H2O2 Participates in the Induction and Formation of Potato Tubers by Activating Tuberization-Related Signal Transduction Pathways
Round 1
Reviewer 1 Report
1. Mark the specific location of histochemical stain with arrows on Figure 3.
2. Can the color of stolons and tubers in CIP-149 be removed with alcohol? The histochemical stain cannot be clearly seen from Figure 3b.
3. It may be better to use letters to indicate the significant differences in Figure 4 and Figure 5.
4. Statistical results on the growth and height of potato plants need to be included in Figure 5 and Figure 9.
5. The authors only selected 9 genes to detect the expression levels in this study. Can this fully explain the problem?
No comment
Author Response
Response to Reviewer 1 Comments
Point 1: Mark the specific location of histochemical stain with arrows on Figure 3.
Response 1: Thanks for this valuable suggestion. We have marked the corresponding location with arrows in Figure 3.
Point 2: Can the color of stolons and tubers in CIP-149 be removed with alcohol? The histochemical stain cannot be clearly seen from Figure 3b.
Response 2: Thanks for pointing out this. The tuber inside of CIP-149 is also dark red. We have treated the tuber with alcohol. The red color cannot be removed with alcohol. Fortunately, the fluorescent probe H2DCFDA can detect total ROS accumulation without little interference with the naturally dark red in CIP-149. The present results showed that the brightest H2DCFDA fluorescence was observed at stage 2 of tuber formation in CIP-178 and CIP-149 (Figure 3a and b), indicating that there is a total ROS accumulation in the hook-like subapical part of stolons.
Point 3: It may be better to use letters to indicate the significant differences in Figure 4 and Figure 5.
Response 3: Thanks for this valuable suggestion. We have used letters to indicate the significant differences in Figure 4 and Figure 5.
Point 4: Statistical results on the growth and height of potato plants need to be included in Figure 5 and Figure 9.
Response 4: Thanks for this valuable suggestion. The statistics results on the growth and height of potato plants in Figure 5b have been included as Figure 5c. The stolon length shown in Figure 9b was not measured at the time, so a figure relating to the stolon length shown in Figure 9b cannot be provided.
Point 5: The authors only selected 9 genes to detect the expression levels in this study. Can this fully explain the problem?
Response 5: Thanks for pointing out this. We have synthesized multiple papers on potato tuberization (Please refer to our review article for details, Lei et al., 2022, Physiological and biochemical basis and molecular mechanism of solanum tuberosum tuberization.), so we have selected these genes. Multiple studies have shown that these genes are important genes that differential expression during the tuberization, and although they are not a global expression profile, they can explain the main problem. However, if more genes were selected, it would be possible to provide a more comprehensive and in-depth explanation for the problem.
Author Response File: 
Author Response.docx
Reviewer 2 Report
The abbreviations were used too much, sometimes mixed with genes designations. Difficult to find meaning of abbreviations. Maybe it worst to develop list of abbreviations?
Author Response
Response to Reviewer 2 Comments
Point 1: The abbreviations were used too much, sometimes mixed with genes designations. Difficult to find meaning of abbreviations. Maybe it worst to develop list of abbreviations?
Response 1: Thanks for this valuable suggestion. We have added a list of abbreviations to the paper.
Author Response File: Author Response.docx
Reviewer 3 Report
This manuscript describes that H2O2 accumulation in potato stolons plays a role acting as a signaling molecule to initiate tuber induction, and StSP6A is a pivotal player in H2O2-induced tuber formation in potato. I think the manuscript is worth publishing after a minor revision.
1、It is necessary to add the content of key genes for potato tuber formation in the Introduction section, especially StSP6Agene.
2、The change of StRboh gene is more significant in Figure 8, but the manuscript does not focus on this gene. Because the function of StSP6A in potato tuber formation is known, while the function of StRboh is unknown (Table2). I think it is more meaningful to study the function of StRboh gene.
Author Response
Response to Reviewer 3 Comments
Point 1: It is necessary to add the content of key genes for potato tuber formation in the Introduction section, especially StSP6A gene.
Response 1: Thanks for this valuable suggestion. We have added the content of these key genes for potato tuber formation in the introduction section. Details can be seen in Line 75-79.
Point 2: The change of StRboh gene is more significant in Figure 8, but the manuscript does not focus on this gene. Because the function of StSP6A in potato tuber formation is known, while the function of StRboh is unknown (Table2). I think it is more meaningful to study the function of StRboh gene.
Response 2: Thanks for this valuable suggestion. In fact, we did genome-wide identification of Rboh gene family and expression analysis during tuber formation in potato, cloning of the StRbohA gene, which was significantly up-regulated during tuber induction and formation, and also verified the effect of StRbohA overexpression on tuberization in diploid potato plants. These follow-up experiment results will be presented in another paper.
Author Response File: Author Response.docx
Reviewer 4 Report
The current manuscript describes the functional characterization of Solanum tuberosum self-prunning 6A (StSP6A) on H2O2-induced tuber formation in potato. Accumulation of different ROS and total ROS were determined under tuber-inducing conditions in tissue culture in two diploid landraces. The exogenous application of H202 and inhibition of internal ROS levels was a nice touch to test the manuscript hypothesis. However, the manuscript needs further experimentation and clarification.
Comments:
1. Authors should discuss why the effect of point and large deletion mutations looks the same in tuber numbers.
2. In order to come up with the final model, I believe the gene expression experiment itself does not provide enough evidence. On top of this, exogenous H2O2 application does not indicate the expression is linking the internal H2O2 signaling to the tuberization. A better experimental design would include determining the expression levels by blocking the H202 production and/or signaling during tuberization.
