Hormonal Signaling in the Progamic Phase of Fertilization in Plants

Round 1

Reviewer 1 Report

The main focus of this review was stated to be an overview of hormone signaling as it relates to male gametophyte formation, cell division, maturation, and pollen tube function.  The paper went into a broad overview of hormones, pollen tube growth, pollen-pistil interactions, programmed cell death.  This paper is lacking in focus, is uneven in level of detail, and is at times both too broad and too focused.  A major rewrite to focus on the topic as stated in the title would be very beneficial. 

 

Main concerns

 

1. The writing needs substantial improvement in organization, focus, clarity, and citations. For an organization and focus example, the abstract begins with a focused topic statement (lines 14, 15) then broadens to include a general statement about hormones in plants (lines 16-17), then refocuses.  Such broad statements detract from the flow of the work.  More citations are needed in several places including (but not limited to) lines 69, 70, 78, 89, 97, 110, 123, 153, 195, 198, 240.  Two examples of focus/clarity are section 2.1 on auxin which compiles all auxin influences on anther formation, pollen grain release and wall composition with no mention of TAP (the main section header), and section 2.4 on ABA, which includes very little direct information and could be cut or rolled into a broader section.  

 

2. The amount of detail on the hormone biosynthesis and signaling pathways is not needed and detracts from focus on pollen and reproductive biology. Unless there is a direct tie in between how the hormone is made and how it acts these sections can be cut or greatly reduced.  The paper could be cut down to just the hormones mentioned in the title (ethylene and cytokinin). 

 

3. The inclusion of information from epidermal, leaf, endosperm, and other non-pollen non-pistil sources is extraneous and can be cut. An exception can be made if the only data is from non-floral non-pollen experiments. 

 

4. What was the inspiration for figures 1 and 2? These are complex figures and are based on a large body of work.  Please provide citations for any prior figures that these ones are based on. 

 

5. Please avoid the use of red and green as informative colors, not all readers can distinguish these colors. Please change the color schemes of figure 1 and figure 2 to improve accessibility. 

 

Minor concerns

 

6. There is a typo in figure 1B (maturing anter instead of anther)

 

7. Figure 2 is lacking in structural detail of the stigma and the ovule. Please show at least the embryo sac and egg in the ovary. 

 

8. The work of others is referred to in an usual manner eg line 606 “the authors assume.”  More typical language would be “propose” or “predict.”  Please be careful in referring to the work of others. 

Author Response

Dear Reviewer,

On behalf of ourselves and the co-authors, we thank you for your appreciation of our manuscript "Hormonal Signaling in the Progamic Phase of Fertilization in Plants: Ethylene and Cytokinin in Male Gametophyte Development and Growth" and for your valuable comments. We are confident that your comments and corrections will make our manuscript better. We made required changes in the manuscript and attempted to answer to all your questions.

Remark 1: The writing needs substantial improvement in organization, focus, clarity, and citations. For an organization and focus example, the abstract begins with a focused topic statement (lines 14, 15) then broadens to include a general statement about hormones in plants (lines 16-17), then refocuses. Such broad statements detract from the flow of the work.  More citations are needed in several places including (but not limited to) lines 69, 70, 78, 89, 97, 110, 123, 153, 195, 198, 240. Two examples of focus/clarity are section 2.1 on auxin which compiles all auxin influences on anther formation, pollen grain release and wall composition with no mention of TAP (the main section header), and section 2.4 on ABA, which includes very little direct information and could be cut or rolled into a broader section.

Response 1: In general, we agree with the valuable comments. We are significantly improvement of organization, focus, clarity, and citations. We have radically changed the content and numbering of sections in the text. All changes are colored in the text (inserts - yellow; deletions - purple).

In our opinion it's just always impossible to focus ONLY on 2 hormones and not include the rest of the phytohormone groups, because it's all about hormonal balance and hormone levels. Indeed, cytokinins and ethylene play a leading role in the development of the male gametophyte, pollination, detection of pollen growth, and arrest of PT in the mechanism of action of the self-incompatibility mechanism, including SI induced PCD. We made a reservation about this in the text (lines 111-117).

