Development of a Predictive Model Linking Electrical Characteristics to Semi-Lethal Temperature in Potted Apple Trees with Validation on Mature Specimens
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsI have read the manuscript, I fine it very interesting, and it meets the guidlines for publication. Please see the handwritten comments in attached file for detailed suggestions.
Comments for author File: 
Comments.pdf
Author Response
Since the reviewers wrote their comments by hand, we are unable to respond point-to-point.We sincerely thank the reviewer for the positive evaluation and for accepting our manuscript. We are truly grateful for your recognition of our work and thoughtful comments, which have encouraged us greatly. Your feedback affirms the value of our research and motivates us to continue our efforts in this field.
Author Response File: Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe manuscript presents a highly interesting study on the development of a predictive model to estimate cold tolerance in apple trees using electrophysiological parameters as predictors of semi-lethal temperature (LT50). A notable strength of the work is the use of two distinct techniques to analyze the electrical characteristics of the studied plants. The experimental approach is robust, involving both young potted plants and validation on mature field-grown trees. The combined use of principal component analysis (PCA), multiple linear regression, and validation using R², MAE, and RMSE reinforces the credibility of the proposed model. The results show strong correlations between electrical parameters and LT50, with good model fitting and predictive performance in tests with mature trees. These findings indicate consistency and practical applicability of the proposed approach. The discussion is comprehensive, well supported by the literature, and appropriately interprets the relationships between electrical parameters and the physiological processes associated with cold tolerance.
Below, I present some points that should be considered to improve the reproducibility of the study and to enhance the clarity of result presentation:
Introduction:
- Lines 101–117: It is important to clarify the differences between measuring plant bioelectricity using techniques such as low-voltage electrophytography—which do not apply any electrical input—and techniques based on electrical impedance. This paragraph currently mixes examples from both approaches, which may cause confusion.
Materials and Methods:
- Line 141: Please also include the name of the country where the experiment was conducted and the plant material was obtained.
- To ensure reproducibility, it is essential to describe the environmental conditions during the experiment, including light intensity, humidity, and temperature.
- Figure 1: Use a lowercase “d” in “design” and provide a clear explanation of the acronym “REL”.
- Line 182: Standardize the terminology—use either relative electrolyte leakage or relative electrolyte conductivity consistently throughout the text.
- The initial part of section 2.1.2 repeats information already described earlier. I recommend reorganizing the text to avoid redundancy. Similar issues can be observed in other parts of the Materials and Methods section.
- Line 255: Clearly justify why a 30-minute waiting period was considered sufficient for electrode stabilization. Several studies suggest that a minimum of 12 hours may be necessary to ensure signal stability.
- Figure 2: The representation of the greenhouse is visually inconsistent with the rest of the diagram. Consider finding an alternative way to illustrate it.
- In general, I recommend revising the figure and table captions to provide additional information that will help readers interpret the data more accurately.
Discussion:
- Line 449: Is LT50 analysis alone sufficient to affirm that one variety is more cold-tolerant than another? Wouldn’t it be necessary to complement it with other analyses, such as gene expression, proline accumulation, and enzymatic and non-enzymatic antioxidant system evaluation? It might be more prudent to state that SH40 appears to exhibit greater cold tolerance, rather than make a definitive conclusion.
Conclusion:
- The conclusion is overly long. I recommend making it more concise by focusing on the main findings without repeating information already discussed in the results and discussion sections.
Author Response
Comment 1: Lines 101–117: It is important to clarify the differences between measuring plant bioelectricity using techniques such as low-voltage electrophytography—which do not apply any electrical input—and techniques based on electrical impedance. This paragraph currently mixes examples from both approaches, which may cause confusion.
Response 1: Thank you for pointing out the differences between measuring plant bioelectricity using techniques and techniques based on electrical impedance. We fully agree with your opinion that mixing the two types of techniques in the same paragraph may indeed confuse readers. Therefore, we have modified the content of this paragraph to avoid confusion about the research methods. Please see lines 109–118.
Comment 2: Line 141: Please also include the name of the country where the experiment was conducted and the plant material was obtained.
Response 2: Thanks for your nice suggestions. We have added the names of the countries where the experiments were conducted and where the plant material was obtained. Please see lines 140-144.
Comment 3: To ensure reproducibility, it is essential to describe the environmental conditions during the experiment, including light intensity, humidity, and temperature.
Response 3: Thank you for the reviewer's valuable suggestions. The experimental part of this study was conducted under natural wintering conditions, aiming to simulate the low temperature stress suffered by apple seedlings in the natural environment. Therefore, environmental factors such as light intensity and air humidity were not controlled and recorded. We added the average temperature information of each month during the experiment in lines 166-169,190-193 of the text to provide environmental background data as much as possible. We will further improve the recording of environmental parameters in subsequent studies to improve the repeatability of the study.
