Effects of Biodegradable Liquid Film (BLF) on Cold Resistance in Grapevine Revealed by Metabolomic Analysis
Round 1
Reviewer 1 Report
This paper, explore the efficacy of treatment with a Biodegradable liquid mulch to stimulate cold resistance in grapevine. The topic covered in the paper is very interesting; results, that was presented in a clear manner using graphical and table means, are supported by appropriate methods and experimental procedures, which are described with enough detail. The significance of results obtained was interpreted and discussed with due reference to previously published studies. I really appreciated this work, in particular the multivariate statistical analysis performed on the collected data. However, I have some concerns regarding the adopted monovariate statistical approach. In particular, the presented experimental design shows a comparison between a treated block and an untreated block. To evaluate differences between these two populations authors used one-way ANOVA and Duncan’s multiple comparison tests. In my opinion this approach is not correct, because ANOVA is a statistical formula used to compare variance across the means of different groups (three or more). Differently, the Student’s t-test is a parametric method that determines whether two populations are statistically different from each other. For these reasons, I suggest the authors review the statistical analysis by reporting the results after applying a Student's t test.
Author Response
Thanks for your suggestions. I agree with your comments. I reported the results by applying a Student's t-test. The statistical analysis resulted in a change in the significance of AsA compared to the one-way ANOVA. I have modified it in Figure 1 and the results.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
This is a very interesting study with advanced omics. However, the only concern I have note is absence of phenotypic data supporting their conclusions and absence of temporal data. No proper rationale was provided for the dates analyzed nor the application schedule. What happened to the orchard after the application of the mixture versus controls? I guess all the indirect inferences stand weak without phenotypic data.
Addressing the above questions in all relevant sections shall make it easy to understand and appreciate the science behind the novel strategy to counter cold stress.
Author Response
Thanks for your suggestions. Our team has been researching BLF spraying for overwintering protection of grapevines for several years. We investigated the effects of BLF on grapevine cold resistance [46,51], including plant survival [43], grapevine transcriptomic analysis [52], and effects on grape quality [43,53]. The results showed that BLF attenuated oxidative stress in grapevines, reduced the semilethal temperature of grapevines, vine, and bud mortality, protected grapes from winter cold, and delayed the date of germination in cold years. Our team has already done a lot of research on cold-resistant phenotypes of grapevine in the previous period. However, this experiment was mainly intended to study the role of BLF by metabolomics approach. Therefore, malondialdehyde (MDA) was selected as an indicator of oxidative stress and superoxide dismutase (SOD), ascorbic acid (ASA) and proline (Pro) as antioxidant indicators, respectively, for the phenotypic assay in this experiment. Carbohydrates and related enzymes were also measured in response to intracellular stability. Exposing plants to non-freezing cold temperatures increases their tolerance to extreme cold temperatures, which is known as cold acclimation [4]. Plants initiated the cold acclimation phase starting in December. In the Penglai region, winter's lowest temperatures were predominantly observed in mid to late January. Therefore, this experiment was conducted between 7 December 2021 and 19 January 2022.
Author Response File: Author Response.pdf
Reviewer 3 Report
This manuscript is supported by a good set of data, providing interesting insight into the metabolic physiology of grape vines in response to cold temperature as affected by a biodegradable liquid mulch. Good technical quality. English requires minor improvement.
Please see below for my comments:
L28: please include a few more keywords, which can be the different parameters being tested
L33: Change severely to significantly
L38: delete “ways”
L78-83: Scientific names need to be italicized; first letter of the genus name should be capitalized. For example Camelia oleifera. Please correct throughout the manuscript.
L99-100: The objectives of this study need to be more specified.
L109: again, italicize scientific names
L118: Control should be not shortened as CK, which is often used as the abbreviation for cytokinins. Please correct the use of CK throughout the text and in figures. Control would be a lot better than CK
L126: remove the extra long
L127: replace refrigerator with -80 C freezer; also the signs used for °C were not consistent throughout the manuscript, please correct
L136: What is EC 1.15.1.1?