Indeed, in the original version of the article, we were strongly limited to references to general terms and statements. We have corrected this oversight according to your comments. We have supplemented the text with relevant references, for example, lines 81-82, 91, 101, 109, 151, 220, 299 etc. (colored in yellow).

We also expanded sections 2.1 and 2.4 as far as possible, since experimental studies are severely limited.

Remark 2: The amount of detail on the hormone biosynthesis and signaling pathways is not needed and detracts from focus on pollen and reproductive biology. Unless there is a direct tie in between how the hormone is made and how it acts these sections can be cut or greatly reduced.  The paper could be cut down to just the hormones mentioned in the title (ethylene and cytokinin).

Response 2: We took into account the comment of reviewer concerning the biosynthesis of hormones and their signaling pathways is not required in article. We have reduced and removed some points (delete 3.1. and 4.1 sections) (lines 199 – 214 and 259-282).

Remark 3: The inclusion of information from epidermal, leaf, endosperm, and other non-pollen non-pistil sources is extraneous and can be cut. An exception can be made if the only data is from non-floral non-pollen experiments.

Response 3:  We agree with the reviewer's remark and excluded from the text dates not related to the progamous phase of fertilization. We made appropriate changes (Lines 482-488; 585-594).

Remark 4, 5, 7: What was the inspiration for figures 1 and 2? These are complex figures and are based on a large body of work.  Please provide citations for any prior figures that these ones are based on. Please avoid the use of red and green as informative colors, not all readers can distinguish these colors. Please change the color schemes of figure 1 and figure 2 to improve accessibility. Figure 2 is lacking in structural detail of the stigma and the ovule. Please show at least the embryo sac and egg in the ovary.

Response 4, 5, 7: We agree with your comments that the figures are complex and based on a large number of experimental data. Our inspiration for the creation of figures 1 and 2 was the systematization of the data array on this issue. We tried to refer to the text of the article, as well as in the captions to the figures. We thank the reviewer's comments on the color of figures. We did not think that not everyone can distinguish between shades of red and green. We have radically changed the color scheme. Additionally, we have detailed the structures of the stigma and ovary in the figure 2.

Remark 6, 8: There is a typo in figure 1B (maturing anter instead of anther). The work of others is referred to in an usual manner eg line 606 “the authors assume.”  More typical language would be “propose” or “predict.”  Please be careful in referring to the work of others.

Response 6, 8: The reviewer's comments have been corrected (lines 56 and 672).

 

We tried to remove out shortcomings in English.

We one more thanks the reviewer for careful analysis of our work and very useful recommendations.

 

Sincerely,

Marat Khaliluev and Ekaterina Zakharova.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments on “Hormonal Signaling in the Progamic Phase of Fertilization in Plants: Ethylene and Cytokinin in Male Gametophyte Development and Growth” by Zakharova et al.

This manuscript reviews the literature relating to hormonal signaling in the progamic phase of fertilization in plants, and it has summarised various sources of the literature. The collection of the literature can be of use for scientific community.  Overall, the manuscript is poorly written. First, the structure of the manuscript is not logical, and sections are not linked well. For example, sections 2,3,4 are about hormones and sections 5 is about developmental stage. The authors should consider to organise the manuscript better. For example, should sections 3 and 4 be the subsections of section 2?  should sections 6 be the subsection of section 5? Second, the text in many places is not organised well. It is more or less like a lab note rather than a written manuscript. For example, lines 422-428 are largely out of context. They could be placed in somewhere else or better linked with the context. The authors need to better organise the text for general readership. Third, the manuscript is not written at a scientific communication level. The authors need to examine their writing carefully throughout the manuscript.  I list some examples on two pages only:

  page 4

The SnRK2-APC/C TE regulatory module antagonistic mediates of GA and ABA pathways

have been simulated as

which downstream regulated of ET receptor and may be component of a MAP ki-164 nase cascade

is supplemented with

page 5

Arabidopsis has CK three

also that the signal initiation on the plasma membrane (PM) may be relevant as well

a membrane-localized sensor kinase, which receive

TFs are highly enriched, supporting a model in which RRs act at the top of a transcriptional cascade (if this sentence should be there, it must be clarified)

expression of a large genes necessary

switch of metabolism

• Need to clarify what switch means in this context?
• is switch of metabolism or switch of signalling?)