Comment 4: Figure 1: Use a lowercase “d” in “design” and provide a clear explanation of the acronym “REL”.
Response 4: Thanks for your suggestion, we have changed Figure 1. Please see line 159.
Comment 5: Line 182: Standardize the terminology—use either relative electrolyte leakage or relative electrolyte conductivity consistently throughout the text.
Response 5: Thanks for your suggestion, we have unified the entire article as relative electrolyte leakage.
Comment 6: The initial part of section 2.1.2 repeats information already described earlier. I recommend reorganizing the text to avoid redundancy. Similar issues can be observed in other parts of the Materials and Methods section.
Response 6: We have carefully considered your opinion that the beginning of Section 2.1.2 is repeated in the previous text. Since Section 2.1.2 is the verification experiment, which is different from the experimental time and experimental materials of the potted plant experiment, we have not deleted the relevant content for the time being, so that readers can clearly understand the independence of the verification experiment and its design logic.
Comment 7: Line 255: Clearly justify why a 30-minute waiting period was considered sufficient for electrode stabilization. Several studies suggest that a minimum of 12 hours may be necessary to ensure signal stability.
Response 7: Thanks for your suggestion, we attach great importance to the effect of electrode stability on the accuracy of impedance measurement. The 30-minute waiting time used in our study is based on the experience and experimental results of existing literature. The referenced literature is as follows:
Zhou, J., Yuan, W., Di, B., Zhang, G., Zhu, J., Zhou, P., ... & Qian, J. (2022). Relationship among electrical signals, chlorophyll fluorescence, and root vitality of strawberry seedlings under drought stress. Agronomy, 12(6), 1428.
Comment 8: Figure 2: The representation of the greenhouse is visually inconsistent with the rest of the diagram. Consider finding an alternative way to illustrate it.
Response 8: Thanks for your suggestion, we have changed Figure 2. Please see line 275.
Comment 9: In general, I recommend revising the figure and table captions to provide additional information that will help readers interpret the data more accurately.
Response 9: Thank you for your helpful suggestion. We have revised the captions of all figures and tables to provide more detailed and informative descriptions, aiming to help readers interpret the data more accurately.
Comment 10: Line 449: Is LT50 analysis alone sufficient to affirm that one variety is more cold-tolerant than another? Wouldn’t it be necessary to complement it with other analyses, such as gene expression, proline accumulation, and enzymatic and non-enzymatic antioxidant system evaluation? It might be more prudent to state that SH40 appears to exhibit greater cold tolerance, rather than make a definitive conclusion.
Response 10: Thank you for your comments. We agree that LT50 alone is not enough to fully determine the difference in cold resistance between varieties. Therefore, we have adjusted the relevant statements in the revised manuscript. At the same time, we also pointed out in the discussion section that in the future, we can further combine antioxidant enzyme activity, osmotic regulation substances and molecular level indicators for comprehensive analysis to more comprehensively evaluate the cold resistance of varieties. Please see lines 496-499.
Comment 11: The conclusion is overly long. I recommend making it more concise by focusing on the main findings without repeating information already discussed in the results and discussion sections.
Response 11: Thank you for your suggestion. We have shortened the conclusion according to your comments, deleted the content repeated in the results and discussion, and made it more concise and clear.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThe authors perform statistical analysis and develop and validate a model to understand the cold tolerance in Apple.
Here are my suggestions to improve the manuscript:
Introduction
L57. Please remove the bold letters in the line
L67. Please remove the white dot
L126. After the introduction of all studies, authors state that “aimed to develop a rapid and non-destructive method”. Based on this, do the authors mean “model” instead of “method”?? This, because according to the abstract and the following lines, several statistical models were applied. Please review this.
Materials and methods
L158. Please state what are the blocks??.. The genotypes? Please specify.
L232, L235. Use “h” instead of “hours”. Please review this in the whole manuscript
L235. Use “min” instead of “minutes”
L239. With “Anaconda Navigator (Anaconda3)”… do you mean python? If phyton was used, please state phyton and using the interface as anaconda… On the other hand, please include the packages used.
L277. Please include any package or at least some commands when using Phyton 3.13
L300. Please state “principal component analysis (PCA)”Furthermore, please include the p and r value for the correlation analysis and the test for the correlation analysis (pearson??)
In the PCA, the data was normalized? Scaled? Standarized? Please include this information
L304. Please state the software or language using for the Stepwise regression… phyton? SPSS? Excel??
L300-305. Due to several softwares and languages were used. Please improve the meaning of the whole paragraph to understand in what software was performed each statistical analysis.
Does the author perform a statistical analysis?
At the beginning of the section, authors mention about a block design. However, there is not evidence about a statistical analysis. Please include one at the end of the section (ANOVA, two-way ANOVA)… tukey test??? LSD test?? Please include this information.
