FmRbohH Mediates ROS Generation and Enhances Pollen Tube Growth in Fraxinus mandshurica
, Shuqi Wang †, Shun Yang, Ying Wu, Zerui Huang, Yunping Liu and Xingguo Lan *Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe paper addresses the topic of ROS signaling and genes involved in that. The paper, although interesting, suffers from many inaccuracies, overinterpretations, it needs much more details to be presented to fully understand what was actually done and how.
Major comments:
Section 3.1: KEGG enrichment analysis was carried out on all DEGs- What does that mean DEGs are only from pairwise comparisons between samples.
Fig 1C: Why you say nothing about other enriched pathways like Citrate cycle, steroid etc , this also relates to the abstract.
How phosphorylation and ROS are related? ROS causes photooxidative damage not phosphor !!
..found 59 genes expressed diferentially in the apoplast, chloroplast, cytoplasm, - 1)You cannot state this because you collected RNA from the entire cell and not from its parts, ribosome etc, 2) Are You sure chloroplasts exist in pollen?
At this point a big question arises how RNA was extracted and if it includes mitochondrial RNA. In this case I would like to see how many mitochondrial RNA is detected in comparison to nuclear RNA. Please provide this in a supplementary material in the form of counts.
Authors must generate the entire list of ROS-related genes and present expression levels for them as on figure 2 and 3 and 4, so that the data is directly comparable. Now it is not possible to compare, for example, MDHAR gene from fig 4 and 3.
What are actually the genes in the oxidative phosphorylation pathway and ROS-Related Genes, give comprehensive lists and definitions.
Define “pollen germination rate in percentage”
Heatmap expression proile FmRboh genes in pollen, pollen tube, stigma, and leaf - where data for leaf come from?
Figure 2 what mean colors (shades of red) in the figure, 1.0 relates to what?
Comments on the Quality of English Language
There are several methodological errors:
phosphorylation pathway was enriched - The pathway cannot be simply enriched, can be enriched with something
Signiicant enrichment of the oxidative phosphorylation pathway was also examined - You cannot investigate enrichment of a pathway by GO/KEGG, inversely - analysis of GO of genes may show a pathway is enriched with genes belonging to this pathway.
Pollens -> pollen
by a Leica - by Leica
Various f Pollen -> Various Pollen
Many ROS-related genes regulate pollen tube growth in diferent ways to keep the ROS level at optimum concentration. - not clear. … genes regulate ROS level to be on the optimal level?
Author Response
We sincerely thank the reviewer for taking time to give constructive comments on our manuscript.
Comment 1. Section 3.1: KEGG enrichment analysis was carried out on all DEGs- What does that mean DEGs are only from pairwise comparisons between samples.
Response: The KEGG is done on DEGs to explore their association with a particular pathway and functions (Cai et al., 2021; Chen et al., 2020). In our study, it was carried out on the DEGs from the pairwise comparisons between samples from stage S1 vs S2, S1 vs S3, and S2 vs S3 respectively. The sentence is modified for clarity. See lines 172 to 173.
Comment 2. Fig 1C: Why you say nothing about other enriched pathways like Citrate cycle, steroid etc, this also relates to the abstract.
Response: We have modified the abstract to include the other important pathways identified, see lines 17 to 20. We have also mentioned the pathways that showed significant enrichment in our results. See lines 172 to 177, and lines 199 to 203. The pathway cluster analysis of citrate cycle was provided in the supplementary figure S1.
Comment 3. How phosphorylation and ROS are related? ROS causes photooxidative damage not phosphor!!
Response: Reactive oxygen species (ROS), superoxide anion and hydrogen peroxide, are generated as byproducts of electron transport chain or oxidative phosphorylation in the mitochondria or via cell signaling-induced NADPH oxidases in the cytosol (Rbohs). For this process to occur in the cell, ATP is generated and transferred along the electron transport chain and phosphorylation of ADP, which resulted in the oxidation of the molecular oxygen to release superoxide anion (O2–) which can be transformed into molecular oxygen and H2O2 by superoxide dismutase 1–3 (SOD2–3) in different cell compartments (Sinenko et al., 2021).
