Canopy-Applied Benzothiadiazole (BTH) during the Pre-Harvest Period as a Tool to Increase Polyphenol Accumulation and Color Intensity in Cabernet Gernischt and Cabernet Franc

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: The study is carried out in the 2022 campaign. In the field, it is difficult to draw conclusions with a single year of results, since many factors can influence, especially meteorological conditions. Do you have data from other campaigns in these two varieties?

Response 1: Firstly, thank you very much for your evaluation of this manuscript. It is indeed difficult to draw any conclusions based on one year's results. The sentiment you expressed resonates strongly with us. In fact, for this whole study, we conducted research over two years (2022 and 2023) due to the consistent results and trends observed in the data from both years, which yielded significant findings. Consequently, we have utilized only one year's worth of data for this paper while intending to incorporate additional content encompassing key enzyme assays and transcriptomic analyses from the 2023 study into another research manuscript. We sincerely apologize for any inconvenience caused by this limitation on data usage. This paper focuses solely on analyzing and discussing the meaningful conclusions drawn from the data collected in 2022. The analysis and discussion of the data obtained in 2023 will be presented in separate manuscripts. We sincerely apologize and kindly request your understanding.

Comments 2: Line 111: The title of the article refers to the foliar application of BTH; however, here, it says that it was applied to the surface of the grapes. How was the application actually made?

Response 2: We apologize for the incorrect description of this section. Thank you for pointing this out, and we have made corresponding revises to the manuscript. The application of BTH is throughout the whole canopy of grape plants; this change in the revised manuscript can be found on lines 103-113.

Comments 3: The authors must specify the amount of BTH solution applied per plant.

Response 3: The original manuscript has been modified and supplemented in accordance with your request. The revised version now specifies that the amount of BTH solution per plant is 15l, and this change can be found on line 112.

Comments 4: The authors say that the sampling was carried out every 14 days but then the graphs do not reflect that. Can you explain it?

Response 4: Thank you for pointing this out. The following content is our explanation: The sampling period for this study was initially designed to be once every 14 days, with a total of 7 samples taken. However, due to the impact of the COVID-19 epidemic, the sampling time for one sample point was adjusted (from 56 DAF to 70 DAF), and the adjusted time did not affect the overall arrangement of the experiment.

Comments 5: Line 134-136: Better explain the methodology used for the determination of phenolic compounds by spectrophotometry.

Response 5: The corresponding supplements have been incorporated into the original draft based on your suggestions. This change can be observed in the revised manuscript (lines 148-196).

Comments 6: Statistical analysis: Heat map not included

Response 6: Thank you for pointing this out. After checking the manuscript, we have annotated this part of the content. This change in the revised manuscript can be found on lines 249-251.

Comments 7: Line 245: "37 Yet" What's that?

Response 7: We have made corresponding revisions in the manuscript. This change in the revised manuscript can be found on line 285.

Comments 8: Figure 1: It does not look good and the statistics used are not well understood. Please, the authors should make the statistical results clearer in the graph.

Response 8: We have made corresponding revisions in the manuscript. This change in the revised manuscript can be found in Figure 1.

Comments 9: Figure 2: Why are there no statistics in these figures?

Response 9: We have made corresponding revisions in the manuscript. This change in the revised manuscript can be found in Figure 2.

Comments 10: Line 273. "22,24,38" What's that?

Response 10: Thank you for pointing this out. The "22, 24, 38" are reference citations and have been corrected accordingly. We have made corresponding revises in the manuscript. This change in the revised manuscript can be found on line 320.

Comments 11: Figure 3: The representation of the graphs corresponding to colour does not clearly show the differences between the different sampling times. I think another format should be chosen to see it more clearly.

Response 11: We have made corresponding revisions in the manuscript. This change in the revised manuscript can be found in Figure 3.

Comments 12: Figure 4: The numbers and letters are barely readable.

Response 12: We have made corresponding revisions in the manuscript. This change in the revised manuscript can be found in Figure 4.

Comments 13: Line 406: Which isomer of resveratrol was identified, cis- or trans-? The authors must identify it.

Response 13: Thank you for pointing this out. We have made corresponding revises in the manuscript. The identified compound in this study is trans-resveratrol, which the revised manuscript this change can be found on line 475.

Comments 14: Line 412: "54" Correct it.

Response 14: We have made corresponding revisions in the manuscript. This change in the revised manuscript can be found on line 485.