3. The model needs revision by understanding the expression patterns better. Authors should decide if they want to draw the model by concentrating on the results of 0 mM H2O2 application or 5 mM H2O2 application
4. A tissue-clearing protocol is essential for Figure 3 since it is difficult to make a complete evaluation of NBT and DAB staining. Since ROS accumulation is increased under other stressful conditions, the authors should have proved that the staining was not due to any other stresses (especially physical damage while staining) by showing at least three different plants side by side in one image.
5. ANOVA + HSD would be a better type of statistical test in understanding the results of Figure 4, Figure 5a, Figure 6, Figure 7, and Figure 8.
6. Figure 5b and 9b need quantification.
7. Since there is no Ca2+ level accumulation experiment included in the manuscript, authors either should modify the model, or they should provide some evidence of Ca2+ acting upstream of StCDPK by citing literature related to tuberization.
Author Response
Response to Reviewer 4 Comments
Point 1: Authors should discuss why the effect of point and large deletion mutations looks the same in tuber numbers.
Response 1: Thanks for pointing out this. This is really a question worth thinking about, and we'll cover it in the discussion section.
Point 2: In order to come up with the final model, I believe the gene expression experiment itself does not provide enough evidence. On top of this, exogenous H2O2 application does not indicate the expression is linking the internal H2O2 signaling to the tuberization. A better experimental design would include determining the expression levels by blocking the H2O2 production and/or signaling during tuberization.
Response 2: Thanks for this valuable suggestion. We conducted exogenous addition of either the ROS inhibitor diphenyleneiodonium chloride (DPI) or H2O2 scavenger catalase (CAT) treatment , which resulted in a decline of tuber formation (Fig. 6 and 7). The results would be better illustrated if various genetic tools could be used to block the H2O2 production and/or signaling during tuberization. We will further study on the problem based on your suggestion. Thank you very much.
Point 3: The model needs revision by understanding the expression patterns better. Authors should decide if they want to draw the model by concentrating on the results of 0 mM H2O2 application or 5 mM H2O2 application.
Response 3: Thanks for this valuable suggestion. We did draw the model with the result of 5 mM H2O2 treatment, but it is not clearly expressed in the paper. Thank you very much for your reminding. We have modified it in the paper.
Point 4: A tissue-clearing protocol is essential for Figure 3 since it is difficult to make a complete evaluation of NBT and DAB staining. Since ROS accumulation is increased under other stressful conditions, the authors should have proved that the staining was not due to any other stresses (especially physical damage while staining) by showing at least three different plants side by side in one image.
Response 4: Thanks for pointing out this. We put fresh plant material directly into the dye solution to minimize any external physical damage. The dyeing process is conducted at room temperature and in complete darkness to eliminate any factors that could be influenced by temperature or light. To ensure the reliability of the staining results, we compared two potato materials, CIP-178 and CIP-149.
Point 5: ANOVA + HSD would be a better type of statistical test in understanding the results of Figure 4, Figure 5a, Figure 6, Figure 7, and Figure 8.
Response 5: Thanks for this valuable suggestion. We indeed used ANOVA + LSD for statistical testing to understand the results of Figures 4, 5a, 6, 7, and 8, but this was not clearly articulated in the paper. This detail has now been added. Thank you very much!
Point 6: Figure 5b and 9b need quantification.
Response 6: Thanks for this valuable suggestion. The statistics results on the growth and height of potato plants in Figure 5b have been included as Figure 5c. The stolon length shown in Figure 9b was not measured at the time, so a figure relating to the stolon length shown in Figure 9b cannot be provided.
Point 7: Since there is no Ca2+ level accumulation experiment included in the manuscript, authors either should modify the model, or they should provide some evidence of Ca2+ acting upstream of StCDPK by citing literature related to tuberization.
Response 7: Thanks for this valuable suggestion. We have incorporated relevant supporting evidence into the manuscript.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
I have no question.
Author Response
Response to Reviewer 1 Comments （Round 2）
Thanks for your valuable suggestion and revision.
Author Response File: Author Response.docx
Reviewer 4 Report
I thank the authors for completing the revisions in a few days. I have small concerns about the following points:
1. Fig 4b: Please double-check the statistical lettering of stage 3. It can be ab instead of b.
2. Indicate the stars representing the statistical difference between the control and treatment in Figs 6, 7, and 9a.
3. There are several studies from species other than plants (even some in plants) with similar results (no phenotypic difference) to the manuscript when there are point and large deletion mutations. Please analyze those and give a brief explanation in the discussion instead of telling that this observation needs further experimentation.
4.
Author Response
Response to Reviewer 4 Comments （Round 2）
Point 1: Fig 4b: Please double-check the statistical lettering of stage 3. It can be ab instead of b.
Response 1: Thanks for this valuable suggestion. We have re-conducted the significance analysis of the differences in the data and have made appropriate modifications.
Point 2: Indicate the stars representing the statistical difference between the control and treatment in Figs 6, 7, and 9a.
Response 2: Thanks for this valuable suggestion. We have made modifications. Detailed changes can be found in lines 351-352.
Point 3: There are several studies from species other than plants (even some in plants) with similar results (no phenotypic difference) to the manuscript when there are point and large deletion mutations. Please analyze those and give a brief explanation in the discussion instead of telling that this observation needs further experimentation.
Response 3: Thanks for this valuable suggestion. We have given the corresponding explanation in the discussion. Details can be seen in lines 500-510.
Author Response File: Author Response.docx