Comment 4. ..found 59 genes expressed differentially in the apoplast, chloroplast, cytoplasm, - 1)You cannot state this because you collected RNA from the entire cell and not from its parts, ribosome etc, 2) Are You sure chloroplasts exist in pollen? At this point a big question arises how RNA was extracted and if it includes mitochondrial RNA. In this case I would like to see how many mitochondrial RNA is detected in comparison to nuclear RNA. Please provide this in a supplementary material in the form of counts
Response: Even though there are few studies to demonstrate the existence of organelles in the pollens, it was previously reported that mitochondria and chloroplasts exist in the pollens of plants and plastids marker genes fused to GFP and YPF were used to detect them during pollen tube germination in Arabidopsis plants (Fujiwara et al., 2012). In our study, we did not specifically isolated RNA from the pollen mitochondria, we could only detect genes associated to mitochondria and chloroplasts in our data which play significant role in the mitochondrial electron chain transport and ROS generation and scavenging, hence our hypothesis of their role during pollen tube growth in F. mandshurica.
Comment 5.
Authors must generate the entire list of ROS-related genes and present expression levels for them as on figure 2 and 3 and 4, so that the data is directly comparable. Now it is not possible to compare, for example, MDHAR gene from fig 4 and 3.
Response: Figures 2B and 3B present a comparative list of genes identified as key players in oxidative phosphorylation, ROS generation, and scavenging. Figure 4, on the other hand, demonstrates the qPCR expression and validation of selected genes representing complexes involved in oxidative phosphorylation during pollen tube growth in F. mandshurica.
Comment 6. What are actually the genes in the oxidative phosphorylation pathway and ROS-Related Genes, give comprehensive lists and definitions.
Response: Section 3.3 presents genes in the oxidative phosphorylation, ROS synthesis and scavenging. Please see lines 222 to 242 which provides the list and full definition of those genes involved in the oxidative phosphorylation pathway and ROS-related genes.
Comment 7: Define “pollen germination rate in percentage”
Response: The pollen tube germination rate and length was clearly defined as suggested by the reviewer. Refer to lines 277 to 279 and lines 310 to 315.
Comment 8: Heatmap expression profile FmRboh genes in pollen, pollen tube, stigma, and leaf - where data for leaf come from?
Response: The heatmap expression profile of FmRboh genes in pollen, pollen tube, stigma, and leaf was generated using quantitative real-time PCR (qPCR), leaf data was included to clearly show the specificity of FmRbohH to pollens and pollen tubes.
Comment 9: Figure 2 what mean colors (shades of red) in the figure, 1.0 relates to what?
Response: The shades of red in Figure 2 is the legend that represent different levels of gene expression. The color scale typically indicates the relative abundance of a specific gene transcript. A value of 1.0 in this context usually signifies the highest fold change observed among the samples being compared and -1.0 signifies the lowest fold change observed among the samples being compared. Lower shades of red would correspond to lower levels of expression. The box represents stage 1, stage 2, and stage 3 of the samples.
Comment 10: phosphorylation pathway was enriched - The pathway cannot be simply enriched, can be enriched with something
Response: We apologize for the confusion. It is correct that a pathway itself cannot be enriched. In our analysis, we found that the oxidative phosphorylation pathway was enriched with differentially expressed genes (DEGs). This enrichment suggests that this pathway is significantly involved in the biological processes under investigation. The paragraph has been modified. See lines 172 to 177
Comment 11: Significant enrichment of the oxidative phosphorylation pathway was also examined - You cannot investigate enrichment of a pathway by GO/KEGG, inversely - analysis of GO of genes may show a pathway is enriched with genes belonging to this pathway.
Response: Both GO and KEGG are valuable resources for pathway enrichment analysis in gene expression data. While they use different approaches, they can often provide complementary insights into the underlying biological processes. In our study, we used KEGG to assess the enrichment of the oxidative phosphorylation pathway by comparing the number of differentially expressed genes mapped to this pathway.
