Agronomic Response of 13 Spanish Red Grapevine (Vitis vinifera L.) Cultivars under Drought Conditions in a Semi-Arid Mediterranean Climate

Round 1

Reviewer 1 Report

This paper discusses the issue related to drought stress in viticulture. Specifically, 13 Spanish vineyards were analyzed in terms of must carbon isotope ratios and agronomic parameters, with the aim to identify which cultivars are most resistant in arid environment. Strength of the article is to have combined many different analyses to get a complete understanding of the plant's physiological response.

General concept comments

I have a principal doubt concerning the methodology of isotopic carbon measurements: the authors say they measured d13C on the must sugars, but in the materials and methods section it is written that they measured directly the whole must. To measure the isotopic signal of sugars, these should be extracted from the must itself and then be measured. It is true that recent studies have shown that there is a strong correlation between the isotopic signal of must sugars and bulk must (see Damiano et al., 2020), but in the text the authors persist in referring to must sugars. Could this point be clarified?

Furthermore, some concepts related to the interpretation of the d13C should be better discussed in the text (see specific comments below).

Specific comments 

P2 L72 I would recommend the inclusion of some bibliography regarding the significance of measuring carbon isotope ratios in must sugars, maybe moving the part in materials and methods (P4, L 129-132) and expanding the explanation about it.

P2 L86 It would be helpful to include a photo or a scheme of how the cultivars are arranged

P3 L114 Irrigation was done with local water, I suppose. Is it right?

P6 L202-203 Please add some references to support this statement

P7 L225-226 Please add some references to support this statement

P9 L275-276 You could add your reference 38 for this sentence

P10 L316-317 The authors exclaim that the oxygen isotope signal of their samples is comparable to that of other non-irrigated cultivars from the same region. But themselves indicated that the cultivars they analyzed were irrigated during particular periods (P3 L114). How can they explain this?

P13 L407-410 Could the authors explain this final concept better, please?

Author Response

Dear reviewer,

The authors thank you very much for your valuable comments. They were considered in the preparation of the new manuscript. The text was modified according your comments.

Regarding your comments, the following were corrected as recommended.

General concept comments

I have a principal doubt concerning the methodology of isotopic carbon measurements: the authors say they measured d13C on the must sugars, but in the materials and methods section it is written that they measured directly the whole must. To measure the isotopic signal of sugars, these should be extracted from the must itself and then be measured. It is true that recent studies have shown that there is a strong correlation between the isotopic signal of must sugars and bulk must (see Damiano et al., 2020), but in the text the authors persist in referring to must sugars. Could this point be clarified?

Although it is true that it is not the same to measure directly d13C in the whole must as in must sugars extracted from the must, it is generally assumed that most of the carbon obtained in the combustion of the whole must comes from the sugars. For this reason, reference was made in the manuscript to the sugars in the must. However, we fully agree with you, in this paper it is more correct to talk about d13C of the whole must, rather than the extracted sugars. This correction is already shown in the new version of the manuscript.

Specific comments 

P2 L72 I would recommend the inclusion of some bibliography regarding the significance of measuring carbon isotope ratios in must sugars, maybe moving the part in materials and methods (P4 L 129-132) and expanding the explanation about it.

As explained in the previous comment, in the new version of the manuscript we have changed the term ‘must sugars’ to ‘must’. Therefore, we think that it is no longer necessary to include new bibliography about measuring carbon isotope ratios in must sugars.

P2 L86 It would be helpful to include a photo or a scheme of how the cultivars are arranged

With all due respect, we do not think it is necessary to include one more figure with a photo or a scheme of how the cultivars are arranged, as it is sufficiently explained in the material and methods section (P2 L86-89). It would be a very simple scheme with lines of 140 vines per cultivar, arranged in parallel.  However, if you think it is necessary, we can include it.

P3 L114 Irrigation was done with local water, I suppose. Is it right?

Yes, you are correct. Irrigation was done with local water from an aquifer well. This explanation has been included in the new version of the manuscript. 

P6 L202-203 Please add some references to support this statement

We assume that you are referring to the figure that supports this statement. A reference has been added.

P7 L225-226 Please add some references to support this statement

As in the previous paragraph, we assume that you are referring to the figure that supports this statement. Two references have been added in the new version.

P9 L275-276 You could add your reference 38 for this sentence

Thank you very much for your suggestion. The reference 38 supports this sentence. It has been added.

P10 L316-317 The authors exclaim that the oxygen isotope signal of their samples is comparable to that of other non-irrigated cultivars from the same region. But themselves indicated that the cultivars they analyzed were irrigated during particular periods (P3 L114). How can they explain this?

In the area where the study was carried out, irrigation water volumes are limited by legislation to 1,350 mm throughout the season. A water volume of 30 mm represents 2.22% of the volumes usually used by the winegrowers in the area and therefore, it can be assumed that the vineyard is rainfed. Three small irrigations of 10 mm each at specific times (after fruit set, when the branches stopped growing, and when the berries reached pea-size) are justified by the need to guarantee the survival of the vines during the three years of the study.  