Comments 15: The authors refer many times in their discussion to the fact that certain enzymes are activated to justify the increase in different phenolic compounds when applying BTH. Wouldn't it have been important to have also analysed these enzymes to be able to explain the results more reliably?

Response 15: Firstly, we appreciate your suggestion and would like to express our gratitude. In order to ensure a more robust interpretation of the findings, it is indeed crucial to analyze these enzymes. However, as previously mentioned, this study was conducted over two years, specifically in 2022 and 2023. The primary investigation focused on examining the impact of BTH treatment on polyphenol changes in 2022. In 2023, while replicating the previous year's results, enzymatic analysis and transcriptome analysis were employed to investigate the underlying reasons. The primary focus of the relevant research content lies in the implementation of 2023 samples. In order to address your concerns promptly, we will expedite the completion of an additional manuscript that offers a comprehensive analysis of current findings. We sincerely apologize for any inconvenience caused and kindly request your understanding and support.

Comments 16: The figures corresponding to the multivariate analyses are very confusing. The Pearson correlation makes it very difficult to elucidate the degree of correlation between the different parameters. On the other hand, in the discriminant analysis part, figure A is understood but the rest of the figures need a more exhaustive explanation to be understood. Finally, if a discriminant analysis has already been done, the heat map is already unnecessary. I believe that it is necessary to choose which multivariate analysis should be used in order to explain the results obtained in the most precise way.

Response 16: (1). Thank you very much for your suggestion. We have provided a more comprehensive description of the results of Pearson correlation analysis (Line 507-534) and OPLS-DA (Line 543-566) in the manuscript.

(2). OPLS-DA analysis can be used to screen differential metabolites between the control group and the treatment group. Through OPLS-DA analysis, a VIP value can be obtained for each metabolite, and the larger the VIP value, the greater the contribution of the substance to distinguishing between the two groups.

(3). The row clustering results of the clustering heatmap reveals that samples with similar feature patterns are clustered together, thereby identifying the similarity between samples. By observing the color distribution of the columns, we can see which features play an important role in different clusters and which features are correlated with each other.

These two multivariate statistical methods have different effects, so this study uses both methods to analyze the data and more comprehensively explore the correlations of the data.

Comments 17: In the conclusions, especially those that refer to multivariate analyses, they should not be so general and should be delved deeper.

Response 17: Thank you for pointing this out. We have provided a more in-depth description in the manuscript. This change in the revised manuscript can be found on lines 610-639.

Comments 1: better to add more about M&M about the concentration and time of application etc.

Response 1: We apologize for not noticing this, and thank you for pointing this out. We agree with this comment. Therefore, we have made corresponding revises in the manuscript. The application concentration and time have been clearly stated in the manuscript. This change in the revised manuscript can be found on lines 13-14.

Comments 2: Line 22-23— from which point?

Response 2: At 70, 84, 98, and 117 DAF, the content of peonidin-3-O-glucoside was higher than that of peonidin-3-O-glucoside. Detailed data comparison can be found in Supplementary Material Table S4.

Comments 3: Line 25-26—before the current conclusion you should add a conclusion based sentence regarding current findings and then for wine....better to give the final conclusion about the wine through better sentences.

Response 3: Thank you for pointing this out. We have, accordingly, modified this part to emphasize this point. This change in the revised manuscript can be found on lines 24-29.

Comments 4: BTH — give it in full form.

Response 4: We have made corresponding revisions in the manuscript. This change in the revised manuscript can be found on line 30.

Comments 5: Line 109: aqueous solutions—only distilled water for BTH aqueous solution was used? any need of temperature or acid for first dissolving?

Response 5: Thank you for pointing this out. BTH aqueous solution was conducted at 50 mg/L BTH with Tween 80 as a surfactant, and to ensure complete dissolution, a small amount of hot water (approximately 100℃) was added during the configuration. This change in the revised manuscript can be found on line 109-111.

Comments 6: Line 113-116: the application day? did you believe in the time exactly after application the changes would be achieved?14 days is too late? why did not you had the sampling at least every week? in total, how many samplings were done? how many clusters did you had for each replication that could cover these volume of samplings in each time? in each sampling, all cluster were picked?

Response 6: Thank you very much for raising these questions. In response to your concerns, we have the following response:

(1). The first application of BTH and the first sampling were both conducted at 21 DAF (June 23, 2022). In addition, according to the results of this study, the application of BTH does have a positive impact on various indicators of grapes.