Author Response

Dear Reviewer,

On behalf of ourselves and the co-authors, we thank you for your appreciation of our manuscript "Hormonal Signaling in the Progamic Phase of Fertilization in Plants: Ethylene and Cytokinin in Male Gametophyte Development and Growth" and for your valuable comments. We are confident that your comments and corrections will make our manuscript better. We made required changes in the manuscript and attempted to answer to all your questions.

In accordance with important reviewer's comments we have corrected the text together with the figures and tried to answer to the questions.

Remark 1: This manuscript reviews the literature relating to hormonal signaling in the progamic phase of fertilization in plants, and it has summarised various sources of the literature. The collection of the literature can be of use for scientific community.  Overall, the manuscript is poorly written. First, the structure of the manuscript is not logical, and sections are not linked well. For example, sections 2,3,4 are about hormones and sections 5 is about developmental stage. The authors should consider to organise the manuscript better. For example, should sections 3 and 4 be the subsections of section 2?  should sections 6 be the subsection of section 5? Second, the text in many places is not organised well. It is more or less like a lab note rather than a written manuscript. For example, lines 422-428 are largely out of context. They could be placed in somewhere else or better linked with the context. The authors need to better organise the text for general readership. Third, the manuscript is not written at a scientific communication level.

Response 1: In general, we agree with the valuable comments. We are significantly improvement of organization, focus, clarity, and citations. We have radically changed the content and numbering of sections. All changes are colored in the text (inserts - yellow; deletions - purple). We hope that the resubmitted version of the article is better structured and logical, as well as more understandable to readers. For example, extended 2.1 and 2.4 sections. Tried to focus exclusively on the progam phase of fertilization, removed information regarding the epidermal leaf, endosperm and other unreasonable ways of male gametophyte development and pollen-pistil interaction. We excluded lines 422-428 from the text.

We have corrected this oversight according to comments of other reviewers. We have supplemented the text with relevant references, for example, lines 81-82, 91, 101, 109, 151, 220, 299 etc. (colored in yellow).

We removed clause 6 and reworked clause 6.1, changed clauses 7 and 8. We absolutely agree with the reviewer that it is more logical to make clauses 3 and 4 subsections of clause 2.

Remark 2: The authors need to examine their writing carefully throughout the manuscript.  I list some examples on two pages only:

  page 4

The SnRK2-APC/C TE regulatory module antagonistic mediates of GA and ABA pathways

have been simulated as

which downstream regulated of ET receptor and may be component of a MAP ki-164 nase cascade

is supplemented with

page 5

Arabidopsis has CK three

also that the signal initiation on the plasma membrane (PM) may be relevant as well

a membrane-localized sensor kinase, which receive

TFs are highly enriched, supporting a model in which RRs act at the top of a transcriptional cascade (if this sentence should be there, it must be clarified)

expression of a large genes necessary

switch of metabolism

• Need to clarify what switch means in this context?
• is switch of metabolism or switch of signalling?)

Response 2: We agree with the reviewer's comments. The improved version of the review does not contain these experimental data, as they are excluded from the text (lines 199-214, 482-488; 585-594 etc. colored purple).

Additionally, we have radically changed the color scheme and detailed the structures of the stigma and ovary in the figure 2.

 

We tried to remove out shortcomings in English.

We one more thanks the reviewer for critical analysis of our work and useful recommendations.

 

Sincerely,

Marat Khaliluev and Ekaterina Zakharova.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Thank you for making many revisions to your manuscript.  The color change for the figures is most appreciated.  The overall manuscript could use some additional improvement to make it more focused. 

 

Some suggestions for improvement

 

1. Add an introduction, something to give bigger picture context and relevance/application for the work. Some additional context or background information to provide more relevance would be helpful.  Even a brief mention of how pollen development and function are key for many crop plants as the desired product is a seed or fruit.  This section could include example of self-compatible non self-compatible species (crop and or/research). 

