Impact on Grape Juice Quality and Phenolic Composition of Greek Autochthonous Grapevine Variety Mouhtaro Under Abscisic Acid Biostimulation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Editor and Authors,

Regarding the manuscript ID: sustainability-3590040, entitled: “Biostimulation of Vitis vinifera L. Mouhtaro grapevine via abscisic acid over two vintages experiment: Effect on grape juice and on grape phenolic compounds”, my comments are the following.

Regarding the introduction, although it provides useful information about the research topic, I believe that information concerning some characteristics regarding the grape variety (shape and size of the leaf and grape, etc.)  is missing and might be useful for the reader.

The experimental part is properly organized, as well as the results and the discussion section. Concerning the data of the compounds analyzed by UPLC-MS, even though the authors give the results through figures, I feel that the exact values of those compounds must be given also in a single table, maybe in the supplementary files, as I believe this is a valuable information of this research.

Finally, English writing is very good although in some points punctuation needs attention and should be corrected.

Based in the above I suggest the acceptance of this study after minor revisions.

Comments on the Quality of English Language

Dear Editor and Authors,

Regarding the manuscript ID: sustainability-3590040, entitled: “Biostimulation of Vitis vinifera L. Mouhtaro grapevine via abscisic acid over two vintages experiment: Effect on grape juice and on grape phenolic compounds”, my comments are the following.

Regarding the introduction, although it provides useful information about the research topic, I believe that information concerning some characteristics regarding the grape variety (shape and size of the leaf and grape, etc.)  is missing and might be useful for the reader.

The experimental part is properly organized, as well as the results and the discussion section. Concerning the data of the compounds analyzed by UPLC-MS, even though the authors give the results through figures, I feel that the exact values of those compounds must be given also in a single table, maybe in the supplementary files, as I believe this is a valuable information of this research.

Finally, English writing is very good although in some points punctuation needs attention and should be corrected.

Based in the above I suggest the acceptance of this study after minor revisions.

Author Response

Dear esteemed reviewer,

we wish to extend our sincerest gratitude for the invaluable insights and constructive critiques that you have graciously provided regarding our manuscript, as your thoughtful observations have significantly contributed to the enhancement of our research paper. The feedback you offered has undoubtedly strengthened our work, thereby allowing us to better align it with the overarching objectives we set out to achieve in our study. In the following section, you will find a comprehensive outline of the revisions we have implemented in response to your thoughtful comments.

On behalf of the authors

Dimitrios Miliordos

1^st reviewer

Dear Editor and Authors,

Regarding the manuscript ID: sustainability-3590040, entitled: “Biostimulation of Vitis vinifera L. Mouhtaro grapevine via abscisic acid over two vintages experiment: Effect on grape juice and on grape phenolic compounds”, my comments are the following.

Regarding the introduction, although it provides useful information about the research topic, I believe that information concerning some characteristics regarding the grape variety (shape and size of the leaf and grape, etc.)  is missing and might be useful for the reader.

We would like to thank reviewer for mentioning. Due to the fact that it’s a minor autochthonous variety no published data are available. Therefore, some details about the grapevine were added at the Introduction section.

Only the Muses Valley in Askri, Central Greece, is home to the grapevine variety "Mouhtaro," which yields few concentrated grapes. The berry is small to medium in size and is found in a medium-sized bunch. Additionally, grapevines are rigorous and distinguished by their late ripening and late bud break. There is currently no available scientific data about DNA genotyping.

The experimental part is properly organized, as well as the results and the discussion section. Concerning the data of the compounds analyzed by UPLC-MS, even though the authors give the results through figures, I feel that the exact values of those compounds must be given also in a single table, maybe in the supplementary files, as I believe this is a valuable information of this research.

We appreciate the reviewer bringing this up. Because there are too many phenolic compound data points (two years of experimentation, three treatments, and three time point sampling), we present all the information in graph bars and PCA figures so that the reader can easily follow the differences between the treatments over the ripening period. We firmly think that if we present all of the data in tables, we should also include a description of each table. As a result, the research's goal can be compromised. Lastly, as we said in the manuscript's conclusion, all data are accessible upon request.

Data Availability Statement: The data presented in this study are available on request from the corresponding authors (pending privacy and ethical considerations).

Finally, English writing is very good although in some points punctuation needs attention and should be corrected.

Based in the above I suggest the acceptance of this study after minor revisions.

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

Manuscript sustainability-3590040 studies the treatment of grapevine with abscisic acid, and the effect on grapes. Interesting study but in some points the results are not well justified.

The text needs to be checked regarding the typing as, there are mistakes in full stops, commas, etc. (wrong place, or absence...). Please check and correct throughout the manuscript.

 

The problem is that there is no line numbering, so it will be difficult to give correct points for revision or clarification. I will try to give more details.