(2). The determination of sampling time is based on the experience of existing laboratory results and adjusted accordingly.

(3). This study conducted a total of 7 samplings, starting from 21 DAF until grape ripening.

(4). There are approximately 100 clusters that can cover the sampling volume of each replication, and in each sampling, almost most of the groups are selected, and this change in the revised manuscript can be found on lines 114-117.

Comments 7: Line 131-136: better to give at least brief explanation regarding each method. not only giving the refs!

Response 7: We appreciate you pointing out this issue, and we have provided a more detailed description of this part. This change in the revised manuscript can be found on lines 132-139 and lines 148-196.

Comments 8: Line 143-144: why choosing the method instead other standard method for color?

Response 8: CIELab is the standard method for determining color, and in this study, we used this method to evaluate color changes. In addition, we also supplemented it with the CIRG method, which has been used in other studies. So, the results obtained by combining these two methods will be more comprehensive and reliable.

Comments 9: Line 146-154: it means that only skin anthocyanin was assayed? why? why anthocyanin of peels were removed? or why did not you assay them separately?

Response 9: We appreciate your question and would like to provide the following explanation: The synthesis and accumulation of anthocyanins are mainly in grape skins, so only anthocyanins in grape skins were measured here. This result is also a supplement to the determination of phenolic substances mentioned earlier. Similar determination methods have also been mentioned in other studies, so we chose to use this determination method to analyze and determine anthocyanins in grape skins.

Comments 10: Line 156-158: grammar point.

Response 10: Thank you very much for raising the grammar issue in this section. We have made the necessary revisions to the manuscript. This change in the revised manuscript can be found on lines 208-212.

Comments 11: Figure 1-6: check the quality of figs and improve them.

Response 11: We are very sorry that the Figures in this study do not reflect the data more clearly. Thank you for pointing out this issue, and we have recently made modifications to address it. This change in the revised manuscript can be found in Figure 1-6.

Comments 12: Line 304-305: why did not you discuss the color parameters? Lack of discussion.

Response 12: We apologize for not discussing this part of the content. Thank you for pointing out this issue, and we have already supplemented it in the manuscript. This change in the revised manuscript can be found on lines 343-366.

Comments 13: Line 354-358: the reasons for the effect could be considered enough but many other studies revealed the effect of different similar compounds on the mentioned phenolic that could be added to discussion.

Response 13: We apologize for not discussing this part of the content. Thank you for pointing out this issue, and we have already supplemented it in the manuscript. This change in the revised manuscript can be found on lines 412-423.

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: In my opinion the title should be: Foliar application of benzothiadiazole (BTH) during the preharvest period as tool to increase polyphenol accumulation and color intensity in grapes of Cabernet Gernischt and Cabernet Franc cv.

Current paper title is not according to presented results, especially title part “A comprehensive study on grape maturation process”.

Response 1: Thank you for pointing this out. We agree with this comment. Therefore, revised based on your proposed Title and incorporating the suggestions of other reviewers, we have accordingly modified the title. This change in the revised manuscript can be found on lines 2-4.

Comments 2: Line 33-36:This is most important influence on human health. This segment is to poor with explanation and it is necessary to add more references.

Response 2: Thank you very much for pointing out the shortcomings of this section and your suggestions. Based on your suggestions, we have provided a more detailed description and supplements to the introduction section. This change in the revised manuscript can be found on lines 36-42.

Comments 3: Line 37-39:it is need to rewrite this reference with out “instrinsic” and “extrsinsic” variables. In this case, to understood better this sentence it it the best to explain concept of terroir and correlation between cultivar, soil, climatic factors and add vineyard management.

Response 3: Thank you very much for your suggestion. We have added additional explanations to this section in the manuscript based on your suggestion. This change in the revised manuscript can be found on line 43-50.

Comments 4: Line 43-46: this par it is necessary to rewrite with point of application or influence on some other plants.

Response 4: Thank you very much for pointing out the shortcomings in this section. Based on your suggestions, we have already supplemented this part of the content. This change in the revised manuscript can be found on lines 46-50.

Comments 5: Line 47-66: This part is to large with point on other fruit and chemical compounds witch is not part of research. Cut or make shorter this part with few references.

Response 5: Thank you for your suggestions. In response to these issues, we have made revises to this part of the content. This change in the revised manuscript can be found on lines 51-62.