L146: the unit of SOD activity doesn’t seem to be correct, or at least the font for “·” sign is not right, please correct
L156: The unit ug/g, is this on a fresh weight or dry weight basis? The same question apply to other units too.
L161: please specify the spectrophotometer that was used to measure absorbance.
All figures: the resolution of all figures need to be higher; I can’t read most of the figures even when the PDF is displayed with 200% ratio
Figure 1-3: why each data points have three dots? Also, use control instead of CK
L339: there is no figure 10b
L382: Correct H2O2
L405: different treatments? There was only one treatment in this study, the BLF
The conclusion section need to be improved to better summary the key fundings from this study.
L532-537: too many sentences fused into one, please rephrase.
minor English editing is needed
Author Response
Point 1: L28: please include a few more keywords, which can be the different parameters being tested
Response 1: Thanks for your comment. I added "oxidative damage" as a keyword.
Point 2: L33: Change severely to significantly
Response 2: Thanks for your comment. It has been changed.
Point 3: L38: delete “ways”
Response 3: Thanks for your comment. It has been deleted. And I rewrote the sentence.
Point 4: L78-83: Scientific names need to be italicized; first letter of the genus name should be capitalized. For example Camelia oleifera. Please correct throughout the manuscript.
Response 4: Thanks for your comment. It has been corrected.
Point 5: L99-100: The objectives of this study need to be more specified.
Response 5: Thanks for your comment. It has been added.
Point 6: L109: again, italicize scientific names
Response 6: Thanks for your comment. It has been corrected.
Point 7: L118: Control should be not shortened as CK, which is often used as the abbreviation for cytokinins. Please correct the use of CK throughout the text and in figures. Control would be a lot better than CK
Response 7: Thanks for your comment. It has been corrected in the manuscript.
Point 8: L126: remove the extra long
Response 8: Thanks for your comment. It has been removed.
Point 9: L127: replace refrigerator with -80 C freezer; also the signs used for °C were not consistent throughout the manuscript, please correct
Response 9: Thanks for your comment. It has been corrected.
Point 10: L136: What is EC 1.15.1.1?
Response 10: Thanks for your comment. It is the EC number of the SOD. The International Commission on Enzymology has standardized the numbering of each enzyme so that there is no confusion as there is only one number for each enzyme.
Point 11: L146: the unit of SOD activity doesn’t seem to be correct, or at least the font for “·” sign is not right, please correct
Response 11: Thanks for your comment. It has been corrected.
Point 12: L156: The unit ug/g, is this on a fresh weight or dry weight basis? The same question apply to other units too.
Response 12: Thanks for your comment. It is on a fresh weight basis.
Point 13: L161: please specify the spectrophotometer that was used to measure absorbance.
Response 13: Thanks for your comment. It has been added in the first appearance, line 136.
Point 14: All figures: the resolution of all figures need to be higher; I can’t read most of the figures even when the PDF is displayed with 200% ratio
Response 14: Thanks for your comment. All figures have been re-uploaded. I can provide the original figures at jpg. if needed.
Point 15: Figure 1-3: why each data points have three dots? Also, use control instead of CK
Response 15: Thanks for your comment. Three biological replications were set up, with 30 vines per experimental unit.
Point 16: L339: there is no figure 10b
Response 16: Thank you. It is in line 377.
Point 17: L382: Correct H2O2
Response 17: Thanks for your comment. It has been corrected.
Point 18: L405: different treatments? There was only one treatment in this study, the BLF
Response 18: Thanks for your comment. It has been modified.
Point 19: The conclusion section need to be improved to better summary the key fundings from this study.
Response 19: Thanks for your comment. It has been revised.
Point 20: L532-537: too many sentences fused into one, please rephrase.
Response 20: Thanks for your comment. It has been revised.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
The manuscript was sufficiently improved to justify publication in Agriculture. I have no other comments to suggest.