Furthermore, the authors should be aligned to journal instructions.

 

Abstract, the last 2 lines: Something is wrong (there are some words in Greek) and the meaning is confused.

Keywords: Usually, the separation of keywords is given by “;”

2.2. sampling and conventional must analysis, lines 4-9: Please make the sentence more understandable. i.e. in lines 4-5 the authors mention the sampling and then in lines 7-8 they repeat the sampling with more information. Also, in line 6 they write “…samples were kept at -80°C and transferred on dry ice.” Please make it clear.

2.3. UPLC–MS-Based Metabolic Profiling: Did the authors make quantification for all the identified compounds? For all the amino acids, flavones etc.? Please be specific.

2.5. Meteorological Data: I think that it needs italics like the above subsections.

Pg. 5, paragraph 2, line 2: It should be read “ ..TSS, expressed as °Brix…”

Regarding the statistical analysis in Table 1 the authors mentioned “different letters in the same column indicate significant differences”. Did the authors mean and in different veraison? I think that this is wrong as during maturation there should be differences. In the same stage of maturation during different treatments I could see or not the significance. But in different stages of maturation the values are quite different. Please explain or make appropriate corrections in Table.

Respectively, in Figures the authors compared in the same way (I mean for one year in different treatments for one stage of maturation). But what about different stages of maturation?

I will propose the authors make further statistical analysis, and the new results may be more interesting.

 

Based on the above I recommend major revision and reconsideration of the manuscript.

 

Author Response

Dear esteemed reviewer,

we wish to extend our sincerest gratitude for the invaluable insights and constructive critiques that you have graciously provided regarding our manuscript, as your thoughtful observations have significantly contributed to the enhancement of our research paper. The feedback you offered has undoubtedly strengthened our work, thereby allowing us to better align it with the overarching objectives we set out to achieve in our study. In the following section, you will find a comprehensive outline of the revisions we have implemented in response to your thoughtful comments.

On behalf of the authors

Dimitrios Miliordos

2^nd reviewer

Manuscript sustainability-3590040 studies the treatment of grapevine with abscisic acid, and the effect on grapes. Interesting study but in some points the results are not well justified.

We would like to thank reviewer  for mentioning. Therefore we have worked in finding more references in order to justify and make stronger our results.

For instance in the manuscript we have added in the Discussion section several sentences in order to give more data. More especially, at the third paragraph of the Discussion we have added the bellow sentences with new references were added

The application of ABA on Savvatiano grapes in previous trial showed that another cru-cial parameter is the cultivar. Studies have shown that ABA treatment can enhance the content of anthocyanins, flavonols, and stilbenes in various grape varieties. For instance, in Malbec grapes, ABA increased low molecular weight polyphenols and stilbenes like trans-resveratrol and piceid [22]. Similarly, in Yan 73 and Cabernet Sauvignon, ABA treatment enhanced anthocyanins and non-anthocyanin phenolics, with varying effects depending on the variety (Luan et al., 2014) (Xi et al., 2013)[46, 47].For instance, contrary to ABA-treated grapes of Mouhtaro which exhibited increased levels of flavan-3-ols and flavonols in 2019 vintage compared to the controls, their concentrations were decreased or unaffected in Savvatiano. On the other hand, E-piceid was increased in the white variety upon ABA application whereas no effect was observed in Mouhtaro [28]. ABA treatment has been consistently shown to increase anthocyanin content in grape berries. For instance, studies on the Savvatiano and Kyoho white grape varieties demonstrated that ABA upregulates genes involved in anthocyanin biosynthesis, such as UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT) and glutathione S-transferase 4 (GST4), leading to higher anthocyanin accumulation [28, 48, 49 ]. Specifically, ABA treatment was found to enhance the production of petunidin- and malvidin-type antho-cyanins, which are associated with deeper berry [49]

46           Luan, L. Y., Zhang, Z. W., Xi, Z. M., Huo, S. S., & Ma, L. N. Comparing the effects of exogenous abscisic acid on the phenolic composition of Yan 73 and Cabernet Sauvignon (Vitis vinifera L.) wines. European Food Research and Technology, 2014, 239, 203-213.

47. Xi, Z. M., Meng, J. F., Huo, S. S., Luan, L. Y., Ma, L. N., & Zhang, Z. W. Exogenously applied abscisic acid to Yan73 (V. vinifera) grapes enhances phenolic content and antioxidant capacity of its wine. International Journal of Food Sciences and Nutrition, 2013, 64(4), 444-451.
48. Guo, S.; Zhang, M.; Feng, M.; Liu, G.; Torregrosa, L.; Tao, X.; Xu, T. miR156b-targeted VvSBP8/13 functions downstream of the abscisic acid signal to regulate anthocyanins biosynthesis in grapevine fruit under drought. Horticulture research, 2024, 11(2), uhad293. https://doi.org/10.1093/hr/uhad293
49. Katayama-Ikegami, A.; Sakamoto, T.; Shibuya, K.; Katayama, T.; Gao-Takai, M. Effects of abscisic acid treatment on berry coloration and expression of flavonoid biosynthesis genes in grape. American Journal of Plant Sciences, 2016. 7(09), 1325.

