Regulatory Effect of Exogenous γ-Aminobutyric Acid on Respiratory Rate through the γ-Aminobutyric Acid Shunt in Malus baccata (L.) Borkh. Roots under Suboptimal Low Root-Zone Temperature
Round 1
Reviewer 1 Report
In this manuscript, the authors investigated how GABA and GABA-T inhibitor treatments affect the root responses under suboptimal low root-zone temperature condition. They evaluated root vitality, metabolite levels, activity of GABA biosynthesis and catabolism enzymes, expression level of GABA-related genes, and root respiration rates and concluded that exogenous GABA treatment enhances the TCAC activity by improving the GABA shunt activity. Although the description of the Discussion is scattered and difficult to follow the author’s arguments, the data presented in this paper are clear except for the PCA.
Major comments:
In PCA, orders of magnitude in root variability or MDA levels are two or one higher than that in respiration rates. Did authors perform the normalization in the PCA? Please explain how the authors performed PCA in Materials and Methods.
The description of Discussion is scattered and it is difficult to follow the author’s argument. For example, the authors explain the importance of pH and Ca2+/CaM from line 471 to line 481. However, I think these descriptions do not help explain how the authors interpreted the results and reached the conclusion. Please consider revising Discussion to more simply describe the process from results to conclusions.
In Results or Discussion, please explain why the authors measured the malondialdehyde levels. What can we infer from the changes in the malondialdehyde levels?
Minor comments:
Line 11, 100, 217, 224, 256, 293, 327, 363, and 424: Baccata -> baccata
Line 56: Replace “, L.J.” with “et al.”.　
Line 217: Add spaces before “(a)” and “(b)”.
Line 225 and 226: “content” were used redundantly.
Discussion: Please cite the Figures that correspond to the description in the text.
Figure 4a: Authors should add the error bars.
Figure 5, 6 and 7 should be combined into a single figure.
Figure title is missing in Figure 8.
Increase the font size of the X and Yaxis labels in Figure 8b.
Author Response
Dear editors and dear reviewers:
Thank you for your letter and the reviewers’ comments concerning our manuscript entitled “Regulation of exogenous γ-aminobutyric acid on respiratory rate through the γ-aminobutyric acid shunt in Malus baccata (L.) Borkh. roots under suboptimal low root-zone temperature” (Manuscript ID: horticulturae-2141256). Those comments are valuable and very helpful. We have read through comments carefully and revised. Revisions in the manuscript are shown using red highlight for additions, and strikethrough font for deletions. I believed that the addressing of these comments has greatly improved the quality of this manuscript. Based on the instructions provided in your letter, we uploaded the file of the revised manuscript. The responses to the reviewer's comments are marked in blue presented following.
We would love to thank you for allowing us to resubmit a revised copy of the manuscript and we highly appreciate your time and consideration.
Best regards,
Sincerely yours,
Huaiyu Ma
E-mail: 2006500038@syau.edu.cn
Response to Reviewer 1 Comments
Major comments:
Point 1: In PCA, orders of magnitude in root variability or MDA levels are two or one higher than that in respiration rates. Did authors perform the normalization in the PCA? Please explain how the authors performed PCA in Materials and Methods.
Response 1: We deeply appreciate your suggestion. The data were standardized and computed in SPSS software. According to the comment, we have added the performance instructions of PCA in the ‘2.8’ part. And, we also added a more detailed instruction in Figure6.
Point 2: The description of Discussion is scattered and it is difficult to follow the author’s argument. For example, the authors explain the importance of pH and Ca2+/CaM from line 471 to line 481. However, I think these descriptions do not help explain how the authors interpreted the results and reached the conclusion. Please consider revising Discussion to more simply describe the process from results to conclusions.
Response 2: We are extremely grateful to you for pointing out this problem. According to your suggestions, now we’ve tried our best to revise the Discussion of manuscript to make our discussion more condensed and concise. Please see ‘Discussion’ part.
Point 3: In Results or Discussion, please explain why the authors measured the malondialdehyde levels. What can we infer from the changes in the malondialdehyde levels?
Response 3: Thank you for the suggestion. We apologize for the lack of clarity about the discription of malondialdehyde (MDA) levels. Many studies have shown that the MDA level is an important parameter reflecting the potential antioxidant capacity of the plant, which can reflect the rate and intensity of lipid peroxidation and also in-directly reflect the degree of tissue peroxidation damage. So, we evaluate the degree of damage to roots caused by suboptimal low root-zone temperatures by MDA content. And,we also use this to judge whether exogenous GABA is effective in this experimental treatment. We have added those explainations in the corresponding places in the ‘Discussion’ part. Please see Lines 451-455.
Minor comments:
Point 1: Line 11, 100, 217, 224, 256, 293, 327, 363, and 424: Baccata -> baccata.
Response 1: We apologize for the mistakes in the manuscript. We have corrected the “Malus Baccata” into “Malus baccata”, and carefully checked the whole manuscript. This revision is highlighted in red (for adding) and strikethrough font (for deleting) in the revised manuscript.
Point 2: Line 56: Replace “, L.J.” with “et al.”.　
Response 2: Thank you for your careful reading. We have revised it according to your suggestion. Please see Line 56. At the same time, we have reviewed the manuscript and corrected similar errors.
Point 3: Line 217: Add spaces before “(a)” and “(b)”.
Response 3: We are so sorry for our careless mistakes. Thank you for your reminder. We have revised it. Please see Line 228.
Point 4: Line 225 and 226: “content” were used redundantly.