RESULTS
L344. There is a p<0.05. However, no information about the statistical analysis or mean test was included
L351. Please include in the legend, the meaning of the lines in the figure.. error standard?? standard deviation?
Moreover, please include letters in the figure to identify significant differences, and state this information in the legend of the figure.
L367. What is the meaning of the asterisk in the correlation figure???
L400. Include the letters if an statistical analysis was performed
L413. Authors should briefly explain why only six predictors were used. Please associated with with the correlation and PCA results in order to explain this
L420. I suggest to substitute the x1, x2, x3… in the equation by the original name predictors
Discussion
L474. The authors state “antioxidant system (SOD, POD)” However, POD, also is included in the oxidant system. Please verify this information.
L492-495. Please include more information about the PCA. I think is relevant information.
Do the authors know which variable is the most important? I suggest include this information in the discussion to improve it
conclusion
L517-518. This was in the method section, not in the conclusion section (normalization and standardized)…
Author Response
Comment 1: L57. Please remove the bold letters in the line.
Response 1: Thanks for your nice suggestions. We have made modifications in the corresponding parts.
Comment 2: L67. Please remove the white dot.
Response 2: Thanks for your nice suggestions. We have made modifications in the corresponding parts.
Comment 3: L126. After the introduction of all studies, authors state that “aimed to develop a rapid and non-destructive method”. Based on this, do the authors mean “model” instead of “method”?? This, because according to the abstract and the following lines, several statistical models were applied. Please review this.
Response 3: Thanks for your nice suggestions. We have made revisions by replacing “method” with “model” to make the context consistent. Please See line 127-128.
Comment 4: L158. Please state what are the blocks??.. The genotypes? Please specify.
Response 4: Thank you for your valuable comment. We have carefully reviewed the manuscript and corrected all mentions of “genotype” to “variety” to ensure consistency and accuracy.
In the context of a randomized complete block design (RCBD), "blocks" refer to groups of experimental units (plots) that are relatively homogeneous. Each block contains all the treatments, and within each block, the treatments are randomly assigned to the individual plots. The purpose of blocking is to account for potential variability among experimental units due to factors such as location, light, temperature, or soil conditions. This design helps reduce experimental error by controlling for variability among blocks.
Comment 5: L232, L235. Use “h” instead of “hours”. Please review this in the whole manuscript.
Response 5: Thanks for your nice suggestions. We have made modifications in the corresponding parts.
Comment 6: L235. Use “min” instead of “minutes”.
Response 6: Thanks for your nice suggestions. We have made modifications in the corresponding parts.
Comment 7: L239. With “Anaconda Navigator (Anaconda3)”… do you mean python? If phyton was used, please state phyton and using the interface as anaconda… On the other hand, please include the packages used.
Response 7: Thanks for your nice suggestions. We have modified this part, please see line 241-243.
Comment 8: L277. Please include any package or at least some commands when using Phyton 3.13.
Response 8: Thank you for your valuable comments. We have revised the manuscript to provide more detailed information regarding the Python packages used, please see line 281-283.
Comment 9: L300. Please state “principal component analysis (PCA)”Furthermore, please include the p and r value for the correlation analysis and the test for the correlation analysis (pearson??).
Response 9: Thank you for your valuable comments. Additional details on the p and r value for the correlation analysis and the test for the correlation analysis have been included in the revised manuscript, please see line 303-309.
Comment 10: In the PCA, the data was normalized? Scaled? Standarized? Please include this information.
Response 10: We performed normalization and standardization of the data for the PCA, and the corresponding calculation formulas have been provided. Please refer to line 336-344 for details.
Comment 11: L304. Please state the software or language using for the Stepwise regression… phyton? SPSS? Excel??
Response 11: Thank you for your valuable comments. We have added this part to the manuscript, please see line 321-324.
Comment 12: L300-305. Due to several softwares and languages were used. Please improve the meaning of the whole paragraph to understand in what software was performed each statistical analysis.
Response 12: Thank you for your valuable comments. We have revised this section and reorganized the content about statistical analysis, please see line 303-324.
Comment 13: Does the author perform a statistical analysis?
Response 13: We conducted a statistical analysis and reorganized this part, please see line 303-324.
Comment 14: At the beginning of the section, authors mention about a block design. However, there is not evidence about a statistical analysis. Please include one at the end of the section (ANOVA, two-way ANOVA)… tukey test??? LSD test?? Please include this information..
Response 14: Thank you for your valuable comments. We have added this part, please see line 316-320.
Comment 15: L344. There is a p<0.05. However, no information about the statistical analysis or mean test was included.
Response 15: Thank you for your valuable comments. We have added information about significant differences in the statistical analysis section. For details, please see 303-309.
Comment 16: L351. Please include in the legend, the meaning of the lines in the figure.. error standard?? standard deviation?
