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The manuscript needs some minor improvements
-Comments and Suggestions for Authors
- The abstract is well written. "To elucidate" is a formal term, you could rephrase it to "To understand" for a slightly less formal tone.
- Introduction is a well-written and informative
- Line 44, replace "an ideal model" with "a valuable model" for slightly different emphasis.
- Line 47, consider replacing "following" with "after" for smoother flow.
- Line 133, consider replacing "contained a T-DNA insertion in the eighth intron" with "had a T-DNA insertion in the eighth intron" for better readability.
- Line 148, remove "Briefly"
- Line 158, following the methodology of (Chang et al., 2021)" can be changed to "as described by Chang et al. (2021)"
- Line 168, "The prepared inflorescence was washed three times with water" can be replaced with "The inflorescence was washed three times with water after preparation."
- Line 214, "revealed that the wild-type (WT) anthers were full of pollen compared to top1α1 anthers, which had few pollen grains" can be rephrased as "revealed that wild-type (WT) anthers contained significantly more pollen grains compared to top1α1 anthers".
- Line 228, Consider combining the sentences "This revealed that meiosis in the mutants appeared largely normal compared to WT, with only minor variations observed in some stages" into "DAPI staining revealed largely normal meiosis in the mutants compared to WT, with minor variations in some stages (Figure 2)."
- Line 233, "bright large blocks (large distances of heterochromatin)" can be rephrased as "bright, large blocks of heterochromatin."
- Line 246, "necessary for correct chromosome segregation" can be rephrased as "essential for ensuring proper chromosome segregation during cell division."
- Line 293, "to clarify these defects in the top1α1" can be rephrased as "to illustrate these pairing abnormalities in top1α1 cells at metaphase I."
- Line 295, "Chiasma visualization during diplotene and diakinesis in Arabidopsis thaliana is hindered by..." can be restructured as "Visualizing chiasmata during diplotene and diakinesis in Arabidopsis thaliana is challenging due to..."
- Line 309, "Conversely, WT displayed a higher frequency of cells with more than 7 chiasmata" can be rephrased as "In contrast, WT had a higher proportion of cells containing more than 7 chiasmata."
- Line 332, "This observation aligns with the known function of TOP1α in resolving torsional stress during DNA transactions" can be rephrased as "This finding aligns with the established role of TOP1α in relieving torsional stress during DNA processes."
- Line 361, "These enzymes allow the passage from single-strand to double-strand DNA and vice versa" can be rephrased as "These enzymes enable the conversion between single-stranded and double-stranded DNA."
The study demonstrates an acceptable experimental design.
No comments
I reviewed the manuscript titled "The Role of DNA Topoisomerase 1α (AtTOP1α) in Regulating Arabidopsis Meiotic Recombination and Chromosome Segregation". This study uncovers novel functions of TOP1α during meiosis in Arabidopsis thaliana and underscores its importance in meiotic recombination.
The study aims to understand the function of AtTOP1α in Arabidopsis meiosis, a crucial process for sexual reproduction. Also, the authors employed various cytological and molecular techniques like DAPI staining, FISH, and PCR to analyze chromosome behaviour, gene expression, and pollen viability. The generation and analysis of the atm×top1α1 double mutant strengthens the role of AtTOP1α in meiotic progression. Overall, this research provides valuable insights into the role of AtTOP1α in Arabidopsis meiosis.
Please find the comments in the attached annotated PDF file
Experimental design is relatively well illustrated
Key Findings:
• AtTOP1α deficiency leads to reduced fertility and abnormal pollen development.
• Meiotic chromosomes display altered heterochromatin distribution and clustered centromere signals in the top1α1 mutant.
• Disruption of AtTOP1α function affects 45s rDNA localization during meiosis.
• AtTOP1α may promote crossover formation during meiosis.
The numbers indicating the authors' addresses are excessive, suggesting that each author works in multiple locations. The 2nd and 3rd paragraphs of the introduction are irrelevant. Moreover, the role of DNA topoisomerase in mitotic activities is already known; although this study is labor-intensive, it does not contribute to science. The last paragraph of the introduction should indicate the purpose of the study, but instead, it states a conclusion. Additionally, the study does not explain what gap it aims to fill and instead prefers to create an information overload. There is no information about the place and year the study was conducted. Table S1 is not available. The statistical and software analyses used are not specified. The results section is only for explaining the results. No discussion or citations should be included there. In the discussion section, differences and similarities should be presented, and assumptions should be made about the reasons for differences or similarities with the literature. The discussion is very weak. Additionally, the conclusion section is unnecessarily long. In the author contributions section, the authors' initials names are provided, which is not meaningful. In the references section, the names of journals and publishers should be italicized. Although this study is labor-intensive, it does not contribute to science. Its publication is not recommended.
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The manuscript explores the role of DNA topoisomerase TOP1a in Arabidopsis male meiosis. The authors analyse fertility and male meiotic chromosome segregation in the Arabidopsis TOP1a mutant and in top1a atm double mutants.
The manuscript reports three exciting observations: 1) a lack of top1a results in reduced fertility in Arabidopsis; 2) male meiotic centromeres cluster in the absence of top1 and 3) chromosomal interlocks are observed during meiosis in top1a mutants.
Literature references and field background are well presented.
The manuscript, however, needs major editing and considerable improvement before it can be published.
I found multiple examples where the manuscript fails to adhere to professional English and terminology - e.g. 'crisp chromosomes' (line 235), 'cell corners (line 324)', 'chromosomal duplication' instead of 'synapsis' (line 673); 'homologous recombination with sister chromatids' (lines 88-89) are just some examples of stylistic and conceptual errors. The sentence starting with 'Recombination nodules... (line 59) is conceptually incorrect. The introduction should be structured more logically - e.g. meiotic chromosomes are mentioned in line 50, but meiosis is not introduced until line 52. Additionally, I would recommend that the authors proof-read the manuscript as I found a lot of typos.