Moreover the last paragraph of the Discussion was rewritten

The combination of a variety's genetics and environmental conditions, including soil, terrain, and climate, determines its distinctive grape and wine quality traits. Given that grapevines have historically been grown in the Mediterranean region, the anticipated global warming scenario puts existing cultivation techniques to the test. As a result, sustainable agronomic practices may be helpful in reducing the adverse consequences of climate change while maintaining the high standards of the historically grown cultivars. Utilizing plant hormones and bioelicitors in viticulture provides a sustainable way to reduce the need for pesticides and fertilizers while improving the qualitative character-istics of grape berries in an eco-friendly way.

Finally, the Conclusion  paragraph was revised and rewritten:

Applications of biostimulants are advantageous and successful viticultural tech-niques for improving grape and wine quality, particularly in view of the difficult climate change scenario. But it's crucial to ascertain the best time and focus for their use. While ABA treatment reduced size and TSS in our two-vintage study, it also enhanced levels of phenolic chemicals, including flavonols, phenolic acids, and flavan-3-ols. As a result, using ABA may be regarded as a viable agronomic approach to improve the quality of grape berries. Further comprehensive studies, incorporating a wider range of indigenous grapevine varieties and local climate conditions, are needed clarifying grapevine reactions to various biostimulants and their unique impacts on each cultivar's metabolic profiles requires taking into account a greater variety of indigenous grapevine varieties and local climate circumstances.

 

The text needs to be checked regarding the typing as, there are mistakes in full stops, commas, etc. (wrong place, or absence...). Please check and correct throughout the manuscript.

We would like to thank reviewer  for mentioning. Therefore, we have checked and revised all the typing mistakes.

The problem is that there is no line numbering, so it will be difficult to give correct points for revision or clarification. I will try to give more details.

Indeed, it is quite apparent that the absence of line numbers significantly complicates the process of articulating precise and constructive feedback on the manuscript in question, thereby underscoring the necessity for such a system in facilitating clearer communication. Consequently, we would like to extend our sincere gratitude to the reviewer for the invaluable insights and critiques they have provided, which are instrumental in enhancing the quality and rigor of our work. Nevertheless, it is important to note that the particular journal to which we are submitting our manuscript does not require authors to include line numbering within the submitted documents, which is a standard practice that diverges from the expectations of many other academic publications.

Furthermore, the authors should be aligned to journal instructions.

We would like to thank reviewer  for mentioning. Therefore, many revisions were done  according to the journal’s instructions. For example,references were revised and typing mistakes.

Abstract, the last 2 lines: Something is wrong (there are some words in Greek) and the meaning is confused.

We would like to thank reviewer  for mentioning. Therefore the revision was done by erasing the forgotten greek words

 Keywords: Usually, the separation of keywords is given by “;”

We would like to thank reviewer for mentioning. Therefore the revision was done by replacing the punctuation.

2.2. sampling and conventional must analysis, lines 4-9: Please make the sentence more understandable. i.e. in lines 4-5 the authors mention the sampling and then in lines 7-8 they repeat the sampling with more information. Also, in line 6 they write “…samples were kept at -80°C and transferred on dry ice.” Please make it clear.

We would like to thank reviewer for mentioning. Therefore the revision was done. 

In order to be clear with all the information and give all the details about the experiment the following changes were done

2.2 Sampling and conventional must analysis was replaced by

2.2 Grape Sampling and Conventional Physicochemical Must Analysis

Moreover, all the paragraph was rewritten

The sampling dates were performed as shown in Table S1 (three distinct maturity levels: at véraison, immediately prior to the 1^rst treatment application, at middle véraison, and at the optimum grape maturity stage) and grapes were harvested on 30^th of August 2018 and on 3^rd of September 2019 respectively. A weekly sample approach was used to track the ripening process and determine the optimum grape level for harvest. Fifty berries were randomly selected from each experimental treatment at each sample point, and the fresh weight of the berries was calculated. Prior to their chemical analysis,grape samples were transported on dry ice and were carefully stored at −80 ◦C... Weekly measurements were made to track the level of grape maturity. The Compendium of International Methods of Wine and Must Analysis's official procedures were followed to evaluate the pH, titratable acidity (g L⁻¹ tartaric acid), and total soluble solids content (◦Brix) [29].

 

2.3. UPLC–MS-Based Metabolic Profiling: Did the authors make quantification for all the identified compounds? For all the amino acids, flavones etc.? Please be specific.

