High Temperature-Induced Spindle Destabilization Results in Aborted Pollen Production in Populus
Round 1
Reviewer 1 Report
The influence of temperature, the study of the tapetum and the study of actin are almost unrelated to each other in discussion and conclusions. The text breaks down into separate facts.
Comments for author File: 
Comments.pdf
Author Response
Dear editors and reviewers,
Thank you for your constructive suggestions, which help us improve the quality of our paper. The authors carefully check the paper, and revised them (marked in red) in the resubmitted version. The response to all the questions concerned by reviewers are listed as follows:
Reviewer 1#
The influence of temperature, the study of the tapetum and the study of actin are almost unrelated to each other in discussion and conclusions. The text breaks down into separate facts.
In the revised version, we delete the study of PtActin gene expression.
Maybe the tapetum delayed helped some pollen grains save viable.
Maybe you are right, but we do not have more evidences.
A reduction in actin gene expression can be considered as the cause of disrupted to the spindle actin, which in turn is the cause of chromosome segregation errors. But the cause of the reduction in actin gene expression and what the high temperature has to do with it remains unknown.
Yes, a reduction in actin gene expression is the cause of disrupted to the spindle action that have reported in a mouse egg (Mogessie et al., 2017) . However, the cause of the reduction in actin gene expression and what the high temperature has to do with it remains unknown.
It would be better to write why triploids are so necessary?
In the new version, author accept suggestions of the reviewer 2#, and this paragraph is deleted.
It is possible to solve such a problem only on the condition that the all multi-flowered inflorescences of the poplar form pollen synchronously!
Generally, all flowers on the same branch of Populus asynchronously develop. However, the collected mature pollen grains can be used to test the fertility of pollen.
Were all the branches collected from the same plant? Did they have the same genotype?
Yes, all the sampled male branches from the same tree. Therefore, they have the same genotype.
Why was the air humidity not controlled?
High temperature treatment is conducted in a chamber. The air humidity is 70%. In the revised version, the authors add it.
Why such a low voltage?
5 kV is a suitable value to abtain the high quality picture of pollen grains.
How did dry air affect the death of buds?
In the paper, all the treatments are conducted in a chamber with 70% air humidity. Therefore, we think that dry air does not affect the death of male buds.
Is all of this positively correlated with aborted pollen?
Yes, a significant correlation (α = 0.049) is found.
This is completely unnecessary work.
After the flowers of Rosa spp. were treated by 36 ℃ high temperature, a large number of pollen grains with fragments of ectexine deposition, result in aborted pollen formation (Pécrix et al., 2011). Therefore, the authors conducted this work.
curios term! sporoderm sculpture?
“ectexine deposition” was also used to describe the ectexine structure of pollen grains in Rosa spp. In the revised version, authors replace “ectexine deposition” with “ectexine structure”.
This result strongly depends on the method of estimating the germination rate.
We have the same idea with you. In this paper, the germination rates of the control and treated groups were estimated by the same method. So the results were reliable.
What happened to the tapetum cells remains unclear. a cytological or ultrastructural study would help.
After treated by high temperature, why delayed tapetum disintegration happened remains unclear. The authors also sure that a cytological or ultrastructural study will help. In the future research, we will design experiments to explain why delayed tapetum disintegration occurs.
There was a noticeable diversity of MKM in one anther in terms of resistance to thermal stress.
Maybe.
This means that the causes of cell death do not coincide with the causes of pollen polyploidization.
Yes
Why do you think so? Explain!
In Populus tomentosa, some aborted pollen formation because the anthers do not dehisced after the anthers mature.
This paragraph is more like a negative review than a conclusion to this manuscript.
The authors accept your advice, and delete this paragraph in the new version.
The influence of temperature, the study of the tapetum and the study of actin are almost unrelated to each other in discussion and conclusions.
In the revised version, the authors delete the study of actin gene.
Thank you again for your help.
Best regards
Pingdong Zhang
Author Response File: 
Author Response.docx
Reviewer 2 Report
The manuscript “High temperature-induced spindle destabilization results in aborted pollen production in Populus”, researched the heat-shock effect on poplar pollen. The paper could have relevance for heat-wave effect on induced male-sterility in poplar. Mechanism description is this species is the main novelty of the paper.
Title, Line 2 – please give species name with author Populus canescens (Aiton) Sm., when first mentioned.
Introduction
Authors present us with more information about temperature effects on female gametes and embryo sac, than state of the art in pollen research. There have been published some good papers both on heat shock as well as long-term mild heat on heat-induced male sterility in various plant species.