 

2. Topical alignment of the title and the content. The title mentions 2 hormones, but the main body of the review goes through several hormones and their possible interplay.  Perhaps the easier option is to change the title to reflect the larger scope of the work.

 

3. Please provide citations for work summarized in the figures.

 

4. Many of the sections can either be greatly reduced or cut. Suggestions are

cut lines 171-174

cut lines 196-198

Greatly reduce lines 298-324

Greatly reduce section 2.2 on pollen pistil interactions

Cut section 2.9

Cut section 2.10              

Cut section 2.11

 

5. The section on self compatible vs not seems to fit better before the section 2.3 information on ET signaling.

 

6. While sections 3.1 and 3.2 are informative, they have no mention at all of hormones. Perhaps they could be moved to an earlier section (before 2.3) introducing the idea of compatibility. 

 

Other minor changes

 

7. One minor but very important change for line 40 is mitosis (not meiosis). The microspore (pollen mother cell) is formed via meiosis (going from diploid to haploid).  From this point forward, cell division leading to grain formation is mitosis.  The microspores divide by mitosis. 

 

8. Please standardize all journal citations regarding journal title (capitalized vs lowercase, full name vs abbreviated).

 

9. Add references to lines 36, 47.

 

10. Define all abbreviations at first usage.

 

11. Add species information in places where it is not indicated (eg line 83, lines 96-98 ,lines 154-155, line 159, 256-257). Note, this is not an exhaustive list, please check other sections as well. 

 

12. Consider a different word choice than monotonous for hormone level changes (eg. Line 188). It is meant to be monumental?  Or continuous? 

 

13. Figure 2 contains stile (steps for going up and over a wall) instead of style (the floral organ)

 

14. The word fertilization in the bottom left of figure 2 either needs a comma before it, or need placing on a different line (right now it reads as an odd phrase, pollen tubes grow to the ovary fertilization)

 

15. The letter s appears by itself in line 396, not sure what was meant to be here, please correct as needed

 

16. The complex interplay between hormones in pollen (sections 2.5 and 2.6) is quite complex, it would benefit from a figure (it would be a nice addition but not essential)

 

17. Move conclusion 3 to the start of the list as it is the first developmental stage

Author Response

Dear Reviewer,

On behalf of ourselves and the co-authors, we thank you for your appreciation of our manuscript. We are confident that your comments and corrections will make our manuscript better. We made required changes in the manuscript and attempted to answer to all your questions. All new changes in the manuscript are colored blue.

1. Add an introduction, something to give bigger picture context and relevance/application for the work. Some additional context or background information to provide more relevance would be helpful.  Even a brief mention of how pollen development and function are key for many crop plants as the desired product is a seed or fruit.  This section could include example of self-compatible non self-compatible species (crop and or/research). 

Answer: We fully agree with the reviewer's recommendation to add an introduction section. We have included additional information (lines 34-76).

 

2. Topical alignment of the title and the content. The title mentions 2 hormones, but the main body of the review goes through several hormones and their possible interplay.  Perhaps the easier option is to change the title to reflect the larger scope of the work.

Answer: We agree with the reviewer's recommendation. We changed the title of manuscript to be more general («Hormonal signaling in the progamic phase of fertilization in plants»).

 

3. Please provide citations for work summarized in the figures.

Answer: We provide citations for work summarized in the figures 1 and 2.

 

4. Many of the sections can either be greatly reduced or cut. Suggestions are

cut lines 171-174

cut lines 196-198

Greatly reduce lines 298-324

Greatly reduce section 2.2 on pollen pistil interactions

Cut section 2.9

Cut section 2.10              

Cut section 2.11

Answer: In general, we agree with the reviewer's comments and removed the redundant information

lines 171-174 – delete

lines 196-198 – delete

lines 298-324 – one paragraph has been removed

Greatly reduce section 2.2 on pollen pistil interactions – lines 348-358 and 383-388, 396-400 removed

Section 2.9 modified

Section 2.10 has been removed

Section 2.11 moved to section 2.3

5. The section on self compatible vs not seems to fit better before the section 2.3 information on ET signaling.

Answer: We don’t entirely agree with this opinion of the reviewer. In our opinion, this section is independent and should be considered separately.