Comment 12: Pollens -> pollen
Response: We have modified this mistake and kept consistent throughout the manuscript.
Comment 13: by a Leica - by Leica
Response: We have modified this mistake. see line 98
Comment 14: Various f Pollen -> Various Pollen
Response: We have modified this mistake. see line 243
Comment 15: Many ROS-related genes regulate pollen tube growth in different ways to keep the ROS level at optimum concentration. - not clear. … genes regulate ROS level to be on the optimal level?
Response: We have modified this mistake see line 245 to 246
Cai, Z., Huang, Z., Wang, Z., Tao, Y., Wu, F., Yu, X., & Luo, J. (2021). Identification of the related genes on the asymmetric root growth of Oryza sativa induced by ethylene through transcriptome sequencing, GO and KEGG analysis. Acta Physiologiae Plantarum, 43, 1-11.
Chen, J., Liu, C., Cen, J., Liang, T., Xue, J., Zeng, H., Zhang, Z., Xu, G., Yu, C., & Lu, Z. (2020). KEGG-expressed genes and pathways in triple negative breast cancer: Protocol for a systematic review and data mining. Medicine, 99(18), e19986.
Fujiwara, M. T., Yoshioka, Y., Hirano, T., Kazama, Y., Abe, T., Hayashi, K., & Itoh, R. D. (2012). Visualization of plastid movement in the pollen tube of Arabidopsis thaliana. Plant Signal Behav, 7(1), 34-37. https://doi.org/10.4161/psb.7.1.18484
Sinenko, S. A., Starkova, T. Y., Kuzmin, A. A., & Tomilin, A. N. (2021). Physiological Signaling Functions of Reactive Oxygen Species in Stem Cells: From Flies to Man. Front Cell Dev Biol, 9, 714370. https://doi.org/10.3389/fcell.2021.714370
Reviewer 2 Report
Comments and Suggestions for AuthorsROS were initially recognized as toxic by-products of aerobic metabolism, removed by antioxidants and antioxidant enzymes. In recent years, it has become apparent that ROS play an important signaling role in plants, controlling processes such as growth, development, response to biotic and abiotic environmental stimuli, PCD, and of course are important for germination and growth of the male gametophyte (both in vitro and in vivo). Rbohs are known to play an important role in a wide range of plant cell development and growth functions. The authors of the paper convincingly demonstrate that FmRbohH is crucial for ROS generation to enhance pollen tube growth in F. mandshurica. I consider the research by Jakada and co-authors to be relevant and interesting from a fundamental point of view.
The applied methods are adequate and modern. The results are clear and objective. However, there are some minor remarks that need to be noted.
1. There is no explanation of the abbreviation Rboh anywhere, especially since you are introducing the innovation FmRboh. Give the explanation in the abstract.
2. The authors do not discuss anywhere why there is a decrease in seed setting in Fraxinus mandshurica (how pollination occurs, self-incompatibility, etc.).
3. How were pollen samples collected from the culture medium for analysis (filtration?)?
Comments for author File: 
Comments.pdf
Author Response
Thank you so much for taking your precious time to make constructive comments to improve our manuscript.
Comment 1: There is no explanation of the abbreviation Rboh anywhere, especially since you are introducing the innovation FmRboh. Give the explanation in the abstract.
Response: The explanation of the abbreviation Rboh has been incorporated into the abstract and introduction as pointed out by the reviewer. See lines 24 and 50
Comment 2: The authors do not discuss anywhere why there is a decrease in seed setting in Fraxinus mandshurica (how pollination occurs, self-incompatibility, etc.)
Response: this important point has been added in the introduction and the discussion section of the manuscript. See lines 78 to 81, and 325 to 330
Comment 3. How were pollen samples collected from the culture medium for analysis (filtration?)?
Response: Liquid media was used for pollen ermination and the pollen were collected collected by centrifugation at 3,000 rpm for 2 minutes. For ROS detection and microscopy, the pollens were treated and then resuspend with PBS buffer. See line 116 to 131.