We wish to emphasize that we have refrained from elaborating on the procedural details within the manuscript owing to concerns regarding plagiarism. In prior communications, we extensively delineated the procedures, yet the editor prompted us to revise them on the grounds of potential plagiarism. Consequently, we reference earlier research publications produced by our research group, which is engaged in the same area of study pertinent to this field. We kindly invite you to consult the references provided below.

15. Martins, V.; Unlubayir, M.; Teixeira, A.; Gerós, H.; Lanoue, A. Calcium and methyl jasmonate cross-talk in the secondary metabolism of grape cells. Plant Physiology and Biochemistry 2021, 165, 228–238. https://doi.org/10.1016/j.plaphy.2021.05.034
16. Miliordos, D. E.; Alatzas, A.; Kontoudakis, N.; Kouki, A.; Unlubayir, M.; Gémin, M.-P.; Tako, A.; Hatzopoulos, P.; Lanoue. A; Kotseridis, Y. Abscisic Acid and Chitosan Modulate Polyphenol Metabolism and Berry Qualities in the Domestic White-Colored Cultivar Savvatiano. Plants 2022, 11 (13), 1648. https://doi.org/10.3390/plants11131648
17. Billet, K.; Delanoue, G.; Arnault, I.; Besseau, S.; Oudin, A.; Courdavault, V.; Marchand, P.A.; Giglioli-Guivarc’h, N.; Guérin, L.; LaNoue, A. Vineyard evaluation of stilbenoid-rich grape cane extracts against downy mildew: A large-scale study. Pest Manag. Sci. 2018, 75, 1252–1257. https://doi.org/10.1002/ps.5237

2.5. Meteorological Data: I think that it needs italics like the above subsections.

We would like to thank reviewer for mentioning. Therefore the revision was done. Moreover, this paragraph was moved in 2.2 instead of 2.5.

Pg. 5, paragraph 2, line 2: It should be read “ ..TSS, expressed as °Brix…”

We would like to thank reviewer for mentioning. Therefore the revision was done

Total Soluble Solids (TSS; expressed as  ^oBrix)

Regarding the statistical analysis in Table 1 the authors mentioned “different letters in the same column indicate significant differences”. Did the authors mean and in different veraison? I think that this is wrong as during maturation there should be differences. In the same stage of maturation during different treatments I could see or not the significance. But in different stages of maturation the values are quite different. Please explain or make appropriate corrections in Table.

We would like to thank reviewer for mentioning. A very important point is the statistical analysis and how we presented. The current Table is reported to record the differences  in each phenological stage among the different ABA treatments. Hence, the above sentence was added.

According to the t-test at the 5% probability level, different letters in the same column and phenological stage indicate significant differences.

Respectively, in Figures the authors compared in the same way (I mean for one year in different treatments for one stage of maturation). But what about different stages of maturation?

We hold a firm conviction that the necessity for an additional and more complex statistical analysis is rendered unnecessary, particularly because the grape berries exhibit the natural and anticipated trends that can be observed within their development. More specifically, in the event that the alterations under investigation are entirely predictable and exhibit a linear pattern—such as a gradual increase in sugar concentration, a natural augmentation in weight, and a decline in acidity levels—it follows that the utilization of descriptive statistics may prove to be adequate, thereby negating the need for a more complex analytical approach. Furthermore, the primary objective of the research conducted was to examine the influence that abscisic acid exerts on the phenolic compounds present at each developmental stage (veraison, mid veraison and harvest), with a particular emphasis on the critical harvest stage. This specific research objective was inspired by findings presented in prior research articles published in various academic journals.

1. Gómez‐Plaza, E., Bautista‐Ortín, A. B., Ruiz‐García, Y., Fernández‐Fernández, J. I., & Gil‐Muñoz, R. (2017). Effect of elicitors on the evolution of grape phenolic compounds during the ripening period. Journal of the Science of Food and Agriculture, 97(3), 977-983.
2. Paladines-Quezada, D. F., Fernández-Fernández, J. I., Moreno-Olivares, J. D., Bleda-Sánchez, J. A., Gómez-Martínez, J. C., Martínez-Jiménez, J. A., & Gil-Muñoz, R. (2021). Application of elicitors in two ripening periods of Vitis vinifera L. cv Monastrell: Influence on anthocyanin concentration of grapes and wines. Molecules, 26(6), 1689.
3. Martins, V., Lopez, R., Garcia, A., Teixeira, A., & Gerós, H. (2020). Vineyard calcium sprays shift the volatile profile of young red wine produced by induced and spontaneous fermentation. Food Research International, 131, 108983. https://doi.org/10.1016/j.foodres.2020.108983
4. Shahab, M., Roberto, S., Ahmed, S., Colombo, R., Silvestre, J., Koyama, R., & de Souza, R. (2019). Anthocyanin Accumulation and Color Development of ‘Benitaka’ Table Grape Subjected to Exogenous Abscisic Acid Application at Different Timings of Ripening. Agronomy, 9(4), 164. https://doi.org/10.3390/agronomy9040164