P13 L407-410 Could the authors explain this final concept better, please?

By this sentence, we mean that genetics of cultivars influence their response to drought. Different cultivars with similar d13C values result in yields with different characteristics, both in terms of yield and quality parameters. Therefore, it is not possible to predict the yield characteristics of cultivars based on their water use efficiency (d13C).

Reviewer 2 Report

This is a sound and needed research. Of course, it would be strengthened and the results would be more robust with more seasons' data ...using the same cultivars, perhaps in another/additional location 

Author Response

Thank you very much for your comments, wich will be taken into account in future studies. The authors would also like to thank for their kind and favorable opinions about the article.

Reviewer 3 Report

The article presents scientific merit and innovation, addressing studies of great importance, which is the adaptation of the vine to drought, especially in climate change scenarios. The information presented can make a valuable contribution to the viticulture of Castilla-La Mancha region in Spain. I didn't find errors or other problems in the manuscript. The text is very clear and properly written, fulfilling all the requirements to be published.

Author Response

Thank you very much for your comments. The authors would also like to thank for their kind and favorable opinions about the article.

Round 2

Reviewer 1 Report

General concept comments

I really appreciate that the authors removed the word “sugars” from the text, since they had analyzed the whole must in their work.

Specific comments

P2 L72 I would recommend the inclusion of some bibliography regarding the significance of measuring carbon isotope ratios in must sugars, maybe moving the part in materials and methods (P4 L 129-132) and expanding the explanation about it.

As explained in the previous comment, in the new version of the manuscript we have changed the term ‘must sugars’ to ‘must’. Therefore, we think that it is no longer necessary to include new bibliography about measuring carbon isotope ratios in must sugars.

What I meant was that there was little explanation of the concept of stable isotopes, whether they were measured in the must sugars or in the whole must. Therefore, when authors are talking about the significance of the higher or lower d13C value, no appropriate physiological explanation has been given upstream. In my opinion, something more could be written about this in the introduction.

P6 L202-203 Please add some references to support this statement

We assume that you are referring to the figure that supports this statement. A reference has been added.

P7 L225-226 Please add some references to support this statement

As in the previous paragraph, we assume that you are referring to the figure that supports this statement. Two references have been added in the new version.

Concerning these last two points: no, I did not mean that authors have to include the reference to the figure, but a bibliographic reference related to the results obtained. The sentences I was referring to are the following (corresponding to the Page and Line that I indicated regarding to the first manuscript version):

“Garnacha Tintorera exhibited the highest mean δ13C, indicating that this cultivar is very efficient in water use”.

“Tinto Velasco had the lowest mean δ18O (8.12 ‰), indicating that this cultivar had the lowest transpiration rates during the seven days before harvest”

All these of my suggestions are again in the context of wanting to give a better explanation of the isotopic results.

The remaining adjustments to the text, in response to my comments, are fine 

Author Response

Dear reviewer,

The authors again appreciate your valuable comments and appreciate your patience. Their suggestions have been considered in the preparation of the new manuscript. The text was modified accordingly.

Comments and suggestions for authors

Comments on general concepts

I really appreciate that the authors have removed the word “sugars” from the text, since they had analyzed all the must in their work.

Specific Comments

Q2 L72 I would recommend the inclusion of some literature on the importance of measuring carbon isotope ratios in wort sugars, perhaps moving the part under materials and methods (Q4 L 129-132) and further explaining it.

As explained in the previous comment, in the new version of the manuscript we have changed the term 'must of sugars' to 'must'. Therefore, we feel that it is no longer necessary to include new literature on the measurement of carbon isotope ratios in wort sugars.

What I meant to say is that there was little explanation of the concept of stable isotopes, whether they were measured in the sugars in the wort or in the whole wort. Therefore, when the authors talk about the importance of the higher or lower value of d13C, an adequate physiological explanation has not been given. In my opinion, something more could be written about this in the introduction.

(P4 L129-132) moved from materials and methods to introduction and added physiological explanation.

P6 L202-203 Add some references to support this statement

We assume that he is referring to the figure that supports this statement. A reference has been added.

P7 L225-226 Add some references to support this statement

As in the previous paragraph, we assume that you are referring to the figure that supports this statement. Two references have been added in the new version.

Regarding these last two points: no, I did not mean that the authors have to include the reference to the figure, but rather a bibliographical reference related to the results obtained. The sentences to which I referred are the following (corresponding to the Page and Line that I indicated with respect to the first manuscript version):

"Garnacha Tintorera exhibited the highest average δ13C, which indicates that this variety is very efficient in the use of water."

“Tinto Velasco had the lowest mean δ18O (8.12 ‰), indicating that this cultivar had the lowest transpiration rates during the seven days prior to harvest”

Regarding these phrases, some bibliographical references have been added to support them.

All of these of my suggestions are again in the context of wanting to give a better explanation of the isotope results.

The remaining adjustments to the text, in response to my comments, are fine.