Reviewer 3 Report
Comments and Suggestions for AuthorsThe manuscript entitled “FmRbohH Mediates ROS Generation and Enhances Pollen Tube Growth in Fraxinus mandshurica” investigates the role of the FmRbohH gene in generating Reactive Oxygen Species (ROS) and its effect on pollen tube growth in Fraxinus mandshurica. Through transcriptomic analysis, 8,734 differentially expressed genes (DEGs) were identified during pollen tube growth, with ROS accumulation shown to promote pollen tube elongation. The study reveals that oxidative phosphorylation and related pathways are significantly enriched during pollen tube development. Knockdown experiments targeting FmRbohH resulted in reduced ROS levels and inhibited pollen tube growth, underscoring the gene's crucial role in the reproductive process of F. mandshurica.
This research offers valuable insights into the role of ROS and FmRbohH in regulating pollen tube growth, contributing to the limited understanding of reproductive biology in Fraxinus mandshurica, a tree species of economic importance. The identification of key pathways, particularly oxidative phosphorylation and ROS generation, provides clear mechanistic insights into how energy metabolism supports pollen tube growth. The experimental evidence convincingly supports the link between ROS accumulation and pollen tube extension. The objectives of this study are well-defined, and the results are of considerable interest. I do not have any major concerns, though I have included a few suggestions below:
Although the role of ROS is undeniably important, the study could benefit from expanding its focus to explore other molecular mechanisms involved in pollen tube growth, such as calcium signalling or cell wall modification. Broadening the discussion in this way would offer a more comprehensive understanding of the process.
The use of CuCl2 and DPI treatments effectively demonstrates the importance of ROS in pollen tube growth. However, the potential off-target effects of these chemical inhibitors have not been fully considered. It would be helpful to address the possibility of non-specific actions and their potential impact on the results.
While 8,734 DEGs were identified, the discussion could place greater emphasis on highlighting and prioritising the most significant genes beyond FmRbohH. A more in-depth analysis of the functional relevance of other upregulated or downregulated genes could provide further insights into the molecular network regulating pollen tube growth.
Comments on the Quality of English LanguageIn terms of English writing, the manuscript is generally well-structured, with a clear progression from the introduction to the methodology, results, and discussion. Each section follows logically, making the paper easy to follow. A few suggestions for improvement are outlined below:
Line 19: Change "H2DCFDA staining confirmed the formation of ROS in the pollens and the pollen tube." to "H2DCFDA staining confirmed ROS formation in the pollens and pollen tubes."
Line 21: Change "Furthermore, expression of FmRbohH was found in the pollen and pollen tube, and the antisense oligodeoxynucleotide assay revealed reduced ROS in the pollen and decreased pollen tube growth in Fraxinus mandshurica." to "Furthermore, FmRbohH expression was detected in the pollen and pollen tube, and an antisense oligodeoxynucleotide assay demonstrated reduced ROS and pollen tube growth in Fraxinus mandshurica."
Line 31: Change "These processes are critical for fertilization in plants." to "These processes are essential for successful fertilization in plants."
Line 36: Change "This process mainly depends on the actin microtubules and cytoskeleton to regulate organelle movement, vesicle trafficking, cytoplasmic flow, and cytoplasm organization in pollen tubes." to "This process is largely dependent on the actin cytoskeleton, which regulates organelle movement, vesicle trafficking, and cytoplasmic organization in pollen tubes."
Line 78: Change "The plant is native to northeast China, southeast Russia, Korea, and Japan." to "F. mandshurica is native to Northeast China, Southeast Russia, Korea, and Japan."
Line 165: Change "The Illumina Hiseq platform revealed 53.4 – 97 raw reads from 9 RNA samples, clean data of 52.2 – 93.6 million clean data was generated, resulting in 95% of total reads." to "The Illumina HiSeq platform generated 53.4–97 million raw reads from 9 RNA samples, with 52.2–93.6 million clean reads retained, accounting for 95% of the total data."