In the corpus of all preceding research articles, the authors consistently employed identical statistical analyses to draw their conclusions. The researchers meticulously conducted comparative analyses among the different treatments administered at each phenological stage with the intention of documenting the effects produced by the different bioelicitors. Moreover, an additional significant objective of the study was to ascertain whether the effects observed occurred during the earlier stages of development or manifested later on. Such findings could provide us with invaluable data regarding the developmental processes of grapes and elucidate the implications of varying treatments—namely low and high doses of abscisic acid—on the biosynthesis of phenolic compounds.

I will propose the authors make further statistical analysis, and the new results may be more interesting.

Based on the above I recommend major revision and reconsideration of the manuscript.

 

Reviewer 3 Report

Comments and Suggestions for Authors

Title can be change according to my comments. Rest of manuscript is well organized. Change text step by step according to comments. 

Comments for author File: Comments.pdf

Author Response

Dear esteemed reviewer,

we wish to extend our sincerest gratitude for the invaluable insights and constructive critiques that you have graciously provided regarding our manuscript, as your thoughtful observations have significantly contributed to the enhancement of our research paper. The feedback you offered has undoubtedly strengthened our work, thereby allowing us to better align it with the overarching objectives we set out to achieve in our study. In the following section, you will find a comprehensive outline of the revisions we have implemented in response to your thoughtful comments.

On behalf of the authors

Dimitrios Miliordos

3^rd  reviewer

suggest new title: Grape juice quality and phenolic composition of Greek autochthonous grapevine varieties Mouhtaro under ABA biostimulation

it is insufficient to put in a title "two vintage experiment". In whole manuscript it is explained duration of experiment. Second, it in unnecessary to write grape vine and Vitis vinifera

From my point of view new title it is shorter and present more clear main objective of paper.

We would like to thank reviewer for mentioning. Therefore, in order to present a more clear title and at the same time present the aim of the research we revised the title to the below

“Impact on grape juice quality and phenolic composition of Greek autochthonous grapevine variety, Mouhtaro under abscisic acid biostimulation”

 

Phyto

We would like to thank reviewer for mentioning. Therefore the revision was done.

rewrite: at veraison (at begining and mid) and harvest...

in three phenological stages it is incorect because veraison it is part (begining) of ripening phenological stages. Like bleeding, bud burst, flowering, ripening...

We would like to thank reviewer for mentioning. Hence, the sentence was revised and rewritten

 

“Μήπως καλυτερα” please translate.... maybe better 

We would like to thank reviewer for mentioning. The greek words were erased.

“plant hormones”

We would like to thank reviewer for mentioning. Therefore the term plant hormone was replaced by the phyto hormone.

delete vitis vinifera. Is not need to stay here

We would like to thank reviewer for mentioning

Greek climate condition?

We would like to thank reviewer for mentioning By using the "term" Greek vineyard in this statement, we hope to convey to the readers the distinctive quality of Greek terroir. Therefore we would like to keep the term Greek vineyard.

improve production and grape quality.

We would like to thank reviewer for mentioning. Therefore the sentence was rewritten into the

“ultimately aimed at ensuring and to improveing production the grape quality of grape production in diverse agricultural settings”

rewrite this sentence...

Experiment carried out in a production vineyard at... Tiva, Central Grecee... Vineyard it is located at GPS N... E..., altitude...

We would like to thank reviewer for mentioning. The sentence was written with the below

“Experiment was carried out in productive vineyard in Muses Valley, near the town of Thiva, in Central Greece. Vineyard is located at GPS 38◦19′30.9′′ (N) and 23◦05′37.7′′ (E), at altitude 450 meters”

Vineyard is planted in 2007, training system is .... with 2.5 m between and 1.2 in rows...

We would like to thank reviewer for mentioning. The sentence was revised and rewritten with the below sentence:

Vineyard was planted in 2007, training system is double cordon and planted on R110 rootstocks with a planting density of 2.5 m between and 1.2 in rows.

All experimental trunks was pruned unique

We would like to thank reviewer for mentioning. The sentence in the manuscript was revised and rewritten

All experimental vines were pruned uniquely, each cordon has three spurs.

three treatments and control

We would like to thank reviewer for mentioning. The sentence ”Three distinct treatments were used in the experiment:” in the manuscript was revised and rewritten in order to describe it in details:

Three treatments were compared in the trial, resulting from combination of two levels of  ABA and the control. More specifically

Explain how you define exact this dosses? It is important to understand next discuses effects of this concentration. Why this concentration were not lower or higher? Explain in discussion effect of low or higher dosses....