Otherwise, the structure is mostly fine, I would recommend putting the ATM experiment under a separate subheading.
Figures are mostly fine, but I would recommend combining Figures 6 and 7 in one figure and labelling wild type and mutant genotypes in all figures, not just in Figure 2.
Raw data not shared.
The overall hypothesis is not very clear, the hypothesis/rationale behind combining top1a and atm mutants is completely missing.
The manuscript represents original primary research which, I believe, is within the Journal's scope.
I am not sure I found that the authors state how research fills an identified knowledge gap.
The reported findings represent an interesting initial observation, but I am not sure how this improves our understanding of the meiotic recombination mechanisms – I believe top1a needs to be investigated further and the manuscripts needs to be considerably reworked to answer this question.
Methods are mostly described in sufficient detail, some detail is missing, for example, how was the centromeric probe designed? How was transcript quantified in Figure 1G?
Conceptually, I would like to see a rationale behind testing how top1a affects meiosis and behind combining top1a and atm mutants. I would recommend adding hypothesis and interpreting the data in the light of these hypotheses.
I believe that the following findings are novel and valid: 1) a lack of top1a results in reduced fertility in Arabidopsis; 2) male meiotic centromeres cluster in the absence of top1 and 3) chromosomal interlocks are observed during meiosis in top1a mutants.
However, I am not sure the authors make it clear how this advances our understanding of the mechanisms of meiotic recombination. I am not sure I agree with all authors' data interpretation and conclusions - I have explained in detail in the 'Additional comments'.
The authors do not provide any information on how many nuclei were analysed for each experiment and what % of nuclei were abnormal.
No statistical analysis is provided - I have listed suggestions for specific improvement of experiments in the 'Additional comments'.
1. Section 3.1 – I suggest reversing the order – starting with the previously published background on the mutant, followed by mRNA analysis, followed by meiotic spreads.
2. Figure 1
a. Panels A and B are out of focus; Please add quantification of viable – non-viable pollen; normal-abnormal tetrads and stats. How is seed set affected in top1a mutants?
b. Panels D, E – please add arrows to indicate microspores.
c. Panel G – how was the transcript quantified? Please add the explanation to Results and/or Materials and Methods
3. Figure 2 – how many nuclei were analysed and what % was abnormal, please add numbers and stats.
a. Panels B-B1, C-C1 – I cannot see any differences between the wt and the mutant
b. Panels D-D1 – please add numbers of nuclei analysed, % abnormalities and stats. Are the authors sure they’re not just looking at early vs late diplotene in D vs D1?
c. Difference in diakineses looks interesting – what % of nuclei were abnormal? How many nuclei were analysed?
d. Metaphase: ‘top1a bivalents were often thin with a few crossovers’ – please use appropriate scientific language to describe the phenomenon and I cannot see this from Figures 2F-F1.
e. I’m not sure I can see ‘chromosome crowding’ at anaphase that is different from the wild type. Please use an appropriate term instead of ‘conglomerate’ – line 241. Figure panels 2G1 and 2H1 look wild type to me.
4. Section 3.3 – To me this looks like centromere clustering in the mutant compared to the wild type. I would recommend replacing strong statements with hypotheses, for example, we can only hypothesise from the observed phenotypes that chromosomes were entangled, and non-homologous recombination has occurred – here and throughout the text, e.g. Figure 6, lines 270-271.
5. Please explain what ‘centromeric probe’ you used.
6. Lines 259-260 – sentence needs editing – I suggest using ‘clustered foci’ or a similar term. Please explain what ‘unusual locations’ mean.
7. Line 262: ‘The number of chromosome signals decreased at the pachytene stage.’ – please indicate whether this refers to the wt or mutant.
8. ‘At diakinesis, the WT had five pairs of homologous chromosomes and five signals’ – please edit to explain what ‘five signals’ refers to – ‘five centromeric foci’?
9. ‘These observations indicate that top1a had abnormalities in pairing and synapsis around the centromeres.’ – needs rewording as a hypothesis, as this is not proven. I recommend describing the observed phenotype as ‘centromere clustering’.
10. Line 286 – should read ‘chromosome pairing’ rather than ‘pairing’
11. Line 290 – should read ‘homologous chromosomes’, not ‘homologue chromosomes’.
12. Section 3.4 – the abnormalities in 45S localisation are most likely the result of abnormal chromosome segregation in the top1a mutant, therefore, I disagree with the authors’ conclusions here. I don’t agree with the statement: ‘This result shows that homolog chromosomes in were incorrectly paired and synapsed easily top1a at the 45S rDNA region.’
13. Figure 5 – either the Results text is incorrect or the Figure is labelled incorrectly – the text states opposite to what’s in the Figure. Overall – I am not sure I agree – what was the total number of chiasmata in the WT and the mutant? Is the difference statistically significant? Were chiasmata counted in all nuclei or only those that did not show abnormalities – otherwise, how can you count crossovers in the metaphase with interlocked chromosomes?
*The article's language was comprehensible. Professional, unambiguous English was employed throughout.
*The references to previous works, adequate background information, and context was given.
*The manuscript was well formatted with figures and tables.
**In line 64: The explanation of "DSB" should be given in first place, not in Line 87.
**In line 152: The word "Breifly" is written twice
*The purpose of the experiment is well defined, explained.
*The questions arised in as aim is explained enough with the findings of the reseach.
The material and method is well explained.
*The underlying data are all available and are controlled, reliable, and statistically sound.
*The conclusions are concise, support the findings, and are connected to the original research topic.
*This research article complies with Peer J's aims and objectives.
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