6. While sections 3.1 and 3.2 are informative, they have no mention at all of hormones. Perhaps they could be moved to an earlier section (before 2.3) introducing the idea of compatibility. 

Answer: One could agree with this remark. However, in our opinion, it is more logical to leave this section as an introduction to the self-incompatibility for further description of the phytohormone roles. 

Other minor changes

7. One minor but very important change for line 40 is mitosis (not meiosis). The microspore (pollen mother cell) is formed via meiosis (going from diploid to haploid).  From this point forward, cell division leading to grain formation is mitosis.  The microspores divide by mitosis. 

Answer: In accordance with the reviewer's comments, we made the appropriate correction (line 89).

8. Please standardize all journal citations regarding journal title (capitalized vs lowercase, full name vs abbreviated).

Answer: In accordance with the reviewer's comments, we standardized references list.

9. Add references to lines 36, 47.

Answer: We added citations in appropriate position.

10. Define all abbreviations at first usage.

Answer: We fully agree with the reviewer's requirement and have made the necessary adjustments.

11. Add species information in places where it is not indicated (eg line 83, lines 96-98, lines 154-155, line 159, 256-257). Note, this is not an exhaustive list, please check other sections as well. 

Answer: We fully agree with the reviewer's requirement and have made the necessary adjustments (lines 129, 141, 143, 180, 182, 199, 207, 231, 304, 411 et al.).

12. Consider a different word choice than monotonous for hormone level changes (eg. Line 188). It is meant to be monumental?  Or continuous? 

Answer: We agree with the reviewer's comment. We chance monotonous on the gradual.

13. Figure 2 contains stile (steps for going up and over a wall) instead of style (the floral organ)

Answer: Thanks for comments. We have fixed the errors (lines 236, 237).

14. The word fertilization in the bottom left of figure 2 either needs a comma before it, or need placing on a different line (right now it reads as an odd phrase, pollen tubes grow to the ovary fertilization)

Answer: Thanks for comments. We made the appropriate correction.

15. The letter s appears by itself in line 396, not sure what was meant to be here, please correct as needed

Answer: Thanks for comments. We made the appropriate correction.

16. The complex interplay between hormones in pollen (sections 2.5 and 2.6) is quite complex, it would benefit from a figure (it would be a nice addition but not essential)

Answer: Thanks for your valuable comments. Indeed, the drawing is appropriate, but it was not possible to systematize this complex data into a general figure or scheme.

17. Move conclusion 3 to the start of the list as it is the first developmental stage

Answer: Thanks for comments. We made the appropriate correction.

 

We one more thanks the reviewer for detail analysis of our manuscript. You have greatly improved our manuscript with your valuable suggestions and recomendation.

Sincerely,

Marat Khaliluev and Ekaterina Zakharova.

Author Response File: Author Response.docx

Reviewer 2 Report

I have no further comments.

Author Response

Dear Reviewer,

On behalf of ourselves and on behalf of the co-authors, we thank you for the review of our manuscript. In the second round of reviews, we tried to significantly improve the manuscript. Corrections are made in the text and highlighted in color.

Generally, we fixed the following:

• We changed the title of manuscript to be more general («Hormonal signaling in the progamic phase of fertilization in plants»).
• Added an introduction section;
• Significantly changed the numbering and content of the sections of the manuscript.
• Unified abbreviations.
• Сhanged the reference list.

Below we also provide a response to the comments of the second reviewer to better understand the changes.

1. Add an introduction, something to give bigger picture context and relevance/application for the work. Some additional context or background information to provide more relevance would be helpful.  Even a brief mention of how pollen development and function are key for many crop plants as the desired product is a seed or fruit.  This section could include example of self-compatible non self-compatible species (crop and or/research). 

Answer: We fully agree with the reviewer's recommendation to add an introduction section. We have included additional information (lines 34-76).

 

2. Topical alignment of the title and the content. The title mentions 2 hormones, but the main body of the review goes through several hormones and their possible interplay.  Perhaps the easier option is to change the title to reflect the larger scope of the work.

Answer: We agree with the reviewer's recommendation. We changed the title of manuscript to be more general («Hormonal signaling in the progamic phase of fertilization in plants»).