We would like to thank reviewer for mentioning. Therefore, a sentence explaining the chosen doses.

These doses and the timing were chosen on the basis of previous studies conducted on other red grapevine varieties (20, 21, 22, 23), as well as according to the suppliers’ instruction.

 

…and

ABA was sprayed only in the grape zone. Hence the "and" is not needed.

….grape.

We would like to thank reviewer for mentioning. Therefore, it was added the above in order to be more precise

(all bunches of the experimental vines)

(81–85, based on the BBCH scale)

We would like to thank reviewer for mentioning. Therefore the correct reference was added

Lorenz, D. H., Eichhorn, K. W., Bleiholder, H., Klose, R., Meier, U., & Weber, E. (1995). Growth Stages of the Grapevine: Phenological growth stages of the grapevine (Vitis vinifera L. ssp. vinifera)—Codes and descriptions according to the extended BBCH scale. Australian Journal of Grape and Wine Research, 1(2), 100-103.

please be precise... Random from one cluster side or from ex an in spalier side, side from direct sun or not? It is imoprtant for results

We would like to thank reviewer for mentioning. Therefore the below sentence was added:

The sampled berries were collected on both sides of each of the ten plants, in both direct sunshine and shade.

Calculated or measured on analytical balance or some other balance?

We would like to thank reviewer for mentioning. Therefore the below sentence was added:

…….the fresh wight of the berries was measured on an analytical balance.

This subsection must be replaced after Experiment design (2.2 subsection) 2.5 Meteorological Data

We would like to thank reviewer for mentioning. Therefore, the current subsection was moved according to reviewers’ comment.

 

here is not completely clear that is quality parameters under effect of ABA doses or vintage/climate condition in harvest period...

We would like to thank reviewer for mentioning. Therefore, below paragraph was rephrased in order to be COMPLETELY clear

Specifically, both doses of applied ABA resulted in significantly higher TA values compared to controls. However, significantly different pH values were recorded only in grapes in which they were applied the higher dose of ABA and the effect was dependent on the vintage (i.e., higher and lower pH values compared to control and ABA low dose in 2018 and 2019, respectively) (Table 1). Contrary to the TSS profile among the two vintages, the TA and pH values were higher in 2018 samples compared to 2019 (Table 1).

In particular, as compared to controls, TA values were noticeably greater for both administered ABA dosages. However, only grapes treated with the higher dose of ABA showed noticeably different pH values, and the impact varied by vintage (i.e., higher and lower pH values in 2018 and 2019, respectively, compared to control and ABA medium dose) (Table 1). In contrast to the TSS profile of the two vintages, the 2018 samples had greater TA and pH values than the 2019 samples (Table 1).

 

can be conclude that accumulation of flavan 3-ols it is stronger affected by ABA concentration, term of treatment or berries ripening level?

The application of ABA did not affect flavan-3-ols level at the véraison and middle véraison stages on 2018 but resulted in a significant increase at harvest (Figure 4 A-C). The same trend was also observed during the 2019 vintage with the higher dose ABA-treated berries recording higher flavan-3-ol concentrations than the control and the lower dose-treated at véraison and harvest stages (Figure 4 A-C).

In order to make it clear, we have mention to the discussion the below.Therefore, we strongly believe that ABA treatment impact is stronger.

More specifically, the ABA-treated grapes exhibited increased concentrations of phenolic acids and fla-van-3-ols at harvest stage, regardless of the vintage, while the flavonols level was in-creased upon ABA treatment only the second vintage of the trial. On the other hand, no significant differences to the controls were observed on amino acids, anthocyanins and stilbenes concentrations. Remarkably, BTH application on Mouhtaro grapes had also no effect on anthocyanins but on the contrary, stilbenes level was increased [35] as well as upon ABA application on Savvatiano [28].

Same question... No significant effect between control and ABA treatments, but higher E piceid concetration it is founded with higher harvest stage?

However, no statistically significant differences were observed between control and ABA-treated vines at harvest stage (Figure 5). Worth mentioning, E-piceid content was higher at harvest stage of 2019 compared to the 2018 (Figure 5).

At the middle véraison stage of both vintages, the treatment of both low and high doses of ABA led to greater levels of E-piceid than the control (Figure 5). At the harvest stage, however, there were no statistically significant changes between the control and ABA-treated vines (Figure 5). Notably, the amount of E-piceid was larger at the 2019 harvest stage than it was in 2018 (Figure 5).

It is clear described in the text how the evolution of E piceid during the two vintages (2018- 2019) is going. No significant effect among  control and ABA treatments was observed.

 

please insert your opinion why only at second vintage?Physiology, ABA concentration, interaction with different climate effect or some else...?