Line 177: Change "The top 15 pathways with the smallest p-value were considered significant, and a strong presence of oxidative phosphorylation was observed, followed by amino acids synthesis and protein processing in the ER, carbon metabolism, pyruvate metabolism, citrate cycle (TCA cycle)-glycolysis/gluconeogenesis." to "The top 15 pathways with the smallest p-values were considered significant, with oxidative phosphorylation showing the strongest presence, followed by amino acid synthesis, protein processing in the ER, carbon metabolism, pyruvate metabolism, and the citrate cycle (TCA cycle)/glycolysis/gluconeogenesis."
Line 183: Change "The oxidative phosphorylation pathway typically occurs in mitochondria, several complexes are employed to produce ATP during oxidative phosphorylation, and a significant amount of ROS by-product is generated in the mitochondria." to "Oxidative phosphorylation occurs in the mitochondria, where several complexes work together to produce ATP, generating ROS as a byproduct."
Line 195: Change "They are important for energy production in the cell and catalyze electron transfer from dihydroubiquinone (QHz) to cytochrome c, and this reaction is coupled to the transfer of a transmembrane proton, at the same time, ROS is produced as a by-product." to "These complexes are crucial for energy production, catalyzing electron transfer from dihydroubiquinone (QHz) to cytochrome c, a process that is coupled to transmembrane proton transfer and ROS generation as a byproduct."
Line 332: Change "This reaction is linked to the respiratory chain, which uses the energy released during chemical oxidation to produce ATP." to "This reaction is part of the respiratory chain, which harnesses energy from chemical oxidation to produce ATP."
Line 359: Change "The differential gene expression in complexes I, II, and III changed, and the trend was relatively consistent, suggesting a change in ROS content during pollen tube growth." to "Differential gene expression in complexes I, II, and III followed a consistent trend, suggesting fluctuating ROS levels during pollen tube growth."
Line 373: Change "Our data showed gene expression changes linked to ROS generation during pollen tube growth in different cellular compartments." to "Our data revealed changes in gene expression related to ROS generation across different cellular compartments during pollen tube growth."
Line 423: Change "Among the ROS-related genes, Rboh genes are essential players in both stress response and reproductive development." to "Rboh genes, among other ROS-related genes, are essential for both stress responses and reproductive development."
Line 446: Change "Similarly, FmRbohH knockdown greatly reduced ROS fluorescence and pollen tube growth." to "Similarly, knocking down FmRbohH significantly reduced ROS fluorescence and impaired pollen tube growth."
Author Response
We sincerely thank the reviewer for taking time to give constructive comments to improve our manuscript.
Comment 1. Although the role of ROS is undeniably important, the study could benefit from expanding its focus to explore other molecular mechanisms involved in pollen tube growth, such as calcium signalling or cell wall modification. Broadening the discussion in this way would offer a more comprehensive understanding of the process.
Response. Although the Rboh genes are involved in calcium signaling, our transcriptome data did not reveal the other components of the core calcium signaling genes such as the Calcium Dependents Protein Kinases (CDPKs), Calcineurin-B-like Proteins (CBLs), CBL-interacting Protein Kinases (CIPKs) and Mitogen-Activated Protein Kinases (MPKs).
Comment 2. The use of CuCl2 and DPI treatments effectively demonstrates the importance of ROS in pollen tube growth. However, the potential off-target effects of these chemical inhibitors have not been fully considered. It would be helpful to address the possibility of non-specific actions and their potential impact on the results.
Response. We used Antisense and sense of oligodeoxyribonucleotides (AS-ODN and S-ODN) to support the CuCl2 and DPI data so that any off target possibility can be ruled out.
Comment 3. While 8,734 DEGs were identified, the discussion could place greater emphasis on highlighting and prioritising the most significant genes beyond FmRbohH. A more in-depth analysis of the functional relevance of other upregulated or downregulated genes could provide further insights into the molecular network regulating pollen tube growth.
Response. While we have explored some of the genes implicated in ROS signaling, a comprehensive understanding of the underlying mechanisms and functional relevance requires transgenic F. mandshurica plants. Unfortunately, genetic transformation of this plant species has not yet been achieved.