Response 16: Thanks for your nice suggestions. We have indicated the meaning of the lines in the legend, please see line 370.
Comment 17: Moreover, please include letters in the figure to identify significant differences, and state this information in the legend of the figure.
Response 17: Thanks for your nice suggestions. We have edited the figure, please see line 368.
Comment 18: L367. What is the meaning of the asterisk in the correlation figure???
Response 18: Thanks for your nice suggestions. We have indicated the meaning of the asterisks in the correlation figures, please see line 387.
Comment 19: L400. Include the letters if an statistical analysis was performed.
Response 19: Thanks for your nice suggestions. We have edited the figure, please see line 418 and 427.
Comment 20: L413. Authors should briefly explain why only six predictors were used. Please associated with with the correlation and PCA results in order to explain this.
Response 20: We first conducted a correlation analysis and identified nine characteristic parameters that were strongly correlated with LT50. These nine parameters were then subjected to principal component analysis (PCA), from which six key parameters with the highest contributions to cold resistance were selected. Finally, these six parameters were used to construct a correlation model with LT50. We have explained it in the article. Please see 378-383 and 389-400.
Comment 21: L420. I suggest to substitute the x1, x2, x3… in the equation by the original name predictors.
Response 21: Thanks for your nice suggestions. We have made changes in the manuscript, please see 432, 439, 440 and 442.
Comment 22: L474. The authors state “antioxidant system (SOD, POD)” However, POD, also is included in the oxidant system. Please verify this information.
Response 22: Thank you for your insightful comment. We agree that peroxidase (POD) can function in both oxidative and antioxidant processes depending on the biological context. However, in the case of cold stress in plants, POD primarily contributes to the antioxidant defense system by scavenging hydrogen peroxide (Hâ‚‚Oâ‚‚), thereby mitigating oxidative damage. Therefore, in this study, POD was referred to as part of the antioxidant system. To avoid ambiguity, we have revised the wording in the manuscript accordingly. Please see 496-499.
Comment 23: L492-495. Please include more information about the PCA. I think is relevant information.
Response 23: Thank you for your helpful suggestion. We agree that a more detailed explanation of the PCA results is valuable. In the revised manuscript, we have added additional information regarding the eigenvalues, the contribution rates of PC1 and PC2, and the loading values of each variable. Please see 511-531.
Comment 24: Do the authors know which variable is the most important? I suggest include this information in the discussion to improve it.
Response 24: Thank you for your helpful suggestion. We have added this information to the Discussion, please see 511-531.
Comment 25: L517-518. This was in the method section, not in the conclusion section (normalization and standardized).
Response 25: Thank you for your suggestion. We agree with your comment and have revised the content accordingly. The relevant technical details, including data normalization and standardization, have now been clearly described in the Materials and Methods section, while the conclusion has been adjusted to avoid redundancy. We appreciate your valuable input, which helped us improve the clarity of our manuscript. Please see 551-554.
Author Response File: Author Response.pdf
Reviewer 4 Report
Comments and Suggestions for AuthorsThe theory behind it seems to be well established in previous studies of other research groups.
Assuming there were no technical problems this study seems to be well organized, and the data thoroughly analyzed.
The text is well written but I have a couple of comments:
Lines 47-50 This sentence talks about cold tolerance during seed germination. Its connection to the topic of this study is vague.
Lines 50-52, 67-68, 436, 446-449 The texts in these places implies that the plants actively “took measures” to increase its cold hardiness. In my opinion, such a teleologiocal style should be avoided.
-other notes:
Which method can be used in order to estimate the cold resistance of various apple varieties?
Topic is It is original and relevant.
As far as I know, up to date there is no accepted method for estimating the cold resistance of apple trees on the basis of a small sample of a branch.
As far as I know, this ms presents a novel way of answering the main question.
As I wrote in my review, I do not have any experience in plant-electropysiology and was not able to assess the accuracy of the methods used in this study.
The method described leads to a parameter (LT50) which is shown to be in direct relationship to the cold resistance if the trees
The figures and references are all relevant and properly presented.
Author Response
Comment 1: Lines 47-50 This sentence talks about cold tolerance during seed germination. Its connection to the topic of this study is vague.
Response 1: Thank you for your valuable suggestion. We agree with your comment and have revised the sentence to establish a clearer connection with the focus of this study. Please see lines 47–50.
Comment 2: Lines 50-52, 67-68, 436, 446-449 The texts in these places implies that the plants actively “took measures” to increase its cold hardiness. In my opinion, such a teleologiocal style should be avoided.
Response 2: Thanks for your nice suggestions. We have made modifications in the corresponding parts. Please see lines 50-52, 68-70, 455-458, 467-473.
Author Response File: Author Response.pdf
Round 2
Reviewer 3 Report
Comments and Suggestions for AuthorsThe authors perform the changes requested. Article can be accepted.