The flavonols levels were increased upon ABA treatment only the second vintage of the trial, while

We would like to thank reviewer for mentioning. Therefore, the below paragraph with our opinion was added.

Additionally, the flavonols may benefit from the lower daily temperatures that were recorded throughout the 2019 ripening season. High temperatures are known to reduce the formation of phenolic compounds, and it has been proposed that some secondary metabolites, such as kaempferol and flavonols in general, may serve as markers of warming-related declines in fruit quality. Under mild climatic circumstances, exogenously applied biostimulants can have a positive impact on grape composition.

They are more affected with higher doses because effect it is more evident in more stress condition. The question is how will be effect with more higher doses than aplayed? Do you find in some other references which dosses it is limit?

We would like to thank reviewer for mentioning, and  try to make it clear. We have mentioned it again provide more references in which Authors recorded the same results. We mentioned it at the Discussion section

Earlier research has demonstrated that ABA application increased the concentration of phenolic compounds in grape berries, leading to improved attributes [21, 22, 23, 39, 40, 41, 42, 43]. However, the diverse effects observed in various studies suggest that the timing, the concentration of ABA application and the environmental conditions are critical factors in optimizing phenolic compounds accumulation in grape berries [12, 44, 45].

And the below references recorded the same results like ours

Villalobos-González, L.; Peña-Neira, A.; Ibáñez, F.; Pastenes, C. Long-term effects of abscisic acid (ABA) on the grape berry phenylpropanoid pathway: Gene expression and metabolite content. Plant Physiology and Biochemistry 2016, 105, 213–223. https://doi.org/10.1016/j.plaphy.2016.04.012

Alonso, R.; Berli, F.J.; Fontana, A.; Piccoli, P.; Bottini, R. Abscisic acid's role in the modulation of compounds that contribute to wine quality. Plants 2021, 10(5) 938 https://doi.org/10.3390/PLANTS10050938

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This paragraph can be replaced to Introduction. Half of them it is repeated.

 

Extinguishing geographical and climatic conditions that have supported grapevine growing since ancient times define the Muses Valley in central Greece. The autochthonous red-colored winemaking variety Mouhtaro represents almost the 90% of the local vineyard. It is an early-harvested red variety and the wine produced is characterized by middle to high color intensity, low astringency, while regarding the aroma is character-ized by caramel, strawberry, vegetal notes and forest fruits aromas [32].

The first paragraph is the intro of the Discussion, so we revised it and made it shorter.

Central Greece's Muses Valley is defined by extinguishing climatic and topographical characteristics that have fostered grapevine cultivation since antiquity. Nearly 90% of the area vineyards are Mouhtaro. It is an early/mid- harvested red variety and the wine produced is characterized by middle to high color intensity, low astringency, regarding the aroma is characterized by caramel, strawberry, vegetal notes and forest fruits aromas [32].

This paragraph can be more shorter or delete them because climate explanation it was presented in 3.1 subsection. Also is not main topic of manuscript.

The variation in climatological conditions across the two years at the vineyard site in-fluenced the maturation process, resulting in a noticeable vintage effect on the grape berry physicochemical characteristics at harvest. For instance, the higher rainfall prior to véraison in 2019 vintage resulted in increased berry size compared to the previous vin-tage. Additionally, both control and ABA-treated grapes were characterized by higher TSS levels and decreased TA and pH values compared to 2018 vintage.

We revised and rewrote the second paragraph as well.

There was a discernible vintage effect on the physicochemical properties of the grape berries at harvest as a result of the two years varying climatic circumstances, which af-fected the maturation process. For example, compared to the previous vintage, the 2019 vintage's berries were larger because to the higher rainfall before véraison

 

Is any other varieties with same or similar research? Please compare results if you can provide some extra references. If you include experience of some other authors quality of manuscript will be better.

We would like to thank the reviewer for his comment. Therefore we added the bellow paragraph in order to provide extra data on this topic

Studies have shown that ABA treatment can enhance the content of anthocyanins, flavonols, and stilbenes in various grape varieties. For instance, in Malbec grapes, ABA increased low molecular weight polyphenols and stilbenes like trans-resveratrol and piceid [22). Similarly, in Yan 73 and Cabernet Sauvignon, ABA treatment enhanced anthocyanins and non-anthocyanin phenolics, with varying effects depending on the variety [46,47].

 

22. Alonso, R.; Berli, F.J.; Fontana, A.; Piccoli, P.; Bottini, R. Abscisic acid's role in the modulation of compounds that contribute to wine quality. Plants 2021, 10(5) 938 https://doi.org/10.3390/PLANTS10050938

46           Luan, L. Y., Zhang, Z. W., Xi, Z. M., Huo, S. S., & Ma, L. N. Comparing the effects of exogenous abscisic acid on the phenolic composition of Yan 73 and Cabernet Sauvignon (Vitis vinifera L.) wines. European Food Research and Technology, 2014, 239, 203-213.