Comment 4. Line 19: Change "H2DCFDA staining confirmed the formation of ROS in the pollens and the pollen tube." to "H2DCFDA staining confirmed ROS formation in the pollens and pollen tubes."
Response. The sentence has been modified as suggested. see line 21
Comment 5. Line 21: Change "Furthermore, expression of FmRbohH was found in the pollen and pollen tube, and the antisense oligodeoxynucleotide assay revealed reduced ROS in the pollen and decreased pollen tube growth in Fraxinus mandshurica." to "Furthermore, FmRbohH expression was detected in the pollen and pollen tube, and an antisense oligodeoxynucleotide assay demonstrated reduced ROS and pollen tube growth in Fraxinus mandshurica."
Response. The sentence has been revised as suggested. Please refer to lines 24 to 26.
Comment 6. Line 31: Change "These processes are critical for fertilization in plants." to "These processes are essential for successful fertilization in plants."
Response. The sentence has been revised as suggested. Please refer to lines 33 to 34
Comment 7. Line 36: Change "This process mainly depends on the actin microtubules and cytoskeleton to regulate organelle movement, vesicle trafficking, cytoplasmic flow, and cytoplasm organization in pollen tubes." to "This process is largely dependent on the actin cytoskeleton, which regulates organelle movement, vesicle trafficking, and cytoplasmic organization in pollen tubes."
Response. The sentence has been revised as suggested. Please refer to lines 38 to 40.
Comment 8. Line 78: Change "The plant is native to northeast China, southeast Russia, Korea, and Japan." to "F. mandshurica is native to Northeast China, Southeast Russia, Korea, and Japan."
Response. The sentence has been revised as suggested. Please refer to lines 76 to 77
Comment 9. Line 165: Change "The Illumina Hiseq platform revealed 53.4 – 97 raw reads from 9 RNA samples, clean data of 52.2 – 93.6 million clean data was generated, resulting in 95% of total reads." to "The Illumina HiSeq platform generated 53.4–97 million raw reads from 9 RNA samples, with 52.2–93.6 million clean reads retained, accounting for 95% of the total data."
Response. The sentence has been revised as suggested. Please refer to lines 162 to 163
Comment 10. Line 177: Change "The top 15 pathways with the smallest p-value were considered significant, and a strong presence of oxidative phosphorylation was observed, followed by amino acids synthesis and protein processing in the ER, carbon metabolism, pyruvate metabolism, citrate cycle (TCA cycle)-glycolysis/gluconeogenesis." to "The top 15 pathways with the smallest p-values were considered significant, with oxidative phosphorylation showing the strongest presence, followed by amino acid synthesis, protein processing in the ER, carbon metabolism, pyruvate metabolism, and the citrate cycle (TCA cycle)/glycolysis/gluconeogenesis."
Response. The sentence has been revised as suggested. Please refer to lines 173 to 177
Comment 11. Line 183: Change "The oxidative phosphorylation pathway typically occurs in mitochondria, several complexes are employed to produce ATP during oxidative phosphorylation, and a significant amount of ROS by-product is generated in the mitochondria." to "Oxidative phosphorylation occurs in the mitochondria, where several complexes work together to produce ATP, generating ROS as a byproduct."
Response. The sentence has been revised as suggested. Please refer to lines 180 to 181
Comment 12. Line 195: Change "They are important for energy production in the cell and catalyze electron transfer from dihydroubiquinone (QHz) to cytochrome c, and this reaction is coupled to the transfer of a transmembrane proton, at the same time, ROS is produced as a by-product." to "These complexes are crucial for energy production, catalyzing electron transfer from dihydroubiquinone (QHz) to cytochrome c, a process that is coupled to transmembrane proton transfer and ROS generation as a byproduct."
Response. The sentence has been revised as suggested. Please refer to lines 191 to 193
Comment 13. Line 332: Change "This reaction is linked to the respiratory chain, which uses the energy released during chemical oxidation to produce ATP." to "This reaction is part of the respiratory chain, which harnesses energy from chemical oxidation to produce ATP."