Comments 6: Line 69: put which BBCH stages by Lorenz et al., 1994.

Response 6: We have made revisions to this part of the content. This change in the revised manuscript can be found on line 65.

Comments 7: Line 72 and 76: Please be more precise how promote accumulation on Merlt and Syraz grape? Same comment like previous.

Response 7: We have made corresponding supplementary explanations in the manuscript. This change in the revised manuscript can be found on lines 67-77.

Comments 8: Line 85-89: This part most be moved to Methods.

Response 8: Thank you very much for pointing out the shortcomings in this section. Based on your suggestions, we have made corresponding supplementary explanations in the manuscript. This change in the revised manuscript can be found on lines 85-92.

Comments 9: Line 96: Add date of harvest for both varieties.

Response 9: We have already supplemented it. This change in the revised manuscript can be found on lines 95-96.

Comments 10: Line 98-102: (1). add altitude of vineyard, training system, type of pruning on experimental plants, management of vineyard etc.(2) Climate and soil condition must be write completely different. 1. Climate condition. Add table or graph with multi year (10-15 year) range of average month temperature. Separately show research year and compare differences. Also, it is necessary to analyze average vegetation and annual temperature, specially temperature and precipitation during months when is applied BTH.Rainfall must be also present in same way.2. Soil condition. It is completely unclear.(3) On which rootstock are grafted experimental varieties?

Response 10: We are very sorry that we overlooked this point. Thank you for pointing out this issue. Climatic conditions for this area for many years (10-15 years) are not available to us, but the average annual temperature and precipitation are described in the manuscript. This change in the revised manuscript can be found on lines 95-102.

Comments 11: Line 107: Add specificity of this change.

Response 11: Thank you for pointing out this issue. We have already supplemented it. This change in the revised manuscript can be found on lines 103-104.

Comments 12: Line 110: Surfactant it is usually using for better application of main chemical, in this case BTH. It is not clear why is use surfactant in control? Control dos not include BTH and in this case is not necessary to apply surfactant.

Response 12: The use of Tween 80 in the control group was to ensure that the conditions in the control and treatment groups were the same so that the only variable in the treatment group was BTH, and a similar application has been demonstrated in the studies of others, as shown in Table 1, which summarizes the study of the use of Tween 80 in the control group.

Comments 13: Line 111: it is applied in zone of grape which is include leaves and bunches? Or it is removed leaves from bunches zone and after defoliation is applied BTH directly on berries?.

Response 13: The application of BTH is the entire canopy application, and the specific content has been supplemented and explained in the manuscript. This change in the revised manuscript can be found on line110-112.

Comments 14: Line 209-110: pH level decrease and increase during the maturation? Usually TA content during maturation decrease followed by pH values. On graph A TA during the berries maturation constantly decrease. Check this statement.

Response 14: Thank you for your suggestions on this section. In the results of this study, the pH level first decreased and then increased. The specific description has been highlighted in red font in the manuscript. This change in the revised manuscript can be found on lines 258-262.

Comments 15: Line 238-242: In this paper is not possible to conclude about influencing of year, precipitation, temperature..., because in paper it is presented data for one research year. For that reason it is necessary to repeat experiment in multi year period and make relevant conclusions.

Response 15: Thank you very much for pointing out the shortcomings of this section. The impact of year, precipitation, and temperature was speculated based on other papers attempting to explain the conclusions of this study. In fact, this indeed requires repeated experiments over many years, so we have rewritten this part of the content to express the results of this study more fully. This change in the revised manuscript can be found on lines 279-282. In addition, we will carefully consider your suggestions and implement them in future research.

Comments 16: Line 269-276: it is need to add more references.

Response 16: We have already supplemented this part of the content in the manuscript. This change in the revised manuscript can be found on lines 316-329.

Comments 17: Line 305: Add some references in this part. Is this results according to other authors or no and why?

Response 17: We have already supplemented this part of the content in the manuscript. This change in the revised manuscript can be found on lines 343-366.

Comments 18: Line 380-381: This part of sentence must be more clear. Rewrite.

Response 18: We have already supplemented this part of the content in the manuscript. This change in the revised manuscript can be found on lines 444-447.

Comments 19: Line 386-398: This part is a bit confusingly written. In order to be better understood, I suggest that it must be rewritten in follow way. Select the days in which the grapes were sampled, which were analyzed, and then emphasize by dates which of the nine mentioned flavonols change their concentration to a greater or lesser degree. Also explain importance of this changing.