Due to this, currently the introduction seems a little disconnected from the entire results section, and digresses from main topic of the paper. The topic of a paper is given by the research conducted. It sounds like authors wanted to study heat stress in female gametes, but instead resumed to male gametes because it is easier as they themselves say at Line 71-72. This is not a scientific and appropriate justification for a study. Please build a strong argument in favor of the topic of your paper, in regards with theoretic importance and practical implication of your study and approach (pollen).
Line 75 - I suggest to change “…aborted pollen forms via high temperature…”, to “…pollen abortion is induced via high temperature…”
Results
Since analysis of variance resulted in significant p values, further post-hoc test could be applied. Line 173 mentions LSD was applied as post-hoc with significant differences found, but these are not indicated/marked in any way in Table 2.
References
I counted only one paper from 2021 (as most recent reference used in your paper!). Please find more recent papers to cite at the discussion section. A paragraph at the end with most recent insights on the topic are fundamental for placing and connecting your paper within current body of literature. What are the perspectives and current trend in pollen-sterility research in recent years on the topic? What other authors say are the future perspectives?
Best regards.
Author Response
Dear editors and reviewers,
Thank you for your constructive suggestions, which help us improve the quality of our paper. The authors carefully check the paper, and revised them (marked in red) in the resubmitted version. The response to all the questions concerned by reviewers are listed as follows:
Reviewer 2#
The manuscript “High temperature-induced spindle destabilization results in aborted pollen production in Populus”, researched the heat-shock effect on poplar pollen. The paper could have relevance for heat-wave effect on induced male-sterility in poplar. Mechanism description is this species is the main novelty of the paper.
Title, Line 2 – please give species name with author Populus canescens (Aiton) Sm., when first mentioned.
We accept your suggestion and revised it in the new version.
Introduction
Authors present us with more information about temperature effects on female gametes and embryo sac, than state of the art in pollen research. There have been published some good papers both on heat shock as well as long-term mild heat on heat-induced male sterility in various plant species.
Due to this, currently the introduction seems a little disconnected from the entire results section, and digresses from main topic of the paper. The topic of a paper is given by the research conducted. It sounds like authors wanted to study heat stress in female gametes, but instead resumed to male gametes because it is easier as they themselves say at Line 71-72. This is not a scientific and appropriate justification for a study. Please build a strong argument in favor of the topic of your paper, in regards with theoretic importance and practical implication of your study and approach (pollen).
We accept your constructive suggestion, and revise them in the resubmitted file.
Line 75 - I suggest to change “…aborted pollen forms via high temperature…”, to “…pollen abortion is induced via high temperature…”
We accept your suggestion and replace this sentence in the revised submission.
Results
Since analysis of variance resulted in significant p values, further post-hoc test could be applied. Line 173 mentions LSD was applied as post-hoc with significant differences found, but these are not indicated/marked in any way in Table 2.
Because dominant meiotic stage (F = 5.75, P < 0.001), temperature (F = 25.71, P < 0.001), and duration of treatment (F = 17.82, P< 0.001) significantly affected the percentages of high temperature-induced aborted pollen, we do not marked the significant differences in table 2, preventing readers from confusion.
References
I counted only one paper from 2021 (as most recent reference used in your paper!). Please find more recent papers to cite at the discussion section. A paragraph at the end with most recent insights on the topic are fundamental for placing and connecting your paper within current body of literature. What are the perspectives and current trend in pollen-sterility research in recent years on the topic? What other authors say are the future perspectives?
The reviewer 1# think that the last paragraph in discussion section is more like a negative review than a conclusion. Therefore, the authors decide to delete this paragraph. However, the authors add some recent references, marked in red.
Thank you again for your help.
Best regards
Pingdong Zhang
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
Comments for author File: Comments.pdf
Author Response
Dear reviewers,
Thank you for your constructive suggestions, which help us improve the quality of our paper. The authors carefully check the paper, and revised them (marked in red) in the resubmitted version. The authors hope the corrections will satisfied with you. The response to all the questions concerned by reviewers are listed as follows:
Reviewer 1#
what do the white arrows mean?
white arrows show aborted pollen grains
g, h, i pictures are not of good quality.
In the revised version, the authors provide three new pictures.
Because of the anther dehisced normally, do you think that the tapetum did not affect the production of aborted pollen.
In the resubmitted version, the authors replace the sentence with “However, no sufficient evidences showed that the delayed degradation of the tapetum was responsible for the production of aborted pollen”.
hypothesis
The authors change this sentence.
Thank you again
Best regards
Pingdong Zhang
Author Response File: Author Response.docx