47. Xi, Z. M., Meng, J. F., Huo, S. S., Luan, L. Y., Ma, L. N., & Zhang, Z. W. Exogenously applied abscisic acid to Yan73 (V. vinifera) grapes enhances phenolic content and antioxidant capacity of its wine. International Journal of Food Sciences and Nutrition, 2013, 64(4), 444-451.

can you give your opinion about more intensive effect on white then red varieties?

We would like to thank the reviewer for his comment. Therefore, we added the bellow paragraph in order to provide more references and strengthen our opinion.

ABA treatment has been consistently shown to increase anthocyanin content in grape berries. For instance, studies on the Savvatiano and Kyoho grape varieties demonstrated that ABA upregulates genes involved in anthocyanin biosynthesis, such as UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT) and glutathione S-transferase 4 (GST4), leading to higher anthocyanin accumulation [28, , 48, 49. Specifically, ABA treatment was found to enhance the production of petunidin- and malvidin-type anthocyanins, which are associated with deeper berry coloration [50].

28. Miliordos, D. E.; Alatzas, A.; Kontoudakis, N.; Kouki, A.; Unlubayir, M.; Gémin, M.-P.; Tako, A.; Hatzopoulos, P.; Lanoue. A; Kotseridis, Y. Abscisic Acid and Chitosan Modulate Polyphenol Metabolism and Berry Qualities in the Domestic White-Colored Cultivar Savvatiano. Plants 2022, 11 (13), 1648. https://doi.org/10.3390/plants11131648
29. Guo, S.; Zhang, M.; Feng, M.; Liu, G.; Torregrosa, L.; Tao, X.; Xu, T. miR156b-targeted VvSBP8/13 functions downstream of the abscisic acid signal to regulate anthocyanins biosynthesis in grapevine fruit under drought. Horticulture research, 2024, 11(2), uhad293. https://doi.org/10.1093/hr/uhad293
30. Katayama-Ikegami, A.; Sakamoto, T.; Shibuya, K.; Katayama, T.; Gao-Takai, M. Effects of abscisic acid treatment on berry coloration and expression of flavonoid biosynthesis genes in grape. American Journal of Plant Sciences, 2016. 7(09), 1325.

it is better ti rewrite that in next research focus will be on interaction of climate change, soil condition, varieties etc.

The distinguishing grape and wine quality characteristics of a certain variety are determined by the combinatorial action of the genotype and environmental factors, such as soil, landscape and climate. Considering that grapevine is traditionally cultivated in Mediterranean region, the predicted global warming scenario challenges the conventional cultivation methods.

We would like to thank the reviewer for his comment. Therefore, we revised and rewrote all the last paragraph of the discussion in order to be focused on the topic of the journal and of the current research.

The combination of a variety's genetics and environmental conditions, including soil, terrain, and climate, determines its distinctive grape and wine quality traits. Given that grapevines have historically been grown in the Mediterranean region, the anticipated global warming scenario puts existing cultivation techniques to the test. As a result, sustainable agronomic practices may be helpful in reducing the adverse consequences of climate change while maintaining the high standards of the historically grown cultivars. Utilizing plant hormones and bioelicitors in viticulture provides a sustainable way to reduce the need for pesticides and fertilizers while improving the qualitative character-istics of grape berries in an eco-friendly way.

Rewrite conclusion according to changing in whole manuscript.

Biostimulant applications are beneficial and effective viticultural practices for enhancing the quality of grapes and wine, especially in light of the challenging climate changing situation. However, determining the optimal timing and concentration of their application is essential. In our two-vintage trial, ABA treatment resulted in decreased size and TSS and on the other hand, increased levels of phenolic compounds such as phenolic acids, flavan-3-ols and flavonols. Therefore, ABA application could be considered as a promising agronomic strategy for the enhancement of grape berry quality. Further comprehensive studies, incorporating a wider range of indigenous grapevine varieties and local climate conditions, are needed clarifying grapevine reactions to various biostimulants and their unique impacts on each cultivar's metabolic profiles requires taking into account a greater variety of indigenous grapevine varieties and local climate circumstances.

We would like to thank the reviewer for his comment. Therefore, we revised the Discussion paragraph and added the below text.

Applications of biostimulants are advantageous and successful viticultural tech-niques for improving grape and wine quality, particularly in view of the difficult climate change scenario. But it's crucial to ascertain the best time and focus for their use. While ABA treatment reduced size and TSS in our two-vintage study, it also enhanced levels of phenolic compounds, including flavonols, phenolic acids, and flavan-3-ols. As a result, using ABA may be regarded as a viable agronomic sustainable approach to improve the quality of grape berries.

 

Author Response File: Author Response.pdf