Response 19: Thank you for pointing out this issue. We have made corresponding modifications to this part of the manuscript. This change in the revised manuscript can be found on lines 452-464.

Comments 20: Line 399-405: Please make connection between Garde-Cerdan and your research. Were is bigger increasing and influencing of BTH treatment? On Tempranilo or on your varieties? How is your explanation?

Response 20: Thank you for pointing out this issue. We have made corresponding modifications to this part of the manuscript. This change in the revised manuscript can be found on lines 465-474.

Comments 21: Line 406-409: Add and other date when is measured resveratrol content. Also, explain why is important resveratrol? Which role has resveratrol in grape metabolism?

Response 21: Thank you for pointing out this issue. We have made corresponding modifications to this part of the manuscript. This change in the revised manuscript can be found on line 475-481.

Comments 22: Line 413-415: Please add some results and compare with your results.

Response 22: We have made corresponding modifications to this part of the manuscript. This change in the revised manuscript can be found on line 483-491.

Comments 23: Line 422-may be: May be yes may be not... This is irrelevant scientific conclusion... Please, delete or rewrite with scientific point of view and without “may be” conclusions. For this reason it is necessary to repeat experiment and make good relevant conclusions.

Response 23: Thank you for pointing out the shortcomings of this section. We explain that this study lacks enzyme content, so the reasons for the relevant results can only be supported by other people’s research. In order to make this description objective, we used the word ‘maybe’. However, the content about enzymes here will be our next focus of work, which we will explore and analyze in our next work.

Comments 24: Conclusion: (1). Change conclusion according to previous comments on results and discussion part. (2). The concentration of compounds shown in the paper can be increased by applying specific ampelotechnical measures during the vegetation period (for example, defoliation). It is necessary to review the importance of the application of these measures and the application of BTH in the discussion, to make a comparison and draw relevant conclusions while giving preference to one of the mentioned measures. Add relevant references. In conclusion, give a brief overview, ie.

Response 24: Thank you for pointing out the shortcomings of this section. We have made corresponding modifications to this part of the manuscript. This change in the revised manuscript can be found on lines 610-639.

Comments 25: Supplementary Material (Table S1 and S2): It may be more practical to show chromatograms with the peaks of the detected compounds instead of a table.

Response 25: Thank you very much for your suggestion. It is indeed more practical to display chromatograms using the peaks of detected compounds instead of tables. However, our determination of anthocyanins and non-anthocyanins is for qualitative and quantitative analysis of compounds. Therefore, the content presented in the table can facilitate the qualitative and quantitative analysis of compounds. Therefore, we will still present these data in the form of tables.

Comments 26: Supplementary Material (Table S3): Why is there a different time interval between the individual measurements? If each sampling is repeated at the same intervals (e.g. after 5,10, 15 or 20 days between terms), the conclusions are more relevant and the dynamics of the accumulation of each of the detected compounds can be more clearly established.

Response 26: Thank you for your question and suggestion. In response to your inquiry, we would like to provide the following explanation. The sampling period for this study was originally designed to be every 14 days. However, due to the impact of the COVID-19 epidemic, the sampling time for one sample point was adjusted (from 56 DAF to 70 DAF), and the adjusted time did not affect the overall arrangement of the experiment. In the revised manuscript, this change can be found on lines 114-117.

Comments 27: Supplementary Material (Table S4): How is it possible that the average values from three measurements for the same compounds are exactly the same, for both varieties. The numbers are exactly the same in decimal. It is impossible that there is no difference in values between varieties...Check it and make correction in paper. Check and previouse table S3.

Response 27: We are very sorry for the data issue caused by our operational error. Thank you very much for pointing out our problem. We have corrected the contents of tables S3 and S4. Once again, we deeply apologize for the inconvenience caused by our operational error. The revised content of this change can be found in the Supplementary Materials (Table S3 and S4).

4. Response to Comments on the Quality of English Language

Point 1: English must be adapted to the professional terminology of the text, are difficult to understand or so difficult to understand that they lose their meaning. Certain parts are not presented in a sufficiently understandable way, despite the author’s good intentions. Look at the commentaries.

Response 1: Thank you for your feedback on this manuscript. We will carefully check the correct use of language so that readers can better understand the content of the manuscript.