Response. The sentence has been revised as suggested. Please refer to lines 336 to 337
Comment 14. Line 359: Change "The differential gene expression in complexes I, II, and III changed, and the trend was relatively consistent, suggesting a change in ROS content during pollen tube growth." to "Differential gene expression in complexes I, II, and III followed a consistent trend, suggesting fluctuating ROS levels during pollen tube growth."
Response. The sentence has been revised as suggested. Please refer to lines 360 to 361
Comment 15. Line 373: Change "Our data showed gene expression changes linked to ROS generation during pollen tube growth in different cellular compartments." to "Our data revealed changes in gene expression related to ROS generation across different cellular compartments during pollen tube growth."
Response. The sentence has been revised as suggested. Please refer to lines 372 to 373
Comment 16. Line 423: Change "Among the ROS-related genes, Rboh genes are essential players in both stress response and reproductive development." to "Rboh genes, among other ROS-related genes, are essential for both stress responses and reproductive development."
Response. Response. The sentence has been revised as suggested. Please refer to lines 418 to 419
Comment 17. Line 446: Change "Similarly, FmRbohH knockdown greatly reduced ROS fluorescence and pollen tube growth." to "Similarly, knocking down FmRbohH significantly reduced ROS fluorescence and impaired pollen tube growth."
Response. The sentence has been revised as suggested. Please refer to lines 439 to 440.
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe paper was modified not sufficiently, most of the comments are still to answer. Following are comments on the replies (original questions see in 1st round):
Comment 1: Put in the text that you pulled all the DEGs from S1, 2 and 3 together. Explain why it would have sense doing this. Usually it is done separately for each pair of comparisons.
Comment 2: Please list pathways according to some metrics for example rich factor. Also remove the following since, it is false: “oxidative phosphorylation showing the strongest presence, followed by “.
Comment 4: The title from 3.3 is misleading. In your case you found genes in mitochondrial genome which are DEGs. they do not exist in other compartments! Title should be “apoplast, chloroplast … have DEGs related to ROS” as example.
Comment 5: I see perfectly what is on Fig 2,3, and 4. I am asking how to compare the results. Till now I see fig 4 does not validate many genes.
Please also provide a list of all genes with counts as a supplementary table. (not DEGs, ALL genes from S1,2,3 in 3 repetitions.). Upload your raw RNA-seq data into NCBI GEO and provide id in the paper.
Comment 7: I can't see any definition, how do you calculate germination rate?
Comment 8: There is nothing in the paper about the qPCR in leaf. Next, according to your picture expression in pollen is the same as in leaves. Therefore, stating the specificity of FmRbohH to pollens is false.
Comment 9:
First of all, and it relates to all comments, you are making explanations not to me, but to all the readers. Therefore you should insert your changes into the text, but not only to comments to me. Please change the text accordingly. Particularly to this comment explain what shades of red mean in the text.
Most important, your selection of “maximum in observed in samples” gives too much arbitrary, which samples? it is not clear what is compared. Define exactly.
Since the number of DEGs is maximum between first and third samples, please redo this figure for the comparison 1 versus 3, and in supplementary material for the other two comparisons (or include it here). For the color shades use ABSOLUTE levels of expression! This way it is evident how strong the difference is.
Comment 10:
Do this not in one place but throughout the text !
Author Response
-
Comment 1: Put in the text that you pulled all the DEGs from S1, 2 and 3 together. Explain why it would have sense doing this. Usually it is done separately for each pair of comparisons.
Response: Thank you for your insightful comment.
We appreciate your suggestion to analyze DEGs separately for each pair of comparisons (S1 vs. S2, S2 vs. S3, and S1 vs. S3). This approach is common and can provide valuable insights into the specific changes between each pair.
However, in our analysis we have added to the paper that S1, S2, and S3 were pulled together for the following reasons:
- Identifying Global Patterns: By pooling the DEGs from all three groups, we aimed to identify global patterns or core changes that are consistently observed across all comparisons. This can provide a broader understanding of the underlying biological processes involved.