Response 4: We are really sorry for such a mistake in the manuscript. We have deleted it. Please see Line 220.
Point 5: Discussion: Please cite the Figures that correspond to the description in the text.
Response 5: Thanks for your professional suggestion. We have cited the Figures that correspond to the description in the text. Please see ‘Disscussion’ part.
Point 6: Figure 4a: Authors should add the error bars.
Response 6: We appreciate your valuable suggestions. We have add the error bars into the Figure 4a.
Point 7: Figure 5, 6 and 7 should be combined into a single figure.
Response 7: Thanks for your thoughtful comments. We have combined Figure 5, 6 and 7 into Figure 5.
Point 8: Figure title is missing in Figure 8.
Response 8: We feel grate thanks for your careful reading. We have rewritten the Figure title. Please see Line 439-449.
Point 9: Increase the font size of the X and Yaxis labels in Figure 8b.
Response 9: We are grateful for the suggestion. We have revised it. Your careful review has helped to make our Figuers clearer. Due to the change of Figure number, it is ‘Figure 6b’ in new manuscript.
Thank you for your careful review. We really appreciate your valuable comments and suggestions in reviewing our manuscript. Your careful review has helped to make our study clearer and more comprehensive. Once again, we appreciate for your enthusiastic and professional review work, and hope our correction will meet with approval.
Author Response File: 
Author Response.docx
Reviewer 2 Report
This is a well written paper on the "Regulation of exogenous γ-aminobutyric acid on respiratory rate through the γ-aminobutyric acid shunt in Malus baccata (L.) Borkh. roots under suboptimal low root-zone temperature".
I have some questions about the experimental design. How many seedlings were employed? Was this a completely randomized block design?
There are some errors reporting the botanical names. It is Malus baccata, not Malus Baccata.
Also, the statistical analysis can be improved. In my opinion this should be a 2-way ANOVA and not a 1-way ANOVA.
Finally, I am little bit confused about the conclusions. The Authors did not see any increase in root vitality when treatments were applied. How can you conclude that treatments were effective?
Author Response
Dear editors and dear reviewers:
Thank you for your letter and the reviewers’ comments concerning our manuscript entitled “Regulation of exogenous γ-aminobutyric acid on respiratory rate through the γ-aminobutyric acid shunt in Malus baccata (L.) Borkh. roots under suboptimal low root-zone temperature” (Manuscript ID: horticulturae-2141256). Those comments are valuable and very helpful. We have read through comments carefully and revised. Revisions in the manuscript are shown using red highlight for additions, and strikethrough font for deletions. I believed that the addressing of these comments has greatly improved the quality of this manuscript. Based on the instructions provided in your letter, we uploaded the file of the revised manuscript. The responses to the reviewer's comments are marked in blue presented following.
We would love to thank you for allowing us to resubmit a revised copy of the manuscript and we highly appreciate your time and consideration.
Best regards,
Sincerely yours,
Huaiyu Ma
E-mail: 2006500038@syau.edu.cn
Response to Reviewer 2 Comments
Point 1: I have some questions about the experimental design. How many seedlings were employed? Was this a completely randomized block design?
Response 1: Thank you for your question. We apologize that our description of the experimental design is not clear and adequate. We cultivated about 1000 seedlings for this experiment. When seedlings had 15 leaves and were determined to be free of diseases and pests, 15 seedlings were selected per experimental treatment on per time point for experimental treatment. That is to say, 255 seedlings were used in the whole experiment. And the total 255 seedlings were designed in a completely randomized block at every time point during the experimental treatment.
Point 2: There are some errors reporting the botanical names. It is Malus baccata, not Malus Baccata.
Response 2: We sincerely appreciate your careful reading and sorry for our calreless mistakes. As suggested by the reviewer, we have checked the whole manuscript and corrected the “Malus Baccata” into “Malus baccata”. This revision is highlighted in red (for adding) and strikethrough font (for deleting) in the revised manuscript.
Point 3: The statistical analysis can be improved. In my opinion this should be a 2-way ANOVA and not a 1-way ANOVA.
Response 3: We sincerely appreciate your suggestion. First of all, please allow us to explain why we use 1-way ANOVA in statistical analysis. In the original manuscript, we focused more on the significant changes between treatments at each time point. Therefore, different letters above the bars in figures indicate significant differences between treatments (P < 0.05). Thanks again for your suggestions on statistical analysis, which further improves the readability of the data in figures. We have supplemented the significance analysis results of the full data in the new manuscript, marked with capital letters, as well as illustrated and annotated in part ‘2.8’ and ‘Figure annotation’.
Point 4: I am little bit confused about the conclusions. The Authors did not see any increase in root vitality when treatments were applied. How can you conclude that treatments were effective?
Response 4: We are extremely grateful to you for pointing out this problem. We apologize that we missed this point in the conclusions. Root vitality is an important indicator to evaluate plant injury caused by stress. Figure 1a in the manuscript shown that suboptimal low root-zone temperature significantly inhibited the root vitality, exogenous GABA application significantly enhanced the root vitality, and pretreated with VGB had an opposite influence on the root vitality. Therefore, we speculated that exogenous GABA was effective in alleviating the damage caused by suboptimal low root-zone temperature. And we have supplemented in part ‘5. Conclusions’. Please see Line 644-650.
Thank you for your careful review. We really appreciate your valuable comments and suggestions in reviewing our manuscript. Your careful review has helped to make our study clearer and more comprehensive. Once again, we appreciate for your enthusiastic and professional review work, and hope our correction will meet with approval.
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
The Authors addressed all my comments and the paper can now be accepted for publication.