 

3. Please provide citations for work summarized in the figures.

Answer: We provide citations for work summarized in the figures 1 and 2.

 

4. Many of the sections can either be greatly reduced or cut. Suggestions are

cut lines 171-174

cut lines 196-198

Greatly reduce lines 298-324

Greatly reduce section 2.2 on pollen pistil interactions

Cut section 2.9

Cut section 2.10              

Cut section 2.11

Answer: In general, we agree with the reviewer's comments and removed the redundant information

lines 171-174 – delete

lines 196-198 – delete

lines 298-324 – one paragraph has been removed

Greatly reduce section 2.2 on pollen pistil interactions – lines 348-358 and 383-388, 396-400 removed

Section 2.9 modified

Section 2.10 has been removed

Section 2.11 moved to section 2.3

5. The section on self compatible vs not seems to fit better before the section 2.3 information on ET signaling.

Answer: We don’t entirely agree with this opinion of the reviewer. In our opinion, this section is independent and should be considered separately.

6. While sections 3.1 and 3.2 are informative, they have no mention at all of hormones. Perhaps they could be moved to an earlier section (before 2.3) introducing the idea of compatibility. 

Answer: One could agree with this remark. However, in our opinion, it is more logical to leave this section as an introduction to the self-incompatibility for further description of the phytohormone roles. 

Other minor changes

7. One minor but very important change for line 40 is mitosis (not meiosis). The microspore (pollen mother cell) is formed via meiosis (going from diploid to haploid).  From this point forward, cell division leading to grain formation is mitosis.  The microspores divide by mitosis. 

Answer: In accordance with the reviewer's comments, we made the appropriate correction (line 89).

8. Please standardize all journal citations regarding journal title (capitalized vs lowercase, full name vs abbreviated).

Answer: In accordance with the reviewer's comments, we standardized references list.

9. Add references to lines 36, 47.

Answer: We added citations in appropriate position.

10. Define all abbreviations at first usage.

Answer: We fully agree with the reviewer's requirement and have made the necessary adjustments.

11. Add species information in places where it is not indicated (eg line 83, lines 96-98, lines 154-155, line 159, 256-257). Note, this is not an exhaustive list, please check other sections as well. 

Answer: We fully agree with the reviewer's requirement and have made the necessary adjustments (lines 129, 141, 143, 180, 182, 199, 207, 231, 304, 411 et al.).

12. Consider a different word choice than monotonous for hormone level changes (eg. Line 188). It is meant to be monumental?  Or continuous? 

Answer: We agree with the reviewer's comment. We chance monotonous on the gradual.

13. Figure 2 contains stile (steps for going up and over a wall) instead of style (the floral organ)

Answer: Thanks for comments. We have fixed the errors (lines 236, 237).

14. The word fertilization in the bottom left of figure 2 either needs a comma before it, or need placing on a different line (right now it reads as an odd phrase, pollen tubes grow to the ovary fertilization)

Answer: Thanks for comments. We made the appropriate correction.

15. The letter s appears by itself in line 396, not sure what was meant to be here, please correct as needed

Answer: Thanks for comments. We made the appropriate correction.

16. The complex interplay between hormones in pollen (sections 2.5 and 2.6) is quite complex, it would benefit from a figure (it would be a nice addition but not essential)

Answer: Thanks for your valuable comments. Indeed, the drawing is appropriate, but it was not possible to systematize this complex data into a general figure or scheme.

17. Move conclusion 3 to the start of the list as it is the first developmental stage

Answer: Thanks for comments. We made the appropriate correction.

 

We one more thanks the reviewer for detail analysis of our manuscript. We hope that the resubmitted version of the article has become better and more understandable to readers.

Sincerely,

Marat Khaliluev and Ekaterina Zakharova.

Author Response File: Author Response.docx

Round 3

Reviewer 1 Report

Thank you for your extensive revisions to your manuscript, the new introduction is particularly helpful in providing context and some key historical background.  The introduction could use some citations in specific locations (such as lines 39, 41, 43, 51).  The citations in the figure legends are very helpful as well.  I have no additional concerns.