- Enhancing Statistical Power: Combining the DEGs can increase the statistical power of our analysis, especially for genes with subtle expression changes that might not be significant in individual pair-wise comparisons.
- Exploring Shared Biological Pathways: We hypothesized that genes involved in shared biological pathways or functions might be consistently differentially expressed across all three groups. By combining the DEGs, we could more effectively identify such pathways. See lines 175 to 185.
Comment 2: Please list pathways according to some metrics for example rich factor. Also remove the following since, it is false: “oxidative phosphorylation showing the strongest presence, followed by “.
Response: The pathways are listed according to the rich factor, the number of genes in the pathway, and corrected p-value metrics.
Regarding the statement about oxidative phosphorylation, we apologize for the inaccuracy. We have removed this statement from our manuscript and carefully reviewed our data to ensure that our conclusions are accurate and supported by our data. See lines 175 to 185
Comment 4: The title from 3.3 is misleading. In your case you found genes in mitochondrial genome which are DEGs. they do not exist in other compartments! Title should be “apoplast, chloroplast … have DEGs related to ROS” as example.
Response: The 3.3 title has been revised as suggested. See line 217 to 218
Comment 5: I see perfectly what is on Fig 2,3, and 4. I am asking how to compare the results. Till now I see fig 4 does not validate many genes.
Please also provide a list of all genes with counts as a supplementary table. (not DEGs, ALL genes from S1,2,3 in 3 repetitions.). Upload your raw RNA-seq data into NCBI GEO and provide id in the paper.
Response: Regarding Figure 4, we acknowledge that it may not validate all genes identified in Figures 2 and 3. This could be due to several factors, such as individual biological replicates can exhibit variability in gene expression, which can affect the validation results. The supplementary table has been added as requested. In addition, we have already uploaded our raw data to the NCBI as mentioned it in the data availability section of the manuscript and can be accessed through the https://www.ncbi.nlm.nih.gov using the Bi-oSample accessions SAMN42883688, SAMN42883689, and SAMN42883690. see line 460 to 461.
Comment 7: I can't see any definition, how do you calculate germination rate?
Response: The pollen germination rate is the number of pollen germination/number of pollen. See lines 126 and 134.
Comment 8: There is nothing in the paper about the qPCR in leaf. Next, according to your picture expression in pollen is the same as in leaves. Therefore, stating the specificity of FmRbohH to pollens is false.
Response: We apologize for the mistake regarding the lack of information about the qPCR data of the leaf. It was transcriptome data mistakenly written as qPCR, the qPCR is figure 6B. We have revised the figure to remove the leaf data and modified the manuscript accordingly. See lines 303 to 308 and 433 to 434
Comment 9:
First of all, and it relates to all comments, you are making explanations not to me, but to all the readers. Therefore you should insert your changes into the text, but not only to comments to me. Please change the text accordingly. Particularly to this comment explain what shades of red mean in the text.
Most important, your selection of “maximum in observed in samples” gives too much arbitrary, which samples? it is not clear what is compared. Define exactly.
Since the number of DEGs is maximum between first and third samples, please redo this figure for the comparison 1 versus 3, and in supplementary material for the other two comparisons (or include it here). For the color shades use ABSOLUTE levels of expression! This way it is evident how strong the difference is.
Response: Thank you for your valuable feedback. We apologize for any confusion caused by our previous responses. We will ensure that all changes are incorporated directly into the text of the manuscript, not just in our comments.
Red shades: In our figure, the shades of red represent the magnitude of differential expression. Darker shades of red indicate larger fold changes between the compared groups. See lines 215 to 216 and 230 to 231.
Maximum DEGs between samples: this is the comparison between the first, second, and third samples, where the maximum number of differentially expressed genes (DEGs) was observed.
Comment 10:
Do this not in one place but throughout the text !
Response: Thank you again for your review and your valuable suggestions. We made modifications based on your suggestions.
