? The abstract still needs to describe the results clearly.
Response 1: We agree with the reviewer's remark. We absolutely agree that this phrase is an incorrect. We have removed this line from the Abstract section.
Remark 2: The Introduction is too long and lacks logic, and it talks about the research progress in general, which fails to grasp the research focus of this paper, and makes it impossible for readers to quickly find the research purpose, main research content and innovation of the manuscript.
Response 2: We thank the reviewer for his comment. We tried to rewrite the introduction section. According to your comment, we have shortened the introduction part (added L 41-92, deleted L 92-109). We also changed some of the cited references [2-7] (L 447-470). We also tried to specify part of the Introduction section, which is highlighted in color in the resubmitted manuscript. In addition, we tried to make the introduction section more logical and understandable to readers. We have tried to ensure that the rewritten introduction section better explains the purpose of this study.
Remark 3: Line 101, Why choose to record the results every hour during 3-h pollen cultivation? Is there any reference?
Response 3: Each hour of cultivation is necessary in order to trace the dynamics of PT growth. In addition, to investigate the effect of ROS on PG germination and PT growth depending on the exposure time. The recording of experimental data for each hour is based on previously conducted experiments. We have added an explanation to the manuscript text (L. 176-178).
Remark 4: There are too few results in the manuscript to support the research content of the manuscript. What about hormones and Ca2+?
Response 4:
We have been studying hormonal balance in the progamic phase of fertilization for many years. In this article we only refer to the role of phytohormones in the text. The purpose of this work was to demonstrate the participation, necessity and importance of ROS for germination and growth of PTs in an in vitro model system. We recently published a review article in the Horticulturae journal on the role of phytohormones in the progamic phase of fertilization ([17] Zakharova, E.V.; Khaliluev, M.R.; Kovaleva, L.V. Hormonal Signaling in the Progamic Phase of Fertilization in Plants. Horticulturae 2022, 8, 365, doi:10.3390/horticulturae8050365). As for the role of Ca2+ in the germination and growth of PTs, it is undoubtedly very significant and has been widely documented in the literature. In our previous experiments, we also partially studied this issue. We continue to study the influence of ROS on the germination and growth of PTs in vivo, as well as their role in the process of arresting the growth of PTs as a result of the gametophytic self-incompatibility. In this article, we only present a small part of the experimental data obtained using an in vitro model system. In this regard, we chose a brief report as the type of manuscript.
Remark 5: The manuscript lacks conclusions, does not lead to accurate data, and cannot find specific results from the study.
Response 5: We agree with the reviewer's remark. We have written a Conclusion section (L 408-417).
Remark 6: Comments on the Quality of English Language. The writing of English needs to be improved.
Response 6: We agree with the reviewer's remark. Additionally, we used the MDPI editing service to improve the English language.
Once again, we are so grateful for your review and valuable comments. In addition, we also send a resubmitted Word document with your and other reviewers' comments. We hope that the resubmitted version of the article has become better and more understandable to readers.
Best regards,
Marat Khaliluev and co-authors.
Reviewer 3 Report
Comments and Suggestions for AuthorsMy congratulations to authors!
Dear colleagues, your paper is very well and it is almost ready for publishing. My congratulations once again.
Comments on the Quality of English LanguageHowever, please just check the grammar because on page 8/10 line 286-288 stays:
"These facts convincingly demonstrate that ROS are the necessary component in regulating the polar growth of male gametophyte; however, the ROS targets (in particular, 287 that of hydrogen peroxide) in the growing PT are still vague."
I think it must be:
These facts convincingly demonstrate that ROS is necessary component in regulating the polar growth of male gametophyte; however, the ROS targets (in particular, 287 that of hydrogen peroxide) in the growing PT are still vague.
Author Response
Dear Reviewer,
On behalf of ourselves and the co-authors, we thank you for your appreciation of our manuscript and valuable comments. We thank the reviewer for his high assessment of our manuscript. We are confident that your comments and corrections will make our manuscript better.
Remark 1: However, please just check the grammar because on page 8/10 line 286-288 stays:
"These facts convincingly demonstrate that ROS are the necessary component in regulating the polar growth of male gametophyte; however, the ROS targets (in particular, that of hydrogen peroxide) in the growing PT are still vague."
I think it must be:
These facts convincingly demonstrate that ROS is necessary component in regulating the polar growth of male gametophyte; however, the ROS targets (in particular, that of hydrogen peroxide) in the growing PT are still vague.
Response 1: We agree with the reviewer's remark. Additionally, we used the MDPI editing service to improve the English language.
Once again, we are so grateful for your review and valuable comments. In addition, we also send a resubmitted Word document with your and other reviewers' comments. We hope that the resubmitted version of the article has become better and more understandable to readers.
Best regards,
Marat Khaliluev and co-authors.
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsApproved for publication after revision.
